47 ¢#?TT200 TOEO O 


ACEI 


IOHM/18IN 


& dit if 


PeRICULIURAL 
Ee CTERIOLOGY 


BY 


JOSEPH E. GREAVES, M.S., Px.D. 


PROFESSOR OF AGRICULTURAL BACTERIOLOGY AND PHYSIOLOGICAL CHEMISTRY IN UTAH 
AGRICULTURAL COLLEGE; CHEMIST AND BACTERIOLOGIST IN UTAH 
EXPERIMENT STATION. 


ILLUSTRATED WITH 48 ENGRAVINGS 


LEA & FEBIGER 
PHILADELPHIA AND NEW YORK 


CopyRIGHT 
LEA & FEBIGER 
1922 


PRINTED IN U.S. A. 


DEDICATED 


IN LOVING REMEMBRANCE OF 


PERNECY 


WHO DURING OUR FEW SHORT YEARS TOGETHER 
CONSTANTLY ENCOURAGED AND 


INSPIRED ME 


- 


PREFACE. 


THE organisms considered in agricultural bacteriology are 
specifically the most numerous, chemically the most active, and 
economically the most important known. This being true, why is 
so much interest shown in the injurious and so little in the beneficial 
bacteria? There are two chief reasons for this condition. When 
an outlaw commits some crime against human society it is heralded 
far and near and the machinery of the law is set in operation to 
apprehend the culprit and bring him to justice. So it is with these 
outlaws in bacterial society. The typhoid, or perchance some other 
disease-producing organism, attacks some individual, or it may be 
an entire community. Ifit be typhoid, we hear of the long-drawn- 
out fight between the human individual on the one hand and the 
invisible enemy on the other. If disease be not checked it spreads 
to other places, and, as in the Dark Ages, sweeps like a prairie-fire 
over a whole continent or, as recently, over the entire world. ‘The 
second reason why we hear more of the disease-producing organisms 
than we do of the beneficial bacteria is that man has learned that 
it is a fight between him and these microbes to determine which 
shall inherit the earth. He has learned that he must protect him- 
self against these enemies. For these reasons man has studied the 
bacterial outlaw, his place and condition of growth. 

On the other hand, though we admire the magnificent structures 
and complex institutions which have been reared by the mind and 
hand of men, we see and pass on. In many cases we do not stop to 
contemplate the countless millions, living and dead, who have 
contributed their mite that things might be as they are. Man does 
not have to protect himself against these honest toilers; hence, they 
go unnoticed. The work of the benefactor lacks the sensationalism 
which is attached to that of the destroyer. So.it is with the count- 
less billions of beneficial bacteria; they toil on day and night, 
generation after generation, accomplishing good for the human race. 
We do not miss them, for they have always helped us. They never 
become discouraged, but work for our good until conditions become 
intolerable, when they die to be in many cases replaced by the 
bacterial outlaw. 

If the following pages help to systematize, to arouse interest, to 
stimulate curiosity or inquiry in even a small degree in this intensely 


vi PREFACE 


interesting and practical subject, the author will feel that his labors 
have not been in vain. 

It is coming to be recognized that agricultural bacteriology and 
agricultural chemistry are at many points intimately associated. 
Hence, the writer has presupposed a knowledge of elementary 
chemistry on the part of the student. However, most of the more 
complex equations have been grouped in one chapter so they may 
be used or omitted as the teacher sees fit. 

It has been more a question of what to exclude than what to 
include. However, the writer has been guided throughout by the 
needs of the student of agriculture, and hence where good, complete 
volumes are available, as is the case with milk, water, sewage, and 
some other subjects, a bare outline is given; so the student should 
consult other works for a more exhaustive treatment. But in the 
case of soils an effort has been made to go more into detail. Even 
in these chapters, however, no attempt has been made to review all 
of the literature. 

In the preparation of this work I have drawn freely from all 
available sources. Much of the material was first written with a 
complete reference to the literature, but it soon became apparent 
that such a procedure would produce a work too large for the purpose 
for which this was written. Hence, all references have been elimi- 
nated. There are, however, listed at the end of most chapters, a few 
select works given in most cases because of the references which 
they contain, and it is to these that the student is referred for 
further details. At the end of the last chapter is given a list of 
additional works which have been consulted in the preparation of 
this book. 

To my friends and colleagues my hearty thanks are offered for 
the valuable encouragement and assistance given in the preparation 
of this book. I am under particular obligation to President E. G. 
Peterson, Dr. I. S. Harris, Dr. B. L. Richards, Professors George 
Stewart, C. T. Hirst, and E. G. Carter for reading parts or all 
of the manuscript and offermg many helpful suggestions, also to 
Mrs. Blanche C, Pittman for her painstaking care in the preparation 
of the manuscript for the press. 

J. E.G. 


Logan, Utan, 1922. 


CONTENTS. 


CHAPTER I. 


DEVELOPMENT OF BACTERIOLOGY 


Spontaneous Generation 
Fermentation 

Smallpox 

Anthrax . 

Other Work of Pasteur 
Other Plagues oes ; 
Lister 

Yellow Fever : 
Agricultural Bacteriology : 
Future Work 

References 


CHAPTER II. 


BAcTERIA AND THEIR PLACE IN NATURE 


Definition of Bacteria . Z 
Divisions of Plant Kingdom . 
Occurrence of Bacteria 

Role of Bacteria in Nature 
Divisions of Bacteriology . 


CHAPTER III. 


MorpuHouoey or BACTERIA 
Bacilli 
Cocci 
Spirilla 
Gradations . 
Pleiomorphism . 
Involution Forms 
Size and Weight 
Brownian Movement 
Organs of Locomotion 
Cell Wall ee ee 
Capsules : 
Sheath 
Zobglea 
Cytoplasm : 
Metachromatic Graniles : 
Spores 4 
Longevity De enters ; 


aT 
Deo 


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44 
45 


Vill CONTENTS 


CHAPTER IV. 


CLASSIFICATION OF BACTERIA 


Migula Classification : 
International Rules of Botanicas Nomenclature 


Classification of American Bacteriological Association 


The Class Schizomycetes 
Order Myxobacteriales . 
Order Thiobacteriales : 
Order Chlamydobacteriales 
Order Actinomycetales . 
Order Eubacteriales 


CHAPTER V. 


CoMPOSITION OF BACTERIA 


Elementary Composition . 
Moisture. : 
Organic Constituents 
Carbohydrates 
Extractives 
Proteins 
Inorganic Gonstitupnts: 
Variation in Composition of Dikerent Parts of Cell 
References 


CHAPTER VI. 


Foop REQUIREMENTS 


Minimum Requirements 
Maximum Requirements . 
Function of Food 

Source of Energy 
Moisture 

Osmotic Pressure 

Kinds of Food oe 
Carbon 

Nitrogen 

Hydrogen 

Sulphur . 

Phosphorus . 

Potassium 

Other Inorganic ‘Substances 
Oxygen Requirements . 
Vitamines . . 
References 


CHAPTER VII. 


BACTERIAL METABOLISM ENZYMES 


‘ 


Early Theories of Fermentation . 
Definition of Enzymes 
Terminology 

Properties of Enzymes. 
Classification : 
Hydrolytic Enzymes 

Oxidizing Enzymes 

References : 


46 
49 
50 
50 
50 
50 
50 
50 
52 


58 
58 
58 
58 
58 
59 
61 
61 
62 


CONTENTS 


CHAPTER VIII. 


BacTERIAL METABOLISM PRODUCTS 


Physiologic Classification . 
Carbohydrate Metabolism 
Acid Production : 
Acetic Acid 
Lactie Acid 
Butyric Acid : 
Other Acid Fermentations 
Oxidation of Organic Acids 
Fats F 
Products from Nitrogenous Compounds 
Indol and Skatol . 
Amins 
Pigments 
Chromophorous Bacteria ; 
Chromoparous, or True Pigment- fornncs Bacteria j 
Parachrome Bacteria 
Heat 
Light 
References 


CHAPTER IX. 


INFLUENCE OF TEMPERATURE AND LIGHT ON BACTERIA 


Temperature and Speed of Reaction 
Relation to Heat : ‘ 
Thermophilic or eat loving Bacteria ; 
Psychrophilic Bacteria 
Mesophilic Bacteria 
Thermal Death Point . 
Cold 
Light 


CHAPTER X. 


EFFECT OF OTHER AGENTS ON BACTERIA 


Radium Rays 
Roéntgen Rays 
Electricity 
Drying 

Osmotic Pressure 
Pressure . 
Shaking . 


CHAPTER XI. 


EFFECT OF CHEMICALS ON BACTERIA 


Chemotaxis . 
Disinfectants : 
Laws Governing the Actions ‘of erica nhs 
Disinfectants of the Chlorine Group 
Formaldehyde ake 
Sulphur Dioxide 
Hydrocyanic Acid Gas 
Mercuric Chloride . 
References . . 


ix 


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x CONTENTS 


CHAPTER XII. 


INFLUENCE OF ARSENIC ON BACTERIAL ACTIVITY 


Occurrence of Arsenic. . RE So Pe OR) ao Peete fee cathy se BIE 
Factors Influencing Solubility co ge ee a Ole oe eA it NETS 
Ammonifiers Sates Slane RPE er isk 8 are MENS neh gt ae ig BS | 
Nitrification cea ee NS SS ie SE eee a eee oo eae 
Nitrogen Fixation .. Bye A ee a Rese eee 2 () 
How Does the Arsenic ‘Act? oth co i SO AR ae ae rns iti Pon ee Raeots 21.2) 
References. 3. *so."se eee ot sts SO See 


CHAPTER XIII. 


Errect oF HEAT AND VOLATILE ANTISEPTICS ON Sort BAcTERIA 


Influence on Plant. . Re Kae ORE ASS ee ere ee 
Effect on Properties of Soil ; iNet Bai ce, mm, 19.1: 
Hypotheses to Account for Observed Phenomena Ut irae dace tee cen IIS i3° 
Koch's" Direct'Stimulation: “@heory sie wee ane salt es eel oes 
Hiltner and Stormer’s indirect) Dheory . 9 ~) 022s) a ee ees 
Russell and Hutchinson’s Protozoan Theory . . . . . . . 1385 
Greig-Smith’s Bacteriotoxin id Me es ae elena, ee ils 
References . . . . RE sae orate ie si cagi ree hes 115%.) 


CHAPTER XIV. 


INFLUENCE OF SALTS ON THE BACTERIAL ACTIVITIES OF THE SOIL 


Calcium Carbonate? 42 yal d ie ec ok Sn Sea sheen eee eo 
Lime ee en ene OUP ae ie! Ma Ne ETS ort Tan kak Otay eeipy aml OE 
Gypsum. . CE Scie ee Mace hs ROY, pier ead rete Sheen nae aL ae 
Calcium Chloride Fetes at ME aon Soe SE en Lh eee sea pe er se Mer ar Foe 57D 
Tron: Sulphate: bese. ete bo pee it cg gk ere eer oer wa eee 
IMsonesium: Sal tsi... : 2s Sak ee ae cee tees hep ot ton ee oaks eee eT hy 
Manvcanese st aia et 5 Si ys Boner a te ee eee sores et 
Potassium Salts ee eet hr ar mn RW WP es Pate ee Ter O Stra mya al li 
Sodium Salts . . Fe, nM RI Rites ieee | Rede 2 tlie ne Bee Ba [155 
Variation in Effect Produced . Phan Cr oo ae ae Ae eens ten AG 
Stimulating Action nik Reaper Tee Ne felt ASL i ba Sern MU ce mee LAL7) 
‘Toxicityiof Various Salts 4-0. |* ne) fe om co aoe neat on I eee 
Reference a0 eat ei Pe ee ere Ls eee 


HAPTER XV.C 


INFLUENCE OF MANURE ON THE BACTERIAL ACTIVITIES OF THE SOIL 


INumiber-*./j.2 7: RENE en a reli EF LUGE 
Ammonification and N itrification APRA tree Aes TSR mE y Ble: 
Loss ‘of Nitrates se Cres ee Os, ee cece nL ches 
Green Manures fo age UT REE ge Dee Sul a ee a eae ee 115 
Reference. <0 -. 502. ae Re ea ee en Oo 


CHAPTER XVI. 


Tue Sort FLtora 


Koch Gelatin-Plate Method . . «DL a See a sae GO) 
Hiltner and Stérmer Dilution Method » = Rae eae ee OO 
Defects of Plate Method . . . ac.) we get Ade a ee hase GO 


Value of Bacterial Counts Pe een es ern nd REO oe Mate IL 


CONTENTS 


Number of Bacteria in Soil 
Factors Influencing Number . 
Kinds of Microérganisms in Soil . 
B. megatherium de Bary . 
Morphology ; 
Cultural Characteristics 3 
Physiology aa 8 

B. mycoides Fliigge, 1886 
Morphology . . - 
Cultural Characteristics ‘ 
Physiology ; 

B. cereus Frankland, 1887. 
Morphology : 
Cultural Characteristics , 
Physiology s k 

Ps. fluorescens (Fliigge) Migula : 
Morphology : : 
Cultural Characteristics : 
Physiology 

Actinomyces 

Reference 


CHAPTER XVII. 


MINERALIZATION AND SOLVENT ACTION OF BACTERIA 


Bacteria as Soil Formers 
Calcium Carbonate 
Phosphorus . 

Sulphur . 

Iron . 

Potassium 

References 


CHAPTER XVIII. 


THE CARBON, NITROGEN, SULPHUR AND PHOSPHORUS CYCLES 


The Carbon Cycle . 
The Nitrogen Cycle 
The Sulphur Cycle 
The Phosphorus oo 
References 


CHAPTERS XIX. 


PUTREFACTION, FERMENTATION AND DECAY 


Definitions . 

Active Agents 3 ; 
Chemistry of the Process : 
References : 


CHAPTERS XxX. 


AMMONIFICATION 
Species and Distribution . 
Methods Sucks 
Material Aramoniteds 


Influence of Soil and Climatic Conditions 


Moisture 

Aération : : 

Lime and Magnesium : 
Phosphorus. . 

Chemistry of the Process: . 
HRELCTENCEB I shi. 6. betes Cs! its 


Xi CONTENTS 


CHAPTER XXI. 


NITRIFICATION 
Early Theories. . g 
The Dawn of the Biological Theory : 
Isolation of Nitrifying Ferments . 
Distribution aia tear te 
Reaction of Media . : 
Food Requirements of Nitrifiers : 
Organic Matter 
Energy 
Metabolism . 
Morphology Soe 
Influence of Moisture . 
Temperature 
Light Rays . : 
Aération and Cultivation ¢ 
Crop and Fallow 
Season . 
Quantity of Nitrates F ommicue 
Loss of Nitrates a oe 
References 


CHAPTER XXII. 


DENTRIFICATION 
Early Theories . 
Organisms oncoorsdes : 
Reaction of the Media 
Food Requirements : 
Metabolism of Denitrifying Organisms : 
Influence of Water : 
Temperature ‘ 
Losses of Nitrates om Mantire and ‘Soil 
Function of Denitrifiers : 


References 
CHAPTER XXIII. 
AZOFICATION 
Historical 
Distribution 


Reaction of the Media : ; 

Food Requirements of the Azofiers ; 

Organic Soil Constituents 

Influence of Colloids 

Sources of Energy for the Azobactéer 

Manure. . : 

Metabolism of A OVGbRUtER Ae: 

Pigments Produced by Azotobacter . . 
Morphology of the Nitrogen-fixing Organisms : 
Methods 

Relation of Azotobacter to Other Organisms 
The Influence of Water 

Temperature. 5 

Light and Other Rays 

Aération : 

Season 

Crop . 

Climate . 

Relationship of eee to Nitrate ‘Acourmmulations 
Soil Inoculation INN dealt ake 

Soil Gains in Nitrogen 

Reference 


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284. 
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287 
289 


CONTENTS 


CHAPTER XXIV. 


SymBiotic NiItrRoGEN FIXATION 


Early Theories . 6 
Early Observations on Boot Tubercles : 
Species .. es é 
Cultural Characteristics 

Morphology of the Colonies 
Morphology of the Bacteria 

Staining . REPL tee 

Bacteroids 

Mode of Fintravice inte: the Host 
Growth of the Nodule 

Relationship to Host : 

Mechanism of Fixation (Metabolism) 
Chemical : 4 | eae ere § 
Source of Energy 

Aération 

Moisture 

Temperature 

Influence of F ertilizers ; 
Legumes Associated with Nowe legumes 
Soil Gains in Nitrogen 5 
Soil Inoculation 

Method Involving the Use of One @onmercial Culture 
Alternative Method 

Commercial Cultures 

References 


CHAPTER XXvV. 


Crop RorTatTion 


Essential Elements : 

Element Added by Legumes : 

Nitrogen y : 

Rothamsted Rotation ; 

Nitrogen Obtained from Atmosphere bee Legumes: 
Distribution of Nitrogen in Legumes : 
Legumes Feed on Nitrates 

Nitrification in Soils d 

How to Maintain Soil Nitrogen : 

References Letts ee 


CHAPTERS XXVI. 


CELLULOSE-DECOMPOSING ORGANISMS 


Cellulose 

Early Observations’ 

Work of Omelianski 

Morphology and Physiology e 
Later Work on Cellulose Wemlcntarion : 
Function 

References 


CHAPTER XXVII. 


BAcTERIA IN AIR 


How Bacteria Enter Air 

Number and Kind ; 

Factors Governing Number anil Kind 
Bacteria in Inspired and Expired Air 
Air-borne Infection 


xi 


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X1V CONTENTS 


CHAPTER XXVIII. 


WATER BACTERIOLOGY 


Classification of Waters ; 
Numbers of Bacteria in Waters . 
Sedimentation 
Light 
Temperature 
Food é 
Classes of Bacteria. 

Soil Bacteria 

Intestinal Bacteria 
Natural Purification of Water 
Artificial Purification of Water 
Chemical Methods : 
Ice : 


CHAPTER XXIX. 


WATER AND DISEASE 


Disease First Definitely Proved as Due to Water . 
Amount of Sickness Due to Water ade 
The Mills-Reincke Phenomenon . 

Cholera . . 

Typhoid 

References 


CHAPTER XXX. 


SEWAGE AND SEWAGE DISPOSAL 


Source, Composition and Quantity of Sewage . 
Bacteria in Sewage ta Daath eM 
Hydrolyzing Bacteria . 

Oxidizing Bacteria . 

Reducing Bacteria . 

Pathogenic Bacteria 4 

Necessity of Sewage Disposal’ : 

What Should Be eee in | Sewage Disposal? 
Methods of pia 

References : 


CHAPTER XXXTI. 


Mitk BActTERIOLOGY 


Milk as a Food 
Classes of Milk 
Bacteria in Milk 


Initial’ Contammatione, |i ace is Soe ok) fe. eee 


Growth of Bacteriain Milk . . ; 

Changes Produced in Milk by Bacteria . 

Abnormal Changes in Milk : 

Classes of Bacteria z 
Acid-forming Bacteria 
Peptonizing Bacteria . 
Bacteria Producing Milk of Unusual Cheracter 
Inert Organisms as 3 : 
Pathogenic Bacteria . 


351 
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CONTENTS 


CHAPTER XXXII. 


MILK AND DISEASE 


Sources of Infection 

Character of Milk-borne Diseases 
Extent of Milk-borne Disease 
The Tuberculin Test 
Pasteurization 

References 


CHAPTER XXXIII. 


BAcTERIA IN OTHER Foops 


Bacteria in Butter . 

Bacteria in Cheese . 

Bacteria in Ice Cream ~ee 
Bacteria in Condensed Milk . 
Bacteria in Bread : 
Bacteria in Eggs 

Bacteria in Meat 

Bacteria in Canned Foods. 
References 


CHAPTER XXXIV. 


BACTERIA AND FOOD-POISONING 


Classes of Food-poisoning 
Poisonous Foods 

Metallic Poisons 5 

Animals Suffering from Tasares 
Typical Paratyphoid Outbreaks . 
Offending Foods s s 
Human Infection 

Ptomain Poisoning 


etree aie: Cee gl eo a es 


Prevention . 
References 


CHAPTER XXXV. 


PRESERVATION OF Foop 


Methods of ers Food . 
Cold ; 
Drying . 

Pressure 

Canning 

Sugar and Salt 
Chemical Preservatives 
References 


CHAPTER XXXVI. 


BACTERIA IN THE ARTS AND INDUSTRIES 


Alcoholic Fermentation 
Vinegar . 

Sauerkraut . 

Ensilage . 

Retting . 

Tanning . 

Vaccines 

References 


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AGRICULTURAL BACTERIOLOGY. 


QAP EE RT; 


DEVELOPMENT OF BACTERIOLOGY. 


a 


 NowHERE in the whole realm of human endeavor has research 
been crowned with more glorious achievements, at least in so far 
as the welfare of the human race is concerned, than in the field of 
bacteriology, and this in face of the fact that bacteriological research 
had a most humble and recent origin. ( Even the dawn of bacteri- 
ology dates back only to the last quarter of the seventeenth century 
to the time when a Dutch linen-draper, Anton van Leeuwenhoek, 
spent his leisure time in grinding lenses. He became so proficient 
in this that his lenses were superior to any made before. Turning 
them on various substances—raindrops, saliva, and many putrifying 
things—he found in all these living, moving forms, which prior to 
this time had been unrecognized. We can imagine his joy and 
surprise from this statement: “I saw with wonder that my 
material contained many tiny animals which moved about in a 
most amusing fashion. The largest of these A (Fig. 1) showed 
the liveliest and most active motion, moving through the water 
or saliva as a fish of prey darts through the sea; they were found 
everywhere, although not in large numbers. A second kind was 
similar to that marked B (Fig. 1) which sometimes spun around 
in a circle like a top. ‘These were present in larger numbers and 
sometimes described a path like that shown in C to D (Fig. 1). A 
third kind could not be distinguished so clearly; now they appeared 
oblong, now quite round. They were so very small that they did not 
seem larger than the bodies marked E, and besides they moved so 
rapidly that they were continually running into one another. They 
looked like a swarm of gnats or flies dancing about together. I had 
the impression that I was looking at several thousand in a given 
part of the water or saliva mixed with a particle from the teeth no 
larger than a grain of sand, even when only one part of the material 
was added to nine parts of water or saliva. Further, the greater 
part of the material consisted of an extraordinary number of rods, 
of widely different lengths, but of the same diameter; some were 

2 


“= 


18 DEVELOPMENT OF BACTERIOLOGY 


curved, some straight, as is shown in F; they lay irregularly and 
were interlaced. Since I had previously seen living animalcules of 
this same kind in water, I endeavored to observe whether there was 
life in them, but in none did I see the smallest movement that might 
be taken as a sign of life.” 

This patient worker, supplied with his crude microscope, gives a 
fairly accurate description of these minute forms of life. But this 
did not awaken the world to even a faint realization of the wonderful 
invisible forms of life which were present in everything and were 
always working for good or evil. It did, however, revive a discussion 
which had waxed long and furious as to whether life can spring 
spontaneously from inanimate matter or whether it is the descendant 
of preéxisting living organisms. - 

A= D 


Fig. 1.—The first drawings of bacteria by Leeuwenhoek. ‘The dotted line C—D 
indicates movement of the organism. (Morrey.) 


Spontaneous Generation.— Back in the sixteenth century a famous 
physicist ahd chemist, van Helmont, stated that mice can be 
spontaneously generated by merely placing some dirty rags in a 
receptible together with a few grains of wheat or a piece of cheese. 
The same philosopher’s method of engendering scorpions is also 
amusing. 

“Scoop out a hole in a brick, put into it some sweet basil. Lay 
a second brick upon the first, so that the hole may be imperfectly 
covered. Expose the two bricks to the sun, and at the end of a few 
days the smell of the sweet basil, acting as a ferment, will change 
the herb into a real scorpion.” 

An Italian, Bouonami, tells of a wonderful metamorphosis which 
he had witnessed. Rotten timber, rescued from the sea, produced 
worms; these gave rise to butterflies; and strangest of all, the butter- 
flies became birds. 

Everyone thought it a self-evident fact that maggots sprang 
spontaneously from decomposing meat or cheese, until an Italian 


SPONTANEOUS GENERATION 19 


poet and physician, Redi, took the simple precaution of screening 
the mouth of jars containing meat so that flies could not enter. 
Flies were attracted by the odor and deposited their eggs on the 
gauze, and it was from these that the so-called “worms” arose. 

The theory of the spontaneous generation of mice, scorpions, and 
maggots had been proved untenable. But how about these micro- 
scopic organisms? They surely could develop directly from organic 
material. For now anyone provided with this new instrument, the 
microscope, could easily demonstrate for himself the spontaneous 
generation of microscopic eels in vinegar, or produce myriads of 
different and interesting living creatures in simple infusion of hay 
or other organic material. 

Needham, a Catholic priest, evolved the theory that a force called 
“productive” or “vegetative” existed which was responsible for 
the formation of organized beings. The great naturalist, Buffon, 
elaborated the theory that there were certain unchangeable parts 
common to all living creatures. After death these ultimate con- 
stituents were supposed to be set free and become active, until, with 
one another and still other particles, they gave rise to swarms of 
microscopic creatures. 

Needham in 1745 took decaying organic matter and enclosed it in 
a vessel; this he placed upon hot ashes to destroy any existing 
animalcule. On examining the contents of the flasks he found micro- 
organisms which he had not noted at first. Later (1769), Spallanzani 
repeated the work. He felt that Needham had not exercised suffi- 
cient care and that the organisms had gotten in from the outside. 
Accordingly he boiled the material for one hour and kept it in 
hermetically sealed flasks. He wrote: “I used hermetically sealed 
vessels. I kept them for one hour in boiling water, and after opening 
and examining their contents after a reasonable interval, I found not 
the slightest trace of animalculz, though I had examined the infusion 
from nineteen different vessels.” 

But the believers in the theory of abiogenesis were not convinced, 
for they claimed that the boiling altered the character of the infusion 
so that it was unable to produce life. Voltaire, with his characteristic 
satire, took up the fight at this point and ridiculed the operations 
of the English clergy “who had engendered the eels in the gravy of 
boiled: mutton,” and he wittily remarks: “It is strange that men 
should deny a creator and yet attribute to themselves the power of 
creating eels.” But this was a controversy to be settled not by 
ridicule but by experimental evidence. 

Spallanzani answered this by cracking one of the flasks so that 
air could enter. Decay soon set in. Even this was not sufficient to 
overthrow a popular belief, for the claim was made that the sealing 
of the flasks excluded the air, and air was essential to the generation 
of these forms of life. This objection was answered by the work of 


20 DEVELOPMENT OF BACTERIOLOGY 


many ingenious investigators. Schulze, in 1836, passed air through 
strong acids and then into boiled infusions and failed to find any 
living organisms in the infusion, whereas Schwann passed the air 
through highly heated tubes with the same results. This was criti- 
cized by their opponents who claimed that the chemical alteration 


4) 


; Wy, 
Mit 


Fic. 2.—Experiment of Schulze: Forcing air through sulphuric acid. (Lafar.) 


of the air subjected to such drastic treatment had been responsible 
for the absence of bacteria in the infusion. The work of Schroeder 
and Dusch (1853) was more convincing, for they found that it was 
sufficient to stopper the bottles «with cotton plugs; the air passed 
in but the microdrganisms were held back by the cotton and the 


rt 


———= Z| = 
CVU UVUVELNGUTVETSVTAVDEOVDOTEOOAOEN ETTORE ATOAA SOOOCOOOL) 


Fic, 3.—Experiment of Schwann: Heating air to make it sterile. (Lafar.) 


contents of the flasks kept in good condition. Every now and then 
the contents of a flask would spoil, even after it had been carefully 
stoppered and boiled. This remained a stumbling block in the way 
of those who maintained that life sprang only from life, until in the 
year 1865 when Pasteur demonstrated that many bacteria may pass 


FERMENTATION vse 


into a resting stage, and while in this condition will withstand con- 
ditions which quickly kill them in the vegetative stage. Eleven 
years later Cohn of Breslau carefully investigated organisms in this 
resting or spore stage, and today forms of microérganisms are known 
which will withstand boiling water for sixteen hours without being 
killed, and others resistant enough even to endure for many hours a 
10 per cent. solution of carbolic acid. 

Fermentation.—Since the dawn of history man has been interested 
in that wonderful process known as fermentation, and although 
many an ingenious theory has been formulated to explain it, little 
more than theory existed until the classic work of Pasteur on fer- 
mentation appeared about 1837. Pasteur claimed that all forms of 
fermentation were due to the action of microscopic organized cells. 
An idea such as this, even at this late date, did not go unchallenged, 
for we find no less illustrious workers than Helmholtz and Liebig 
opposing it. Liebig scoffed at such an idea, writing: “Those who 
pretend to explain the putrefaction of animal substance by the 
presence of microdrganisms reason very much like a child who 
would explain the rapidity of the Rhine by attributing it to the 
violent motions imparted to it in the direction of Burgen by the 
numerous wheels of the mills of Venice.” 

However, Pasteur’s carefully planned experiments soon demon- 
strated that without the micro6rganisms there would be no fermen- 
tation, no putrefaction, no decay of any tissue, except by the slow 
process of oxidation. The care with which his experiments were 
planned and executed are well shown in the experiments with grape 
sugar, concerning which he wrote: “I prepared forty flasks of a 
capacity of from two hundred and fifty to three hundred cubic 
centimeters and filled them half full with filtered grape-must, per- 
fectly clear, and which, as is the case of all acidulated liquids that 
have been boiled for a few seconds, remains uncontaminated, 
although the curved neck of the flask containing them remains 
constantly open during several months or years. 

“Tn a small quantity of water, I washed a part of a bunch of 
grapes, the grapes and the stalks together, and the stalks separately. 
This washing was easily done by means of a small barber’s hair- 
brush. The washing-water collected the dust upon the surface of the 
grapes and the stalks and it was easily shown under the microscope 
that this water held in suspension a multitude of minute organisms 
closely resembling either fungoid spores or those of alcoholic yeast, 
or those of Mycoderma wni, ete. This being done, ten of the forty 
flasks were preserved for reference; in ten of the remainder, through 
the straight tube attached to each, some drops of the washing-water 
were introduced; in a third series of ten flasks a few drops of the 
same liquid were placed after it had been boiled; and finally in the 
ten remaining flasks were placed some drops of grape-juice taken 


22 DEVELOPMENT OF BACTERIOLOGY 


from the inside of perfect fruit. In order to carry out this experi- 
ment the straight tube of each flask was drawn out into a fine and 
firm point in the lamp, and then curved. This fine and closed point 
was filed round near the end and inserted into the grape while 
resting upon some hard substance. When the point was felt to 
touch the support of the grape it was by a slight pressure broken off 
at the file mark. Then if care had been taken to create a slight 
vacuum in the flask, a drop of the juice of the grape got into it; the 
filed point was withdrawn and the aperture immediately closed in 
the alcohol lamp. This decreased pressure of the atmosphere in the 
flask was obtained by the following means: After warming the 
sides of the flask, either in the hands or in the lamp flame, thus 
causing a small quantity of air to be driven out of the end of the 
curved neck, this end was closed in the lamp. After the flask was 
cooled, there was a tendency to suck in the drop of grape-juice in 
the manner just described. 

“The drop of grape-juice which enters into the flask by this 
suction ordinarily remains in the curved part of the tube, so that to 
mix it with the must it was necessary to incline the flask so as to 
bring the must into contact with the juice and then replace the flask 
in its normal position. The four series of comparative experiments 
produced the following results: 

“The first ten flasks containing the grape-must boiled in pure air 
did not show the production of any organisms. The grape-must 
could possibly remain in them for an indefinite number of years. 
Those in the second series, containing the water in which the grapes 
had been washed separately and together, showed without exception 
an alcoholic fermentation which in several cases began to appear 
at the end of forty-eight hours when the experiment took place at 
ordinary summer temperature. At the same time that the yeast 
appeared, ‘in the form of white traces, which little by little united 
themselves in the form of a deposit on the sides of all the flasks, 
there were seen to form little flakes of Mycelium, often as a single 
fungoid growth or in combination, these fungoid growths being 
quite independent of the must or of any alcoholic yeast. Often, also, 
the Mycoderma vini appeared after some days upon the surface of 
the liquid. The vibria and the lactic ferments, properly so-called, 
did not appear on account of the nature of the liquid. 

“The third series of flasks, the washing-water of which had been 
previously boiled, remained unchanged, as in the first series. Those 
of the fourth series, in which was the juice of the interior of the 
grapes, remained equally free from change, although I was not 
always able, on account of the delicacy of the experiment to eliminate 
every chance of error. These experiments cannot leave the least 
doubt in the mind as to the following facts: 

“Grape-must, after heating, never ferments on contact with air, 


FERMENTATION 23 


when the air has been deprived of the germs which it ordinarily 
holds in a state of suspension. 

“The boiled grape-must ferments when there is introduced into 
it a very small quantity of water in which the surface of the grapes 
of their stalks have been washed. 

“The grape-must does not ferment when there is added to it a 
small quantity of the juice of the inside of the grape. 

“The yeast, therefore, which causes the fermentation of the grapes 
in the vintage-tub comes from the outside and not from the inside 


(aa 


Fia. 4.—Tyndall’s box. One side is removed to show the construction. The 
bent tubes at the top are to permit a free circulation of air into the interior. The 
window at the back has one corresponding in the front (removed). Through these 
the beam of light sent through from the lamp at the side was observed. The three 
tubes received the infusion and were then boiled in an oil bath. The pipette was 
for filling the tubes. (Popular Science Monthly, April, 1877.) 


of the grapes. Thus it destroyed the hypothesis of MM. Trecol and 
Fremy, who surmised that the albuminous matter transformed 
itself into yeast on account of the vital germs which were natural 
to it. With greater reason, therefore, there is no longer any ques- 
tion of the theory of Liebig of the transformation of albuminoid 
matter into ferments on account of tbe oxidation.” 

Pasteur’s work did not stop here, for he soon proved that a disease 
that was attacking the silkworm was caused by bacteria. And from 
this there developed the idea that disease in general is due to bacteria. 


24 DEVELOPMENT OF BACTERIOLOGY 


If there were any doubts left in the minds of the scientific world 
as to the fallacy of the theory of spontaneous generation, after the 
work of Pasteur, they were dispelled by the work of Tyndall. 
Tyndall proved that in an atmosphere devoid of dust, as on the 
tops of mountains and in some ingeniously constructed boxes used 
by him, perishable substances, such as beef tea, if sterile when placed 
in such an atmosphere, will keep for an indefinite period. 

Smallpox.—Smallpox was formerly looked upon as practically 
unavoidable by all members of the human family, as is seen from a 
popular saying current in Germany in the eighteenth century: 
“von Pocken und Liebe bleiben nur wenige frei,” from smallpox and 
love few remain free. 

Concerning smallpox Macaulay wrote in referring to the death of 
Queen Mary from the disease in 1694: “The havoc of the plague 
had been far more rapid; but plague had visited our shores only 
once or twice within living memory, and the smallpox was always 
present, filling the churchyards with corpses, tormenting with 
constant fears all whom it had not yet stricken, leaving in those 
whose lives it spared the hideous traces of its power, turning the 
babe into a changeling at which the mother shuddered, and making 
the eyes and cheeks of the betrothed maiden objects of horror to the 
lover.” 

For the different condition which exists today in civilized countries 
where the fear of smallpox is nearly as remote as that of leprosy, 
Edward Jenner (1749-1823) is chiefly to be thanked. His attention 
was at first directed to the subject by the remark of a young girl: 
“T cannot take smallpox for I have had cowpox.” After consider- 
able labor and opposition he developed and gave to the world, 
without monetary consideration, his vaccine which has all but 
banished from the world the dreaded disease—smallpox. 

Anthrax.—As early as 1863 investigators had seen in the blood 
of some animals that had died of a disease known as anthrax, a very 
small rod-like organism which permeated all the capillaries. Their 
experiments showed that the blood from such an animal, when 
injected into the veins of a second animal, caused it to die of the 
same disease. But they found that there were times when the organ- 
ism could not be discovered in the blood of the dead animal, although: 
injection with blood from this animal would cause the death of 
another. ‘This fact left a doubt in the minds of thinking men aggto 
whether this rod-shaped organism was thé cause of the animal’s 
death or whether it was “some invisible element in the blood.” 
Not until thirteen years later was this fully settled by the work of 
Robert Koch. He not only saw the rod-shaped organism, but 
obtained it free from all other substances, and proved that it was the 
specific cause of the disease. This was followed by many other dis- 
coveries, until today it is known that practically all diseases are due 


ANTHRAX 25 


to microscopic organisms. Yes, even many of the changes taking 
place in the body and associated with old age are attributed by some 
writers to the products generated by bacteria. 

The workers in this field are not satisfied with knowing the cause 
of a disease, but they wish to know how they may ward off disease 
and how to cure it when once it gains access to the body of an animal. 
Pasteur soon announced that he had found a preventive for anthrax. 
His statement was immediately challenged by the president of an 
agricultural society in such a way that it was brought to the atten- 
tion of the entire civilized world. He suggested that the subject be 
submitted to a decisive public test and offered to furnish fifty sheep, 
half of which should be protected by the attenuated virus prepared 
by Pasteur. Later they were all to be infected by the disease- 
producing organisms and if the vaccine were a success the protected 
ones were to remain healthy, the unprotected ones to die of the dis- 
ease. Pasteur accepted the challenge and suggested that for two 
of the sheep there should be substituted two goats, and that there 
be added to the herd ten cows, but he stated that these latter animals 
should not be considered as falling rigidly within the test, for his 
experiments had not yet been extended to cattle. Before this time 
the fame of Pasteur had been considered firmly established, but now 
all the world looked on with doubt to think that any man should 
make such a preposterous claim. On May 5 the animals to be pro- 
tected received their first treatment with the vaccine and a second 
two weeks later. Virulent cultures of the disease-producing organ- 
ism were then inoculated into the animals. The results of the test 
were indeed dramatic. 

“Two days later, June 2, at the appointed hour of rendezvous, a 
vast crowd, composed of veterinary surgeons, newspaper corre- 
spondents, and farmers from far and near, gathered to witness the 
closing scenes of this scientific tourney. What they saw was one of 
the most dramatic scenes in the history of peaceful science, a scene 
which Pasteur declared afterward, ‘amazed the assembly.’ Scattered 
about the enclosure, dead, dying, or manifestly sick unto death, lay 
the unprotected animals, one and all, while each and every protected 
animal stalked unconcernedly about with every appearance of per- 
fect health. Twenty of the sheep and the one goat were already 
dead; two other sheep expired under the eyes of the spectators; the 
remaining victims lingered but a few hours longer. ‘Thus, in a 
manner theatrical enough, not to say tragic, was proclaimed the 
unequivocal victory of science.” 

It has been estimated by conservative writers that Pasteur’s dis- 
covery of the means of preventing or curing anthrax, silkworms’ 
disease, and chicken cholera, adds annually to the wealth of France 
a sum equivalent to the entire indemnity paid by France to Germany 
after the War of 1870. 


—_ 
= 


26 DEVELOPMENT OF BACTERIOLOGY 


Other Work of Pasteur.— This was only a part of the work of this 
great man, for in 1885 he announced a cure for hydrophobia. Prior 
to this time the disease developed in at least 16 per cent. of the 
individuals bitten by mad dogs, and of this 16 per cent., 100 per cent. 
died. Since Pasteur’s discovery the number of deaths from this 
cause has been reduced almost to zero. ‘The profound importance 
of his work has been well summarized by Lord Lister: ‘Truly there 
does not exist in the entire world any individual to whom the medical 
science owes more than they do to you. Your researches on fermen- 
tation have thrown a powerful beam which lightened the baleful 
darkness of surgery and has transformed the treatment of wounds 
from a matter of uncertain and too often disastrous empiricisms 
into a scientific art of sure beneficence. Thanks to you, surgery has 
undergone a complete revolution which has deprived it of its terrors 
and has extended almost without limit its efficacious powers.” 

And Tyndall writes: ‘“We have been scourged by miserable 
throngs, attacked from impenetrable ambuscades, and it is only 
today that the light of science is being let in upon the murderous 
dominion of foes.” 

Other Plagues Conquered.—In the realm of medicine one discovery 
after another has followed in rapid succession during the last few 
years, until today diphtheria instead of having a death-rate of over 
30 per cent. has one of less than 3. Typhoid fever is all but con- 
quered. Asiatic cholera and the yellow fever have been nearly 
wiped from the face of the earth, thus making possible the building 
of the Panama Canal. 

Lister.— Thanks to the wonderful work of Lord Lister we no longer 
have that terrible suppuration, which before his time followed even 
slight wounds. At the close of the nineteenth century it was asserted 
that “Listerism’’ had saved more lives than had been sacrificed by 
all the wars of the nineteenth century. Although continually 
brought in contact with suffering and misery, this truly great man 
did not lose his tender-hearted nature and love of children, as is 
shown by the following story related by one of Lister’s students. 

“One day when Lister was visiting his wards in the Glasgow 
Royal Infirmary, there was a little girl whose elbow-joint had been 
excised, and this had to be dressed daily. Lister undertook this 
dressing himself. The little creature bore the pain without com- 
plaint, and when finished she suddenly produced from under the 
clothes a dilapidated doll, one leg of which had burst, allowing the 
sawdust to escape. She handed the doll to Lister, who gravely 
examined it; then asking for a needle and thread, he sat down and 
stitched the rent, and then returned the doll to its gratified owner.’ 

Yellow Fever.—'The investigators in some of these fields have gone 
into it not only with a knowledge of the fact that failure may be 
their lot, but they even risked their lives in the work, as is shown 


FUTURE WORK 97 


in the fight against yellow fever. Dr. Lazear, an American army 
surgeon, allowed himself to be bitten by a mosquito in an infected 
ward. He soon acquired yellow fever in the most terrible form and 
died a martyr to science and a true hero. He gave up his life for 
others; the plain record of his sacrifice is recorded thus upon a 
tablet erected to his memory: ‘ With more than the courage and 
devotion of the soldier, he risked and lost his life to show how a 
fearful pestilence is communicated and how its ravages may be 
prevented.”” That this is conveyed only by the bite of the mosquito 
was shown by the following: Three brave men slept for twenty 
nights in a small, ill-ventilated room screened from mosquitoes but 
containing furniture and clothing smeared with the excretion of 
yellow fever patients—some of whom had died of the disease. None 
of the men contracted yellow fever, thus indicating the disease was 
not of a contagious nature. 

Agricultural Bacteriology.—In 1883 Burrill, by the discovery -of 
the organism which causes fire- or pear-blight, opened up a similar 
interesting and practical field in the plant kingdom which even at 
the present day is only in its infancy. 

It may appear from the preceding that bacteria are all enemies 
of man, but this is not true, for there are many more beneficial 
bacteria than injurious ones. 

Even in the field of agricultural bacteriology rapid advances have 
been and are being made. To Beijerinck, Hellriegel, Wilfarth, Lip- 
man, and a host of others, we owe our knowledge concerning the 
morphology and physiology of the nitrogen-fixing organisms. In 1888 
Winogradsky isolated the nitrifying organisms which grow on a 
medium devoid of all organic matter and since that time there is an 
ever-increasing volume of work on this phase of the subject. Han- 
sen’s Investigation in industrial fermentation is also important. 

Future Work.—One may think from the preceding that in this 
field of science there is little to be done, but this is not the case, for 
there are diseases still unconquered. The great “White Plague” 
still claims its millions each year. There are diseases which are 
sapping the very life-blood of the nation, yet they go unchecked. 
Science as yet has not come to the aid of the unfortunate victims. 

As regards the beneficial organism we have only just started to 
realize their great possibilities. In the soil are five great classes of 
organisms which deal with the transformation of nitrogen. One 
class carries on putrefaction, changing the insoluble proteins into 
ammonia, another picks the ammonia up as formed, transforming - 
it into nitrites, and even this must be changed into nitrates before 
plants can use it. Under what condition are these changes carried 
on at a maximum rate? What influence has moisture, temperature 
crop, and method of tillage on this change? Some of these questions 
are being answered by the work now being conducted, but there are 


28 DEVELOPMENT OF BACTERIOLOGY 


many yet unanswered. Still they are vital questions, for, in many 
cases, the crop yields will be determined by the skill with which 
these various changes are controlled. There is another set of organ- 
isms in the soil, the function of which is to take the practically 
valueless nitrogen of the atmosphere and change it into forms such 
as the higher plants can feed upon. How may we control them for 
maximum yields? For if treated properly they will never tire, but 
toil on forever. Then again it is possible that bacterial action may 
be used as a measure of soil fertility and methods so perfected which 
are more sensitive than any now in use. Truly, in this field great 
things have been accomplished, but there remains yet to conquer 
fields richer by far than the workers of the past have ever dreamed. 


REFERENCES. 


Locy: Biology and its Makers. 

Paget: Pasteur and after Pasteur. 

Gregory: Discovery—The Spirit and Service of Science. 
Vollery-Radot: The Life of Pasteur. 

Libby: History of Science. 


CHAR LER TI, 
BACTERIA AND THEIR PLACE IN NATURE. 


BacTERIOoLoey in the strictest sense is that branch of science 
which deals with the distribution, morphology, classification, and 
function of bacteria. However, it is often used more general to 
include bacteria, yeasts, molds, and protozoa. A better term where 
all four groups are included is “microbiology.” Many of the modern 
writers use this term. 

Definition of Bacteria.—Bacteria are extremely minute, simple, 
unicellular organisms which multiply with great rapidity, usually 
by transverse fission, and.are devoid of chlorophyl. Although they 
contain nuclear material which is usually diffused throughout the 
cell body in the form of larger or smaller granules, they possess no 
definite organized nucleus. They are generally accepted as belong- 
ing to the vegetable kingdom. This is not without some opposition, 
due to the inherent difficulty of the subject, as is so admirably 
pointed out by Fischer: “The terms ‘animal’ and ‘plant’ are collec- 
tive terms invented by laymen to describe familiar living things, 
insects and elephants, mosses and oak trees, and they date from a 
time when such minute beings as bacteria were quite unknown. It is 
therefore as superfluous as it is futile to attempt, as many have done, 
to detect the distinguishing characters of the ‘animal’ and the 
‘vegetable’ kingdoms among organisms for which these terms were 
never intended. For this reason, Haeckel and others have proposed 
to establish a third dominion, that of the Protista, which shall 
include all those forms in which differentiation has not been pro- 
nounced on the lines of either animal or plant development. The 
new group would take up Radiolarians, Flagellata, and Infusoria 
from the animal side, and the Cyanophyceex as well as some low 
forms of Algze and Fungi from the plants. The border-line between 
protista on the one hand and plants and animals on the other is— 
it must be confessed—artificial. To these protista, which embrace 
approximately all those forms of life we commonly call micro- 
organisms or microbes, the bacteria belong.” 

It is generally stated that the plant cell differs from the animal 
cell by the possession of a firm and well differentiated wall, wholly 
distinct from the containing protoplasm, whereas the boundary 
surface of the animal cell is more often an outer layer of the proto- 
plasm and not separable from it. Moreover, the typical cell wall 


30 BACTERIA AND THEIR PLACE IN NATURE 


of plants is usually made up of cellulose or one of its derivatives; the 
outer membrane of the animal cell is nitrogenous and where there 
is a heavy cell wall it is chitinous. Both of these distinctions break 
down in the case of the lower forms of plant and animal life. 

The “blue-green” alge, or Schizophycez, possess chlorophyll and 
are. obviously plants. Structurally, many of these are practically 
identical with bacteria. This constitutes a strong argument for the 
plant affinities of the bacteria. 

Nor is it an easy task to differentiate nicely between bacteria, 
yeasts, and molds. Generally speaking, typical bacteria, yeasts, and 
molds may be distinguished from each other as follows: Bacteria 
are unicellular, devoid of a definite organized nucleus but con- 


ag ; 


@ 

GAA 

an } ; 
14,64. 7 i “ll 5 

ME bet ite ; 
i 

a SS 


Zz 


Fic. 5.—To illustrate the close relationship of the bacteria to the blue-green alge. 
The figures to the left (A) are blue-green alge, those to the right (B) bacteria. Those 
forms most closely resembling each other are lettered alike. A, blue-green alge: 
a, Aphanocapsa; b, Merismopedia; c, Gleotheca; d, Spirulina; e, Phormidium; //, 
Nostoc. (All adapted from West.) B, bacteria: a, Micrococcus; b, Sarcina; c, 
Bacillus; d, Spirillum; e, Bacillus in chains; /, Streptococcus. (Buchanan’s Household 
Bacteriology.) » 


taining nuclear material. They multiply by transverse fission. At 
times they are united into filaments or masses, but are usually 
easily separated. Yeast cells are usually, though not always, larger 
than bacteria. Although unicellular they contain a definite organ- 
ized nucleus. They may remain united after cell division, but each 
cell constitutes a definite entity. Most yeasts multiply by budding 
—only a few by simple fission. Molds are multicellular, nucleated 
organisms which are usually made up of a mass of interwoven or 
radiating threads consisting of chains of cells. 

Divisions of Plant Kingdom.—Plants are divided into four great 
groups: Spermatophytes or seed plants, Pteridophytes or fern plants, 
Bryophytes or liver-worts and mosses, and Thallophytes or thallus 
plants. This last group has little or no differentiation of vegetative 


OCCURRENCE OF BACTERIA ol 


organs, such as stems and leaves. Two groups stand out conspicu- 
ously—known as alge and fungi, but there are other groups whose 
relationship is not so clear. The main divisions of the Thallophytes 
are (1) Myxomycetes, commonly known as slime molds, or slime 
fungi, which combine characters of plants and animals; and (2) 
Schizophytes or fission plants, characterized by cell divisions occur- 
ring in rapid succession which is their only method of reproduction. 
They consist of two groups: the Cyanophycee, or blue-green alge, 
and the Schizomycetes, or bacteria. 
The relationship is shown diagrammatically below: 


Myxomycetes—slime molds, or slime fungi. 
Cyanophyces—blue-green 


Schizophytes—fission plants algee. 
Thallophytes—simple, _ Schizomycetes—bacteria. 
undifferentiated plants; | Algse, including seaweeds, pond scums, water-silks, etc.; 
do not develop roots, contain chlorophyll. 
stems or leaves. Yeasts. 
Molds. 
Fungi without chlorophyll. 4 Mildews. 
Smuts. 
| Rusts, etc. 


Occurrence of Bacteria.—Bacteria are ubiquitous, occurring as 
they do nearly everywhere. They are found in soil to great depths, 
their number decreasing with the depth and nature of the soil, being 
more numerous in soil containing organic matter than in those 
practically devoid of it. Although they occur in the atmosphere, 
it is not their normal habitat, for growth and multiplication cannot 
take place in it under ordinary conditions. The number and kind 
found in air vary with a number of factors, chief among which is 
locality. The air of some high mountains is practically devoid of 
bacteria; city and country air also differ from each other in the 
number and kind of bacteria they contain. Other controlling 
factors are moisture, presence or absence of injurious substance, and 
minute particles in the atmosphere. 

Most natural waters contain great numbers of bacteria. In 
sewage and polluted water they are especially numerous, but occur 
only in small numbers or not at all in deep wells and springs. The 
kind of organism varies with the composition of the water and with 
the original contamination. Milk as secreted by the milk glands 
of cows is practically free from bacteria, but the vessels in which it 
is handled so contaminate it that it rapidly gains in bacteria. Often 
by the time it reaches the consumer it contains millions in every 
cubic centimeter. In short, all food except that recently cooked 
contains bacteria, the number and kind of which vary with the 
nature and age of the food. 

Living as we do in a world which is teeming with bacteria, we can 
expect to find them on the surfaces of the skin and mucous mem- 


32 BACTERIA AND THEIR PLACE IN NATURE 


brane: Normally, the infant enters the world free from bacteria, 
but they soon begin to settle on the skin; they penetrate the nose 
and mouth; the first respiratory movements and cries carry them 
into the respiratory passages; and between the tenth and seven- 
teenth hour they have reached the intestines. 

Ordinarily, the deeper respiratory passages contain but few 
bacteria, but it has been proved that even the tubercle bacillus can 
penetrate with the inspired air to the bottom of the pulmonary 
alveoli. 

On account of its acidity, yeasts and molds flourish better in the 
stomach than do bacteria. However, at least thirty species of 
bacteria (occurring in the stomach) have been described, many of 
which have attracted special attention on account of the belief that 
their presence may favor other more injurious species. 

The intestines, on account of their alkaline reaction and the partly 
digested condition of their contents, are a great reservoir of bacterial 
activity. Metchnikoff and others have given an immense amount 
of work to a consideration of their function within the body and the 
probable result in their absence. The only conclusion which is 
possible at present is that. living as we are in a world filled with micro- 
organisms, life without them is impossible. All that can be done is 
to make conditions such that the injurious species are suppressed 
and the beneficial ones favored. Out of this has grown sour-milk 
therapy. 

The normal tissues of plants and the blood and tissues of animals 
are free from bacteria. They are rarely found on certain healthy 
mucous membranes, such as those of the kidney, bladder, and 
lungs. Occasionally they pass through the skin or the mucous 
membrane of the digestive tract after which they may be found for 
a short time in the blood. This is especially the case during the 
height of digestion and it probably accounts for the large number 
of leukocytes which swarm in the intestinal mucosa and which have 
been thought to be in some way associated with the process of fat 
absorption. 

In certain diseased conditions the blood and many of the tissues 
of the human body are found to contain numerous bacteria. Soon 
after death even the saprophytes rapidly invade and decompose the 
body tissues. 

Réle of Bacteria in Nature.— Bacteria play a wonderful réle in the 
many transformations going on in this world. It is difficult to con- 
ceive of life without them and their help. For the beneficial ones 
we turn first to the soil, for from this—either directly or indirectly— 
man largely draws his food, clothing, and other necessities of life. 
The soil is not, as many think, a dead, inert mass, but it is teeming 
with life! Both microscopic plants and animals inhabit it by the 
millions. These have been at work within it long before man began: 


ROLE OF BACTERIA IN NATURE 33 


to till the soil. In the formation of soil from the primitive rock, 
bacteria played no small part, the changes wrought by the elements 
first giving them a foothold. 

Changes in temperature tear loose huge rocks and break them 
into fragments. It is well known that most substances when heated 
expand and contract in such varying degrees that parts are put 
under a strain. This strain at times is sufficient to cause cracks of 
various sizes to occur in the rock, a result which may be illustrated 
by the sudden cooling of hot glass or the sudden heating of cool 
glass. Throughout the long, hot days of summer rock is heated to 
a comparatively high temperature, as the boy who has chased bare- 
foot over their surface in quest of grasshoppers or butterflies will 
testify. At night they cool. This is repeated day after day. This 
continued heating and cooling gradually causes small crevices to 
appear in even the most resistant. ‘These become filled with water 
and dust; when the cold nights of autumn come the water freezes. 
In freezing, the water expands and the rocks are broken into pieces. 
So it continues day after day and year after year, until the rock 
becomes a fine powder. Even then, however, the plant-food is still 
insoluble and cannot be taken up by the plant. Long before it has 
reached the form of powder, bacteria begin to grow upon the surface 
of the rock and in the crevices. In their growth they form acids 
which act upon the insoluble plant-foods, rendering them soluble. 
Bacteria continue their work long after the rocks have been changed 
to soil, each day liberating a little more plant-food for the growth 
of plants during that day. During the year the bacteria are able in 
a fertile soil to liberate enough plant-food for the production of a 
good crop. When manure is applied, it not only supplies food for 
the growing crop, but it also supplies food for the microérganisms, 
and they in turn liberate more of the insoluble constituents of the 
fine rock particles of which the soil is mainly composed. There are 
millions of them in every ounce of soil, struggling, to be sure, for 
their very existence, but always rendering a little more mineral 
plant-food available. 

One of the essential elements for crop production, and the one 
which is usually in the soil in the smallest quantities, is nitrogen. 
This, unless it be applied to the soil in the form of the costly fer- 
tilizer, nitrates, must be prepared for the plant by bacteria. The 
farmer finds his crops are limited directly by the speed with which 
these organisms prepare the food for his growing crop. If they 
are active, other things being favorable, he will get a good crop; 
but if they do not play their part, though everything else may be 
ideal, yet there is no crop. 

Bacteriological examinations of cultivated soils have shown that 
usually those that are richest contain the greatest number of bac- 
teria. ‘The number in the soil is dependent upon the quantity and 
—- 


34 BACTERIA AND THEIR PLACE IN NATURE 


character of food the bacteria find in the soil. If the soil is rich in 
plant residues— barnyard manures and the like—many bacteria will 
be found there pulling these substances to pieces, liberating gases 
and acids which act upon insoluble particles of the soil and render 
them soluble. One class of organisms changes the protein constitu- 
ents of the soil into ammonia. This type is called “ammonifiers.”’ 
A person can often detect their activity from the odor of ammonia 
coming from manure heaps. 

Most plants cannot, however, use nitrogen in the form of ammonia; 
it must be in the form of nitrates. This transformation is brought 
about by two distinct types of organisms. One of them feeds upon 
the ammonia produced and manufactures nitrous acid. Should the 
change cease at this point and nitrites accumulate in the soil in 
large quantities, plants would not grow upon it, for this is a poison 
to plants. But in soils properly cared for only minute quantities 
of nitrites accumulate. As soon as they are formed another type 
of organism feeds upon them and manufactures nitric acid for the 
growing plant. This, when formed, reacts with other constituents 
of the soil, such as limestone. It is then ready to be taken up by the 
plant and manufactured into nourishing food, beautiful flowers, or 
fragrant perfumes for the human family. 

Were it not for bacteria the world in time would be filled with 
never-changing organic matter. The plant residues, trees, and 
animal bodies would remain stored up in the soil, and with it that 
element—carbon—which, in the form of carbon dioxid, is required 
by all chlorophyl plants. Bacteria, in getting the energy which they 
require in their life activity, are continually liberating carbon so 
that it may start again on its journey of construction. If carbon 
and nitrogen could but speak, what tales of wonderment they would 
tell! The chemist, the bacteriologist, and the farmer would each be 
wiser, for many of the changes through which carbon and nitrogen 
pass, due either to the action of the lower plants—bacteria—or that 
of the higher plants are so complex that even the scientist with his 
apparently magical methods cannot follow them. 

So far only the plant-food in the soil and the changes through 
which it passes have been considered. The farmer, however, is 
usually more concerned with that substance his soil lacks and which 
' must be supplied 1 in order to get good crops. In many cases the 
lacking element is nitrogen. One notes from the fertilizer quotations 
that fis elements will cost fifteen cents a pound or over if purchased 
in the form of sodium nitrate, ammonium sulfate, or dried blood. 
If one stops to make a simple calculation he finds that it would cost 
fifteen dollars for enough to produce 100 bushels of corn, eleven 
dollars for enough to produce 50 bushels of wheat, and seven dollars 
and fifty cents for enough to produce one ton of alfalfa hay. In 
these calculations it has been assumed that one could get back in 


ROLE OF BACTERIA IN NATURE 30 


the form of corn, wheat, or alfalfa every pound of commercial nitro- 
gen that has been applied to the soil, which, on the face of it, is an 
utter impossibility. So we have to look to other means of getting 
nitrogen for our growing crop, and here again bacteria come to our 
rescue. 

There are seventy-five million pounds of atmospheric nitrogen 
resting upon every acre of land. None of the higher plants, however, 
have the power of taking this directly out of the air. One family 
of plants, the Leguminos, in which are included peas, beans, alfalfa, 
clover, and many others, if properly infected by bacteria have the 
power of using this atmospheric nitrogen. Under this condition 
and with these plants nitrogen no longer remains the limiting ele- 
ment of crop production. For these microscopic organisms which 
live within small nodules upon the alfalfa are master chemists. 
Within their tiny laboratory they can bring about changes which 
man can imitate but imperfectly with costly machinery and under 
the action of powerful electric currents. In some of the experiments 
carried on at the Illinois Experiment Station these minute organisms 
were found to be able to increase the value of the first cutting of 
alfalfa hay $27.80 an acre, if the nitrogen in the alfalfa be counted 
only at the same price as we would have to pay on the market for 
an equivalent quantity of nitrogen in the form of a commercial 
fertilizer! If these crops be plowed under the fertility of the soil 
would be increased to just that extent. One writer has said of them: 
“They not only work for nothing and board themselves, but they 
pay for the privilege.” This is strictly true, for all they require is a 
plant on which to grow and a well-aérated moist soil containing 
limestone. ~) They cannot work in an acid soil. 

There is another class of nitrogen-gathering organisms within the 
soil which differs from the above in that they live free in the soil 
and gather nitrogen. Under ideal conditions they may gather 
appreciable quantities. 

It is quite possible that much of the benefit derived from the 
summer fallowing of land is due to the growth within the soil of this 
class of organism which stores up nitrogen for future generations 
of plants. It has been found that they are more active and found 
in greater numbers in such a soil. All the work that the farmer puts 
upon the soil to render it more porous reacts beneficially upon these 
organisms, because they not only love atmospheric nitrogen and 
oxygen, but must have them. These elements are absolutely essen- 
tial to their life activities and they must be obtained from within 
the soil since the minute organisms cannot live upon the surface for 
the direct rays of the sun kills them in a short time. 

But these are only a few of the many that help the farmer. They 
are at work in his silo rendering the feed more palatable and nutri- 
tious for his cattle. They are working in his milk and cream, and 


36 BACTERIA AND THEIR PLACE IN NATURE 


if they be the right kind they give to butter and cheese a desirable 
flavor. They take part in the tanning of leather, the retting of flax, 
the curing of tobacco, and, in short, they help us in a hundred and 
one ways we little suspect. One of the most fascinating and instruc- 
tive tasks set for man is to learn how to increase the work of the 
beneficial bacteria and to suppress or entirely weed out the injurious 
bacteria. 

Divisions of Bacteriology.—Bacteriology, although one of the 
youngest of sciences, is no Jonger confined to one branch which can 
be adequately covered by one text or its whole field covered by any 
one individual, but is, as are the other sciences, divided into a 
number of divisions each dealing with a certain phase of the subject. 
The main divisions are: 

1. Agricultural bacteriology which deals with the bacteria of the 
soil and their relation to plant life. 

2. Dairy bacteriology which deals with the Seat of milk and 
faaie relations to dairy products such as pure milk, butter, and 
cheese. 

3. Industrial bacteriology, which considers the use of bacteria in 
the arts and which also deals with methods of suppressing injurious 
bacteria and favoring the beneficial. 

4, Plant pathology which deals with the cause and prevention of 
those diseases that attack plants by invading their tissues. 

5. Animal pathology which deals with bacteria in relation to the 
diseases of the lower animals. 

6. Human pathology which deals with the distribution, mor- 
phology, physiology, and pathological changes produced by bacteria 
which are pathogenic to man. 


Lp i evel ots Ded See ad 
MORPHOLOGY OF BACTERIA. 


In shape, bacteria have the very simplest conceivable structure, 
and although there are thousands of different kinds differing in 
properties, they all have one of three general forms: rod-shaped, 
spherical, or spiral. 

Bacilli.—The rod-shaped organisms, which may be compared to 
a lead pencil, are cylindrical organisms in which a longer and shorter 
dimension may be recognized. They are the bacilli (sing. bacillus). 
The ends of the organisms may be convex, less often flat or even 
concave. The size also varies, some being so short that it is next 
to impossible to tell whether they are rods or globular organisms; 
others are comparatively long. 


GD 
ar ao 
ele) 16 
ee ae 
o/s 


Fic. 6.—The normal types of bacteria. 1-6, cocci; 7-13, bacilli; 14-16, spirilla; 
1, micrococcus; 2 and 8, diplococci; 4, tetracoccus; 5, sarcina; 6, streptococcus (the 
lower chain includes an arthrospore); 7 and 8, bacilli; 9, 10, 12 and 13, bacilli with 
various granules; 11, streptobacillus; 14, vibrio; 15, spirillum; 16, Spirocheta trepo- 
nema. (Kendall.) 


Cocci.—The cocci (sing. coccus) are typically spherical and may 
be likened to a ball or at times to an egg. They may in the early 
stages of cell division appear temporarily as bacilli with convex ends. 
They often occur in pairs, diplococci, in which case usually their 
proximate surfaces are flattened. This flattening of the organism 
may at times be accompanied by an elongation of the axis of the 
organisms parallel to the plane of opposition. This leads to the 
coffee-bean shape exemplified in the gonococcus and miningococcus. 
At other times we have the flattening perpendicular to the plane of 
the flattened surface as seen in the “lance-shaped”’ pneumococcus. 
The cocci may be large or small and group themselves in various 
ways. 


38 MORPHOLOGY OF BACTERIA 


Spirilla.—The third group is the spirilla (sing. spirillum) and may 
be likened unto a corkscrew. ‘The spiral may be loosely or tightly 
coiled or there may be one, two, or many coils. At times the curve 
may be so slight that the organism viewed under the microscope 
appears “comma-shaped.”’ 

More bacilli are known than cocci and more cocci than spirilla. 
Migula enumerates 833 bacilli, 343 cocci, and 96 spirilla, a total of 
1272. Other workers have tabulated more with a similar propor- 
tional distribution among the various groups. 

Gradations.— The difference between these fundamental types is 
at times very slight. In fact the cocci often merge into the bacilli 
and the bacilli into the spirilla. It is often difficult accurately to 
distinguish between the various groups, as is exemplified by the 
fact that at times B. prodigious has been described by one investi- 
gator as a coccus and at another time by a different worker as a 
bacillus. This same condition holds for the pneumonia germ and the 
one causing pear blight, whereas the cholera organism has been 
described both as a bacillus and a spirillum. 

Pleiomorphism.—By pleiomorphism is meant a permanent or 
semipermanent change in the normal form of the organism. ‘The 
organism may at one time represent a coccus, at another a bacillus, 
and at still another a spirillum. This led the early writers to believe 
that there was a mutability of species. The condition is especially 
likely to occur among some soil organism and much light has been 
thrown on the subject by Lohnis who finds the life history of bacteria 
to be only slightly less complex than that of other organisms. 


Fia. 7.—Involution forms from bacilli. (From Fligge.) 


Involution Forms.— Although the form of bacteria is quite constant 
under normal conditions, yet there is a tendency with many organ- 
isms, especially when grown for some time on artificial media, to 
show abnormal or bizarre forms. Such organisms are known as 
involution forms. Some of the rod-shaped organisms may appear 


SIZE AND WEIGHT OF BACTERIA 39 


as clubs many times larger than the ordinary, or they may appear 
as crosses or stars. ‘The formation of large club-shaped organisms 
is very characteristic of the organism which causes diphtheria, 
whereas the formation of crosses, stars, and the like is characteris- 
tic of the organism which grows in the roots of alfalfa. Some writers 
have considered them degenerate forms and compared them to the 
“Jame and halt” in the human species: This, however, is hardly an 
apt illustration, for these peculiar shaped organisms have all of the 
powers possessed by others and if they found their way into the 
body of an animal, they would be just as likely to produce the dis- 
ease which is characteristic of the organism as would the ones with 
the normal shape. 

In some cases this characteristic has served as a valuable aid to 
the differential diagnosis of the organism. ‘This is especially true 
with the plague bacillus which, when grown on nutrient agar con- 
taining from 2.5 to 3.5 per cent. of sodium chlorid, is prone to give 
rise to a are forms. 

Size and Weight.—'The unit of measurement in microscopy is the 
micron (u), or Taercron eter, This is 0.001 of a millimeter or 
approximately 5:4), of an inch. The majority of the organisms 
vary from 0.24 up to 30u or 40u. They are smallest in the case of 
the cocci and largest in the case of the spirilla. 

Although there is a great variation in the size of bacteria, all are 
extremely small; even ‘the largest are not visible to the naked eye. 
The smallest are beyond the range of our most powerful micro- 
scopes, and others appear as mere dots. The Pfeiffer bacillus, the 
one which was thought to cause influenza, are rod-shaped organisms, 
and if they be placed end to end it would take fifty thousand of 
them to reach one inch, or it would require about fifteen thousand of 
the bacteria which cause typhoid to form a line one inch in length. 
Of the very largest known it would require seven thousand to reach 
an inch. We often magnify bacteria one thousand times and then 
they appear as dots under the microscope, but if we would magnify 
a man to that extent he would appear to be six thousand feet tall 
and fifteen hundred feet wide. Bacteria are so small that at times 
we find five millions in a small drop of milk and yet they have 
plenty of room to move about, for it would require one hundred and 
twenty-five billion to weigh the same as a drop of milk. 

A person may wonder, since bacteria are so small, how they can 
bring about such enormous changes, for it takes but a short time 
for them to tear to pieces the body of a large animal that has died. 
All know how fast various plants and fruits decay under appro- 
priate conditions. Decay is due to bacteria. One organism could 
of itself bring about only a small change, but they multiply with 
almost inconceivable rapidity. The bacilli grow until they have 
reached a certain length, then divide into two, and these in turn 


40 MORPHOLOGY OF BACTERIA 


grow to maturity and then divide. Some of them may remain 
linked together, and hence appear as long chains. 

In the case of the cocci, they may divide into two and remain 
linked together as diplococci or a great many may remain connected 
together, thus giving the appearance of a string of beads; Strepto- 
coccus. ‘This is the characteristic of the common blood-poison 
organism. Other spherical shaped organisms divide alternately in 
two planes and when they remain connected together and great 
masses are formed they resemble a bunch of grapes; Staphylococcus. 
This is a characteristic of the common boil-causing organism. Still 
others of the spherical organisms divide alternately in three planes 
and when they remain connected appear very similar to a bale of 
cotton; Sarcina. This is a characteristic of many of the organisms 
found in air. 

It has been estimated that if bacterial multiplication went on 
unchecked, the descendants of one cell would in two days number 
281,500,000,000, and that in three days the descendants of this 
single cell would weigh 148,356,000 pounds. It has been further 
estimated by an eminent biologist that if proper conditions could 
be maintained for their life activity, in less than five days they 
would make a mass which would completely fill as much space as 
is occupied by all of the oceans on the earth’s surface, if the water . 
has an average depth of one mile! 

Even in the face of these assumptions one need not fear, for bac- 
teria have been on this earth, and have been multiplying probably 
long before the advent of man, and as yet the earth has not been 
filled by them. This is due to there being a struggle among them, 
just as there is among higher plants and animals. One knows that 
if wheat be sown too thick, none of it will mature. Sometimes it is 
a lack of food, other times a lack of sunshine, at still other times it 
is a lack of moisture which prevents the growth. So it is with 
bacteria, the food or water may give out, but more often it is the 
products which they form that prevent them from continuing to 
multiply. 

Brownian Movements.—If one examines under a microscope a 
suspension or colloidal solution containing particles about lu in 
diameter, they are seen to be in motion oscillating through a dis- 
tance about equal to their own diameter. With smaller particles 
the oscillation is much greater proportionately. When the diameter 
is about 4u the motions are hardly perceptible. The mean velocity 
for a particle of platinum weighing 2.5 x 10°" gm. has been estimated 
to be 3x 10°? cm. per second at ordinary temperature. These 
smaller particles often travel in straight lines and suddenly change 
their direction. Zsigmondy, describing the movement of the gold 
particles in a gold hydrosol, compared them to a swarm of dancing 
gnats. ‘This interesting phenomenon is called “Brownian Move- 


, 


BROWNIAN MOVEMENTS 41] 


ment” from Robert Brown (1773-1858), an English botanist, who 
first observed them in 1827 when studying grains of pollen. Observa- 
tions made by ingenious methods upon the Brownian movements 


Fie. 8.—Spirillum of Asiatie cholera, Fie. 9.—Spirillum volutans, showing 
showing single flagellum. (Kolle and flagella at either end of the bacterium. 
Zetnow.) (Herzog.) 


of colloidal suspensoids are exactly what the kinetic theory indi- 
cates would be the behavior of molecules of that size. Both dead 
and non-motile bacilli show this movement as do also small particles 
freely suspended in the liquid. However, many bacteria show a 


Fic. 10.—Bacillus proteus vulgaris, showing numerous flagella around the entire body 
of the bacterium. (Herzog.) 


true independent motion and if watched the organism will be found 
to change its position with relation to other organisms. This is 
known as “vital movement.” 


42 MORPHOLOGY OF BACTERIA 


The speed with which they travel, being magnified to the same 
extent as are the organisms, makes them appear to be travelling 
with enormous speed, insomuch that Leeuwenhoek, who first 
described it, stated that “they seemed to tear through each other.”’ 
The actual speed, however, is not great for the typhoid bacillus 
may travel a distance of 4 mm. or about 2000 times its own length 
in one hour, whereas the cholera spirillum has been known to attain 
a speed of 18 cm. per hour. Some organisms are motile if grown on 
one cultural media, and non-motile if grown on another; for example, 
the colon bacillus is usually motile if examined from young cultures 
grown on gelatin or agar, but non-motile if taken from boullion. 

Organs of Locomotion.— The protoplasmic threads called organs 
of locomotion are flagella or cilia. A cilium differs from a flagellum 
in that the former has a simple curve whereas the latter has a com- 
pound curve, like a whip lash. The size, the number, and the 
arrangement of the flagella are characteristic of the organism. 
Most bacteria possess flagella rather than cilia. Differences exist in 
respect to the number and position of the flagella on the cell body. 
Some forms possess only a single flagellum at one pole and are 
called monotricha, others a flagellum at each pole (amphitricha), 
others a tuft of flagella at one pole (lophotricha), others flagella 
projecting from the whole body of the cell (peritricha); and still 
others possess no flagella and are known as atricha. | 


Fig. 11—Pneumococci with unstained capsules. From pneumonia sputum, 
stained with carbol-fuchsin and differentiated with weak acid alcohol. Magnifica- 
tion 1000. (Karg and Schmorl.) 


Cell Wall (Ectoplasm).—The cell wall is the slightly differentiated 
outer portion of the cell substance. Many writers prefer to call it 
“ectoplasm.”” Early in the history of bacteriology it was con- 
sidered that the absence of cellulose in bacteria indicated that they 
belonged to the animal rather than to the plant kingdom. But 
cellulose or hemicellulose has been identified in bacteria from pus, 


METACHROMATIC GRANULES 43 


B. subtilis, tubercle bacilli, and diphtheria bacilli. However, the 
great majority of the organisms contain chitin, a substance which 
on hydrolysis yields glucosamin, CH,OH(CHOH);CHNH.CHO, 
and acetic acid. Chitin is typically animal in origin and for this 
reason some have argued that the bacteria belong to the animal 
and not to the plant kingdom. The flagella probably originate from 
the ectoplasm. 

Capsules.— Many bacteria possess a capsule which is an outgrowth 
of the cell membrane and is composed of mucin. In stained cultures 
it usually appears as a halo surrounding the organism. The forma- 
tion of a capsule is not confined to only a few species, some writers 
arguing that under appropriate conditions all organisms form them; 
yet the so-called capsulates are especially prone to do so. Some 
organisms produce capsules when grown on one media, but not if 
grown on another. Milk especially favors the formation of capsules. 

Sheath.— Often a distinct tube is formed in which is inclosed the 
chain of cells; to this tube is given the name “sheath.” It is espe- 
cially characteristic of some of the trichobacteria as crenothrix in 
which there is a deposition of iron. Sometimes these become fossil- 
ized, occurring in hugh deposits in ferruginous water. 

Zodgleea.— Often the gelatinous material of the cell causes great 
masses of cells to adhere to each other, to which condition is given 
the name “zoégloea.” This is especially characteristic of the nitri- 
fying bacteria. . 

Cytoplasm.— Chemical analysis of the cytoplasm of the bacteria 
cell shows it to be richer in nitrogen and phosphorus than are the 
cells of higher plants. Moreover, on being stained the cytoplasm 
appears as a homogeneous mass filling the whole cell, thus making 
it certain that bacteria do not possess a nucleus in the ordinarily 
accepted sense of the term. But the fact that the organisms stain 
so readily with the ordinary nuclear stains has led some to believe 
that the organisms are made up mainly of nuclear material. This 
is the view held by Zettnow who has succeeded in staining some large 
spirilla in a living motile condition. Hence the idea held by the 
majority of workers at the present time is that the bacterial cell is 
composed of small quantities of cytoplasm in which is imbedded 
large quantities of fragmented, irregularly distributed chromatin. 

Metachromatic Granules.— Some bacteria contain various granules 
within the cell which stain differently from the substance of the cell 
body; these are known as “metachromatic” granules or “Babes- 
Ernst” granules, or because of their frequent position at the ends of 
bacilli as polar bodies. Microchemical examination has shown them 
to be composed of various substances: fat, sulphur granules, gly- 
cogen, lecithin, and protein-like compounds. 

Their function has been variously interpreted. Some have com- 
pared them to the centrosomes of more highly specialized cells. 


44 MORPHOLOGY OF BACTERIA 


Others consider there to be a relationship between the richness of 
the cell in granules and its virulence. Hill, however, considers that, 
inasmuch as nitrates increase the nitrogen assimilated by Azotobacter 
and the number and size of the volutin bodies, they bear some 
relationship to the organisms’ power to fix nitrogen. Although it 


Fie. 12.—Successive stages in division of Bacillus diphtherie, showing relation of 
line of division to metachromatic granule. Continuous observation of living bacillus 
drawn without camera lucida. (Williams.) 


is quite possible that they may possess various functions in differ- 
ent organisms, the majority of them would seem to be, as suggested 
by Meyer, reserve food materials which occur in the cytoplasm of 
the cells of various bacteria. They are most numerous in rapidly 
growing young cultures and usually disappear when the food becomes 
scarce. 


Of A, 


Fig. 13.—Types of bacterial spores. (Kendall.) 


Spores.— Bacteria possess the power of mobilizing the vital parts 
of their body into a much smaller space than they occupy during 
their normal life. They exclude all of the excess moisture and sur- 
round themselves by a tough resistant coat. In some respects 
this form of the organism resembles the seed of the higher plant 
and we speak of it as a spore. While in this stage they will with- 
stand many conditions which would quickly prove fatal to growing 
bacteria. Some of them can withstand the temperature of boiling 
water for many hours, or they may survive treatment with strong 
carbolic acid. For the time being they have lost the power of mul- 
tiplying, but they are still alive and if they are brought into appro- 
priate surroundings they will change back into normal bacteria just 
as a kernel of wheat changes into the young plant when placed in 
moist soil. It is indeed fortunate for mankind that but few of the 
disease-producing organisms form spores. ‘There are, however, 
many of the bacteria which cause fruit, meat, and various other 
food products to spoil, which do form very resistant spores and this 


LONGEVITY OF BACTERIA 45 


is why many food products have to be heated for such a long time, 
or to such a high temperature to keep them. 

The manner of formation of spores within the body of the organ- 
ism is characteristic. They develop within the cell body and hence 
are called ‘‘endospores.’’ They are formed by the bacilli and spirilla, 
but not by the cocci. The beginning of spore formation is marked 
by a granulation of the cell contents. As the process proceeds the 
granules become larger and eventually fuse and collect at one por- 
tion of the cell which is then surrounded by a spore wall. The spore 
may be either smaller or larger than the mother cell. In the latter 
case there is a bulging of the mother cell. The spore may be equa- 
torial, polar, or intermediate within the cell depending on its position. 
When situated equatorially and larger than the mother cell it gives 
to it a boat-shape appearance (clostridia). If situated at the pole 
and large, we have the capitate or drumstick appearance. When 
bacteria are found in chains and spores form in the end, there is a 
tendency for them to occur in adjacent ends of contiguous cells. 
A cell usually forms only one spore; hence, this cannot be considered 
a process of reproduction. 

When the spores are brought under favorable conditions of food 
supply, temperature, and moisture they germinate. The process 
differs according to species. In some species the spore ruptures at 
the pole and the young cell emerges in such a way that its long axis 
is in the same direction as the long axis of the spore, thus leaving 
the spore membrane still visible at one of the poles. In other species 
the spore germinates equatorially and the young cell emerges with 
its long axis at right angles to the long axis of the spore. In still 
other species there is no rupturing of the spore, but germination 
occurs by a gradual elongation and absorption of the spore. 

Longevity of Bacteria.—Due to their method of multiplication 
there is no such condition as old age among bacteria since both 
daughter cells are similar in age and composition. It is well known 
that while in the spore condition many organisms can survive for 
over two decades. Both the spore-forming and non-spore-forming 
organisms have been obtained from soil which had been kept in 
bottles in an air dry condition for more than fifty years. Recently 
Sarcina lutea and other well-known air organisms have been obtained 
from a Mastodon uncovered by the recession of the ice in Siberia. 
This animal must have been covered for hundreds of years. This 
would, therefore, seem to indicate that the longevity of bacteria 
may be extremely great. 


CHAPTER IV. 
CLASSIFICATION OF BACTERIA. 


Tue difficulties inherent in the classification of bacteria are 
numerous and, due to the small simple structure of the organism, 
cannot be worked out on a purely morphological basis as is the 
case with the higher plant. Moreover, physiological characteristics, 
such as pigment production which at first sight may appear useful 
are not constant. Even morphology of bacteria was not considered 
constant until 1872 at which date Cohn established upon mor- 
phological bases a classification which with minor changes has been 
retained until the present. |Bacteria play a part in many fields of 
activity, and hence the criteria eria whereby they _are_recognized vary 
greatly according to the art or science in which they are studied,| 
This has led to considerable confusion in classification and nomen- 
clature as is so admirably pointed out by Jordan. 

“The present nomenclature of bacteriology may be ariGebeed 
on two grounds: first, as already pointed out, for the unwieldy 
size that certain ‘genera’ have been allowed to assume; and second, 
for the haphazard way in which trinomial and even quadrinomial 
names have been bestowed. Such names can be properly employed 
only with reference to subspecies or varieties; and designations, like 
B. coli communis, Granulobacillus saccharobutryicus mobilis non- 
liquefaciens and Micrococcus acidi paralacticr liquefaciens Halensi, 
are both cumbersome and unscientific. The use of a single genus 
name for a multitude of organisms is in fact responsible for the 
tendency toward trinomial nomenclature, and the remedy for both 
conditions would seem to lie in the abandonment of such a term as 
Bacillus for the name of a genus and the frank establishment of 
new genera on the basis of physiological characters, such, for example 
as distinguish the colon-typhoid group or the diphtheria group of 
bacilli. Until some such reform in nomenclature is brought about 
the names used to designate different kinds of hacteria will fail to 
make clear the group relationships which undoubtedly exist, and 
will continue to be a stumbling block to all students of the subject.” 

The classification most commonly accepted at the present day 
is that formulated by Migula. This, with certain modifications, is 
given below. 

Bacteria, Schizomycetes, fission fungi (chlorophyll-free), cell divi- 
sion in one, two or three planes; many varieties possess the power 
of forming endospores. Whenever motility is present, it is due to 
flagella, or more rarely to undulating membranes, 


, 
/ 
AN 


CLASSIFICATION OF BACTERIA AT 


Famity I.—Coccacee—cells in free state spherical; division i in one, 
two or three planes; endospore formation rare. 

Genus I. — Streptococcus— cells divide in one plane only for which 
reason; if they remain connected after fission bead-like chains may 
be formed; no organs of locomotion. 

Genus II.—WMicrococcus (Staphylococcus)—cells divide in two 
planes, whereby, after fission, tetrad and grape-like clusters may be 
formed; no organs of locomotion. 

Genus III.—Sarcina—cells divide in three planes, whereby, after 
fission, bale-like packets are formed; no organs of locomotion. 

Genus IV.—Planococcus—cells divide in two planes, as in Micro- 
coccus; possess flagella. 

Genus V.— Planosarcina—cells divide in three planes, as in Sar- 
cina; posses flagella. 

Famity I].—Bacteriacee—cells long or short; cylindrical, straight 
never spiral; division in one plane only, after preliminary elongation 
of the rods. 

Genus I.— Bacteriwm—cells without flagella; often with endospores. 

Genus II.—Bacillus—cells with peritrichal flagella; often with 
endospores. 

Genus III.—Pseudomonas—cells with polar flagella; endospores 
occur in a few species but are rare. 

Famity III.—Spirillacee—cells spirally curved or representing 
a part of a spiral curve; division in one plane only, after elongation 
of cell. 

Genus I.—Spirosoma—cells without organs of locomotion; rigid. 

Genus IJ.—Microspira—cells rigid, with one or more rarely, two 
or three polar undulated flagella. 

Genus III.—Spirillum—cells rigid, with polar tufts of five to 
twenty flagella usually curved in semicircular or flat undulating 
curves. 

Genus IV.—Sprrocheta—cells sinously flexible; organs of . loco- 
motion unknown, perhaps a marginal undulating membrane. 

Famity 1V.—Chlamydobacteriacee—Forms of varying stages of 
evolution, all possessing a rigid sheath, which surrounds the cells; 
cells united in branched or unbranched threads. 

Genus I.— Streptothrix—cells united in simple, unbranched threads; 
division in one plane only; reproduction by non-motile conidia. 

Genus II.—Cladothrix—cells united or pseudodichotomously 
branching threads; division in one plane only; vegetative multipli- 
cation by separation of entire branches; reproduction by swarming 
forms with polar flagella. 

Genus III.—Crenothrix—cells united in unbranched threads; 
division at first in one plane only. Later the cells divide in all three 
planes; the daughter cells become rounded and develop into repro- 
ductive.cells. 


48 CLASSIFICATION OF BACTERIA 


Genus IV.— Phragmidiothrix—cells at first united in unbranched 
threads, dividing in three planes, thus forming a rope of cells; later 
some of the cells may penetrate through sheath and thus give rise 
to branches. 

Famity V.—Beggisatoacea—cells united in sheathless threads; 
division in one direction of space only; motility by undulating mem- 
brane as in Oscillaria. 

Genus I.— Thiothrix—unbranched, non-motile threads, inclosed 
in fine sheaths; division of cells in one plane only; cells contain 
sulphur granules. 

Genus I1.—Beggiatoa—cells with sulphur granules. 

The difficulties inherent in this classification and especially the 
needs of reform to the agricultural bacteriologist are seen from the 
following: 

“Many workers in medical bacteriology and in other special 
fields of applied microbiology, who deal with only a few well- 
recognized species, may perhaps feel no need for any change in 
current practice. Few can deny, however, that it is a serious 
inconvenience for such names as B. welchw, B. sporogenes, B. per- 
fringens to be used by various workers, sometimes for the same, 
sometimes for different organisms, or for the same form to be 
described as Bacteriwm lactis aérogenes or Streptococcus lacticus 
when it is isolated from milk and as Streptococcus salivarvus or 
Str. fecalis when it is isolated from the human mouth or intestine.”’ 

‘When one passes from a study of the practical effects of the 
activity of some particular microbe to a consideration of its relation- 
ship to other forms it becomes essential not only to have a name 
for each kind of organism but to have also a system of nomenclature 
which will make it possible to express such relationship with reason- 
able clearness and accuracy.\ 

“This need is met by the Linnaean system of classification uni- 
versally adopted by all biologists outside our own limited and sys- 
tematically undeveloped fields. According to this Linnaean system 
each recognizable kind of plant or animal receives a binomial 
Latinized name, the first half designating the genus or group to 
which it belongs and the second half the particular kind or species 
to which the name applies. The genera in turn are grouped in 
tribes, the tribes in families, the families in orders, and the orders in 
classes. These divisions will often be artificial and often of greatly 
unequal size and importance in different groups. They make it 
possible, however, to express in a simple manner the essential facts 
of biological relationship—the fact that A, B, and C are more 
nearly related to each other than are any of them to D, E, and F; 
and that the series A-F exhibits common relationships closer than 
any similarities which its members bear to G or H. 

“Tf such a system is accepted it is in the next place important to 


CLASSIFICATION OF BACTERIA 49 


make sure that each group, from species to class, shall bear a single 
universal name. The name need not be appropriate; it need only 
be stable. It is an arbitrary label, not a description. If the door 
be once opened to criticism on the ground of inappropriateness, 
stability must disappear. 

“Tt is in order to ensure uniformity and stability of nomenclature 
that the International Codes referred to have been formulated; and 
it is to the International Rules of Botanical Nomenclature (1910) 


that we, as _as bacteriologists, should nat turally turn for guidance. 

Leaving out a great many minor rules and recommendations, 
the most important of the rules which would affect bacteriological 
practice may be cited as follows: 

Chapter I, Article 7.—‘“‘Scientific names are in Latin for all 
groups.” 

Chapter II, Article 10.—“ Every individual plant belongs to a 
species (species), every species to a genus (genus), every genus to 
a family (familia), every family to an order (ordo), every order to a 
class (classis), every class to a division (divisio).” 

Chapter III, Section 1, Article 15.—“Each natural group of 
plants can bear in science only one valid designation, namely, the 
oldest, provided that it is in conformity with the rules of Nomen- 
clature and the conditions laid down in Articles 19 and 20 of 
Section 2.” 

Chapter III, Section 2, Record iii.—“ Orders are designated 
preferably by the name of one of the principal families, with the 
ending ales.” 

Chapter III, Section 3, Article 21.—‘‘Families (familie) are 
designated by the name of one of their genera or ancient generic 
names, with the ending ace.” 

Chapter III, Section 3, Article 23.—‘‘Names of subfamilies 
(subfamilie) are taken from the name of one of the genera in the 
group, with the ending oidee. The same holds for the tribes 
(tribus) with the ending ee and for the subtribes (subtribus) with the 
ending ine.” 

Chapter III, Section 3, Article 24.—“Genera receive names 
(substantive adjectives used as substantives) in the regular singular 
number and written with a capital letter which may be compared 
with our own family names. These names may be taken from 7 


Samet Pi; Section 3 3, Article 26.—“ All species, even those that 
singly constitute a genus, are designated by the name of the genus to 
which they belong, followed by a name (or epithet) termed specific, 
usually of the nature of an adjective (forming a combination of two 
names, a binomial or binary name).”’ 


Chapter III, Section 3, Article 26, Record yiii,—“The specific 
4 


50 CLASSIFICATION OF BACTERIA 


name should in general give some indication of the appearance, the 
characters, the origin, the history or the properties of the species. 
If taken from the name of a person it usually recalls the name of the 
one who discovered or described it, or was in some way concerned 
with it. 

Chapter III, Section 3, Record x.—“‘Specific names begin with a 
small letter except those which are taken from names of persons 
(substantives or adjectives) or those which are taken from generic 
names (substantives or adjectives).”’ 

The classification suggested by the Committee of the Society 
of American Bacteriologists has many points of merit to the agri- 
cultural bacteriologist. The classification in brief is as follows: 


THE CLASS SCHIZOMYCETES. 


Minute, one-celled chlorophyll-free, colorless, rarely violet-red or 
green-colored plants, which typically multiply by dividing in one, 
two, or three directions of space. The cells thus formed are usually 
spherical, cylindrical, comma-shaped, spiral, or filamentous and are 
often united into filamentous, flat, or cubical aggregates. Filament- 
ous species often surrounded by a common sheath. The cell plasma 
generally homogeneous without a morphologically diiferentiated 
nucleus. Reproduction by simple fission. In many species resting 
bodies are produced, either endospores or gonidia. Cells may be 
motile by means of flagella. 

A. Order Myxobacteriales.—Cells united during the vegetative 
stage into a pseudoplasmodium which passes over into a highly- 
developed cyst-producing resting stage. 

B. Order Thiobacteriales.— Cells free or united in elongated fila- 
ments. Typically water forms, not cultivable on ordinary media. 
Life energy derived mainly from oxidative processes. Cells typi- 
cally containing either granules of free sulfur or bacteriopurpurin 
or both, usually growing best in the presence of hydrogen sulphid. 

C. Order Chlamydobacteriales.— Cells normally united in elongated 
filaments, often showing false but never true branching. Typically 
water forms. Sulphur and bacteriopurpurin are absent. Iron often 
present and usually a well-marked sheath. 

D. Order Actinomycetales.—Cells usually elongated, frequently 
filamentous and with a decided tendency to the development of 
branches, in some genera giving rise to the formation of a definite 
branched mycelium. Cells frequently show swellings, clubbed, or 
irregular shapes. No pseudoplasmodium. No deposits of free sul- 
phur or iron. No bacteriopurpurin. Endospores not produced, but 
conidia developed in some genera. Usually Gram-positive. Non- 
motile. Some species are parasitic in animals or plants. Not 
water forms. Complex proteins frequently required. As a rule 


CLASSIFICATION OF BACTERIA 51 


strongly aérobic (except for some species of Actinomyces and the 
genera Fusiformis and Leptotrichia) and oxidative. Growth on 
culture media often slow; some genera show mold-like colonies. 

Faminy I.—Actinomycetacee.— Filamentous forms often branched 
and sometimes forming mycelia. Conidia sometimes present. 
Some species parasitic. 

Genus 1.—Actinobacillus.— Filament formation, resembling strep- 
tobacilli. In lesions no mycelium formed, but at peripheries finger- 
shaped branched cells are visible. Gram-negative. Not acid-fast. 
Type species, Act. Lignieresi. 

Genus 2.—Leptotrichia.— Thick, long, straight or curved threads, 
unbranched, frequently clubbed at one end and tapering to the other. 
Gram-positive when young. ‘Threads fragment into short, thick 
rods. Anaérobic or facultative. Non-motile. Filaments sometimes 
granular. No aérial hyphe or conidia. Parasites or facultative 
parasites. Type species, Lep. buccalvs. 

Genus 3.—Actinomyces.—Organism growing in form of a much- 
branched mycelium which may break up into segments that func- 
tion as conidia. Sometimes parasitic, with clubbed ends of radiating 
threads conspicuous in lesions in animal body. Some species are 
micro-aérophilic or anaérobic. Non-motile. Type species, Act. 
bovis Harz. 

Genus 4.— Erysipelothriv.—Rod-shaped organisms with a ten- 
dency to the formation of long filaments which may show branching. 
The filaments may also thicken and show characteristic granules. 
No spores. Non-motile. Gram-positive. Do not produce acid. 
Micro-aérophilic. Usually parasitic. Type species, Bacillus 
rhusiopathie suis Kitt 1893; Mycobacterium rhusiopathie Chester 
1901; Erysipelothrix porct Rosenbach 1909, the causal organism of 
swine erysipelas. 

Famity Il.—Mycobactervacee.— Parasitic forms. Rod-shaped, 
frequently irregular in form but rarely filamentous and with only 
slight and occasional branching. Often stain unevenly (showing 
variations in staining reaction within the cell). No conidia. 

Genus 1.— Mycobactertwm.—Slender rods which are stained with 
difficulty, but when once stained are acid-fast. Cells sometimes 
show swollen, clavate, or cuneate forms, and occasionally even- 
branched cells. _Non-motile. Gram-positive. No endospores. 
Growth on media slow. Aérobic. Several species pathogenic to 
animals. Type species, Mycobacterium tuberculosis. 

Genus 2.—Corynebacterium.—Slender, often slightly curved, rods 
with tendency to club and pointed forms, branching cells reported 
in old cultures. Barred uneven staining. Not acid-fast. Gram- 
positive. Non-motile. Aérobic. No endospores. Some pathogenic 
species produce a powerful exotoxin. Characteristic snapping 
motion is exhibited when cells divide. Type species, Cornynebac- 
tervum diphtherie. 


52 CLASSIFICATION OF BACTERIA 


Genus 3.— Fusiformis.—Obligate parasites. Anaérobic or micro- 
aérophilic. Cells frequently elongate and fusiform, staining some- 
what unevenly. Filaments sometimes formed; non-branching. 
Non-motile. No spores. Growth in laboratory media feeble. 
Type species, Fusiformis termitidis Heelling. 

Genus 4.— Pfeifferella.—Non-motile rods, slender, Gram-nega- 
tive, stain poorly, sometimes forming threads and showing a ten- 
dency toward branching. Gelatin may be slowly liquefied. Do 
not ferment carbohydrates. Growth on potato characteristically 
honey-like. Type species, Pfeifferella mallet. 

E. Order Eubacteriales.—The order Eubacteriales includes the 
forms usually termed the true bacteria, that is, those forms which 
are considered least differentiated and least specialized. The cell 
metabolism is not primarily bound up with hydrogen sulphid or 
other sulphur compounds, the cells in consequence containing neither 
sulphur granules nor bacteriopurpurin. The cells apparently do not 
possess a well-organized or well-differentiated nucleus. These 
organisms are usually minute and spherical, rod-shaped or spiral, 
in most genera not producing true filaments, and rarely branching. 
The cells may occur singly, in chains, or other groupings. They 
may be motile by means of flagella, or non-motile, but they are never 
notably flexuous. Cell multiplication occurs always by transverse, 
never by longitudinal, fission. Some genera produce endospores, 
particularly the rod-shaped types. Conidia are not observed. 
Chlorophyll is absent, though the cells may be pigmented. The 
cells may be united into gelatinous masses, but they never form 
motile pseudoplasmodia nor develop a highly specialized cyst- 
producing fruiting stage, such as is characteristic of the Myzo- 
bacteriales. 

Famity 1.—Nitrobacteriacee.—Organisms usually rod-shaped 
(sometimes nearly spherical in Nitrosomonas and possibly in Azoto- 
bacter). Cells motile or non-motile. Branched involution forms in 
Rhizobium and Acetobacter. Endospores never formed. Obligate 
aérobes, capable of securing growth energy by the direct oxidation 
of carbon, hydrogen, or nitrogen, or of simple compounds of these. 
Non-parasitic (except in Genus Rhizobiwm)—usually water or earth 
forms. 

Tribe 1.—Nitrobacteree.—Organisms deriving their life energy 
from oxidation of simple compounds of carbon and nitrogen (or of 
alcohol). / 

Genus 1.—/1ydrogenomonas.—Monotrichie short rods capable of 
growing in the absence of organic matter and securing growth energy 
by the oxidation of hydrogen (forming water). Kaserer (1905) who 
first described the organism states that his species will also grow well 
on a variety of organic substances. Type species, Hydrogenomonas 
pantetropha (Kaserer 1906) Orla-Jensen. Nikleuski (1910) described 
two additional species, H, vitrea and H. flava, 


CLASSIFICATION OF BACTERIA 53 


Genus 2.—Methanomonas.—Monotrichic short rods capable of 
growing in the absence of organic matter and securing growth 
energy by the oxidation of methane (forming carbon dioxid and 
water). ‘Type species, Meth. methanica. 

Genus 3.—Carborydomonas.— Autotrophic rod-shaped cells cap- 
able of securing growth energy by the oxidation of carbon monoxid 
(forming carbon dioxid). Type species, Carb. oligocarbophila 
(Beijerinck and van Delden (1903) Orla-Jensen is described as non- 
motile. 

Genus 4.— Acetobacter.—Cells rod-shaped, frequently in chains, 
non-motile. Cells grow usually on the surface of alcoholic solutions 
as obligate aérobes, securing growth energy by the oxidation of 
alcohol to acetic acid. Also capable of utilizing certain other carbo- 
naceous compounds, as sugar and acetic acid. Elongated, filament- 
ous, club-shaped, swollen, and even-branched cells may occur as 
involution forms. Type species, Ace. aceti. 

Genus 5.— Nitrosomonas.—Cells rod-shaped or spherical, motile, 
or non-motile, if motile with polar flagella. Capable of securing 
growth energy by the oxidation of ammonia to nitrites. Growth on 
media containing organic substances scanty or absent. Type species, 
Nitro. europea Winogradsky. 

Genus 6.— Nitrobacter.—Cells rod-shaped, non-motile, not grow- 
ing readily on organic media or in the presence of ammonia. Cells 
capable of securing growth energy by the oxidation of nitrites to 
nitrates. Type species, Nitro. Winogradskyt. 

Tribe 2.—Azotobacteree (Nitrogen-fixing organisms). 

Genus 7.—Azotobacter.—Relatively large rods, or even cocci, 
sometimes almost yeast-like in appearance, dependent primarily 
for growth energy upon the oxidation of carbohydrates. Motile or 
non-motile; when motile, with tuft of polar flagella. Obligate 
aérobes usually growing in a film upon the surface of the culture 
medium. Capable of fixing atmospheric nitrogen when grown in 
solutions containing carbohydrates and deficient in combined nitro- 
gen. ‘Type species, Azotobacter chrodcoccum Beijerinck. 

Genus 8.— Rhizobium.—Muinute rods, motile when young. Invo- 
lution forms abundant and characteristic when grown under suitable 
conditions. Obligate aérobes, capable of fixing atmospheric nitrogen 
when grown in the presence of carbohydrates in the absence of 
compounds of nitrogen. Produce nodules upon the roots of legu- 
minous plants. Type species, R. leguminosarum Frank. 

Famity II.— Pseudomonadacew.—Rod-shaped, short, usually 
motile by means of polar flagella or rarely non-motile. Aérobic and 
facultative. Frequently gelatin liquefiers and active ammonifiers. 
No endospores. Gram stain variable though usually negative. 
Fermentation of carbohydrates as a rule not active. Frequently 
produces a water-soluble pigment which diffuses through the medium 


54 CLASSIFICATION OF BACTERIA 


as green, blue, purple, brown, etc. In some cases a non-diffusible 
yellow pigment is formed. Many yellow species are plant parasites. 

Genus 1.—Pseudomonas.—Characters, those of family. Type 
species, Ps. aéruginosa (Schroeter) Frost? 

Famity III.—Spirillacee.—Cells elongate, more or less spirally 
curved. Cell division always transverse, never longitudinal. Cells 
non-flexuous. Usually without endospores. As a rule motile by 
means of polar flagella, sometimes non-motile. Typically water 
forms, though some species are intestinal parasites. 

Genus 1.—Vibrio.—Cells short bent rods, rigid, single, or united 
into spirals. Motile by means of a single (rarely two or three) polar 
flagellum which is usually relatively short. Many species liquefy 
gelatin and are active ammonifiers. Aérobic and anaérobic. No 
endospores. Usually Gram-negative. Water forms, a few parasites. 
Type species, ’. comma (Koch 1884) Schroeter 1886. 

Genus 2.—Spirillum.—Cells, rigid rods of various thicknesses, 
length, and pitch of the spiral, forming either long screws or portions 
of a turn. Usually motile by means of a tuft of polar flagella (5 
to 20) which are mostly half circular, rarely wavy-bent. These 
flagella occur on one or both poles; their number varies greatly and 
difficult to determine; since in stained preparations several are 
often united into a common strand. Endospore formation has been 
reported in some species. Habitat: water or putrid infusions. Type 
species, S. wndula (Mueller 1786) Ehrenberg. 

Famity IV.—Coccacee.—Cells in their free conditions, spherical; 
during division somewhat elliptical. Division in one, two, or three 
planes. If the cells remain in contact after division they are fre- 
quently flattened in the plane of division and form chains, packets, 
or irregular masses. Motility rare. Endospores absent. Metab- 
olism complex, usually involving the utilization of amino-acids 
or carbohydrates. Pigment often produced. 

Tribe ‘A.— Neisserea.—Strict parasites, failing to grow or growing 
very poorly on artificial media. Cells normally in pairs. Gram- 
negative. Growth fairly abundant on serum media. 

Genus 1.—Neisseria.—Characters, those of tribe. Type species, 
N. gonorrhee ‘Trevisan. 

Tribe B.—Streptococcee.— Parasites (thriving only or best on 
or in the animal body) except genus Leuconostoc. Grow well under 
anaérobic conditions. Many forms grow with difficulty on serum- 
free media, none very abundantly. Planes of fission usually parallel 
producing pairs, or short, or long chains, never packets. Generally 
stain by Gram. Produce acid but no gas in glucose and generally in 
lactose broth. Pigment, if any, white or orange. 

Genus 2.— Diplococcus.— Parasites, growing poorly, or not at all, 
on artificial media. Cells usually in pairs of somewhat elongated 
cells, often capsulated, sometimes in chains. Gram-positive. Fer- 


CLASSIFICATION OF BACTERIA — 55 


mentative powers high, most strains forming acid in glucose, lactose, 
sucrose, and inulin. ‘Type species, D. pnewmoniew Weichselbaum. 

Genus 3.— Leuconostoc.—Saprophytes usually growing in cane 
sugar solutions. Cells in chains or pairs united in large zodgleal 
masses. Some types at least Gram-negative. Type species LD. 
mesenteroides (Cienkowski) van Tieghem. 

Genus 4.— Streptococcus.—Chiefly parasites. Cells normally in 
short or long chains (under unfavorable conditions sometimes in 
pairs and small groups, never in large packets). Generally stain by 
Gram. Capsules rarely present, no zodgleal masses. On agar 
streak, effused translucent growth often wih isolated colonies. In 
stab culture little surface Seach. Many sugars fermented with 
formation of large amount of acid, but inulin is rarely attacked. 
Generally fail to liquefy gelatin or reduce nitrates. Type species, 
S. pyogenes Rosenbach. 

Genus 5.— Staphylococcus.— Parasites. Cells in groups and short 
chains, very rarely in packets. Generally stain by Gram. On agar 
streak good growth, of white or orange color. Glucose, maltose, 
sucrose, and often lactose, fermented with formation of moderate 
amount of acid: Gelatin often liquefied very actively. ‘Type species, 
S. aureus Rosenbach. 

Tribe C.—Micrococcee.—Facultative parasites or saprophytes. 
Thrive best under aérobic conditions. Grow well on artificial media, 
producing abundant surface growths. Planes of fission often at 
right angles; cell aggregates in groups, packets, or zo5gleal masses. 
Generally decolorize by Gram. Pigment yellow or red. 

Genus 6.—Micrococcus.—Facultative parasites or saprophytes. 
Cells in plates or irregular masses (never in long chains or packets). 
Generally decolorize by Gram. Growth on agar abundant, with 
formation of yellow pigment. Glucose broth slightly acid, lactose 
broth generally neutral. Gelatin frequently liquefied, but not 
rapidly. Type species, M. luteus (Schroeter) 1872b, Cohn. 

Genus 7.—Sarcina.—Sarcina differs from Micrococcus solely 1 in 
the fact that cell division occurs under favorable conditions in three 
planes, forming regular packets. Type species, Sarcina ventricult 
Goodsir. 

Genus 8.— Rhodococcus.—Saprophytes. Cells in groups or regular 
packets. Generally decolorize by Gram. Growth on agar abundant 
with formation of red pigment. Glucose broth slightly acid, lactose 
broth neutral. Gelatin rarely liquefied. Nitrates generally reduced. 
Type species, R. rhodochrous Zopt. 

Famity V.—Bacteriacee.—Rod-shaped cells without endospores. 
Usually Gram-negative. Flagella when present peritrichic. Metab- 
olism complex, amino-acids being utilized and generally carbo- 
hydrates. 


56 CLASSIFICATION OF BACTERIA 


Tribe 1.—Chromobactere.—Water bacteria producing a red or 
violet pigment. 

Genus 1.— Erythrobacillus.—Small aérobic bacteria, producing a 
red or pink pigment, usually a lipochrome. Gram stain variable. 
It is possible that related yellow and orange chromogens should be 
included here as well. Type species, E. prodigiosus (Ehrenberg). 

Genus 2.— Chromobacterium.— Aérobic bacteria, producing a violet 
chromoparous pigment, soluble in alcohol but not in chloroform. 
Motility and Gram reaction variable. ‘Type species, Chr. violecum 
Bergonzini. 

Tribe 2.— Erwinee.—Plant pathogens. Growth usually whitish, 
often slimy. Indol generally not produced. Acid usually formed in 
certain carbohydrate media, but as a rule no gas. 

Genus 3.—Erwinia.—Characters those of the tribe. Type species, 
E. amylovora. 

Tribe 3.—Zopfee.—Gram-positive rods, growing freely on arti- 
ficial media. Not attacking carbohydrates. 

Genus 4.—Zopfius.—Long rods occurring in evenly curved chains. 
Gram-positive. Motile. Proteus-like growth on media. Faculta- 
tive anaérobes. Carbohydrates and gelatin not attacked, hydrogen 
sulphid not formed. Type species, Z. zopfi (Kurth) Wenner and 
Rettger. 

Tribe 4.—Bacteree.—Gram-negative rods growing freely on 
artificial media. Generally forming acid from carbohydrates and 
often gas composed of CO, and Hp. 

Genus 5.—Proteus.—Highly pleomorphic rods, filaments and 
curved cells being common as involution forms. Gram-negative. 
Actively motile. Characteristic ameboid colonies on moist media. 
Liquefy gelatin rapidly and produce vigorous decomposition of 
proteins. Ferment glucose and sucrose (but usually not lactose) 
with formation of acid and gas (the latter being CO, only). Type 
species, P. vulgaris Hauser. 

Genus 6.— Bacterium.—Gram-negative, evenly staining rods. 
Often motile, with peritrichic flagella. Easily cultivable, forming 
grape-vine leaf or convex whitish surface colonies. Liquefy gelatin 
rarely. All forms except B. alcaligenes and the Bb. abortus group 
attack the hexoses and most species ferment a large series of carbo- 
hydrates. Acid formed by all, gas (CO, and He) only by one series. 
Typically intestinal parasites of man and the higher animals although 
several species may occur on plants, and one (B. aérogenes) is widely 
distributed in nature. Many species pathogenic. Type species, 
B. coli Escherich. 

Tribe 5.—Lactobacillee.—Rods often long and slender, Gram- 
positive, non-motile, without endospores. Usually produce acid 
from carbohydrates, as a rule Jactic. When gas is formed it is CO; 


CLASSIFICATION OF BACTERIA 57 


without H». The organisms are usually somewhat thermophilic. 
As a rule micro-aérophilic; surface growth on media poor. 

Genus 7.—Lactobacillus.—Generic characters those of the tribe. 
Type species, L. caucasicus (Kern?) Beijerinck. 

Tribe 6.— Pasteurellee.—Gram-negative rods, showing bipolar 
staining. Parasitic forms of slight fermentative power. 

Genus 8.— Pastewrella.—Aérobic and facultative. Powers of car- 
bohydrate fermentation slight; no gas produced. Gelatin not 
liquefied. Parasitic, frequently pathogenic, producing plague in 
man and hemorrhagic septicemia in the lower animals. Type 
species, P. cholere-gallinarum (Fliigge) 1886 Trevisan. 

Tribe 7.—Hemophilew.—Minute parasitic forms growing only in 
presence of hemoglobin, ascitic fluid or other body fluids. 

Genus 9.—Hemophilus.—Minute rod-shaped cells, sometimes 
thread forming and pleomorphic, non-motile, without spores, strict 
parasites, growing best (or only) in presence of hemoglobin, and in 
general requiring blood serum or ascitic fluid. Gram-negative. 
Type species H]. influenze (Pfeiffer 1893). 

Famity VII.— Bacillacee.—Rods producing endospores, usually 
Gram-positive. Flagella when present peritrichic. Often decom- 
pose protein media actively through agency of enzymes. 

Genus 1.—Bacillus.—Aérobic forms. Mostly | saprophytes. 
Liquefy gelatin. Often occur in long threads and form rhizoid - 
colonies. Form of rod usually not greatly changed at sporulation. 
Type species, B. subtilis Cohn. 

Genus 2.—Clostridiwum.—Anaérobes or micro-aérophiles. Often 
parasitic. Rods frequently enlarged at sporulation, producing 
clostridium or pleotridium forms. Type species, C. butyricum. 
Prazmowski. 


CHAPTER V. 
COMPOSITION OF BACTERIA. 


THE elementary composition of bacteria is the same as that 
of the higher plants. This is also true concerning the main chem- 
ical constituents composing their body. But the proportions of 
these latter at times vary quite widely. Moreover, some micro- 
organisms contain constituents not found in higher plants. 

Elementary Composition.— Bacteria on analysis yield carbon, hy- 
drogen, oxygen, nitrogen, potassium, phosphorus, sulphur, calcium, 
magnesium, iron, aluminum and manganese. As to whether the 
last two are essential to normal development is not certain. In 
some species they are known to be non-essential, whereas in others, 
for instance the Azotobacter, they seem to play an important part. 

Moisture.— Moisture is essential for all plant and animal life and 
is always abundant in actively growing cells; hence, we expect, 
and do actually find, large quantities of water in bacteria. The 
quantity present in the actively growing cell varies from as low as 
70 per cent. to as high as 90 per cent. Generally speaking, young 
cultures contain less moisture than do older cultures; this appears 
to be true until the spore stage is reached, after which the quantity 
of water greatly decreases. The temperature at which the cultures 
are grown also governs in a measure the quantity of water present, 
this being less when grown at 37° C. than when grown at 20° C. 
The cultural media undoubtedly play’a great part in determining 
the moisture content of the cells. It is probably rather low in 
bacteria obtained from alkali soil or from saline waters. 

Organic Constituents.—The bacterial cell contains carbohydrate- 
like bodies, proteins, extractives (fats, fatty acids, waxes and 
lipoids), and enzymes. In addition to these some bacteria also 
contain pigments, toxins and possibly ptomains. The quantity 
and quality of each, especially of the last four, vary greatly with 
the class of organisms and the conditions under which they are 
grown. . 

Carbohydrates are really conspicuous by their absence in most 
bacterial cells, but the following members of the carbohydrate 
group have been recognized in varying quantities in some bacteria: 
cellulose, hemicellulose, dextrin, starch, glycogen and a number of 
the sugars. 

Extractives, although found to a limited extent in all microérgan- 
isms, are found in larger quantities in the tubercle bacillus and other 


~ 


ORGANIC CONSTITUENTS 59 


members of the acid-fast group. In some members of this group 
the extractives vary from 26 to 40 per cent. of the total dry residue. 
In the early studies of the chemistry of bacterial cells it was assumed 
that the alcoholic and ethereal extracts consisted of fats exclusively. 
Tributyrin, tripalmatin, tristearin, triolein, lecithin and various 
waxes have been recognized. 

Klebs found in the tubercle bacillus 20.5 per cent. of a red fat 
melting at 42° and 1.14 per cent. of a white fat melting above 50°, 
the latter being insoluble in ether but soluble in benzol. De Schwei- 
nitz and Dorset concluded that the fat of the tubercle bacillus con- 
tains palmitic and arachidic acids, while that of the glanders bacillus 
contains oleic and palmitic. They also obtained a crystalline acid, 
for which they suggested the name tuberculinic acid. This is quite 
different from Ruppel’s nucleic acid. It had an elementary com- 
position of C7H,O.. The authors called attention to the similarity 
in composition and properties of this body and tetraconic acid. 
They suggest that it may be the substance which is responsible 
for the coagulating necrosis and reduction in temperature. 

Kresslig extracted tubercle bacilli successfully with ether, chloro- 
form, benzol and alcohol, and obtained 38.95 per cent. of fatty and 
waxy substances. Repeated extraction with chloroform gave a 
dark brown mass of the consistency and color of beeswax and melting 
at 46°. He found 14.38 per cent. of free fatty acid, 77.25 per cent. 
of neutral fat and esters of fatty acids, and some volatile fatty 
acid, probably butyric. He concluded that the fat of the tubercle 
bacillus is quite different from that obtained from any other source. 

The fats and waxes are probably both intra- and extracellular, 
for extraction of the intact cell yields some and the crushed cell 
yields still more. The quantity found within the cell varies greatly, 
depending on the media upon which the organism is grown. Meyer 
found that the fat in Bacillus twmescens gradually increases until 
spore formation occurs, when it disappears; the spores are also free 
from fat. This, however, is not general for the spores of some 
organisms contain proportionally more fat than do the vegetative 
forms. 

Proteins.—The bulk of the dry matter of the bacterial cell 1s com- 
posed of proteins. The following analysis reported by Ruppel 
indicates the composition of the tubercle cell: 

Nucleic (tuberculinic acid) 8 
Nucleoprotamin PE tape hee ae Piet eee pelle ee 
INcleoprotelding lin: genet ak nN hee Mere: Wap re oF 
Albuminoids 8 

6 


Tater Ce we We ee ce ene ee AT DRI gM ray aR 
EAS ite pe i omer tL ae le eevee ketamine, 9 te LEA er geek MQ) yf a8 


The wonderful synthetic reaction catalyzed by the Azotobacter cell 
has directed the attention of workers to this specific organism. 


60 COMPOSITION OF BACTERIA 


Therefore, our knowledge of the composition of this organism is 
probably more nearly complete than it is of many other species. 

Berthelot early recognized that the nitrogen fixed by the Azoto- 
bacter is insoluble in water, thus indicating its protein nature. 
Lipman found there was a small but appreciable quantity of nitro- 
gen in both young and old cultures of A. vinelandii not precipi- 
tated by lead acetate and a large proportion not precipitated by 
phosphotungstic or tannic acid. Further work indicated that the 
substance was either amino-acids or comparatively simple peptids. 
He considered that one of the early substances synthesized by these 
organisms was alanin. An analysis of the Azotobacter membrane 
gave the following: 


INitrogenas ammonia ye ee acct eee ke oe ere 0.9 oper cent. 
Basie Nitrogen a)" j. yee hh nee Rs ae ee ne tee O 
Non-basic nitrogen ‘ LE AOR Nah Sah aaa oe ae are ONO 
Nitrogen in MgO precipitate ay wie 1 len erences ecm CA, t 
Total nitrogen . . ee eer i ier ice OSA ¢ 


This, he finds, corresponds remarkably closely to legumin. That 
it is complex is indicated by the fact that it is not readily assimilated 
by plants. 

Stoklasa found the Azotobacter cell to contain 10.2 per cent. of 
total nitrogen and 8.6 per cent. of ash. The ash was from 58 to 
62.35 per cent. phosphoric acid. The nitrogen and phosphorus 
were mainly in the forms of nucleoproteins and lecithin. The 
percentage of both nitrogen and phosphorus in the cell increases 
with age. 

The most complete analysis of the Azotobacter cells, so far 
reported shows them to contain, when grown on dextrin agar and 
rapidly dried at 30° C., 12.92 per cent. of protein. The protein is 
similar to other plant proteins. It contains 10 per cent. of ammonia 
nitrogen, 26.5 per cent. of diamino-nitrogen, and 60 per cent. of 
mono-amino-nitrogen. It contains the amino-acids normally found 
in proteins but the quantity of lysin present is high, whereas the 
histidin is present only in traces. 

An examination made by Nishimura of a pure culture of a water 
bacillus gave the following as the composition of the dry matter in 
the bacillus. 


Albumin fo Sie op ea) DMA Wake ita op op eat Oro OUDEL cent. 
Carbohydrates ia) ) 20 tums be Re werd onl as te. oo ewerttalacee 
Alcoholextract (20 Wate on We ease Dore 3.2 sg 
Mther extract: vet mer el aah a ae ee ee en OTC “ 
Ash iis. Ss re a es ae en aoe ak hee es eat MELE) or 
Hecithin 2/7 aos | A ee Wao tle cee ee OL OS: if 
EXianthin seule ee pee hie so) ee Le lee ane RTL “s 
Gann 8 ee A, a eo ca) oe ee ee mC nen EO ok S 


Adenin. sy Blass, | bee a Shere et. get ee ee ee ee es es 


VARIATION IN DIFFERENT PARTS OF THE CELL 61 


The organism is, therefore, extremely rich in protein, and, 
although the albumin predominates it is not free from nucleoprotein, 
as is seen from the presence of the purine bases, xanthin, guanin 
and adenin. 

Inorganic Constituents.—The ash-content of the bacterial cell is 
not far different qualitatively from that of the higher plants. 
Quantitatively, however, there is a marked difference, the ash- 
content of bacteria being comparatively high. The ash-content 
of the cell is subject to wide variation, depending on the specific 
organisms and especially on the media upon which it is grown. 
This may be seen from the following results by Cramer who grew 
the Cholera wbrio on various media. 


1 per cent. soda | Phosphate | : 
| bouillon (regular | bouillon (regular GReriee poe 
broth + 1 percent. broth + 1 percent. 4 pe Gait Nach: 


Composition of medium in 
which organisms were 


Erown. NaOH). | sod. phosphate). 
Ash-content of bacteria in dry sub- 

SUANCC eee he i ye ne Fh 9.30 22.30 25.90 
Ash-content of moist mass 1.34 Date Seo 
Ash-content of medium in moist 

TN SSSo aA ee AE ee, Lh A 1.25 P45) Al? 
Phosphoric acid in bacterial ash . | 28.70 | 34.80 | 10.90 
Phosphoric acid in media ash ._. | 7.90 39.80 2.10 
Chlorin in bacterial ash hte, we 16.90 Gaon 40.70 
Chiorin'im mediaash . 9. . ..| 23.00 11.40 49.20 


Analyses have been reported in which the phosphoric acid-con- 
tent reaches as high as one-half the total ash-content of the cell. 
It is quite probable that a great proportion of this is combined with 
the nucleic acid in the nucleoproteins. 

Variation in Composition of Different Parts of the Cell.—As has 
been pointed out, the bacterial cell is not homogeneous but is made 
up of fairly distinct parts, namely, ectoplasm, capsule and cyto- 
plasm and nuclear material. These constituents vary noticeably 
in their chemical composition. Although the ectoplasm at times 
contains in some species of bacteria small quantities of cellulose 
and hemicellulose, yet the predominating substance is chitin, a 
substance which may be considered as an intermediary compound 
between the carbohydrates and proteins. When pure, chitin yields 
over 80 per cent. of its weight as glucosamine. It yields first acetic 
acid and chitosan: 


CisHs0oN201n + 2H2O = 2CHsCOOH + CrusHogN2O10 
Chitin. Acetic acid. Chitosan. 


Chitosan on further hydrolysis yields acetic acid and glucosamine: 


CiusHosN2010 + 2H20 = CHsCOOH + 2CH2OH(CHOH)s;sCHNH2CHO 
Chitosan, Acetic acid, Glycosamine. 


62 COMPOSITION OF BACTERIA 


The cell membrane is, therefore, more nearly that of the animal 
than the plant. The brown color obtained on staining some 
bacteria with iodin has led observers to believe that they contain 
glycogen, whereas the blue color with the same reagent is attributed 
to starch. 

The capsules contain comparatively large quantities of mucin. 
These are protein-like substances which may be precipitated by 
alcohol. They give most of the protein reactions and, in addition, 
when heated with an acid, acquire the property of reducing Fehling’s 
solution, thus showing them to contain a carbohydrate complex in 
addition to the protein. 

The cytoplasm consists largely of bacterial proteins which appear 
to be specific in character for any given species. Within this are 
large quantities of the nucleoproteins, for on hydrolysis large 
quantities of the purine bases are obtained. Vaughan, Wheeler 
and Leach conclude that the bacterial cytoplasm contains carbo- 
hydrates, nuclein bodies, and polymers of mono- and di-amino-acids. 
They are glyconucleoproteins. Spores differ from the vegetative 
organism in that they contain but small quantities of water. 


REFERENCES. 
Vaughan: Protein-split Products in Relation to Immunity and Disease. 


Kendall: Bacteriology—General, Pathological, Intestinal. 
Kruse; Allgemeine Microbiologie. 


CHAPTER. V1. 
FOOD REQUIREMENTS. 


Foop is any substance which bacteria can utilize in obtaining 
either building material or energy for the cell activity. The quan- 
tity and quality of food necessary vary widely with the different 
species. However, all foods must contain certain essential ele- 
ments. Our knowledge at the present time indicates these ele- 
ments to be carbon, hydrogen, oxygen, phosphorus, potassium, 
nitrogen, sulphur, calcium, iron and magnesium, or, using the key 
for remembering as suggested by Dr. Hopkins, we have C. Hopk’ns 
CaFe Mg - - - “C. Hopk’ns Cafe - - mighty good.” 

Minimum Requirements.—In considering the food used by bacteria 
a minimum and a maximum requirement must be recognized. 
These two extremes differ greatly, for although the minimum quan- 
tities appear inconceivably small the maximum ones are enormous. 
One may obtain a fair idea of the minimum requirements from the 
following calculation made by Rahn: “The quantity of organic 
and inorganic matter just sufficient to support a very weak growth 
is certainly very small, since a few species will multiply to some 
extent in ordinary distilled water. Such water, after having stood 
for some time, is found to contain several thousand bacteria per 
cubic centimeter. It may seem to the laymen that in such water 
it would be possible to detect easily the organic and inorganic matter 
of the microérganisms so that it could not be considered distilled 
water. An estimate of the weight of bacteria demonstrates, how- 
ever, that this is not the case. If we suppose the average bacterial 
cell to be a cylinder whose base measures 1 square micron and 
whose height is 2 microns (which is a high estimate). The volume 
of such a cell would be 1 X 1 X 2 cubic microns = 0.001 X 0.001 
< 0.002 mm. = 0.000,000,002 cumm. The specific gravity of 
bacteria being very nearly one, the weight of one bacterium would 
be 0.000,000,002 mg. One hundred thousand cells per cubic centi- 
meter means 100,000,000 cells per liter, which would weight 0.2 
mg. Of this total weight, at Jeast four-fifths is water and only one- 
fifth is solid matter. The total solid matter in 1 liter of water 
containing 100,000 bacteria per cubic centimeter amounts to the 
immeasurable quantity of 0.04 mg. Such water will pass the test 
for distilled water. How much food the bacteria in distilled water 
have used is impossible to say, since, besides the traces of minerals 


64 FOOD REQUIREMENTS 


in the water, they obtain some food from volatile compounds of 
the air, like carbon monoxid (CO), carbon dioxid (CO.), ammonia 
(NH;), hydrogen (H) and perhaps methane (CH,). Under all 
circumstances the amount of food used is very small.” 

Maximum Requirements.—The maximum quantity of food which 
may be decomposed by bacteria is often enormous. They quickly 
decompose the body of an ox after its death. Tons of material 
run into the septic tanks of large cities, all of which is rapidly 
decomposed by bacteria. It is, however, usually the case that the 
speed of the reaction is great at first, but soon slows up or comes 
to a complete stop. This is due to the fact that the accumulation 
of end-products interferes with the growth of bacteria. This is 
true in milk where at first the lactose is rapidly changed to lactic 
acid, which if not neutralized soon becomes concentrated enough, 
to slow up the reaction. This is also true with the changes going 
on in sauerkraut and silage. 

Function of the Food.—The food utilized by bacteria has two 
functions, namely, the furnishing of energy and the acting as cellular 
building material. The quantity required by each bacterial cell 
for building material is not great, for MacNeal and his associates 
found that the dry matter of 550,000,000 cells of B. colt weigh 
only 0.1 mg. Others have estimated the weight of a single colon 
bacillus to be 0.000,000,163 mg., or it would require 1,600,000,000 
colon bacilli to weight approximately 1 mg. The waste products 
and repair material would make the cellular requirements slightly 
greater than this, but from these figures it is evident that the actual 
quantity required by a cell for building material is extremely small. 
Even this, however, is not immaterial, for Conn starting with the 
assumption that the period of generation is a half hour makes the 
following calculation. “If we take a single bacillus measuring 
2u in length and ly in breadth, with a weight of 0.000,000,001,571 
mg., it’ will increase, according to the aforesaid assumption, at 
such a rate that in two days’ time its progeny will amount to 
281,000,000,000, and will occupy a volume equal to about 4 liter 
(30.51 cu. in.); within a further three days the quantity would 
increase to a mass sufficient completely to fill the beds of all the 
oceans of the globe.’’ Due to the accumulation of waste products 
they never continue to multiply long at such a rate, but the numbers 
in suitable media often become hundreds of millions per cubic 
centimeter before retardation occurs. 

Source of Energy.—Animals and plants require energy in their 
life activity, the former obtaining it directly from the kinetic 
energy of the sun which they store up as potential energy. 
This is liberated by the animal in the process of oxidation. Now, 
bacteria do not possess the powers of the higher plants to utilize 
directly the energy of the sun, but, like the animals they are 


MOISTURE 65 


dependent on the stored energy of the plant and animal kingdom. 
From their method of oxidation it is necessary to recognize two 
groups of bacteria: (1) Those which completely oxidize their 
food, the carbon and hydrogen occurring in the final products as 
carbon dioxid and water; (2) those which only partly decompose 
their food, thus leaving much of the energy still within the food. 
Now the actual food requirements of the two classes of organism for 
the accomplishment of the same end, in so far as energy is con- 
cerned, is materially different. For instance, the complete oxida- 
tion of glucose to carbon dioxid and water as brought about by some 
yeasts according to the equation CsH1».O0,; + 60, = 6CO, + 6H,O + 
674 cal.; whereas, when only partly oxidized to alcohol it would be 
CsH2O; = 2C.H;OH -f- 2CO; +- 22 eal. 

The energy obtained in the first case is over thirty times that 
obtained in the second, and the quantity of food decomposed would 
be relatively greater in the latter than in the former. It has been 
estimated that the lactic acid bacteria decompose their own weight 
of sugar in one hour. 

Although all organisms require the elements listed at the begin- 
ning of this chapter, yet the nature of the organic compound required 
varies greatly with different species. 

Moisture.—Moisture may be considered the most important 
factor of life. “It is little short of astounding that living matter 
with all its wonderful properties of growth, movement, memory, 
intelligence, devotion, suffering and happiness should be composed 
to the extent of from 70 to 90 per cent. of nothing more complex or 
mysterious than water. Such a fact as this is most perplexing, espe- 
cially when all experiments show that this water is playing a pro- 
foundly important part in the generation of the vital phenomena. 
Any interference with the amount normally present makes a change 
at once in the activities of the cell. In fact we might say that 
‘all living matter lives in water,’ as Claude Bernard put it. For 
not only is this obviously true in the lower and simpler forms of 
animals and plants, which are little more than naked masses of 
protoplasm living in water, but it is no less true of the higher 
forms, since in all of them an internal medium, or environment, of 
a liquid nature, the lymph, the blood or sap, is found which is the 
immediate environment of the cells. Water is the largest and one 
of the most important constituents of living matter, and if organisms 
are carefully examined the most various devices are found to assure 
the regulation of the water content of the cells of the body. The 
younger, the more vigorous, the more alive, the more actively 
growing, the more impressible cells are, the more watery are they.” 

Water enters very largely into the composition of the bacterial 
cell, since they consist of from 70 to 95 per cent. water; moreover, 
it enters into nearly every change which they bring about. When 

5 


66 FOOD REQUIREMENTS 


bacteria decompose the carbohydrates, one or more molecules of 
water are taken up; when they synthesize, water is eliminated. 
The hydrolysis of fats requires for every molecule three of water; 
when they are synthesized from glycerin and fatty acids three 
molecules are eliminated. The digestion of the proteins by bacteria 
is usually hydrolysis in which a number of molecules of water are 
caused to enter the large protein molecule, thus causing it to break 
down into elementary diffusible foods. When the bacteria build 
their own proteins from the peptones and amino-acids, it requires 
that water be eliminated. Thus water plays an all-important part 
in all bacterial syntheses and decompositions. 

Water accelerates or is essential in all reactions taking place in 
the cell. It has a higher inductive capacity, or dielectric constant, 
than any other liquid, except possibly hydrogen dioxid. “It is a 
good insulator. It does not in itself, at ordinary temperatures, 
conduct the current readily. In virtue of this property it happens 
that when electrical disturbances occur in a cell they are not 
instantly compensated, so that oppositely charged particles may 
coéxist in water. It is probably because of this property that 
water forms such a good ionizing medium. At any rate, this 
property may account for the undoubted fact, whatever explanation 
we may choose to give of that fact, that substances dissolved in 
water interact with greater ease and speed than when dissolved in 
any other nedium. It has the property then, so important for the 
cell, of accelerating all kinds of chemical reaction. Thus hydro- 
gen and oxygen will not unite, except at very high temperatures, 
unless some water is present. Hydrochloric acid and sodium 
hydrate react vigorously in the presence of water, but not when they 
are quite dry. Chlorin and hydrogen do not form hydrochloric 
acid, except at very high temperatures, unless water be present, and 
everyone knows that the rusting of iron does not occur unless water 
is there too. Water has, then, this fundamental property of 
making reactions go on between bodies dissolved in it or wet by it. 
This property is believed by many to be correlated with its ionizing 
powers and with the fact that its solutions conduct electrical currents 
more than those of any other solvent.” 

Another very remarkable property of water is its power of solu- 
tion. No other solvent surpasses it. All substances dissolve in it 
to some extent. It is a solvent for salts, carbohydrates, protems 
and even for fats to some extent. This universal solvent power 
has not yet been fully explained, but it is probable that it is cor- 
related with, or due to, the extra valances on the oxygen atoms 
which are perhaps able to unite with the extra valances on the dis- 
solving molecules and thus to produce solution. But be the expla- 
nation what it may, it is well known that its solvent action con- 
tributes much to life. Bacteria are able to absorb their food only 


KIND OF FOOD REQUIRED 67 


when in solution, while in solution it reacts and after it has served 
its purpose the waste products are carried from the cell in solution. 

Osmotic Pressure.—If a cell be placed in a strong salt solution, 
there is a shrinking of the cell which may result in plasmolysis. 
If, on the other hand, a cell be suspended in pure water the cell 
greatly increases in size and finally bursts. This is the case when 
any solution is separated from pure water or from a less-concen- 
trated solution by a membrane which to the dissolved substance is 
impermeable but to the water of the solution permeable. The solu- 
tion exerts pressure on the membrane and the water passes through 
the membrane into the solution. The pressure is called osmotic 
pressure, and depends not upon the percentage of solute, but upon 
the number of particles—molecules and ions—per unit volume. The 
amount of this pressure varies in different cells, but for mammals 
it is supposed to be about that of a 0.9 per cent. sodium chlorid 
(NaCl) solution, since in such a solution the tissue neither gains nor 
loses weight. This is about 7.1 atmospheres. 

However, some bacteria and many molds can survive and even 
grow in salt solution which would be fatal to the life of the cell of 
higher plants. Penicilliwm and Aspergillus have been known to 
thrive in solutions, the osmotic pressure of which is equivalent 
to a 20 per cent. potassium nitrate solution. Bacillus anthracis 
flourishes on agar containing as much as from 8 to 10 per cent. of 
sodium chlorid. Since turgidity is essential to growth, it follows 
that these organisms must have some means of altering the pressure 
of their cell contents according to the concentration of the sur- 
rounding medium; only in this way can plasmolysis be avoided. 
The plasmotic membrane in the case of many bacteria is highly 
permeable; this would be the case, especially with those organisms 
which grow in brines. Even some pathogenic bacteria possess the 
power of accommodating themselves to high osmotic pressures. 
Bacillus cholere are temporarily plasmolyzed by salt and sucrose 
solutions but not at all by a glycerin solution, the cell membrane 
being permeable to the latter. The plasmolysis produced by the 
salt and sugar disappear in the course of an hour or two as a rule, 
showing that even salt and sugar slowly penetrate the plasmotic 
membrane. 

Kind of Food Required.—The quality of the food required by 
bacteria varies greatly with the species. This is well exemplified 
in Jensen’s classification of bacteria which is based upon the sources 
of nutrition and distinguishes the following groups: 

“1. Bacteria which, like green plants, need neither organic 
carbon nor organic nitrogen. These so-called ‘autotrophic bacteria’ 
can build up both carbohydrates and proteins out of carbon dioxid 
and inorganic salts. 

“2, Bacteria which need organic carbon compounds, but can 


68 FOOD REQUIREMENTS 


dispense with organic nitrogen. These bacteria are able to synthe- 
size protein substances out of carbohydrates (or organic acids) and 
ammonia, nitrogen or nitrates. 

“3. Bacteria which, like the higher animals, require both organic 
carbon and organic nitrogen compounds. These bacteria cannot 
accomplish either carbohydrate or protein synthesis out of inorganic 
substances.” 

Carbon.—The carbon dioxid of the air cannot be utilized by 
bacteria as a source of energy since it is already fully oxidized. 
There are, however, some organisms which possess the power of j 
utilizing both carbon monoxid and methane. On the contrary, 
the carbon of carbohydrates, fats and proteins are readily utilized 
by bacteria. The hydrocarbons of both the aliphatic and aromatic 
series are resistant to bacteria, but those compounds which contain 
oxygen in addition to the carbon and hydrogen are more readily 
attacked. Many organic acids and oxy-acids are used by some 
bacteria. Only a few bacteria can use the simpler alcohols. The 
more complex alcohols, like glycerin and mannite, are utilized 
by many. The carbohydrates are especially valuable to most 
bacteria, those containing six or twelve carbon atoms being the 
most valuable. 

Nitrogen.—The nature of the nitrogen requirements of bacteria 
are extremely different, depending upon the specific organism. 
Some organisms, such as the symbiotic nitrogen-fixers and the 
azofiers, are able to obtain all the nitrogen required from the 
atmosphere. The nitrosomonas obtains its nitrogen from ammonia, 
whereas the nitromonas obtain it from nitrites. The majority of 
bacteria obtain their nitrogen from peptones, proteoses, and even 
amino-acids. Rettger concludes from oft-repeated experiments on 
animal and vegetable proteins that bacteria are unable to derive 
nourishment from native proteins, and that in a medium in which 
there is'no possible source of nitrogen other than the proteins 
themselves they will thrive no better than in a chemically pure 
saline solution. When proteolytic enzymes are present the com- 
plex protein molecules are broken up and, at least in part, made 
available for cell nutrition. It would appear that “it is as essen- 
tial to break down complex nitrogenous food substances into their 
simple components, before they can be utilized, as it 1s to reduce the 
walls of an old church brick by brick before they can be made 
over into a modern schoolhouse.” The more strictly pathogenic 
organisms, as the gonococcus and the leprosy bacillus, may require 
nitrogen in the form of highly specific tissue proteins. As a rule, 
animal proteins are more readily utilized than are plant proteins. 

Hydrogen. —Hydrogen is obtained from many organic compounds 
containing hydrogen and oxygen, such as the carbohydrates, fats 


OXYGEN REQUIREMENTS 69 


and proteins, but usually not those compounds which contain only 
carbon and hydrogen, such as methane and its homologues. 
Sulphur.—Sulphur is required by all bacteria possibly for the for- 
mation of the proteinaceous material of their bodies. In addition 
to this, some organisms use it as a source of energy. [or instance, 
the Beggiatoa sometimes use two to four times their own weight of 
hydrogen sulphid in a day, under which conditions the sulphur grains 
may be seen in the cell-protoplasm and may be looked upon as an 
intermediate stage in the oxidation process, the reaction proceeding 


as follows: 
2HS + Or = Meals 
28 + 302 + 2H 


Some bacteria may get their required sulphur from sulphates, sul- 
phites or thiosulphates, but probably the great majority of them 
obtain it from the proteins. 

Phosphorus.— Phosphorus is used by bacteria in large quantities, 
being essential for the building of the nucleoproteins and phospho- 
proteins in which the unicellular organisms are especially rich. 
The form and quantities required by the organisms vary greatly 
with the species. The Azotobacter are able to utilize it from most 
organic and inorganic sources, some, however, being much more 
valuable than others. 

Potassium. — Potassium is essential to the higher plants and cannot 
be replaced entirely by related substances, yet Gerlach and Vogel 
early reached the conclusion that potassium and magnesium are 
not essential to Azotobacter. Their results, however, were con- 
sidered for a long time to be erroneous. But if these elements are 
essential to Azotobacter it must be in extremely small quantities. 
Potassium does, however, favor their developmentyand is probably 
valuable, if not essential, to all bacteria. Most inorganic potassium 
compounds can be utilized. 

Other Inorganic Substances.—The other inorganic constituents 
are required by bacteria only in small quantities and are obtained 
from either organic or inorganic compounds, depending upon the 
specific organism. 

Oxygen Requirements.— Bacteria, like all other plants and animals, 
require oxygen in their life activity. The various classes of organ- 
isms are not indifferent as to the form in which they obtain their 
oxygen. One great class requires that their oxygen be furnished 
free; to these is given the name “aérobic.”” Another requires their 
oxygen in the combined form; they are called “anaérobic.”’ Some 
organisms grow best in the presence of free oxygen but may become 
adapted to combined oxygen; these are known as “facultative 
anaérobes.”’ Others grow best in the absence of free oxygen but 
may become adapted to it; they are known as “facultative aérobes.” 


70 FOOD REQUIREMENTS 


Few bacteria are true aérobes or anaérobes, but many gradually 
blend from one class into another, as some will withstand small 
quantities of free oxygen but not a full atmospheric pressure of it. 
Vitamines.— The extracts of animal organs, as well as those of 
some plant tissues, are valuable nutrient material for bacteria which 
it is as yet impossible to supply in any medium of known chemical 
composition. The composition of these more or less unstable 
but highly nutritive substances is a matter of purest speculation. 
For want of a better name they are termed “vitamines” or “acces- 
sory growth factors.” ‘These accessory bodies are moderately 
heat-stable and are soluble in alcohol and in water. They are 
rapidly absorbed from solution by filter paper, but not by glass 
wool. They increase the reaction velocity of the proteolytic metab- 
olism of the meningococcus and are essential to many other organ- 
isms. After the first or primary cultivation some organisms become 
independent of these substances. This phase of bacterial nutri- 
tion, which is only just beginning to receive attention, is beset by 
many difficulties. The work being done, however, gives promise 
of so clearing up the field that much that was impossible of expla- 
nation in the past will be readily explained. But the present 
status of the case is well summarized by Rettger when he stated: 
“We are as yet in the dark regarding the real food requirements 


of bacteria.” 
REFERENCES. 

Marshall: Microbiology. 

Kendall: Bacteriology—General, Pathological and Intestinal. 

Kruse: Allgemeine Microbiologie. 

Berman, Nathan, and Rettger, Leo F.: Bacterial Nutrition—Further Studies 
on the Utilization of Protein and Non-protein, Jour. Bacteriol., 1918, iii, 367-388. 

Berman, (Nathan), and Rettger (Leo F.): The Influence of Carbohydrates on 
the Nitrogen Metabolism of Bacteria, Jour. Bacteriol., 1918, iii, 389-402. 


CHAP TE RV II. 
BACTERIAL METABOLISM—ENZYMES. 


It was pointed out in the last chapter that bacteria require food 
for at least two purposes—building material and the liberation 
of energy. In fulfilling these functions the foods are profoundly 
changed; at times they are broken up into comparatively simple 
products, after which they are built into the complex molecules 
composing the bacterial cell; at other times they are split and the 
energy utilized; at still other times they are completely oxidized, 
the organisms thus obtaining all the stored potential energy. The 
sum of all these changes which the food undergoes, including the 
deterioration of the cell, is called metabolism. ‘These changes con- 
sist of two separate processes; the one—construction of new cells 
or parts of cells—is a process of synthesis and is called anabolism. 
The other is analytical or the breaking-down of the cell and is 
called katabolism. Although these two processes are usually going 
on simultaneously in the cell, yet it is true that during the first few 
hours after inoculation of a culture the anabolic aspect predominates; 
later the katabolic phase predominates. That this should be the 
case can be readily seen, for the bacterial cell must be morphologi- 
cally complete before it can bring about its characteristic energy 
transformations, which continues until the death of the cell. 

Moreover, recent investigations have demonstrated that it is 
just as true of bacteria as of animals that “it is as essential to 
break down complex nitrogenous food substances into their simple 
components before they can be utilized, as it is to reduce the walls 
of an old church brick by brick before they can be made over into 
a modern schoolhouse.” 'The development and present status of 
our knowledge of this represents one of the most interesting and 
valuable chapters of bacteriology. 

Early Theories of Fermentation.—Even as early as 1595 the great 
medical chemist, Labavius, considered fermentation a process akin 
to digestion, and von Helmont (1648) stated that out of the ferment 
something passes into the fermenting liquid which grows in it as 
a seed. But it was the great chemist, Liebig, who first developed 
the purely chemical explanation of fermentation. It was he who 
developed the idea of catalysis, a word already invented by 
Berzelius. Liebig compared fermentation changes to the action 
of finely divided platinum which possesses the power of bringing 
about the union of gases at low temperatures. The ferment he 


72 BACTERIAL METABOLISM—ENZY MES 


considered to be in a state of unstable equilibrium or decomposi- 
tion. This is communicated to its surroundings, producing chemical 
changes. This was opposed by Pasteur and Tyndall who showed 
that in the absence of microérganisms fermentation does not take 
place. 

There were certain changes which they proved to be due to 
bacteria and yeast; others which were brought about by pepsin, 
tripsin, etc. This led to the classification of ferments as organized 
and unorganized. Under organized ferments were grouped such 
substances as some bacteria and yeasts, which, when examined 
under the microscope, possess a definite organized structure and 
which act by virtue of vital processes. The unorganized ferments 
included amylase, pepsin, rennin, ete., and were described as 
“non-living unorganized substances of a chemical nature.” iihne 
designated this last class of substances, enzymes. This classifica- 
tion into organized and unorganized ferments was generally accepted 
and practically unquestioned until overthrown by Biichner (1897) 
in his epoch-making investigation of yeast. He carefully mixed 
1000 grams of brewers’ yeast with an equal weight of quartz sand 
and 250 grams of infusorial earth generally known as Kieselguhr. 
This mixture was ground together until plastic; 100 ¢.c. of water 
was added and wrapped in a press cloth and filtered in a press cap- 
able of exerting a pressure of from 400 to 500 atmospheres. The 
juice was clarified by shaking with Kieselguhr and filtering. The 
liquid so obtained is slightly heavier than water and possesses a 
pleasant odor. On boiling, a quantity of protemaceous matter 
separates and the liquid becomes nearly solid. 

The unboiled juice possesses all the power of the yeast cell in so 
far as fermentation is concerned. However, the action is not 
stopped by chloroform nor by the passage of the liquid through a 
Berkefeld filter nor through a dialyzing membrane. The enzyme 
which is. present in the solution has been termed by Biichner 
zymase. Later the lactic acid- and the acetic acid-producing bacteria 
were subjected by Biichner to similar treatment to that given the 
yeast cells, and the active intracellular enzymes were obtained. 
Since that time the list of unorganized ferments or enzymes has 
continued to grow at the expense of the organized ferments until 
it is generally conceded today that all fermentations are due to 
enzymes, there being only this difference—that some are formed 
and readily diffuse out of the body of the cell during its life and are 
known as extracellular ferments, whereas others remain in the cell 
and are known as intracellular ferments. 

Definition of Enzymes.—Enzymes have been defined as “unor- 
ganized, soluble ferments, which are elaborated by an animal or 
vegetable cell and whose activity is entirely independent of any 
of the life processes of such a cell.” 


DEFINITION OF ENZYMES 73 


Enzymes act by catalysis and hence are often stated to be “select- 
ive colloidal catalysts, present in living cells and destroyed by 
heat.” A catalyzer is “a substance which alters the veloc ity of 
a chemical reaction without undergoing any apparent phy sical or 
chemical change itself and without becoming a part of the product 
formed.” It is a well-known fact that the speed of many chemical 
reactions is accelerated by catalyzers; for example, the inversion of 
cane sugar by acid and the numerous reactions affected by platinum. 
Negative catalysis is not as common, but the stopping of the slow 
oxidation of phosphorus in air by a trace of ether vapor may be 
taken as an example. The general characteristics of catalysts are 
admirably illustrated by Bayliss: 

“There are certain phenomena which, at first sight, might be 
confused with those of catalysis, but which must be carefully dis- 
tinguished from them. A mechanical model will serve to make 
this clear. If a brass weight of, say 500 grams, be placed at the 
top of an inclined plane of polished plate-glass, it will be possible 
to find a slope of the plane such that the weight will slowly slide 
down. This represents any reaction taking time to complete. 
If now the bottom of the weight be oiled (oil-catalyst) the rate of 
its fall will be greatly increased. We see, that in either case, the 
weight if placed at the top of the plane does not remain there, but 
sooner or later reaches the bottom. It may, however, be kept at 
the top by some kind of catch or trigger arrangement, in which case 
it will remain there indefinitely until the catch is released. The 
amount of energy lost by the weight in its fall, being the product 
of its weight and the vertical height from which it has fallen, is in 
no way affected by the work required to remove the obstacle pre- 
venting its fall, nor is the rate at which it falls when set free. A 
typical instance of such a ‘trigger’ action is that of supersaturated 
solutions, which remain for any length of time unchanged unless 
infected with a crystal. It has, moreover, been shown by B. 
Moore (1893) that the rate at which the solidification of supercooled 
glacial acetic acid moves along a tube is independent of the quantity 
of crystals placed at one end to start the process. Not so with 
true catalytic action; although the work done by our sliding weight 
is in no way affected by the amount of catalyst (oil) used, the rate 
of the fall is, within limits, directly proportional to it, and this is a 
property of catalysts in general. 

“Tt cannot be expected that a rough model of this kind would 
show all of the characteristics of catalytic phenomena, but there 
are two instructive points shown by it in addition to those already 
spoken of. The first is the disappearance of the catalyst by stick- 
ing to the glass as the weight slides down. An analogous phe- 
nomenon is often met with in catalytic processes, as will be seen 
later. The second point is one of importance with regard to 


74 BACTERIAL METABOLISM—ENZYMES 


certain enzyme actions; it consists in the fact that, although the 
presence of the catalyst neither adds to nor subtracts from the 
total energy change in the reaction, the form of this energy may be 
altered. When the weight falls slowly by itself, nearly the whole 
of the energy appears as heat due to friction along the glass plane, 
so that the weight arrives at the bottom with very little kinetic 
energy; on the contrary, when oiled, nearly the whole of the energy 
is present in the weight at the end of its fall as kinetic energy, very 
little friction having been met with in its descent. We may notice, 
also, comparing the effects of different amounts of oil, that small 
amounts produce a much more marked result than the subsequent 
addition of further quantities. This is also characteristic of 
enzymes, as we shall see later. 

“From what has been said it follows that a catalyst is merely 
capable of changing the rate of a reaction already in progress. In 
opposition to this it may reasonably be said that a reaction does 
sometimes seem to be initiated. Such a case is that of a mixture 
of oxygen and hydrogen gases caused to combine by spongy plati- 
num. Now there are reasons for the belief that an extremely slow 
combination is taking place at ordinary temperatures without 
catalysis. One thing to be considered in reference to this belief 
is the enormous acceleration of chemical reactions by rise of tem- 
perature, the majority being about doubled by a rise of 10° C. In 
this way a reaction having a velocity of 1 at 0° would reach one 
of 2 at 10°, 4 at 20° and 1 X 2'° = 1024 at 100°. At the tempera- 
ture of 500° there is appreciable formation of water in the case in 
point, and Bodenstein (1899) has shown that if the velocity at 689° 
be represented by 163, that at 482° has already sunk to 0.28; so that 
at room temperature the velocity would be quite incapable of 
detection by chemical means, since centuries would be needed 
to produce a fraction of a milligram of water. Grove’s gas battery 
also proves that the two gases are not in equilibrium at ordinary 
temperatures, since electrical energy is obtained by their slow com- 
bination. 

“To take another case of a reaction which progresses at a slow 
rate when left to itself: When methyl acetate is mixed with water 
at ordinary temperatures it is very slowly hydrolyzed to alcohol 
and acetic acid until a certain proportion of it is decomposed, so 
that a state of equilibrium is finally arrived at. This process takes 
many days for its completion, but the time may be shortened to a 
few hours by the addition of a small amount of hydrochloric acid. 

“The objection may be made to the former of these two examples 
that the combination of oxygen and hydrogen does not take place 
except in the presence of water vapor, which probably acts as a 
catalyst. Similarly, the hydrolysis of esters by water may be said 
to be due to the hydrion present therein. This point of view does 


TERMINOLOGY 75 


not, however, in reality, affect the reasoning, since the reactions 
can be enormously accelerated by other bodies, which act as addi- 
tional catalysts and may be investigated independently. It is, in 
fact, a matter of considerable difficulty to discover a slow reaction 
which is definitely known to take place in the complete absence of 
any catalyst. 

“Moreover, it must not be forgotten that, as J. J. Thomson and 
others believe, a catalyst may possibly start a reaction. This is 
not, theoretically, in disagreement with the view taken by 
Ostwald. To return to our mechanical illustration, the ‘friction’ 
between the weight and the glass plane may be sufficiently great 
to prevent movement altogether, until oil is applied. But the use 
of the name ‘friction’ implies the idea of movement and the exist- 
ence of forces tending to produce it. One may indeed suppose 
that the weight actually does move for an infinitesimal distance, 
but is at once arrested by the resistance met with. From this 
point of view the definition of a catalyst would be expressed some: 
what thus: A substance which changes the rate of a reaction which 
is actually in progress, or which is capable of proceeding without 
any supply of energy from without, if certain resisting influences 
are removed. The difference between diminution of friction by 
oil and the removal of a catch is that, in the former case the action is 
continuous throughout the fall of the weight, whereas in the latter 
case the action is only momentary, at the commencement of the 
fall, on the rate of which it has no further effect.” 

Terminology.— Within recent years attempts have been made to 
systematize the terminology used in referring to enzyme action. 
The name of the substance on which the enzyme acts is called 
substrate. 

As to the names of the enzymes themselves it is customary to 
use the termination “ase” which denotes an enzyme and this 
termination should be added to the root of the word which names 
the substrate; for example, lactase is the enzyme accelerating the 
hydrolysis of lactose, sucrase of sucrose, maltase of -maltose, etc. 
Unfortunately, in many cases old names have become so fixed that 
it is not desirable to replace them, as, for example, pepsin for the 
acid proteinase and trypsin for the alkali proteinase. At other 
times the enzymes are incorrectly named from the simpler substance 
in place of the more complex substrate; for example, invertase for 
the ferment which inverts sucrose. 

It is the custom with many writers to speak of the enzymes which 
attack, say, starch or protein, as “amylolytic” or “proteolytic,” 
respectively; but Armstrong has pointed out that these names 
are incorrectly formed. “Amylolytic” in analogy with “electrolytic” 
should mean a decomposition by means of starch. To avoid this 
misuse of words he advocates the use of the termination “clastic” 


76 BACTERIAL METABOLISM—ENZYMES 


instead of “lytic,” giving us terms such as “amyloclastic,” “proteo- 
clastic,” “lipoclastic,” ete. 

Enzymes ordinarily do not occur active within the cell, but 
are present in the form of a zymogen or mother substance. This 
substance, when acted upon by a specific substance, becomes 
active and the process is termed “activation.” The agency which 
is instrumental in activating a zymogen is termed “zymo-excitor” 
or kinase. 

Properties of Enzymes.— Enzymes are known from the reactions 
which they catalyze and they are found to follow quite definite 
laws in their reactions. Some of the more important are as follows: 

1. An enzyme does not initiate a chemical reaction but only 
alters its velocity; nor does it appear in the final products of the 
reaction which it accelerates. We must, therefore, assume that 
substances are slowly changing and that the catalyst does nothing 
more than alter the speed of this reaction. The state of affairs is, 
therefore, similar to that of a mixture of oxygen and hydrogen 
gases catalyzed by platinum in which there is evidence that the 
combination takes place at room temperatures, although at an 
unmeasurable rate. Salicin, which is readily hydrolized by ptyalin 
and emulsin to glucose and saligenin slowly decomposes in water 
at 150° C. It would, therefore, be inferred that the process also 
takes place at room temperature. Starch solutions slowly undergo 
a spontaneous change into dextrin and sugar and solutions of 
ammonium caseinogenate increase in electrical conductivity when 
left to themselves, a change similar to that which occurs when 
they are acted upon by trypsin. Taylor has shown that an appreci- 
able proportion of pure sterile globulin kept in distilled water at 
ordinary temperature for eighteen months is hydrolyzed to protease 
and that leucin may be recovered from a sterile suspension of casein 
in pure water and that arginin may be recovered from a solution 
of protamin sulphate in pure water. True, the reaction is slow and 
the products have accumulated only in small quantities after the 
lapse of a year; nevertheless, it is evident that the process is slowly 
occurring in the absence of the catalyzer. 

It is hkely that the ferment enters temporarily into chemical 
combination with the substance acted upon. This assumption is 
made on the ground that the sensitiveness of the enzyme often 
changes when brought in contact with the substrate and may at 
first be hard to separate. Moreover, it is definitely known that in 
some simple catalytic processes the catalyzer does temporarily 
combine with the reacting substance. This is the case in the 
manufacture of sulphuric acid, where steam, sulphur dioxid, oxygen 
and the oxids of nitrogen are introduced simultaneously into a 
large chamber when the following reactions probably occur. 


SO2 + N:0s = SOs + 2NO 
SOs + H:O H2SO, 
2NO + Oz 2NO2 


PROPERTIES OF ENZYMES Tah 


Thus it is that the oxids of nitrogen serve to convert the sulphur 
dioxid to the trioxid and in the presence of air reverts to the original 
condition and again repeats the cycle. While in the Gay-Lussac 
tower the nitrosul-sulphuric acid is formed: 


N203-+ H2SO4 = 2NO2HOSO2+ H2O 
2NO2HOSO:+ 2H2O = 2H2SO1+ N2Os 


Where there are a number of steps in a reaction, as is the case with 
the above, it is necessary, as pointed out by Ostwald, that the sum 
of all the reactions in the catalyzed system are more rapid than are 
the changes in the uncatalyzed. 

The classic illustration of an organic reaction of this type is that 
afforded by the production of ether from alcohol. In this process 
sulphuric acid is employed as catalyzer and as well known this first 
combines with alcohol with the formation of ethyl-sulphuric acid. 


HO CoH;O 
ae ‘ a 
CoH;OH + SO: = HOH + SOz 
H HO 
Alcohol. Sulphuric acid. Ethyl-sulphuric acid. 


The ethyl-sulphuric acid reacts with another molecule of alcohol 
forming ether and regenerating sulphuric acid. 


C:H;0 HO 
BSS 
SO. + CHOH = bar + GH; —- O — GHs 


H 
Ethyl-sulphuriec acid. Alcohol. Sulphuric acid. Ether. 


Similar combinations occur with the enzymes, for it is found that 
sucrase will withstand uninjured a temperature 25° C. higher in 
the presence of sucrose than in its absence. It is difficult to see 
how this could happen unless the enzyme entered into some sort of 
union with the sugar. 

Intimately connected with the subject of combination of enzyme 
with substrate is that of specificity, an example of which is seen 
in the fact that certain enzymes act only on carbohydrates, others 
on fats, and still others on proteins. The group of those trans- 
forming carbohydrates is further subdivided into specific enzymes 
each of which has the power of acting alone upon only one sugar. 
This property is so specific that in many cases the enzyme will 
act upon one optically active compound leaving the opposite optical 
isomer untouched. This led Fischer to the formulation of his 
famous simile of the “lock and key” relationship. In this he 
considers that the enzyme and its substrate must have an inter- 
relation, such as the key has to the lock; otherwise, the reaction 


78 BACTERIAL METABOLISM—ENZYMES 


does not occur. By means of this-theorem it has been possible to 
foretell the structure of many complex substances and explain 
~ hitherto obscure points in biology. 

2. The chemical change brought about by an enzyme in infinite 
time is independent of the concentration of the enzyme, but for 
shorter periods it is clearly and usually a definite function of the 
concentration of the enzyme. This means that a small quantity 
of enzyme will bring about as much change as a large one, provid- 
ing unlimited time is given: In this regard, then, enzyme reac- 
tions differ from ordinary reactions in that they do not follow the 
law of mass action. This may be illustrated by the carrying of 
brick to the top of a building by men. Give one man sufficient 
time and he would be as able to transfer the whole pile to the top 
as would a group of men, but in the latter case the time occupied 
would be inversely proportional to the number of men working. 
So it is with enzymes; the intensity is almost directly proportional 
to the concentration of the enzymes. In certain instances where 
this generalization has been found not to hold, attempts have 
been made to apply the Schutz-Borissow Law—that the intensity 
of enzyme reaction is directly proportional to the square root of 
the concentration. But even this generalization does not hold, for 
there are a number of factors which tend to retard or accelerate 
enzyme action. Chief among these which retard are (a) reversi- 
bility, (b) combination of enzyme with products, (c) negative 
autocatalysis, which with the previous factor leads to reversible 
inactivation of the enzyme, (d) destruction or similar drastic changes 
in the properties of the enzyme. Those which accelerate are as 
follows: (a) combination of the whole of the enzyme with the sub- 
strate when the latter is in relatively large excess, (b) positive 
autocatalysis. 

3. Reactions which are catalyzed by enzymes are reversible. 
It has been conclusively shown in the case of many reactions and 
is generalized for others that where a reaction is being catalyzed by 
enzymes it is, unless the products so formed are removed from the 
reaction medium, reversible. This is illustrated by the saponifica- 
tion of ethyl-butyrate by means of lipase. 


C3H;COOC:H;s + H:O = C3sH;COOH + C2H;0H 
Ethyl-butyrate. Butyric acid. Ethy] alcohol, 


Starting with a definite quantity of ethyl-butyrate, after a time 
we find in the reacting media ethyl-butyrate, butyric acid and 
ethyl alcohol; commencing with butyric acid and ethyl alcohol, we 
obtain the same products as in the first case. This implies that the 
synthetic reactions which are going on in the cell are catalyzed by 
the same enzymes as are the analytic reactions; hence reactions 
that are catalyzed by enzymes are never complete unless the result- 
ing products are removed as fast as formed. 


HYDROLYTIC ENZYMES 79 


4, Enzymes are usually characterized by great sensitiveness to 
comparatively low temperatures and to many poisons. ‘This prop- 
erty formerly was used to determine whether or not a reaction was 
being catalyzed by an enzyme; but there are known a few cases in 
which the enzyme is not destroyed by boiling water. The great 
majority of all enzymes are, however, destroyed by a temperature 
somewhat below 100° C., many even as low as 60° C. This property 
is no doubt due to the colloidal nature of the ferment which, on 
being heated, coagulates—probably much as does a protein, for it 
is well known that enzymes are more sensitive in the presence of 
water than in its absence. 

Although the addition of hydrocyanic acid or formaldehyde to 
a media in which reactions are being catalyzed by enzymes puts 
a stop to the reaction, yet the concentration necessary is usually 
greater than that which can be borne by the living protoplasm. 
This makes it possible to kill the cell and still have the enzyme 
reactions going on in the medium by carefully adjusting the con- 
centration of the antiseptic used. 

Variots methods are used in the extraction of enzymes. Some 
readily diffuse out of the cell and may be taken up with water; 
others are extracted with glycerin or acids; in still other cases it 
is necessary to decompose completely the cells as did Biichner 
in obtaining zymase. The resulting product is then often purified 
‘by alcoholic or other precipitants. This drastic treatment, how- 
ever, often impairs the activity of the ferment. 

Classification.—Fuhrmann has classified enzymes of bacterial 
origin into four types as follows: ; 

A. Schizases (hydrolytic) cleavage enzymes: 
1. Carbohydrate-splitting enzymes. 
. Glucoside-splitting enzymes (synaptase). 
. Fat-splitting enzymes. Lipases (esterases). 
. Proteases, protein-splitting enzymes, pepsin, trypsin 
(lysins, coagulases). 
B. Fermentation enzymes: 
Zymase urease, lactic-acid enzyme. 
C. Oxidizing enzymes: 
Tyrosinase, acetic bacteria, oxidase. 
D. Reducing enzymes: 
Reductase. 

Hydrolytic Enzymes.—As a type of the hydrolytic enzymes which 
act upon carbohydrates, we may take maltase which converts 
maltose into dextrose according to the following equation: 


He CO bo 


CrHx2Ou + HeO = CeHwOs + CoHi2O0c 


Maltose. Dextrose. Dextrose. 


Maltase is an enzyme which occurs in yeast, many bacteria, and 
numerous other cells. It is of special interest inasmuch as it is 


80 BACTERIAL METABOLISM—ENZY MES 


the first case of reversible action that was studied. Craft Hill 
found that the addition of maltase to a very concentrated solution 
of dextrose resulted in the formation of a disaccharid. This he at 
first thought was a simple reversion of dextrose into maltose, but 
later work showed that the sugar formed was an isomer of maltose. 
The essential fact, however, remained that the one enzyme possessed 
both synthetic and analytic properties. 

Emulsion is an enzyme which possesses the power of decompos- 
ing mandelic-nitrile-glucose into glucose, benzaldehyde, and hydro- 
cyanic acid. The mandelic-nitrile-glucose is obtained by the action 
of maltase upon the glucoside amygdalin. The total change 
brought about by the two ferments is indicated by the following 
equation: 

CoHaNOn + 2H = CeH;CHO + HCN + 2CsH20s 
'  Amygdalin. Benzaldehyde. Hydrogen cyanid. Glucose. 

Lipases act upon the neutral fats and are widely distributed in 
both plant and animal cells. They bring about a reaction which 
may be expressed by the following general reaction, where R = 
the residue of a fatty acid. 


CH:—R CH:0H 
CH—R + 3H:0 = 3RH + CHOH 
CH:—R - CH.0H 


One molecule of neutral fat is split into three molecules of fatty 
acid and one of glycerin. This is the general reaction which occurs 
in the spoiling of butter or fat due to bacterial activity. 

Proteases, which possess the power of splitting proteins, are 
widely distributed in bacteria, as is exemplified by their gelatin- 
liquefying powers. This also is a hydrolytic reaction in which a 
number of molecules of water is caused to enter the protein molecule 
with its subsequent breaking down into proteoses, peptones, and 
finally amino-acid. Even this, as complex a reaction as it is, has 
been shown to be reversible in at least two cases. 

Zymases, which occur in the yeast cell, are endo-enzymes and 
their function is to liberate energy for the use of the cell, as is shown 
by the following table from the work of Rahn: 


ENERGY LIBERATED FROM 1 GRAM OF SUBSTANCE. 


Soluble enzymes. ‘ Endozymes. 
Pepsin, trypsin . . . Ocalories Lactacidase . . . . 80calories 
Dt pasoiale pose Ge mk? Bt toe “¢ Alcoholase, 354.9) =) 4120 S 
Maltase sucrase . . . 10 « Urease tags big. we MaOU as 
Lactase led He tiat O vt Vinegar oxidase . . .2500 . 


The first zymase isolated from a microérganism was that of 
urease, or the ferment which converts urea into ammonium car- 


OXIDIZING ENZYMES 81 


bonate, and which was shown by Musculus to be present in the dead 
cells of Micrococcus wree which develops in putrid urine. Zymase 
was obtained by Biichner through the pressing of the ground yeast 
cells, as has been described. This same method was later applied 
to the lactic acid bacteria and the lactacidase obtained. 

Oxidizing Enzymes.—The most typical example of an oxidizing 
enzyme is the vinegar oxidase, the action of which is fairly well 
known. The reaction may be written in the simple form 


CH;CH-OH + O: = CHsCOOH + HO. 


Since, however, many side reactions may occur, the bacterial oxida- 
tion of alcohol is not in reality capable of so simple an expression. 

Reducing enzymes are the most common of ferments. ‘They are 
formed by practically all plants and animals and contained by all 
but a very few bacteria, Strept. lacticus being one of the few excep- 
tions. In this case the absence of the enzyme is used as a diagnostic 
test for the organism. One of the most important reductases is 
the peroxidase which reduces hydrogen peroxid to water with the 
liberation of oxygen. 


2H202 + peroxidase = 2H20 + On. 


Others which reduce nitrates to nitrites of particular interest to 
students of agriculture are 


2KNO3; = 2KNO2 + Oz. 


Or at times they may reduce the nitrite to elementary nitrogen: 
2Ca(NOs)2 = 2CaO + 2Ne2 ‘+ 502. 


Under appropriate conditions the important element, nitrogen, 
may thus be lost from the soil by denitrification. In a similar way 
sulphates are reduced to hydrogen sulphid: 


HSO, = HS + 202. 


REFERENCES. 


Bayliss: The Nature of Enzyme Action. 

Euler: General Chemistry of the Enzymes. 

Falk: The Chemistry of Enzyme Actions. 
Robertson: The Physical Chemistry of the Proteins. 


CHAPTER VIL: 
BACTERIAL METABOLISM PRODUCTS. 


Bacreria are able to bring about enormous changes in their 
media in a very short time. This is due in no small measure to their 
method of metabolism which differs from that of the animal, in 
most cases, in being a process of incomplete oxidation, whereas that 
of the animal is a process of complete oxidation. For this reason, 
many of the organisms of especial economic importance often leave 
products of considerable commercial value. 

Physiologic Classification.— From a physiologic viewpoint Jordan 
divides the substances produced by bacterial metabolism into four 
classes: 

1. The secretions, or those substances which serve some purpose- 
ful end in the cell economy. These may either be retained within 
the cell or may pass out into the surrounding medium. 

2. The excretions, or those substances that are ejected because 
useless to the organism; the ashes of cell metabolism. 

3. The disintegration products, or those bodies that are produced 
by the breaking down of food substances. Their nature is deter- 
mined partly by the chemical structure of the nutrient and partly 
by the specific bacteria concerned in the disintegration. Some of the 
most conspicuous, if not the most important, of bacterial products 
belong to this class, enzyme activity being largely responsible for 
their existence. 

4. The true cell substance. To this class belongs the cell proto- 
plasm, those products which are being built up into cell protoplasm, 
and those substances which are being broken down but have not 
yet reached the stage where they are cast off from the cell. 

The great objection which may be brought against such a classi- 
fication is that although many products can be definitely placed, 
others, for instance pigments, cannot. 

Carbohydrate Metabolism.— Products from carbohydrate metab- 
olism vary greatly, depending upon the species of bacteria, age, 
medium, and whether grown in the presence or absence of oxygen. 
Some writers distinguish six types of microédrganisms, depending 
upon the change which they produce in their media, namely: 

1. Complete oxidation which occurs’ only to a limited ‘extent 
among bacteria and then only where there is a ready supply of 
oxygen, as is the case in a well-aérated soil in filters or on the 
surface of decaying substances. 

2. Partial oxidation is much more common among microérganisms 
than is complete oxidation. The product formed is also often of 
considerable commercial value. This is the case in the oxidation 


CARBOHYDRATE METABOLISM 85 


of alcohol to acetic acid. On the other hand, the products formed 
may serve as food to other microdrganisms and thus be completely 
oxidized. Acetic acid, if not too strong, may be further oxidized to . 
carbon dioxid and water, as sometimes occurs, resulting in a decrease 
in the strength of vinegar. 

3. Alcoholic fermentation is brought about by yeast; yet there 
are bacteria which possess the power of producing alcohol, but none 
of them are of economic value. Such organisms have been obtained 
from hay (B. fitizianus) and sheep manure (B. ethaceticus). The 
Bact. pneumonie of Friedlander is not only a pathogenic organism, 
but also possesses the power of decomposing sugar solutions with the 
formation of ethyl alcohol and acetic acid. 

The reaction as brought about by yeast is due to the endo-enzyme, 
zymase, first isolated by Biichner. The reaction is dependent upon 
a readily available supply of phosphate, and according to Harden 
this forms an intermediate product with glucose, thus: 

I 
2CeHi206 + 2POs:HR2e = 2CO2 + 2C2H-O + 2H2O + C6Hi1004(POsR2) 2 


II 
CeHi004(POsRe)2 + 2H2O = CeHwOs + 2POs1HRe 


According to equation (I), two molecules of glucose are concerned 
in the change, the carbon dioxid and alcohol being equal in weight 
to one-half of the sugar used, and the hexosephosphate and water 
representing the other half. In the second equation the phosphate 
is again liberated, and the hexose presumably fermented. 


I II III 
CHO H CHO CHO 
| | | | 
CHOH — OH C(OH) CO 

| 

CHOH = CH CH: 
| | | 
CHOH iiies = CHOH 
| 
CHOH CHOH CHOH —> 
CH.0OH CH2OH CH2O0H 
Glucose. Enol form. Keto form. 

IV V VI | Vo ” VII 

* Methylglyoxal. 
CHO COOH CO2 
| 
CO + H:0 as CHOH — CH2OH 
| | 
CHs3 CHs CHs 
CHO Fal CHO CHO COOH CO2 
| | | | 
CHORE OFM COR, —72cO CHOH — CH.OH 
| | | | 
CH.OH CH» CH: + HO + CH: CH3 
Glyceraldehyde. Methyl-glyoxal. Lactic acid. Alcohol and carbon 


dioxid. 


84 BACTERIAL METABOLISM PRODUCTS 


Wohl has developed a theoretical scheme of reactions by which 
the process of alcoholic fermentation could be represented. In the 
first place the elements of water are removed from the « and B 
carbon atoms of glucose (I) and the resulting enol (II) undergoes 
conversion into the corresponding keton (III), which has the consti- 
tution of a condensation product of methylglyoxal and glyceral- 
dehyde, and hence is readily resolved by hydrolysis into these com- 
pounds (IV). The glyceraldehyde passes by a similar series of 
changes (V, VI) into methylglyoxal, and this is then converted by 
addition of water into lactic acid (VII), a reaction common to all 
ketoaldehydes of this kind. Finally, the lactic acid is split up into 
alcohol and carbon dioxid (VIII). 

In alcoholic fermentation there also results small quantities 
(0.1 to 0.7 per cent.) of fusel oil. This contains normal propyl 
alcohol, primary isobutyl alcohol, primary iso-amyl alcohol, and the 
optically active (primary) iso-amyl alcohol. It was thought at 
one time that these resulted from the fermentation of the glucose, 
but Ehrlich in a series of masterly researches, shows conclusively 
that their origin is the amino-acid which result from the hydrolysis 
of the proteins, the reactions of which may be given as follows: 


I 
(CH3)2—CH—CH:—CHNH:—COOH + H:0-—>(CHs)2—CHCH2CH20H +CO:+NHs 


Leucine. Primary isobutyl alcohol, 
II 
CH;:CH(C2H;)—CH(NH2)COOH + H2O — CH3sCHC:H;sCH2OH + COs + NHs 
Isoleucine. Primary iso-amyl alcohol. 


Succinic acid also occurs among the products resulting from alco- 
holic fermentation of sugar and has its origin in the amino-acids. It 
results when aspartic acid is acted upon by putrefactive bacteria- 


COOH—CH:—CH—NH:2COOH + H2=COOH—CH:—CH:—COOH + NHs 


This, however, differs from the first reaction in that it is a process 
of partial reduction and not hydrolysis. 

Other bacteria have been studied which possess the power of pro- 
ducing butyl and amyl alcohol from carbohydrates. It is still an 
open question to what extent the amyl alcohol (fusel oil) produced 
during an impure alcoholic fermentation is due to bacteria, for it 
is known that some alcohol yeasts possess the power of decomposing 
two protein decomposition products, leucin and isoleucin, with the 
production of fusel oil. 

4. Acid Production.—In general it may be stated that an acid 
reaction is caused by the fermentation of one of the sugars, glycerin, 
or a similar substance in the nutrient media. It is one of the more 
constant physiologic characteristics of bacteria, and in addition to 


ACID PRODUCTION 85 


being of considerable economic value is often used advantageously 
to distinguish closely related species, notably in the groups of para- 
typhoid and dysenteria bacilli. In addition to the acid produced 
by the fermentation of carbohydrates, many bacteria hydrolyze 
proteins with the formation of an acid reaction. Formic acid is 
the simplest organic acid which can be formed. It is produced 
by B. typhosus, the causative agent of typhoid fever. B. typhosus 
does not form gas, but B. coli does; and in this latter case it may 
result from the decomposition of the formic acid. 


HCOOH = He + COs... 


Acetic acid is one of the most important acids formed by bacteria. 
Bact. acetti and Bact. pasteurianum are two of the more important 
acetic acid-forming bacteria. They occur in fermenting fruit juices 
and convert the alcohol into acetic acid. 


CH;—CHos—OH >) 02" = CHECOOH -- H.0: 


Many other species also possess the power of transforming 
alcohol and other substances containing the characteristic radical 
—CH,CH,OH—into acetic acid. This fermentation, however, can 
take place only within certain limits of concentration, and even then 
there must be available nitrogen in the form of proteoses, peptones 
or amino-acids, and mineral elements, especially phosphorus in the 
form of a phosphate. Acetic acid is produced on the commercial 
scale by a number of processes. Two of the best are the Orleans 
and the Quick, or German, methods. 

Lactic Acid.—This product is formed by a great number of bac- 
teria. The chief species, however, is the Streptococcus lacticus 
which produces only a scanty growth on agar, but an excellent 
growth in milk, bringing about a solid curdling in a few days. The 
lactose of the milk is first inverted forming two hexoses— dextrose 
and galactose. 


CreH»On + H:O = CeHwOe + CeHi20¢ 


Lactose. Dextrose. Galactose. 


The hexose in turn is decomposed yielding two molecules of lactic 


acid. 
Cuore OCHO: 


Dextrose. Lactic acide 


In actual experience the reaction occurring is not as simple as 
written in the equation, but there are other products formed. For 
instance, B. coli ferments glucose with the formation of alcohol, 
carbon dioxid, hydrogen, lactic acid, succinic acid, and other 


86 BACTERIAL METABOLISM PRODUCTS 


products. The mechanism of the fermentation as outlined by 
Harden is illustrated by the reaction. 


CH:OH CH:sCH2OH + 3C0O2*+ 3He 
CH OH CH:,0H COOH 
(____ |-—_, 
CH OH CH OH CHs CHz 
| | 
CHOH CHOH = CHOH + CH: 
| \ 
CH OH CH OH COOH COOH 
ae ee 
CHO CHOH 
| 
CHO : 
Glucose. Lactic acid. Suceinic acid. 


_ Unless some base be added to neutralize the acid as formed, the 
lactose of milk is never completely converted into lactic acid because 
the accumulation of the acid is sufficient to stop most bacterial 
growth, as is seen by the fact that meat placed in buttermilk will 
keep for some time. 

butyric Acid.—Under certain conditions a further decomposition 
of the lactic acid may occur with the formation of butyric acid 
according to the following equation: 

2C3HsO3 =. CaHsO2 + 2CO2 + 2He 
Lactic acid. Butyrie acid. 

However, the butyric acid bacteria possess the power of ferment- 
ing sugar with the formation of butyric acid. 

CsHwOs + O2: = CHsCH»CH2>COOH + 2CO. + 2H:0 
Glucose. Butyric acid. ° 

Although the number of organisms which possess the power of 
producing butyric acid are large, they are not as numerous as those 
which possess the power of forming lactic acid. They are usually 
anaérobic spore-bearers with a tendency to form spindle-shaped 
cells, for which reason they have been given the name Clostridium. 
Many members of this group possess the power of fixing nitrogen; 
they probably play an important part in maintaining the nitrogen 
supply of the soil. B. botulinus, the causative agent in meat poison- 
ing, forms butyric acid as does also B. tetant. 

In the great majority of cases bacteria produce a number of differ- 
ent products; for instance, Azotobacter chrodcoccum produces from 
the carbohydrates, ethyl alcohol, glycocoll, acetic acid, butyric acid, 
lactic acid, carbon dioxid, and hydrogens ‘The quantity and quality 
of the different products vary with the species and with the carbo- 
hydrate used. 

Bacterium pasteurianum grows in wine and cider vinegars. It 
produces a variety of products, depending upon the specific sub- 
stance acted upon. It produces gluconic acid CH,OH (CHOH).- 
COOH from dextrose, propionic acid (C;H;COOH) from propyl 


PRODUCTS FROM NITROGENOUS COMPOUNDS 87 


alcohol (C;H;OH), and acetic acid (CH;COOH) from ethyl alcohol 
(C2H;OH). 

Other Acid Fermentation.—In addition to the acids described 
many others have been identified among the products of carbohy- 
drate fermentation. Many molds, especially, possess the power of 
fermenting dextrose with the formation of citric acid, the general 
reaction being as follows: 


CH2COOH 
/ 
2CseHwO6 + 202 = 2HOC—COOH + 2H2O 
CH2COOH 
Dextrose. Citrie acid. 


Patents have been taken out on this method and attempts made 
to produce citric acid on a commercial scale, but so far without any 
great success. 

Oxalic acid is also produced by certain molds in sugar solutions 
and where care has been taken to neutralize the acid so formed 
one-half the calculated theoretical yield has been obtained from 
cane sugar. Formic (H COOH) and valeric acids (C,H,COOH) 
are also produced by some microérganisms. 

Oxidation of Organic Acids.—The organic acids formed in the 
various processes of carbohydrate fermentation are often further 
oxidized by bacteria, yeasts, or molds to simpler products. Ordi- 
narily the process consists of a complete oxidation. This may be the: 
case In sauerkraut, ensilage, pickles, etc. Thus Oidiwm lactis destroys 
the lactic acid of sour milk with the formation of carbon dioxid 


and water. 
C3HsOs; + 302 = 38CO2 + #£3H20. 


At times the spoiling of pickles is due to the oxidation of the acetic 
acid by yeasts which grow in a thin white scum over the surface. 

Fats.—A comparatively few microérganisms attack fat. When 
they do the decomposition of the fat is comparatively simple. One 
molecule of the neutral fat is split with the formation of one mole- 
cule of glycerin and three of fatty acids. 


ae plea: 
H ea + 3H2O HCOH + 3C1;H3:i—COOH 
H2COOC—Ci;Ha1 H2COH 

Neutral fat. Glycerin. Fatty acid. 


The glycerin is readily used by the microérganism, whereas the 
fatty acids are but very slowly oxidized and that by only a few 
species. 

Products from Nitrogenous Compounds.—Proteins are complex 
organic substances composed of carbon, hydrogen, oxygen, nitrogen, 


88 BACTERIAL METABOLISM PRODUCTS 


and generally, but not always, sulphur, and sometimes phosphorus. 
The proportion of these constituents is approximately C, 50-55 
per cent.; H, 6-7.3 per cent.; O, 19-24 per cent.; N, 15-19 per cent. 
5, when present, 0.3-2.5 per cent.; and P, 0.4-0.8 per cent. They are 
substances which in the main consist of combinations of * amino- 
acids or their derivatives. The decomposition products of proteins 
include proteoses, peptones, peptides, carbon dioxid, ammonia, 
hydrogen sulphid and amino-acids. The amino-acids constitute a 
long list of important substances which contain nuclei belonging 
either to the aliphatic, carbocylic, or heterocyclic series. The 
present list includes glycocoll (glycin) alanin, serin, phenylalanin, 
tyrosein, cystin, tryptophan, histidin, valin, argin, leucin, isoleucin, 
lysin, aspartic acid, glutamic acid, prolin, oxyprolin, and norleucin. 

Many, especially of the saprophytic bacteria which occur in 
the soil, have the power of breaking down native proteins with the 
formation of the various amino-acids. Undoubtedly the complex 
organic compounds which are being isolated from the soil, and 
which are assumed by some to play such an important part in soil 
fertility, have just such an origin. But it is usually the case in all 
media that the bacterial catabolism carry the substance much farther 
than the amino-acid. The extent of this change varies greatly with 
the species of microérganisms and the conditions under which they 
are acting. Kendall summarizes some of the further changes which 
may occur as follows: 

1. The reductive deaminization of amino-acids to fatty acids with ° 
the same number of carbon atoms. 


RCH:;CHNH:COOH + H: = RCH:CH:COOH + NH: 

2. Hydrolytic deaminization of amino-acids to oxyacids with the 
same number of carbon atoms. 

R—CH;—CHNH:—COOH + H:0 = R—CH:—-CHOH—COOH + NH: 

3. Oxidative deaminization of amino-acids to keto-acids with the 
same number of carbon atoms. 

R—CH:—CHNH:—COOH + 0: = R—CH:—CO—COOH + NH: 

The above reactions may be taken as types of the last stages of 
the reactions brought about by the ammonifying bacteria within 
the soil. : 


4. Carboxylic decomposition of amino-acid to amine with one 
less carbon atom. 


R—CH:—CH—NH:2—COOH = R—CH:—CH:—NH2 + CO: 


5. Carboxylic decomposition with the formation of fatty acids. 


R—CH:—CH:—COOH = R—CH:—CHs + CO: 


PRODUCTS FROM NITROGENOUS COMPOUNDS 89 


6. Carboxylic decomposition with the formation of a fatty acid 
with one less carbon atom. 


R—CH:—CH:COOH + 30 = R—CH:—COOH + CO: + HO 


Some of these changes are often produced either within food or in 
the alimentary canal and are of considerable clinical significance. 
The most important of these are indol, skatol and the amins, the 
simplest of which is trimethylamin. 

Indol and skatol are substances produced in the intestinal tract 
from tryptophan chiefly by B. coli and B. proteus. They are also 
formed in putrifying proteins and it is to indol and skatol that putri- 
fying substances owe their intensely disagreeable odor. Indol 
gives a rose color with nitrites in acid solution and this is used as a 
method of identifying certain bacteria. The tryptophan is deam- 
inized with the formation of indol propionic acid. This is oxidized 
to indol-acetic acid. From this latter there is split off acetic acid 
with the formation of indol. The reactions are as follows: 


@EH 
VA 
HC C—— io H 
| | | 
H (e CH CH—NH: — 
See Ss | 
C N COOH 
H H 
Tryptophan. 
CH 
VA 
HC C— C—CH2 
| I I | 
HC C CH CH: > 
Nie ot a | 
C N COOH 
H 
Indol-propionic acid, 
CH CH 
Po oN Yj 
HC iC C— CH: HC C—— CH 
| i | | Se. 2| | | 
HC CH COOH HC C CH 
NAS See SO ONS OTIS Ge 
CH NH (G; 
Indol-acetie acid. Indol. 


Often the bacteria split out carbon dioxid from the indol-acetic 
acid with the formation of skatol: 


CH CH 
Va Yi 
el ah ee 
HC G CH COOH HC © CH 
Rae es Wee se 
CH -. NE CH) na, NEE 


Indol-acetie acid. Skatol. 


90) BACTERIAL METABOLISM PRODUCTS 


These substances when formed in the intestinal tract are absorbed 
and carried to the liver where they are conjugated with the formation 
of indican which is then eliminated by the kidneys. The stages 
through which indol passes in forming indican are as follows: 


H H 
C C 
bias es 
HC C CH HC C COH 
| | | sie oe ae | | 5 
HC C CH HC C CH 
Ser oe ENE iat aie A 
CH NH C NH 
H 
Indol. Indoxyl. 
H H 
C C O—SO3sK 
Vi Vl x 
HC C CO—S0;H HC Cc ——C 
HSO: | | | ap ele | | 
HC C CH HC C J 
ie Sra 
NH 
Indoxyl] sulphurie acid. Indican, 


Amins.—The simplest member of this series is methylamin 
(CH;NHe2) which is produced in small quantities in the decomposi- 
tion of nitrogenous organic matter. It occurs in herring brine along 
with dimethylamin (CH3)2NH and trimethylamin (CH3;)3;N. When 
alinin is acted upon by the carboxylase the carboxyl group of the 
amino-acid is split off with the formation of ethylamin according to 
the following reaction: 

CH:CHNH:COOH = CH;CH:NH, + COz 
Alanin. Ethyl amin, 

Others of special interest which may be due to bacterial activity 
are: 

1. Cadaverin from lysin: 


CH.—CH.CH:—_CH:—_CH—COO0H CH2—CH2C H2CH2CH2 
| _— 
NHe NH: NH: NH. + CO: 
Lysin. Cadaverin. 


2. Putrescin from ornithin: 
CH:—CH:—CH.—CH—COOH CH: —CE:—_CH: CH, 

| | = | | + COs 
N He NH2 NH2 NHo2 


Ornithin. Putrescin. 


3. Beta-imidazole ethylamin from histidin: 


TAO a Cae het 

G 0 cw 7 

én = a + CO: 
fess ae 

an 


Histidin,. Beta-imidazole ethyl-amin. 


PRODUCTS FROM NITROGENOUS COMPOUNDS 9] 


Vaughan considers that beta-imidazole-ethylamin is the active 
principle of the protein molecule. Some of these amins are strong 
stimulants of the heart or vasodilators. It is quite likely that their 
hberation by bacterial activity in the intestinal tract and their 
subsequent absorption may result in severe constitutional symptoms. 
These compounds belong to a group of substances called “ptomains.”’ 
They are alkaloid-like bodies of basic character and of more or less 
well-known structure. Some of them are harmless, while others are 
apparently violent poisons. It is interesting to note that in the 
majority of cases the poisonous properties decrease or at times 
entirely disappear as purification proceeds thus indicating that the 
poisonous principle in some cases at least is an impurity associated 
with them. Their production is not limited to any one special class 
of bacteria, for Zinsser defines ptomains as “poisons elaborated 
by all bacteria that are capable of producing protein cleavage, if 
planted on suitable nutrient materials under conditions favoring 
growth. The matrix of these poisons is the protein nutriment; they 
are not products of intracellular metabolism specifically characteris- 
tic of the bacteria which produce them.” 

Bacterial toxins, in contradistinction to the ptomains, are specific 
bacterial poisons which are characteristic of each individual species 
of bacteria and are truly the products of bacterial metabolism in 
that they emanate from the cell itself either as a secretion or excre- 
tion during cell life, or as an inherent element of the cytoplasm 
liberated after death. 

Enzymes which are true products of bacterial metabolism have 
been considered in detail in the preceding chapter. 

Urea, uric acid, and hippuric acid are the forms in which the 
waste nitrogen is excreted by the higher animals. There are a 
great number of organisms occurring widely distributed which 
possess the power of changing urea into ammonium carbonate. 
This is a simple hydrolysis. 


NH» NH,O 
CO + 20:0 = CO 
NH: NH:O 


Uric acid can be changed in several ways by bacteria, that is, it 
may be hydrolyzed with the formation of dialuric acid and urea. 


HN—C—O HN—CO NH: 
| [hia] X 
Of Ga NES 5 2H2O —> OC CHOH + (@0) 
I we — | 
HN—C—NH HN—CO NH, 
Uric acid. Dialurie acid. Urea. 


On oxidation uric acid yields various substances, alloxan, urea, 
oxalic acid, carbonic acid, tartronic acid, allantoinic and uroxanic 


92 BACTERIAL METABOLISM PRODUCTS 


acid. If uric acid is given to man the greater portion of it is prob- 
ably destroyed by bacteria in the alimentary tract, but the liver or 
kidneys of some animals secrete a uric acid-destroying enzyme or 
uricolytic enzyme, called uricase. It is presumably through the 
formation of such an enzyme that bacteria are able to decompose 
uric acid. 

Hippuric acid is hydrolyzed by certain bacteria with the forma- 
tion of benzoic acid and glycocoll. 


COOH 
C—C—NH—CH:—COOH C 
Pee 
HC CH HC CH 
| | +HO—> | || + CH:NH,COOH 
HC CH HC CH 
ee" 
Cc C 
Jal H 
Hippuric acid. Benzoic acid. Glycocoll 


The glycocoll may then be deiminized with the formation of 
ammonia and acetic acid. Many extremely complex transforma- 
tions of organic substances occur in the soil, due to bacterial 
activity. In this medium many of the changes considered above 
occur. These have been summarized diagrammatically for the 
carbohydrates, proteins, oils, and waxes by Russell. 


Carbohydrates; 


Proteins. cellulose. > Orgs Waxes. 
ir 1 ks (eral 
a capa | Anas | it 
NH; Other anleaee {| Undecom- 
_ acids. | “Humus.” Calcium posed. 
Gaseous _Nitrites. { | salts. 
N. I Calcium salts, | CO. i CO: 
Nitrates. Tes CO, CaCO; 
CaCO; 


Products from mineral compounds may be either oxidized or 
reduced by bacteria. Some of the important oxidations are the 
oxidation of ammonia to nitrites, and these in turn to nitrates. 


NH; .+ 30 = HNO: + 4:0 
HNO: + O = HNO; 


These changes are of especial interest to the student of soils and 
are brought about by the nitrosomonas and nitromonas, respectively. 

Ferrous salts may be oxidized to ferric, while sulfur may be 
oxidized to sulphuric acid. 


So 30" -— 8:0) = Febes: 
The important reduction reactions are the ones which occur in 
denitrification wherein the nitrate is changed to nitrite. 
Ca(NOs)2 = Ca(NOz)2 + O2 


PRODUCTS FROM NITROGENOUS COMPOUNDS 93 


Or the nitrate may be completely reduced with the liberation of 
gaseous nitrogen, thus completely removing it from the soil. 


2Ca(NOs)2 = 2CaO + 2N2 + 502 


Sulphates may in a similar manner be reduced to hydrogen sulphid. 


HeSO, = HS + 202 


Hence, water containing calcium sulphate if shut off from air may 
give rise to that ill-smelling gas, hydrogen sulphid. 

Pigments.—Many bacteria produce pigments, among which are 
practically all the colors of the spectrum—violet, indigo blue (B. 
violaceus, B. janthinus, B. cyanogenes, B. pyocyaneus), green (B. 
fluorescens), yellow (Staphylococcus aureus, Sarcina lutea), orange 
(Sareina aurantiaca), and red (B. prodigiosus). Usually oxygen is 
essential to the production of pigments and their intensity varies, 
depending upon the media upon which the organism is grown. 

The phenomenon of pigment production has long attracted the 
attention of bacteriologists, and many attempts have been made to 
explain their occurrence; but so far none of the explanations would 
seem to be. wholly satisfactory. The pigment seems to be of no 
material advantage to the organism, for colorless strains may be 
cultivated which possess all of the properties of the original strain 
with the exception of pigment production. ‘There is no evidence 
that they protect the organism against light, nor is there anything 
that would lead to the belief that (analogous to hemoglobin) they 
form a loose combination with the oxygen which under certain 
circumstances may be liberated. The pigment does not make it 
possible for the organisms to assimilate carbon dioxid as does the 
chlorophyll of the higher plants in the majority of cases. The best 
evidence, therefore, points to the conclusion that they are mere 
by-products that have no particular meaning to the organism. 

Beijerinck divides chromogenic bacteria into three classes: 

1. Chromophorous bacteria, in which the pigment remains within 
the cell and has a certain biological significance analogous to the 
chlorophyll of higher plants. To this class belong the green bacteria 
and the red sulphur bacteria, or, purple, bacteria. 

2. Chromoparous, or true pigment-forming bacteria, which set free 
the pigment as a useless excretion, either as a color-body or as a 
leuco-body which becomes colored through the action of atmospheric 
oxygen. The cells themselves are colorless and may under certain 
conditions cease to produce pigments. ‘To this class belong B. 
prodigiosus and others. 

3. Parachrome bacteria which form their pigment as an excretory 
product but retain it within their body, as B. janthinus and others. 

The chemical nature of pigments is not well understood, but it is 
known that they differ in solubility and are usually classified 
according to solubility in water, alcohol, chloroform, ether, benzol, 


94 BACTERIAL METABOLISM PRODUCTS 


and other solvents. The pigment produced by Azotobacter chro6- 
coccum is insoluble in all of these solvents but dissolves in alkalies 
undergoing decomposition with the formation of a dark brown 
solution. 

Heat.— Probably all bacteria liberate energy as heat in their 
metabolic process and there are a number which liberate it in suffi- 
cient amount perceptibly to change the temperature of the media 
in which they grow. This is exemplified in the heating of fermenting 
silage, manure, and hay. At times the temperature is raised to the 
kindling point with the result that spontaneous combustion may 
occur in hay and grain stacks. Bacteria generate considerabie of 
the heat, but other chemical processes are also active. 


Fic, 14.—Photogenic bacteria colonies on a plate photographed by means of their 
own light. (Lafar.) (Buchanan’s Household Bacteriology.) 


Light.—Sometimes one sees on the surface of decaying wood, 
fish, or various meats a bright illuminated surface which at times 
may be sufficient for the photographing of objects in an otherwise 
dark room. This is due to the growth of certain light-producing 
bacteria. Other organisms produce a beautiful phosphorescence. 
The organisms producing light are especially prone to occur in saline 
waters and are invariably aérobes. 


REFERENCES. 
Marshall: Microbiology. 
Taylor: Digestion and Metabolism. 
Lafar: Technical Mycology. 
Kendall: Bacteriology—General, Pathological and Intestinal. 


CHAPTER IX. 


INFLUENCE OF TEMPERATURE AND LIGHT ON 
BACTERIA. 


TEMPERATURE influences life phenomena in two ways—chemi- 
cally and physically. Chemically, heat influences powerfully the 
reacting velocity within the cell and the aggregate condition of the 
molecules, or coagulation. Physically, temperature influences the 
viscosity of the liquids composing the cell. 

Temperature and Speed of Reaction.— According to the law of 
Van’t Hoff and Arrhenius, a chemical reaction is increased two or 
more times its original speed whenever the temperature is increased 
10° C. This holds good for the reactions in living organisms, within 
certain limits of temperature, as well as for non-living, as may be 
seen from the following table given by Clausen in which is recorded 
the number of milligrams of carbon dioxid produced by 100 grams 
ot lupine seeds in one hour: 


Carbon dioxid Increase 
Temperature, produced. ror 10° C. 
0° Thee ah 
5 13.87 
10 itoje IML 10.84 
15 34.37 
20 43.55 25.44 
25 58.76 
30 85.00 41.45 
35 100.00 
40. 115.90 30.90 
45 104.45 
50 46.20 69.70 
55 Li cO 


The above table shows that for temperatures below 40° C. there 
is a general increase in the speed of the reactions with increases in 
temperature. However, at higher temperatures the amount of 
carbon dioxid diminishes rapidly with further increase in tempera- 
ture. This is very generally observed in enzymatic processes, as at 
temperatures over 60° C. enzymes are rapidly decomposed and 
many become immediately inactive when they are heated up to 
63° to 65° C. This may be due to the fact that the enzymes them- 
selves undergo hydrolysis which also would follow the temperature 
law of Van’t Hoff and Arrhenius. Furthermore, enzymes are prob- 
ably protein and would undergo heat coagulation. ‘This would 
reduce the reacting areas between enzymes and fermentable sub- 


96 INFLUENCE OF TEMPERATURE ON BACTERIA 


stance, and hence decrease proportionally the speed of the catalyzed 
reaction. 

The protoplasm composing the bacterial cell consists of carbo- 
hydrates, lipins, proteins, and ash having a definite structural 
arrangement within the living cell. The cellular protoplasm is, 
therefore, a colloid existing during life in the soluble condition, but 
when heated there occurs an irreversible reaction with the forma- 
tion of a gel. This heat-coagulation of the protein is explained by 
Robertson as essentially a phenomenon of dehydration, the first 
stage of which consists of internal neutralization through the loss 
of the elements of water from end-groups (— NH: and —COOBH), 
thus: 

H:N—RCOH—N—R—COOH = HN—R—COH—N—R—CO +H.0 

pe aera es 


In the second stage, or true coagulation, there is a polymerization 
of the amino-acids with the formation of the irreversible gel. 


2H2.N—R—COH—N—R—COOH = H2N—R—COH—N—R—COH—N 
R—COH—N—R—COOH + H:0 


Relation to Heat.—From the above theoretical consideration we 
should expect to find, and do actually find, an upper temperature 
limit at which all organisms cease to function. This upper limit 
varies considerably with the species of bacteria and the condition 
under which it is being held. B. phosphorescens will not grow above 
37° C., B. tuberculosis above 42° C., B. thermophilis above 72° C., 
and Setchell has found bacteria living in the water of hot springs 
at a temperature of 89° C. 

This great variation in temperature requirement of bacteria has 
led to their division into four classes: 

1. Thermophilic, or heat-loving bacteria, are those that develop 
at relatively high temperatures, usually above 45° to 50° C. These 
organisms occur in the water of hot springs, in decaying piles of 
compost or manure, in fermenting ensilage, in the intestinal contents 
of man and animals. ‘To this class belong the non-motile bacilli 
isolated by Miquel from .the Seine, which grew rapidly at tem- 
peratures around 70° C., as does also the so-called “ Mudedinus 
thermophiles,’ described by ‘Tsiklinsky, which develop readily at 
temperatures slightly above this. Most of the thermophiles are 
spore-bearing bacilli of little or no practical importance. 

2. Psychrophilic bacteria are those which grow best at relatively 
low temperatures, usually below 10° C. ‘They are most common in 
cold waters such as those of springs, wells, the depths of lakes or 
oceans and the soils of arctic regions. Forster has described certain 
phosphorescent bacteria, which he isolated from sea water which 
grow readily at 10° C. Many bacteria of the soil must belong to this 
class, as Conn and Brown have repeatedly shown that soil bacteria 


RELATION TO HEAT 97 


increase in number near the freezing-point. Some bacteria of this 
type probably play important réles in soil fertility and in the decay 
of foods in cold storage. 

3. Mesophilic bacteria are those whose optimum temperature is 
between these two extremes. They comprise the great group of 
pathogenic organisms occurring in the bodies of men and animals. 
To this class also belong many of the decay and putrefying organisms 
found in the soil. All of the more important bacteria belong to 
this group. 

Three temperature limits may be distinguished for bacterial 
growth: (a) Minimum, the lowest temperature at which bacterial 
growth will occur. This for the true thermophiles is about 40° C., 
for some pathogenic 29° C., and for the mesophilic as low as 0° C., 
or in solutions which do not solidify it may be even lower than this. 
(b) Optimum, that of most luxuriant growth. This, like the minimum 
temperature, varies greatly with the species. (c) Maximum, the 
highest temperature at which growth and multiplication can take 
place. This may be a few or many degrees above the optimum. 
For the thermophilic it may be as high as 89° C., whereas for the 
pathogenic bacteria it lies between 40° and 50° C. The growth of 
some pathogenic organisms at a high temperature for some time 
causes them to lose their virulence or disease-producing power, and 
is, therefore, made use of in the preparation of vaccines. 

The temperature relations are seen from the following table 
reported by Fischer: 


Temperature. 
Species. 
| Minimum. | Optimum. | Maximum. 
Psychrophilic bacteria. 0 15-20 30 Many water bacteria. 
Mesophilic bacteria. . 15-25 37 43 Pathogenic bacteria 
and others. 
Thermophilic bacteria. 25-45 50-55 85 Spore-bearing bac- 
teria from soil, feces 
and thermal springs. 


The growth temperature range of an organism is the number of 
degrees difference between the minimum and maximum. ‘This is 
very small with some bacteria like the gonococcus, the pneumococcus, 
the tubercle bacillus, and others which are highly susceptible to 
temperature changes and have the power of growing only within 
limits varying but a few degrees from the optimum. However, 
most pathogenic bacteria may grow at temperatures ranging 
between 20° C. and 40° C. Others, like the colon bacilli group, the 
Bacillus anthracis, and the Spirillum cholere asiatice, may develop 
at temperatures as low as 10° C. and as high as 40° C. or over. The 

7 


98 INFLUENCE OF TEMPERATURE ON BACTERIA 


range of temperature at which saprophytic bacteria, including soil 
organisms, may develop is usually a far wider one. These points 
are well illustrated by the following table taken from Fischer: 


Temperature. | 
Difference between Mini- 
mum and maximum. 
Minimum. | Optimum. | Maximum. 
B. phosphorescens . . 9 20 38 29 
B. fluorescens RE. rs 5 20-25 38 33 
B. subtilis BN OS Aarne 6 30 50 44 
iB danthracis) ss eo, oe 12 37 45 33 
Vibrio cholere . . . 10 37 40 | 30 
B.diphtherie . . . 18 33-37 45 27 
Mic. gonorrhee . ... 25 37 39 14 
Bact. tuberculosis . . 30 37 42 12 
B.thermophilis . . . 40 60° 80 40 


The fatal temperature may be even somewhat higher than this. 
It will vary with a number of factors, the condition of the organism 
playing a great part. For instance, Duclaux found that certain 
bacilli (Tyrothrix) found in cheese are killed in one minute at a 
temperature of from 80° to 90° C., whereas for the spores of the same 
bacillus a temperature of from 105° C. to 120° C. was required. 

Duclaux considers it erroneus to speak of a definite temperature 
as a fatal one; instead he considers it better to speak of it as deadly. 
This is due to the fact that the length of time an organism is exposed 
to a high temperature is important. This is illustrated by the experi- 
ments of Christen on the spores of the bacilli of the soil and of hay. 
The spores were exposed to a stream of steam and the time noted 
which was necessary to kill the spores at the various temperatures. 


Temperature. Time reqired to kill spores. 
100° over 16 hours. 
105-110 2 to 4 hours. 
115 30 to 60 minutes. 
125-130 5 minutes or more. 
135 1 to 5 minutes. 
140 1 minute. 


Moist heat is much more effective as a germicide than is dry 
heat. The probable explanation of this is that where dry heat is 
applied it must be high enough to decompose the organic constit- 
uents of the cell, the proteinaceous substance being in the form of 
the anhydride which can, in the presence of moisture, take up water 
according to the following equation: 


HN—R—COH—N—RCO + H:20 = H2N—RCOH—N—R—COOH 
[Be SS ee eet | 


Two or more molecules of this hydrated protein would then con- 
dense with the formation of the non-reversible gel. 


2H2NRCOHNRCOOH = H:N—R—COH—N—R COH—N—R COH NR— 
COHNR—COOH + H:20 


THERMAL DEATH POINT 99 


That moisture is essential for coagulation of proteins is illustrated 
by the following table from the work of Hiss and Zinsser: 


Ege albumen in dilute aqueous solution coagulates at 56° C. 
a with 25 per cent. water coagulates at 74°-80° C. 

“ (a3 a3 18 “ “ce 8) o= 90° CG 

“ c cc 6 “ce “ “ 145° @ 


Absolute anhydrous albumin, according to Haas, may be heated 
to 170° C. without coagulation. 

Moreover, moist heat is much more penetrating than is dry heat. 
This is illustrated by an experiment carried out by Koch and his 
associates. Small packages of garden soil were wrapped with vary- 
ing thicknesses of linen with thermometers so placed that the tem- 
perature under a definite number of layers could be determined. 
These were exposed to hot air and steam for four and three hours, 
respectively, with the following results: 


Temperatures reached within thicknesses of linen. 


| Time of 
Temperatures. Sea Vs 
application. | . ce ene 
a ees ao ine | 100 thicknesses. 
| F | 
Hot air . | 180°-140° C. | 4 hours 86° 72° | Below Incomplete 
| he Oe sterilization 
Steam . . | 90°-105.3° C.| 3 hours 101° | 01° | 101.5° Complete 
| | sterilization 


The comparatively low specific density of the steam enables it to 
displace the air from the interior of materials. Furthermore, when 
the steam comes in contact with the substance to be sterilized it 
condenses with a liberation of heat. This in the case of water vapor 
amounts to 536.6 calories. 

Although the spores of certain bacteria of the soil can withstand 
live steam for several hours, they may be destroyed in a few minutes 
or even instantaneously in compressed steam ranging in USUI OIGNAL NS 
from 120° to 140° C. 

The germicidal action of the great majority of disinfectants is 
due to a chemical reaction taking place between the protoplasm of 
the bacterial cell and the germicide. This reaction follows the 
temperature law of Van’t Hoff and Arrhenius. Hence, the mere 
raising of the temperature a few degrees of a sugar, salt, acid or 
alkali solution makes of it a disinfectant. 

Thermal Death Point.—'The thermal death point of an organism 
is the lowest temperature that will certainly destroy it under definite 
conditions. These conditions are time (which is generally taken as 
ten minutes), amount of moisture present, the reaction and com- 
position of the medium in which the organism is heated, and the 


100 INFLUENCE OF TEMPERATURE ON BACTERIA 


presence or absence of spores. The thermal death point of bacteria 
varies with the specific character of the organism (some organisms 
being much more resistant to heat than are others) and the age of 
the culture, young cultures being more resistant than older cultures 
which have not formed spores, especially when heated in the prod- 
ucts resulting from their metabolism. 

Cold.—It has been shown that the criterion for death is the non- 
reversibility of the change brought about by the agency in question. 
Now, does the lowering of the temperature bring about irreversible 
changes in the protoplasm as does the raising of the temperature? 
It is known that even intense cold does not cause these irreversible 
reactions in proteins. Where death does occur in cold-blooded 
animals and in plants, it must be due to the formation of ice crystals 
in the cells which may mechanically injure and kill them. This 
seems to be the case in the freezing of plants. Another irreversible 
change is connected with the thawing of the cells which have been 
frozen. Barring these two secondary and mechanical complications, 
the lowering of the temperatures does not seem to bring about 
irreversible changes i in the condition of the protoplasm which are 
incompatible with life. 

When the temperature of the protoplasm becomes sufficiently 
low, for example, approximately 0° C., the velocity of the chemical 
reaction becomes so small that the manifestations of life cease. The 
same is the case where the water content is sufficiently decreased. 
This is the reason why seeds of higher plants and spores of bacteria 
can be kept alive so long. Lack of water may reduce the reaction 
velocity of the hydrolytic processes in these at ordinary tempera- 
ture to such an extent that it may become practically zero. So 
resistant are bacteria to low temperature that they may be frozen 
solid and kept in this condition for days and even weeks, and many 
survive. Many bacteria, including the typhoid and colon bacilli, 
will survive freezing for twenty-four hours in lquid hydrogen 
(252° G:) and dev elop v igorously when brought into suitable 
media at an optimum temperature. Bacteria do not lose their 
virulence when exposed to low temperatures, as is the case when 
exposed to comparatively high temperatures. There is, however, 
a tendency for the number of organisms gradually to decrease as 
they are kept in the frozen condition. When typhoid bacilli are 
frozen in water, approximately 90 per cent. of them die during the 
first week, 95 per cent. succumb by the end of four weeks; but from 
four to six months’ continuous freezing is required to kill all of the 
organisms. The speed with which bacteria disappear from a frozen 
medium varies greatly with the nature of the medium. It is very 
slow in colloidal substances and much faster in erystalloids. Alter- 
nate freezing and thawing in colloids is much less disastrous to 
bacteria than the same treatment in aqueous solutions. It is prob- 


LIGHT 101 


able that the crystals formed in the freezing of water play a great 
part in the mechanical injuring of the bacteria. The freezing of the 
soil increases not only the number of bacteria within it, but the 
ammonifying and nitrogen-fixing powers of the soil. Whether or 
not this will vary with the water content of the soil has not yet been 
answered, but it is likely that as the moisture content increased 
the greater would be the injurious influence of the low temperatures. 

Light.—That light greatly affects the metabolism of the living 
cell is well known. However, bacteria are even more sensitive to 
light than are most cells. Diffused daylight exerts a hindering effect 


Fie. 15.—Thickly sown plate culture of typhus bacilli on agar-agar. Covered 
with paper letters and exposed to the sun’s rays for one and a half hours, then kept 
twenty-four hours in the dark, whereupon development of thickly congregated 
whitish colonies was found only at the parts covered by letters. (After H. Biichner.) 


upon bacterial growth and metabolism, whereas direct sunlight is 
highly injurious to certain bacteria, many microérganisms being 
killed almost instantly when exposed to the full action of the sun’s 
rays. The different colors of the spectrum do not act alike. The 
longer rays, from red to green, are practically without influence 
upon bacteria, but the blue and violet rays have the most marked 
germicidal power. 

Since light has no effect upon bacteria in a vacuum, it has been 
inferred that the changes brought about in the bacterial cell are 
primarily oxidation changes which are incompatible with the life 
of the cell. This reaction is brought about more rapidly in those 


- 402 INFLUENCE OF TEMPERATURE ON BACTERIA 


cells which contain considerable fat, and this may be the reason 
why some spores which contain an oily substance are especially 
sensitive to light. Death of the cell in many cases may be due to a 
type of coagulation like heat coagulation, for Bovis has shown that 
protein solutions in quart vessels are coagulated by exposure to 
ultra-violent light. This consists of two stages—first, that of dena- 
turation, and second, agglutination, or flocculation. 


CHAPTER -X. 


EFFECT OF OTHER AGENTS ON BACTERIA. 


Radium Rays.—Fernan and Pauli have shown that the exposure 
of proteins (serum albumin) in acid or alkali solution to radium 
radiations causes their coagulation. It is well known that the expos- 
ure of living tissue to these rays cause their destruction, and attempts 
have been made to treat certain bacterial diseases by their use, but 
so far without any great degree of success. The sterilization of milk 
and other foods by this method has been suggested, but its practical 
application appears to be improbable on account of the cost and 
uncertainty of the results. 

The fixation of elementary nitrogen by A. chrodécoccwm is dis- 
tinctly increased when the air is activated by pitchblend, somewhat 
better results being obtained with weak than with stronger radio- 
active intensity. Attempts have been made to force higher plants 
by its use, but so far without any practical success. 

Rontgen Rays.— Although réntgen rays are used in the treatment 
of microbial diseases of the scalp and skin, it has been conclusively 
shown that they are not even inhibitory, let alone fatal to the cells. 
This is seen from the results by Zeit, who found that bouillon 
and hydrocell-fluid cultures in test-tubes of non-resistant forms of 
bacteria was not killed réntgen rays after forty-eight hours’ ex- 
posure at a distance of 20 mm. from the tube. Tubercular sputum 
exposed to these rays for six hours at a distance of 20 mm. from the 
tube caused acute miliary tuberculosis of guinea-pigs inoculated 
with it. The hopes that were entertained of being able to disinfect 
the diseased body by this means have not been realized. The clinical 
results which are sometimes obtained must be explained by factors 
other than their direct germicidal influence, possibly by the pro- 
duction of ozone, hypochlorous acid, extensive necrosis of the deeper 
layers of the skin and phagocytosis. 

Electricity.—The influence of electricity itself upon micro- 
organisms is probably very slight, but it is often difficult nicely 
to differentiate between purely electrical effects and chemical 
changes which are produced in the media by the electric current. 
A direct current passing through a nutrient medium will cause an 
electrolysis which is usually manifest by the generation of acid on 
the positive electrode and alkali on the negative. The passing of 


104 EFFECT OF OTHER AGENTS ON BACTERIA 


an electric current through a sodium chlorid solution brings about 
an extremely complex change, as indicated in thefollowing equations: 


2NaCl 

2Na + 2HOH 
4Cl + 2HOH 
2NaOH + 2Cl 
3NaClO 


2Na + Ch 

2NaOH + He 

4HCl1 + Or 

NaClO + NaCl + H:0 
NaClO3; + 2NaCl 


Many of the products so formed, if in sufficient concentration, are 
good germicides and would be, therefore, the agents causing death 
instead of the electricity doing it. Moreover, the passing of an 
alternate current through a medium may heat it sufficiently to kill 
many bacteria. When the solution is properly cooled the action 
of the current is practically zero. 

Zeit, who has made a very careful study of the effect of electricity 
upon bacteria, summarizes his findings as follows: 

“1. A continuous current of 260 to 320 milliampéres passed 
through bouillon cultures kills bacteria of low thermal death points 
in ten minutes by the production of heat —98.5° C. The anti- 
septics produced by electrolysis during this time are not sufficient 
to prevent growth of even non-spore-bearing bacteria. The effect 
is a purely physical one. 

“2. A continuous current of 48 milliampéres passed through 
bouillon cultures for from two to three hours does not kill even 
non-resistant forms of bacteria. The temperature produced by such 
a current does not rise above 37° C. and the electrolytic products 
are antiseptic but not germicidal. 

“3. A continuous current of 100 milliampéres passed through 
bouillon cultures for seventy-five minutes kills all non-resistant 
forms of bacteria even if the temperature is artificially kept below 
37° C. The effect is due to the formation of germicidal electrolytic 
products in the culture. Anthrax spores are killed in two hours. 
Subtilis spores were still alive after the current was passed for three 
hours. 

“4. A continuous current passed through bouillon cultures of 
bacteria produces a strong acid reaction at the positive pole, due 
to the liberation of chlorin which combines with oxygen to form 
hypochlorous acid. The strongly alkaline reaction of the bouillon 
culture at the negative pole is due to the formation of sodium 
hydroxid and the liberation of hydrogen in gas bubbles. With a 
current of 100 milliampéres for two hours it required 8.82 mg. of 
sulphuric acid to neutralize 1 c.c. of the culture fluid at the negative 
pole, and all the most resistant forms of bacteria were destroyed 
at the positive pole, including anthrax and subtilis spores. At the 
negative pole anthrax spores were killed also, but subtilis spores 
remained alive for four hours. 


DRYING 105 


“5. The continuous current alone, by means of DuBois-Ray- 
mond’s method of non-polarizing electrodes and exclusion of chemi- 
cal effects by ions in Kruger’s sense, is neither bactericidal not anti- 
septic. The apparent antiseptic effect on suspensions of bacteria 
is due to electric osmose. The continuous electric current has no 
bactericidal nor antiseptic properties, but can destroy bacteria 
only by its physical effects—heat—or chemical effects—the produc- 
tion of bactericidal substances by electrolysis. 

“6, A magnetic field, either with a helix of wire or between the 
poles of a powerful electromagnet has no antiseptic or bactericidal 
effects whatever. 

“7, Alternating currents of a three-inch Ruhmkorff coil passed 
through bouillon cultures for ten hours favor growth and pigment 
production. 

“8. High frequency, high potential currents—Tesla currents— 
have neither antiseptic nor bactericidal properties when passed 
around a bacterial suspension within a solenoid. When exposed to 
the brush discharges, ozone is produced and kills the bacteria.” 

The electric current is used in the purification of sewage, the 
sterilization of milk, the improvement of wines, and the purification 
of water. In all of these cases the effect is due to a chemical pro- 
duced by the electricity. The purification of water is due to the 
ozone formed, which in turn acts as an oxidizing agent toward the 
bacteria. Although expensive, it is one of the most effective means 
of rendering water safe. 

Drying.— The results which have been reported on the influence 
of drying upon bacteria are exceedingly divergent. This is due 
mainly to the fact that the influence exerted by drying varies with 
a number of factors, chief among which are: 

1. Light.—Bacteria that are killed in a few minutes in direct 
sunlight may live for weeks in a dark place or even in diffused 
light. 

2. Oaygen.—Pauli and his associates consider death through 
drying as due to an oxidation process. They found that bacteria 
die much faster in pure oxygen than in air. Moreover, they found 
that the number of bacteria dying in unit time under constant con- 
ditions is proportional to the number surviving, therefore, com- 
parable with the simplest chemical processes, the monomolecular 
reactions. 

3. Thickness and Nature of the Medwum in Which They Are 
Dried.—In a dried medium bacteria usually die quickly but may 
survive long in sputum or feces. Moreover, bacteria suspended in 
the extract from a rich clay loam before being subjected to desicca- 
tion in sand live longer than if subjected to desiccation after sus- 
pensions in a physiological salt solution. 

4. The More Complete the Drying the Shorter the Life.— Alternate 
drying and moistening is unfavorable. 


106 EFFECT OF OTHER AGENTS ON BACTERIA 


5. The Higher the Temperature the Sooner the Bacteria Perish.— 
Death due to drying is probably in some cases due to a non-reversible 
reaction which follows the well-known temperature law of Van’t 
Hoff and Arrhenius. In other cases it is undoubtedly due to the 
increased osmotic pressure produced by the removal of the moisture. 

6. Old cultures, unless they be spore bearers, succumb sooner to 
drying than do young cultures. 

7. The influence of drying upon bacteria varies greatly with the 
species. Whereas the gonococcus, pneumococcus, spirochete of 
syphilis, cholera spirilla, and Pfeiffer bacillus can withstand drying 
only a few hours,.the typhoid, diphtheria, and tubercle bacilli may 
survive days; and tetanus, anthrax, and many soil organisms may 
survive drying for months or even years. Ammonifying, nitrifying, 
and nitrogen-fixing bacteria have been isolated in great numbers 
from soils which have been kept in tight bottles air-dry for more than 
fifty years. Even the non-spore-forming types of Azotobacter will 
withstand desiccation over sulphuric acid for a considerable time. 

Osmotic Pressure.—Bacteria vary greatly in their ability to with- 
stand great osmotic changes. Some. are quickly plasmolyzed in 
solutions having low osmotic pressure, whereas others can grow in 
strong sugar or salt solutions. This factor plays a great part in the 
preserving of fruits by means of sugar, of pickles and cabbage by 
means of salt, and many fruits by drying. Those fruits which have 
the highest carbohydrate content, such as grapes and prunes, are 
especially easy to preserve by drying. , 


3 “ 
a aD 


Fic. 16.—Plasmolysis of various bacterial cells. (Buchanan's Household 
Bacteriology.) 


Probably the great osmotic pressure in the soil solution of alkali 
soils plays a great part in retarding the bacterial activity of these 
soils. In this case, however, there is also a physiological factor in 
which the living protoplasm of the cell is so changed in its chemical 
and physical properties that it cannot function normally. It is 
found that equivalent osmotic concentrations of sodium and potas- 
sium salts act very differently upon some bacteria. 

Pressure.—Bridgman found that the application of very great 
hydrostatic pressure resulted in the coagulation of white of egg. 


SHAKING 107, 


He applied the pressure very slowly to avoid any rise in temperature 
due to the compression. That the effect is not due to heat is further 
demonstrated by the fact that it is more easily obtained at 0° C. 
than at 20° C. The application of five thousand atmospheres pro- 
duces stiffening of the white of egg; six thousand atmospheres 
applied, for thirty minutes, produced an appearance of the white 
resembling that of curdled milk; and seven thousand atmospheres’ 
pressure brought about complete gelatination. 

These facts seem to indicate that high pressure is fatal to many 
bacteria. Experiments have shown this to be the case. B. anthracis, 
B. pseudodiphtheria, M. pyogenes, var. aureus, and Oidiwm lactis 
survived after being subjected to a pressure of 2000 atmospheres for 
ninety-six hours. The pigment production and virulence of patho- 
genic organisms were either diminished or completely lost after such 
treatment. 

Successful attempts have been made to preserve fruit and vege- 
tables by exposing them to high pressure. Apple juice subjected 
to 4000 to 6000 atmospheres’ pressure for thirty minutes did not 
later develop gas. Peaches and pears exposed to this pressure did 
not spoil for five years. Those vegetables on which are found resist- 
ant spores could not be preserved by such pressures. It therefore 
appears that pressure high enough for the coagulation of the proteins 
is fatal to the less resistant bacteria. 

The power of resisting and actually functioning under high 
pressure is especially necessary for the denitrifying bacteria which 
live at the bottom of the ocean and return to the atmosphere the 
thousands of tons of combined nitrogen which is carried each year 
to the ocean from the soil and in the sewers. 

Shaking.—It is well known that proteins may be coagulated by 
shaking and that proteolytic enzymes undergo important modifica- 
tions under the influence of shaking. An active solution of proteo- 
lytic enzyme introduced into a reaction tube and agitated for two 
minutes may lose as much as 75 per cent. of its activity. After five 
minutes the disappearance is almost total. The effect of shaking 
varies with the speed, temperature, and reaction of the medium in 
which the ferment is placed. This phenomenon is known to be due 
to a coagulation or absorption of the substance, and it is quite 
possible that part of the influence exerted by shaking upon bacteria 
is due to this factor. It is known, however, that bacteria may be 
broken into the finest particles by the rapid shaking of cultures 
causing death at times by a disintegration of the cell body. 


CHAPTER XI. 
EFFECT OF CHEMICALS ON BACTERIA. 


Chemotaxis.—It has been repeatedly demonstrated that bacteria, 
like other free-moving organisms, are apparently attracted by cer- 
tain chemical substances in solution (positive chemotaxis), and 
repelled by others (negative chemotaxis). 

Pfeiffer, who was the first to study this phenomenon, developed a 
very simple and efficient method of studying it with bacteria. A 
capillary tube, sealed at one end and from 5 to 10 mm. long, is 
filled with a 5 per cent. slightly alkaline solution of Liebig’s beef 
extract or of peptone. The outer surface of the glass is carefully 
cleaned from any traces of the bouillon and is placed in a drop of 
water containing bacteria. In a few seconds the bacteria are found 
to thickly congregate around the open end of the capillary tube. 
According to the view held by Jennings, the swarming of bacteria 
around any point, where faverable nutrient conditions exist is not to 
be looked upon as due to a definite attraction exerted upon the 
bacterial cell, but as caused simply by the tendency to remain at 
those points where the conditions are favorable. But this does not 
seem to be the true explanation, for had the capillary tube been 
filled with sugar, or glycerin, which are the best foodstuffs and richest 
sources of energy, there would have been no such gathering of the 
the bacteria at the end of the tube. Moreover, a solution of 0.019 
per cent. potassium chlorid plus 0.01 per cent. mercuric chlorid 
attracts bacteria by reason of the potassium which it contains, but 
they rush into the tube only to meet their death from the mercury 
salt. 

The explanation given by Loeb seems to be more reasonable: 
“Theoretically, we may assume that if substances diffuse in air or 
in water, the particles move in a straight line away from the center 
of diffusion. If they strike an organism whose surface is affected 
by the diffusing substance on one side only, the contractile proto- 
plasm, or the muscles, turning the tip or the head of the whole 
organism toward that side, are thrown into a different state of con- 
traction from their antagonists. ‘The consequence is a turning or 
binding of the tip of the head until symmetrical points of the chem- 
ically sensitive surface of the body are struck by the line of dif- 
fusion (or the diffusing particles) at the same angle. As soon as 
this occurs the contractile elements on both sides of the organ or 
organisms are in an equal state of contraction, and the animal will 


CHEMOTAXIS 109 


bend or move in the direction of the lines of diffusion.”” Why one 
substance should act positive and another negative is at present 
quite inexplicable. 

Chemotaxis can take place only in media which permit free 
movement and its sphere of action is comparatively small. Different 
kinds of bacteria by no means react in the same way to the same 
substance. Furthermore, the action, whether positive or negative, 
chemotaxis or neutral, varies with the chemical. The salts of potas- 
slum are among the more active positive chemotactic substances, 
followed by sodium and rubidium. The alkaline earths are less 
effective. The influence of a salt is attributed mainly to its electro- 
positive constituent; asparagin and peptone are strongly chemo- 
tactic, whereas sugar and glycerin are inactive. 

Negative chemotaxis is noted when capillary tubes are filled 
with free acids and alkalies or with alcohol. In some salts the action 
of the acid radical and that of the base neutralize each other 
(ammonium carbonate and monobasic potassium phosphate). In 
this case the bacteria are neither attracted nor repelled by the sub- 
stance. 


E 8 x 


Fig. 17.—Oxygen-loving bacteria infesting a thread of alga lying in the micro- 
spectrum. The chlorophyll granules contained in the alga cells are not shown, but 
the spectrum lines are given to denote the position of the spectrum. Mag. 200. (After 
Engelmann.) 


Engelmann ingeniously made use of this phenomenon as a test 
for oxygen and the effect exerted upon assimilation by the different 
parts of the solar spectrum. If a thread of alge and some aérobic 
bacteria are placed under an air-tight cover-glass, the bacteria are 
active; but if the preparation is kept in the dark the action of the 
bacteria will cease, showing that all the oxygen has been consumed. 
If brought back to the light as the alge assimilate carbon dioxid 
with the elimination of oxygen the bacteria again become active. 
If exposed to the spectrum the greatest aggregation of bacteria 
occurs at the red end of the spectrum, indicating that the maximum 
assimilative activity of the alge protoplasm is proceeding at this 


110 EFFECT OF CHEMICALS ON BACTERIA 


point. By means of this highly sensitive test as little as one-billionth 
part of a milligram of oxygen may be detected. 

It is quite possible that the phagocytes which play such a part 
in freeing the body of bacteria are directed or guided in their choice 
and perception by chemotaxis to the bodies which they ingest. 
The attraction of leukocytes toward the point of bacterial invasion 
is, in part at least, due to the properties of the bacterial proteins. 
This attraction is sometimes increased by injecting into the tissues 
at the point of infection some bland substance, such for instance as 
bismuth subnitrate. 

Disinfectants.—Of great interest are those substances which in 
minute quantities destroy the life of the cell. These substances 
when considered in their effects upon man and animals are called 
poisons. But when considered from the standpoint of micro- - 
organisms they are called germicides. Analogous with the general 
term germicide, are the terms bactericide and fungicide. A disin- 
fectant is a substance which destroys the causative agent of infec- 
tion. Although disinfection may occasionally mean sterilization, in 
the majority of cases it does not. It implies the destruction of those 
minute forms of life which cause disease. 

Antiseptics prevent decomposition and decay. ‘They do not 
necessarily destroy microérganisms; they prevent their growth and 
activity. One and the same substance may be a disinfectant under 
one condition and an antiseptic under another. Formalin in the 
proportion of 1 to 50,000 is an antiseptic, whereas it requires from 
3 to 10 per cent. solution to be a disinfectant in a reasonably short 
time. Mercuric bichlorid in the proportion of 1 to 300,000 will 
sometimes prevent the germination of anthrax spores. Yet it 
requires a 1 to 1000 solution to kill them. 

The term preservative is usually applied to those substances 
which are added to foods, feeding-stuff, and substances of similar 
origin with the intention of preventing decomposition or decay. 
These may be either comparatively poisonous—benzoic acid, boric 
acid, salicylic acid, formalin, or sulphates—or the non-poisonous 
substances—common salt or sugar. The method of action of the 
two is markedly different, the first combining with the protoplasm 
of the cell, the second acting through increased osmotic pressure. 

Deodorants are substances which have the power of destroying 
or masking unpleasant odors arising from putrifying or fermenting 
organic matter. Deodorants destroy odors, disinfectants destroy 
germs. A deodorant may or may not be a disinfectant. Formalin 
is a good disinfectant and deodorant, whereas charcoal is a good 
deodorant but has no value as a disinfectant. 

The classification of disinfectants is difficult, inasmuch as we do 
not understand in many cases their complete mode of action. 
Moreover, almost any compound, if used in sufficient concentration, 
may act as an antiseptic if not as a disinfectant. ‘The methods 


DISINFECTANTS 111 


most often used in classification are according to either composition 
or mode of action. The simplest method is by chemical structures 
and qualities under which are distinguished the following natural 
groups: acids, alkalies, metallic salts, hydrocarbons, alcohols, 
aldehyds, anesthetics, essential oils, and oxidizing and reducing 
agents. The first three—acids, alkalies, and salts—are distinguished 
from the rest by being electrolytes. The strength of acids and alka- 
lies is dependent upon the hydrogen or hydroxy] ion concentration 
with the metallic salt; the action is dependent upon the nature of 
the metallic ion and the degree of electrolytic dissociation. 

Rosenau classified disinfectants according to mode of action as 
follows: (1) Those compounds which destroy by oxidation, as ozone, 
chlorinated lime, potassium permanganate, and the halogens. 
(2) The destruction by ionic poison with coagulation, as the metallic 
salts, mercury, and lead salts. (3) Destruction by coagulation and 
poisoning not ionic in character, as carbolic acid and its derivatives. 
(4) Destruction by emulsoid action, that is, through Brownian 
movement and adsorption; soap solutions and creolin. 

Laws Governing the Action of Disinfectants.—These have been 
mainly worked out by Chick who found that disinfection is an 
orderly time-process, which may be considered analogous with a 
chemical reaction, viz., a reaction between the bacterium on the 
one hand and the disinfectant on the other. In the ideal case disin- 
fection proceeds in accordance with some rule analogous to the mass 
law, so that if the disinfectant is present in large excess, disinfection 
rate at any moment is proportional to the concentration of bacteria 
— = = Kn, where n is the concentration of bacteria at the time , 
and K is a constant, depending on the temperature concentration 
of disinfectant, etc.). 

The velocity of disinfection increases with rise in temperature in 
an orderly manner according to the well-known equation of Arrhe- 
nius. Some idea of the magnitude of the effect of temperature may 
be gained from the fact that with metallic salts the mean velocity 
of disinfection increases two- to four-fold for a rise in temperature 
of 10° C., whereas with phenol it was as high as eight-fold, using 
B. paratyphosus as the test organism in each case. Hence, the use 
of a disinfectant at a comparatively high temperature, other things 
being equal, is more effective than its use at a low temperature. In 
reality, a solution which at one temperature is only an antiseptic 
may become a disinfectant by a small increase in temperature. 

The efficiency of a disinfectant varies with the moisture. A dry 
poison has but slight action on microérganisms. For this reason, 
dry formaldehyde gas is practically without effect. In a similar 
manner absolute alcohol has not nearly the same germicidal power 
as has 50 to 70 per cent. alcohol. This is probably due to the 
absolute alcohol coagulating the outer membrane of the organism 


112 EFFECT OF CHEMICALS ON BACTERIA 


and thus prevents the poison from diffusing into the vital part. 
The burning of sulphur in a dry atmosphere has little if any effect 
upon bacteria, but in the presence of moisture there is formed 
sulphurous acid which is a rather efficient disinfectant. 

The germicidal property of salts of the heavy metals, acids, and 
alkalies is governed in a large measure by the degree of ionization. 
Mercurie chlorid in water is a good disinfectant, but in alcohol has 
little or practically no germicidal properties. The addition of sodium 
chlorid to mercuric chlorid increases the solubility of the latter and 
yet decreases its germicidal power. This is due to the fact that there 
is formed a double salt: 

2NaCl + HgCh = aati 


This is Poon dissociated by steps into Na +N abecl, Na - 


Hel li, me + 4C a, The number of Hg ions formed is very small, 
therefore, in the presence of sodium chlorid. 

As a general rule the addition of a common negative ion decreases 
the number of ions of the metal going into solution. If mercuric 
chlorid is shaken with water, the salt dissolves until there is an 
equilibrium between the solid phase and the undissociated molecules 
in solution. As the molecules dissociate, the equilibrium is dis- 
turbed and more of the solid dissolves to restore it, until a second 
equilibrium is established between the ions and the molecules. 
These equilibria may be expressed by the equation 

[Hge"] x [CIP 


[HgCls] = a constant; 


since the concentration of the undissociated molecules is constant. 
So long as there is any undissolved salt, the equation becomes 


Hes >< Ch y= salvconstant: 


If we add NaCl, the Cl will increase the concentration of the Cl 
ions which will combine with the Hg, giving HgClh, which will 
crystallize out. Moreover, in this case the NaCl combines with the 
HgCl, giving the NazHgCl, which greatly decreases the Hg ions in 
solution. The effect of this on the disinfecting power of different 
dissociated salts of mercury on anthrax spores is indicated in the 
following from Paul and Kronig: 


Bat. | \Conoentesiia | Oelgnen ser 20 | “Uolenieeiee Se Co ee 
HgCh. . 1/64 mol. 7 0 0 
HegBrz ‘ 1/64 mol. 34 0 0 
HgCne : 1/16 mol. 0 33 0 
KeHgCh . 1/16 mol. va We 0 
KeHgBrs . 1/16 mol. an £4 5 
Koliolg, 7. 1/16 mol. Li 3 389 
K,HgCn, . 1/16 mol. ahs Ee 1035 


DISINFECTANTS 113 


The power of a disinfectant to kill bacteria is dependent in a 
remarkable degree upon the nature of the medium in which bacteria 
are present when the germicide is applied. Almost invariably the 
greatest germicidal activity is shown when the substance acts upon 
the bacteria freed from all contaminating culture media and sus- 
pended in distilled water or salt solution. The presence of proteins, 
peptones, and similar substances usually cause a great reduction in 
the germicidal powers of the substance. ‘This is also the case in the 
presence of pus, many of the organisms being partly digested in 
the body of dead leukocytes. This property is illustrated by the 
following table reported by Dakin and Dunham. The — sign 
indicates sterilization as indicated by negative subcultures, and the 
+ sign incomplete sterilization. 


Staphylococci in 50 per 


Antiseptic. Staphylococci in water. | ‘aan, [Ges RENTED 
1:250— 1:50— 
Phenol 1:500-+ 1:100-+ 
Re? é 1:2,500 — 1:100— 
Salicylic acid 1:5,000-+ 1:250-4 
See 1:3,500 — 1:1,700— 
Hydrogen perioxid . S| 1:8,000-+ 1:2,000-+ 
ae: 1:100,000 — 1:1,000— 
pees Za 1:1,000,000+ 1:2,500-+ 
: : 1:5,000,000 — 1:25,000 — 
Mercurie chlorid 1:10,000,000 + 1:50,000-+ 
Sily strat 1:1,000,000 — 1:10,000 — 
rab treeecs eile 1:10,000,000 + 1:25,000+ 
: ney Sis d 1: 6500,000— 1:1,500— 
Sodium hypochlorite 1:1,000,000-+ 1:2,000-+ 
Chloramin T 1: 500,000— 1:2,000 — 
Soe 1:1,000,000+ 1:3,000+ 


This decreased efficiency in the presence of a protein is variously 
explained. In the case of such disinfectants as phenol and the dye- 
stuffs, it is frequently stated that the disinfectant is ‘‘ quenched” 
or “fixed” by the protein medium, Adsorption in some cases may 
play a part, but in the case of salts of the heavy metals, they com- 
bine with the protein giving an insoluble non-ionizing proteinate. 
The low germicidal action shown by most antiseptics against pus 
is due in part no doubt to the mechanical difficulties of penetrating 
the mucoid particles in the pus. 

Young cultures of bacteria are usually more resistant than are 
older cultures. This is especially true when the disinfectant is 
applied to cultures living in the products resulting from their 
metabolism. Cultures, the organisms of which form spores, become 
more resistant to disinfectants as the spore stage is reached. 

Emulsions as a rule have greater germicidal power than have 
solutions. According to Chick and Martin, emulsions or soapy 

8 


114 EFFECT OF CHEMICALS ON BACTERIA 


preparations of the coal-tar acids exhibit active Brownian motion. 
Often the bacteria are considerably larger than the mean diameter 
of the emulsified particles. These bombard the bacteria and in this 
way frequently bring them into intimate contact with the undiluted 
particles of the disinfectant, which would not occur in solutions. 
Emulsions act upon bacteria first through physiochemical adsorp- 
tion and second through chemical combination. But-other particles 
held in suspension also possess this power of adsorption, and hence 
the strength of emulsions is rapidly reduced. Thus the value of 
phenol is barely impaired by the presence of organic matter in solu- 
tion, whereas emulsified disinfectants are reduced to ene gine or 
one-half of their original value. 

Disinfectants of the Chlorin Group.—'T’o this group belong many 
of the more active disinfectants. They are all characterized by a 
chemical instability in the presence of organic matter. ‘The mem- 
bers of this group contain active chlorin in distinction to inert 
chlorin, such as that in common salt. The phrase “active chlorin”’ 
does not, however, necessarily imply that free chlorin is contained 
in the substance or liberated by it. The active agent may be hypo- 
chlorous acid or some other compound containing chlorin. 

It used to be assumed that they acted mainly by the liberation 
of nascent oxygen. Hypochlorous acid decomposes thus: 


4HCIO = 2H2O + 2Cl + Or 


The chlorin then reacts with water, liberating more nascent 


oxygen: 
2Cch + 2H.O = 4HCl + Or 


Dakin, however, defines a substance as possessing active chlorin 
when it will part with chlorin either free or combined in such a way 
that it can effect the chlorination of bacteria and other proteins. 

All proteins are made up of amino-acids in which the amino- 
group of the one has reacted with the carboxyl group of the other 
with the elimination of water. This gives imino NH— groups. It 
is assumed by some that the chlorin replaces the hydrogen in this 
group, thus: 


R R 
renee He eee 
: =O + 2 = C=0O + HCl 
ae NCl 
eee H—t—cool 


R R 


DISINFECTANTS OF THE CHLORIN GROUP 115 


Or it may be that the extra bonds of the nitrogen in the imino 
group is utilized: 


i i 
| 
tae ey 
| H 
NH NZ ] 
| \cl 
H—C—COOH ‘Dip aes 
| 
R R 


according to this explanation hypochlorous acid may react without 
decomposing into chlorin: 


» R 
| 
ae Be ory 
| 
C=O. JE BOGE. f= he 
| /H 
NH NZ 
| | \o—cl 
H_¢ COOH H—C—COOH 
R R 


In any case the chemical and physical properties of the protoplasm 
would be so changed as to be incompatible with the life processes of 
the microorganisms. 

Compounds containing the group— NCl—belong to the class of 
chloramins. Their chlorin is still active and they are themselves 
active germicides. Such compounds have been studied thoroughly 
by Dakin who used them extensively in the disinfecting of wounds 
in the great European war of 1914-18. 

Chlorinated lime, or bleaching powder, may be taken as a type 
of the chlorin disinfectants. Its precise chemical composition is 
not known although calcium oxychlorid (CaOCl.) is now generally 
accepted as being the essential agent of dry bleaching powder and 
calcium hypochlorite (Ca(OCI)z) to be the active germicide of the 
solution. Although the reactions which occur are quite complicated, 
it is certain that the active substances are nascent oxygen, chlorin, 
and hypochlorous acid, and are probably formed as follows: 


2CaOCle = Ca(OCl)2 + CaCle 

Ca(OCl)2 + H2COs = CaCO; + 2HOCI 
2HOCI = 2HCl + Os 

2HOCI = H:0 a ly + O 


The substance is extensively used in the disinfection of sewage, 
outhouses, cellars, and for miscellaneous purposes. Since 1908 it 
has been used rather extensively in water purification. In practice 


116 EFFECT OF CHEMICALS ON BACTERIA 


from 5 to 12 or more pounds of bleaching powder is used to each 
million gallons of water. It cannot be detected by the sense of taste 
provided the amount does not exceed 25 pounds to 1,000,000 gallons. 
Waters containing considerable organic material of any kind give 
rise to amins, chloramins, and other compounds with unpleasant 
flavors. The method, however, is cheap, reliable, efficient, harmless, 
and easy of application. 

Formaldehyd is one of the best volatile antiseptics. If used in 
sufficient concentration and under proper conditions it can be 
depended upon for surface disinfection. Although more penetrating 
than sulphur dioxid, it is not sufficient to depend upon in deep layers 
of cloth and similar bodies. It does not rot nor bleach fabric nor 
tarnish metal as does sulphur dioxid. Moreover, formaldehyd 
unites with nitrogenous substances forming new chemical compounds 
which are both sterile and odorless. It is, therefore, good both as a 
germicide and as a disinfectant. 

Although there are numerous methods of using, one of the best 
is that recommended by the Pennsylvania Department of Public 
Health: 


Sodiumy dichromate wk Gee) cake) a ee ee ee ee nee ORO Ze 
ovmalines’ Fs) ees re ke es Tig Bees ON rena Ming SO meaner Lad RMOIZS 
Commercialsulphurieyscid. 2a) ee ete eee a ee One ee 14 oz. 


The sulphuric acid is added to the formalin and the mixture poured 
over the crystals of sodium dichromate causing immediate liberation 
of formaldehyd gas. Five hundred c.c. of formalin and 250 gm. of 
sodium dichromate should be used for each thousand cubic feet of air. 
The floor should be protected against the heat by placing the bucket 
upon a brick or other suitable device. 

Sulphur Dioxid.— Sulphur dioxid is not very efficient as a germicide; 
it is, however, an effective insecticide. It is also good to use against 
diseases spread by rats, mice, flies, fleas, mosquitoes, etc. 

Its action as a germicide depends upon the presence of moisture. 
The dry gas is practically inert against bacteria. It cannot be 
depended upon where penetration is required, its action being merely 
upon the surface. It does not kill spores. Moreover, it is a bleaching 
agent and tarnishes metals. In sterilization by means of sulphur, 
time is an important factor. The things to be disinfected should 
be exposed for eight hours to an atmosphere of at least 4 per cent. 
by volumes of sulphur dioxid gas in the presence of water. This 
requires the burning of 4 to 5 pounds of sulphur for every 1000 cubic 
feet of air space. About one-fifth of a pound of water should be 
volatilized for every pound of sulphur used. 

One method of using it follows: The required quantity of sulphur 
is placed in a pan which is put into a second larger pan containing 
water. The sulphur is made into little craters and liberally soaked 


MERCURIC CHLORID 7 


with alcohol. It is well to place the generator on a table or box as 
sulphur dioxid is heavier than air and hence tends to sink and would, 
therefore, extinguish the flame if placed on the floor. 

Hydrocyanic acid gas is an extremely powerful insecticide, but 
a poor germicide. It is used rather extensively against mosquitoes, 
lice, bedbugs, and roaches, but on account of its highly poisonous 
nature it must be used with extreme caution. It is effective against 
bacteria no hardier than those of diphtheria and typhoid, but it 
cannot be depended upon as a general disinfectant. 

Mercuric chlorid is one of the best known and most effective of 
the metallic salt disinfectants. A solution of 1 to 1000 is ample 
for the destruction of all non-spore-bearing bacteria, provided it 
comes in direct contact with the organisms for some time. It is 
especially valuable for disinfecting the hands and for washing floors, 
woodwork, and furniture. It attacks metals and hence cannot be 
used to disinfect them; it is rendered inactive by protein substances; 
it acts on bacteria by a coagulation of the protoplasm. 

Its germicidal value as usually given is too high. This is due to 
the fact that it may inhibit the growth of bacteria and in the planting 
of the cultures the metallic salt is carried over into the new medium, 
there preventing growth but not necessarily killing the organism. 
The explanation of this is given by Miss Chick who found that if 
bacteria are subjected to the action of 1:1000, 1:10,000, or even 
weaker solutions of mercuric chlorid, there is an interval during 
which some at least of them may be resuscitated by the timely 
administration of an antidote—in this case a sulphid solution. If, 
however, this antidotal treatment is not employed, no amount of 
subsequent dilution beyond the limits of inhibition can prevent the 
death of the organism. 


REFERENCES. 
Loeb: The Dynamics of Living Matter. 


McClendon: Physical Chemistry of Vital Phenomena. 
Dakin and Dunham: Handbook of Antiseptics. 


CHAR TE ho xg 
INFLUENCE OF ARSENIC ON BACTERIAL ACTIVITY. 


Occurrence of Arsenic.— Kunkel showed the presence of arsenic 
in many rocks and water, while Czapek states that traces are nearly 
always present in soils. Herzfeld and Lange found arsenic in certain 
German raw sugars and traced it to the lime which had been used 
in the manufacture of sugar. Headden found some virgin prairie 
soils relatively rich in arsenic, an observation in accord with my own 
experience. I have found arsenic to the extent of 4 parts per million 
in virgin soil; and, as in the cases referred to by Headden, it did not 
result from smelter fumes or any such source, but was derived from 
the decay of native rocks. On the other hand, Headden found 
arsenic in some cultivated orchard soils to the extent of 138 parts per 
million. He claims that in many places arsenic from spray is accu- 
mulating in sufficient quantities to become injurious to vegetation. 
Francois, however, thinks there is little danger of the soil’s becoming 
unfit for vegetation from the proper use of insecticides. Grunner, 
who found arsenic to the extent of from 0.026 per cent. to 1.426 per 
cent. in the Reichenstein soil, is not so optimistic. An extensive 
analysis of the sprayed orchard soils of western America showed 
arsenic to be present in all of those soils and varying from mere 
traces to 500 pounds an acre. In some cases it occurred to a depth 
of three or four feet. The most interesting fact is that in some of 
these soils there were as much as 17 pounds per acre of water-soluble 
arsenic. It is not, however, always the case that the greatest 
quantity of water-soluble arsenic is found in those soils which con- 
tain the greatest total quantity of arsenic, for often soils are found 
which contain only a few pounds to the acre-foot, probably two- 
thirds of which is in a soluble form. So the conclusion has been 
reached that some virgin and many cultivated soils contain arsenic 
in large quantities, but the proportion in a soil is no index of the 
amount that is soluble in water. The latter is probably governed 
by many factors; for example, kind of soil, water-soluble salts, and 
form in which the arsenic was applied to the soil. 

Factors Influencing Solubility.— That the form in which the arsenic 
is applied govern largely its solubility is shown by an experiment in 
which 100 grams of arsenic in the form of lead arsenate was applied 
to a soil, and to another portion of the same soil was added 100 
grams of arsenic in the form of Paris green. To still another soil 


NITRIFICATION 119 


was added enough arsenic in the form of zinc arsenite to make 100 
grams of arsenic. These were carefully mixed and allowed to stand 
for some time, after which an examination was made for soluble 
arsenic. The analysis revealed the fact that 14 per cent. of the lead 
arsenate, 30 per cent. of the zinc arsenite, and over SO per cent. of 
the Paris green were in the water-soluble form. 

Arsenic being in the soil, some soluble and some insoluble, very 
naturally suggests the question as to what effect it has upon the 
bacteria of the soil. Any factor which influences the bacterial 
activities must indirectly influence the crop yield. 

Extensive studies have been made on the influence of various 
arsenic compounds upon the bacterial flora of the soil with the result 
that arsenic was found to be a stimulant in low concentration and 
toxic only in larger quantities. The extent of stimulation and toxic- 
ity varies greatly with the specific type of organism and the form 
in which the arsenic is applied. 

Ammonifiers.— Experiments on ammonifiers show that this class 
of bacteria are not at first poisoned by the arsenic, but their speed 
of action is increased. The actual results showed that whereas the 
untreated soil produced in unit time 100 parts of ammonia, soil to 
which 60 pounds of arsenic an acre was applied produced 103 parts 
of ammonia in the same length of time. And it was not until 2500 
- pounds of arsenic an acre was applied to the soil that the production 
of ammonia was reduced to one-half. The Paris green, on the other 
hand, retarded the action of this class of bacteria even in the lowest 
concentration added, and by the time,600 pounds an acre had been 
applied the ammonia produced in unit time had been reduced to 
one-half normal. This poisonous action of arsenic on bacteria is in a 
direct relationship to its solubility. An extremely large quantity 
of lead arsenate would have to be applied to a soil before it would 
interfere with ammonification. 

Nitrification.— The nitrifying flora of a soil are more resistant and 
are stimulated to a greater extent by arsenic than are the ammoni- 
fiers. Tests made in soil have shown that whereas untreated soil 
produced 100 parts of nitrates in unit time, the same soil to which 
had been added arsenic in the form of lead arsenate at the rate of 
120 pounds an acre produced 178 parts of nitrates. In other words, 
in place of being injured by the arsenic, the bacteria were nearly 
twice as active in the presence of this quantity of arsenic as they 
were in its total absence. It was not until more than 700 pounds 
of arsenic, in the form of lead arsenate, an acre, had been applied 
to the soil that the bacterial activity fell back to 100. Even when 
arsenic in the form of lead arsenate was applied at the rate of 3500 
pounds an acre there was 68 per cent. as much ammonia produced 
as in the untreated soil. The Paris green gave similar results. The 
untreated soil produced 100 parts of nitrates in given time, while 


120 INFLUENCE OF ARSENIC ON BACTERIAL ACTIVITY 


similar soil to which arsenic in the form of Paris green was added 
produced, under the same condition, 129 parts of nitrates. When, 
however, higher concentrations of arsenic in the form of Paris 
green were added it became toxic, and eventually stopped all 
bacterial activity; but the quantity added had to be so large that 
it is not likely that sufficient would ever occur under agricultural 
practice. 

Arsenic, then, does not injure the ammonifying or nitrifying 
organisms of the soil. But how about the other beneficial bacteria 
of the soil? What effect has it upon them? 

Nitrogen Fixation.— There are 75,000,000 pounds of atmospheric 
nitrogen resting upon every acre of land, but none of the higher 
plants have the power of taking this directly from the air. Certain 
bacteria, however, can live in connection with the legumes and 
assist them to take nitrogen from the air. Then there is another 
set of nitrogen-gathering organisms which live free in the soil, and 
which may, under ideal conditions, gather appreciable quantities 
of nitrogen. It is rather possible that much of the benefit derived 
from the summer fallowing of land is due to the growth of this class 
of organisms in the soil and storage by them of nitrogen for future 
generations of plants. In such soils they are both more active and 
are also found in greater numbers. All the work put on soil to 
render it more porous reacts beneficially upon these organisms. 
They not only require atmospheric nitrogen and oxygen, which are 
absolutely essential to their life activities, but they must obtain 
them from within the soil, for the minute organisms cannot live 
upon the surface of the soil because to them the direct rays of the 
sun means death. How does arsenic influence this class of organisms 
which are so beneficial to the soil, but which are so much more 
sensitive to adverse conditions than are the other kinds of bacteria? 
Arsenic in the form of lead arsenate, zinc arsenite, and arsenic 
trisulphid stimulate these bacteria. When arsenic in the form of lead 
arsenate was applied to the soil at the rate of 500 pounds an acre, 
the nitrogen-fixing organism gathered twice as much nitrogen in 
unit time as it did in the absence of arsenic. The Paris green, 
however, is poisonous to this group of organisms in the minutest 
quantities. This is most likely due to the copper rather than to the 
arsenic in the compound. 

How Does the Arsenic Act?—It may, therefore, be concluded that 
arsenic stimulates all the beneficial bacteria. But how does it act? 
Will it stimulate for a short time and then allow the organism 
to drop back to its original or to a lower level as does alcohol and 
various stimulants when given to animals? Will it act as does 
caffeine—continue to stimulate? From the results on men and 
horses the former might be expected, for although the arsenic eaters 
of India and Hungary maintain that the eating of arsenic increases 


HOW DOES THE ARSENIC ACT 121 


their endurance, and there is considerable evidence to indicate this, 
it is only for a short time. If the use be discontinued the arsenic 
eaters cannot endure as much physical exertion as can others who 
are not addicted to the drug. Many European horse dealers place 
small quantities of arsenic in the daily corn given to the horse, for 
they find it improves the coat of the horse. Ifa horse, however, has 
been doped on arsenic for a long time it seems necessary to continue 
the practice; otherwise, the animal rapidly “‘loses his condition.”’ 

Similar results might be expected with the bacteria, and experi- 
ments have shown that although during the first few weeks the 
bacterial activity of soils containing small quantities of arsenic is 
much greater than it is in a similar soil without arsenic, this activity 
continues to get less and less, until at the end of several weeks it is 
no greater than in soil containing no arsenic. It is interesting to 
note that if proper aération is maintained bacterial activity never 
becomes lower than in untreated soil. 

Now why this stimulating influence of arsenic upon soil bacteria? A 
similar condition has been found to exist when soils are treated with 
carbon bisulphid, chloroform, or other disinfectants, or even when 
the soil is heated. Many theories have been offered to account for 
it, but probably the most interesting is the one held by Russell and 
Hutchinson. They maintain that within the soil are microscopic 
plants, bacteria, and also microscopic animals, protozoa. The 
minute animals are continually feeding upon the minute plants, 
with the result that the bacterial plants cannot multiply as they 
could in the absence of the protozoa. Now when a weak solution 
of an antiseptic is applied to the soil it kills many of the protozoa, 
and the bacteria being no longer preyed upon by their natural foe 
rapidly multiply. As the antiseptic evaporates the few remaining 
protozoa start to multiply and soon are able to keep in check the 
bacterial flora of the soil. So within the soil one species preys upon 
another. It is possible that microscopic forms of life wage within 
the soil battles as terrific as those waged by the higher forms of life 
upon the earth’s surface. 

It is likely that this is one of the ways in which arsenic stimulates 
the bacterial activities of the soil. It acts more readily upon the 
protozoa than upon the bacteria. After the arsenic has been in the 
soil for some time it may become insoluble or some of it may be 
changed by molds into a gas arsine and pass into the air. Then the 
few protozoa which have not been destroyed by its presence rapidly 
multiply and soon hold the bacteria in check. 

This, however, is not the only way in which arsenic acts, for pure 
cultures of the Azotobacter have been obtained from these soils, and 
it is found that these are so stimulated that they bring about greater 
changes in the presence of arsenic than they do in its absence. This 
is due to the action of the arsenic upon these minute specks of living 


122 INFLUENCE OF ARSENIC ON BACTERIAL ACTIVITY 


protoplasm, causing them to utilize their food more economically 
in the presence of arsenic than in its absence. This is similar to the 
influence of the arsenic upon the cells within the horse. 

Other experiments have demonstrated that the addition of arsenic 
to a soil increases the liberation of the insoluble plant-foods of the 
soil, especially of the phosphorus. Thus arsenic by various means 
stimulates all the bacterial activities of the soil, and these increased 
activities, as experiments have shown, are reflected in greater crops. 
This increased growth must be looked upon as due to a stimulus 
and not to the direct nutritive value of the substance added. Soils 
so treated would produce larger crops and wear out more quickly 
than would untreated soils. It is interesting and important to know 
that arsenic has to be applied to a soil in enormous quantities before 
it retards microscopic plant life, and probably before it retards the 
growth of higher plants. 

The data available prove conclusively that the arsenical com- 
pounds, with the single exception of Paris green, stimulate the 
nitrogen-fixing organisms of the soil and that this influence varies 
qualitatively but not quantitatively with the various soils. The 
results also bring out the fact that both the anion and the cation of 
the compounds have a marked influence upon the growth of the 
organisms. With some compounds both the anion and cation act 
as stimulants, but with other compounds one stimulates and the 
other retards. It is likely that little or no influence is exerted upon 
the nitrogen-gathering organisms by the sodium of sodium arsenate 
and that the stimulating influence noted with dilute solutions and 
the toxic influence exerted with more concentrated solutions are due 
entirely to the arsenic. It is rather likely that the stimulating 
influence which Riviere and Bouilhac have found sodium arsenate 
to have upon wheat and oats is an indirect effect which is exerted 
upon the bacterial flora of the soil and which in turn influences the 
yield of the various grains. 

Both the anion and cation undoubtedly act as stimulants in the 
lead arsenate. Stoklasa has shown that lead when present in soil 
stimulates the growth of higher plants. This he ascribes to the 
catalytic action of these elements on the chlorophyll. The results 
reported indicate that it is due to the influence of the compounds 
upon the biological transformation of the nitrogen in the soil. The 
fact that the lead plays no small part in the stimulating influence 
is borne out by the work of Lipman and Burgess who found lead to 
stimulate nitrifying organisms. 

Paris green is toxic to the nitrogen-fixing organism in the lowest 
concentration tested. This is due to the copper and not to the 
arsenic, as it is-well known that the copper ion is a strong poison 
to many of the lower plants. Brenchley found it to be toxic to 
higher plants when present in water to the extent of one part in 


HOW DOES THE ARSENIC ACT . 123 


4,000,000,000. Although Russell states that it is not as toxic in 
soil as in water, Darbishire and Russell found it to be toxic in soils, 
and they failed to get a stimulating influence with it. Monte- 
matini has noted a stimulation with copper sulphate when used in 
dilute solutions. This, however, may have been due to the anion 
and not to the cation, as sulphates do stimulate plants by their action 
on insoluble constituents of the soil. The same interpretation could 
be placed upon the results obtained by Lipman and Wilson and also 
those reported by Voelcker in which they noted a stimulation with 
copper salts. Clark and Gage have found that very dilute solutions 
of copper have an invigorating influence upon bacterial activity. In 
order that the stimulation may be noted the copper must be present 
in small quantities. Jackson found that 1 part of copper sulphate 
in 50,000 parts of water kill Bacillus coli and Bacillus typhosus. 
Kellermann and Beckwith found that the common saprophytic 
bacteria are more resistant to copper than is B. colt. There is con- 
siderable evidence that copper stimulates the ammonifying and 
nitrifying organisms of the soil, but these results show the nitrogen- 
fixing organisms of the soil to be very sensitive to copper, and if it 
is to act as a stimulant it must be in extremely dilute solutions. The 
toxicity of the copper in the Paris green is great enough in the 
dilution of 10 parts in 1,000,000 to offset the great stimulating 
influence of the arsenic in combination with it. 

The marked stimulating influence noted where the arsenic trisul- 
phid is used is very probably due to the stimulating action of both 
the arsenic and sulphur. Demolon attributed much of the fertilizing 
action of sulphur to its action upon bacteria, and Vogel found that 
sulphur decidedly increased the activity of the nitrogen-fixing organ- 
isms. The results which Russell and Hutchinson obtained with 
calcium sulphid are interesting in this connection. They found that 
after thirty days there were five times as many organisms in a soil 
to which calcium sulphid had been added as in an untreated soil, and 
the yield of ammonia and nitrates in the same length of time was 
one-third greater in the treated soil than in the untreated soil. This 
in turn reacts upon the crop harvested, as shown by Shedd. 

The first part of the curve for zinc arsenite nearly coincides with 
that of sodium arsenate, save that zinc arsenite stimulates in greater 
concentrations than does sodium arsenate. This is partly due to 
the difference in solubility of the two compounds, but there is 
another factor—that the zine also acts as a stimulant. Latham 
found that small quantities of zine stimulated alge. The same 
results have been obtained by Silberberg in working with higher 
plants. Ehrenberg concludes that zine salts are always toxic when 
the action is simply on the plant, but that they may lead to increased 
growth through some indirect action on the soil. He found that zine 
stimulated plant growth in soils, but when the soil was sterilized the 


124 INFLUENCE OF ARSENIC ON BACTERIAL ACTIVITY 


zinc became toxic. Lipman and Burgess have shown that it stimu- 
lates the nitrifying organisms and that the influence is shown in the 
crop yield. ‘The great variation in the results reported by the differ- 
ent investigators for zinc, arsenic, and lead is probably due to the 
fact that it modifies the bacterial flora of the soil. When heated 
soil or water cultures are used a different result is noted. This, 
however, is not the only factor, for these results show a marked 
difference in soil and in water culture. The lead arsenate stimulates 
the nitrogen-fixing organisms when placed in soils but becomes 
highly toxic to the same organisms when placed in nutritive solutions. 

The difference is due in part to adsorption by the soil, but this 
would have to be attributed to the silica compounds of the soil, for 
the nitrogen-fixing organisms are stimulated by arsenic in quartz 
sand. free from organic colloids. In this case the arsenic becomes 


PULLIGREUIS OF N FIXED 


QAWS /NCUSGATLO 


Fia. 18.—Graph showing the effect of aération on the nitrogen-fixing activity of soil- 
containing compounds of arsenic. (Soil Science.) 


concentrated at the surface, layers of the silica leaving the inner 
part of the water film comparatively free from arsenic, in which 
the micro6érganisms multiply and carry on their metabolic processes. 
This being the case, it is probable that a water solution weak enough 
to stimulate bacteria could be found. A great difference, however, 
between the solution and the sand-culture method is the greater 
aération in the latter than in the former. That the aération of a 
cultural medium does play a great part in determining the activity 
of the nitrogen-fixing powers of a soil is strikingly brought out in 
Fig. 18. 

It is remarkable how the aération of the soil or the filtering of the 
soil extract can prevent the high loss of nitrogen which is noted at 
first in the unaérated soil. This cannot be attributed directly to the 
denitrifying organisms; otherwise, it would not be removed by filtra- 


LAPLLANATION 
FULTERED SOL. SOLUTION, (VO LEAD pepper teary 
, SU SLUT 728 Bh LEAD 
—20, 
(-) 20 dO 60 gO 100 120 /IO fons os 


4 


HOW DOES THE ARSENIC ACT 125 


tion. The graphs also bring out the fact that adding arsenic and 
filtering the soil only shift for the time the equilibrium within the 
soil, which later tends to regain its old equilibrium. This is a condi- 
tion that coincides well with what would be expected if the limiting 
element were some other microscopic forms of life. The filter 
would not separate them quantitatively, and it is possible that the 
arsenic has only a selective influence. Later, many of the organ- 
isms become accustomed to its presence; or, what is more likely, the 
arsenic becomes fixed within the soil. 

That this limiting factor is a thermolabile body is brought out 
more clearly by Fig. 19. The quantity of nitrogen fixed by the 
unheated soil receiving no arsenic has been taken as 100, the heated 
soil with and without arsenic being compared with this. 


320 % 


300 % 


280% 


260 % 


240 % 


220 % 


200 % 


180 


160% 


140 % 


120% 


100% 


NOT 50> 55> 60° 65° 70° 15° 80° 85° 
HEATED 


Fic. 19.—Graph showing the effect of the heat on the nitrogen-fixing power of soil 
treated and not treated with arsenic. 


The heating of the soil extract to 50° C. for fifteen minutes has 
exactly the same influence measured in terms of nitrogen fixed as 
does 0.0728 gm. of lead arsenate. The stimulating influence of heat 
is noted even in the presence of arsenic and reaches its maximum 
effect in the absence of arsenic at 60°, and in the presence of arsenic 
at 65° C. Above these temperatures there is a decline in the nitrogen 
fixed. Even soils inoculated with solutions which had been heated 
to a temperature of 85° fixed nitrogen; at least there is more nitrogen 
accumulated in such soil than in that inoculated with the untreated 
soil solution. The results indicate that many of the organisms which 
take part in the gathering of nitrogen in soils are very resistant to 
heat. It is also significant that the greatest stimulating influence 


126 INFLUENCE OF ARSENIC ON BACTERIAL ACTIVITY 


is exerted in soil which had been inoculated with solutions heated 
just above that point which Cunningham and Loéhnis found to be 
the thermal death point of soil protozoa. 


REFERENCES. 


Greaves, J. E.: The Occurrence of Arsenic in Soils (Bichem. Bull., 1913, ii, 519- 
523). 

Greaves, J. E.: Some Factors Influencing Ammonification and Nitrification in 
Soil (Centr. f. Bakt., Bd. xxxix, Abt. II, 1913, 542-560). 

Greaves, J E.: Stimulating Influence of Arsenic upon the Nitrogen-fixing Organ- 
isms of the Soil (Journal Agricultural Research, 1916, vi, 389-416). 


CHAPTER XILT, 


EFFECT OF HEAT AND VOLATILE ANTISEPTICS ON 
SOIL BACTERIA. 


SoIs are often heated, steamed, or treated with volatile or non- 
volatile antiseptics both for experimental and practical purposes. 
The process is not sufficient to destroy all forms of life within the soil. 
It only destroys some of the weaker species and the aim is usually to 
destroy an injurious species. Yet the process is often referred to 
as sterilization. In view of the fact that they fail to render the soil 
sterile, some workers prefer the terms partial sterilization or pas- 
teurization, which more accurately describe the process. 

Although it was well known that the kiln-burning of clay produced 
a far-reaching chemical and physical effect, yet soil investigators 
considered that the process of sterilization produced no change 
either in the mechanical nature or chemical composition of a soil 
until the work of Frank appeared in 1888. He found that heated 
soils contained a great deal more soluble matter than unheated soil, 
peaty soils containing more than twice as much and heated sandy 
soils not quite twice as much. This increased soluble matter he 
considered sufficient to account for the increase in crops which was 
‘often found to follow the heating of a soil. 

A great impetus was given to the work in 1894 by Oberlin in 
Germany and Girard in France who found that the application of 
carbon bisulphid increased the crop-producing power of the soil. 
Oberlin found that vineyards treated with carbon bisulphid to kill 
phylloxera showed greatly increased productivity after the treat- 
ment, and he founded on this his system of grape culture, where 
fallowing and rotation could be dispensed with in the resetting of 
vineyards. Girard noticed that soil treated with carbon bisulphid 
for the purpose of combating a parasitic disease of sugar-beet was 
more productive than it was before such treatment. The beneficial 
influence of the treatment extended even into the second year. 
These facts stimulated investigation and created much discussion, 
particularly as to the manner of its action. No working hypothesis 
was, however, formulated until 1899 when Koch announced his 
direct “stimulation theory,” since which time numerous theories 
have been formulated to account for the noted phenomena. 

Influence on Plant.—'The use of carbon bisulphid at the rate of 2904 
pounds an acre resulted in a gain of 15 to 46 per cent. in the yield 


128 EFFECT OF HEAT ON SOIL BACTERIA 


of wheat grain and of 21 to 80 per cent. in wheat straw. The yield 
of potatoes was similarly increased by 5 to 38 per cent. and that of 
beets from 18 to 29 per cent. Although the yields of the legumes 
were not always increased, yet some fields of clover treated with 
carbon bisulphid gave increases of 119 per cent. 

Wollny clearly showed that the application of carbon bisulphid to 
a soil within the growing season may lead, according to the amount 
introduced, to a complete destruction of the growing crop, or to 
a temporary retardation merely, involving a greater or slighter 
depression in the production of plant substance. Its application 
several months before planting increases the fertility of the soil 
to a considerable extent. This influence is felt, according to the 
amount of carbon bisulphid used, through one or several growing 
seasons, after which if no manure or fertilizer has been applied a 
marked decrease in the yields becomes evident. 

There was the dark green color and the vigorous development of 
the plants together with the decided tendency of grain crops to 
lodge just as if too great quantities of nitrogen were at their disposal. 
These facts led Heinze to conclude that on the whole we must seek 
the cause of the beneficial effect of carbon bisulphid on the soil in 
the enormous increase of soil organisms at the proper time, thus: 
rendering available, or possibly increasing, the nitrogen supply to 
growing plant. 

The large amounts of nitrogen thus made available to the crops 
are derived partly from the soil and partly from the atmosphere. 
Kruger and Heinze not only demonstrated that soils treated with 
carbon bisulphid showed an increase in their total nitrogen content, 
but also that the increase was the result of the more vigorous growth 
of the nitrogen-fixing Azotobacter species. This, Heinze considers, 
resulted from the initial suppression of amid-ammonia formation 
and nitrification which would create favorable conditions for the 
development of nitrogen-fixing flora. Later there would be more 
intense transformation of the bacterial proteins and of other nitrog- 
enous organic substances into amino- and ammonia compounds 
which would result in a more vigorous nitrification, thus placing 
at the disposal of the plant an abundant and uniform supply of 
soluble nitrogen compounds. The various organic materials in the 
soil—such as plant residues, pectins, pentosans, humic substances, 
and the like, together with the rapid growth of algee and molds— 
may furnish the carbon food for the Azotobacter species. 

Effect on Properties of Soil.— Egorow, who investigated the effects of 
carbon bisulphid upon the physical properties of the soil, found that 
(1) the capillary rise of water in the soil treated with carbon bisul- 
phid to be slower than in the untreated; (2) the moisture content 
is reduced considerably, especially in peaty soils; and (3) the water- 
holding capacity of the soil is decreased. Thus, he concludes that 


EFFECT ON PROPERTIES OF SOIL 129 


the treatment of soils with carbon bisulphid acts unfavorably upon 
the water content of the soil. 

Other characteristic effects of treatment with volatile antiseptics 
reported by yarious investigators are: 

1. An initial decrease in the number of bacteria followed by a 
long-continued increase. A careful piece of experimentation illus- 
trating this is that of Fred who used loam soil (mixed with sand) 
and found that 2 per cent. carbon bisulphid has little effect upon 
the moisture content of the soil. With varying percentages of ether 
(together with 2 per cent. of sugar) in the soil, he finds an initial 
depression in bacterial numbers followed by a considerable increase 
in eight hours, 4 per cent. giving the maximum count. 

2. A disturbance of the equilibrium of the bacteria, by which 
certain types multiply more rapidly than others. Hiltner and 
Stérmer found that under normal conditions there is a certain 
equilibrium established among the various groups of soil bacteria, 
and that the organisms capable of growing on meat extract gelatin 
are composed of Streptothrix species 20 per cent., gelatin-liquefying 
species 75 per cent., and the non-liquefying species 5 per cent. 

When carbon bisulphid is applied to a soil, its bacterial inhabitants 
are injured, though not completely destroyed, the injury varying 
with the changing conditions of temperature, moisture, and amount 
of carbon bisulphid applied, as well as with the duration of its action. 
Not all of the bacterial species are depressed in their development 
to an equal extent, the injury being most pronounced in the strepto- 
thrix species and least pronounced in the gelatin-liquefying species. 
The depressing action of carbon bisulphid disappears after a shorter 
or longer interval and is followed by a rapid multiplication of the 
microdrganisms in the soil. The equilibrium having been destroyed, 
however, the new development follows along different channels, 
and there occurs not only an enormous increase in the total number 
of soil bacteria, but also an abnormal predominance of certain 
species. The new conditions thus established for a time favor a 
more ready utilization of the stores of soil nitrogen, and likewise the 
fixation of atmospheric nitrogen by certain bacterial species. These 
conclusions are borne out by the work of Lipman and Brown who 
examined abnormal soil after applying carbon bisulphid in various 
quantities alone, and in combination with muriate of potash and 
acid phosphate. They then determined the ammonifying, nitrifying, 
denitrifying, and nitrogen-fixing powers of the soil. They concluded 
that in normal soil flora the different groups occur in fairly definite 
relations which are evidently disturbed by the addition of carbon 
bisulphid, which, destroying the bacterial equilibrium prepares the 
way for an entirely new bacterial development whereby certain 
species become far more prominent than previously. This applies 
especially to the nitrifying and nitrogen-fixing bacteria. 

9 


130 EFFECT OF HEAT ON SOIL BACTERIA 


3. A slight initial increase in ammonia content, followed by a 
considerable increase in the production of ammonia. This, although 
noted by the majority of workers, is especially brought out by the 
work of Russell and Hutchinson, as is illustrated by the following 
results from their work: 


Nitrogen pres- | Nitrogen present | Total nitrogen present as 


| 
At | After Atbegin- After Atbegin- After | Gain in 


a 23 days.| ning. |23days.| ning. | 23 days. | 23 days. 


Untreated soil vi gttee 
Soil heated 2 hours at 98°C. 
Soil treated with toluene 
which was then evapor- 
Btodyibs th a he ip Re Ci eUOAl ears | 1270 12.0 WO | 39.8 22.8 
Soil treated with toluene | | 
which was not removed. | 7.2) 14.5 Hike(D, | Oso 18.2 | 45.5 6.3 


or oo 


aie NA 16.0 13.8 WIZ. 3.9 
8 13.0 | 12.0 19.5 | 55.8 | 36.3 


aor 


4. A depression of the processes by which ammonia is converted 
into nitric acid, and a very slow recovery of the activity of the bac- 
teria concerned, as a result of which ammonia accumulates in the 
soil. Warington, in his early investigation on the biological nature 
of nitrification, observed that when air containing carbon bisulphid 
was passed through the soil the process was inhibited, whereas C. 
de Briailles noted that during the winter the carbon bisulphid seemed 
to exert a harmful influence on the accumulation of nitrates. How- 
ever, with the first open weather in spring the reverse seemed to be 
true—the carbon bisulphid caused a marked increase in nitrates over 
the untreated. : 

5. An increase in the rate at which oxidation takes place in the 
soil. In a study of oxidation in soils and its relationship to produc- 
tiveness, Darbishire and Russell found that the absorption of 
oxygen by soil is mainly brought about by the action of micro- 
organisms and is greatly diminished if the soil has been previously 
heated to 120° C. When heated to 95° C., it was found that the 
rate of oxidation on a sand, two loams, and a chalky soil, instead of 
being reduced was considerably increased, as was the case after 
treatment with and removal of volatile antiseptics, such as toluene, 
chloroform, carbon bisulphid, and other volatile antiseptics. 

The rates of oxidation of heated soils were as follows: 


Milligrams of oxygen absorbed in 


days. 6 days. 9 days. 
Hop garden soil, unheated : aie ff 5.2 7.0 
Hop garden soil, heated to 95° C. 6.0 8.2 12.0 
Garden soil, unheated A te a ke ice 7.5 10.2 15.5 
Garden soil, heated to 95°C. . 16.9 2022 BBE 


EFFECT OF PROPERTIES OF SOIL 1351 


6. It has been repeatedly demonstrated by many workers that 
both heat and antiseptics destroy all or part of the protozoa found 
in the soil, depending on the degree of heat applied or the strength 
of antiseptic used. . 

7. Some workers have found antiseptics and heat to depress 
denitrification in soil. Both Wagner and Morgan found that carbon 
bisulphid kills denitrifying organisms. 

8. Especially significant is the fact that there is a considerable 
increase in the soluble matter in the heated soil, not only of inorganic 
matter, as phosphorus and potash, but even more in the organic 
matter made soluble. Stoklasa holds that the plants are able to get 
more phosphate-ions from a soil as a result of the disintegration of 
the bacteria killed by the treatment with carbon bisulphid. 

Fred found that the application of carbon bisulphid to a soil 
increases the insoluble compounds of nitrogen and sulphur as well as 
the bacterial activities. Lyon and Bizzell determined the effect of 
sterilizing soils by steam on the water-soluble material and found 
that steaming the soil at two atmospheres reduced the nitrates to 
nitrites and ammonia, but that most of the ammonia is formed from 
organic nitrogen in the soil. 

9. Although the majority of workers report an increase in nitrogen 
fixed in a soil treated with carbon bisulphid, yet Koch reports cases 
in which carbon bisulphid added to a soil containing fairly large 
quantities of cane sugar has resulted in a weakening rather than in 
a strengthening of their nitrogen-fixing powers. The increase in 
nitrogen fixation may at times be very pronounced, as may be 
seen from the following experiments in which tumblers containing 
soil were all carefully sterilized and half of them placed in the 
incubator in the sterile condition. To the others was added a soil 
extract prepared by shaking one part of soil with two parts of sterile 
distilled water for three minutes. After standing for about five 
minutes the liquid was decanted and 10 c.c. portions were used to 
inoculate the soil. Before inoculating, this extract was placed in 
thin-walled test-tubes in 10 ¢.c. portions and then kept at the 
required temperature for exactly fifteen minutes before adding to 
the soil. The moisture content was made up to 18 per cent. and 
the whole incubated for twenty days. The milligrams of nitrogen 


fixed under the varying treatments were as follows: 
Milligrams 


Temperature of soil extract (° C.). nitrogen-fixed. 
ENO CTT Nanette oe Nee oN tte! eT RE MED el eh SS veh mes Be ital 
50 Be ee ERT te) sa ord e tate See PL, | me eine Wat 9.00 
55 Ac 2G aoe PRE tire: Wire etree <A Otk kee Ma a ee, AA 
60 i: PEI PET, ics ae man Er, we ee. tl gee Geom os 
65 Fe eRe ee Tee CPR tae Fk gh ak Bl ee a Be ae a ait lle She oe 
70 Sra MESS te ee ee ee ees hp tees Ble a Oy eh A LoniOe 
75 ip hats § Scary tea sa eg CR ec AEP gt SUN Ren 2 tear RE Se Per ae Oe 
80 Sea seme oe ene oie le Oe a Stee a camera et tos Te es et Bre TOO G 


Sc) SP EM tcc Aare een aroun Peete ae ed ts Tate. ge gn OKO 


132 EFFECT OF HEAT ON SOIL BACTERIA 


10. Although strongly contested by many experimenters, there 
are some workers who have produced strong evidence that the heat- 
ing of a soil destroys toxins which have been formed in it due either 
to bacterial activity or to plant growth. Fletcher grew plants in 
a nutrient solution until he considered it to be made toxic by plant 
excreta. He then steamed it at a pressure of 150 pounds for two 
hours. Flakes of an organic substance were thrown down, which 
did not dissolve on removal of pressure. This substance he con- 
sidered to be the toxic excreta. 

The principal changes produced in and by sterilizing soil are thus 
summarized by Johnson: 

I. Destruction of Life: 

(a) Normal soil flora and fauna, desirable and undesir- 
able forms of bacteria, fungi, protozoa, and higher 
animals. , 

(b) Plant parasites, especially pathogenic bacteria, fungi, 
nematodes, and injurious soil-infection insects. 

(c) Propagative organs of higher plants, especially weed 
seeds. 

II. Immediate Chemical Action (formation of toxic and bene- 
ficial compounds): 

(a) Decomposition of organic material resulting in the 
formation of ammonia, carbon dioxid, and various 
new and complex organic compounds. 

‘(b) Decomposition of inorganic material, reduction of 
nitrates and nitrites to ammonia and increased solu- 
bility of potassium, phosphorus, and other salts. 

IIT. Biochemical Action: 

(a) Increased ammonification, particularly, and modified 
nitrification, denitrification, and nitrogen-fixation. 

IV. Physical Action: 

(a) Absorption capacity of soil modified for water, gases 
and salts. 

(b) Increased concentration of soil solution. 

(c) Modified capillarity, colloidal state, and mechanical 
condition. 

(d) Modified color and odor. 

VY. Action on Organisms Growing in Sterilized Soils: 

(a) Lower organisms. 

1. Increased development due to reduced competi- 
tion, increased food supply, destruction of 
“bacterio-toxins,” “stimulation,” by products 
added or formed, or other causes. 

2. Retardation in growth in rare cases due to inju- 
rious conditions produced. 


HILTNER AND STORMER’S “INDIRECT” THEORY 133 


(b) Green plants. 

1. Injurious action as indicated by retarded rate 
and percentage of seed germination and by 
retarded rate of plant growth. 

2. Beneficial action as shown by increased rate and 
percentage of seed germination and increased 
rate and amount of plant growth. 

3. Modified in form, color, and other “qualitative” 
changes. 

Hypotheses to Account for Observed Phenomena.—A number of 
hypotheses have been formulated to account for the increased plant 
growth and for the many changes produced in soils by treatment 
with heat and volatile antiseptics. A number of these theories 
are considered, but it must be borne in mind that there is a wide 
disagreement among workers as to the real cause. No single 
hypothesis yet formulated can be said to fully account for all of the 
observed phenomena. 

Koch’s ‘‘Direct Stimulation” Theory.— The first theory offered to 
account for the increased yield obtained from soils treated with an 
antiseptic was the “direct stimulation” theory advanced by Koch 
in 1899. He considered carbon bisulphid to have a direct stimulat- 
ing effect on the plants themselves. He later found ether to have 
a similar effect. In experiments dealing with the addition of ether 
to the soil Koch found that the increased yield was pronounced on 
the first crop, whereas the residual effect was slight, as with carbon 
bisulphid the beneficial effect increases with the amount of applica- 
tion. He further found that soils sterilized with heat produced 
better crops when treated with carbon bisulphid than when not so 
treated and concludes that the effect of the antiseptic, therefore, 
cannot be due to its effect on bacteria. 

The theory of Koch has been supported by Fred who fertilized 
soil with an abundant supply of sodium nitrate and found that in 
every case in which carbon bisulphid was added the growth and 
yield of crop were much superior to those in the corresponding pots 
not treated with that substance. He concludes that as there was no 
lack of plant-food and other conditions were favorable to plant 
growth, the effect of the antiseptic must have been directly upon the 
plant. There is ample evidence to prove that many of these anti- 
septics in dilute solutions stimulate the plants directly, yet there 
is no evidence which will substantiate the claim that this is the only 
or even the principal influence on the plant and soil. 

Hiltner and Stormer’s ‘‘indirect’’ theory of antiseptic action is 
outlined by them as follows: 

“1. By destroying the existing bacterial equilibrium in the soil, 
the carbon bisulphid opens the way for an entirely new bacterial 
development. This is achieved through the unequal retardation 


134 EFFECT OF HEAT ON SOIL BACTERIA 


in the growth of the different groups of bacteria. Hence, certain 
groups become disproportionately prominent, while others are 
almost entirely suppressed. 

“9. The rapid increase in the numbers of the bacteria is followed 
by a more intense transformation of plant-food substances. Decom- 
position and fixation processes result in an accumulation of readily 
available nitrogen compounds utilized by the crops. Hence, the 
action of carbon bisulphid is in the nature of nitrogen action. 

“3. The initial suppression of the nitrifying species becomes of 
advantage in that the nitrogen compounds, simplified by other 
species, are prevented from being rapidly changed into nitrates 
and being leached out of the soil. 

“4. The more or less permanent suppression of the denitrifying 
organisms must be regarded as an additional factor favoring plant 
growth. 

“The introduction of the poison into the soil at first decimates 
its bacterial flora, but with the disappearance of the injurious 
carbon-bisulphid vapors it also encourages a vigorous and long- 
continued increase of the organisms, resulting in an increase of the 
store of more readily available nitrogen. It is still to be deter- 
mined whether this increase is largely due to the fixation of atmos- 
pheric nitrogen or to the unlocking of the vast store of combined 
nitrogen in the soil. It is most probable, however, that even though 
one of these processes predominates the other is surely more exten- 
sive than it would be in normal soil. The nitrogen thus secured is 
not at once made accessible to the higher plants, but is at first laid 
fast in the bacterial bodies. This assumption would best explain 
the fact that plants growing upon a soil treated with carbon bisulphid 
show retarded growth, even some time after the application of the 
latter, and the explanation hitherto accepted that the injury results 
from the direct action of the poison seems hardly reasonable after 
our discovery that the most intense bacterial activities are asserting 
themselves just at that time. The nitrogen fixed in the bacterial 
bodies is gradually rendered soluble by decomposition processes, 
and thereby made accessible to nitrification and the higher plants. 
Hence, when the carbon bisulphid is applied in the fall, there is 
enough time left until the planting of the following spring crop for 
the mineralization of the bacterial nitrogen. It is quite evident, of 
course, that the nitrogen combined in the bodies of generations of 
bacteria is not all made soluble within a single year, but only in the 
course of several growing seasons, so that we may readily account _ 
for the increased harvests secured for two or more successive years 
after strong applications of carbon bisulphid, even though the bac- 
terial transformations had by that time declined. The exhaustion 
of the soil finally manifesting itself after a shorter or longer time 
may be explained by the deep-seated changes in the bacterial soil 


RUSSELL AND HUTCHINSON’S PROTOZOAN THEORY 135 


flora, which does not return so easily to its normal state. It is quite 
possible that the return to the normal conditions is prevented by 
the exhaustion for years to come of the more available portions of the 
plant nutrients.” 

Evidence corroborating this theory has been brought forward by 
Heinze, Stoklasa, Lipman, and Brown, whereas Sirker furnishes 
evidence in the cultivation of the mulberry which opposes it. He 
found that the addition of carbon bisulphid to a completely fertilized 
mulberry plant increases the vegetation 44 per cent., whereas a 
heavy application of sodium nitrate was of slight value. 

Russell and Hutchinson’s Protozoan Theory.—They consider that 
the microscopic flora of the ordinary arable soil includes a wide 
variety of organisms performing very different functions which 
may be divided roughly into two classes: (a) saprophytes, tending 
to increase the fertility of the soil, for example, producing ammonia, 
fixing nitrogen, and similar changes; and (b) phagocytes and large 
organisms ininiical to bacteria which limit fertility. Between these 
two classes of organisms there is an equilibrium under natural 
conditions, but when partial sterilization takes place the phago- 
cytes are killed but the bacterial spores are not; and subsequently 
the latter develop with great rapidity, since they are freed from the 
attacks of their enemies, and there is an increase not only in 
ammonia but likewise in crop production. 

In support of this theory they point out the following: “In 
untreated soil there is no accumulation of ammonia, whereas the 
‘toluene evaporated’ soil, as well as the soil heated to 98° C., show 
an increased production of ammonia. That this is mainly the work 
of microérganisms is proved by the following considerations: (a) 
The curves belong to the type associated with bacterial, rather than 
with purely chemical activities. (b) Soil which has been heated to 
125° C. (at which temperature all organisms are killed) behaves 
altogether differently; after the first production of ammonia due 
_ to heating there is no further change. (c) If the toluene is left in 
the soil there is only a slow production of ammonia, and never a 
rapid rate; the curve is more nearly linear. The action of micro- 
organisms is here excluded, but enzymes may still act. (d) The 
rapid period sets in only when the soil is sufficiently moist. Thus 
the two significant changes induced by partial sterilization are, 
(1) an increase in the amount of ammonia; and (2) cessation of the 
nitrifying process. 

“It now becomes necessary to determine the part played by 
bacteria, and why they can increase so much more rapidly in the 
partially sterilized soil (which accounts for the increased ammonia 
production) than in the untreated soils. That the comparative 
inertness of the bacteria in the untreated soil cannot be caused by 
any bacterial factor is evidenced by the following considerations: 


136 EFFECT OF HEAT ON SOIL BACTERIA 


(a) If a filtered soil extract containing bacteria from an untreated 
soil is added to a toluened soil, there is an increase in the rate of 
ammonia production, and also in the number of bacteria.  (b) 
However, if untreated soil is added to toluened soil, there is no 
increase, but on the contrary a reduction. (c) As pointed out above, 
an extract of the toluened soil is more active than an extract of 
untreated soil. (d) But when the extract of toluened soil is added 
to the untreated soil there is no increase in ammonia production. - 

“The conclusion drawn is that ‘the untreated soil contains a 
factor, not bacterial, limiting the development of bacteria, this 
factor being put out of action by toluening or heating.’ 

“Having determined the presence of a limiting factor in untreated 
soils an examination of its nature reveals that: (a) it is not a toxin, 
for if it were it would be sure to affect the nitrifying bacteria most; 
(b) barley seedlings grown in aqueous extracts of untreated and 
toluened soils showed no difference in growth over a period of four 
weeks; (c) the limiting factor is probably biological, for when 
untreated soil is added to toluened soil the reduction in the rate of 
ammonia is not at once operative. It is also a large organism, since 
it is only in the soil and not the filtered extract of the untreated soil 
that is effective in reducing the rate of ammonia production in 
toluened soil. An examination of treated and untreated soil was 
made, and the latter revealed the presence of large organisms, 
protozoa, etc., which constitute the factor, or one of the factors, 
limiting the bacterial activity, and therefore the fertility of untreated 
soil. Direct evidence is furnished by inoculating toluened soil or 
soil extract with cultures of large organisms and studying the 
effect produced—which is a consequent depression in the rate of 
ammonia formation.” 

Although accepted by many workers, there are many of what 
appear to be fatal objections that have been brought against this 
theory: (1) It has been demonstrated that the soil contains many 
species of fungi which are capable of producing considerable quan- 
tities of ammonia‘and these would withstand the actions of the anti- 
septic or partial sterilization by heat and may develop later and 
produce large quantities of ammonia. (2) There may be a great 
difference in the physiological efficiencies of the surviving ammoni- 
fiers. (3) The work of Russell and Hutchinson does not consider 
the probability of the protozoa being in soil as cysts. (4) The 
direct laboratory work of Fred and Gainey cannot be interpreted in 
the light of this theory. Kopeloff and Coleman analyze the work 
of Fred in the light of the protozoan theory as follows: 

“In order to test the validity of Russell and Hutchinson’s con- 
clusion that the absence of protozoa (by treatment with toluene) 
is responsible for increased production of ammonia, Fred, using 
ether instead of toluene, subjected one series of flasks containing 


RUSSELL AND HUTCHINSON’S PROTOZOAN THEORY 137 


compost soil to 100° C. moist heat for an hour and used a similar 
series, unheated, as a check. All the flasks received 0.2 per cent. 
ammonium sulphate—some of the flasks received 2 per cent. and 5 
per cent. ether. In order to obtain vigorous nitrification 170 c.c. 
of amebe-free extract was inoculated into all the flasks. (This 
was prepared by leaching 2 kg. of compost soil and 4 liters of sterile 
water and filtering through filter paper; the microscopic examination 
revealed the presence of no amebze.) 

“The analyses for nitrate nitrogen were made at the beginning 
of the experiments and at the end of 100 and 150 days, respectively; 
the results showed that heating the soil to remove amebe did not 
have a beneficial effect upon nitrate formation, contrary to Russell 
and Hutchinson’s work—although the addition of a small amount 
of ether increased nitrification in the flasks containing amebze, and 
had the opposite effect in the soil free from amebe. This, the 
authors believe, may be accounted for by the stimulating effect 
upon the nitrifying bacteria, since the heated soil not treated with 
ether showed no such increase. 

“Fred concludes (in addition to the above mentioned observa- 
tions) that ether and carbon bisulphid cause an increased fixation of 
nitrogen in pure cultures of Azotobacter. The development of deni- 
trifying organisms is hindered for only a short time, because of 
treatment with antiseptics. Both Azotobacter and denitrifying 
organisms are insignificant in a normal soil. Nitrification is at first 
inhibited and later accelerated by antiseptics, while toxins remain 
unaffected by treatment. Carbon bisulphid and ether cause an 
increase in crop yield under sterile conditions. 

“He holds that the increased growth of plants following the use 
of antiseptics in the soil depends essentially upon the stimulation 
to the plant itself, in combination with a similar effect on the lower 
organisms. 

“Fred’s work is highly suggestive, but the determination of 
nitrogen produced is in the form of nitrates alone, and no data are 
set forth concerning ammonia. That this might affect his results is 
evident when one takes into consideration the fact that most investi- 
gators have proved that nitrification is depressed by antiseptics. 

“Furthermore, like many other experimenters he does not con- 
sider the possibility of protoze cysts passing through the filter 
paper in the preparation of ‘ameb-free extract.’ And we have 
found in our experimental work that cysts do pass through several 
thicknesses of high-grade filter paper. 

“Tn much the same manner, Gainey concludes that investigations 
relative to the effect of toluol and carbon bisulphid upon the micro- 
flora and fauna of the soil, that: (a) small quantities of carbon 
bisulphid, toluol, and chloroform, such as have been used practically 
and experimentally, when applied to soils studies, exert a stimulative 


138 EFFECT OF HEAT ON SOIL BACTERIA 


rather than a diminishing effect upon the total number of bacteria 
present; (b) an application of such quantities of toluol and carbon 
bisulphid does not have an appreciable effect upon the number of 
types of protozoa present; (c) a very marked increase in yield may 
be noted following such an application when no evident change 
occurs in the total number of bacteria present.” 

Greig-Smith’s Bacteriotoxin Theory.—A widely different theory 
from any so far considered is that advanced by Greig-Smith. He 
considers that when disinfectants are added to the soil their action 
is two-fold: They kill the less resistant bacteria and dissolve from 
the surface of the soil particles a waxy covering to which he has given 
the name “agricere.”’ The surviving bacteria, which he assumes 
are the beneficial ones, are then able to function much more rapidly 
on account of the exposure of the food due to the removal of the 
“agricere.” 

Moreover, he considers that there is a toxin contained in the soil 
which is soluble in dilute saline, partially destroyed by heating to 
94° C., and rapidly decayed in aqueous solution; boiling water 
converts it into a nutrient, or by destroying the toxin enables the 
nutrients dissolved in the saline to act. Thus, heating the soil 
destroys the bacteria toxin, which accounts for enhanced fertility. 
Bottomley and others also claim to have found soluble bacterio- 
toxins in soils. Russell and Hutchinson, on the other hand, obtained 
wholly negative results, and conclude that soluble bacteriotoxins 
are not normal constituents of soils, but must represent unusual 
conditions wherever they occur. Not only could no experimental 
evidence of the existence of bacteriotoxins be obtained, but Russell 
and Thaysen showed that the assumption of toxins leads to difficul- 
ties. It is necessary to suppose that heating fresh soil for fifteen 
minutes is sufficient to produce toxins but not to destroy them, 
whereas heating for sixty minutes both produces and destroys them, 
and in the case of air-dried soils fifteen minutes’ heating causes their 
decomposition. 

REFERENCES. 


Vorhees, E. B. and Lipman, J. G.: A Review of Investigations in Soils Bacteri- 
ology (U.S. D. A. Off. Exp. Sta. Bul. 194). 

Lohnis: Handbuch der Landwirtschaftlichen Bakteriologie. 

Kopeloff, Nicholas and Coleman, D. A.: A Review of Investigations in Soil Pro- 
tozoa and Soil Sterilization, Soil Science, 1917, ili, 197-269. 

Johnson, James: The Influence of Heated Soils on Seed Germination and Plant 
Growth, Soil Science, 1919, vii, 1-103. 


CHAPTER: XIV. 


THE INFLUENCE OF SALTS ON THE BACTERIAL 
ACTIVITIES OF THE SOIL. 


Sats that occur naturally in soils and those applied to them in 
various operations influence the number, species, and activity of 
the soil microflora. These factors are in turn reflected by yields 
obtained. Some substances applied to a soil serve as food for the 
growing plant; others increase plant growth but not through the 
direct furnishing of food. This latter effect may be due to a change 
brought about by the salt on the physical, chemical, or bacterial 
properties of the soil. The substance may alter the physical proper- 
ties of the soil to such an extent that the bacterial flora is modified; 
this in turn may increase or decrease the crop produced upon the 
soil. Other substances may react chemically with constituents 
within the soil and in so doing liberate substances which can be 
directly utilized by the growing plant. Again, they may directly 
modify the microflora and microfauna of the soil both as to numbers 
and physiological efficiency. In some cases all three changes may 
be wrought by the same salt. The question, therefore, arises as to 
what effect this or that fertilizer or soil amendment is going to have 
upon the bacterial activity of the soil. Furthermore, there are 
millions of acres of land in arid America which contain varying 
amounts of soluble salts. Some of these soils contain such large 
quantities of these so-called “alkalies” that no vegetation is found 
upon them. Other soils contain only a medium amount of soluble 
salts and the vegetation is composed chiefly of alkali-resisting plants. 
Still other soils contain much smaller quantities of soluble salts and 
they become injurious only when the soil is improperly handled. 
The reclaiming of the heavily charged soils and the maintaining of 
the others in a productive condition can be carried on successfully 
only when we understand the influence of salts upon the growing 
plants and their action upon the biological, chemical, and physical 
properties of the soil. 

Calcium Carbonate.—Much work has been done to determine the 
influence of calcium carbonate, especially when applied to acid soils, 
on the bacterial content and activity of the soil, but the conclusions 
reached have not always been concordant. Withers and Fraps 
found that calcium carbonate added to a soil greatly accelerated 
nitrification and that it is especially desirable that it should be 


140 INFLUENCE OF SALTS ON THE SOIL 


added where ammonium sulphate is being used as a fertilizer. Lip- 
man’s work showed that calcium carbonate stimulated nitrification 
more than did gypsum, that sodium chlorid was injurious to nitrify- 
ing organisms, and that ferrous sulphate in amounts from 10 to 100 
mg. per 100 gm. of soil was without effect. Later, he and Brown 
decided that both ammonification and nitrification were promoted 
by magnesia lime to a more marked extent than they were by non- 
magnesia lime. This, however, was to a certain extent dependent 
upon the treatment and crop growing on the soil. Both ammonifi- 
cation and nitrification were accelerated by sodium nitrate. In a 
more recent work Lipman, Brown, and Owen found that smali 
applications of calcium carbonate stimulated bacterial activity, 
whereas large applications had a detrimental effect upon ammonifi- 
cation. 

In Owen’s experiments, magnesium carbonate was more efficient 
in promoting ammonification and nitrification than was either 
calcium or potassium carbonate. According to Engberding ammo- 
nium sulphate, sodium nitrate, potassium nitrate, and caustic lime 
all increase the bacterial content of the soil, but decrease its nitrogen- 
fixing powers. Kruger’s work indicated that calcium carbonate 
was more effective in promoting nitrification than was lime, the 
reverse being true with regard to the putrefactive bacteria. The 
formation of ammonia from peptone was especially favored by 
calcium carbonate. Lyon and Bizzell found that lime favored 
nitrification, as did also certain nodule-bearing legumes. Fischer 
concluded that the presence of calcium carbonate in a nutritive 
solution favored the formation of protein nitrogen, but magnesium 
carbonate lessened the transformation of ammonia into protein 
nitrogen. Calcium oxid, however, exerted a much greater influence 
upon soil bacteria than did calcium carbonate. 

Kellermann and Robinson’s results are of especial interest as 
they indicate that magnesium carbonate, applied in amounts 
exceeding 0.25 per cent. to a soil fairly high in magnesium carbonate, 
positively inhibited nitrification, whereas calcium carbonate up to 
2 per cent. favored it, thus indicating that the lime-magnesia ratio 
is of great importance with regard to bacteria as well as the higher 
plants. These results have been confirmed by C. B. Lipman and 
Burgess in whose experiments magnesium carbonate was highly 
toxic both in soil and in solution to Azotobacter chrodcoceum, while 
calcium carbonate was never toxic even in quantities up to 2 per 
cent. Furthermore, calcium carbonate exerted a protective influ- 
ence against the toxic properties of magnesium carbonate. The 
optimum ratio varied, depending upon the medium. : 

Peck studied the influence of a number of salts upon bacterial 
activity when applied to the soil, with the result that the carbonate, 
sulphate, and phosphate of calcium were found to stimulate ammoni- 


LIME 141 


fication, while sodium nitrate depressed it; both potassium sulphate 
and calcium carbonate accelerated nitrification in soil. Brown, 
working with a typical Wisconsin drift soil, found that the applica- 
tion of ground lime up to 3 tons an acre increased the number of 
bacteria in the soil, and also the ammonifying, nitrifying, and nitro- 
gen-fixing powers of the soil. The increase was in every case nearly 
proportional to the limestone applied. 

At times the increase noted in ammonification is due to the 
retention of the volatile ammonia by the carbonate, as is shown by 
Lemmermann’s results where the addition of calcium carbonate to a 
soil up to 1 per cent. reduced the volatilization of ammonia, but 
calcium oxid had the opposite effect. Both calcium chlorid and 
calcium sulfate reduced the loss of ammonia, but the chlorid was 
the only salt of magnesium tested which had this effect. Potassium 
and sodium chlorids, sulphates, and carbonates all reduced the 
absorptive powers of the soil. Paterson studied the influence of a 
number of substances upon nitrification with the result that caustic 
lime was found practically to stop all nitrification. Calcium car- 
bonate promoted it, as did also magnesium carbonate; gypsum was 
less effective, while ferric hydrate had a decidedly favorable effect. 
Sodium chlorid, on the other hand, had a distinctly injurious effect. 

Kelley studied the effect of calcium and magnesium carbonate 
alone and in combination upon ammonification and _ nitrification. 
In his work calcium carbonate only slightly stimulated ammonifi- 
cation of dried blood, but it had a marked stimulating effect upon 
nitrification. The magnesium carbonate was found to be toxic 
to both groups of organisms. No antagonism was found to exist 
between calcium and magnesium. Later, when working with 
Hawaiian soils, he reports a stimulation for both. The results, 
however, varied with different soils, and he considers the lime- 
magnesia ratio of little importance as regards the ammonifying 
and nitrifying organisms. Allen’s conclusion is that large quantities 
of limestone must be applied to a non-caleareous soil in order to 
bring its nitrifying powers up to those of natural calcareous soils. 

Lime.— Peterson and Wollny found that lime increased the carbon 
dioxid given off by soils, and Ebermayer, Hilgard, and Hartwell 
and Kellogg proved conclusively that lime increases the decay taking 
place in a soil. 

Chester showed that lime increased the number of bacteria in 
soil, the increase being proportional to the lime applied up to 4000 
pounds an acre. He considered the effect as being due to the lime 
giving to the soil a more favorable reaction for the growth of bac- 
teria and not to its direct action upon the organisms themselves. 

Lime not only increases the number of organisms in a soil, but 
it increases the ammonifying powers of the soil, as is seen from the 
work of Remy, Ehrenberg, Vorhees, and Lipman, 


142 INFLUENCE OF SALTS ON THE SOIL 


The literature dealing with the influence of lime upon the nitrify- 
ing organisms is so voluminous that no attempt is made here to 
refer to all of it. In most cases the experiments were conducted on 
soils which were acid and the lime supplied neutralized the acidity 
of the soil, thus giving the necessary neutral medium for the action 
of the nitrifying organisms. Such results give little if any idea of 
the direct stimulating or toxic influence of calcium. Furthermore, 
the work has recently been summarized by Brown who concludes 
that the application of lime increased nitrate production from 
ammonium sulphate and dried blood, the gain being almost prepor- 
tional to the quantity of lime applied. This, in turn, was found to 
bear a close relationship to the number of organisms developing on 
synthetic agar. 

Gypsum.—Gypsum is a strong soil stimulant and in most cases it 
greatly increases the crop yield. The beneficial effect may be due 
to its liberating potassium or supplying sulphur for the direct nutri- 
tion of the plant. At other times it may react with the ammonia 
formed by the ammonifying organisms, the ammonium sulphates 
formed being readily nitrified. There is also the possibility that 
calcium sulphate acts as a direct stimulant to the microérganisms of 
the soil. The literature is meager and inconclusive on this phase 
of the subject. 

Severin concludes from his work that gypsum not only prevents 
the loss of ammonia from manure, but it increases the speed of 
decomposition from 10 to 20 per cent., while Paterson states that 
gypsum slightly increases nitrification in soil, as determined by 
laboratory experiments. Prior to this Pichard had shown that the 
sulphates of calcium, potassium, and sodium promote nitrification. 

Opposite results are reported by Dezani who found that gypsum 
in amounts varying from 0.5 to 2 per cent. had no appreciable 
effect on nitrification. The results obtained by Lipman and others 
varied and were inconclusive. 

Calcium Chlorid.— According to Lipman calcium chlorid in solutions 
accelerated the action of ammonifiers. It is interesting to note that 
in a later work he failed to find antagonism between either calcium 
and magnesium or calcium and sodium. The chlorids of calcium, 
magnesium, potassium, and sodium were found to be toxic in the 
order named. Marked antagonism exists between calcium and 
potassium magnesium and sodium, and potassium and sodium. 
Sea water was found to be a physiologically balanced solution for 
Bacillus subtilis. 

Iron Sulphate.—Many writers have made great claims for iron 
sulphate as a fertilizer. A goodly number of these have been made 
by individuals who wished to profit by its sale, but even when these 
cases are ignored there are still many cases in which it has produced 
good results. The composition of the crop usually indicates that 


MANGANESE 145 


iron sulphate influences the phosphorus metabolism of the plant. 
It is hard to see how this is possible unless it be that the iron stimu- 
lates the bacterial activity, which in turn liberates phosphorus 
from its insoluble forms within the soil. It has already been noted 
that Lipman found ferrous sulphate to have but little effect upon 
nitrification, but his results were not conclusive. Guffroy found 
sulphate of iron decidedly beneficial to oats, less beneficial to rye, 
without action on rye-grass, and harmful to wheat. He concluded 
that its action must be due to its influence on the biological processes 
of the soil. According to Paterson and Scott, ferric hydrate has a 
distinctly beneficial effect upon nitrification. In this latter case 
its action could be due to its serving as a base. According to 
Lipman and Burgess the ammonifiers are more sensitive to iron 
sulphate than are the nitrifiers, for though small amounts of iron 
sulphate stimulated the latter, it was toxic to the former in all con- 
centrations tested. 

Magnesium Salts.—Magnesium compounds usually stimulate 
bacterial activities to a greater extent than do calcium compounds, 
as has been noted in some of the literature already cited, Eng- 
berding’s results, however, showed that, while magnesium sulphate 
stimulated bacterial activities, it was not as effective in this regard 
as was ammonium sulphate, sodium nitrate, or potassium sulphate. 
The work of Makrinov is of interest since he found pure magnesium 
carbonate a very suitable substance on which to grow the nitrous 
organism. Furthermore, magnesium carbonate had a_ strongly 
beneficial effect on the physiological action of the organism. [eller- 
mann and Robinson, on the other hand, found that magnesium car- 
bonate when applied to a soil already rich in magnesium carbonate 
positively inhibited nitrification if the quantity added exceeded 
0.25 per cent. This is an apparent contradiction, but it may be 
due to the different conditions of the experiments, since one investi- 
gator was working with culture of the organisms whereas the other 
was using the soil with its complex flora. Furthermore, it is quite 
possible that magnesium carbonate may be without effect upon or 
even accelerate the growth and activity of the N2trosomonas and 
yet inhibit the Nitromonas. 

Manganese.—Some experiments by Skinner and Sullivan demon- 
strate that manganese acts in various ways as a fertilizer. It is 
often without influence, occasionally injurious, but usually bene- 
ficial, its effect depending apparently upon the composition and 
character of the soil. The oxidation in soils under treatment with 
manganese salts was also studied and it was found that an increase 
in oxidation and growth frequently occurred in aqueous extracts of 
poor, unproductive soils. Although oxidation was increased in 
fertile soils, growth was decreased, the plants showing indications 
of excessive oxidation. Field experiments showed practically no 


144 INFLUENCE OF SALTS ON THE SOIL 


effect due to the manganese salts, but the soil was acid, a condition 
which may have accounted to a considerable degree for the nature 
of the results. 

It is suggested that when the action of manganese is beneficial, 
“it 1s probably due (1) to the increased oxidation produced in the 
plant roots whereby the plant is stimulated to greater activity and 
to increased absorption of the material useful for its growth and 
general metabolism; (2) to the stimulation of the activity of micro- 
organisms in the sovl; (3) to an increased oxidation within the soil.”’ 

The same authors also suggest that when large applications of 
manganese have been found to be injurious, the injury is 
undoubtedly due to the “excessive stimulation and excessive oxida- 
tion in microérganisms and in the plant, with a resulting change in 
the biochemical activities of plant and microérganisms and in the 
conditions of inorganic and organic soil constituents, the ultimate 
result of which change is injurious to the growing crop.” 

Montanan found that manganese carbonate, sulphate, and dioxid 
greatly stimulated nitrification, but attributed it to either the direct 
or the indirect furnishing of oxygen to the nitrifying organisms, 
and not to any catalytic effect of the manganese itself. Beijerinck 
observes that some soil organisms have the power of oxidizing 
manganous carbonate. Olaru found that manganese in the propor- 
tion of one part to 200,000 parts of nutritive media greatly increases 
nitrogen fixation. He also considers it rather likely that the 
increased yield obtained after the application of manganese com- 
pounds to a soil is due to its accelerating the action of the nitrogen- 
fixing organisms of the soil. 

Leoncini found that the application of manganese oxid to the 
soil at the rate of 0.035 to 2.2 per cent. favored nitrification, whereas 
larger amounts had no effect. 

Brown's work, which is probably the most extensive study of the 
influence of manganese compounds on bacterial activities so far 
reported, shows that the influence of the manganese salt upon bac- 
terial activities varies with (1) the kind of salt added—manganese 
chlorid and sulphate acting in low concentrations as stimulants to 
both ammonifying and nitrifying bacteria, but in greater concentra- 
tion being highly toxic; (2) its action varies with the specific class 
of organisms which were being studied, the ammonifiers being more 
resistant than are the nitrifiers. 

Potassium Salts.— Because potassium is essential for the nutrition 
of both the higher and lower forms of plant life, it is to be expected 
that when added to a soil medium poor in potassium it will increase 
bacterial growth, but, like many other true nutrients, may become 
toxic if present in too high concentrations. As already noted, 
Engberding states that potassium sulphate caused a slight increase 
in the bacterial content of a soil. While Peck found it to increase 


SODIUM SALTS 145 


nitrification in soils, Renault claims that slow ammonification and 
subsequent nitrification is always accompanied by a low percentage 
of potash. Dumont’s experiments showed that potassium car- 
bonate, added to a soil at the rate of from | to 2.5 gm. per 1000 gm. 
_ of soil, markedly increased nitrification, but that larger applications 
of the salt progressively diminished the rate of nitrification, while 
the addition of S gm. to 1000 gm. of soil completely checked it. 
Lumia concluded that potassium chlorid and sulphate were nearly 
as effective in promoting the activity of alcoholic ferments as were 
phosphates. 

Fred and Hart found that both calcium and potassium sulphates 
increased ammonification in solution and that the sulphates of potas- 
sium, calcium, and magnesium each increased the volume of carbon 
dioxid from soil. From the results obtained with different salts, 
they conclude that the addition of the potassium ion did not mate- 
rially increase ammonification in the soil examined. 

Sodium Salts.—Sodium salts are often used as fertilizers and with 
good results. Furthermore, many alkali soils contain sodium salts 
in quantities sufficient to be toxic to both the higher and lower 
plants. For these reasons many investigations have been conducted 
to determine the influence of sodium compounds upon higher 
plants, and many have had as their object the determination of their 
influence upon soil bacteria. 

As early as 1884 Warington showed that the presence of 0.052 
per cent. of sodium bicarbonate distinctly retarded nitrification, 
and that in the presence of 0.096 per cent. nitrification was very 
slight. Schlésing had added various salts to the soil in quantities 
not exceeding 485 parts per million with no apparent effect upon 
nitrification. However, Dehérain found that common salt com- 
menced to be harmful when it exceeded one-thousandth of the weight 
of soil, and when larger quantities are applied nitrification almost 
ceased. According to the same observer sodium nitrate may stop 
nitrification for a time, but later it recommences. Lipman and 
others found that sodium nitrate increased the accumulation of 
nitrates in a soil. They found, however, a certain periodicity in 
the accumulation of nitrates which would account for the different 
results reported by various investigators. In later investigators they 
concluded that at times sodium nitrate stimulates ammonification. 
McBeth and Wright found that carbonates, chlorids, and sulphates 
inhibited nitrification and that the former were more injurious than 
the latter. 

The most far-reaching and systematic work which has been 
reported on the influence of salts upon bacterial activity is the 
excellent work by C. B. Lipman who demonstrated that ammonifi- 
cation is inhibited by sodium chlorid, sodium sulphate, and sodium 
carbonate. The points at which the salts became toxic are: for 

10 


146 INFLUENCE OF SALTS ON THE SOIL 


sodium chlorid, between 0.1 per cent. and 0.2 per cent.; for sodium 
sulphate, 0.4 per cent.; and for sodium carbonate, 2.0 per cent. A 
stimulating influence was noted in the case of sodium carbonate, 
but not in the case of the sulphate or the chlorid. The points at 
which they became toxic to nitrifiers were found to be: for sodium 
carbonate, 0.025 per cent.; for sodium sulphate, 0.35 per cent.; 
and for sodium chlorid, less than 0.1 per cent. 

All except sodium chlorid acted as a stimulant in lower concen- 
trations. Later Lipman and Sharp found the point at which sodium 
chlorid became toxic to nitrogen-fixing organisms in soil to be from 
0.5 to 0.6 per cent.; sodium, sulphate, at 1.25 per cent.; and sodium 
carbonate, at 0.4 to 0.5 per cent. Sodium chlorid was the only one 
which acted as a stimulant. Recently Lipman has demonstrated 
that there exists, as measured by ammonification, a-true antagonism 
between sodium chlorid and sodium sulphate; between sodium 
chlorid and sodium carbonate; and between sodium sulphate and 
sodium carbonate. 

Brown and Hitchcock found nitrification to be stimulated by 
small amounts of sodium chlorid, sodium sulphate, and magnesium 
carbonate, and large amounts of calcium carbonate. The large 
quantities, however, became toxic, the point at which toxicity and 
probably stimulation occurs varying with the different soils. 

Variation in Effect Produced.—It is quite evident from the litera- 
ture reviewed that the addition of a salt to a soil may produce a 
number of different effects, depending upon the nature and quantity 
of the salt added: (1) The salt may stimulate some or all of the 
bacterial activities of the soil; (2) it may be without effect; (3) it 
may be toxic to some and without effect or stimulating to others; 
(4) it may be toxic to all of the bacteria of the soil and hence either 
kill the organisms or, what is more often the case, materially 
decrease their metabolic activity. 

These factors are well illustrated in an extensive study carried 
on by the author on the chlorids, nitrates, sulphates, and carbonates 
of sodium, potassium, calcium, magnesium, manganese, and iron. 
They are especially interesting in that it indicates the influence of 
these twenty-four salts on the ammonifying and nitrifying powers of 
a soil all tested under similar conditions. Though the results are 
not absolute, they do represent rather nearly the relative action of 
the various salts, as may be seen by comparing them with the 
results reported by others. One fact, however, which the student 
must bear in mind with these, as with all other results, is that an 
accumulation of a specific substance within the soil may represent - 
either an acceleration of the activity of the organisms which form 
that compound or a decrease of the efficiency of the organism which 
destroys it. 


STIMULATING ACTION 147 


Stimulating Action.—Only six of the twenty-four compounds 
tested failed to stimulate the ammonifying organisms at some con- 
centration. ‘Those which did not stimulate were calcium chlorid, 


oe = nm fp) coe & 
Soe ee ee 
bal Sues oa aR 
Ke ep & S S a 
osc, SiS ay oll atl eal -s! 
a ; 
Na sO, | | nn 
a 
| al| Baan 
Na,CO, [FF S 
et | ny 
T ee Si 
CaCO, 2s 
KoSO4 
ate | z z 
| = 
KsC Onan aaa | Zoe as 
lk ga x 
Fe(NOs), 4 | | 7 oh 
} o42 
Pes Pe 
ue bar os 
Fes(SO4)s 9 a= 
aoe 
MnSO4 I 
MnCl» 
KNO3 
MgSO, 
Mg(NOg)o 
KCI 
Mn(NOg)o 
Ca(NO,), 


’ 


Fic. 20.—Graphs showing molecular concentrations at which the various salts exert 
greatest stimulation on bacteria in soil. 


148 INFLUENCE OF SALTS ON THE SOIL 


calcium nitrate, potassium chlorid, potassium sulphate, magnesium 
nitrate, and sodium sulphate. 


a EXPLANATION 
190 NITRIFICATION aes 
AMMONIFICATION CJ 
180 
170 
, 160 
Zz 
wW 
© 150 
oe 
uw 
* 140 
130 
120 
110 
me = PS a ~ 
ee Ent epee ee eee oe °° 
SBSdSSSSRSS 29S S EP Zao = GB 
a 2 On O Zeothe —E ss ~~ 00% 9 OZ OF 6 1OL.a 
es oS 8NaS) ey Sol te 1G OUR a ay te CO OE a ne wi O an OS on eg tems es 
Sanh Se gs AEN wale nes MEN Sas oe a a Mas a Eh S) ob) 


Fic. 21.—Graphs showing the percentage of stimulation at the above noted molec- 
ular concentrations (See Fig. 20), the untreated soil being counted as producing 


100 per cent. of nitric nitrogen. 


x10 A aiixtlOr 6x 10-3 6x 10% 4x 104 
65 x 107° fF EXPLANATION [ 
60 x 107° |" NITRIFICATION [amma 
55 x 107° : AMMOIFICATION L___ 
5) x 107 
45 x 1071 | 
4)x 10° IE 
33x 107° a 
3) x 10°57 
25x 1075 
20x 107% 
150 x 10 5% 
100 x 107° 
500 x 10°'| | 
0 = | 
Zao ES = 4 a 
MG) Gaon SOL oe POM Saleh De ae ee es oy 
G220-5 RE cic 2 OR 95. oC om oon e. 
Cee ege mS LS Jet es ery Tee rh oy) CEH he OM nay preva (Oh SEs wrt (OY Cs 
Ou use ae SSO) Oye ee ee 


Fie. 22.—Graphs showing the molecular concentrations at which the various salts 
are toxic to ammonifying and nitrifying organisms in the soil, 


. 


‘There were also six which failed to stimulate the nitrifiers, but 
it is quite evident from the results given in Fig. 20 that in the 
majority of cases these are different from the ones which failed to 
stimulate the ammonifiers. This is remarkable when we remember 


TOXICITY OF VARIOUS SALTS 149 


that the speed of the nitrification process is controlled and dependent 
upon the speed of ammonification. The results clearly indicate that 
there are other side reactions taking place which are influenced by 
the salts but which are not measured by the ordinary bacterio- 
logical method. 

It is evident, however, from the results reported in Fig. 21 that 
those compounds which are most active as stimulants to the higher 
plants are also most active in stimulating bacteria. It is likely that 
the effect upon the plant is due in a large measure to the action of 
the compound upon the bacteria, which in turn render available 
more plant-food. 

Toxicity of Various Salts.—'There is an extremely wide variation in 
the concentration at which various salts become toxic to soil bac- 
teria (Fig. 22). Some must oecur in soils in large quantities before 
becoming toxic, whereas others are toxic when present in only minute 
quantities. . The toxicity of the salts to ammonifying organisms are 
controlled largely by the electronegative ion, but this is not as 
pronounced in the case of the nitrifiers. The latter class of organisms 
is, however, more sensitive to salts than are the ammonifiers. The 
ammonifiers represent more nearly the higher plant. 

It is apparent from these results that the increased osmotic 
pressure exerted by a salt within the soil plays a part in retarding 
the bacterial activity of such a soil, but it is not the only factor. 
The main factor is probable a physiological one, due to the action 
of the substance upon the living protoplasm of the cell changing 
its chemical and physical properties in such a way that it cannot 
function normally. 

REFERENCE. 


Greaves, J. E.: ‘‘The Influence of Salts on the Bacterial Activity of the Soil”’ 
(Soil Science, 1916, ii, 443-480). 


CHAPTER XV. 


INFLUENCE OF MANURE ON THE BACTERIAL 
ACTIVITIES OF THE SOIL. 


THE application of barnyard manure to a soil brings about a 
far-reaching change within the soil. It has been found that, on the 
average, one ton of barnyard manure contains 10 or 12 pounds each 
of nitrogen and potassium and 2 or 3 pounds of phosphorus. It also 
carries other substances of less importance which may be directly 
utilized by the growing plant or which may react with substances 
within the soil, changing their solubility. This direct and indirect 
nutritive value of a manure is not its only function, for it greatly 
changes the physical structure of the soil. It improves the tilth of 
a clay soil by increasing the granulation within it, while in a sandy 
soil it tends to bind the particles together, making it Jess porous. 
Each of these changes react upon the water-holding capacity and 
the capillarity of the soil, greatly altering the aération of the soil 
and with the aération the temperature. 

The biological changes which the manure produces in the soil, 
especially when small quantities are added, may be even more far- 
reaching than either the chemical or physical changes which it 
produces. Every pound of manure carries with it to the soil millions 
of bacteria. Many of these will find the new conditions unsuited for 
their growth, but some will continue to multiply, and in so doing 
not only: will decompose the constituents of the manure but also 
will greatly alter other organic and inorganic substances of the soil. 
The bacterial content of the soil is, therefore, changed both quanti- 
tatively and qualitatively. There are added with the manure many 
new species; the changed physical and chemical conditions of the 
soil due to the manure will greatly modify those already present, 
for the microflora and microfauna originally present in the soil were 
due to specific soil properties. 

This changed flora and fauna will in turn change the chemical 
and physical properties of the soil still more. Acids are generated, 
which react with insoluble constituents, rendering them soluble. 
Gases are formed, which change the air within the soil; in these 
reactions heat is generated, thus changing the temperature of the 
soil. The metabolism of the bacterial cell requires nutritive sub- 
stances, among which are water and the elements essential to plant 
growth. Some soluble constituents will be changed to insoluble and 


NUMBER 151 


some inorganic to organic. All of these changes are reflected in the 
crop yield. 

Number.—That the addition of manure to a soil increases the 
number of bacteria has been shown by Remy and Fischer. 

Caron found that the number of bacteria present depends not 
only upon the manure added but upon the cultural methods and the 
crop grown on the soil. Fabricius and von Feilitzen found that 
bacteria increased in the soil on the addition of manure and that a 
direct relationship existed between the temperature of a soil and 
the number of bacteria found in it. That the temperature of the 
soil is influenced by the addition of manure is shown by Wagner who 
observed that manure increased the temperature of soil from 1 to 
2.8° C., depending on the kind and condition of manure added. 
Troop noted an average increase of 5° in temperature of soil receiving 
25 tons an acre of manure over an unmanured soil. Petit, however, 
claimed that, while there was at first an increase in the temperature 
of manured soils, later it became lower than the unmanured. Stigell 
concluded that bacteria under favorable conditions for de *elopment 
retarded the conduction of heat in soils and thereby reduced the 
temperature changes due to the variation in the outside temperature. 
This, in a way, might neutralize the effect of manure, for Hecker 
found that although the temperature of soil to which well-rotted 
manure had been added was higher than adjacent unmanured soil 
during the day, the opposite was true during the night. Grazia 
stated that manures greatly increase the temperature of the soil. 
King found that a definite increase in bacterial activity occurred 
with increased temperature, but that an excessive moisture content 
greatly reduced the number of bacteria in a soil. Engberding 
claimed that manure increased the number of bacteria in a soil, but 
he considered that the moisture content had a greater influence on 
numbers than did temperature. That the moisture content greatly 
influenced bacterial activity was shown by Dehérain and Demoussy, 
who found that the bacterial action of a soil was at its maximum 
when a rich soil contained 17 per cent. of water, but that it decreased 
if the proportion of water fell to 10 per cent. or rose to 25 per cent. 
With soils less rich in humus a somewhat higher proportion of water 
was necessary to retard oxidation to any marked degree. In a 
manured soil the coarse manure tended to cause the surface soil to 
dry out, while fine manure prevented evaporation. King observed 
that manured land contained more moisture throughout the year 
than unmanured, and this was reflected in both a greater number 

of bacteria and in a larger crop. The bacteria themselves may 
play a small part in this difference in moisture content, as was 
shown by Stigell, who found that bacteria decreased the speed of 
evaporation of water from Petri dishes. Hiltner and Stérmer’s 
results indicate that the addition of manure to a soil brought about 


152 INFLUENCE OF MANURE ON THE SOIL 


a marked change in the number of bacteria. The temperature, 
cultural methods, and crop had an influence, but it was not nearly 
so pronounced as that produced by the manure, as is seen from the 
following in which is given the number, stated as millions per gram, 
of bacteria found in manured and unmanured soils at different 
seasons. 


1901. 1902. 


10 May. | 27 Aug.| 18 Oct. | 1 Feb. | 12 June.| 18 Aug. 


Cropped land, grass and clover . 8.3 3.2 6.4 6.6 eal 4.9 
Cultivated fallow, unmanured . 8.0 4.2 4.0 4.1 Say 4.1 
LOM Oss. 11.0 9.3 tae 8.4 


Cultivated fallow, manured . . itil. 


Brown, in a study of the influence of manure on the bacterial 
activities of a loam soil, found that applications of manure up to 16 
tons an acre increased the number of bacteria and also the ammoni- 
fying and nitrifying powers of the soil. The greatest increase in the 
processes was brought about by small applications of manure, 8 to 12 
tons to the acre. He observed a close relationship between the 
ammonifying powers of the soil, the bacterial content, and the crop 
produced on the soil. 

Temple stated that the addition to a soil of 10 tons an acre of cow 
manure greatly increased the number of bacteria in the soil, but 
that a greater increase occurred when a sterilized manure was applied. 
This, however, is not in keeping with the results obtained by other 
investigators. Hellstrém concluded that manures possessed a 
fertilizing effect aside from the quantities of fertilizer constituents 
contained within them; and this, he maintained is due to their great 
bacterial content. Stoklasa found that manure increased the bac- 
terial content and activity of a soil, the increase being greater with 
small, frequent applications of manure than with large applications 
made at longer intervals. Moreover, Lipman and others observed 
that the bacteria conveyed to soil in small quantities of manure 
were valuable in bringing about a more rapid decomposition of a 
green-manure crop. Briscoe said that a direct relationship existed 
between the organic matter added to a soil and the bacterial count, 
and that a light dressing of manure with green manure produced a 
marked increase upon both the yield of the crop and number of 
bacteria. Bacterial cultures added with the green manure gave just 
as pronounced an effect as did the stable manure. Lemmermann 
and Einecke, however, obtained no increase on adding stable manure 
with green manure. This may be due to the different kind of manure 
used, for Emmerich and others maintained that a more favorable 
effect was obtained from the use of well-rotted manure than from the 


LOSS OF NITRATES 153 


use of fresh manure. ‘This, they maintained, was due to the pro- 
duction in the latter of formic, acetic, and butyric acid, indol, skatol, 
and hydrogen sulphid, which are toxic to the plant. Under some 
conditions, the large quantities of carbon dioxid liberated from the 
rapidly decomposing fresh manure may be valuable in rendering the 
plant-food soluble. Bornemann found that soil constantly supplied 
with carbon dioxid through a pipe buried in the ground gave an 
increase in yield of 12.2 per cent. over the crop grown on untreated 
soil. Wollny has shown that manure greatly increased the carbon- 
dioxid production in a soil. 

Ammonification and Nitrification.— Moll considered that the season 
of the year, and not the kind of fertilizer used, nor even the weather 
conditions, is the principal factor in determining the extent of pep- 
tone decomposition, nitrification, and nitrogen fixation of a soil. 
According to Wohltmann, Fischer, and Schneider, ammonification, 
nitrification, and nitrogen fixation were all more or less increased 
by the application of manure. Heinze found that manure was 
especially beneficial to the nitrifying organisms. Warington reports 
that much more nitric nitrogen was found in the soil of plots which 
had received annually for thirty-eight years a dressing of 14 tons of 
manure to the acre than in any of the other manured or unmanured 
plots. Stevens found that nitrification was much more active in 
manured than in unmanured soil, but Frankfurt and Duschechkin 
observed an increase in nitrification only on those manured plots on 
which the yield had increased. Welbel has shown that the chief 
factors controlling nitrification in fallow soil were the humus and 
the humus-nitrogen content, the nitrification having increased 
directly with the humus. He noted, however, a certain amount of 
denitrification at first, but later in the summer nitrification became 
more rapid on the manured than on the unmanured soil, the effect 
of the manure being still perceptible after four years. Some investi- 
gators have reported a reduction of nitrates, but the quantity of 
manure applied was excessive, or else of a very coarse nature, or the 
soil poorly aérated. Barthel found that nitrification did not take 
place in the presence of soluble organic matter, but he considered 
it unlikely that sufficient quantities of soluble organic constituents 
occurred in normal agricultural soils to interfere greatly with nitri- 
fication. Niklewski maintained that nitrification occurred in solid 
stable manure when there was not much liquid present. He stated 
that on the first day some nitrite bacteria were present and at the 
end of four weeks there were 10,000 in each gram. Associated with 
these were nitrate bacteria which were identical with those isolated 
by Winogradsky. Millard, however, was unable to find many nitrify- 
ing bacteria in manure. 

Loss of Nitrates.— Many of the cases in which individuals have 
reported a disappearance of nitrates in soil are due to synthetic 


154 INFLUENCE OF MANURE ON THE SOIL 


reactions, the nitrates being built up into complex proteins. For 
Gerlach and Vogel have shown that there are several varieties of 
bacteria in the soil which have the power of converting ammonia, 
nitrites, and nitrates into insoluble proteins. 

It is evident from the literature cited that there is a wide variation 
in the ideas held concerning the influence of manure upon the 
bacterial flora of the soil. This is due to a number of factors, chief 
among which are: (1) variation in the physical and chemical com- 
position of the soil; (2) a great variation in the composition of the 
manure used; and (3) the manure added may have influenced either 
‘beneficially or injuriously the water content of the soil. The results 
noted may have been due to the moisture factor and not to the 
manure. An experiment was conducted at the Utah Experiment 
Station both in pots and under field conditions. Each ton of the 
manure added was partly rotted and contained in one ton 734 pounds 
of dry matter, 3.04 pounds of phosphorus, 13.7 pounds of potassium, 
and 16.08 pounds of nitrogen. 

The conclusions reached follow: A calcareous soil kept in pots 
with varying amounts of manure and different percentages of moist- 
ure gave on bacteriological analysis at the end of four months the 
following results: The temperatures of the manured and unmanured 
were practically the same for the period, but the temperature of the 
soil with 12.5 per cent. of water was 1° C. higher than soils with 
22.5 per cent. of water. The greatest number of organisms devel- 
oped on synthetic media from the soils receiving the greatest quan- 
tity, 25 tons, of manure. There were more colonies developed from 
the soil receiving 12.5 per cent. of water than from any of the other 
soils receiving higher quantities of water. 

The nitrifying powers of the soil increased as the manure and water 
applied increased up to 25 tons of manure and 22.5 per cent. of 
water. 

The nitrogen-fixing powers of the soil were greatest in those pots 
receiving manure at the rate of 10 tons an acre. Increasing the 
water above 12.5 per cent. but not above 22.5 per cent. slightly 
increased the nitrogen-fixing powers of the soil. Nothing in the 
results indicated that the application of manure up to 25 tons an 
acre and of water up to 22.5 per cent. caused denitrification in the 
soil. 

Bacteriological analyses of fallow field soil receiving no manure, 
5 tons, and 15 tons an acre and receiving no water, 5 inches, 10 inches, 
20 inches, 30 inches, and 40 inches of irrigation water, indicated that 
the maximum number of bacteria were obtained from the soil 
receiving 15 tons of manure. The application of irrigation water 
up to 20 inches increased the bacterial count, the increase being 
most noticeable in the soil receiving the greatest quantity of manure. 

If the ammonifying power of the unmanured soils is considered 


LOSS OF NITRATES 155 


as 100 per cent. and that of the unirrigated as 100 per cent., the 
manured and irrigated soils then become with 5 tons of manure, 147 
per cent.; with 15 tons of manure, 188 per cent.; 5 inches of water, 
106 per cent.; 10 inches of water, 117 per cent.; 20 inches of water, 


O78 


LLY LLL LLL 


EXPLANATION 

Me OPER CENT OF GRAIN 
FE] sPER CENT OF STOVER 
[] - PER CENT OF BACTERIA 

500 i PER CENT OF BACTERIA (FALLOW) 
, PER CENT OF AMMONIA 

fA PER CENT OF AMMONIA (FALLOW ) 
480 PER CENT OF NITRIC NITROGEN 


2 


f PER CENT OF NITRIC NITROGEN ( FALLOW) 
200 

180 \ by 
160 i; 
= Yes : 
140 BN HE : 
BIGZIN WalGe i 
120 ra EVAN GZING 
HIGe iY SW EIGzIN EH V/4zA WR 

100 Hs AN PETIA ZING otf 
HNGe EE Va Na ZIN% 
Bl a SEIN BNG 
80 BVA le Ve aN 
NG GANS AN 
Zi RY | CA IN MY 
: 60 YAN BET VAN ANG 
. ANG BPEIEAN& Ni 
aN | BNI ZN 
Ha AY EVAN ZINt 
40 /; i IGANG ANS: 
Vans His Ni ZING 
FA NR =| HVE Na oy 
ZN EWEIGAN : 
20 EN BEIEVEN : 

NO MANURE 5 TONS 15 TONS 
OF MANURE OF MANURE 


Fie. 23.—Diagram showing the influence of manure on the yield and bacterial activi- 
ties of a soil. The unmanured soil is expressed as 100 per cent. 


108 per cent. Large quantities of irrigation water produced the 
greatest depressing effect in the presence of 15 tons of manure to the 
acre. 

Fewer organisms develop on synthetic agar from a cropped than 
from a fallow soil. The application of manure to a cropped soil 


156 INFLUENCE OF MANURE ON THE SOIL 


increases the bacterial count of the soil. The greatest number of 
organisms developed from the soil receiving 10 inches of irrigation 
water. 

The ammonifying power of the cropped soils was slightly lower 
than similarly treated fallow soils. ‘The application of 5 and 15 
tons of manure on each acre increases the ammonifying power of the 
soil. The application of irrigation water up to 30 inches increases 
the ammonifying power of the soil. The greatest increase resulted 
in those soils receiving 15 tons of manure to the acre. The applica- 
tion of 40 inches of irrigation water to corn land, especially to that 
receiving 15 tons of manure an acre, depresses the ammonifying 
power of the soil. 

The nitrifying power of fallow soil was higher than similarly 
treated cropped soils. The application of manure to a cropped soil 
greatly increases the rate of nitrification. The application of irriga- 
tion water up to 30 inches, especially to a soil receiving 15 tons of 
manure, greatly increases its nitrifying power. 

Green manures are rapidly taking the place of bare fallows in all 
of the better systems of agriculture where the rainfall will permit of 
the practice. The practice of green-manuring consists, essentially, 
of the turning under of green crops for the benefit of succeeding 
crops. In addition to the various legumes, which are preferred on 
account of their ability to take nitrogen direct from the air, crops 
like rye, wheat, oats, buckwheat, mustard, rape and even turnips 
have been used more or less extensively as green manures. 

The plowing under of green manure produces either a beneficial 
or injurious effect, depending upon the nature of the soil to which it 
is applied, the kind of manure added, and the season of the year 
when applied. Some of the beneficial influences noted are:  ” 

1. They carry to the soil large quantities of organic matter which 
on decaying yield humus, and this in turn changes materially the 
physical and chemical composition of the soil. Schultz started in 
1855 on an extremely poor, coarse-grained, sandy soil, and gradually 
improved it by the use of lime, phosphoric acid, and potash in con- 
nection with such green-manuring crops as lupines, serradella, and 
field-peas, until he could produce three hundred to four hundred 
bushels of potatoes to the acre. Neale noted a marked gain in the 
yield of corn when crimson clover was used as a green manure. He 
believed the nitrogen thus applied to be much more economical than 
when nitrate of soda is used. For sandy soils the results of Delwiche 
would indicate cowpeas, hairy vetch, soy beans, and crimson clover 
to be best, and Pfeiffer’s results indicate that it is the open, sandy 
soils which give the best results with green manures. The actual 
effect produced, however, varies with the time of application. 
Bassler recommends that where lupines, serradella, crimson clover, 
and hairy vetch are used for green manure on sandy soils they should 


£ 


LOSS OF NITRATES 157 


be turned under as late as possible in the life of the plants and not 
in the hot summer when the plants are green. This agrees with the 
findings of Causemann who recommends that they be turned under 
late in September. It has been observed by Bredemann that the 
addition of organic matter, such as hay and sugar, produces a harm- 
ful effect the first year and a beneficial effect the two following years. 

2. Green manures change the number and kind of organisms 
occurring in the soil. Hill found the total number of bacteria in 
soils treated with green manures to be much greater than in those 
receiving no green-manure treatment. Legumes gave in most cases 
a greater increase than non-legumes. Miintz considers the value of 
green manures proportional to the rapidity with which their nitrogen 
is converted into nitric nitrogen. Heinze, on contrasting stall and 
green manures, found that the latter carried but few organisms which 
would break down the insoluble material. ‘The decomposition of 
green manure was found to be due to dust organisms and to organ- 
isms found in soil. For this reason the number and kind of organisms 
in a given soil determine in a great measure the influence of green 
manures on succeeding crops. Decomposition is rapid in an open, 
sandy soil rich in bacteria and relatively slow in a soil poor in bacteria. 
In an open, sandy soil the nitrogen of the green manure may pass 
over into nitrates and be washed out, whereas in a heavier soil the 
nitrogen becomes available more slowly and is not washed out so 
rapidly. For this reason in heavy soils green manures often give the 
best results the second year. 

Kkoch thinks the good effects produced when green manure is 
added to stable manure may be due to an increased nitrogen fixation 
by Azotobacter, the organism using the cellulose of the manure as a 
source of energy; whereas Heinze considers that the results which he 
obtained from studying the action of carbon bisulphid on soils may 
help us to understand the peculiar effects produced at times by the 
turning under of mustard, buckwheat, rye, and other non-leguminous 
crops. It has been noted repeatedly that these crops when plowed 
under in a green state lead to a better growth of the following cereal 
or root crops on nitrogen-poor soils. As Heinze points out, there 
may have been more or less justification for this belief, so far as the 
indirect influence of mustard is concerned. It would seem at times 
that the action of mustard is not unlike that of carbon bisulphid in 
affecting the bacterial flora of the soil, and it really appears from 
facts already known that the green mustard substance in the soil 
retards the development of the acid-forming species and encourages 
the growth of the nitrogen-fixing Azotobacter species. Heinze, 
therefore, thinks that further study may enable us to make extensive 
use of mustard as an indirect source of combined nitrogen, and tries 
to find theoretical support for this belief in the fact that allyl 
isothiocyanid mustard oil, C;H; — N = C =5S, which is a constit- 


158 INFLUENCE OF MANURE ON THE SOIL 


uent of the mustard plant, may be regarded as a derivative of 
carbon bisulphid and probably would have a similar influence upon 
the soil microflora. 

3. The growing of a crop during the season of the year when the 
heavy rains would wash much of the nitric nitrogen beyond the 
roots of the plants prevents this loss, for the nitrogen is stored in 
the body of the plants and is later liberated by decomposition for 
succeeding plants. 

4, The growth of the legumes may actually lead to an increase in 
the nitrogen of the soil. The method, extent, and conditions under 
which this occurs is considered in detail in later chapters. 

The results, however, following the use of green manures are not 
uniformly beneficial, for. the following ill effects have at times 
followed its use: 

1. The physical condition of the soil may be injured. It becomes 
too loose and open, decomposition being thereby decreased and 
leaching increased. This was probably the reason why Brown did 
not always find an increase in the bacterial activities following the 
application of green manure. This is also true with regard to the 
work reported by Laurent. 

2. Engberding, studying the effect of straw and sugar upon the 
number of bacteria in the soil, found at first an increase, followed by 
a decrease. The ammonifying and nitrogen-fixing groups of bacteria 
showed an increase, but the nitrifying group was retarded. 

Fischer, in his paper on the changes undergone by nitrogen in 
sandy and clayey soils, offers an explanation for the loss of nitrates 
in a soil to which carbohydrates have been added in the following 
reaction: 


24 NaNOs + 5 CeH20¢ = 24 NaOH a 12 Noe + 30 COz ++ 18 H.0 


The oxygen of the nitrate is used by the ferments for the oxidation 
of the carbohydrate and the nitrogen is liberated as a gas. 

Frankfurt and Duschechkin state that green manure under field 
conditions caused a diminution of the nitrate content. Both legumes 
showed this effect, but they consider it as due to the action of the 
manure upon the soil moisture. . 

Stevens and Withers give two reasons why the activity of nitrify- 
ing organisms, in pure culture under laboratory conditions, cannot be 
compared with their activities under conditions in the field: (1) “In 
mixed culture their symbiotic and physiologic relations are so 
different from those obtained in pure cultures that their metabolic 
processes are with difficulty expressed;” (2) “the presence of large 
amounts of solid matter, sand, or earth, in contact with the liquid 
medium, so alters its relation to the nitrifying organisms that their 
physiologic activities and metabolic products are different.” These 
authors say in conclusion; “In the light of the facts set forth, the 


LOSS OF NITRATES 159 


direct application of Winogradsky’s conclusions to the field must be 
abandoned and with them any practices based upon them, and the 
activities of these soil bacteria must, in the future, be studied more 
largely under their natural environments.” 

Lipman, commenting on these earlier views, believes that “the 
exact relation of organic matter in the soil to the activities of nitrify- 
ing bacteria is but beginning to be properly understood. Earlier 
observation made it manifest that heavy applications of animal 
manures, or green manure, may not only retard nitrification but 
may actually cause the disappearance of a part or of all of the nitrate 
in the soil. Subsequent experiments by Winogradsky and Omelianski 
showed that in pure cultures the presence of even slight amounts 
of soluble organic matter may depress or even suppress the develop- 
ment of the nitrifying bacteria. It was, therefore, concluded by 
these authors that relatively small amounts of soluble organic 
matter may inhibit nitrification. These conclusions, based on the 
study of liquid cultures only, were given a very broad application 
by many writers on agricultural topics. More recent experiments 
make it certain, however, that in the ‘soil itself small amounts of 
soluble matter, for example, dextrose, are not only harmless but 
may really stimulate nitrification. It was shown, likewise, that 
humus and extracts of humus may, under suitable conditions, stimu- 
late nitrification to a very striking extent.” 


REFERENCE. 


Greaves, J. E., and Carter, E.G.: Influence of Barnyard Manure and Water upon 
the Bacterial Activities of the Soil (Journal Agricultural Research, 1916, vi, 889-926). 


CHAPTER AANA, 
THE SOIL FLORA. 


Two methods are in general use for the quantitative determina- 
tion of bacteria in the soil: the Koch gelatin-plate method and the 
Hiltner and Stormer dilution method. 

Koch Gelatin-plate Method.—The determination by the gelatin- 
plate method is made as follows: Samples of soil are carefully mixed, 
usually by shaking, and 100 grams of the mixed soil weighed on sterile 
paper or watch-glasses into 200 c.c. of sterile water. ‘This is shaken 
for one minute. Ten c.c. of this is transferred to 90 c.c. of sterile 
water. This is continued until the proper dilution is obtained, 
usually 1 to 20,000 and 1 to 200,000, then they are plated on gelatin 
or some other solid media. The number of colonies developing is 
counted at the end of four or seven days. The longer period is pref- 
erable for many of the important soil organisms are missed in the 
shorter incubation period. 

Hiltner and Stormer Dilution Method.— The Hiltner and Stormer 
method uses solutions prepared to favor in each case the develop- 
ment of one of the various groups of bacteria (ammonifying, nitrify- 
ing, nitrogen-fixing, etc.). The soil is inoculated into the appro- 
priate media in constantly decreasing quantities usually 100, 10, 1, 
0.1, 0.01, 2, and 0.001 mg. of soil. The material is incubated and 
after the appropriate time the gain in ammonia, nitrites, nitrates, ete. 
determined. By this procedure a point is finally reached where the 
small quantity of soil employed contains none of the specific organ- 
isms, and hence fails to give rise to the specific physiological reaction. 
By this method a fairly accurate estimation may be made of the 
number of different microdrganisms in the soil. 

Lohnis clearly demonstrated that the Hiltner'and Stormer method 
is more exact than is the gelatin-plate method. He found that 
a soil which yielded 1,270,000 bacteria per gram by the gelatin- 
plate method, by the Hiltner and Stérmer method yielded 3,750,000 
peptone-decomposing bacteria, 50,000 urea-decomposing bacteria, 
50,000 denitrifying bacteria, 7000 nitrifying bacteria, and 25 
nitrogen-fixing bacteria, making a total of 3,857,025, which is over 
three times the number obtained by the plate method. 

Defects of Plate Method.—The number of organisms found in soil 
by this method varies with the media used and the period elapsing 
between plating and counting. But even under the most favorable 


NUMBER OF BACTERIA IN SOIL 161 


conditions, the numbers are far below the number actually occurring 
in the soil. Some of the reasons for this are as follows: 

1. Even many of the peptone-decomposing bacteria fail to grow 
on the gelatin plates. This may be due to overcrowding or to the 
sudden change of conditions and the resulting osmotic disturbances. 

2. The nitrifiers do not grow on the ordinary organic laboratory 
media; moreover, they have not been found in soil in sufficient 
numbers to occur on plates dilute enough to show the more abundant 
organisms. ‘The nitrogen-fixing organisms—both symbiotic and 
non-symbiotic—usually occur in soil in too small a number to be 
noted by the ordinary plate method. 

3. The strict anaérobes which occur in soils in vast numbers do 
not grow on the gelatin plate under the ordinary conditions of 
aérobic culture. 

4. No medium yet devised resembles the soil in composition and 
structure, and hence the plate does not necessarily reflect the flora 
active in the soil. Moreover, it is impossible to tell which of the 
forms developing on the plate are active and which are spores in the 
soil. 

A third method for the determination of the bacteria in the soil 
is the direct microscopic count. This method, however, has not 
been used sufficiently as yet to permit a conclusion as to its relative 
value. 

Value of Bacterial Counts.—The methods for determining the 
number of bacteria in soil are admittedly faulty; yet they have the 
advantage of showing whether the number is high or low and whether 
they are increasing or decreasing. The counts show fairly accurately 
whether any given treatment of the soil has raised or lowered the 
number of bacteria in the soil. But numbers alone furnish only 
meager information, for, as pointed out by Remy, the number of 
colonies of aérobic soil bacteria appearing on plates show no direct 
relationship to the ammonifying, nitrifying, or denitrifying powers 
of the corresponding soil. Léhnis is even more emphatic than Remy 
in designating mere quantitative methods as untrustworthy. He 
points out that it is quite possible that two million very efficient 
ammonia-producing bacteria present in 1 gram of soil will accomplish 
more work than five million less efficient ones will in another soil. 

This same principle is brought out by Chester when he states 
that a soil may be low in the total number of bacteria, but contain 
such a bacterial flora, or combination of bacterial species, which 
are known to be favorable to the rapid digestion of plant-food, as 
to give it what might be termed a high bacterial potential. In other 
words, he holds that we should consider not alone numbers but also 
physiological efficiency. 

Number of Bacteria in Soil.—'The number of bacteria, as deter- 
mined by the plate method, in good arable soil well supplied with 

iil 


162 : THE SOIL FLORA 


organic matter usually ranges from three to forty million. Tight, 
non-porous soils, as well as alkali and soils low in humus and 
moisture, yield low bacterial counts. 

The number of microérganisms in light, sandy, very tight clay, 
desert and forest soils is usually much smaller, other things being 
equal, than in normal cultivated soils. Cultivation of soils tends 
to increase greatly the number of bacteria. The average of several 
hundred determinations made on cultivated soil of the arid regions 
gave a bacterial count of 4,452,000, whereas the average of a similar 
number of samples taken from adjoining virgin soils was 2,270,000. 

Factors Influencing Number.—Optimum moisture, organic matter 
and aération tend to increase the number as does also the addition 


of sugar and certain antiseptics: 
Bacterial number after 50 days. 
Soil treated with antiseptics, 


Control soil. millions per gram. 
Cane sugar (p. 25 percent.) . . . 21 51 
Amy] alcohol (0.1 per cent.) . . . 30 85 
Phenol (m/200 per kilo) . . . . 27 101 
Hydroquinone (m/200 per kilo) . . 16 55 


There is a direct relation between the number of bacteria found 
in the soil and the quantity of organic manure added. ‘This is 
illustrated in results obtained by the author and given in tabular 
form below. The unmanured soil is taken as 100 per cent. 


Treatment. Fallow soil. Cropped soil. 
INOIMAMTTC cual ss tn eat sence acon ae ea OD 100 
OECONSOL Manure 1s atewe a ee ee eee a 123 
TPDACONSOL aN Ure wea eae ca pee ee hee kn acca lind 129 


The aération of the soil often increases manyfold the number of 
bacteria, and according to Conn the greatest numerical increase 
occurs in the group of non-spore-forming bacteria. 

The number of bacteria found in the soil varies considerably 
with the season of the year. A very interesting phenomenon, noted 
by Conn and confirmed by Brown, was that the number of bacteria 
in soil increase on freezing. This fact is illustrated in the following 
table from the work of Conn. In it are listed a series of gelatin- 
plate counts made from a single soil plate at intervals throughout 
the course of three years. 

During the three years over which the sampling was conducted 
the plate count of non-spore-formers varied from 5,000,000 to 
44,000,000 per gram, whereas the spore-forming bacteria and 
Actinomycetes varied only from 3,200,000 to 10,500,000. The 
increase which occurs during the winter is in the slow-growing 
bacteria and not in those which liquefy gelatin rapidly or in the 
Actinomycetes. Conn tries to account for the noted phenomenon 
by assuming two groups of bacteria—winter and summer bacteria. 
The latter, he thinks, prevent the former from multiplying rapidly 
in warm weather. Hence, the increase in the frozen soil is due to the 
depressing effect of the cold upon the summer bacteria. ‘There is, 


FACTORS INFLUENCING NUMBER 163 


however, the possibility that the freezing breaks up the clumps of 
bacteria and the noted increase is apparent and not real. Little 
relationship has been found between the moisture and cropping of a 


Number of colanics per gram of dry 
Moisture eh 
Baie Pee eae | Other colonies eee 
- | Non-spore-form- Oe 
| “ing bacteria. | Sore formers and 
| 

Nov. 24,1911 . | 2080) 22,500,000 5,500,000 
Jane, LOZ 40.0 28,500,000 7,500,000 Frozen 8 days. 
Jan. 238, 1912 37.0 31,000,000 9,000,000 Frozen 18 days. 
Feb. 13, 1912 43.0 11,000,000 10,000,000 Frozen 39 days. 
Mar. 1, 1912 | 37.0 20,500,000 6,500,000 Frozen 57 days. 
Apr. 24, 1912 23.4 18,000,000 4,500,000 
May 6, 1912 40.0 20,000,000 9,000,000 
June 5, 1912 20.4 19,200,000 4,500,000 
Sept. 23, 1912 22.5 28,500,000 10,500,000 
Oct. 25, 1912 24.5 14,500,000 5,500,000 
Deez — 3; 1912 262 28,500,000 | 6,500,000 
Jane or ols 39.5 14,000,000 | 6,000,000 Frozen 7 days. 
Feb. 5, 1913 22,0 22,300,000 7,700,000 Frozen 4 days. 
Feb. 14, 1913 PAD 44,000,000 | 10,000,000 | Frozen 13 days. 
Mar. 11, 1913 38.8 | 22,000,000 7,000,000 | Partly frozen. 
Apr. 4, 1913 22.8 | 19,300,000 7,700,000 | 
July 10, 1913 17.0 | 16,800,000 5,200,000 | 
Nov. 26, 1913 Diesen ee eS 00000 4,200,000 
Dee. 15, 1913 19.6 7,800,000 3.200,000 | 
Jan. 16, 1914 31.2 | 15,500,000 3,500,000 | Frozen 9 days. 
Jan. 30, 1914 24.6 | 26,000,000 7,000,000 | Thawed 1 day. 
Heb, 7, 1914 . 27.7 | 22,700,000 | 7,300,000 Partly frozen. 
Feb. 26, 1914 BO) || SHINO) 7,000,000 | Frozen 21 days. 
Apr. 15, 1914 20.0 13,700,000 7,300,000 | 
Apr. 29, 1914 20.0 11,800,000 | 4,200,000 | 
Aug. 7, 1914 9.0 5,000,000 | 4,000,000 | 
Aug 20.0 | | 


Os UO 


18,000,000 


4,700,000 


The number of organisms in the soil vary greatly with the depth. 


Due to the lack of moisture and the germicidal action of light, the 
number of bacteria in the uppermost inch or two of soil is lower than 
in the layers of soil immediately below. Beyond the depth of eight 
or nine inches, the number diminish rapidly, and in humid regions 
the number below two feet is extremely low. This is illustrated by 
results reported by Chester: 


Depth. Number of colonies. 
2 inches pes 
4 inches 1,632,00 
6 inches 1,623,000 
12 inches 73,000 
18 inches 21,000 
~24 inches 4,000 


The conditions, however, are quite different in the arid regions 
where we have the slow formation of clay substances in the soil, 
and, therefore, the absence of the cementing substances in the soil. 


164 THE SOIL FLORA 


This results in greater aération, and hence often ideal conditions 
for bacterial growth to a great depth. Moreover, the roots in search 
for water penetrate to a great depth in the soils of the arid regions. 
This results in an aération of the soil and the supplying of organic 
matter to bacteria at a great depth. Lipman found the ammonify- 
ing organisms at all depths to the tenth foot and at times the nitrify- 
ing organisms to a depth of eight feet. The nitrogen-fixing organ- 
isms seldom occurred below the third or fourth foot. I have found 
great numbers of bacteria in both dry-farm and irrigated soils of 
the arid regions in the second and third foot. The average of several 
hundred such determinations is given below: 


Number of colonies. 


Depth. Trrigated soil. Dry-farm soil. 
LHOG hear ia poe ee ee ee ree “O24 0000 4,372,000 
DiCOGus Mes MAM ise Lo ia tice = Saati se OOCO00 1,267,000 
LCOU Eee tc Care ca att ae Pes ee ea ae OOO 1,174,000 


The larger number found in the irrigated soil is due to the pres- 
ence of a better supply of organic matter and not to the moisture 
supplied. 

Kinds of Microérganisms in Soil.—The work so far done in this 
field clearly establishes the fact that soils have a definite bacterial 
flora as do water and cheese. The work done on the soil so far is 
meager and has been carried on by a few investigators— Hiltner and 
Stérmer in Germany, and Chester, Harding, and Conn in this 
country. By far the best and most extensive piece of work is that 
of Conn, and it is on his work that the main points of the following 
are based. 

Hiltner and Stérmer found that normally soil contains 5 per cent. 
of liquefiers, 70 per cent. of non-liquefiers, and 20 per cent. Strepto- 
thrix. The 5 per cent. liquefiers include the B. subtilis and Ps. 
fluorescens groups. Chester showed that the relative abundance of 
these three groups is nearly constant in normal soil and that any 
external influence which disturbed the equilibrium of the soil flora 
would be indicated by a change in the relative abundance of these 
three groups. This conception of the soil microérganisms as being 
normally in a state of equilibrium has proved of considerable value 
in interpreting soil phenomena. 

Conn divides soil bacteria into the following groups: 


1. Spore-producers : 

2. Non-spore-producers “Rapid liquefiers” 
Liquefaction rapid 
Liquefaction slow or none 

Rods 
Yellow chromogenic “Slow growers” 
Non-chromogenic 
Cocci 
3, Actinomycetes Actinomycetes 


CULTURAL CHARACTERISTICS 165 


He found the relative number of these organisms occurring in 
soils to be as follows: 

1. From 5 to 10 per cent. spore-formers (the B. swbtilis group). 
Nearly all the colonies of these bacteria, however, seem to come from 
spores instead of from active organisms. 

2. Under 10 per cent. rapidly liquefying, non-spore-forming, 
short rods with polar flagella (principally Ps. flworescens). 

3. From 40 to 75 per cent. slowly liquefying or non-liquefying, 
non-spore-forming, short rods. 

4. A few micrococci. In cultural characteristics these are almost 
identical with the last mentioned group. 

5. From 12 to 50 per cent. Actinomycetes. 

The most abundant spore-formers found in the soil and described 
by Conn were B. megatherium De Bary, B. mycoides Fliigge, B. 
cereus Frankland, and B. simplex Gottheil. 

“B. megatherium De Bary, 1884.—This species is to be distin- 
guished from B. mycoides and B. cereus by the larger average size 
of its spores, by its poor growth in liquid media, its failure to grow 
in the closed arm of fermentation tubes of dextrose broth, and by 
its comparatively slow liquefaction of gelatin. 

“Morphology.— Very young cultures (under twelve hours) consist 
of large rods about 1 to 1.5 w in diameter and about 3 to 6 yu 
long. They often occur in chains with connecting threads be- 
tween the rods, resembling strings of sausages. In older cultures 
the rods generally become swollen and are sometimes full of highly 
refractive globules (fat drops?). One of the most distinctive 
characteristics is the presence in cultures a day or more old of 
large ovoid bodies about 2 by 4u in size, which seem to have 
heavy walls and stain much more lightly than the young rods. 
Only the young rods are motile, and they are not vigorously so. 
The flagella are difficult to stain, and the best preparations made 
show comparatively few flagella on each rod. Spores are formed in 
the center of the rods and immediately become free from all trace of 
the sporangium wall. They are oval to ovoid (or occasionally 
reniform) and vary considerably in size, from 1.3 to 2 « in diameter 
and from 1.5 to 3 u in length, both extremes often occurring in the 
same preparation. 

“Cultural Characteristics.—Growth in broth flocculent or none, 
with no surface growth. Gelatin colonies under 10 mm. in diameter, 
center white, opaque, flocculent or granular, surrounded by a clear 
liquefied zone. Growth on agar streak cultures smooth, soft, glisten- 
ing, cream-color, typically with minute drop-like areas of lighter 
color. 

“Physiology.—'The typical group number is B. 111.4442074. As 
indicated by this group number, there is ordinarily no growth in 
sugar and glycerin broths. This does not mean, however, that no 


166 THE SOIL FLORA 


acid is produced from sugar or glycerin. Some cultures have pro- 
duced growth and have acidified one or even all of the sugars. This 
suggests that the irregularity may be due to the poor growth in 
liquids, tested on “slants” of litmus agar, in fact, B. megatherium 
has been found to produce acid from dextrose and sucrose quite 
regularly. Similarly, its poor growth in broth raises a doubt as to 
whether the second figure of its group number (denoting it to be a 
strict aérobe) may be correct; for it grows so poorly even in the open 
arm of a fermentation tube that its failure to grow in the closed arm 


4. QQ00000 


Fic. 24.—B. megatherium. X 1000 diameters. (After Conn) 


does not necessarily prove its inability to grow in the absence of 
oxygen. It is also possible that its failure to reduce nitrates may 
be due merely to the fact that it grows poorly in nitrate broth. 

“B. mycoides Fliigge, 1886.— This is the most easily recognized of 
all the soil bacteria. It can readily be distinguished by its rhizoid 
growth on agar. 

“Morphology.— Young cultures consist of rods about 0.8 to 1.3 by 
2to 6 y. They occur in long chains which often lie parallel and 
show false branching, an arrangement which gives the colonies 


rw 
REBIR 
on. Er 
oD oom 
(ep Oe 
0600066 
Fie. 25.—B. mycoides. XX 1000 diameters. (After Conn) 


their rhizoid structure. The very young rods are apparently 
slightly motile, but no success has been obtained in staining flagella. 
Gottheil describes several peritrichic flagella. In older cultures, 
highly refractive globules that do not take ordinary stains (probably 
fat drops) appear within the rods, particularly if growing on dex- 
trose agar, sometimes causing the rods to swell to extremely large 
size and to lose all resemblance to their original form. The lightly 
stained ovoid bodies that characterize B. megatheriwm have never 
been observed. Spores are borne centrally and the remnants of the 


CULTURAL CHARACTERISTICS 167 


sporangium wall persist for some little time at either end of the 
spores. Spores are oval to cylindrical 1.0 to 1.6 by 2 to 2.5 uw often 
in fairly long chains. 

“Cultural Characteristics.—Growth in broth vigorous, flocculent, 
with no persistent surface growth. Gelatin colonies rapidly liquefy- 
ing, filamentous to rhizoid. Growth on agar streak rhizoid, mostly 
beneath the surface of the medium. 

“Physiology.—The typical group number is B. 121.23230?2. It 
shows less variation than does the group number of B. megatheriwm, 
probably because B. mycoides grows better in the media used for 
making the tests. The same acid reactions are obtained in broth 
culture and on litmus agar. 

“B. cereus Frankland, 1887.— This type can be distinguished 
from B. megatheriwm by the smaller size of its spores and by its 
more vigorous growth in liquid media, and from B. mycoides by the 
absence of rhizoid growth on agar. 


SQ0RDOCO 
00000 
COS 


Fic, 26.—B. cereus.  X 1000 diameters. (After Conn) 


‘“Morphology.—In morphology it is scarcely to be distinguished 
from B. mycoides. Young rods are 0.8 to 1.3 by 2. to 6yu form- 
ing long chains, but unlike B. mycoides they are very actively 
motile and are easily shown to be surrounded with numerous flagella. 
Older rods are often swollen and contain unstained globules. Oval 
to cylindrical spores, 1.2 to 1.6 by 2. to 2.54, are produced cen- 
trally, retain the remnants of the sporangium wall for a short time 
and often cling together in chains. — 

“Cultural Characteristics.— Growth in broth vigorous, with uniform 
turbidity, sediment and a surface pellicle. Gelatin colonies quite 
large, ordinarily round, with entire margin, and covered with a 
pellicle that generally shows concentric rings, although under some 
conditions the colonies are filamentous and resemble those of B. 
mycoides. Growth on agar streak raised, ordinarily rugose, soft 
to membranous, generally dull; never rhizoid or beneath the surface 


168 THE SOIL FLORA 


of the medium like B. mycoides or full of clear drop-like areas like 
B. megatherium. 

“Physiology.—The typical group number is the same as that for 
B. mycoides, B. 121.23230?2, although considerable variation has 
been observed, particularly in the production of acid from sugars 
and from glycerin. In the earlier work, two subtypes were recog- 
nized, basing the distinction upon the production of acid from 
glycerin; but no such distinction is now recognized, because of the 
inconsistent results obtained in regard to acid production. Dextrose 
is always acidified by this organism; sucrose is generally acidified; 
glycerin less frequently, and lactose very seldom. A strain that 
acidifies lactose has always been found to produce acid from all 
three of the other compounds. The production of acid from lactose 
may be a better basis for subdividing the type than acid-production 
from glycerin. Out of 130 cultures studied, 19 acidified lactose; 
but it seems unwise to consider them as constituting a separate 
species, in view of the variation that has been found when cultures 
have been retested. 

“Although many of the spore-formers are active ammonifiers in 
solution and occur in soils in comparatively large numbers, yet 
it is doubtful if they play any very important role in soil fertility. 

“Although of considerable importance, except for the nitrifiers and 
some other organisms concerned with the transformation of nitrogen, 
scant consideration has been given to any non-spore-forming bac- 
teria found in soil.” 

Ps. fluorescens which belongs to this group is described by Conn 
as follows: 

“Ps. fluorescens (Flugge) Migula.—The most striking character- 
istic of this type is its fluorescence, which is observed in broth, 
beef-extract-peptone agar, and sometimes in gelatin. Ability to 
produce fluorescence is often lost, however, and then the type must 
be recognized by other characteristics, such as rapid liquefaction 
of gelatin, uniform turbidity in broth, cloudy, structureless colony 
in gelatin, and acid production from dextrose. 

‘“Morphology.— Rods 0.4 to 0.8 by 0.8 to 1.5 microns in old cultures 
nearly the same shape and size as in young cultures. Flagella 3 to 
6, arranged in a clump at one pole. Motility great. Rods do not 
form chains. 

“Cultural Characteristics. — Good growthin broth; no surface growth, 
uniform turbidity, causing distinct cloudiness of medium; sediment 
scant or none. Gelatin colonies liquefying with great rapidity; 
round to irregular in shape, cloudy, structureless, occasionally 
fluorescent. Growth on agar streak cultures, smooth, soft, glistening 
generally causing the medium to show a green fluorescence. 

“Physiology.—The typical group number is Ps. 211.2332138. 
Fairly consistent results can be obtained in determining its group 


CULTURAL CHARACTERISTICS 169 


number, although certain variations occur. Occasionally there is 
no acid from dextrose. Some of the cultures reduce nitrates. The 
power of producing fluorescence is often lost. These variations may 
indicate the existence of separate species that are now grouped 
under this one head.” 

Actinomyces from 12 to 50 per cent. of the organisms found in 
soil are the Actinomycetes. This genus, the Actinomyces Harz, em. 
Gasperini, is characterized by the possession of a mycelium composed 
of hyphz which show true branching, like those of the higher fungi 
(seldom measuring over 2 microns in diameter), and judging by 
their staining reactions resemble true bacteria in their protoplasmic 
properties. Their growth is not wholly within the agar or gelatin 
medium upon which they have been inoculated; for when condi- 
tions favor, an aérial mycelium is produced. In the aérial mycelium 
“conidia” are formed. These conidia are sometimes round, some- 
times oval, and sometimes rod-shaped. They resemble bacteria 
closely in size, shape, and staining properties. They are generally 
between 0.6 and 1.5 microns in diameter, and if oval or rod-shaped, 
between 1 and 2 microns long. They stain readily with ordinary 
bacterial stains, and in a microscopic preparation which does not 
contain any hyphe, often cannot be distinguished from true bac- 
teria. In many cases deep-stained granules show at the poles, 
strongly suggestive of the metachromatic granules of the diphtheria 
organism. According to Sanfelice, some of the actinomycetes are 
acid-fast like the tubercle organism. The diphtheria and tubercle 
organisms, moreover, sometimes produce branching forms, and some 
writers place these two organisms in the same group with Actino- 
myces. 

The growth of actinomycetes on solid media is very characteristic. 
The mass of growth is generally of a tough, leathery consistency, 
sometimes smooth, sometimes wrinkled, and often piled high above 
the surface of the medium, Often the mass is brilliantly col- 
ored, and the color produced varies greatly with differences in the 
composition of the medium, but with constant composition of the 
medium, the color of the growth may be characteristic of the species. 
The aérial mycelium, often produced above this growth, may also 
be brilliantly colored and of an entirely different color from the mass 
of growth beneath it. Sometimes the aérial hyphe are short and 
give the growth a chalky or mildewy appearance; but often they are 
long enough to cover the growth with a light, delicate nap, 1 or 2mm. 
thick. Some species produce pigments that diffuse through the 
medium, coloring it gray, yellow, brown, red, blue, or green. The 
color varies with the species and with the composition of the medium. 
It is not so definitely characteristic of the species as is the color of 
the growth itself or of the aérial mycelium; but with a medium of 
constant composition, the color produced is of considerable value 


170 THE SOIL FLORA 


in the recognition of species. On gelatin there is less diversity of 
growth than on agar. The growth is generally gray, brown, or 
colorless; the aérial my celium i is often lacking, and if present is 
white, gray, or colorless; and if the medium itself is colored, it 
generally becomes a reddish brown. 

The growth in liquid media is also characteristic. The medium 
remains Clear except for small colonies that may sink to the bottom, 
remain in suspension, float on the surface, or adhere to the walls 
of the tube. The surface colonies often grow together and become 
covered with a mass of aérial mycelium, sometimes forming a firm, 
wrinkled membrane that strongly suggests the surface membrane 
of the tubercle organism growing on broth. Pigments are often 
produced in liquid culture, the pigment varying with the composition 
of the medium and with the species growing in it. 

Nearly all liquefy gelatin and ammonify proteid, Miinter main- 
taning that ammonification is their chief function. Nitrate reduc- 
tion has often been observed, as has the decomposition of cellulose. 
Some are animal pathogens, and at least one a plant pathogen. 
Other important physiological activities will undoubtedly be worked 
out when the technic for studying them is further developed. It 
is not impossible that they are as diverse in physiology as are the 
true bacteria. 

One of the most common characteristics of many members of this 
group is their peculiar odor. They have a pungent, musty odor, 
difficult to describe, but impossible to mistake after once having it 
brought to the attention. It is sometimes spoken of as an earthy 
odor, but it would be more correct to say that soil often has an 
actinomyces-odor, as the odor of the cultures is much stronger than 
that of soil, and the soil odor is undoubtedly due to the actinomy- 
cetes it contains. The odor seems to be associated with the aérial 
conidia, and does not seem to be produced by cultures that do not 
possess aérial mycelium. Not all species of Actinomyces have this 
odor, however, even when an abundant aérial mycelium is produced. 

Various functions have been ascribed to the actinomycetes— 
ammonification, nitrate reduction, and cellulose-decomposition— but 
enough work has not been done to enable a definite statement as 
to whether these are their functions in the soil. Conn has demon- 
strated that the addition of grass roots to a soil materially increases 
this group of organisms and Waksman and coworkers consider 
that inasmuch as the actinomyces are strong cellulose decomposers 
and weak producers of ammonia their probable role in soil fertility 
lies in the formation of humus. 


REFERENCES. 


Conn, H. Joel: Soil Flora Studies, New York Agr. Exp. Sta. Tech. Buls., 57, 58, 59, 
and 60. 

Waksman, Silman A.: Cultural Studies of Species of Actinomyces. Soil Science, 
1919, viii, 71-215. 


CHAPTER XVIiL 


MINERALIZATION AND SOLVENT ACTION OF 
BACTERIA. 


SoILs are the earthy material in which plants have their anchorage, 
and from which they obtain their water and part of their food. 
They are in reality disintegrated rock intimately mixed throughout 
with varying quantities of decaying plant and animal residues. 
They are derived from the native rocks by a complex process known 
as weathering. ‘The agents at work to bring this about are changes 
of temperature, the action of air, water, ice, and plant and animal 
life. 

Bacteria as Soil Formers.—Early in the history of soil formation 
bacteria appear and play an essential part in rendering the soil 
fertile. Their life activities result in the production of carbon 
dioxid, organic and inorganic acids, and alkalies. These in turn 
react with the constituents of the rock particles, thereby changing 
their solubility. When water becomes charged with substances 
from the soil, its solvent powers are greatly increased. Especially 
is this true when it becomes filled with carbon dioxid either from the 
atmosphere or from the decay of plants and animals. 

Common limestone is one of the rocks most actively attacked by 
carbonated water; none are wholly resistant to its action. Even 
quartz is slowly dissolved. Granite and related rock are rather 
quickly acted on by water due to the feldspar minerals which it 
contains. The bases—potash, soda, lime, and alumina—are 
dissolved out. ‘The last is deposited as clay, the first as beneficial 
or injurious soil constituents, depending on the kind and the con- 
centration left in a particular soil. In a similar manner the sulphur, 
iron, and phosphorus of the soil are changed to available forms. 

Moreover, bacteria play a very important part in the mineraliza- 
tion of plant and animal residues which continually find their way 
into the soil. The phosphorus, sulphur, iron, calcium, magnesium, 
and potassium in the plants and animals are mainly in the form of 
organic compounds and as such are not available to other plants. 
Bacteria act upon them, liberating carbon dioxid, ammonia, hydrogen 
sulphid or sulphur with the liberation of the plant-food. In this 
manner, the biological activities become of the utmost importance in 
the transformation and migration of mineral substances in Nature. 
The bacteria act merely as the link between the living and the dead. 


172 MINERALIZATION AND SOLVENT BACTERIA 


Calcium and magnesium occur in soils mainly as carbonate, sul- 
phate, silicate, and as the cation in the salts of organic acids which 
have resulted during the breaking down of the organic plant and 
animal residues. 

Soil bacteria in their life processes are continually forming large 
quantities of carbon dioxid, nitrous, nitric, and sulphuric acid, 
together with organic acids which are in the main combined with 
calcium or magnesium of the soil, with the formation, often, of a 
soluble compound. ‘The waters carry these to the lakes, seas, and 
oceans—there to be taken up by marine life. In the course of time 
these are deposited as coral reefs, chalk cliffs, and marl beds. At 
times the speed with which the lime is taken from the waters by 
marine life is faster than it is carried into a lake by its tributaries. 
The result is that, in spite of the evaporation and concentration 
which is going on, the main body of water contains less lime than 
does its tributaries. This is the case with Bear Lake, Utah, the 
tributaries of which have an average lime content of 101.7 parts 
per million, whereas the lake contains only 13.2 parts per million. 

Calcium Carbonate.—The loss of calcium carbonate from a soil 
varies with (1) the methods of agriculture, intense methods increas- 
ing the loss; (2) with the application of animal manures and green 
manures, which increases the bacterial activity and also the solubility 
of the calcium carbonate; (3) with the addition of commercial fertil- 
izers added to a soil which hasten the loss of calcium in drainage 
water. 

The carbon dioxid generated by bacteria reacts with the calcium 
carbonate forming the much more soluble calcium bicarbonate: 


or 
CaCOzs + COz2 + H20 = Ca(HCOs)2 


. Ammonium sulphate resulting either from ammonification or from 
the addition of a fertilizer changes the calcium from an insoluble 
to a soluble form: 

(NHa)2SO4 + 2CaCOz + 402 = Ca(NOs)2 + CaSO, + 4H2O + 2COs2z 
(NHi)2S8O, + CaCO; = (NHs)2CO; + CaSOsz 


The addition of acid phosphate or potassium chlorid also helps 
deplete the soil of its calctum carbonate: 
CaHa(POs)2 + 2CaCOzs = Cas(POs)e + 2H20 + 2COr 
2KCl + CaCOs3 = K:COs3 + CaCle 


The absolute amount of calcium and magnesium lost from a soil 
varies with the aridity of a region as well as with the composition 
of the soil. Hall estimates that the annual loss from the Rotham- 
sted soil, which contains about 3 per cent. of calcium carbonate, is 
from 800 to 1000 pounds an acre annually, whereas in some parts 


PHOSPHORUS 173 


of Scotland, where liming has been practised for some time, the loss 
is from 500 to 600 pounds. Inthis country, where liming is necessary, 
the farmers usually provide for a loss of 400 pounds an acre annually. 

Bacteria are also responsible for the restoration of varying 
amounts of carbonates. In the weathering of complex silicates, 
carbonates and silicic acid may be formed in considerable quantities: 


CaAlSix0s + COz2 + 2H20 = AbSPO;(OH)s + CaCOs 


According to Nadson, soil bacteria may cause the formation of 
calcium carbonate from calcium sulphate through the reacting of 
ammonium carbonate formed in the decay of protein substances 
with calcium sulphate: 


(NHa)2COs + CaSO, = (NH4)2S0O, + CaCOs 


Or even after the sulphate has lost its oxygen through the action of 
reducing bacteria, calcium carbonate may be formed through the 
action of carbon dioxid and water on the calcium sulphid: 


Case  CO:) —) HsO> — CaCO; -— Eas 


Denitrifying bacteria may act on calcium nitrate with the forma- 
tion of calcium carbonate: 


2Ca(NOs)2 + 2CO2. — 2CaCOzs + 2Ne + 502 


Calcium carbonate may also be formed in the soil due to the 
action of bacteria upon humates and calcium salts of simpler organic 


acids: 
(RCO0):Ca. =: CaCO; + RCOR 


Cunningham has demonstrated that Azotobacter chroécocewm is 
capable of growing in solution of calcium oxalate with the formation 
of calcium carbonate, as were also six other types of organisms 
isolated by him. The presence of oxygen is essential for the process. 
He considers that an equilibrium is set up by which the withdrawal 
of calcium carbonate is balanced by the results of another set of 
reactions which restores the base to the soil. This enables many 
soils which contain only very small quantities of lime to retain their 
neutral reaction and so to produce fair crops. This, however, is not 
always the case, as is witnessed by the acid soils occurring in many 
agricultural districts. 

Phosphorus.— Phosphorus occurs mainly in the form of the calcium, 
iron, or aluminum phosphate; in any soil the quantity soluble is 
small. Moreover, as soluble phosphorus compounds are applied to 
the soil they become fixed as insoluble compounds. Hence, the loss 
through leaching of this element from the soil is small under any 
conditions. 


174 MINERALIZATION AND SOLVENT BACTERIA 


There are also varying amounts of organic phosphorus in soil. 
This occurs in the form of lecithin, phospho-proteins, and nucleo- 
proteins. Little has been done to determine the action of bacteria 
upon these compounds, but it is to be expected that they would be 
hydrolyzed by bacteria as they are by ferments. 

Lecithin yields on hydrolysis glycerin, two molecules of fatty 
acid, phosphoric acid, and cholin: 


CwHssaNPOos + 4H20O = CisHsuO2 + CisH2O2 + CsHsO3 + 
lecithin water oleic acid palmitic acid glycerol 


H3PO, + CsHisNO2 
phosphoric acid cholin 


The phospho-proteins yield on hydrolysis amino-acids and phos- 
phoric acid, whereas hydrolytic cleavage produces from nucleo- 
proteins carbohydrates, phosphoric acid, purin and pyrimidin bases, 
with the intermediate formation of nucleins and nucleic acid, as 
may be represented by the following scheme: 


nucleo-proteins 
l 


= 
proteins nucleins 

ia a 
proteins nucleic acid 

is fea in FUl-y. + a BP | 
carbohydrates phosphoric purin bases pyrimidin bases 
pentoses acid adenin thymin 
hexoses guanin cytosin 
unidentified xanthin uracil 

hypoxanthin 


Schettenhelm has shown that nearly all of the nuclein substances 
of feces disappear as they undergo autoputrefaction. He and 
Schroeter showed that bacteria may bring about a deep cleavage of 
yeast nucleic acid. Plenge showed that some bacteria have the 
power to liquefy the sodium salt of nucleic acid from thymus. 

It seems reasonable, therefore, to believe that phosphorus would 
be liberated by soil bacteria in a somewhat similar manner. It is 
known that the bacterial flora of the soil play a highly important 
role in rendering the phosphorus of the inorganic phosphates avail- 
able to the higher plant. 

Brown found that twelve out of twenty-three bacteria isolated 
from soil exerted a definite solvent action on difficultly soluble 
plant-food. One organism which produced no gas but a large 
amount of acid showed the greatest solvent action upon calcium 
carbonate, whereas other organisms which produced gas—largely 
carbon dioxid—but not as much acid as the former, gave an action 
more marked than that of the stronger acid-producer upon the 
dicalcium and tricalcium phosphates. B. subtilis, B. mycordes, 
B. proteus vulgaris, and B. coli communis, as well as several agar 
cultures from garden soil, were found to be capable of dissolving 


PHOSPHORUS 175 


the phosphates of bone and to a less extent that of mineral phos- 
phates. The greatest solvent action was exerted in media contain- 
ing sodium chlorid, potassium sulphate, and ferrous sulphate. Even 
yeast may be important in dissolving phosphates. As suggested 
by Krober the life activity of the bacteria, that is, assimilation of 
phosphorus by the living organism, probably plays little or no direct 
part in dissolving the phosphates, but it is due to the action of the 
organic acids and of the carbon dioxid produced. 

The acids produced by bacteria act upon all phosphates, convert- 
ing them into the soluble monophosphate, but the rate of solution 
varies widely with the different phosphates. Tricalcium phosphate 
in precipitated form, dicalcium phosphate, and tetracalcium phos- 
phate of Thomas slag are much more rapidly dissolved than the 
crystalline or the so-called amorphous phosphates. The general 
reaction is as follows: 


2RCOOH + Cas(PO.s)2—>Ca2H2(POs)2 + (R COO):Ca 


The reaction takes place most rapidly in soils containing large 
quantities of organic matter due to the active fermentation taking 
place in such soils. 

Grazia considers enzyme action to play a part in the dissolving 
of phosphates in soil, for he found the addition of chloroform to a 
soil reduced bacterial activity and decreased the acid produced, but 
at the same time the solution of phosphates was increased. This is 
in keeping with the finding of Bychiklin. 

The presence of ammonium chloride and sulphate in the cultural 
media is especially effective in increasing the solvent action of 
bacteria, according to Perotti, who considers the successive steps 
in the solution or decomposition of phosphorus compounds by 
bacteria asfollows: (1) generation of acids, (2) secondary reactions 
in the solution, and (3) production of a soluble phosphorus contain- 
ing organic substance. The first two of these are the.result of 
the activity of the bacteria on the phosphorus, and the last is due 
to the metabolic assimilation of the microérganisms. 

The oxidation of sulphur by soil bacteria may at times generate 
sufficient acid to play a very important réle in dissolving soil phos- 
phorus. Hopkins and Whiting, however, consider that the nitrite 
bacteria are of the first importance in rendering phosphorus and 
calcium soluble when they oxidize ammonia into nitrites: 


(NH4)2COs + 3802 = 2HNO. + HCO; +. 2H.O 


The resulting nitrous acid then reacts with the raw rock phosphate, 
rendering it soluble, thus: 


Ca3(PO4)2 + 4HNO. = CaHa(PO4)2 + 2Ca(NOz)e2 


176 MINERALIZATION AND SOLVENT BACTERIA 


The actual ratio found showed that about one pound of phos- 
phorus and about two pounds of calcium are made soluble for each 
pound of nitrogen oxidized, aside from the action of the acid radicals 
associated with the ammonia. The carbonic acid would play an 
important part also in this reaction: 


4H2CO3; + Cas(POs)2 = 2Ca(HCOs)2 + CaH,(POs)2 


They found that neither ammonia-producing bacteria nor nitrate 
bacteria liberated appreciable quantities of soluble phosphorus from 
insoluble phosphates. 

Whereas this would readily occur in soil poor in calcium carbonate, 
in those rich in calcium carbonate there would be only small quanti- 
ties of phosphorus liberated, according to Kelley. But where the 
soluble phosphorus is being rapidly removed by the growing plant, 
or even by bacteria, there is little doubt that the various soil organ- 
isms play an important part in rendering phosphorus soluble, for 
results obtained at the Utah Experiment Station show there to bea 
relationship between the increased nitrification produced by various 
salts, and the quantity of water-soluble and organic phosphorus in 
the soil. This is illustrated by the following results which give the 
nitric nitrogen, water-soluble and organic phosphorus in a soil after 
various treatments, the untreated soil being considered as 100 per 
cent. 


PER CENT. NITRIC NITROGEN WATER-SOLUBLE AND ORGANIC PHOS- 
PHORUS OCCURRING IN SOIL RECEIVING VARIOUS SALTS. 


Water- 

Nitric soluble Organic 

nitrogen. phosphorus. phosphorus. 
Treatment. Per cent. Per cent. Per cent. 
None ta ait eel none tees EeLOO RO 100.0 100.0 
312 x 10-, mol. MgSO, . . . 101.2 105.2 PG 
26'x 10-7 mol. Fes(SOs)3 eee O20 94.3 142.3 
625 x 10-7 mol. Ca(NOs)2 «=O -«~Ss «102.0 114.3 97.5 
156x 10-; mol. KNO; - . . 106.4 108.1 103.3 
625° 10—; molOKCl . Teen =. W0GrD 105.8 107.3 
312 x 10-7 mol. Mg(NOs)2 . . 106.5 115.5 95.1 
125x10-. mol. MnCOz:. . . 108.4 107.5 162.6 
156x10-7mol. MnCle . . - 112.9 100.2 98.4 
78x10-7mol. MnSQ, . . . 1138.2 94.3 107.9 
13 xi10=.anolsHeCOs se. aoe DH S4 105.6 94.8 
25x10-;mol. MgCl . . . 1238.2 109.7 96.5 
625 x 10-7 mol. Mn(NOs)2 . . 125.4 84.8 87.9 
84 x 10-5 mol. FeCls ty an loses 105.6 94.8 
295x10-; mol. MgCOs . . . 140.7 98.2 72.2 
1 x 10-3 mol. NaCl sie te ELA ZO 109.3 138.7 
1 x 10-3 mol. CaCly A el Gin, 114.9 88.2 
Duy 10 san Ol LORS Oa Etre n mlo oer. iaeo 103.3 


Moreover, it is evident that Azotobacter in their metabolism trans- 
form soluble inorganic soil constituents either into soluble or into 
insoluble organic forms. This is especially true of phosphorus which 


PHOSPHORUS bay 


is found in the ash of these organisms in such large quantities. ‘The 
phosphorus, on the death of the organism, would be returned to the 
soil in a readily available form, for Stoklasa has found that 50 per 
cent. of the nitrogen of these organisms is nitrified within six weeks, 
and there is no reason for believing that the phosphorus would be 
liberated much more slowly. Then there is the possibility that many 
of the constituents of the bacterial cell may become available, 
through the action of autolytic enzymes without the intervention 
of other bacteria. 

It is further evident that an organism which possesses the power, 
when growing under appropriate conditions, of generating 1.3 times 
its own body weight in carbon dioxid during twenty-four hours, as 
does the Azotobacter, must greatly change the composition of the 
media in which it is growing. Water charged with carbon dioxid 
is a universal solvent and will attack even ordinary quartz rock. 
Granite and rocks related to it are rather quickly attacked, with the 
liberation of potassium and other elements. Carbonated water 
would act upon the tricalcium phosphate of the soil with the forma- 
tion of more readily soluble phosphates, for this substance is four 
times as soluble in water charged with carbon dioxid as it is in pure 
water: 


Caz(POs2 + 2CO2 + 2H2:0 = CaszH2(POs)2 + Ca(HCOs)2 


Moreover, the nitrogen-fixing organisms form, among other 
products, formic, acetic, lactic, butyric, and other acids. ‘The kind 
and quantity of each depends upon the specific organisms and upon 
the substance on which they are acting. ‘These substances are sure 
to come in contact with some insoluble plant-food which may be 
rendered soluble, for they have a highly solvent action on the 
insoluble phosphates. The resulting salts of calcium would be 
further attacked by bacteria, with the formation of calcium car- 
bonate. 

Whether these processes will give rise to an increase in the water- 
soluble plant-food of the soil depends upon whether the products 
of the second, the analytic reactions, exceed the products of the 
first, the synthetic reactions. It must not be forgotten that, 
although many of the organic phosphorus constituents may not be 
soluble in pure water, they may be more available to the living plant 
than are the constituents from which they were at first derived 
through bacterial activity. . 

This being the case, variations in the results reported from 
laboratory tests are to be expected. Stoklasa found that bacterial 
activity rendered the phosphorus of the soil more soluble, whereas 
Severin, in his early work, found the opposite to be true. Others 
have found that the solvent action of bacteria for insoluble phos- 
phates is in direct proportion to the acid secreted by the organism. 

12 


178 MINERALIZATION AND SOLVENT BACTERIA 


In a later work, Severin obtained different results. He used three 
soils—one sterile, a second sterilized and inoculated with pure 
cultures of Azotobacter, and a third sterilized and inoculated with 
cultures of Ps. radicicola and Azotobacter. The solubility of the 
phosphorus increased 8 to 14 per cent. over that in the sterile soil. 
The acid-producing organisms, due to the acid secreted and their 
intimate contact with the soil particles, possess the power of dissolv- 
ing silicates. Moreover, arsenic greatly stimulates nitrogen fixa- 
tion, and there is a relationship between this increased bacterial 
activity and the form and quantity of phosphorus found in a soil. 

Although the metabolic activity of Azotobacter gives rise to large 
quantities of phosphate solvents, yet these organisms transform 
phosphorus into organic phosphorus compounds less rapidly than 
do the ammonifiers. There are, however, cases in which bacterial 
activity has decreased the water-soluble phosphorus of the soil and 
of raw rock phosphate. This does not mean, however, that it is 
less available, for, as pointed out by Truog, the mixing of floats with 
manure caused an immediate decrease in the solubility of the phos- 
phorus in 0.2 per cent. citric acid solution, yet when thoroughly 
mixed with the feeding area of the soil its availability was increased 
to such an extent that some species of plants were apparently able 
to secure almost an adequate supply of phosphorus from this 
material. ‘The addition of manure to a soil greatly increased the 
carbon-dioxid production, and for a short time measurably increased 
the solvent action on floats. Where there is for-a time a decrease 
of water-soluble phosphorus in fermenting media, it is probably due 
to the formation of phospho-proteins within the bodies of the 
bacteria which would later be rendered soluble due either to further 
bacterial activity or to autolytic enzymes. 

Sulphur.—Sulphur is an essential element for all plants, but the 
quantity required is relatively small and most soils contain sufficient 
for maximum crop preduction. It occurs within the soil mainly as 
sulphate or organic sulphur, and these substances are often materially 
changed by bacterial activity. 

Bacteria act on sulphur compounds in three ways: (1) on complex 
organic compounds with the production of hydrogen sulphid or 
mercaptans, (2) the oxidation of sulphur compounds occurring in the 
soil, and (3) the oxidation of sulphur compounds, especially hydrogen 
sulphid by the true sulphur bacteria, with the production of metallic 
sulphur, sulphuric acid, and eventually mineral sulphates. 

Hydrogen sulphid is produced by the majority of the common 
laboratory forms of bacteria. Lafar states that this faculty is even 
very common among the pathogenic bacteria and was absent in not a 
single one of 37 species examined. Other bacteria possess the 
power of reducing sulphates. Beijerinck found inf{soil an organism 
which he named Spirdllum desulphuricans and which Van Delden 


SULPHUR 179 


later classified as Microspira desulphuricans which possessed the 
power of reducing sulphates. Another sulphate-reducing organism 
is) Msp aestuarti. These organisms act only in the presence of 


organic matter: 
MSO, + 2C = 2CO. + MS 


The true sulphur bacteria possess a directly opposite physiological 
action to the reducing bacteria. There are two genera of the 
true sulphur bacteria recognized— Beggiatoa and Thothrix. Beggia- 
toa is filamentous, motile, and morphologically resembles the blue- 
green alga, Oscillaria. Thiothrix is not filamentous nor motile and 
possesses a sheath and forms spores. The sulphur bacteria contain 
in their protoplasm highly refractive inclusions of amorphous sulphur. 
According to Winogradsky, a single Beggzatoa thread used in a day 
two to four times their own weight of hydrogen sulphid with the 
production of sulphur: 


4H29S + 202 = 4H20 + 458 


The sulphur seen within the cell protoplasm is to be looked upon as 
an intermediate state in the oxidation process, for if the organisms be 
transferred to fresh water these soon disappear with the formation 
of sulphuric acid: 

2S -+ 30:2 + 2020 = 2H SO, 


This reacts with a base, usually calcium carbonate, with the forma- 
tion of calcium sulphate: 


CaCOs3 + HeSO4 = CaSO.4 + H:0 +t. CO. 


There arealso organismsin soil that can oxidize sulphur to sulphuric 
acid which in turn would act as a solvent for plant-food. Moreover, 
small quantities of sulphur added to a soil will increase ammonifica- 
tion. It is likely that much of the benefit resulting from sulphur 
fertilization is due to these factors. 

Brown has recently shown the power of oxidizing sulphur to vary 
with different soils. Aération and optimum moisture favor it, 
whereas the addition of carbohydrates, depresses the process. He has 
elaborated a method of measuring the speed of sulphur oxidation in 
soils and given to it the name of sulphofication. 

According to Lafar, the importance of the sulphur bacteria in the 
economy of nature is unmistakable. In codperation with the sulphate- 
reducing bacteria they insure that the sulphur cycle pursues an un- 
interrupted course, the elements being taken up by the higher plants 
in the condition of sulphates and deposited in the cells in the torm of 
organic compounds from which, in the course of putrefaction, sulphur 
is liberated as hydrogen sulphid, and finally reconvertedinto sulphates 
by the sulphur bacteria. It then recommences its course through the 
higher plants. 


180 MINERALIZATION AND SOLVENT BACTERIA 


Iron.— The iron bacteria resemble the sulphur bacteria greatly in 
their metabolic activity. The best known of these organisms are 
the Crenothrix polyspora, Chlamydothrix ochracea, and Spirophyllum 
ferrugineum. Winogradsky considers that the iron is deposited in the 
sheath of the organisms due to a physiological reaction, the organ- 
isms oxidizing ferrous to ferric compounds: 


4FeCO; + 6H20 + Of = 2Fe(OH)s + 4COr 


The energy so liberated is utilized in their growth. However, the 
investigations of Molisch, Adler, and Ellis show that they grow well 
in a medium devoid of iron and that the precipitation of the iron is 
due to chemical and mechanical processes independent of the physio- 
logical activity of the organism. They play a great part in the 
deposition of bog-iron, though not the only cause, for Molisch con- 
siders that well-known physio-chemical agencies often play an 
important part in the process. Manganese may at times be found 
in the sheath of Crenothrix, in large quantities. 

Potassium.— This element is required by all plants in compara- 
tively large quantities, and the total supply in nearly all soils is 
exceedingly large as compared to crop requirements. Yet potas- 
sium is quite extensively used as a fertilizer, and this with beneficial 
results. This is due to the fact that its addition to a soil well 
supplied with available potassium results in the liberation of other 
more deficient plant-food elements. Moreover, it may be applied to 
soils having a large quantity of total potassium, but a small quantity 
available to plants. Therefore, one of the problems which is con- 
fronting the farmer is how to render available as needed by plants 
the large supply of potassium in the soil. 

The potassium occurs in the soil mainly as silicates and is rendered 
soluble by the nitrous, nitric, sulphuric, acetic, lactic, and butyric 
acids, and by carbon dioxid. The last may react with inert potas- 
slum resulting i in the formation of available potassium according to 
the following equation: 


AloO3K20. 6Si02 fe CO2 + 2H20 — AlOs 2Si02 + 2H2O a 
K2COz; + 48i02 


Hence, the addition of animal manure, green manures, com- 
mercial fertilizers, or even soil amendments may increase bacterial 
activity and in a similar degree increase the soluble soil potassium. 


REFERENCES. 


Lafar, Franz: ‘‘ Handbuch der Technischen Mykologie,’’ Dritter Band. 

Greaves, J. E., Carter, E.G.: ‘‘The Action of Some Common Soil Amendments” 
(Soil Science, vol. vii (1919), pp. 121-160). 

Kossowicz, Alex.: ‘‘Agrikulturmykologie, I Bodenbakteriologie.”’ 

Ellis, David: Iron Bacteria, London, 1919. 


CHAPTER XV ITLL 


THE CARBON, NITROGEN, SULPHUR, AND 
PHOSPHORUS CYCLES. 


PLANTS contain ten essential elements, and these elements found 
in the body of the plants or animals today are the same as those 
which constituted the organic world thousands of years ago. But 
between these dates they may have played many parts, or, in the 
words of Duncan, ‘‘ We believe—we must believe in this day—that 
everything in the universe of world and stars is made of atoms, in 
quantities x, y, or z, respectively. Men and women, mice and 
elephants, the red belts of Jupiter and the rings of Saturn, are, one 
and all, but ever-shifting, ever-varying swarms of atoms. Every 
mechanical work of earth, air, fire, and water, every criminal act, 
every human deed of love or valor; what is it all, pray, but the rela- 
tion of one swarm of atoms to another? 

“Here, for example, is a swarm of atoms, vibrating, scintillant, 
martial—they call it a soldier— and, anon, some thousands of miles 
away upon the South African veldt, that swarm dissolves—dis- 
solves, forsooth, because of another little swarm—they call it lead. 

“What a phantasmagoric dance it is, this dance of atoms! And 
what a task for the master of the ceremonies! For, mark you, the 
mutabilities of things. These same atoms may come together again, 
vibrating, clustering, interlocking, combining, and there results a 
woman, a flower, a blackbird, or a locust, as the case may be. But 
tomorrow again the dance is ended, and the atoms are far away; 
some of them in the fever germs that broke up the dance, others are 
the green hair of the grave, and others are blown about the Antipodes 
on the wings of ocean, and the eternal everchanging dance goes on.” 

In this building up and breaking down, bacteria play an all- 
important part. The higher plants build up the carbon and nitrogen 
into complex organic compounds. This same end is also accom- 
plished to a lesser degree by the animals which, however, mainly 
act as analyzers of organic matter, but the master analysts are the 
bacteria which are continually resolving into simple and often 
elementary constituents, the plant and animal débris. Were this 
not true, all the carbon and combined nitrogen of the world would 
soon become locked up in the dead bodies of animals; plants would 
starve and die, and animals would likewise become extinct. There- 
fore, bacteria are the link between the living and the dead. ‘The 


\ 


182 THE CARBON, NITROGEN, AND PHOSPHORUS CYCLES 


absence of bacteria is incompatible. with life on this earth, or, as 
stated by Pasteur, “they are the important, almost the only, agents 
of universal hygiene. They clear away more quickly than the dogs 
of Constantinople or the wild beasts of the desert, the remains of 
all that has had life; they protect the living against the dead; they 
do more; if there are still living beings, if, since the hundreds of 
centuries the world has been inhabited, life continues, it is to them 
we owe it.” 

The Carbon Cycle.— Carbon occurs free in the earth as coal to the 
extent of over 500 billion tons. Chemically combined, it is found 
in far larger quantities in limestone, chalk, marble, and dolomite— 
rocks which form such a considerable portion of the surface of the 
earth. According to Pettenkofer, a man weighing 154 pounds 
contains 26.4 pounds of carbon; no less than 257 million tons’ weight 
of it 1s, therefore, stored up in the bodies of men and women living 
upon the earth at the present time, to say nothing of the far greater 
quantities occurring in the tissues of trees, plants, and lower animals. 

Carbon dioxid occurs in the atmosphere to the extent of three 
parts in 10,000. This is the equivalent of 600 billion tons of carbon. 
Moreover, the ocean is a vast reservoir of carbon dioxid, which is 
partly in solution and partly combined. Between the surface of 
the sea and the atmosphere there is a continual interchange, each at 
times losing and at times gaining the gas. 

Carbon dioxid is being added to the air from several sources: 
the combustion of fuel, the respiration of animals, and the decay of 
organic matter. It is also being evolved in enormous quantities 
from mineral springs and volcanoes. Krogh estimates that the 
annual consumption of coal adds yearly to the atmosphere about 
one-thousandth of its present content in carbon dioxid. Were 
there no factors offsetting this increase in atmospheric carbon dioxid 
animal life would soon become extinct. 

On the other hand, there are two large factors at work removing 
carbon from the atmosphere—first, the decomposition of carbon 
dioxid by plants with the liberation of oxygen, and second, the 
consumption of carbon dioxid in the weathering of rocks. No 
precise valuation can be given to either of these factors, although 
various writers have attempted to estimate their magnitude. Cook 
computes that leaf action alone more than compensates for the 
production of carbon dioxid. Chamberlain estimates that the 
amount of carbon dioxid annually withdrawn from the atinosphere 
is 1,620,000,000 tons, and that the greater part of this is taken up 
by the weathering of mineral. This is continually being returned 
to the atmosphere by the factors considered in the preceding chapter. 
There are then two compensating sets of factors—decay, respiration, 
and combustion liberating carbon; plant growth and rock weather- 
ing fixing it. These balance each other, thereby completing 


THE NITROGEN CYCLE 183 


the carbon cycle and rendering the carbon-dioxid content of the 
atmosphere nearly constant. 

The Nitrogen Cycle.—Since nitrogen occurs as an essential part of 
the structure of every plant and animal, it is found in all crops and 
crop residues. It occurs in the top soil in proteins, protein decom- 
position products, ammonia, nitrites, and nitrates. It is not found 
in the mineral matter of the earth except in shales and other deposits 
containing the residues of plant and animal bodies. Hence, the 
quantity in the combined form is not great when compared with 
other essential elements. Yet it is required by all living organisms 
in large quantities. Many of these are returning it to its inert 
atmospheric form. This fact led Sir William Crooks, in his famous 
address before the British Association for the Advancement of 
Science in 1898, to predict dire calamity to the human race if science 
were not able to utilize atmospheric nitrogen. 

In the free form, nitrogen occurs in enormous quantities; four- 
fifths of the atmosphere is composed of it. Dr. Hopkins has pointed 
out that the total supply of nitrogen over each acre of the earth’s 
surface, if available, would meet the needs of a hundred-bushel crop 
of corn every year for 500,000 years, whereas the supply of carbon 
is sufficient for such crops for only two years. Nevertheless, 
carbon has no commercial value as plant-food, while nitrogen in 
available form is worth from 15 to 20 cents a pound on the market. 

The same atom of nitrogen at different times plays many different 
roles. One of the triumphs of agricultural bacteriology is the 
advancement which it has made in following nitrogen through its 
cycle. 

Nitrogen occurs in the plant and animal mainly in the form of 
protein. The plant protein may be eaten by the animal and produce 
animal protein. Either may reach the soil and decay. ‘The nitro- 
gen eaten by animals may be deposited as tissues of the animal or 
excreted as urea, hippuric or uric acid. These products are acted 
upon by bacteria with the formation of ammonia. 

Either the plant or animal proteins may reach the soil where 
decay sets in with the formation of albumoses, proteoses, peptones, 
peptids, and amino-acids. The amino-acids are then deaminized 
with the formation of an acid-and ammonia. The process is spoken 
of as ammonification. 

The ammonia does not accumulate in the soil, but is acted upon 
by other bacteria, the nitrosomonas, with the formation of nitrous 
acid. This is quickly taken up by the nitrobacter and oxidized 
to nitric acid which reacts with bases in the soil with the formation 
of nitrates. The nitrates are the main source of nitrogen for the 
plants which build from. them and carbon dioxid, amino-acids, 
peptids, peptones, proteoses, albumoses, and finally plant proteins— 
and the nitrogen has completed its cycle. If this were the whole 


184 THE CARBON, NITROGEN, AND PHOSPHORUS CYCLES 


story the quantity of combined nitrogen in the world would remain 
constant. But it is not—there are many leaks in the cycle. Some 
of the plants and animals may be burned with the liberation of free 
nitrogen. Millions of pounds of it reach sewers, and from here 
rivers, lakes, and oceans. In time this is broken down and the 
nitrates so formed are reduced by denitrifying bacteria with the 
liberation of gaseous nitrogen. ‘The processes of decay continually. 
going on may also liberate free nitrogen. Furthermore, millions of 
pounds of nitrogen are returned to the air by explosives. So the 
combined nitrogen would continue to grow less were it not that other 
factors are at work in nature causing it to combine. Every flash 
of lightning causes some nitrogen to combine as oxids, but the 
quantity of combined nitrogen thus formed is relatively insignificant. 
The major factors are biological. There are within the soil two 
great groups of bacteria which possess the power of fixing nitrogen. 
The first—the non-symbiotic nitrogen-fixing organisms living free 
in the soil—are able, with the energy they obtain from the oxidation 
of organic carbon, to build up complex organic nitrogen compounds. 
There are two groups of these organisms—the aérobic and the anaé- 
robic, the first being the more important. The other class of 
nitrogen-fixers is the symbiotic; these live in conjunction with 
legumes and obtain from them carbonaceous material, and in return 
give combined nitrogen. In either case the combined nitrogen 
becomes available for higher plants. Then it again starts on its 
journey through the living and the dead. 

The Sulphur Cycle.—Sulphurisan essential element for all plantsand 
animals, but the quantity required for normal growth and develop- 
ment is relatively small even when compared with the small per- 
centage found in soil. It occurs in the soil as organic and inorganic 
sulphur. The former is derived from the plant and animal residues. 
These are acted upon by microérganisms with the liberation of 
hydrogen sulphid, sulphur dioxid, and sulphates. Some of the hydro- 
gen sulphid is carried into the ocean or soil by the first rain; some of it 
reacts upon the iron silicates of the soil and forms pyrite or marca- 
site, but most of it is oxidized by bacteria with the formation of - 
sulphates. The sulphur dioxid is also further oxidized to sulphates, 
when they are again taken up by plants and start anew upon their 
wonderful journey through bacteria, higher plants, and animals. 

The Phosphorus Cycle. — Phosphorus occurs in the soil in the form 
of calcium, aluminum, and iron phosphate, also as organic phos- 
phorus. It is also found in places as huge deposits of rock phos- 
phate. It is an integral part of every living plant and animal cell. 
In these it occurs in two forms—organic and inorganic. ‘The organic 
phosphorus occurs in the nucleo-proteins, phospho-proteins, and 
phospho-lipins. 

The mineral phosphates of the soil are rendered soluble through 


THE PHOSPHORUS CYCLE 185 


bacterial activity, as outlined in a preceding chapter. This is taken 
up by the living plant and deposited either as organic or Inorganic 
phosphorus compounds within the plant tissues. The plant tissues, 
if eaten by animals, yield phosphor us to the animal to be laid down 
in the body of the animal as organic or inorganic compounds. ‘The 
excreta of animals always contain phosphorus in both organic and 
inorganic forms. ‘The inorganic phosphorus is readily utilized by 
plants and again starts on its cycle. However, the organic and 
animal residues must be mineralized by bacteria before they can be 
utilized again by plants. Micro6rganisms split off the carbonaceous 
material and the phosphorus is liberated mainly in the form of 
phosphates. Under some conditions mold action may give rise to 
small quantities of phosphin which must be again oxidized before 
being available to higher plants. In either event, the resulting 
phosphate is now ready to start on its cyclic journey through fe 
plant and animal organism. This is dramatically outlined: for a 
phosphorus atom by one writer as follows: 

“Where was I born? Ah, that I cannot tell you. It was far, 
far away from here, deep in the endless abyss of space, at an epoch 
so distant that even the earth on which you live had not been formed 
as yet; not even the great sun, now blazing in his glory, nor any of 
the innumerable multitudes of stars of the great universe now 
shining in the sky, had as yet come into being. No, they were mere 
cold whiffs of invisible vapor, scattered over all space, remnants of 
worlds vanished zeons before this great universe began. Out of the 
vast I came, born into that great sea of ether which stretches 
unbroken from star to star through all the endless depths of space. 
Some vast change, some murmuring and stirring of gigantic forces 
in its bosom, forces scarce known, scarce dreamt of, but working 
there in irresistible might, first brought me into being, and I hung 
suspended in the great void. It was utterly cold and utterly dark, 
and gleaming afar in the distance I could see the myriad fires of the 
great worlds and suns of space shining at me through the darkness. 
How long I hung in the void I know not. It was millions upon 
millions of years. Then atoms began to gather round me, stream- 
wise, coming from afar in phosphorescing torrents, and I perceived 
that I already formed part of a mighty mass of gas, a huge nebula, 
which stretched its gigantic arms out for millions of miles, like vast 
flaming swords, through the darkness of space. And so I hung for 
sons of time, while atom after atom in an endless stream flashed 
past me in the gloom, while the great nebula slowly drew together 
in its glory, and began to take shape and form. Then the tempera- 
ture began to rise in leaps and bounds, it grew stifling hot, and great 
lightnings flashed and quivered about me, and we atoms crowded 
more and more together, colliding, whirling, flying. Each second I 
smote a thousand million atoms and at each collison my motion grew 


186 THE CARBON, NITROGEN, AND PHOSPHORUS CYCLES 


more and more violent, until after millions upon millions of years of 
this tumult, I found myself part of an immensely hot flaming mass 
of gas, part of an embryo sun. There in the whirl and roar of this 
elemental flame I remained for unthinkable ages, but at last vast 
thunders beneath and around me made me aware that something 
tremendous was happening. It was a world—my first world— 
gradually condensing out of the fire mist, and the gigantic explosions 
which occurred from time to time were just great seas of boiling rock 
leaping upwards. I will spare you the account of how I entered 
into that world, and saw it slowly form and develop into a fair planet, 
covered with wonderful swarming masses of living creatures, with 
great cities filled with busy life, and wonderful civilizations. Nor 
will I tell you of how that world grew old, and passed into a vast 
desert, and finally, after wandering for eons of time in darkness 
and silence, burst suddenly forth into flame, the victim of a great 
cosmical catastrophe, and, like a bubble, vanished, exploding into 
incandescent gas. Nor will I tell you of how, far flung, I fell upon 
another world, and saw this world too in time perish; and of how 
I passed from world to world, and formed part of world after world, 
wandering in mighty migrations through space, until at last I joined 
the fire mist from out of which, ultimately, this present world of 
yours condensed amidst titanic convulsions. You will, therefore, 
see that even before your world began, I was old, immensely old. 
I will pass over all this and come to a time quite recent, when I 
found myself forming part of the molten fire underground. Here I 
lay for age after age, while the land above me was being eaten away 
by wind and rain and storm, and was buried—continent after 
continent crumbling into ruim—into the great ocean waiting 
patiently to receive it. Now I was urged upward by vast forces, 
slowly, steadily, for thousands of years, until I finally was uplifted 
to form part of a hard, cold rock, which soon reared itself into a 
mighty cliff, beaten upon by wind and rain and storm; I have a dim 
recollection of looking out from the cliff face upon a widespread blue 
sea, filled with strange vast monsters, which have long since vanished 
from the earth. But at last the cliff was washed away and I passed 
into the great body of the sea, and was absorbed into a tiny plant, 
living beneath the salt waters; but this was devoured by a glittering 
gorgeous fish, and so I entered his body. Then this fish was 
devoured by a reptile, which, creeping out of the water, entered a 
swamp and died, and its huge body decaying, I was washed into the 
soil, and there meeting with the rootlet of a plant, I entered into and 
formed part of it; and this was eaten by an animal; and so I entered 
into its body and formed part of it; and this was eaten by an animal; 
and so I entered into its body and formed part of his bones. While 
we were crossing a ravine one bright sunshiny day, millions of years 
ago, a green monster flashed out upon us and slew my master and 


THE PHOSPHORUS CYCLE 187 


devoured me. After a time my new host was also slain in a similar 
manner, and his body, decaying in the rank grass and vegetation of 
the swamp, I was ultimately washed out to sea in a sudden flood, 
which, coming down from the hills, swept me away. Here I 
mingled with the mud at the bottom of the sea, and stayed there for 
millions of years, and became covered over with mighty layers of 
mud and sand, and sank ever deeper and deeper into the earth, and 
at last once more felt the glow of the nether fires. Here in the great 
gleaming-turnaces of the deep I remained for many millions of years, 
while miles above me the world changed and developed, mountains 
came and went, new and strange creatures evolved, developed, 
filled all the earth, and died out again. One day, I was hurled forth 
amidst vast thunderings through the throat of a great voleano, and 
formed part of a molten lava stream, which in time became a fertile 
field covered with waving crops and golden grain. ‘Then I entered 
into a grain of corn, and was devoured by a man living thousands of 
years ago, a mere savage you would term him, wild and fierce. From 
him I passed to earth once more, and since then have been passing 
in a ceaseless round of change through the bodies of living creatures. 
I have flown through the air in a bird, I have swum in the sea in a 
fish, I have roamed over the earth in a beast, I have formed part of 
innumerable plants. But the full tale would only weary you, 
wonderful as itis. One day, a few years ago, I was devoured by an 
ox while forming part of a piece of grass, and soon by the mysterious 
chemical forces of its body I was made to form part of its bone. 
The great beast was slaughtered by men, and his flesh eaten, and 
his bones burnt to a fine white dust ina furnace. Out of this dust, 
I, the tiny phosphorus atom, was distilled in a furnace and found 
my way to a match factory, and am now in this little match-box 
lying in the table before you. Is my journey finished? Oh dear no, 
far from it. I shall go on changing and journeying and dancing, 
age after age, even until the world fades away like a mist, and long 
after all that you see and hear around you has crumbled away and 
vanished into the awful maw of time. I have been taking part in 
the great dance of atoms which forms the basis of all passing things 
and events, for millions upon millions of years, and shall continue 
to do so for millions and millions of years to come. I may, indeed, 
see this world perish, and may yet dance in worlds as yet unborn. 
My future will be probably even more strange than my past.” 


REFERENCES. 


Kossowicz, Alex.: ‘ Agrikulturmykologie, I Bodenbakteriologie.” 
Lafar, Franz: ‘‘Handbuch der Technischen Mykologie,”’ Dritter Band. 


CHAPTER XIX. 
PUTREFACTION, FERMENTATION, AND DECAY. 


PUTREFACTION, fermentation, and decay are in reality terms which 
are essentially distinct although they have been greatly confused 
and used synonymously even by professional men. As pointed out 
by Kendall, this confusion is attributed partly to the use of terms 
to designate certain processes which occur in nature before these 
changes were studied either biologically or chemically. 

Definitions.— Fischer considers the term fermentation, as it should 
be used in bacteriology, as the biochemical decomposition of nitro- 
gen-free compounds, chiefly carbohydrates, due to the action of 
microorganisms, and putrefaction as the biochemical decomposition 
of nitrogenous organic compounds by the action of microérganisms. 

The distinction which is usually drawn between decay and putre- 
faction—as the decomposition of nitrogenous organic substances in 
the presence of oxygen on the one hand, and the absence of oxygen 
(or with a limited supply) on the other—is not always sharply 
defined. The end products in both cases may be quite similar. 
Nencki found that in the decomposition of gelatin at 40° C. in the 
presence of air, there were formed in four days for every 100 parts 
of the original substance 9.48 parts of ammonia, 24.2 parts of 
volatile fatty acids, 12.2 parts of glycocol, 19.14 parts of peptone, 
and 6.45 parts of carbon dioxid, the other 28.53 parts being unde- 
termined. Jeannert repeated these experiments with the exclusion 
of air and found as the decomposition products of gelatin, carbon 
dioxid, ammonia, a gas smelling like carbon bisulphid, acetic, butyric, 
and valeric acids, glycocol, leucin, and a colloidin base-like substance. 
He concluded from these and other experiments that (1) the decom- 
position of nitrogenous substances and of carbohydrates may be 
accomplished with access or exclusion of air; (2) in the latter case 
the decomposition is considerably less rapid, and complete decompo- 
sition requires a period six times as long; and (3) the more simple 
chemical products formed are in the two cases identical. 

Nor is it a safe criterion to state that putrefaction is accompanied 
by the formation of ill-smelling substances, for this is usually a 
quantitative and not a qualitative difference. Moreover, Hirschler 
has pointed out that the putrefaction of protein substances is modi- 
fied by the presence of carbohydrates. The addition of various 
carbohydrates, glycerin, and calcium carbonate changed the decom- 


ACTIVE AGENTS 189 


position of meat so that aromatic products of putrefaction could not 
be detected. From this he drew the conclusion that the decomposi- 
tion of protein substances in the presence of cane sugar, starch, 
dextrin, glycerin, or lactic acid may not be accompanied by the 
formation of the characteristic putrefaction products—indol, phenol, 
and oxyacids. Nevertheless, there is a marked quantitative dif- 
ference in the two processes—decay and putrefaction. ‘The former 
is marked by the volatilization of the organic constituents— either 
protein or non-protein—while the non-volatile mineral constituents 
are left behind in a form largely available. Putrefaction is the rapid 
and intense decomposition of nitrogenous (for the most part protein) 
bodies by certain bacteria, usually with the formation of large 
quantities of gaseous, ill-smelling products. There may result as 
intermediate products, basic substances often having highly toxic 
properties. These substances have been named ptomaines by 
Brieger. Many of them contain only carbon, hydrogen, and 
nitrogen, and are ammonia-substitution products. Some of the 
simpler ones are: 


Mothsiaminga sy meee ies eee ewe se ae PCs) Nie 
Dimethylamin re wee. ees Are oe. ta PCCM) ENE 
dininebhylaminve ae eene time leo eter ly) sce th nce 4 (©) aN 
PUbreRChniae eee gee ee eTrust o(@ He) «Nino 
Cadavertnen =) eek armas cer eens) oso ec ses NIB (@H ENE: 


They are usually protein-cleavage products, sometimes resulting 
from the mere removal of carbon dioxid from the carboxyl of the 
amino-acid. Putrescin may be formed from ornithin thus: 


CH:—CH2:—CH:—_CH—COOH CH2—CH:—CH:—CH)» 
| | = | | + COs 
NH: NH: NH» NH: 
Ornithin Putrescin 
and cadaverin from lysin: 
CH2—CH:—CH:»—CH:—_C H—COOH CH:—CH:—CH2—CH:2—CHs» 
| | = | ae er OOs 
NH: NH: NH: NH: 
Lysin Cadaverin 


In putrefying mixtures the ptomaines appear on or about the 
fifth or seventh day after putrefaction sets in, and disappear, by 
further cleavage, more or less rapidly, yielding less complex nitrog- 
enous substances that are non-toxic. 

Active Agents.—Liebig and the early workers considered these 
changes to be purely chemical processes. The ferment was to them 
an extremely alterable organic substance which decomposed, and 
by decomposing set in motion its own elements. The momentum 
thus engendered is sufficient to tear to pieces the fermenting sub- 
stance. ‘This in turn then possesses the power of imparting to other 
compounds this same property, or, in other words, they considered 


( 


190 PUTREFACTION, FERMENTATION, AND DECAY 


fermentation a true chemical process. This was overthrown by 
Pasteur, who proved fermentation to be due to living microscopic 
organisms, and it came to be generally believed that putrefaction 
was due to a certain microdrganism— Bacterium termo. Cohn 
wrote in 1872 that through his own experiments, as well as through 
those of other investigators, he was convinced that Bact. termo was 
the ferment of putrefaction in the same way that yeast is the alcoholic 
ferment. He considered that other bacteria may play a secondary 
role, but that Bact. termo is the primary cause of putrefaction. How- 
ever, bacteriologists soon came to realize that Bact. termo was only 
a general name given to the many species of rod-shaped organisms 
occurring in decaying substances. In 1884 and 1885 Hauser 
isolated three distinct species of bacteria capable of causing putre- 
faction— Proteus vulgaris (B. proteus, B. vulgaris, B. zopfi), Proteus 
mirabilis, and Proteus zenkert. ‘The first two are capable of liquefy- 
ing gelatin, while the last is not. Many different bacteria are 
encountered in a spontaneously putrefying substance. Among the 
most active which have been studied are, according to Effront: 
the family of Proteus, B. putrificus colt (Bienstock), B. perfringens 
(Veillon and Zuber), Micrococcus flavus liquefaciens (Fluegge), 
B. gracilis putidus (Tissier and Martelly), B. bifermentans sporo- 
genes B. diplococcus griseus non-liquefaciens (‘Tissier and Martelly), 
B. cola communis (Escherich), Streptococcus pyogenes (Doleris and 
Pasteur), and Staphylococcus pyogenes albus (Rosenbach). These 
bacteria are very widely distributed, B. proteus being especially apt 
to occur in substances undergoing decomposition. Its presence is 
constant in rotten meat, is very frequent in manure, and is met with 
in large numbers even in normal dejecta. The putrefying bacteria 
are usually anaérobic, but there are often very active aérobes. 

H. Martelly made a careful study of the bacterial flora of putrefy- 
ing material and found that it changed from period to period. He 
found at first Micrococcus flavus, Staphylococcus albus, B. coli, and 
Diplococcus griseus. ‘Then at the end of three or four days B. 
perfringens, B. sporogenes appeared, at the end of eight to ten days 
he detected the presence of B. putidus, B. putrificus, and Proteus 
zenkeri, and after three months there remained only B. putrificus, 
B. putidus, and Dzplococcus griseus. 

Products of Putrefaction and Decay.—Due to the trypsin and 
erepsin secreted by the bacteria the proteins are broken into albu- 
moses, peptones, proteoses, and amino-acids, and even in very 
advanced putrefaction nitrogenous substances are always found 
which give the protein reactions. The amidases secreted by 
bacteria give rise to volatile acids, amins, phenol and indol deriva- 
tives. Effront summarizes the products formed as follows: (1) 
Ammonia and amins—ethylamin, propylamin, and trimethylamin; 
(2) volatile acids, comprising all the members of the fatty series up 
to caproic acid; (3) aromatic acids and oxyacids, like phenylpro- 


ACTIVE AGENTS 191 


plonic, oxyphenylacetic, and oxyphenylpropionic acids; (4) phenol, 
indol, skatol, pyrrol, and its derivatives, these bodies sometimes 
being in very small quantities or even completely absent; (5) sulphur 
derivatives like methyl-mercaptan; (6) various amino-acids, leucin, 
tyrosin, tryptophan, and sometimes glycin, creatinin, etc.; (7) 
various ptomains, like putrescin and cadaverin, the guanidins, 
cholin, and nurin, pyridin, hydrocolloidin, ete. 

In the process of decay the carbon and hydrogen is liberated as 
carbon dioxid, methane, water, and other volatile products with the 
result that the carbon in the soil tends to fall off relatively to the 
nitrogen and the ratio -;-, which in the original plant material 
is about 40, is reduced in the soil to 10. This carbon-nitrogen ratio 
varies with climatic conditions, also with soil type and previous 
treatment. Lawes and Gilbert, as quoted by Lipman, give the 
following carbon-nitrogen ratio in the organic matter of different 
soils: 


Cerenlerootsaimdsstub blew Aste sc). ta ty ee an ee eee xe 6 ABO 
IrecuminOusiseubDleme ta cee a ae aed 1 athe ets 2880 
Dung 5 WS oh UN an Ca RE a al CR Ue Ms ei at Rr RM kT 0210 | 
Wervaoldtorass) and vrei tes: 2 8) ee Os ind een ke weed LIBET 
IM emavital om joyerneystorll oo, PS ee, ea eee en leat) 
iPasiiiresrecentiyslaigvdowm 9%. 2). so «ee Me AME 
PAT Ao CESO1 Marans ne ene ee a ee Reh Be UP ww Uy ek SLOT 
Clay subsoil Ee er eR AE eee pal fy ek OE eee | 6.0 


Other things being equal, a wide carbon-nitrogen ratio indicates 
a more fertile soil than a narrow carbon-nitrogen ratio. But 
this must always be interpreted with regard to the climatic condi- 
tion. In the arid regions the carbon-nitrogen ratio is narrow when 
compared with soils of the humid regions, yet the bacterial activity 
of the former is just as active as that of the latter. 

The organic substances found within the soil are called humus and 
result from the action of bacteria upon the plant residues. The 
composition of the substance varies with the products from which 
it has been formed, also the degree of humification which has taken 
place. Moreover, the quantity and speed with which humus is 
formed depends upon the nature and condition of the material used 
and the physical, chemical, and biological conditions of the soil. 
Hilgard thinks that in the humid regions one part of normal soil 
humus may be formed from five to six parts of dry-plant débris, 
whereas in the arid regions from eighteen to twenty parts of the 
same material would be required. Snyder allowed various organic 
substances to humify for one year with the following results: 


Per cent. 

nitrogen 
1 part fresh cow manure yielded 33 parts humus containing 6.16 
1 “ green clover OS sete 85: CS és e 8.24 
1 “ meat scraps ss ee bi > 48 4 Us 10.96 
1 “ sawdust oY oe al Olenes Se 0.30 
1 ““ oat straw “< TG 6 “ “ “ 2.50 


192 PUTREFACTION, FERMENTATION, AND DECAY 


Humus is mainly valuable because of its physical effect upon the 
soil and because of its content of nitrogen, potassium, and phos- 
phorus which are slowly liberated by bacteria. The beneficial effect 
of organic matter upon the bacterial flora of the soil and soil 
fertility, however, is mainly exerted before it reaches the stage of 
humus. 

Chemistry of the Processes.—The primary and secondary products 
resulting from the decay of organic matter in the soil are classed ‘as 
humus. ‘They are not, as was once believed, a few comparatively 
simple organic compounds but are a heterogeneous mixture of 
colloidal and crystalline organic compounds resulting from the 
action of bacteria upon plant residues. 

The chemical composition of the end products being in many 
cases unknown, the chemistry of the process is still to be explained, 
but we have some very suggestive information due to the fact that 
acids and alkalies when they act upon carbohydrates yield brown 
humus-like substances very similar to, if not identical with, the 
substances found in the soil and resulting from bacterial activity. 

It is known that the aldehyd group of a carbohydrate easily 
opens its double bonds between carbon and oxygen and adds water 
to form a polyhydric alcohol, as follows: 

OH 
y 
Son =O + HO = RON 


[ap OL 
H H 


This reacts with sodium hydroxid with the formation of the 
following salt: 


H H on H 6B oNna 
| iy , ee 

Stak see 4 aNaOHus = Para EEO 
OH On OH OH 


This salt is unstable and the molecule forms enols: 


HH HH HH H H -OH Si 
[> Cleese weil: Sales a enlaeyea 
H—C—C—C—C—C—C—ONa = HO—C—C = C—C = ape + 2H20 


(ieee Celt | 
OH OH OH OH HO HO H H OH 


These break apart at the double bonds: 


lef del OH H OHH 
7 (eal So sea mt aap Pg Ze S80 
I on 
Ve Maat aL OH H OH OH OH 
Hoses = oe ee nee 


CHEMISTRY OF THE PROCESSES 


193 


By this process pieces having various numbers of carbon atoms 
are formed, all of which are very reactive in their nascent state due to 
the free open bonds on the carbon atom. ‘These react with each 
other and give rise to long chain compounds, the more complex of 
which have a brown color and other physical and chemical charac- 


teristics of soil humus. 


Soil humus also contains nitrogen which would come through the 


action of bacteria upon proteins. 


The products resulting through 


such action are numerous and varied, but the work of Schreiner and 
his associates has shown them to be the following: 


Arginin (Ce6Hi402N,4) 

Adenin (CsH;Ns) 

Agroceric acid (C2H4203) 

Acrylic acid (C3H4O2) 

Agrosteral (C2H20H20) 
Cytosin (Cs4H;0N3H20) 

Cholin (CsHisO2N) 

Creatinin (C4H7ON3) 

Creatin (CsH9O2N3) 
Dihydroxystearic acid (CisH3604) 
Hentriacontan (Cs:Hes) 

Histidin (CesH 02Ns3) 
Hypoxanthin (CsHsONs) 
Lignoceric acid (C2sH4sO2) 
Monohydroxystearie acid (CisH360s3) 
Mannite (CeH140¢) 


Nucleic acid (constituents unknown) 
Oxalie acid (C2H20.) 

Picolin carboxylic acid (C7H702N) 
Paraffinic acid (C2sH4sO2) 
Phytosterol (C2sHasO.H20) 
Pentosan (CsHsQ,) 

Quanin (CH;N3) 

Rhamose (CsH14010) 

Succinie acid (CsH¢6Os.) 

Saccharic acid (CsHsOv) 

Salicylic aldehyd (CsHsCHOOH) 
Trimethylamin (C;H»9N) 
Trithiobenzaldehyd (CsHsCSH)3; 


REFERENCES. 


Vorhees, Edward B. and Lipman, Jacob G.: 


‘A Review of Investigations in Soil 


Bacteriology,’ U. S. Dept. Agr., Off. Exp. Sta. Bul. 194. 


Fuller, George W.: 
Effront, Prescott: 


‘ 


15 


“Sewage Disposal.” 
“Biochemical Catalysts in Life and Industry. 


” 


CHAPTER XxX. 
AMMONIFICATION. 


In the preceding chapter it was shown that one of the final prod- 
ucts resulting from putrefaction, fermentation, and decay is 
ammonia. The production of ammonia through the intervention of 
microorganisms is known as ammonification. The speed with which 
this ammonia is formed within a soil varies with the physical and 
chemical composition of the soil together with the number and 
physiological efficiency of the various organisms taking part in the 
process. 

Although it has been known for some time that small quantities 
of ammonia occur in all arable soil, its formation was not known 
to be due to a biological process until 1893 when Muntz and Coudon 
demonstrated that ammonia is no longer formed in soils sterilized 
by heat. They, together with Kayser, isolated from soil two species 
of Bacterium, one of Bacillus, two of Micrococcus, and two of molds 
—all of which produced ammonia in veal bouillon, and all but one 
(a micrococcus) gave the same results in soil. From these results 
they concluded that the formation of ammonia in the soil is the 
result exclusively of the conjoint activity of numerous lower organ- 
isms of very widely different characters. 

This conclusion was confirmed the same year by Marchal who 
isolated from the soil the species of microérganisms (molds, yeast, 
and bacteria) which were the most prevalent, and determined which 
of these had the power of transforming nitrogenous material into 
ammonia. Of 31 species tested, 17 displayed a strong ammonify- 
ing power. Most of the others displayed a smaller but none the 
less distinct ammonifying power. Molds and yeast were also found 
to produce ammonia. On inoculation into a solution containing 
1.365 gms. of organic matter per liter the various organisms were 
found to transform the following proportion of nitrogen into am- 
monia in twenty days: 


B. iyeoides ¢.. 00 AU 5 ee ee, pe ee oe ee 
1 i ee oN | ee a Sle Pena ee ee lite, 39 
Proteus vulvaris’ ¢ -- “Ne  eee, , Caen ome 
B. mesenterius vulgatus.<: <<. 2: os ols ee ee 
Sarcina luteas rc cos eee a ay Glee ne a ie eT 
B. janthinus: MY} a-dees ah Gra Pe 8 kee 23 
B. subtilia. o-oo aga RE Bae eee ee tam 


The B. mycoides was selected by him for special investigation. 
This organism is found very widely distributed in nature. It is 


AMMONIFICATION 195 


always present in the surface layers of cultivated soil, and is found 
frequently in manure, vegetable mold, composts, and in the humus 
of forests. It occurs at times in air and natural waters. He found 
that when inoculated into a neutral solution of albumen the medium 
soon becomes strongly alkaline due to the accumulation of ammo- 
nium carbonate; simultaneously there was a corresponding decrease 
of albumin. The analysis of the atmosphere in which the culture 
was confined showed a marked decrease of oxygen with a corre- 
sponding increase of carbon dioxid. Hydrogen and nitrogen were 
not among the gaseous products. The quantity of carbon dioxid 
and ammonia formed in the respiration of this organism were nearly 
in the proportion in which they are formed in the complete combus- 
tion of albumin. In addition to these two substances, there were 
found in the solution small quantities of peptones, leucin, tryosin, 
and formic, butyric and propionic acids. Marchal considered that 
in the life processes of B. mycoides atmospheric oxygen is made to 
combine with the constituents of albumin, its carbon being trans- 
formed into carbon dioxid, its sulphur into sulphuric acid, a portion 
of the hydrogen into water, the ammonia appearing as a residual 
product. He assumed the following equation: 


CrHi2NisSO2 + 7702 =. 29H2O + 72CO2 + SOs; + 18NH; 


The best conditions for the activity of the organisms are: (1) A 
temperature of about 30° C., (2) thorough aération, (3) a slightly 
alkaline medium, and (4) a dilute solution of protein. It was also 
found that this organism can ammonify not only albumin but also 
casein, fibrin, legumin, glutin, myosin, serin, peptones, creatin, 
leucin, tyrosin, and asparagin, but was unable to utilize urea, urea 
nitrate, or ammonium salts. In the main these results have been 
amply confirmed by a great number of investigators. 

C. B. Lipman and Burgess, however, have demonstrated that B. 
mycoides is by no means always the most efficient ammonifying 
bacterium, for even this organism varies greatly in its activity, 
depending upon the chemical and physical conditions of the sub- 
strata. They make the following critical statements concerning 
Marchal’s findings: “First, the results of solution cultures are no 
criterion as to the results to be obtained in soils. Secondly, that 
no two forms of organic nitrogen are attacked and ammonified with 
the same vigor by any one organism. ‘Thirdly, that different soils 
will modify an organism’s power to ammonify any one given form 
of nitrogen very markedly, so that it may be efficient in one case and 
feeble in another. Fourthly, that the ammonifying efficiency of 
organisms is greater in sandy soil, and possibly in others, than in 
solutions, for we have obtained a transformation of 41.98 per cent. 
of peptone nitrogen and 36.06 per cent. of bat guano into ammonia 


196 AMMONIFICATION 


by Sarcina lutea and B. mycoides, respectively, in twelve days at 
temperatures between 27° C. and 30° C., while Marchal only 
obtained similar transformation in thirty days at 30° C. in albumin 
solutions.” 

Species and Distribution.—As was pointed out by Marchal, the 
ammonifying organisms are very widely distributed in nature. 
The power to split off ammonia from protein is a characteristic of 
the majority of soil bacteria. Gage noted the production of 
ammonia in thirteen out of twenty cultures of sewage bacteria tested 


Fic, 27.—Ammonifying bacteria.—1. Bacterium mycoides; X 3,000. (Nadson.) 
2. Bacterium mycoides; involution forms; X_ 3,000. (Nadson.) 3. Bacterium 
twmescens. (Myer.) 4. Proteus vulgaris; X 3,000. (Nadson.) 5. Proteus vul- 
garis; involution forms; X 3,000. (Nadson.) (Lipman’s “Bacteria in Relation to 
Country Life.’’) 


by him. He further found that the gelatin liquefiers have an 
ammonifying power nearly twice as great as the non-liquefiers. 
Chester found all but one of the organisms tested by him capable 
of producing ammonia. C. B. Lipman tested the following fifteen 
organisms in soils: B. mesentericus vulgatus, Ps. putida, B. vulgatus, 
B. megatherium, B. mycoides, B. subtilis, B. tumescens, Sarcina 
lutea, B. proteus vulgaris, B. icteroides, B. ramosus, Streptothria, 
sp., Ps. fluorescens, B. vulgaris (navy strain), and Mic. tetragenus as 
to their ammonifying powers of dried blood, tankage, cotton-seed 


SPECIES AND DISTRIBUTION 197 


meal, sheep and goat manure, peptone, fish guano, and bat guano. 
He found that while all produced ammonia, their efficiency varied 
greatly, depending upon the nature of the soil and the nitrogenous 
material to be ammonified, B. twmescens, however, on the whole 
appeared to be the most efficient organism tested. 


Fic. 28.—Ammonifying bacteria.—1. Proteus rulgaris; X 2,600. (Rodella.) 
2. Bacillus megatherium; X 2,600. (Hinterberger.) 3. Bacillus mycoides; X 2,600. 
(Emmerling.) 4. Bacillus cereus; X 2,600. (Wilhelmy.) Lipman’s “ Bacteria in 
Relation to Country Life.”’ 


Moreover, many fungi, as shown by McLean and Wilson, possess 
the power of rapidly ammonifying cotton-seed meal and dried blood. 
Fungi belonging to the Moniliacee were more active ammonifiers 
than were members of the Aspergillacee, Mucoracee, or Dematiacee. 

An idea as to the number of ammonifying organisms which must 
occur in soils may be gleaned from the fact that Conn found about 
10 per cent. of a soil’s flora to be rapid liquefiers, principally Ps. 


198 AMMONIFICATION © 


fluorescens, and many others are slow liquefiers. ‘This indicates 
that the class of organisms must play a very important role in the 
degradation of the nitrogenous material of the soil. 

It is quite likely that the organisms are even more efficient in the 
soil in the mixed cultures than they are in the pure cultures. For 
the transforming of protein nitrogen to ammonia is a complex 
process which must proceed by steps and some organisms must be 
more efficient than are others in specific phases of the reaction. 
But so far we have little definite information on this subject. 

Methods.—Two methods are in general use for the determination 
of the ammonifying powers of the soil. The one in which a definite 
portion of soil is inoculated into a liquid media and after a given 
time the ammonia determined; in the other the nitrogenous sub- 
stance is incorporated into the soil and after a definite period the 
ammonia determined. The latter method would appear to approach 
more nearly field conditions, but both methods have their advocates. 
It is not my purpose to go into the claims made for each, but suffice 
it to state that Léhnis, who has made a careful study of each, finds 
the more important factors in both to be: (1) Nature and quantity 
of material used as substrata; (2) concentration and distribution of 
the substrata in the medium; (3) aération; (4) diffusion, absorption, 
destruction or evaporation of metabolic products; (5) reaction of the 
medium; (6) temperature; and (7) duration of the experiment. 

As was pointed out by Pagnoul in 1895, the formation of ammonia 
in the soil is only a transition state of organic nitrogen in passing 
to the nitrates. So that with either the solution or soil method, 
what we measure is the accumulation of ammonia in the media and 
not the actual quantity formed. Various factors may enter and slow 
down the quantity of ammonia formed. This would be indicated 
by a smaller quantity of ammonia in the soil, or the speed with which 
the ammonia is transformed into nitrates may decrease, and hence the 
ammonia accumulates while the actual quantity found is the same. 
Moreover, it is well known that many microérganisms possess the 
power of transforming ammonia into protein nitrogen, and this 
factor may either increase or decrease with a corresponding change 
in the ammonia of the soil. Where large quantities of ammonia are 
being formed, part of it may be lost from the medium by volatiliza- 
tion. The extent of this loss varies with the soil. Lemmermann 
and Fresenius found the addition of calcium carbonate to a soil to 
the extent of 1 per cent. reduced the volatilization of ammonium 
carbonate and increased the absorptive power of the soil for ammonia. 
Calcium sulphate and chlorid and magnesium chlorid have a similar 
effect. Caustic lime has the opposite effect. The zeolites are very 
effective in reducing the loss of ammonia from soil, and according to 
Pfeiffer and coworkers the nitrogen so fixed is so firmly held that it 
does not become available to plants during the first season. 


METHODS “199 


Material Ammonified.— The speed with which ammonia is formed 
within a soil varies greatly, depending upon the nature of the 
material to be ammonified. Lipman and his associates found the 
following proportions of nitrogen were transformed into ammonia 
in six days: 


Concentratedittankaze ~.. 7 | =. =. 3 = . «*. 56:66 percent: 
GrounGensiiaiiete MME OeL Reeve aa ee ee Cr ne AAO é 
Cow manure, solid and liquid excreta . . . . . . 82.60 
Mrredebloodas te te Hee ee ee en ee ye G4 
Bonegnen lees: ae Ome ee ees omc et Se G65 
Cowsmanure, solidvexcreta . 355° 2 © . =. . 5.39 is 
Gotton-seed-ineals.. 5-7. eles oe) se 4.95 * 


However, this order is not always maintained, for C. B. Lipman 
has found it to vary with the soil and with the bacterial flora. 
Lipman and Brown consider the carbon-nitrogen ratio important in 
determining the rate of ammonification of nitrogenous materials, 
and then the modification of this ratio by soluble carbohydrates or 
by other soluble compounds may lead to changes in the numbers 
and species of the microdrganisms in the soil or culture solutions 
and a consequent depressed or intensified ammonification, depend- 
ing on the character of the nitrogenous fertilizer. 

The addition of dextrose, sucrose, lactose, maltose, and mannite, 
according to Lipman and Brown, decrease the accumulation of 
ammonia in the soil. Kelley found that by adding 1.586 gms. of 
starch to 1.072 gms. of casein the quantity of ammonia in the soil 
at the end of nine days was decreased 50 per cent. 

In the presence of the carbohydrates the decrease may be either 
real or apparent. The true decrease may be due to the carbohydrate 
which causes the organism to use only sufficient protein to meet its 
nitrogen metabolism when only a small quantity of ammonia would 
accumulate. 

The apparent decrease is probably due to an acceleration of the 
speed with which ammonia is transformed into protein nitrogen. 
Inert organic substances in general, such as starch, cellulose, and 
peat, usually decrease the speed of ammonification. This is due, 
according to Rahn, to the substance making some of the soil moisture 
unavailable to the bacteria, for he found that when the moisture is 
sufficiently great cellulose acts as a stimulant to ammonification, 
probably by holding the sand particles farther apart and thus in- 
creasing aération. Dzierzbicki has found that small amounts of 
some humic acid salts increase ammonification. 

The addition of manure to a soil greatly increases the ammonia 
produced in a soil. This is illustrated by the following results 
obtained by Greaves and Carter. In the first column is given the 
per cent. of ammonia found, the untreated soil being taken as 100 
per cent. The various quantities of manure were applied to the 


200 AMMONIFICATION 


soil in pots and after four months the ammonifying powers deter- 
mined. In the second and third columns are given the results from 
actual field soil receiving each year the designated quantity of 
manure. 


Pot Field Experiment 

Treatment Experiment Fallow Cropped 
WoMmanUTéy! <a ON recast ae eeLOO 100 100 
DitOns Of TAanUrG we, es 147 129 
LOT eerie ra deere eet a0 
TDi Sos vse “f eh ARCA OP rer Ups Ps 188 183 
ZOe aca’ + SPE. ri MLO 
Dow anes cz le et ee MEY G| 


Crops grown on a soil decrease its ammonifying powers. In the 
order of their decreasing ammonifying powers came fallow, potato, 
corn, oats, and alfalfa. 

Influence of Soil and Climatic Conditions.—Lipman points out 
that the ammonia production in a soil is affected by (1) its mechani- 
cal and chemical composition, (2) by fertilizer treatment, and (3) 
by methods of tillage and cropping. He further states that the 
mechanical composition of the soil determines the proportion of 
aérobic and anaérobic organismsina soil. If the latter predominate, 
the production of ammonia is comparatively rapid. Yet Fischer 
reports a more intense ammonification of dried blood in light than 
in heavy soils, but under this case it is possible that the loss of 
nitrogen from the sandy soil was sufficient to account for the 
observed difference. C. B. Lipman has shown that the order within 
various soils differs with the various ammonifying organisms. 
Moreover, he and Waynick found the removal of California soil to 
Kansas increased the ammonifying powers about one-third, but 
removal to Maryland had little effect. Whereas Kansas soil re- 
moved to California loses greatly in its ammonifying power, yet 
Kansas soil transported to Maryland suffers little change. We must, 
therefore, conclude that climate exerts a great influence on the 
ammonifying powers of the soil. 

The quantity of ammonia in a soil varies from season to season 
and from period to period. Green found that as regards the 
ammonification of the organic manures—flesh meal, horn meal, and 
blood meal—the bacterial activities rise from August to October, 
with a tendency to fall or remain constant in November, rising to a 
maximum in December. This was followed by a minimum in 
February and a low maximum in April, and from April to J uly there 
was a slight fall which probably continued to a summer minimum 
commencing in August. 

Moisture.—'The influence of moisture on the ammonia formed in 
the soil is very great. Lipman and Brown found ammonification 
in a loam soil increased with increased water content even up to 
35 per cent. of the weight of the soil. However, later they and 


MOISTURE 201 


Owen found ammonification to increase as the water added increased 
up to a certain percentage, which varies with the physical nature 
of the soil, but larger quantities of water reduced the ammonia 
recovered. The work clearly demonstrated that the optimum 
moisture content for maximum ammonification is higher than it is 
for maximum nitrification. The quantitative difference between 
the two processes in the same soil was found by Sharp. Ammoni- 
fication was most rapid with a 25 per cent. moisture content and was 
not markedly affected by 3 per cent. differences. Nitrification was 
at its maximum when the soil contained 19 per cent. of water. When 
it was increased to 25 per cent., the rate of nitrification was décreased 
50 per cent. 

The work of Greaves and Carter shows that ammonification is 
at its maximum in a soil containing 60 per cent. of its water-holding 
capacity, as is illustrated in Fig. 29 which gives the results obtained 
with twenty-two soils. Moreover, according to Briggs the moisture- 
holding capacity, the wilting coefficient, the moisture equivalent, 
and the hygroscopic coefficient are related by linear equations: 


Q 

Il I 

ee WS) 
wae 
qas 
stat te 
bo BDO bo 
ed 


where C is written for the moisture capacity as defined by Hilgard, 
W for wilting coefficient, / for moisture equivalent, and H for 
hygroscopic coefficient. Therefore, taking the maximum ammoni- 
cation at 0.6 of the water-holding capacity we could relate these 
other soil constants to maximum ammonification by a similar set ot 
linear equations. Thus: 


Ma. = .942 
Vises mise len aeeeVig leet 
Mas i= 2epon ele eet dionG, 


writing Ma for per cent. water for maximum ammonification. 

Variation in moisture content of a soil changes its ammonifying 
flora, for soils held at various moisture contents for several months 
show different ammonifying efficiency, as may be seen from the 
following results obtained by Greaves and Carter. These soils 
were held at the indicated moisture content for three months and 
then all brought to 20 per cent. moisture and after four days the 
ammonia determined. 


Per cent. Ammonia 


Moisture Added Produced 
LEA OW ETACEIIGS WA LeL es) wis aera iealulay el win ver te oe cit Bs oy oaalOO 
OM OSPEICeb ew alelie aiainae hee rea eet. wee tie as ptr! a eeretiiy 
li PrOgDeTy COM ber ALCL Sn is seed steals centree ce Nt yee ieee ey Ups) Niece Ne a LS 
YOROSPerscenianwaletac: hay Pict oe twists lop aan ao (ene DS 


ZUR OVDeL COMO Wal lelinsau cereal Lemp nL a cae Be lecid ie, ot Ge) | ae ILO 


202 AMMONIFICATION 


This increased ammonification with increased moisture content is 
due, according to Lipman, to the suppression of the aérobic decay 
bacteria and an acceleration of the anaérobic putrefactive bacteria. 


Fic. 29.—Average percentages of ammonia produced in soils receiving varying 
quantities of water. The quantity produced at 60 per cent. is taken as 100; on the 
ordinate is given the per cent. of ammonia formed whereas on the abscissa is given 
water applied as per cent. of water-holding capacity. 


Aération.—Carbone considers oxygen essential as a rule to the 
most efficient action of organisms in decomposing organic matter, 
and he points out that it is not possible to segregate the agents of 
decomposition strictly into aérobes and anaérobes. ‘The aérobes 
are the more active agents in the decomposition of carbonaceous 
material with the formation of humus. But Dzierzbicki found a 


PHOSPHORUS 203 


strong aération decreased—at least in many cases—the quantity of 
ammonia split off from peptone solutions which had been inoculated 
with soil. Plimmer, working with a Dunkirk clay loam, failed to 
find any optimum oxygen content for the maximum production of 
ammonia. Under purely anaérobic conditions, caused by an 
atmosphere of pure carbon dioxid, there was somewhat less ammonia 
produced than when oxygen was present at the beginning, but even 
under these conditions ammonia was formed in rather large pro- 
portions. This would probably vary with the specific bacterial 
flora of the soil, for the work of Marchal demonstrated that the 
formation of ammonia is favored by the unhindered access of oxygen, 
and in the process considerable quantities of oxygen are used up and 
a nearly corresponding quantity of carbon dioxid produced. There 
is the possibility of the carbon dioxid resulting from side reactions, 
the oxidation of the carbon chain compounds which have been 
deaminized, and not due to the main process of ammonification. 
For theoretically, at least, ammonification can be considered as a 
true hydrolytic reaction. The microérganism, however, gets its 
energy from the oxidation of the carbon, and where conditions are 
favorable ammonia production follows very closely the evolution 
of carbon dioxid. But Gainey found unfavorable conditions to 
have a more detrimental effect upon the formation of ammonia than 
upon the production of carbon dioxid. 

Lime and Magnesia. — These exert a marked effect on ammoni- 
fication, and Vorhees, Lipman, and Brown found magnesium lime 
usually superior to non-magnesium lime in this respect. Its effect 
varied, depending upon the character of the organic matter to be 
ammonified and the crop grown upon the soil. Lipman, Brown, 
and Owen found lime carbonate in which the large per cent. of 
boron proved to be the factor which hindered decay bacteria. 

The increased ammonia resulting from the use of the magnesium 
lime may be due to an apparent and not to a real increase in ammoni- 
fication. For Fischer has noted that calcium carbonate increased 
the speed with which ammonia sulphate is transformed into protein 
to a greater extent than did magnesium carbonate. Moreover, 
Lipman and Green found that magnesium carbonate interferes with 
the speed with which nitrite is converted into nitrate which would 
slow down the action of the nitrosomonas. In either case this 
would increase the accumulation of ammonia in the soil, which may 
be interpreted as increased ammonification. 

Phosphorus.— In experiments on ammonia cleavage by Dzierzbicki 
in peptone solutions, it was found that the intensity of such cleavage 
depends not only on the bacterial flora of the soil but more so on its 
chemical composition and especially on the presence of phosphoric 
acid. Monocalcium and dicalcium phosphate are equally effective, 


204 AMMONIFICATION 


according to Lipman, in stimulating the activities of the decay 
bacteria. Thomas slag increases the production of ammonia to 
a considerable extent. Where acid phosphate is applied to a soil 
the increased ammonification resulting, according to McLean and 
Wilson, is due to fungi rather than to bacteria, but this would 
probably vary with the magnesium and calcium carbonate content 
of the soil. 

Chemistry of the Process.— Rettger and his associates have shown 
that bacteria are unable to attack or bring about the decomposition 
of proteins without the aid of enzymes or other proteolytic agents, 
whereas Itano using the formal titration method of Sdrenson has 
shown that B. swbtilcs produces a gradual increase of formal-titrating 
nitrogen for a period of two hundred and forty hours. The greatest 
proteolysis took place toward the optimum hydrogen-ion concentra- 
tion. He, therefore, suggested that the enzyme is probably tryptic- 
like in nature, and endocellular. Moreover, as was seen in the preced- 
ing chapter, a number of amino-acids have been isolated from the soil. 
Hence it is most natural to assume that the disintegration of pro- 
teins in the soil is primarily protein hydrolysis catalyzed by endo- 
or exo-enzymes secreted by the decay bacteria. The enzymes— 
pepsin, trypsin, erepsin, and probably other protoclastic enzymes— 
are capable of hydrolyzing proteins with the formation of some 
elghteen amino-acids. The number and quantity of each depends 
upon the specific protein hydrolyzed. Incomplete hydrolysis 
results in the production of a number of intermediate substances 
variously designated in the order of decreasing complexity— 
proteoses, peptones, and polypeptids. 

Taylor suggests the scheme given on page 205 as indicating the 
stages in the hydrolysis of the protein molecule. 

There are reasons for believing that in the process of ammonifica- 
tion the hydrolysis is similar to this, for Marchal showed that the 
ammonifiers are capable of hydrolyzing proteins, proteoses, and 
peptones. Moreover, many of the protein hydrolytic products are 
found in the soil, and Miyaka has shown from a mathematical 
analysis that the process of ammonification is an autocatalytic 
chemical reaction in which the increase of ammonia in the process 
is in accordance with the formula: 


Log x 


= K(t-ti) 


A-x 


Where 2 is the amount of ammonia which has been produced at 
time (¢), a is the total amount of ammonia produced during the 
process, KK is a constant, and ¢, is the time at which half of the total 
amount of ammonia is produced. 


205 


CHEMISTRY OF THE PROCESS 


Splov-oulMy 
Splov-ourme<—_pr}dog 


\ pdedsjog 
Hee pydod4jog 


Sploe-ourmle<—_prydag 


Splov-oulMmly 
sploe-ourme<—_pydog PHeeea ed 
Splov-oulmly 
Splov-ourme<—prydog } pagedsied 
Splov-Oululy 
Splov-ourme<—pr}dogq 


Splov-oululy 
Splov-ourme<—_prydog 


} pydedajog 


} pydedsjog 


Sploe-oullnly 
sploe-outme<—prydog 


splok-ourmy<—pr}dedAjog 


euojydog 


9800} 01g 


| ' aseo}01q 


9uo0}deg 


} pyded4sjog 
euo}deg t 
sploe-outmre<—_pydog 


sploe-ouray | 
sploe-ourme<—prydog { 


pudedAjog 


splov-ourmry | 


sploe-ourmre<—prydeg f{ PHeomsd 


splv-ourumy \ 
splov-ourme<—prydeg { 


sploe-oulary | 
sproe-ourmree—prydog f{ 


9800} 01g 
asoojorg | 
era doa u19}01g 
ploe<ule} 
-o1d 9ATVEN 
pydedsjog 
9uoj}dag 
pydedsjog 


206 AMMONIFICATION 


The quantity and quality of the products formed would depend 
upon the specific enzyme or enzymes secreted by the microérgan- 
isms. Where pepsin is the main ferment large quantities of proteoses 
and peptones would be formed, while if trypsin is the active agent 
the amino-acids would also occur, and with erepsin the amino-acids 
would be formed even more rapidly. It is not unreasonable to 
suppose that in a soil having a high ammonifying efficiency the 
ammonifying flora are not only numerous but they are active 
secreters of proteoclastic ferments. Moreover, this disintegration 
of the protein molecule which eventually converts nearly the whole 
of the organic nitrogen into ammonia results from the combined 
action of numerous microérganisms of different species. ‘The 
products elaborated by one class probably serve as the point of 
attack for another. 

Potter and Snyder found there was no tendency for the amino- 
acids to accumulate in the soil, but they fluctuated with the ammonia, 
thus indicating a connection between the two. Jodidi added 
various amino-acids and acid amids, including glycocol, leucin, 
phenylalanin, asparagin, aspartic acid, glutanic acid, tyrosin, 
alanin, cadaverin, acetimid and propionamid to the soil, and after 
from two to ten days determined the ammonia, although the 
transformation was not quantitative, probably due to other reac- 
tions occurring simultaneously. Yet it was evident that the 
amino-acids and acid amids examined readily undergo in the soil 
the process of ammonification, and, all other things being equal, the 
rate of transformation is greatly influenced by the chemical struct- 
ure so that amino-acids and acid amids of equal structure yield 
about the same proportion of ammonia. 

Effront in 1905 demonstrated that there are produced by soil 
bacteria amidases. The process of deamination was at first 
thought to be one of simple hydrolysis, as follows: 


R CH NEP — COOH -++ HOH = R — CHOH — COOH + NH&; 


But Neubauer and Fromberz concluded that the primary pathway 
of deamination within the animal organism was oxidative and not 


hydrolytic: 
R R R 
ea (SORT | 
a Oy ee oz = 9C = 0°4 INE: 
|\NH2 |\NH2 | 
COOH COOH COOH 
Amino-acid Oxy-amino-acid Keto-acids 


The amino-acids containing sulphur would probably in the 
presence of sufficient oxygen first oxidize the sulphur atom forming a 
derivative of sulphuric acid: 

CH:SH CH.2SO03H 


| 
2CH NH: + 30: = 2CH NH» oa 


COOH COOH 


CHEMISTRY OF THE PROCESS 207 


And this would be deaminized: 


CH2S0;3H CH2S0;:H CH2SO;H 
paesal (OF 
CK 0s" = (C7 = 2¢ = O + 2NH: 
| \NH2 | \NH2 
COOH - COOH COOH 


In any case the resulting ammonia would be fixed within the soil 
by the zeolites, acid radicals, or lost through volatilization. That 
which is retained in the soil is later taken up by plants or trans- 
formed by bacteria into protein or nitrate nitrogen. 

For each molecule of ammonia formed there would result a corre- 
sponding molecule of volatile acid, or oxy-acid, or else of alcohol 
which may serve as a source of carbon for other microérganisms, 
possible for the azofiers. 


REFERENCES. 


Lohnis, F.: ‘‘ Handbuch der Landwirtschaftlichen Bakteriologie.”’ 

Lafar, Franz: ‘Handbuch der Technischen Mykologie,’’ Dritter Band. 

Voorhees, Edward, B., and Lipman, J. G.: ‘‘Review of Investigations in Soil 
Bacteriology” (U.S.D.A. Off. Exp. Sta. Bul. 194). 


CHAPTER XXII. 


NITRIFICATION. 


Tue term nitrification refers to the oxidation of ether ammonia 
or nitrites to nitrates. It is often used in a broader sense to imply 
the production of nitrates from decomposing organic material. 
The process of nitrification was made use of in the manufacture of 
saltpeter to supply the large quantities of gunpowder consumed 
in the almost incessant wars of Europe. In the eighteen century 
the artificial production of saltpeter in beds of decaying organic 
matter reached a high degree of perfection. Especially was this 
true in Sweden, Switzerland, and France, where nitre was collected 
as a part of each farmer’s tax. In the year 1777 the French Govern- 
ment issued special instructions for manufacture of saltpeter. In 
these there was given special attention to the form of pit to be 
used, the covering of the organic matter, the arrangement for the 
free entry of air, the necessity of a,minerai base, and the optimum 
amount of moisture which was best supplied from the drainage ot 
stables. 

Early Theories.— Even though the process had reached such a 
high state of development, the underlying principles were entirely 
unknown until the last third of the nineteenth century. At this 
time attempts were made to explain the oxidation of ammonia to 
nitric acid, on the strength of certain chemical reactions which 
could be brought about in the laboratory. These were the experi- 
ments of Kuhlmann and Dumas. The first investigator found, 
on passing ammonia and air through a heated tube containing 
a platinum sponge, that they combined with the formation of 
ammonium nitrate, while Dumas found that nitric acid was pro- 
duced when air and ammonia were heated to 100° C. with moist- 
ened lime. It was considered possible that the porosity of the 
soil could act as did the platinum sponge or the lime of the soil 
act in a manner similar to that used in Dumas’ experiments. After 
the discovery of ozone by Schénbein this substance was used _ to 
explain all natural processes of oxidation, and hence naturally the 
case with nitrification. Mulder stated that investigations had 
shown that ozone is capable of oxidizing ammonia to nitric acid 
and water, and that it is probable that the same reaction could 
take place in the soil, the soil acting merely as a catalyzer in the 
reaction, 


THE DAWN OF THE BIOLOGICAL THEORY 209 


It may be seen that in all the early theories it was supposed 
to be a purely chemical process; it was not until the time of Pasteur 
that the biological explanation for the formation of nitrates received 
any support. 

It is interesting, however, to note the careful work of Bous- 
singault in the years between 1860 and 1878, on the natural occur- 
ring saltpeter beds, especially those of Peru and Ecuador. He 
raised the question: ‘‘ Have not the nitrates in these natural deposits 
resulted from the breaking down of organic substances rich in 
nitrogen?” for it had long been the practice to use blood, urine, 
and other animal refuse for the production of nitrates. For this 
reason, Boussingault did not think it likely that the gaseous nitro- 
gen of the air played a very great part in the process of nitrifica- 
tion. In order to test this, he placed soil with known nitrogen 
content in 100-liter jars and allowed them to remain for eleven 
years. At the end of this time they were analyzed, and, in spite 
of the fact that a very active nitrification had taken place, there 
was no increase in the total nitrogen of the soil. From this he 
concluded that free nitrogen takes no part in the formation of 
nitrates, but that they result from the organic matter of the soil. 
In another set of experiments he added organic manure to soil, 
sand, and chalk, and left them to nitrify. He obtained an active 
nitrification in the soil, but none in the sand or chalk. This fact 
had a great influence on the old theories of nitrification. Why this 
difference if the soil acts merely as a catalyzer? It may be said 
that this was the beginning of the end of the old chemical theories 
of nitrification. 

The Dawn of the Biological Theory.—At this time (1878) the 
work of Pasteur was beginning to take firm root. There had 
appeared a series of reports on fermentation, -one of the earliest 
(1862) being on the fermentation of acetic acid.. This was similar 
to nitrification, for it was known that alcohol could be oxidized 
to acetic acid by use of the platinum sponge. In fact in this early 
publication Pasteur suggested that nitrification was due to a fer- 
ment, and soon after Miiller observed that the ammonia in sewerage 
is rapidly changed into nitrates, but no corresponding change takes 
place in pure ammonia solution. He suggested that the sewage 
probably contained a ferment which was absent from the pure 
solution prepared in the laboratory. He, however, took no steps 
to prove that the process was a true fermentation. Between 
1871-75 Gilbert noted that the drainage waters from the Rothamsted 
experiment fields contained more nitrates as the ammonium salts 
applied to the soil increased. 

To the chemists, Schlésing and Miintz, patece the credit of 
establishing by experiment the fact that nitrification is a biological 


process. ‘They were trying to ascertain if the presence of humus is 
14 


210 NITRIFICATION 


essential in the purification of sewage by soil. They filled a glass 
tube one meter long with ignited quartz sand and powdered lime- 
stone. Sewage passed through his filter at first unchanged, but 
later nitrates began to appear, and soon the filtrate contained 
no ammonium salts. They suspected microérganisms as being 
the active agent, and hence treated the contents with chloroform 
vapor. Nitrification entirely ceased and was not renewed for 
seven weeks, although the supply of chloroform was suspended. 
A water extract of fresh garden soil added to the tube soon restarted 
the process. 

These experiments were immediately repeated by Warington who 
confirmed the results of Schlésing and Miintz and showed that: 
(1) The power of nitrification could be communicated to media 
which did not nitrify by simply seeding them with a nitrifying 
substance; (2) the process of nitrification in garden soil is entirely 
suspended by the presence of the vapor of chloroform or carbon 
disulphid. Since these early experiments much additional proof 
has been furnished by investigators showing that the process of 
nitrification both in soils and waters is undoubtedly due to living 
microorganisms. Some of these are the limits of temperature 
within which nitrification 1s possible, the necessity of a suitable 
food, and finally the isolation of specific organisms having the 
power of producing nitrates. 

Schlésing and Miintz were unable to isolate any specific ferment 
capable of causing nitrification, but the true nature of the process 
not being known, many investigators turned to this phase of the 
work and the race to see who would first reach the coveted goal be- 
came intensely interesting. Celli-Zuco and Heraeus, in 1886, suc- 
ceeded in isolating from water rich in nitrates a number of forms of 
bacteria which they considered possessed very feeble nitrifying 
properties. Inasmuch as the nitric acid in their cultures may have 
been absorbed from the air, and as they did not succeed in isolat- 
ing and proving any organism to be capable of nitrification their 
experiments were considered to be inconclusive. Frank attempted 
a similar isolation but without success, and he even concluded 
that nitrification was not due to the direct action of microdrganisms 
but was a purely chemical process. But this view was opposed 
by a number of writers, notably Landolt, Platt, and Baumann. 

Warington and Frankland studied a large number of soil organ- 
isms, but neither was able to find any which produced active 
nitrification. Frankland continued to maintain that the nitrify- 
ing organism was present in soil, and in 1890 succeeded in isolating 
a spherical organism about 0.8 « in diameter which possessed the 
power of converting ammonium salts into nitrites, but not into 
nitrates. The separation was made by means of the dilution 
method, using only inorganic salts. 


ISOLATION OF NITRIFYING FERMENTS 211 


Isolation of Nitrifying Ferments.—The only definite result which 
had been reached up to 1890 was that there must exist in soil 
microorganisms which possess the power of nitrifying. However, 
up to the time Winogradsky took up the subject all attempts to 
isolate such organisms had proved futile. His previous experience 
had been such as to confirm his belief in such organisms. He had 
discovered microdrganisms which oxidized hydrogen  sulphid 
(sulpho-bacteria) and an iron compound (ferro-bacteria). He 
reasoned that it was extremely probable that organisms should exist 
in water and insoil capable of availing themselves of the abundant 
energy which would come from the oxidation of the ammonium 
compounds contained therein. He considered that the number of 
such species would be small and that the way to secure and study 
such organisms would be: (1) To find a medium and condition 
under which they would thrive and by which the growth of deni- 
trifying organisms would be discouraged; (2) to continue the culti- 
vation by such a method long enough to: eliminate for the most 
part other organisms; and (3) when the cultures of the oxidizing 
organisms should have been obtained reasonably pure and their 
nitrification of ammonia active, to proceed to isolate the various 
organisms and study the character, and especially the nitrifying 
power of each in pure cultures. He knew that all previous attempts 
to isolate the organism on gelatin plates had failed and he con- 
sidered that probably the organism would not grow on gelatin. 
He began by working with two soils, one rich in organic matter, 
the other poor, but both rich in carbonate, and soon he learned that 
organic matter hindered nitrification. After considerable work he 
finally adopted a medium having the following composition: 


Waterarlake:Zurichw oul fcgie sks gion wa oso) LOOO'CIC, 
AMMONIUM Sulphate «ke A OL eae A 1 gm. 
Rotassum mhosph ate ees tore led ie a) Serr aw satan | 1 gm. 
Maenestim: carbonate cau. lina. is, bt.) Se amine us 0.5-1 gm. per 100 c.c. 


When such a medium was inoculated with suitable material, 
nitrification was so active that after fifteen days every trace of 
ammonia had disappeared, whereas check solution contained only 
slight traces of nitric acid. 

Cultures were continued and repeated in this medium for three 
months, when he considered that only the organisms suited to 
this medium had survived. These were inoculated on to gelatin 
plates and five species of organisms were found to form colonies— 
three bacteria, one oidium, and the fifth a peculiar organism which 
he designated as “fungus.” None, however, possessed the power 
of converting ammonia into nitric acid. 

In the liquid cultures it was observed that a very thin film gradu- 
ally formed on the surface of the culture and at times a slight 


212 NITRIFICATION 


cloudiness of the solution was noted. The latter disappeared 
after a time and a microscopic examination showed it to be due 
to the presence of an oval somewhat spindle-shaped organism 
which moved about very rapidly. Nitrification was at the same 
time very active. It was thought that the film on the surface 
might contain the nitrifying organisms as the acetic acid bacteria 
and other oxidizing ferments work at the surface where plenty of 
oxygen can be obtained, but tests with this gave negative results. 

The plan of work was then somewhat changed. The attempt 
was made to cultivate the nitrifying organisms more abundantly. 
To this end a quantity of ammonium sulphate was added to the 


Fic. 30.—Surface colonies of nitrosomonas on silicic acid gelatin, stained with 
Fuchsin without removal from the gelatin. XX 1200. (After Gibbs: Soil Science.) 


nitrifying cultures, and the process of nitrification thus continued 
in the culture for some time. A change was noticed in the mag- 
nesium carbonate at the bottom of the solution which gradually 
assumed a grayish color and a gelatinous consistency. By shaking 
the solution vigorously this mass was broken up into small flakes, 
which a microscopic examination showed to consist of transparent 
particles of the salt covered with a mass of oval bacteria, identical 
in form with those which had previously been noticed as the cause 
of the cloudiness. These bacteria seemed to be on the particles 
exclusively and not on the walls of the flask, and slowly enveloped 
the salt which was finally dissolved. 


ISOLATION OF NITRIFYING FERMENTS PA 3) 


A culture medium was then prepared which was free from every 
trace of organic matter. On being inoculated, nitrification took 
place in this energetically, and it was found to contain large num- 


Pe aaa ee 8", @ 
a pee & > er 
®>s * 
ek, ea 
> Pd “pe 
R “e > & 
t oF ‘ e 
o* w& * 3  » 
J ? 5 > ee 
& od - ox : 
7.8 = g ¢. Bh 
of id - i: 4 ¢ 
a “ > & 8 2 ry} 
an ay ok Tc vas wf 
* oy. Tt « 
se? 269,32 te: ¢ 4% 
ie ° 78 Sp *¥ " 
o? % * ¢ 
ee : sd N 
? wee bed »y 
¢ ! 4 
| e? 3 3 © Ah Py 
: tae a 
cee ¥ 
ad - * . 
an 2 s o 
* ee 


Fig. 31.—Nitrobacteria from cultures in liquid medium stained with carbol Fuchsin. 
x 2500. (After Gibbs: Soil Science.) 


bers of the oval bacteria, as well as the fungus form previously 
noticed, but the other forms had all disappeared. The fungus 
remained constant, and all attempts to cultivate it out were unsuc- 
cessful. 


214 NITRIFICATION 


The research was thus brought to the stage where it seemed 
probable that the oval bacteria might be the nitrifying agents. 
To test their nature and action satisfactorily the removal of the 
sprouting fungus was called for. ‘To accomplish this, Winogradsky 
resorted to a very ingenious though a simple device. The fungus 
would develop in gelatin; the bacteria would not. Small particles 
of the carbonate, more or less enveloped by the bacteria, were 
taken from the bottom of the culture flask by means of a capillary 
tube and placed in a large flask of sterilized water. The contents 
of the flask were then well shaken and a gelatin plate inoculated 
with drops of the liquid, the particles of carbonate serving to indi- 
cate the places where the gelatin had been inoculated. In some 
of these the fungus developed. Inoculations of the culture liquid 
from the other spots failed to yield the fungus but developed the 
bacteria. By this method of “inverse gelatin culture” the bacteria 
were obtained pure. Liquid cultures inoculated with the bacteria 
oxidized ammonia rapidly. The inference was that the bacteria 
were the nitrifying organisms of the soil. 

Winogradsky describes the nitrite-forming organisms as of oval 
form, about 1.1 to 1.8 » long and 0.9 to 1 uw wide, usually at rest but 
at times capable of motion and dividing perpendicularly to the 
longest axis. He places it in a genus by itself, which he calls 
Nitrosomonas. 

The nitrate-forming organism, nitrobacter, is 0.3 to 0.4 u wide 
and about 1 w long. The cells occur singly or in pairs and occa- 
sionally in threes. They are spindle-shaped, non-motile, and 
possess a capsule which makes them difficult to stain. 

By way of comparing the activity of the nitrobacter with that 
of the ferments as they actually occur in soil, Winogradsky made 
a series of experiments to compare the amount of nitrification in 
his culture liquid with that observed by Schlésing in a soil to 
which, however, more oxygen had access than was the case with 
Winogradsky’s liquid. 

While in Schlésing’s experiments by the use of 200 grams of 
earth, 3.4, 9, and 4.1 mg. of nitrogen, respectively, were nitrified, 
Winogradsky’s pure cultures of bacteria nitrified 860 mg. of ammo- 
nium sulphate in twenty-seven days and 930 mg. in thirty days. 
Therefore, during the period at which nitrification was most ener- 
getic there would be formed about 7.2 mg. of nitrogen per day. 

Winogradsky further investigated the interesting and very 
remarkable fact previously cited, that the nitrobacter, although 
containing no chlorophyll, grows and is able to multiply in a solu- 
tion entirely free from organic matter. ‘To prove this fact beyond 
doubt he prepared a culture medium absolutely free from every 
trace of organic matter by using twice distilled and tested water 
and salts which had been carefully purified by recrystallization. 


ISOLATION OF NITRIFYING FERMENTS 215 


He thoroughly removed all organic matter from the glass dishes 
and apparatus to be used, and inoculated separate portions ot 
the medium with the nitrobacter. The cultures developed nor- 
mally in the dark as well as in the light. To gain an idea of the 
extent of the assimilation of carbon, the carbon in the organic 
matter which had been formed by the organism in its growth was 
determined by analysis. Four cultures contained 10.2, 7.1, 4.6, 
and 4.8 mg., respectively, of assimilated carbon, and in these 
cultures 928, 604, and 83.5 mg., respectively, of nitric acid had 
been formed. This seemed to leave no doubt that nitrobacter is 
able to assimilate the carbon of carbonic acid. 

Later, in 1891, Warington, in a solution containing mineral 
salts, obtained after repeated generation a culture which nitrified 
vigorously. This contained no organisms which would grow on 
gelatin and was regarded by him as containing only nitrifying 
bacteria. The organisms thus obtained were oval in form and 
seldom 1 micromillimeter thick and only slightly longer. 

At this time Winogradsky made a decided improvement in the 
separation of the nitrifying organism from solutions by use of the 
Kiihne gelatin silica medium. The nutrient basis of this medium 
as used by Winogradsky was composed of ammonium sulphate, 0.41 
grams; magnesium sulphate, 0.05 grams; potassium phosphate, 0.1 
gram; sodium carbonate, 0.6-0.9 gram; calcium chlorid, a trace; 
and water, 100 c.c. 

The inoculation of the plates took place either by mixing the 
inoculating material with the above solution before the addition 
of gelatinous silica, or it was made as a streak or smear culture 
on the already hardened material. In this way the nitrifying 
organisms developed distinct colonies from which pure cultures 
were obtained. 

The investigations of Winogradsky and simultaneously of War- 
ington showed the following: (1) That in the soil the nitrifying 
process was effected by two distinct but closely related organisms, 
_the one converting ammonia into nitrous acid and nitrite and the 
other changing the nitrites into nitrates. (2) That these two 
processes follow one another in such rapid succession that the 
production of nitrites is only a transitory phenomenon, so that 
if both the nitrite and nitrate organism be added to sterilized soil 
the process is completed in the natural way, only the merest traces 
of nitrous acid appearing. 

If to a mineral solution containing ammonium salts, a pure cul- 
ture of rfitrosomonas be added, only nitrites will appear and these 
will remain unchanged in the absence of the nitrobacter. If, 
however, the two organisms be added simultaneously nitrates will 
be rapidly formed. 


216 NITRIFICATION 


According to Kaserer there is an organism which can oxidize 
ammonia direct to nitric acid, but so far this has not been confirmed. 

In 1892 Winogradsky studied the nitrifying organisms of the 
soil from a number of different localities. Those from several 
parts of Europe, from Africa, and from Japan, which he considers 
to be the same organism, he names Nitrosomonas europea. A second 
form from Java soil differing from the first, he names Nitrosomonas 
javenensis. Both of these comprise the nitrate ferments of Wino- 
gradsky, the second nitrate ferment was isolated by Winogradsky 
from Quito soil and differs from the first not only as to size, as 
above mentioned, but also by entirely lacking the motility common 
to the latter. 

In 1895 Burri and Stutzer isolated from soil a nitrate organism 
with properties akin to the Quito bacillus of Winogradsky. It was 
a motile organism, 0.75 — 1.5 x 0.5. micromillimeters, growing 
on gelatin which it liquefied, said organism, according to these 
workers, being able to convert nitrites into nitrates, but losing 
such power when grown on organic media. 

The results of Burri and Stutzer, so contrary to those of Wino- 
gradsky, brought forth a vigorous rejoinder from the latter. In 
this Winogradsky stated that he tested the same earth used by 
Burri and Stutzer and isolated therefrom his own nitrosomonas, and 
that the latter when tested in bouillon, meat peptone, gelatin, and 
agar failed to grow. He, therefore, regards the German work as 
erroneous. 

In 1897 Stutzer and Hartleb appeared with a still more startling 
series of discoveries in which they not only maintained the ability 
of the nitrifying organisms to grow in organic media, but also 
showed that the latter possessed a polymorphic habit never imag- 
ined in this or any other like group in the whole domain of mycol- 
ogy—the ability of simple coccoid or réd-shaped forms to develop 
into filaments or even into branched forms, with the further pro- 
duction of true gonidia and other even more highly organized 
fructification bodies. 

Girtner discussed the work of Burri, Stutzer and Hartleb on the 
polymorphism of the nitrifying organism, and from presumably 
pure cultures of the latter’s nitrifying ferment was able to isolate 
thirteen different microédrganisms, including a fungus form (Schim- 
melpilz), thus proving their impure character. Furthermore, 
Giirtner showed that these several organisms, when once separated 
in their pure state, retained their fixed character, with no tendency 
to polymorphism, and indicated none of those transition stages 
from bacteria to fungi noted by Stutzer. Again, none of these 
isolated organisms possessed the power to convert ammonia into 
nitrites. C. Fraenkel simultaneously isolated from Burri and 
Stutzer’s cultures 11 different organisms, including 7 bacilli 


DISTRIBUTION 217 


2 streptothrices, and 2 fungi (a Fadenpilz and a Schimmelpilz). 
These showed no polymorphism, but all retained constant char- 
acters. 

In 1902 Chester summarized the knowledge on nitrification as 
follows: 

1. That nitrification in the soil is caused by a distinct or rather 
by two distinct organisms possessing certain definite characters. 

2. That these organisms will not grow in the presence of any 
considerable amount of organic matter, and that all reported 
attempts, to cultivate them on ordinary organic media are without 
authentication. 

3. That the above nitrifying organisms are found abundantly 
in all cultivated soils and in ordinary soil water containing a due 
proportion of ammonium carbonate, sulphate, etc., they find a favor- 
able medium for their development. 

4. That the result of such development is: (a) The conversion 
of ammonia into nitrous acids through the agency of the nitrous 
organism; and (b) the immediate conversion of the previous nitrous 
into nitric acid by means of the equally abundant nitric ferment. 

Distribution.— Probably the nitrifying bacteria were some of the 
first living organisms to appear upon this planet, and even yet 
they act as the pioneers preparing the soil for other plants. Miintz 
has found the decayed rocks of Alpine summits, where no other 
life exists, swarming with the nitrifying ferments. The limestones 
and micaceous schists of the Pic du Midi, in the Pyrenees, and the 
decayed calcareous schists of the Faulhorn, in the Bernese Ober- 
_land, offer good examples of this kind. The organisms draw their 
nourishment from the nitrogen compounds brought down in snow 
and rain; they. convert the ammonia into nitric acid, and this in 
turn corrodes the calcareous portions of the rock. Stiitzer and 
Hartleb have observed a similar decomposition of cement by 
nitrifying bacteria. 

The nitrifying bacteria appear to be very widely distributed 
Miintz and Aubin have observed their presence not only in all 
cultivated soils which they have examined, but also in those of 
deserts. They are not usually found in the air or in rain water. 
River water and sewage contain them. ‘They are usually present 
in well waters. In the case of deep wells their origin is due to 
surface soil or to drainage from the surface soil which has found 
its way into the well, the water of deep wells not being their natural 
habitat. Thomasen found the nitrite organism in samples of 
ooze from the bottom of the Mel Fjord, but not in the sea water 
nor on the Plankton or the fixed alge. It was also found in similar 
samples of soil from the vicinity of Helgoland and in slime from 
the bottom of the Bay of Naples, but eal in samples taken near 


the land. 


218 NITRIFICATION 


Warington failed to find the nitrifying ferments in a clay soil 
below eighteen inches, and this is in keeping with the findings of 
Ladd at North Dakota. For a long time it was considered that 
they are found only in the surface soil, but in 1906 Welbel pre- 
sented results with soils where nitrification is almost as active in 
the subsoil as in the surface soil when the subsoil is aérated. In 
1912 C. B. Lipman found them often to a depth of five or six feet 
in soils of the arid regions. In one case soil from the eight-foot 
depth showed a vigorous nitrifying power. The author found soil 
from second and third foot-sections to nitrify dried blood quite 
readily, as is shown below: 


Irrigated Dry-farm 
Depth Soil Soil 
Hirst inwelveanehes 42% 29m gay sees wanes Lee SES 45) 
Second twelve inches eA A iad WE TON is A 2.70 2.41 
Phirdstwelyeunchess se) ies ie es le a ee 1.98 1.56 


These are the averages of several hundred examinations, and 
many soils which were fairly heavy clays showed active nitrifica- 
tion in the second and third foot-sections. This great difference 
observed in the arid regions is due mainly to a better aération of 
these subsoils which, because of the peculiar climatic conditions, 
the arid soils are not as rich in clay as are the subsoils of the humid 
regions. Moreover, the plants in the arid regions root to a great 
depth in search of water. These decaying roots loosen up the 
subsoil and also furnish food for bacterial growth. 

Reaction of Media.— Boussingault long ago observed that many 
forest soils do not contain nitrates, and later this was verified by 
Bréal and others. We now know that the absence of nitrates is 
due to the acid reaction of a soil which contains an excess of organic 
matter. The nitric ferment does not act in an acid medium; hence, 
we have the explanation of the great benefit derived from the use 
of basic substance. 

Experiments by Wiley and Elwell in which solutions containing 
calcium chlorid and water were seeded with nitrifying ferments 
continued to nitrify until the medium contained an acidity equiva- 
lent to 4 c.c. of normal acid per 100. 

Dumont and Crochetelle’s experiments are of the same order. 
They took soil which had been in grass from time immemorial and 
which contained 6.84 per cent. of humus. This was treated with 
variable quantities of potassium carbonate. It was stirred and 
watered several times during the experiment and after one month 
the nitrates were extracted with the following results: nitric nitro- 
gen, per 1000 grams, of soil without addition of potassium car- 
bonate, 70 mgs.; with 1 gram of potassium carbonate, 160 mgs.; 
with 2 grams of potassium carbonate, 230 mgs.; with 3 grams, 250 
mgs.; with 4 grams, 130 mgs.; with 5 grams, 73 mgs. In similar 


REACTION OF MEDIA 219 


experiments, Kochenayski demonstrated that potassium carbo- 
nate is more efficient in this regard than is calcium carbonate, 
probably because the potassium acts as a food in addition to the 
neutralizing of the acid. Owen has found magnesium carbonate 
even more efficient than potassium carbonate, and this is in keep- 
ing with the findings of Lyon, and Bizzell and White. Pangan- 
iban’s findings appear to differ from these, for he claims that liming 
greatly increases nitrification only when the limestone contains little 
magnesium carbonate. The soil of the Utah Greenville farms con- 
tains 16.88 per cent. of lime (CaO) and 6.1 per cent. magnesium 
(MgO), and they nitrify ammonium sulphate, dried blood, and 
cottonseed meal readily. 

The carbonates are not the only substances in the soil which 
serve as bases for nitrification, since, according to Ashby, a marked 
nitrification of ammonium salt can be brought about in the presence 
of ferric hydrate, either in the freshly precipitated state or as 
“iron rust.’’ In solutions, however, nitrification is not completed 
where iron is the only base, probably because the ferric nitrite or 
nitrate formed dissociates and the solution becomes acid. 

The double ammonium combination formed by the absorption 
of ammonium salts by modelling clay can most probably be nitri- 
fied in the absence of any base, but the corresponding combination 
with peat undergoes no nitrification in the absence of a base. 

One of the functions of the base in nitrification is to form ammo- 
nium carbonate, and the facility with which nitrification is set up 
by different carbonates depends upon the rapidity with which 
they can react with a neutral ammonium salt to produce ammo- 
nium carbonate. This reaction is greater with magnesium car- 
bonate than with calcium carbonate, but is almost absent with 
copper carbonate. 

The quantity of lime which must be added to a soil for maximum 
nitrification varies with the original reaction of the soil and the 
fertilizer to be nitrified; ammonium sulphate requires more than 
bone meal, cottonseed meal, or dried blood. 

There should always be an excess of the base present, for Fischer 
found that the theoretical amount of lime (200 grams of calcium 
carbonate) required for the nitrification of ammonium sulphate 
(182.7 grams) was not sufficient for complete nitrification, but 
about three and one-half times the theoretical amount was required. 
Even much larger quantities of either magnesium carbonate or 
- calcium carbonate may be used without ill effect, but large quan- 
tities of quicklime may cause a rapid burning out of the organic 
matter and even volatilization of ammonia and may even stop 
nitrification. For, while nitrification takes place in a feebly alka- 
line medium, yet the presence of anything beyond a small quan- 
tity of an alkaline salt is a hindrance to the process, and a large 


220 NITRIFICATION 


amount will check it entirely. Thus Warington found that the 
presence of 0.052 per cent. of bicarbonate of soda distinctly retarded 
nitrification, and in the presence of 0.096. per cent. nitrification was 
only barely possible.. The same author also showed that the 
presence of 0.0477 per cent. of ammonia in urine rendered it unnitri- 
fiable. Dumont and Crochetelle found that potassium carbonate 
added to soil at the rate of from 1 to 2.5 grams per 1000 grams of 
soil markedly increased nitrification, but larger applications of the 
salt progressively diminished the rate of nitrification, and that the 
addition of 8 grams per 1000 grams of soil completely checked it. 
A heavy dose of lime by unduly increasing the alkalinity of the 
soil may at first check or suspend nitrification until the said lime 
has been converted into carbonate. This, however, takes place, 
rapidly, diminishing in turn its strong alkaline properties and per- 
mitting nitrification to commence more actively than before. 

Food Requirements of Nitrifiers.—'The nitrifying organisms require 
the same elements as do other bacteria, and hence will be considered 
in this chapter only in a very general way, except in regard to 
the source of the required elements. 

Winogradsky found that the nitrosomonas were able to grow in 
a medium consisting of 2.25 grams of ammonium sulphate, 2 grams 
of common salt, and 1 grams of magnesium carbonate in 1 liter of 
well water. For the nitrobacter the ammonia is replaced by 
sodium nitrite. In media such as the above, devoid of organic 
carbon, the nitrifying organisms are able to function in the dark 
and form from the inorganic carbon, organic carbon compounds. 
He proved by numerous quantitative determinations that during 
nitrification an increase in the amount of carbon compounds takes 
place. “Since this bound carbon in the cultures can have no 
other source than the carbon dioxid and since the process itself 
can have no other cause than the activity of the nitrifying organ- 
ism, no other alternative was left but to ascribe to it the power of 
assimilating carbon dioxid. 

‘“Since the oxidation of ammonia is the only source of chemical 
energy which the nitrifying organisms can use, it is a priori that 
the yield in assimilation must correspond to the quantity of oxi- 
dized nitrogen. It turned out that an approximately constant 
ratio exists between the values of assimilated carbon and those of 
oxidized nitrogen.” This is illustrated by the following results: 


No. 5. No. 6. No. 7. No. 8 
Oxidized nitrogen Se awe) 506.1 928.3 815.4 
Assimilated carbon. 19.7 15e2 26.4 22.4 
Ratio—nitrogen: carbon 36.6 33.3 BOR 36.4 


It is evident that 1 part of assimilated carbon corresponds to 
about 35.4 parts of oxidized nitrogen or 96 parts of nitrous acid. 


FOOD REQUIREMENTS OF NITRIFIERS 221 


More recently, Coleman using pure cultures of nitrate producers 
obtained ratios varying from 40 to 44. 

Now there are two sources of carbon dioxid which are available 
to the nitrifying organisms—one, the carbonate, which is present 
in the soil; the other, the carbon, in the air. According to Wino- 
gradsky, the carbonate supplies the carbon for the bacterial growth, 
it being liberated by means of the acids, which they produce. On 
the other hand, Godlewski considered that it is chiefly from the 
atmosphere that the carbon dioxid requisite for the construction of 
new cellular substance is derived. He found that development 
did not occur in cultures containing magnesium carbonate when 
air free from carbon dioxid was admitted, and concluded that: 
(1) Nitrosomonas placed in a pure mineral solution are unable to 
assimilate the carbon of magnesium carbonate; (2) it is very improb- 
able that the nitrobacter derive their carbon from the organic 
substances of the air; (3) it is very probable that these organisms 
find the carbon which they need in the free carbonic acid or in the 
‘carbonic acid of bicarbonates. But Owen, after careful experi- 
ments in which he used a specially devised flask for the elimination 
of the carbon dioxid of the air, concluded that “the nitrifying 
organisms of the soil do not depend to any appreciable extent on 
the carbon dioxid of the air for their carbon supply.” Hence, the 
evidence seems to be that the organisms under appropriate condi- 
tions possess the power of utilizing either source of carbon. 

The nitrite bacteria obtain their nitrogen both for oxidation in 
the production of energy and as building material from ammonia 
preferably in the form of ammonium carbonate. They are, how- 
ever, according to Ashby, able to utilize the double ammonium 
combination formed through the absorption of ammonium salts 
by modelling clay, but the corresponding combination with peat 
undergoes no nitrification in the absence of a base. However, 
according to Marcille, the nitrogen of ammonium phosphate is not 
so readily transformed into nitrous acid as is that of ammonium 
sulphate. Yet the phosphate appears to furnish a much more favor- 
able medium for the transformation of nitrites into nitrates than 
does the sulphate. » 

While calcium cyanamid is nitrified when added to a soil, it is _ 
not until it has been transformed into ammonia by other bacteria, 
chief among which are, according to Léhnis, B. putidum, B. 
mycoides, B. vulgare var., B. zopfii lepsiense (n.sp.), B. kirchnerr 
(n. sp.), B. megatherium, B. fluorescens, B. subtilis, B. ellenbachensis 
and B. vulgare. According to Boullanger, the nitrous organism 
does not attack hydroxylamin hydrochlorid. 

Excessive quantities of ammonia or ammonium salts hinder the 
multiplication{of ,nitrifying organisms but do not interfere with 
the action of those already present, Boullanger and Massol found 


222 : NITRIFICATION 


the minimum retarding amount of ammonia to be about 2 parts 
per million. It is seldom sufficient ammonia accumulates in soils 
under natural conditions to interfere with the multiplication of 
nitrifying bacteria. 

Just as all organic nitrogen must be ammonified before it can 
be changed by the nitrosomonas to nitrous acid, so all ammonia 
compounds must be oxidized to nitrous acid before the nitrobacter 
can convert them into nitric acid. The nitrite organism readily 
oxidizes nearly all nitrites in solutions containing 0.5 to 1 gram 
per liter, but larger quantities of the nitrites are toxic even to the 
nitromonas. 

Organic Matter. —Winogradsky early learned that the nitrifying 
organisms will not grow in a medium containing soluble organic 
matter, and since that time numerous experiments have been 
made to account for this apparent discrepancy. It was well known 
that nitrification takes place in the soil and compost which contains 
organic material. Hence, the theory was soon advanced that 
organic matter in the form of humus is not injurious and may 
actually be beneficial, as is illustrated by the work of Smirnov: 


Humus Nirric Nirrocen IN 100 Grams Soin 

Per cent. At beginning After 19 Days After 36 Days After 73 Days 
0.42 TH mak Dane. 14.0 mg. 25.5 mg. 28.0 mg. 
3.55 vat eee eNOS 21.0 38.0 53.0 


Miintz later concluded that humus even in larger quantities 
does not interfere with nitrification, but on the other hand it is 
favorable to it. Nor is an abundance of humus a necessary condi- 
tion to nitrification, since soils poor in this constituent gradually 
develop intensive nitrification. He considers that the humus 
favors the multiplication of the nitrifying organisms and a soil 
which contains a large amount of humus is more abundantly sup- 
plied with these organisms and more apt to enter into rapid nitri- 
fication. : 

Coleman found dextrose, cane sugar, glycerin, and lactose, in 
small amounts, to favor nitrification, and in some cases even as 
much as 1 per cent. of dextrose has proved beneficial. This con- 
clusion has been confirmed by numerous other workers. Where 
larger quantities of sugars are used there is usually a disappearance 
of nitrates. This is probably due to its favoring other organisms 
which produce protein from the nitrates rather than interfering 
with nitrification or accelerating to a great extent denitrification. 
The optimum amount of organic matter for most rapid nitrification 
varies with the moisture and nature of the‘soil. Fischer found 
even peat extract to favor nitrification, while Niklewski claims 
that nitrification occurs in solid stable manure when there is not 
much liquid manure mixed with it, and that on the first day nitrite 
bacteria are found in the manure coming originally not from the 


METABOLISM C 223 


stock but from the straw, particles of earth, etc., that stick to the 
manure. ‘These bacteria increase in number until at the end of 
four weeks there may be 1000 per gram of substance associated 
with these. Hence, we may conclude that the absence of nitri- 
fication which has been noted by various workers when organic 
matter-is present may be due to some of the following factors: 
(1) Excessive quantities of soluble organic matter. This has been 
repeatedly found-to be the case where excessive quantities of 
carbohydrates have been added to the media. (2) A low per- 
centage of potassium as suggested by Renault. (3) The physical 
and chemical properties of the medium, as noted by Stevens and 
Withers. (4) The presence of organic acid, as is the case in peats 
and forest soils. In this condition it is the acid reaction which 
interferes with the process and not the organic matter present. 
(5) A substance may be toxic when tested by the solution method, 
whereas in the soil it may be inert or actually beneficial. 

Energy.—The nitrifying organisms are devoid of chlorophyll 
and function best in the dark, yet they synthesize from the carbon 
dioxid complex organic compounds. The energy necessary for 
this synthesis is obtained by the nitrosomonas from the oxidation 
of ammonia: 


2NH; + 3802 = 2HNO: + 2H20 +. 157.6 Cal. 


and by the nitrobacter from the oxidation of nitrous acid: 


2HNOz: + O2 = 2HNOzs + _ 36.6 cal. 


Lafar points out that if the quantity of nitrogen oxidized per 
unit of time be taken as the standard for measuring the chemical 
energy of these organisms, the nitrosomonas will be found the 
most active of the two. From this fact he concludes that the 
conversion of the trivalent nitrogen of nitrous acid into pentay- 
alent nitric nitrogen requires the expenditure of a greater amount 
of internal force than is needed for the first step in the oxidation. 

Metabolism.—The metabolism of these organisms has, therefore, 
been the subject of considerable study. Winogradsky early sug- 
gested that the ammonium carbonate in the first place probably 
gives rise to an amid, somewhat similar to the transforming of 
ammonium carbonate into urea: 


NH,O NH,:O NH2z. 
ms 
CO — eo = co 
NH:O/ - NH: / NH2/ 
ammonium ammonium urea 


carbonate carbamate 


224 NITRIFICATION 


It is quite likely that all organic compounds are first trans- 
formed into ammonia by other organisms before they are nitrified. 
Demoussy found this to be true of monomethylamin, trimethylamin, 
anilin, pyridin, and quinolin, and, according to Léhnis, calcium 
eyanamid. This is also true for carbamid, thiocarbamid, uric 
acid, acetamid, anilin sulphate, methylamin sulphate, ammonium 
oxalate, asparagin and ammonium sulphate, which, with the excep- 
tion of thiocarbamid and anilin sulphate, are readily transformed, 
according to Busley, into ammonia by other bacteria and then 
nitrified. Hence, the early conclusion reached by Winogradsky— 
that pure cultures of nitrifying bacteria are incapable of nitrifying 
organic nitrogen—has been borne out by other investigators. 
Where contrary results have been reported it has been due to 
the presence of other organisms by which the nitrogen has been 
converted into ammonia and then nitrified. The process is cata- 
lyzed by oxidizing enzymes which must be specific in their action, 
for Omelianski found the nitrifying organisms unable to oxidize 
mineral compounds such as sodium sulphite and phosphite. 

Oxidation in this case cannot be regarded as being of a violent 
nature and it scarcely seems conceivable that the nitrosomonas 
should be able to oxidize ammonia direct to nitrous acid without’ 
passing through intermediate stages of oxidation. Most workers 
consider it probable that in the oxidation of the ammonium radical 
there are formed certain intermediate substances which must be 
regarded as more or less hydroxylated ammonium radicals. 

Mulford, in a study of the bacterial oxidation of aqueous solu- 
tions of ammonium salts on experimental filters inoculated from 
actively nitrifying sewage filters found that the oxidation proceeded 
in a series of stages compatible with the hypothesis that the hydro- 
gen atoms are successively hydroxylated with the subsequent elimi- 
nation of water. Hydroxylamin salts and salts of hyponitrous 
acid and nitrous acids were found as intermediate compounds. 


Ke) 
Va 
JN 
Ss caanty 
vA H 
in hyponitrous 
Ye acid 
H H H O 
v4 x Va Va 
N—H -— N—H > N—OH >N 
AS. \ Pos 
H OH OH fh OH 
Ne OH nitrous 
ammonia hydroxylamin pee fs acid 
\, N—OH 
/ 
dihydroxylamin OH 
trihydroxyl- 


amin acid 


MORPHOLOGY 225 


There are, however, two serious objections to these conclusions: 
(1) It is not evident that these initial changes noted by Mulford 
were due to the nitrifying organisms, as a mixed culture was used; 
(2) Boullanger and Massol found that while the nitrous organism 
accommodates itself to all ordinary carbonates, it does not attack 
hydroxylamin hydrochlorid. 

The majority of workers have reported a loss of nitrogen in the 
nitrification process, there never being the theoretical yield of 100 
per cent. of the ammonia transformed into nitrous acid, but this 
may be due to side reactions Lafar considers that the loss may 
be due to the reaction of the nitrous acid on the undecomposed 
ammonia in accordance with the equation: 


N20; + 2NH3; = 38H:O + 2N2 


The whole subject of the metabolism of nitrifiers is indefinite 
and in need of careful investigation using the latest refined methods. 
The only fact that does seem to be well established is that the 
process of nitrification goes in two stages from ammonia to nitrous 
acid and from nitrous acid to nitric acid. That these two steps 
are due to two classes of organisms is the claim of most  investi- 
gators. However, Kaserer considers that there is an organism, 
B. nitrator, which can oxidize ammonia direct to nitric acid, the 
reactions being as follows: 


NE: --) HeCO; +O: = HNO; == E:O == CH3O = 41 Cal: 
CH;O + O2 = H:2CO3 + 132 Cal. 


It is interesting to note that the reaction catalyzed by the nitri- 
fying ferments are similar to reactions catalyzed by ultraviolet 
rays. Gaudechon exposed solutions at temperatures of 35° to 50° 
C. for from three to nine hours at a distance of 3 to 6 em. from a 
lamp of 110 watts. Under these conditions the ultraviolet rays 
oxidized solutions of ammonia in the presence of oxygen to nitrites. 
Nitrates were in no case formed. Ammonium salts were also 
oxidized to nitrites, the reaction being slower in the case of the 
sulphates and chlorids than the carbonates. Urea was first con- 
verted into ammonia and then into nitrites. Other organic nitro- 
gen compounds, for example, ethyl- and methylamin, guanidin, 
hydroxylamin, acetamid, and acetonitril behaved similarly. 

Morphology.— Winogradsky described two varieties of the organ- 
isms capable of changing ammonia to nitrites. One of these in 
several species was found in all the soils of the Old World (Asia, 
Africa and Europe) and is known as nitrosomonas. The second 
is peculiar to the soil of the New World and has received the name 
of nitrosococcus. 

He described a single species of the nitrosomonas from European 
soils, namely, Nitrosomonas europea. This organism is provided 

15 


226 NITRIFICATION 


with a single short flagellum and in the early stages of the culture it 
exhibits active powers of locomotion. It appears as short rods 
1.2-1.8 w long and 0.9-1.0 uw broad. The cells of Nitrosomonas 
javanica obtained from the Botanical Garden at Buitenzorg, near 
Batavia, are globular and only attain a diameter of 0.5-0.6 », but 
they have a long flagellum, at times measuring as much as 30 u. 
Those obtained from Tokio soil ( Nitrosomonas japonica) and from 
Africa ( Nitrosomonas africana), are very similar to the European 
species, differing only in that they are somewhat smaller. 

Observations by Burri and Stutzer on impure cultures in mineral 
media led them to believe that there was a difference in oxidizing 
powers in organisms derived from different sources. By this means 
they distinguished five classes from German and one from African 
soil. 

Joshi has recently described a new species from the soils of 
India which differ morphologically from others hitherto described. 

The different species show a variation in sensitiveness to heat. 
Beddies found one species to live for one minute in steam at a 
temperature of 100°. The other two were more sensitive but 
survived for several minutes in dry heat of 80° to 100° C. 

The genus, nitrococcus, found in the New World do not possess 
cilia nor do they form zodglea.. The one obtained from Quito 
(Ecuador) is a coecus 1.5-1.7 uw in diameter. The species, N7tro- 
sococcus braziliensis, obtained from Brazil soil is much larger, being 
2 uw in diameter. 

The nitromonas or nitrobacter differ from those already described 
in physiological properties in that they oxidize nitrites into nitrates. 
Morphologically, they differ in being smaller and more slender. 
They are elongated, oval, mostly pear-shaped, 0.5 w in length and 
0.15-0.25 » in breadth. In liquid cultures they develop a thin 
mucinous skin which adheres firmly to the walls of the vessel. 

From: the variation in sensitiveness to heat, Beddies isolated 
four forms of nitrobacter, one of which was capable of resisting 
the action of steam at 100° C. for two minutes. But Burri and 
Stutzer’s comparative experiments with nitric organisms derived 
from different localities showed no essential difference in physio- 
logical action. 

Neither nitrosomonas nor nitromonas have been observed to 
form spores, but their resistance to drying and to heat, as shown by 
Beddies, makes it appear possible that some species may form spores. 

Influence of Moisture.—Long before the process of nitrification 
was known to be due to microérganisms, the underlying principles 
governing the speed of the reaction had been investigated nation- 
ally by France, Germany and Sweden. Among other things, they 
had learned that there must be a certain proportion of water, and, 
in order that the maximum yield of nitrates be obtained, that this 


INFLUENCE OF MOISTURE 227 


must be diminished as the soil becomes richer in nitrates. As early 
as 1887 Dehérain found that the most active nitrification took place 
when the soil was allowed to become partially dry between the 
applications of water, and later he found that there was a rela- 
tionship between the speed of nitrification and the moisture con- 
tent of fallow soil, the nitrification increasing with the water. 
Boussingault taught that when soils contain as much as 60 per 
cent. of water they lose in a few weeks the greater part of their 
nitrates. This teaching gave rise to the general belief that deni- 
trification may take place to a great extent in soils, but recent 
work has amply demonstrated that it is only extremely abnormal 
conditions where this becomes an important factor. 

Dehérain and Demoussy found that the bacterial action of a 
soil was at its maximum when a rich soil contained 17 per cent. 
of water, but that it decreased if the proportion of water fell to 
10 per cent. or rose to 25 per cent. With soils less rich in humus 
a somewhat higher proportion of water was necessary to retard 
oxidation to any marked degree. 

The optimum moisture content for nitrification, according to 
Dehérain, is 25 per cent. An insufficient supply of moisture 
checked both nitrification and nitrogen fixation. This occurred 
when the water had been reduced to 16.5 per cent. This, however, 
would vary with the soil, for Schlésing found bacterial activity 
less in fine-grained soils than in lighter, coarse-grained soils. In 
order that nitrification be equally active in both light and heavy 
soils, the latter must have a higher percentage of water than the 
former, a difference in moisture content of soil of 1 per cent., accord- 
ing to Dafert and Bollinger, being sufficient to produce a marked 
change in the oxidation going on in the soil. 

Fraps found that the number of nitrifying organisms in a soil 
varies with the moisture and that their activity was periodic, rapid 
nitrification being preceded and followed by periods of less activity. 
Later he found nitrification to be at its height in soil containing 
59.6 per cent. of its water-holding capacity. Excessive quantities of 
water practically stopped nitrification and were much more injur- 
ious than too small a quantity. The water requirements, however, 
varied considerably with the soil. Coleman’s work with a loam 
soil showed nitrification to be most active when the soil contained 
16 per cent. of water. It was greatly retarded when the water 
content was reduced to 10 per cent. or increased to 26 per cent. 

Patterson and Scott’s work is interesting in that they found 
nitrification to be inactive in sand and clay soils which still con- 
tained about three times as much moisture as in their average air- 
dry condition. At the lower limits of moisture less water starts 
nitrification in sand than in clay. At the higher limits of moisture 
less water stops nitrification in sand than in clay, while the opti- 


228 NITRIFICATION 


mum amount of water probably varies for each soil; it is higher 
for clay, yet for both soils it lies within the range of from 14 to 
18 per cent. A rise above the optimum amount of water is more 
harmful than an equal fall below it. 

The work of the Utah Experiment Station demonstrated that 
the application of irrigation water to a soil has a distinct beneficial 
effect upon nitrification, being greatest where 15 inches of water 
were applied when the nitric nitrogen formed amounted to 28.5 
pounds per acre-foot of soil. The greatest benefit per inch of 
water, however, was obtained where only 7.5 inches of water were 
applied, resulting in 3.8 pounds of nitric nitrogen per inch of water, 
while where 15 inches were applied it was 1.1 pounds of nitric nitro- 
gen per inch of water applied, and when 25 inches of water were 
applied to the soil the nitric nitrogen produced was only 0.7 pound. 

Miinter and Robson found that hornmeal decomposed more 
rapidly in dry sandy soil than in clay or loam, whereas with higher 
moisture content there was little difference. Ammonia sulphate 
transformation increased with a higher water content. The best 
nitrate formation from hornmeal occurred in sandy soils. In clay 
and loam it was best with a medium water content. Sharp found 
that the water content most favorable for ammonification was 
not the optimum condition for nitrification. The former was 
most rapid with a 25 per cent. water content and was not markedly 
affected by 3 per cent. differences. Nitrification was at its maxi- 
mum when the soil contained 19 per cent. of water. When it was 
increased to 25 per cent. the rate of nitrification was decreased 
50 per cent. 

McBeth and Smith found a slight variation in the number and 
nitrifying powers of soil, depending upon the moisture content. 
However, Gainey considers that among the factors controlling the 
bacterial activity of a soil the available moisture probably plays 
a leading part. But the author has reported results which indicate 
that the nitrous nitrogen content of a soil is independent of the 
irrigation water applied up to 37.5 inches a year. Results recently 
published by the Utah Experiment Station clearly demonstrate 
that the influence exerted by water upon ammonifying, nitrifying, 
and nitrogen-fixing activities of the soil varies greatly with the 
organic matter in the soil and is much more marked in effect on 
soils recently manured than on those which have received no 
manure. 

From the literature cited it may be seen that the nitrifying power 
of the soil is a function of the moisture content of the soil, and 
that the optimum varies with the physical and possibly with the 
chemical properties of the soil. Recent work at the Utah Experi- 
ment Station shows a close correlation between the nitrifying 
powers of a soil and its water-holding capacity and varies only 


INFLUENCE OF MOISTURE 229 


slightly with the physical properties of a soil. ‘Twenty-two soils 
varying widely in physical properties yielded maximum nitrification 
when the soil contained from 50 to 60 per cent. of their water- 
holding capacity, as indicated in Fig. 30. Furthermore, the opti- 
mum moisture content for maximum nitrification is correlated 


06 00/ 


08 


OL 


09 


DE ae CEN OZ 0G: 


O/ 


S 
ine 

a 
co 
ce 
ea 
e 
ae 
a 


Ome OnE IO HeZO NO 60 TO BO" 90 > 00 


Fic. 32.—Average percentages of nitric nitrogen produced in soil receiving various 
quantities of water. The quantity produced at 60 per cent. is taken as 100; on the 
ordinate is given the per cent of nitric nitrogen formed, whereas on the abscissa is 
given water applied as per cent. of water-holding capacity. 


with the other soil constants with a set of equations similar to 
those given for ammonification, page 201. ‘Thus, 


Mn = 55). XG; 

Va .8525 EH + 11.55 
Mn) = 13472 W + 11.55 
Wit ) BGS) et Se abil ain 


230 NITRIFICATION 


Mn is written for per cent. of water for maximum nitrification, 
C for moisture capacity as defined by Hilgard, W for wilting co- 
efficient, / for moisture equivalent, and H for hygroscopic 
coefficient. 

Temperature.—The temperature is a factor which controls in a 
great measure the quantity of nitrates produced in unit time. 
Schlésing found nitrification very slow at 7.5° C., quite marked at 
11°, reached its maximum at 37°, and ceased entirely at 55.° 
Dehérain found nitrification almost ceased at 5° C. and begins very 
slowly in soils which have been frozen, yet Conn found the freezing 
of soil increases its nitrifying powers. These temperatures are 
questioned by some, for example Warington states that he was 
unable to start nitrification at 40° C. 

Hutchinson gives the optimum temperature for nitrification in 
Pusa soil at 35° C. No nitrates were formed at 40°, nor did nitri- 
fication take place in soil which had been kept at 40° C. when its 
temperature was afterward reduced to 30° C. These apparent 
contradictions may be due to different strains of the organisms 
varying in sensitiveness to heat. Beddies isolated four stable 
forms of nitric and three of nitrous ferments. One of the nitric 
forms was capable of resisting the action of steam at 100° C. for 
two minutes and one of the nitrous bacteria lived for one minute 
in steam at the same temperature. ‘The other two nitrous ferments 
could not withstand steam but survived for several minutes in a 
dry heat of 80° to 100° C. Moreover, Bazarewski found the most 
favorable temperature for nitrification in soils to be between 25° and 
27° C., or about 10° C. lower than in pure cultures in artificial 
media. 

King, in his work, found that there was 1.26 times as much 
nitric nitrogen formed at 9° C. as at 1° C., 2.76 times as much at 
20°, and 6.24 times as much at 35°, asat 1°. The significance of 
these figures is brought out more fully when we examine the amounts 
of nitric nitrogen obtained in some cases. At 1° C. there were 
formed 120 pounds per acre; at 9°, 150 pounds per acre; at 20°, 329 
pounds per acre; while at 35° there were formed 747 pounds per acre. 

Light Rays.—The nitrifying organisms are heat-loving and light- 
avoiding. ‘They are dependent on the heat of the earth or of the 
sun, but they carry on their activities best in the absence of sunlight. 
Direct sunlight, partly due to the coagulation of the bacterial col- 
loids by the rays of the ultraviolet light, soon proves fatal to them. 

Aération and Cultivation.— The nitrifying bacteria are all aérobic; 
hence, nitrification is best—other things being equal—in a well- 
aérated soil. This is illustrated by the work of Schlésing who 
exposed soil for four months to an atmosphere containing different 
percentages of oxygen. Soil which contained 1.5 per cent. of 
oxygen yielded 45.7 mg. of nitric nitrogen, that containing 6 per 


CROP AND FALLOW 231 


cent yielded 95.7, that containing 11 per cent. yielded 132.5 mg., 
whereas that containing 16 per cent. of oxygen yielded 246.6 mg. 
of nitric nitrogen. 

Plummer found there to be an optimum mixture of carbon 
dioxid and oxygen for the best production of nitrates. This he 
found to be one containing from 35 to 60 per cent. of oxygen. But 
Hutchinson found complete nitrification of ammonium sulphate took 
place under semi-anaérobic conditions in which no nitrification of 
oil cake occurred. 

Stirring and pulverizing the soil is, therefore, of great importance, 
as further shown by the experiments of Dehérain. A number of 
pots were filled with soil. Part of them were allowed to stand 
undisturbed, while the others were poured out upon the floor and 
frequently stirred. Those stirred invariably contained from ten 
to forty times as much nitrates as did the unstirred. 

The work of King also shows that the stirring of the soil affects 
nitrification. He further found land plowed in the fall contained 
a different amount of nitrates than did the unplowed land, the 
difference being apparent throughout the following summer. 

Crop and Fallow.— Even as early as 1855 the work at Rothamsted 
had demonstrated that the beneficial effects of fallowing lies in 
the increase brought about in the available nitrogen compounds 
of the soil. Dehérain and Demoussy’s work indicated that there 
is a larger production of nitrates in fallow than in cropped soils, 
and Pfeiffer considers fallowing an extreme form of soil robbery, 
for he found that it promotes the activity of the soil organisms, and 
hence hastens the exhaustion of the nitrogen supply. But, as it 
is so clearly pointed out by Warington, these results may not hold 
in a dry climate or during dry seasons; for here bare fallow may 
not necessitate this loss and much is to be gained by its practice. 
But it must always be borne in mind that if there be sufficient 
moisture the loss may be great. For instance, Schneidewind, 
Meyer and Miinter record a loss in fallow plats of 85.5 pounds 
per acre, which even exceeded the nitrogen removed by the growing 
plant on the cropped soil. 

On the other hand, McBeth and Smith claim that plats con- 
tinuously cropped to alfalfa, potatoes, oats, and corn all show a 
higher nitrifying power than do corresponding fallow plats and 
that the stimulating effect of crop production on the nitrifying 
power of a soil is most marked in alfalfa soil. This is in keeping 
with the recent findings of Welbel, but is contrary to the findings 
of many other investigators, for Heinze found fallow to increase 
the pectin, cellulose and humus fermenters and also the ammoni- 
fiers, nitrifiers and Azotobacter. Russel finds that late summer 
fallow land is richer in nitrates than is cropped, even after allowing 
for the nitrogen taken up by the crop; and Heinze shows that 


232 NITRIFICATION 


repeated cultivation of fallow soil increased the number of organ- 
isms in the soil, while Hiltner maintains that no nitrification occurs 
in soils where legumes are growing vigorously and fixing large 
quantities of nitrogen. This latter view, however, is the extreme, 
as is shown by much of the literature on the subject. 

Welbel and Winkler found that fallow not only increased the 
assimilable nitrogen, but also the available phosphoric acid of 
the soil, and that the increased yield of wheat after fallow is due 
to these factors. But Bychikhin and Skalski point out that fall 
fallow is even more wasteful of soil nitrates than is summer fallow, 
for here the excessive rains wash the soluble nitrates from the soil 
as fast as formed. ‘The cultivating of fallow further increases the 
nitrate content, as was shown by- Richardson. Nitrification is 
related to fallow and crop, as may be seen from the following results 
obtained by the author: 


Milligrams » Milligrams 

nitric nitrogen nitrogen 
formed. fixed. 
Cultivated ti fee tuasor cae ge ee ee AEG 14.28 
Waren soil) ocak oe eae 09 6.99 
Wheatsolt "oJ ie ons, Seah e eer 00 11.83 
Alfalfa: soil 2420 vee ak an ee Oke eee 12.24 
Fallow soil, potato fallow, etc. hee tet GRO ; 22.88 


The results reported under milligrams of nitrogen fixed indicate 
that in an arid soil the increased nitrogen fixation in a fallow soil 
more than offsets the loss of nitrates, even though rapidly formed, 
for little, if any, would be lost in the drainage waters. These 
results have recently been confirmed by Reed and Williams. More- 
over, the number of organisms in the soil and the rapidity of the 
bacterial activity within the same is going to vary greatly with 
the thoroughness and time of cultivation, as shown by Dehérain, 
Neish, King and Whitson, Chester and Quiroga, while the number 
and activity of the organisms in the soil may in a degree determine 
the speed with which the water evaporates from a soil. 

The work at the Rothamsted station early demonstrated that 
the nitrates in the drainage water from the various plats varied 
greatly, depending upon the crop growing upon the soil, thus indi- 
cating a relationship between the available nitrogen in a soil and 
crop growing upon the soil. Since that time many experiments 
have confirmed this conclusion. Furthermore, King and Whitson 
found 22 per cent. more nitrogen developed from soil after clover 
than from soil after corn, and 13 per cent. more than after oats. 
Later work by them showed that there are greater quantities of 
nitrates throughout the entire season in soil under corn or potatoes 
than in soil under clover and oats. Stewart and Greaves found 
that different plants show a marked difference in their demands 


CROP AND FALLOW 233 


upon the nitrate content of the soil, there being a steady decrease 
in the concentration of the nitrate content of potato and corn 
lands as the season progressed, while that of fallow and alfalfa 
remained. practically constant, the nitrate content of the latter 
being uniformly low through the season. According to Lyon and 
Bizzell, soil that had produced alfalfa for five years was higher in 
nitrates than soil that had grown timothy during the same period. 
Furthermore, the former nitrified ammonium sulphate more readily 
than did the latter. 

Brown found that the rotation of crops caused an increase in 
number of organisms in a soil, also greater ammonifying, nitrifying 
and nitrogen-fixing powers than continuous cropping to either corn 
or clover. Furthermore, the crop on the soil at time of sampling 
was of more importance from the bacterial viewpoint than the 
previous crop. However, the preceding crop has a marked effect 
upon the nitrate content of the soil, as is seen from the work of 
Lyon and Bizzell, where plats that had been planted to certain 
crops were kept bare of vegetation in the early part of the growing 
season of 1911. Nitrate determinations of the soil were made and 
the nitrate present showed a distinct and characteristic relationship 
to the nitrate content found under the several varieties of plants 
previously grown upon the soil. Later they showed that alfalfa 
soil nitrified more rapidly than timothy soil, both in the soil on 
which the crops had been grown continuously and in that from 
which they had been removed and the soil kept bare for two seasons. 
The author has shown that the nitrifying powers of alfalfa soil, 
while slightly higher than that of virgin soil, is very low when 
compared with either wheat or potato and fallow soil. Further- 
more, the extensive work which has been conducted at the Utah 
Experiment Station demonstrates that there is a very pronounced 
_ relationship between the crop growing upon a soil and its nitrate 
content. However, in this work the nitrate content of the alfalfa 
and oat soil is very low, while that of potatoes and fallow is high, 
and we find the nitrifying powers of alfalfa and potato soil high as 
compared with fallow. 


Nitric nitrogen Nitrifying 
Crop. in soil. powers. 
IRallow insets ka a oi est oe Gar > LOO 100 
Alfalfa SL aN Od Rake eee ae Sa oon 36 148 
FO@Sts wether yuh a Mia comin, | ceyekn ce 36 103 
(GOs eked dace Gat than ea ek een go 33 161 
IPGtatOes eral Sa) oad GRE oes ee 99 21 


Hence, we can conclude that alfalfa not only feeds closer upon 
the soluble nitrates of the soil but also makes a much greater drain 


1 Fallow taken as 100. 


234 NITRIFICATION 


upon the insoluble nitrogen of the soil by increasing its nitrifying 
powers. 

Season.— The season of the year has a marked influence upon 
the bacterial activities of the soil, but it is not necessarily corre- 
lated with the nitrate content of the soil. Schlésing found the 
nitrates in the drain water from both manured and unmanured soil 
high in spring, as compared with midsummer, fall, or winter, thus 
confirming the results obtained at the Rothamsted station. Shutt 
reports nearly five times the quantity of nitrates in fallow and 
cropped soil during June as during November. He does, however, 
find more during June than during May. The exact season of 
the year at which the maximum nitrate content is reached will 
vary with a number of factors, chief among which is the kind of 
crop growing on the soil, for King and Whitson found that the 
nitrates in the surface foot start in the spring comparatively low 
and increase rapidly until June 1 on clover and oat ground, and 
until July on corn and potato ground. From these dates they fall 
more or less rapidly and the work at the Utah Station demonstrates 
conclusively that there is a seasonal variation, depending upon 
temperature, crop and quantity of irrigation water applied to 
the soil. 

Moreover, André has shown that the insoluble nitrogenous 
compounds of the surface soil are largely transformed into soluble 
compounds during the summer, and these are widely diffused 
through the deeper layers of soil during the winter, so that in the 
spring the lower layers of soil contain more soluble nitrogen than 
the surface soil. At the end of summer, however, the distribution 
is quite uniform. ‘This finding has been amply verified by the 
results reported by Stewart and Greaves, Welbel, Jensen, and 
Lyon and Bizzell. The results will vary, however, with different 
soils, as shown by Russell who reports the fluctuations in nitrates 
more marked on loams than on clays or sands. Moreover, he 
found the bacterial activities much greater in early summer than 
later. 

Moll even goes so far as to claim from his work that the season 
of the year is the principal factor in determining the biochemical 
transformation in a soil, and Heinze found that the number of 
organisms in a soil was highest in the summer months and lowest 
in the fall and spring. As already pointed out, the highest nitri- 
fying power of a soil is not necessarily correlated with the highest 
nitrate content. The latter is highest in spring or early summer, 
while Vogel found the former to be highest in October and Novem- 
ber, after which there was a falling off until April, when it rose 
again, but not so high as in autumn. This corresponds fairly well 
with the findings of Green, for the ammonifying powers of the 


QUANTITY OF NITRATES FORMED 235 


soil. ‘These findings, however, are contrary to those of Wojtkie- 
wiez, who found the maximum number of organisms to occur in 
soil during the spring and the minimum in the winter. He also 
notes a correlation between bacteria present and the amount of 
nitrates in the soil. 

Climate influenced the nitrifying powers of the soil, and Hilgard 
taught that the nitrifying powers of the arid soils are superior to 
those of the humid soils, but the extensive work by C. B. Lipman, 
both by laboratory and field experiments, in which soils have been 
transported from humid to arid districts, and vice versa, has shown 
just the opposite to be true—namely, that the biological activities 
of a soil are more pronounced under humid than under arid 
conditions. 

Quantity of Nitrates Formed.—The quantity of nitrates produced 
in a given soil varies with all of the factors which have been con- 
sidered; hence, any results obtained must be interpreted with 
thisin mind. The greatest rate of nitrification noted by Warington, 
when working with an ordinary arable soil from the Rothamsted 
farm, yielded 0.588 parts of nitrogen per million of air-dried soil 
a day. Similar soil supplied with ammonium chlorid. nitrified 
about 0.924 parts per million in the same time. 

Lawes and Gilbert, working with the far richer Manitoba soils 
and with a higher temperature, obtained in two cases (soils from 
Selkirk and Winnipeg) average daily rates of nitrification of 0.7 
parts of nitrogen per million during three hundred and thirty-five 
days, the rates during the early portion of this period being as high 
as 1.03, 1.24, 1.36 and 1.72 per million. 

Dehérain, working with a soil containing 0.16 per cent. of nitro- 
gen, obtained daily rates of nitrification varying from 0.71 to 1.09 
per million in ninety days. Working with a richly manured soil 
containing 0.261 per cent. of nitrogen, he obtained a maximum 
daily rate of nitrification during forty days of 1.48 of nitrogen 
per million of soil. 

At times the difference in nitrification noted in different soils 
may be due to a difference in physiological efficiency of the nitrify- 
ing ferment, as Marcille compared the nitrifying powers of three 
different soils and found that the poorest yielded an organism 
nitrifying less rapidly than the others. Some soils nitrify ammonia 
more readily, while others nitrify cotton-seed meal more rapidly. 
This must be due to differences in the metabolism of the organism 
found in the various soils. 

Hutchinson considers this variation at times due to toxins which 
develop under anaérobic conditions produced by water saturation. 
Subsequent aération removes the toxic condition and the formation 
of nitrates takes place. He also found copper had a decided 


236 NITRIFICATION 


influence in neutralizing the toxic action. Several other observers, 
including Greig-Smith and Bottomley, claim to have found soluble 
bacteria toxins in soil. Russell and Hutchinson, on the other hand, 
obtained wholly negative results and concluded that soluble 
bacterio-toxins are not normal constituents of soils, but must 
represent unusual conditions wherever they occur. But, as pointed 
out by Russell the possibility of the existence of toxins soluble in 
water still remains. 

Loss of Nitrates.—The loss of nitric nitrogen from a soil may be 
either great or small, depending upon certain factors, the more 
important of which are as follows: 

1. The rapidity of nitrification. Nitric nitrogen may be pro- 
duced in some soils so rapidly that even luxuriant vegetation will 
not remove it as fast as formed, whereas in another soil it may be 
formed so slowly that it will not suffice for even meager growth. 
The loss in the first case may be very large, while that of the second 
would be nearly zero. 

2. The nature of the soil. A tight soil, other things being equal, 
would retain the nitric nitrogen to a greater extent than would 
a loose porous soil, and a deep soil than a shallow soil. 

3. The amount and distribution of rainfall. All other condi- 
tions being equal, thirty inches of precipitation throughout the 
year would remove more nitric nitrogen from the soil than would 
fifteen inches similarly distributed. But if the fifteen inches came 
within a short period, while the thirty was distributed through- 
out the year, the fifteen inches :of rainfall under these conditions 
may remove more nitric nitrogen from the soil than would the 
thirty. 

4. The rapidity with which the nitric nitrogen is removed by 
the growing crop. Alfalfa, oats and wheat are heavy feeders upon 
nitric nitrogen and in most soils remove it as fast asformed. Hence, 
little is left to be washed out by the drainage. Moreover, crops 
such as these rapidly remove the water from the soil and hence 
diminish the drainage from such soils. Moreover, crops growing 
during the rainy season tend to conserve the nitric nitrogen where 
fallow soils rapidly lose nitric nitrogen during this period. 

5. The rapidity with which nitric nitrogen is transformed into 
protein nitrogen by soil microérganisms. It is now known that 
there are within the soil many microérganisms which transform 
nitric nitrogen into protein nitrogen, and the speed with which 
this change occurs may at times become important; work at the 
Utah Experiment Station indicates that this may at times reach 
twenty or thirty pounds per acre yearly. 

The factors must always be kept in mind in an attempt to reach 
general conclusions from any special cases, yet it is instructive to 


LOSS OF NITRATES 237 


examine a few results from the Rothamsted Experiment Station, 
as compiled by Dr. Hopkins: 


NITROGEN IN DRAINAGE WATERS. ROTHAMSTED EXPERIMENTS 
AVERAGE OF 12 YEARS (OR MORE). 


- 


Bare Soil—60-inch Gauge. Whest 1 Land. 
in vi Than 
Month. Rainfall eke Nitrogen. | Nitrogen. 

(inches). (inches). = arg a. Wh ara eS 

Per Million | Pounds | Per Million 

of Water. | per Acre. | of Water. 
January . 7 is! 1.93 8.9 3.88 Si5ll 
February 2.16 ee 74. 9.1 315 5% 4.0 
March We) 0.94 8.9 1.89 2.0 
April 2s) 0.79 9.0 1.61 1.9 
May . 2.48 0.79 9.1 1.63 0.9 
June . 2.59 0.78 9.1 1.60 0.1 
July . 2.85 0.62 11.8 1.66 0.1 
August 2.69 0.76 13.83 2.28 0.1 
September Pati 4 0.82 13.4 2.50 3.9 
October) ES. «| See | 1.68 11.9 4.53 | 4.6 
November . . . 3.20 De, Tila! SAOS ey] 3.6 
December 2.34 1.88 10.6 “Nl 4.8 
January—April ... | 8.24 5.40 9.0 10.95 28 
May—August.. 10.61 2.95 10.6 7.17 0.3 
Sept._December . 36 G70 sy 11.8 7252 4.2 

—— | | 

January-December 30.21 15.05 10.5 35.64 2.4 


| 
| 
| 


It does not necessarily follow that all of the nitric nitrogen 
which is carried to a depth of sixty inches is lost to the growing 
plant, for in work at the Utah Experiment Station the author and 
coworkers have found in the spring a nitrate belt as low as the 
seventh and eighth foot-section. These nitrates had been carried 
to this depth by the winter and spring water. It was noted that 
later in the season as the water was brought to the surface by 
capillarity the nitrates also returned, and by June, July or August, 
depending upon the crop grown upon the soil and the quantity of 
irrigation water applied, the nitrate belt which in the spring was 
in the seventh and eighth foot-section had reached the surface 
foot-section. Moreover, the deep-rooted plants of the arid regions 
probably feed from lower depths than do the shallow-rooted plants 
of the humid regions. 

The practical conclusion to be reached from these results is that 
the method of reducing the loss by leaching is by growing plants, 


238 NITRIFICATION 


the roots of which may absorb the plant-food as rapidly as it is made 
available. 

The loss of nitric nitrogen from irrigated soil may be prevented 
by the judicious use of irrigation water. Experiments at the Utah 
Station covering a period of fourteen years have demonstrated that 
the application of fifteen or twenty inches of irrigation water, 
distributed throughout the season, to deep soil causes little, if any, 
loss of nitric nitrogen from such a soil, whereas applications of from 
twenty-five to thirty-seven inches similarly distributed causes consid- 
erable diminution in the crop yield. This decrease in crop yield due 
to excessive quantities of water, up until the soil becomes water- 
logged, is largely due to the rapid washing of the nitric nitrogen 
beyond the feeding area of the plant roots. 


REFERENCES. 


Lohnis: ‘Handbuch der Landwirtschaftlichen Bakteriologie.”’ 
Lafar: ‘‘Handbuch der Technischen Mykologie,’’ Dritter Band. 
Kossowiez: ‘ Agrikulturmykologie,’’ I—Bodenbakteriologie. 


Warington, Robert: ‘Six Lectures on the Investigations at the Rothamsted 
Experimental Station,’’ U. 8S. Dept. Agr. Off. Exp. Sta. Bul. 8. 
Voorhees, Edw. B. and Lipman, Jacob .G.: ‘A Review of Investigations in Soil 


Bacteriology,’’ U. 8. Dept. Agr. Off. Exp. Sta. Bul. 194. 

Chester, Frederick D.: ‘Bacteria of the Soil in Their Relation to Agriculture.” 
Penn. Dept. of Agri. Bul. 98. 

Greaves, J. E., Stewart, R., and Hirst, C. T.: ‘Influence of Crop, Season, and 
Water on the Bacterial Activities of the Soil.” Jour. Agr. Rsch., vol. ix, pp. 293-341. 

Gibbs, W. M.: The Isolation and Study of Nitrifying Bacteria. Soil Science, 
1919, vol. vili, pp. 427-481. 


CHEAP Bee Ss LT. 


DENITRIFICATION. 


Ir has been known for a long time that under conditions which 
were not fully understood, there may and often does result a loss 
of soil nitrogen. Most of this is due to the loss of nitrates in the 
drainage water, but occasionally there are losses which cannot thus 
be accounted for. This has been attributed to various causes, 
namely: (1) the liberation of elementary nitrogen in the process 
of decay as the complex protein is broken down into simple products, 
(2) the reduction of nitrates or nitrites with the production of 
ammonia or elementary nitrogen, (3) the transforming of nitrates 
and ammonia into complex proteins through the action of micro- 
organisms. 

Often the losses from all of these processes have been grouped 
together and considered as denitrification. This vague usage of the 
term has led to considerable confusion and often erroneous con- 
clusions. But the term denitrification in its proper and more 
limited sense refers only to the complete reduction of nitrates with 
the evolution of elementary nitrogen. It is, however, often applied 
in a broader sense to include all deoxidation processes whereby 
nitrates are partly or wholly reduced. But, as pointed out by Lip- 
man, for practical agriculture the differences are of some moment. 
The partial reduction of nitrates to nitrites or to ammonia does not 
necessarily involve a loss of soil nitrogen, whereas the complete 
reduction of nitrates, wherever it occurs, must of necessity involve 
such losses. Hence, there is some justification for referring to the 
partial reduction of nitrates as denitrification. But it is not justifi- 
able to classify under the head of denitrification all bacterial activi- 
ties in the soil which lead to the disappearance of nitrates or even 
to the diminution in the total store of soil nitrogen. For it has been 
repeatedly demonstrated that the nitrates may completely disappear 
without involving any loss of nitrogen. 

Early Theories.—We have seen that the early investigators 
attempted to explain nitrification by purely chemical theories. 
This was also true with denitrification. Kuhlman, as early as 1846, 
expressed the belief that nitric nitrogen may be reduced in the soil 
to ammonia by the fermentation of organic substances. This same 
idea was brought out twenty-one years later by both Froehde and 
Angus Smith, and it also appears prominently in the writings of 
Johnson in 1870, and Davy called attention to the fact that gaseous 
nitrogen was set free from decomposing organic matter in the soil, 


240 DENITRIFICATION 


The splitting-off of free nitrogen, theoretically, could be due to the 
reduction of nitrates or the action of nitrous acid on ammonia or 
amins: 

2HNO; = 2H,0 
NH; oo HNO: 
CH3;NH2 + HNO: 


202 + Ne 
No + 2H2O 
Noe + CH;0H + H:O 


alles 


Lawes, Gilbert, and Pugh showed that losses of nitrogen often 
take place when nitrogenous. organic matter was made into an 
“agelutinated condition” with water and allowed to decompose in 
the presence of air. Practically no ammonia could be detected. 
Three possible reactions were suggested by Lawes and Gilbert: (1) 
an oxidation analogous to that of the action of chlorin on ammonia 
by which free nitrogen is evolved; (2) a reduction similar to that of a 
great number of substances upon the oxygen compounds of nitrogen, 
by which the oxygen is appropriated and the nitrogen set free; (3) 
the two actions may operate in succession, the one to the other. 

Organisms Concerned.—Gayon and Depetit, however, were the 
first to announce that the nitrogen originated from the nitrates. 
They found that the ferments which possess this power need organic 
matter for their development and that part of the organic nitrogen 
is transformed into ammonia and perhaps also into amido-deriva- 
tives of organic substance. 

In 1886 they isolated two organisms— B. denitrificans, a and 6 
—capable of reducing nitrates with the evolution of gaseous nitrogen. 
They also encountered a number of bacteria that could reduce 
nitrates to nitrites, and since that time the denitrifiers have been 
found very widely distributed. 

The discovery by Bréal that many substances of organic origin, 
and especially straw, are the carriers of denitrifying organisms was 
of far-reaching importance. ‘These organisms are, therefore, carried 
with the litter to the manure and later with the manure to the soil. 
It was found by Kunnemann that horse manure as a rule contains 
denitrifying organisms and these are usually of two species, one of 
them also being found on straw. ‘The organism found only in 
manure reduces nitrates in symbiosis with B. coli and is B. denitrifi- 
cans I of Burri and Stutzer; the organism occurring in both manure 
and straw is the B. denitrificans II of the same authors. The denitri- 
fying organisms are less frequently present in cultivated soil and 
are usually a different kind. Yet they are abundant in the upper 
layers of the soil. Bazarewski found them irregularly distributed 
in the deeper layers of the soil, but frequently they occurred abun- 
dantly at a depth of one meter. They have also been found to a 
great depth in the Nebraska soils. Putnam examined 201 species 
and 139 were found to reduce nitrates to nitrites, while the other 
species did not effect this reduction. Burri and Stutzer called atten- 
tion to the fact that while there are many bacteria which will reduce 


REACTION OF THE MEDIA 241 


nitrates to nitrites, those capable of reducing nitrates to ammonia 
or of setting nitrogen free are not very numerous. Severin isolated 
32 different organisms from horse manure and studied 29 of these. 
Of this number eight species were capable of complete reduction of 
nitrates, provided the nitrate concentration be not too great. Nine 
of the other species were able to reduce nitrates to nitrites. 

Stoklasa divides the denitrifying organisms present in soils and 
manures into two principal groups. The first group contains 
Clostridium gelatinosum, Proteus vulgaris, P. zenkeri, B. ramosus 
n. liquefaciens, B. mycoides, B. megatherium, B. subtilis, and B. 
prodigiosus, and others. The characteristic of these organisms is 
that they reduce nitrates to ammonia without the formation of 
elementary nitrogen. The second group contains Bac. hartleb, 
B. fluorescens liquefaciens, B. pyocyaneum, B. stutzeri, B. filefaciens, 
B. mtrovorum, B. centropunctatum, B. denitrificans, B. coli communis, 
B. typhi-abdominalis, and others. These organisms as a rule reduce 
nitrates to elementary nitrogen. | 

Beer yeasts (Laurent), especially those of Duclaux, reduce 
nitrates at 20°C. Penicillum glaucum, mucor racemosus, and similar 
organisms also have a reducing power. 

It is, therefore, true that whereas active nitrogen fixation is a 
characteristi¢ possessed by only a limited number of microérganisms, 
the opposite—denitrification—appears to be a characteristic pos- 
sessed by many widely dissimilar organisms. 

Reaction of the Media.—The denitrifying organisms are similar 
to the nitrogen-fixing organisms in that they require a slightly 
alkaline medium in which to function. Von Caron considers that 
with a sugar concentration of more than 1 or 2 per cent., a depres- 
sion of denitrification occurs. This probably is due to the formation 
of fatty acids by the butyric acid ferments of the soil. When it 
first became known that denitrification may take place in manure 
heaps, the practice became prevalent to add to the manure sulphuric 
acid to prevent denitrification. It was found that sulphuric acid is 
extremely active in preventing denitrification and 0.17 per cent. in 
the cultural medium was sufficient to prevent the development of 
the denitrifying organisms. 

Ampola and Garino found that the addition of ground peat 
showing an acidity of 9.85 per cent. checked the activity of the 
denitrifying organisms as well as that of other ferments. ‘The 
organisms, however, were not killed and commenced their activity 
again as soon as the acidity was neutralized. The soil conditions 
are favorable to the neutralization of the acid of the peat, and thus 
the restraining effect of the latter on the denitrifying organisms is 
nullified. Moreover, an acid condition which would restrain deni- 
trifiers in soil retards the other beneficial bacteria and higher plants. 
Hence, while acids may be used at times with some success on 
manures, it is not necessary nor practical to add it to soils. 

16 . 


242 DENITRIFICATION 


An excessive alkaline reaction is also inimical to the growth and 
activity of denitrifying organisms, as was early shown by Pfeiffer, 
but the application of such large quantities of caustic lime to soil 
as he found necessary to check denitrification tends to “burn out” 
the nitrogenous organic matter, as has been amply demonstrated 
by the work in Pennsylvania. 

Food Requirements.— The food requirements of the denitrifiers 
are quite similar to those of other soil organisms. They can, accord- 
ing to Richards and Rolfo, survive in purely mineral media, but in 
such media the reduction of nitrates takes place very slowly and 
incompletely. Jensen found a certain relationship between the 
nitrate destroyed and the carbon compounds used. No denitrifica- 
tion takes place without a source of carbon. The optional relation 
between the carbon and the nitrate used was found by von Caron 
to be for two strong denitrifiers— B. pyocyaneus and B. fluorescens 
liquefaciens, a 1 per cent. dextrose to 1.6 per cent. potassium nitrate. 
Reduction of the nitrate supply far below that of carbon greatly 
reduces the intensity of the process. Furthermore, the destructive 
fermentation of nitrates depends to a great extent on the character 
of the organic substances in the nutritive medium, some being 
much better adapted than others to furnish the necessary energy 
for the breaking down of the nitrates. Stoklasa claims that most 
of the denitrifying bacteria causes no reduction of nitrates in media 
where chemically pure d. levulose and d. galactose are present. Nor 
is denitrification favored by glucose in nutritive solutions (Stutzer), 
but is promoted by the presence of salts of organic acids, like potas- 
sium lactate, or sodium citrate. The reason for this is that glucose 
is not as suitable for furnishing the molecular energy required for 
the breaking down of the nitrates as are the salts of organic acids. 
Stutzer tried four different organisms and found that they possessed 
the power of denitrification in a different degree. Their action on 
the different meat extracts on the market is also variable.  B. 
hartlebit was the only organism tested by him which could destroy 
nitrates in a medium containing Liebig’s beef extract. He suggested 
that this phenomenon may be due either to a difference in chemical 
constitution of the compounds or a difference in ionization. The 
knowledge we now possess concerning the specificity of enzymes 
would lead us to believe the former to be the true explanation. 

Certain of the most widely distributed carbohydrates in soil and 
manures, as for example xylose and arabinose, are not especially 
good nutrients for denitrifying bacteria, according to Stoklasa and 
Vitek. The quantitative relationship which they found to occur is 
widely different with the various carbohydrates. Of the bacteria 
which reduce nitrates to nitrites and finally to ammonia, B. mycoides 
reduced 20.69 per cent. of the nitrate nitrogen present to ammonia 
in the presence of glucose 1.9 per cent. in the presence of levulose, 
1.72 per cent, in the presence of galactose, and 1.91 per cent, in the 

z ! 


METABOLISM OF DENITRIFYING ORGANISMS 243 


presence of arabinose; B. subtilis, 2.41 in the presence of glucose, 
6.55 per cent. in the presence of levulose, and 6.22 per cent. in the 
presence of galactose; Clostridium gelatinosin, 45.55 per cent. in the 
presence of arabinose, and 9.68 per cent. in the presence of xylose; 
and B. prodigiosus, 2.58 per cent. in the presence of xylose. The 
reaction was in all cases relatively slow and was not alike for all the 
sugars. 

Of the. organisms which reduced nitrates to free nitrogen, B; 
hartlebii set free 93.97 per cent. of the nitric nitrogen in the presence 
of glucose, 87.59 per cent. in the presence of levulose, 84.66 per cent. 
in the presence of galactose, 66.38 per cent. in the presence of arab- 
inose, 83.38 per cent. in the presence of xylose, 84.48 per cent. 
in the presence of sucrose, and 77.15 per cent. in the presence of 
lactose; B. centr pes 5.17 per cent. in the presence of glucose. 
B. flitrovorum, 5 .17 per cent. in the presence of levulose; B. colt 
communis, 5.34 per cent. in the presence of galactose; and Bact. 
fluorescens liquefaciens, 7.08 per cent. in the presence of arabinose. 
The reaction was as a rule very intense both with the sugars and 
with the salts of organic acids, especially of lactic acid, and was 
accomplished by a gradual breaking up into carbon dioxid and 
hydrogen or into carbon dioxid and water. The hydrogen produced 
was thought to play a very important reducing role. 

Xylan and araban, the most abundant and widely distributed 
carbohydrate materials in soils and manures, yields on hydrolysis 
xylose and arabinose which are very poor sources ot carbon and 
energy for denitrifying organisms. However, Stoklasa and Vitek 
found that the typical denitrifying organism, B. hartlebiz, assimilated 
33.6 per cent. of the total nitrate nitrogen in a nutritive solution 
containing arabinose and converted it into protein compounds. 

Sodium citrate, sodium acetate or glycerin added to a soil greatly 
increase denitrification, and it is generally considered that the addi- 
tion of starch, straw, rape cake, compost, etc., to a soil favors deni- 
trification, whereas well-rotted manures, rape cake, and composts 
are much less apt to have this effect. 

Metabolism of Denitiifying Organisms.—Dehérain found that 
reduction was more rapid in closed flasks than in the open air, the 
nitrogen escaping mainly in the form of protoxid. From this he 
argued that the organisms, being deprived of the necessary oxygen 
of the air, were forced to appropriate that contained in the nitrates 
and thus accomplish their reduction, but we now know that the 
denitrifiers do not necessarily require anaérobic conditions for deni- 
trification, but do require a readily oxidizable carbohydrate. More- 
over, as pointed out by Stoklasa, there are two classes of denitrifiers 
—one which reduces nitrates to elementary nitrogen, the other 
which reduces it only to ammonia. Probably in both groups of 
organisms the first steps in the process are the same. The carbo- 
hydrates are broken down under the influence of the microérganism 


244 DENIT RIFICATION 


into lactic acid, aleohol, and carbon dioxid. The nitrate is reduced 
to nitrous acid and this in turn is reduced to ammonia or element- 
ary nitrogen. The oxygen so obtained is utilized by the microérgan- 
isms for the further oxidation of the carbohydrates, and it is in this 
manner that the organism obtains its requisite energy. 

Stoklasa and Vitek believe that nitrous acid is always the inter- 
mediate product in the reduction of nitrates. They consider that 
carbon dioxid and hydrogen are produced from the carbohydrates 
or organic acids of the cultural media and the nascent hydrogen 
combines with the oxygen of the nitrates to form water and thus 
reduces the latter to nitrites. Gayon and Depetit give this formula: 

5CsHi0s + 24KNO3 = 24KHCOz; + 6COz2 + 18H2O0 + 12Ne 


The process is probably due to enzymes. Fred was able to demon- 
strate the production of both oxidases and peroxidases by B. 
denitrificans. Hulme considered that reduction may be divided into 
two parts: the bacterial reduction and the enzymatic reduction. 
However, we are led to doubt whether either is due to a true enzyme, 
for the enzymes which have been obtained in impure forms are not 
affected by heat and the reducing substances are not specific, as 1s 
the case with most enzymes, for they reduce chlorates to chlorids, 
arsenates to arsenites, and ferricyanids to ferrocyanids in the same 
manner as nitrates are reduced to nitrites. 

Influence of Water.— Many of the results obtained on denitrifica- 
tion were with the use of liquids, and it is now known that denitrifica- 
tion in soils progresses differently from that in liquids, depending 
upon the nature of the bacteria and the physical conditions of the 
medium in which they are situated. In liquids and very wet soils 
from which oxygen is excluded, the bacteria take their oxygen from 
the nitrates present in the soil and thus liberate nitrogen, but in 
well aérated soils this does not occur, as the bacteria can use the 
oxygen of the air. 

The author failed to find any evidence of denitrification in a highly 
calcareous soil to which had been added from 0 to 25 tons per acre 
of manure and from 12.5 to 22.5 per cent. of moisture, as may be 
seen from the following: 


Nitric Gain in 
nitrogen total nitrogen 
Treatment. (per cent.) (per cent.). 
12.5 per cent. of water! Le eee wee OO 100 
15.0 oe a 5) Waele See AS 108 
i Aas s - So tem ieee Ieee Mala 102 
20.0 is ¥ Penn OT ere ae Aw eo 104 
22.5 4 es 123 108 


The results may vary with different soils, but Lemmermann found 
that in three greatly dissimilar soils it was greatest when the soil 
was saturated. 

Therefore, when the moisture exceeds certain limits, it may 


1 The soil containing 12.5 per cent. of water was taken as producing 100 per cent. 


LOSSES OF NITRATES FROM MANURE AND SOIL 245 


promote denitrification. Variations in the moisture within the 
usual limits, however, have little influence upon the process. 

Temperature.— Kruger considers that the factors which exert the 
greatest influence upon denitrification are temperature and the 
mechanical condition of the material which furnishes the food for 
the organisms. These organisms function best at a temperature 
which is high enough to greatly retard nitrification. They act very 
energetically at a temperature of 37° C. in pure cultures, but there is 
some evidence (von Bazarewski) that in mixed cultures they func- 
tion better at a lower temperature. These factors make it probable 
that laboratory results on denitrification are high as compared with 
field conditions even where all of the other conditions are optimum. 

The denitrifying bacteria are more resistant to light and drying 
than are the nitrifying or nitrogen-fixing organisms. Ampola 
found sunlight to have no effect upon two denitrifiers isolated by 
him— B. denitrificans V. and B. denitrificans VI. In pure distilled 
water these organisms were capable of surviving for seven months. 
When dried, B. denitrificans V died within eight weeks and B. 
denitrificans VI was alive and active at the end of five months. 

Losses of Nitrates from Manure and Soil.—The finding of the deni- 
trifying bacteria on straw and in manure, together with the estab- 
lishment of the fact that they can under appropriate conditions 
decompose nitrates with the evolution of gaseous nitrogen, led 
Wagner in 1895 to emphatically declare that the application of cow 
or horse manures to a soil is often not only unprofitable but harmful, 
that when applied together with nitrates they cause, by virtue of the 
microérganisms contained in them, the destruction of the nitrates. 
More than that, the baneful effects do not stop here, for the nitrates 
as they are gradually formed from the organic matter of the soil are 
also attacked by the denitrifying bacteria and their nitrogen set 
free. In reality, then, the animal manures applied are not only 
useless in themselves but are harmful because of their destructive 
effects on the oxidized nitrogen derived from other sources. 

These conclusions were criticized by Warington who pointed out 
that they were based on experiments in which the dressings of dung 
were enormous and the same would not occur under ordinary prac- 
tice. The next year a serious attempt was made to solve the problem. 
When the German Agricultural Association called for a united effort 
on the part of the German experiment stations, offering to place the 
necessary means at their disposal, the Experiment Stations of Augs- 
burg, Darmstadt, Jena, Rostock, Bonn, and Géttingen responded. 
The questions to be answered were as follows: 

1. How are the great losses of nitrogen that take place in the decay 
of organic substances to be explained? How much of the nitrogen 
is liberated in the elementary state and how much as ammonia? 

2. What means do we possess of checking these losses, and how 
does the substance thus employed act? 


246 DENITRIFICATION 


The published reports of the various stations are voluminous and 
only the general conclusions reached can be considered here. They 
were as follows: 

1. The losses of ammonia from manure are comparatively slight, 
but the setting free of elementary nitrogen which is due to micro- 
organisms and not chemical means may be considerable. 

2. With a limited supply of air in manure, the loss of elementary 
nitrogen and of organic substance are not extensive, but the greater 
the access of air the greater the loss of nitrogen, in some cases 
becoming as great as 40 or 50 per cent. 

3. In ordinary conservation materials when applied in the usual 
quantities, do not stop entirely the loss of nitrogen, but burnt lime 
is quite effective in stopping denitrification. Solid excreta and straw 
lose their nitrogen very slowly and no conservation material is 
needed. It is only the nitrogen of urine which requires conservation. 

It is sometimes found that the addition of large quantities of 
organic matter to a soil cause a decrease in crop yield. This is 
especially true with regard to the carbohydrates and it has often 
been interpreted as indicating rapid denitrification, but Pfeiffer 
and Lemmermann have pointed out that there are at least three 
factors which may play a part, namely: (1) direct injury to the 
growing plants by large quantities of organic matter; (2) fixation 
of soluble nitrogen by the increased activity of different organisms; 
(3) denitrification proper. 

It is quite probable that the last is of the least importance, for 
Voorhees and Lipman after ten years’ investigations under care- 
fully controlled conditions conclude “that at least with cow manure, 
used at the rate of sixteen tons per annum for a period of ten years, 
no destruction of nitrogen takes place. In view of the long duration 
of the experiment and of the comparatively large amounts of 
manure used in the course of the ten seasons, we must assume that 
denitrification is not a phenomenon of economic importance in 
general farming and under average field conditions. We have no 
hesitation in emphasizing again the view expressed above—that 
under the wide range of field conditions, denitrification is not a 
phenomenon of economic significance to the general farmer.” 

Moreover, at Rothamsted a plot of ground, 0.001 acre in extent, 
has been kept free from vegetation by hoeing for thirty-five years. 
During this time it has lost one-third of its original stock of nitrogen, 
but all except 110 pounds of this is accounted for by the nitrates 
in the drainage water, as may be seen from the following: 


Pounds nitrogen : 
; : -overe : 
per acre. Nitrogen recovered Nitrogen unaccounted 


: 
1870 1905 Loss si Ge for. 
35 years. = 


Nitrogen in soil. 
1870 1905 


3500 2450 1050 940 110 


| 
| 
| 
| 
| 


FUNCTION OF DENITRIFIERS 247 


Russell, commenting upon the results, states: ‘‘The experiment 
is not fine enough to justify any discussion of the missing 110 pounds, 
but it shows that the loss of nitrogen is mainly due to leaching out 
of nitrates.” 

It is even doubtful if denitrification goes on to any appreciable 
extent in a well aérated soil even though it contains considerable 
nitrates. The nitrates, however, may disappear as seen from the 
following results in which the author mixed 2 grams of dried blood 
and 3724.8 parts per million of various nitrates with 100 grams of 
soil made the moisture up to 18 per cent. and after twenty-one 
days’ incubation at 30° C. recovered the various percentages of 
nitric nitrogen. The untreated soil was taken as 100 per cent. 


Per cent. of nitric nitrozen found in the presence of 


NO3 added = =X : 
(p. p-m.) , | 
*NaNOz KNOs3 | Ca(NOz)2 | Mg(NOs)2 | Mn(NOsz)2 | Fe(NOs)2 
aa ies 2 | — nay? 
None Peden. 100 100 100 100 100 100 


SLES e nee ls. 9 —41.2 —20.2 | -354.7 =17.8 if ste: 


This indicates a loss of nitrates where sodium, potassium, calcium, 
magnesium, and manganese nitrates had been applied to the soil. 

An analysis of the soil for total nitrogen showed a loss only where 
the potassium nitrate was applied to the soil, and in this case it 
was only 5.96 mgms. in place of 41.2 per cent., as was indicated by 
the first results. 


Gain (+) or loss (—) in 
nitrogen over soil receiving 


Treatment. no nitrate. 

Dredsbloods! mas -NOMIUTALes suet swe ean sn my fe 0.00 

5 * + 84.06 mg. N.N. as KNO; oe ee ogo mas: 

a ‘ +: 84.06mg.N.N.asNaNOs ... . 1.34 

k 2 + 84.06mg. N.N.asMg(NOs)2 . . . 25.14 

‘: % + 84.06 mg. N.N. as Fe(NOs)s. . . . 387.04 

i - + 84.06 mg. N.N. as Ca(NOs)2 Ee i CSE 

3 + §84.06mg.N.N.asMn(NOs)2 . . . 48.54 


Function of Denitrifiers.— Huge quantities of organic and inorganic 
nitrogen find their way into the septic tanks of large cities, and 
much of this is returned to the atmosphere by these bacteria. More- 
over, that which reaches the lakes and oceans is also acted upon by 
denitrifying bacteria; hence, they play a part, although of minor 
importance in the nitrogen cycle. 


REFERENCE. 


Voorhees, Edward B., and Lipman, Jacob G:: ‘‘A Review of Investigations in 
Soil Bacteriology.”” U.S. Dept. Agr. Off. Exp. Sta. Bul. 194. New Jersey Exp. Sta. 
Ann. Rpts. 1901 and 1902. 


CHAPTER: XXITI. 
AZOFICATION. 


THE maintenance of the nitrogen supply of the soil is the phase 
of soil fertility which has received greatest consideration both from 
the scientist and from the practical agriculturist. Nitrogen is one 
of the more expensive commercial fertilizers and is, in the majority 
of soils, the limiting factor of crop production. The supply of com- 
bined nitrogen on the earth is comparatively small and it is possible 
to calculate approximately the time necessary for its exhaustion. 
Basing his conclusion on such a calculation, at least one scientist 
has predicted dire calamity to the human race were science not able 
soon to solve this problem. Science has measured up to its require- 
ments in this regard, for the synthetic production of combined 
nitrogen has been accomplished, and this in a manner so highly 
satisfactory that it is able to compete successfully with the product 
of natural deposits. Advancements have also been made in our 
knowledge of the underlying principles influencing the natural proc- 
esses which govern the fixation of nitrogen in the soil. Although 
there is much yet to be learned in this field it is upon the control 
of these natural processes that ultimate success will be based. 

Historical.— It has been known for generations that uncropped soils 
increase in fertility. Less ancient, however, is the knowledge that 
this increase may be due to a gain of nitrogen in the abandoned 
soils. Even more recent than this is the knowledge that it may be 
due to bacteriological action. 

In the middle of the nineteenth century Boussingault wrote: 
“Vegetable earth contains living organisms—germs—the vitality 
of which is suspended by drying and reéstablished under favorable 
conditions as to moisture and temperature.”’ He also hinted at the 
fact that these microdrganisms take part in the process of nitrogen 
fixation. He spread out thinly 120 gm. of soil in a shallow glass dish 
and for three months moistened it daily with water free from 
nitrogen compounds. At the end of this time analysis showed that 
it had lost carbon, but had gained nitrogen. It was not until thirty 
years later that Hellriegel and Wilfarth made their discovery of 
nitrogen-fixation by symbiotic organisms. At that time the labora- 
tory technic of modern bacteriology was still undeveloped. Since 
then, however, we have learned much concerning the relationship 
of plants to free and combined nitrogen of the air and of the soil. 


HISTORICAL 249 


We know that soil gains in nitrogen are often due to microérganisms, 
either living free in the soil or in company with the higher plants. 
The production of nitrogen compounds out of atmospheric nitrogen 
by bacteria independent of higher plants is designated non-symbiotic 
nitrogen-fixation, or azofication. When fixation is accomplished by 
bacteria living in connection with and receiving benefit from higher 
plants, it is called symbiotic nitrogen-fixation. 

As early as 1883 Berthelot undertook the study of soils as 
regards their relationship to free and combined nitrogen, and as a 
result of these studies he was the first definitely to recognize that 
gains which occur in bare unsterilized soils are due to microscopic 
organisms. He found that when 50 kgm. of arable soil were exposed 
to air and to rain in a vessel for seven months, after allowing for the 
small amount of combined nitrogen brought down by the rain, there 
was a gain in nitrogen of more than 25 per cent. In another experi- 
ment in which the soil was first washed free from nitrates, there was 
a gain of 46 per cent. Many other experiments showed gains from 
10 to 15 per cent. Berthelot was not content with the bare knowl- 
edge that nitrogen is fixed in the soil by living organisms, but con- 
tinued his work with the idea of isolating some of these organisms. 
With the aid of Guignard, he made soil inoculation into sterile 
bouillon and from this prepared gelatin plates. Cultures were taken 
from the colonies growing on the plates and bacteria were tested 
for their nitrogen-fixing power. His results were conclusive that 
there exist within the soil chlorophyll-free bacteria capable of fixing 
atmospheric nitrogen. His work had shown that these organisms 
act best at summer temperatures, between 50° and 104° F., in the 
presence of a good supply of oxygen, a proportion of water in the 
soil not exceeding 12 to 15 per cent. and not falling below 2 to 3 
per cent. 

They require carbon, hydrogen and enough combined nitrogen 
to promote initial growth. The nitrogen, gained by the soil was 
proteinaceous in nature, being insoluble in water. Although some 
of his soils had gained large quantities of nitrogen, he considered 
that the fixation of atmospheric nitrogen by microérganisms has 
its limits, since the organisms isolated drew from the atmosphere 
only so long as the amount fixed in the medium was not great. Heat- 
ing the soil to 230° F. immediately stopped the process. 

Prior to this a number of chemists, notably Konig and Niesow, 
Armsby, Birner, Kellner, Dehérain and Avery had found that when 
organic matter in one form or another undergoes fermentation there 
is frequently an increase of nitrogen in the fermenting substance. 
Armsby states it thus: “We must conclude that decaying organic 
substances in the presence of caustic alkali are able to fix free 
nitrogen without the gain being manifest as nitric acid or ammonia, 
and probably without the formation of these bodies.” His explana- 


250 AZOFICATION 


tion of the process was that the nascent hydrogen evolved during 
the fermentation process reacted with the free nitrogen of the air. 
Others considered that the active agents were compounds of iron, 
manganese, and lime existing in the soil and in some way acting 
as catalytic agents. 

Berthelot’s discovery interested Winogradsky who commenced 
work which eventually bridged the chasm. He employed, as a 
medium, a nutritive solution free from combined nitrogen, but con- 
taining mineral salts and dextrose. Fifteen separate species of soil 
bacteria were isolated, but only one—a long sporebearing bacillus 
which developed normally in the absence of combined nitrogen and 
seemed to produce butyric fermentation—fixed nitrogen to any 
appreciable degree. Quantitative tests showed that the maximum 
fixation was attained where no combined nitrogen was purposel 
added, and that on the addition of such, fixation of nitrogen was 
diminished. For example, several determinations gave the following 
results: 


N as NH; in dextrose solution 
N fixed 


“I bo 
So tb 


The presence of combined nitrogen tends to decrease fixation. 
He concluded that in order for any gain to be made, the ratio of the 
combined nitrogen to the sugar should not exceed 6:1000. Because 
of the characteristic formation of clostridia in his cultures, Wino- 
gradsky named the organism Clostridium pasteurianum. The con- 
clusion which the author reached, however, was that the power of 
fixing nitrogen is not general among microdrganisms, But confined 
to a few special forms. 

Following Winogradsky, C aron made some erenen discoveries. 
He found that soils under leafy crops contain greater numbers of 
bacteria than those under grasses. He also observed that the bac- 
terial flora of soils in the spring are different from those in the fall 
both quantitatively and qualitatively. He used in vegetation 
experiments pure cultures of the bacteria most frequently 
encountered in natural soils. Some soils were inoculated with 
bouillon culture, whereas others received only sterile bouillon. 
The crop yields were usually in favor of the inoculated plots, but 
showed variations from season to season. Exceptionally good results 
were obtained with a sporebearing bacillus which he termed Bacillus 
ellenbachensis. 

Caron’s work led to the commercial exploitation of his cultures, 
one of which, “alinit,’’ was the subject of much study and contro- 
versy. This culture was found to contain, according to Severin, two 
closely -related bacilli which he chose to designate as B. ellenbachensis 
Aand B. These had the power to fix nitrogen to some extent. Tests 


HISTORICAL 251 


with “alinit,” however, have not confirmed to any great extent the 
claims of its exploiters. 

In 1901 Beijerinck’s investigations led to an extremely important 
addition to the history of non-symbiotic nitrogen-fixation. He 
described a new group of large aérobic bacilli to which he gave the 
generic name Azotobacter. 

In an early paper published by Beijerinck and van Delden, they 
maintain that Azotobacter are incapable of fixing appreciable 
quantities of nitrogen in pure culture, but are dependent to a large 
extent on Granulobacter, Radiobacter, Aérobacter. They considered 
that in mixed cultures the Granulobacter, Radiobacter, and Aérobacter 
possess the power of fixing nitrogen in the presence of Azotobacter, 
which grows at the expense of the combined nitrogen escaping from 
them into the solution. 

A little later Gerlach and Vogel succeeded in isolating from soil 
the Azotobacter of Beijerinck and in showing that in pure cultures 
and in the presence of salts of organic acids, Azotobacter are capable 
of active nitrogen-fixation. They obtained a fixation of 9 mgm. 
of nitrogen in a | per cent. solution of grape sugar. But Beijerinck 
challenged this assertion, claiming that their cultures were not pure 
but were mixed with other forms difficult to separate. The claims 
of Gerlach and Vogel were substantiated by the work of Freuden- 
reich, Koch and Lipman. The latter not only showed that the 
Azotobacter possess the power of fixing nitrogen in pure cultures, 
but he explained the failures recorded by others. 

Although not necessary, the presence of other organisms often 
proves advantageous. Lipman found that in the presence of such 
forms as B. radiobacter and B. levaniformus the nitrogen-fixation is 
faster and goes on at a more regular rate. 

To the two species of Azotobacter— A. chrodcoccum and A. agilis— 
described by Beijerinck and van Delden, Lipman, added A. vine- 
landii, A. beijerinckii, and A. woodstownii. Later Lohnis and 
Westermann described A. vitrewm, and after a study of 21 cultures 
of various Azotobacter concluded that they represented only four 
types. A. chrodcoccum is most widely distributed in the soils so far 
studied. 

The discussion of the subject thus far has been more or less con- 
fined to the Azotobacter, but investigations of Beijerinck and van 
Delden, Léhnis, Moore, Chester, Bredemann and others have 
brought to light other microérganisms having the power to fix 
nitrogen. Among these are B. mesentericus (which fixes appreciable 
quantities of nitrogen), B. pnewmonie, B. lactis viscosus, B. radio- 
bacter, B. prodigiosus, B. asterosporus and B. amylobacter. 

Bredemann, after a careful study of the morphological and physio- 
logical characteristics of eleven “original species” of other investi- 
gators and of sixteen cultures prepared by himself from various soils, 
concluded that all belong to the single species B. amylobacter of 


252 AZOFICATION 


van Tieghem. Since this, however, there has been described at 
least one aérobie clostridium. Moreover, Omelianski considers 
that the Clostridium pasteurianum, isolated from the Russian soils, 
is clearly a morphologically distinct race. An idea of the activity 
of some organisms in fixing nitrogen may be obtained from the fol- 
lowing results reported by Léhnis. In. every 100 ¢.c. of 1 per cent. 
mannite, or grape sugar soil extract, there was fixed, in the course 
of three weeks, nitrogen as follows: 


Mg. 
IBactchrysoploes. tec ste trie ae Smee eae tee Be 1.4 
Bact: ttactaricus 3 Fy, see hae ae eee ey See ee 0.3 
Bact. lipsiense EWE: Eee tas cy hee Tee 0.2 


Gage vs Lipman esha 18 organisms, iotudiag Be, pseudo- 
yeasts, and molds, nearly all of which showed a more or less pro- 
nounced power of fixing atmospheric nitrogen. 

Pringsheim has isolated from ordinary garden soil certain thermo- 
philic organisms which fix from 3 to 6 mgm. of nitrogen per gram otf 
dextrose when incubated at 61° C. in a Winogradsky’s solution to 
which a little soil extract was added. Duggar and Davis have 
recently investigated the subject of the fixation of nitrogen by the 
filamentous fungi, Aspergillus niger, Macrosporium commune, Peni- 
cullium digitatum, Pexpansum, Glomerella, Gossypii, and Phoma 
bete,-and of these only the last-named was definitely proved to be 
able to fix nitrogen. Itis thus seen that the power of fixing nitrogen 
is a characteristic possessed by many microérganisms, in contradic- 
tion to the supposition of Winogradsky that this power is limited 
to a particular, or, at most, a few species. This is especially empha- 
sized by the recent work of Emerson who examined soil which 
contained 2,400,000 organisms per gram which would develop on 
nitrogen-free media. Of these, 97 per cent. possessed the power 
of fixing nitrogen; they constituted at least four distinct groups. 
Nevertheless, the most important group yet discovered is the 
Azotobacter, and it is with these mainly that this chapter deals. 

Distribution.— The nitrogen-fixing organisms are widely distributed, 
occurring in most soils. Lipman and Burgess, who studied the nitro- 
gen-fixing flora, especially those of the Azotobacter group, of 46 soils 
from Egypt, India, Japan, China, Syria, the Hawaiian Islands, 
Guatemala, Costa Rica, Spain, Italy, Russia, Mexico, Asia Minor, 
Canada, Unalaska, Samoa, Australia, Tahiti, Belgium, Queensland, 
and the Galapagos Islands, found every soil possessed the power of 
fixing nitrogen in mannite solution. About one-third of the soils 
contained Azotobacter; frequently the same soil showed the presence 
of two or three different species of Azotobacter. A. chrodcoccum, 
however, was the most prominent. It was also found most widely 
distributed in the various soils. Groenewege found Azotobacter in all 
but one of a series of Java soils. 

Several hundred Utah soils have been examined and all found to 
fix nitrogen, many of them without the addition of carbohydrates. 


DISTRIBUTION 253 


Aérobice Azotobacter are present in nearly all Utah soils. Hutchinson 
found the Azotobacter in all the Indian soils examined. They occur 
in cultivated more frequently and in greater numbers than in virgin 
soils. This probably accounts for the much higher nitrogen-fixing 
power of cultivated soils. 

Azotobacter were found in only two out of 64 localities in the soils 
of Danish forests. Both of the soils which gave positive tests were 
from beechwood forests and contained calcium carbonate. Although 
the soils of these forests rarely contain enough carbonate to effervesce 
they are usually neutral or slightly alkaline. They contain calcium, 
but in forms other than the carbonate. It is generally understood 
that Azotobacter occur commonly in soils which contain sufficient 
calcium carbonate to effervesce when acid is added and that they 
scarcely ever occur in acid soils. Their disappearance from soil 1s 
usually due to the absence of basic substances, especially of calcium 
and magnesium carbonate, and not to the presence of toxic sub- 
stances. However, they are frequently not present in peaty soils, 
where their absence cannot be attributed to a lack of lime. 

The aérobic nitrogen-fixers are probably more widely distributed 
in soils than are the anaérobiec, for, although both groups are gener- 
ally found in the Russian soils, the aérobic are found in the sands 
of Kirghese steppes and in the peat soils of the Province of Arch- 
angel in which the anaérobic forms are absent. Anaérobic nitrogen- 
fixers are, however, quite widely distributed in soils and are at times 
found on the leaves of forests trees. 

The nitrogen-fixing organisms are confined almost entirely to the 
first three feet of soil, although they have been found in soil at all 
depths down to the tenth foot in the very favorably constituted 
loose soils of Nebraska. 

They are most active in the upper few inches of soil, as is indicated 
by results obtained by Ashby. 


Average 

Depth nitrogen fixed. 
Soil. inches. mgm. 
IhittlesHoOs an eee we os inact ee SLO 9.23 
NeiGtlevnlOOSe erate eta ween) CL ease Osim e720 7.29 
ittles El OOSHit Manage cares a, Po Wee pelea eee ase WOO 4.60 


Reports on some Hawaiian soils show them to be equally active 
at all depths to 4 feet, but this must be considered an exception, 
for the examination of numerous soils in Utah has shown a gradual 
decrease in nitrogen-fixing powers with depth. ‘The average of 
several hundred determinations, in both solution and soil media, 


are given below: 
Nitrogen fixed in 


Nitrogen fixed in 100 cc. of Ashby’s 
100 gm. of soil + solution with 1.5 
1.5 gm. of mannite. gm. of mannite. 
Depth of sample. mgm. mgm. 
ITSO tsa ret Tee ee eee hen oes Pa salal 
SecondHtootis Aha ce ee ae 0.77 


iRhindtootmur akan iat. une ee ne a OO 0.58 


254 AZOFICATION 


These samples were collected with such great care that there was 
no possibility of the mixing of one foot section with another. It is 
interesting to note that while the actual gain in nitrogen per gram 
of mannite is over twice as great in the soil as in the solution, yet 
the relative gain per foot section is the same in both. There is 
about one-half as much nitrogen fixed in the second as in the first 
foot, and one-fourth as much in the third as in the first. 

The nitrogen-fixing organisms are not confined to the soil alone, 
for Beijerinck and van Delden first isolated Azotobacter agilis from 
canal water in Holland. Azotobacter chroécoccum and B. Clostridium 
pasteurtanum are both found in many fresh ard salt waters, living 
on alge and plankton organism. 

Reaction of the Media.—The distribution and the physiological 
efficiency of the nitrogen-fixing organisms, especially of the Azoto- 
bacter species, are governed by the physical and chemical properties 
of the soil, foremost among which is the basicity of the soil, namely, 
its calcium or magnesium carbonate content. Ashby bases his 
method for obtaining pure cultures of Azotobacter upon this property, 
for he finds that by picking out the crystals of the carbonate from 
the soil and seeding them into nitrogen-free media the likelihood of 
obtaining the organism is greatly increased. The addition of calcium 
carbonate to a soil often increases its azofying power, the extent of 
which increase depends on the lime requirements of the soil and on 
the fineness of the added limestone. 

Christensen has suggested that the Azotobacter be used as an index 
to the lime requirements of a soil. The test should include both a 
search for the organism in the soil and a test of their ability to grow 
when inoculated into the soil. He and Larson examined more than 
one hundred soils of known lime requirement. They determined 
the carbon dioxid set free by acids, the amount of calcium dissolved 
by an ammonium chlorid solution, the behavior of the soil toward 
litmus, and the biological test. The result of this test was that the 
biological test agreed with the known condition in 90 per cent. of the 
cases, the ammonium chlorid in 50 per cent., the litmus in 40 per 
cent., and the carbon dioxid failed more often than not to indicate 
the correct condition of the soil. 

Fischer failed to find Azotobacter in a heavy loam soil containing 
only 0.145 per cent. of lime, while adjoining limed plots had an 
Azotobacter flora. The quantity of calctum carbonate which must 
be added to obtain maximum fixation varies with the soil. 

A West Virginia Dekalb silt loam, which required 0.175 per cent. 
of calcium carbonate to render it neutral by the Veitch method, 
gave greatest nitrogen fixation when 0.375 per cent. of calcium 
carbonate was added. Above. this concentration azofication 
decreased, but when phosphorus was applied with the lime it was not 
toxic even when present in quantities as great as 0.5 per cent. It is 


REACTION OF MEDIA 255 


certain that large quantities of calcium carbonate may be present 
in soil without injury to the azofiers. 

The author found numerous Azotobacter and a very active nitro- 
gen-fixation in a soil 43 per cent. of which was calcium and mag- 
nesium carbonate. 

The organisms develop normally in the presence of either calcium 
or magnesium carbonate, but in liquid cultures the film develops 
earlier and it contains less foreign organism in the presence of mag- 
nesium carbonate than in the presence of calcium carbonate. The 
actual nitrogen fixed, as reported by Ashby, is also greater where 
the magnesium carbonate is used. This he attributes to the sup- 
pression by the magnesium of foreign organisms, especially of the 
butyric acid ferments. 

There is, however, a marked difference in the action of caletum 
carbonate and magnesium carbonate when they are applied in large 
quantities. Lipman and Burgess found the calcium carbonate stimu- 
lating and never toxic to Azotobacter chroécoccum in concentrations 
up to 2 per cent. in mannite solution. The magnesium carbonate 
was sharply toxic in higher concentrations up to 2 per cent. in 
mannite solution. The magnesium carbonate was sharply toxic 
in higher concentrations above 0.1 to 0.2 per cent. in such cultures. 
The calcium salt is without effect when added to most soils up to 
1.4 per cent., but the magnesium carbonate is even more toxic in 
soils than in solutions. Moreover, their work indicates that calcium 
exerts a protective influence, in both soils and solutions, against 
the toxic influence of magnesium. ‘The best ratio of calcium to 
magnesium varies with solution and soil. ‘ 

In many soils lime increases the nitrogen fixed, for Krzemieniewski 
found limed soil to fix in ten days 17.52 mgm. of nitrogen, whereas 
adjoining unlimed soil fixed only 7.15 mgm. There is, however, the 
possibility of applying too large a quantity of the caustic lime and 
thereby decreasing nitrogen-fixation, a condition which has never 
been experienced in the use of the carbonate. 

Von Feilitzen, however, found neither a direct relationship be- 
tween lime content of moor soil and the development of Azotobacter, 
nor relationship between their development and the reaction of the 
soil. But this only serves to illustrate the fact that although lime 
and neutral or slightly alkaline media are essential, they will not 
ensure a rich Azotobacter flora in a soil unless all other conditions 
are optimum. Remy found sodium and potassium carbonate less 
favorable for nitrogen-fixation than was calcium or magnesium. 

So far as the writer is aware, Krainsky is the only worker who has 
found sodium carbonate more favorable than calcium carbonate. 
This may have been due to the sodium carbonate’s liberating plant- 
food which was in the soil in an insoluble form but which was essen- 
tial to the development of Azotobacter. Mockeridge has found that 


256 AZOFICATION 


the presence of sodium salts is unnecessary and depressing at least 
to the growth of Azotobacter. The beneficial effect ascribed to sodium 
chlorid solution in inoculating agar plates is due to the fact that 
this liquid is isotonic with the cell content solution, but the sodium 
hydroxid is a far less advantageous neutralizing agent than is cal- 
clum or magnesium carbonate. Furthermore, Lipman failed to 
stimulate the azofiers with any of the sodium salts. 

Food Requirements of the Azofiers.—'These organisms probably 
require for their nutrition the same elements as do the higher plants, 
namely carbon, hydrogen, oxygen, nitrogen, potassium, phosphorus, 
sulphur, calcium, magnesium, and iron, and possibly aluminum and 
manganese. 

They obtain their carbon and hydrogen from organic compounds, 
preferably from carbohydrates, which are considered in detail under 
sources of energy. Oxygen is obtained either from the atmosphere 
or from combined sources depending on the species and the condi- 
tions under which they are grown. 

A marked difference between these and the higher plants is that 
they possess the power of obtaining their nitrogen from the air, but 
in the presence of combined nitrogen they obtain but little from the 
air. Lipman, Stranak, Heinze, and Stoklasa found that small 
quantities of nitrates stimulated Azotobacter, whereas large quanti- 
ties discouraged nitrogen-fixation since the organisms live on the 
nitrates. This is the case whether the nitrates are added to the soil 
or to the solution in which nitrogen-fixation is taking place. Cole- 
man considers this action as due to several different factors: namely, 
(a) a direct toxic action of the salt, (b) antagonism of other organ- 
isms which it favors, (c) the using up of the energy supply by 
these organisms, and (d) the discouragement of fixation by the use 
of sodium nitrate. The last would seem to be the most important 


factor when viewed in connection with the following results reported 
by Hills: 


Relative per cent. of nitrogen fixed. 


Relative number of organisms. == 
Sterilized soil. Unsterilized soil. 
Treatment | 
nitrate. 
Mem. | KNOs NaNOs | Ca(NOs)2 NaNOz | Ca(NOs)2 NaNOs | Ca(NOs)e2 
0 100 100 100 100 100 100 100 
10 348 191 362 100 105 240 219 
50 8210 3150 4528 342 371 500 444 
150 12 117 763 
200 | 0 0 0 352 467 879 557 


FOOD REQUIREMENTS OF THE AZOFIERS 257 


The number of organisms developing and the nitrogen"fixed in 
the one receiving no nitrate is taken as 100 per cent. 

It is quite evident from these results that although nitrates cause 
more active multiplication of Azotobacter, it greatly reduces their 
physiological efficiency. The organisms used by Hills had probably 
grown for a long time on media poor in nitrogen, and their ability 
to fix nitrogen was, therefore, high. But would they continue to 
exert this power if grown on media rich in nitrogen? The evidence 
points strongly to the conclusion that they would not. It is certain, 
however, that the nitrates are toxic in comparatively low concentra- 
tions. Nitrates and ammonium sulphate are rather effective in stimu- 
lating nitrogen-fixation when the Azotobacter are grown in connection 
with the cellulose ferments. Even here, however, large quantities 
decrease this power. In pure cultures ammonium sulphate seriously 
retards nitrogen-fixation, whereas the nitrogen of humus, even in 
large quantities, appears to have no serious retarding influence. 
Nevertheless, a high nitrogen content of soils seems to be unfavor- 
able to vigorous nitrogen-fixation. 

Whether this would be the case where the nitrate content of the 
soils is kept low but with the readily decomposable protein nitrogen 
high, is yet to be answered. Hiltner and Stérmer consider that when 
the nitrogen content of the soil passes beyond a certain limit, the 
decay bacteria increase rapidly, and in the struggle for existence 
they are able, with the advantage at their disposal, to suppress the 
more slowly growing Azotobacter. 

Potassium is essential to the higher plants and cannot be replaced 
entirely by related elements, yet Gerlach and Vogel early reached 
the conclusion that potassium and magnesium are not essential to 
the Azotobacter. Their results were, however, generally considered 
erroneous, for while as much nitrogen was fixed in twenty days 
without as with potassium, after forty days there was no further 
fixation in the solution without potassium, but in its presence the 
nitrogen gain nearly doubled. It was, therefore, argued that the 
traces of potassium left in the chemicals and dissolved from the 
glass during sterilization had been enough to permit development 
for a time. If these elements are essential, it must be in extremely 
minute quantities, for Vogel, using the purest chemicals obtainable, 
was able to prepare potassium-free media in which the Azotobacter 
developed. He did find, however, that potassium favors their 
development. 

Phosphorus is required by these organisms, large quantities being 
used for the building of the nucleo-proteins and phospho-proteins in 
which their bodies are extremely rich. Moreover, it greatly acceler- 
ates the reaction and economizes the carbohydrates; hence it is 
rather evident that phosphorus plays a very essential part in Azoto- 
bacter metabolism. Possibly in the early stages of the process a 

ily 


258 AZOFICATION 


definite chemical reaction occurs between the phosphate and the 
carbohydrate similar to that occurring in alcoholic fermentation. 


I. 2CséHi2006 + 2ReHPOs: — 2CO2 + 2H20 + CeHiO04(POsRe)2 + 
2CoHeO 
II. CeHiOs(POsR2)2 + 2H2O — CsHw2O6 + 2ReHPO, 


The Azotobacter are able to utilize the phosphorus of di- and 
tri-basic sodium and potassium phosphate and of dibasic calcium 
phosphate. 

Mockeridge obtained an increase of 23 per cent. in nitrogen fixa- 
tion with basic slag. There were two maxima, one with 0.4 per cent. 
the other with 1.0 per cent. slag. This is attributed to the stimulat- 
ing effect of the iron and manganese in the slag, the maximum effect 
of one being produced at 0.4 per cent., the other at 1.0 per cent. 
The tribasic calcium phosphate—bone ash, iron, and aluminum 
phosphate—all serve only as difficultly available sources of phos- 
phorus. Raw rock phosphate and bone meal fail entirely to furnish 
enough available phosphorus for the development of Azotobacter. 

The addition of phosphorus to a soil often greatly increases azofi- 
cation. 


Without With 
phosphorus. phosphorus, 
Treatment. mgm. mgm. 
IN Opis leds ity ey aed rd ewe TA tS te Pea EG 0.9 
LGstan SW aioe Ch ween? ink Alm asian Pere man ed, Pee AL is° 4.6 


Moreover, Christensen has found soils in which phosphorus is the 
limiting element in Azotobacter growth. He entertains the hope that, 
in view of the relationship between Azotobacter growth and lime and 
phosphorus, it will become eventually possible by the determin- 
ation of bacterial food requirements to secure a general expression 
for the soil content of plant-food available to crops. He further 
suggests that where a mannitol solution free from phosphorus 
produces a vigorous growth of Azotobacter after inoculation with a 
soil, it may be assumed that the soil is not deficient in available 
phosphorus. Dzierzbicki notes that if soils are deficient in available 
lime, phosphoric acid, or potash, nitrogen-fixing bacteria, such as 
Azotobacter, are either entirely absent or present only in small 
numbers. 

There is a definite relationship between the carbon and phos- 
phorus content of a soil and the nitrogen assimilated. According 
to Stoklasa, Azotobacter assimilates from 5.0 to 5.7 grams of free 
nitrogen for every gram of phosphorus used. Although these 
organisms are directly dependent upon a readily available supply 
of phosphorus to promote grow th, they do not change it into the 
organic form as rapidly as do the ammonifying bacteria. 

Sulphur is required by the azofiers possibly for the formation of the 


ORGANIC SOIL CONSTITUENTS 259 


proteinaceous material of their bodies. It is certain that the benefit 
derived by Azotobacter from the sulphates of iron and calcium is due 
ina large measure to the sulphur which these compounds supply. No 
evidence has as yet been produced which would lead us to believe 
that the organisms can use sulphur as a source of energy. 

Calcium carbonate and calcium oxid, in addition to furnishing a 
base which neutralizes the acid formed in the metabolic processes 
of the Azotobacter, also furnish calcium to the organism. Christensen 
brought out the fact that Azotobacter can derive their calctum from 
dibasic calcium phosphate and some calcium salts of organic acids. 
They could not, however, utilize the calcium of tribasic phosphate, 
of calcium chlorid or sulphate. 

Tronis essential and either the ferric or ferrous sulphate is especially 
beneficial. Rosing found the amount of nitrogen fixed increased 
from 2.23 mgms. to 10.3 mgms. per gram of mannite when iron 
sulphate was added to the cultural media. This is due, in a great 
degree, to the iron which serves as food for the organisms, yet its 
colloidal nature may play a part, for both organic and inorganic 
colloidal substances have an especially favorable action on Azoto- 
bacter, although the action of the inorganic colloids is fully manifest 
only in the presence of organic colloids. If used alone, large quan- 
tities of the ferric hydroxid are essential for the maximum effect, 
but in the presence of organic colloids very small quantities of iron 
are effective. This has been attributed to the action of the colloidal 
iron which adsorbs the nitrogen and oxygen of the air and brings 
them into more intimate contact with the Azotobacter. This would 
not only accelerate the normal processes of the aérobic Azotobacter 
by furnishing them with nitrogen and oxygen, but it would tend to 
suppress the anaérobic processes which are extremely wasteful of 
the food. According to Kaserer, these organisms also require 
aluminum. Although this may accelerate, it has not been proved 
to be essential to their growth. 

While not essential to the organisms, manganese is an extremely 
active catalyzer in increasing proportions up to 6 mgm. per 100 c.e. 
of media. Above this concentration the reaction falls off rapidly, 
and at 20 mgm. it is less than in the absence of manganese. It is 
oxidized by Azotobacter, and in the proportion of 1 part to 200,000 
parts of soil it is an active stimulant. Olaru considers it likely that 
the increased yield obtained after the application of manganese 
compounds to a soil is due to its accelerating the action of the 
nitrogen-fixing organisms of the soil. 

Organic Soil Constituents.— Reed found urea, glycocol, formamid, 
and allantoin active in depressing nitrogen-fixation. This he 
attributes to the compounds furnishing the Azotobacter an available 
source of combined nitrogen and not to a direct toxicity. But 
Walton found that the addition of urea, peptone, acetamid, aspar- 


260 - AZOFICATION 


agin, and casein to culture media had only a slight influence on the 
fixation of nitrogen by Azotobacter. 

Caffeine, alloxan, betain, trimethylamin, legumin, cinnamic acid, 
aspartic acid, asparagin, hippuric acid, creatin, creatinin, xanthin, 
and hypoxanthin, are all toxic to Azotobacter even in small quantities. 
Only the first two have been tested in concentrations dilute enough 
to stimulate, which is remarkable, as many of these compounds 
stimulate the higher plants and some can be utilized directly by 
the plant. 

Ksculin, vanillin, daphnetin, cumarin, pyrocatechin, heliotropin, 
arbutin, resorcin, pyrogallol, phloroglucin, hydroquinon, salicylic 
aldehyd, oxalic acid, quinic acid, dihydrostearic acid, rhamnose 
and borneol, on the other hand, do not stimulate in any concentra- 
tion. Nor are they toxic until fairly large quantities have been 
added. In this regard the nitrogen-fixing organisms appear to differ 
widely from the nitrifying bacteria and higher plants. The resist- 
ance of the nitrogen-fixers to various chemicals has likewise been 
called to our attention by Lipman in his study of the influence of 
alkalies on nitrogen-fixation. 

Influence of Colloids.—It was recognized early in the study of 
nitrogen-fixation that when sterilized soil is added to a nutritive 
medium it greatly increased the quantity of nitrogen fixed. This 
condition is due to several factors and is partly explained by 
Krzemieniewski’s results wherein he found that nitrogen-fixation is 
decidedly increased by the addition of soil humus, either as free 
humic acid or as salts of potassium, sodium or calcium. Kaserer 
maintains that this is due to the inorganic nutrients, especially to 
aluminum and silicic acid supplied to the microérganisms through 
the humus. This is probably true in part, for the fixation varies with 
the humus derived from different sources. Moreover, artificial 
humus, prepared by boiling sugar with acids, fails to stimulate. 

That much of the beneficial effect is due to the constituents in the 
humus appears likely from the results obtained by Séhngen who 
found that colloidal iron oxid, aluminum oxid, and silicon oxid all 
greatly stimulated the nitrogen-fixing powers of Azotobacter chroé- 
coccum. ‘This he attributed to the absorption of oxygen and nitrogen 
by the colloid, which he maintains would make them more readily 
available to the organisms. The boiling of natural humus with 
hydrochloric acid would either remove the foreign material or change 
it from the colloidal form, and thus, as has been found to be the 
case, render it inert. Léhnis and Green take exception to this 
explanation, for they found no adsorptive action exerted by humus 
on either the nitrogen or the oxygen. Furthermore, Rosing found 
that he could stimulate just as effectively with iron as with humic 
acids. But much larger quantities of colloidal iron are required 
when it is used singly than when used in conjunction with an organic 


SOURCES OF ENERGY FOR THE AZOTOBACTER 261 


colloid. The extent of the stimulation resulting varies with the form 
in which the iron is applied and is most effective in the form of the 
hydroxid and in the presence of cane sugar. In this case it is 
probable that the saccharate is the active substance. Hence, the 
contradictory results reported may be due to the different min- 
eral constituents of the humus. 

These facts make it certain that colloids of the metals act as 
stimulants to nitrogen-fixing bacteria, as does also crude humus. 
Carefully purified humates do not possess this property, but it 1s 
possessed by the aqueous extract, the alcoholic extract, and the 
phosphotungstic fraction of the aqueous extract from “bacterized”’ 
peat. Whether this influence is due to a catalytic effect, as suggested 
by Séhngen, or whether the substance furnished a direct source of 
nutritive material is not clear at the present time. 

Moreover, the colloid may act as a protection to the organism 
against poison; for, when 10 parts per million of soluble arsenic is 
maintained in a soil, it acts as a stimulant to Azotobacter. If, how- 
ever, this proportion is added to the Ashby nutritive solution it stops 
all nitrogen-fixation. This is due in part to the adsorption of the 
arsenic by the soil. This adsorption would have to be attributed 
largely to the silica compounds, for the nitrogen-fixing organisms are 
stimulated by arsenic in quartz free from organic colloids. This 
could readily be due to the arsenic becoming concentrated at the 
surface layers of the silica, leaving the inner part of the water film 
comparatively free from arsenic, in which part of the water film the 
microdrganisms multiply and carry on their metabolic processes. 
This being the case, one should and probably could find a water 
solution weak enough to stimulate bacteria. A great difference, 
however, between the solution and the sand-culture method is the 
greater aération in the sand. That the aération of a culture medium 
does play an important part in determining the activity of the 
nitrogen-fixing powers of a soil is strikingly brought out in Fig. 18, 
page 124. 

Sources of Energy for the Azotobacter.—'The nitrogen-fixing organ- 
isms differ widely from other plants in their energy requirements. 
This is due to the fact that they are carrying on endothermic reac- 
tions in which nitrogen is concerned. This necessitates a greater 
supply of energy than is required by other bacteria. They are 
similar to most other bacteria in that this energy must be supplied 
by an organic compound, preferably one of the carbohydrates. 

Berthelot in his early work maintained that the gains in nitrogen 
noted in some soils were due to the action of biological agents on 
the humus of the soil. This was followed by the observation of 
others that when forest leaves are allowed to decompose in soil there 
is an increase in its nitrogen content. Koch in 1907 increased 
nitrogen-fixation by the addition to soil of dextrose, cane sugar or 


262 AZOFICATION 


starch, but there was practically no increase when straw, filter 
paper or buckwheat was applied. Yet Stoklasa showed that the 
decomposition products of these substances acted as a valuable 
source of energy to the Azotobacter, and Stranak considered that the 
pentosans of the soil are of the greatest importance in the assimila- 
tion of nitrogen by soil bacteria. 

A fair idea of the great variety and relative efficiency of substances 
which may serve as a source of energy to the azofiers may be obtained 
from the work of Léhnis and Pillai. They inoculated a nutritive 
solution with 10 gm. of soil and after ten days determined the gain 


in nitrogen. 
Nitrogen fixed 


Substance added. after10 days 

mgm. 
Mannite 9.40 
Xylose 9.54 
Lactose 9.12 
Levulose 8.52 
Inulin . pike 
Galactose 7.86 
Maltose 7.44 
Arabinose 7.62 
Dextrin “18 
Sucrose 8.60 
Dextrose . 4.62 
Starch ae 3.36 
Sodium tartrate . 2.82 
Glycerin’... = 1.68 
Sodium succinate 2.96 
Calcium lactate . 2.49 
Sodium citrate 1.42 
Sodium propionate 1.10 
Potassium oxalate Dee 
Calcium butyrate 0.02 

—0.96 


Humus 


Other workers have noted larger gains of nitrogen than those 
noted by Léhnis and Pillai, but they can readily be attributed to the 
time of incubation—in this case, ten days being far too short for the 
complete utilization of the carbonaceous substance applied; the 
species of nitrogen-fixers which are bringing about the change; and 
whether pure or mixed cultures are used. The order of effectiveness 
noted above, however, is that recognized by most workers. Brown 
and Allison, however, do report results in which greater fixation 
was obtained with dextrose than with mannite. But in this case, 
calcium or sodium carbonate seems to be even more necessary than 
it is with the mannite. Moreover, some species utilize one carbo- 
hydrate most effectively and another species a different one. To 
this list may be added malate, gum tragacanth, ethylene glycol, 
methyl, ethyl, and propyl alcohols, lactic, malic, succinic and gly- 
collic acids. Fatty acids are readily utilized, the amount of nitrogen 
fixed being greater with the increased molecular weight, from 1.47 
mgm. with formic acid, to 6.08 mgm. with butyric acid. Most of the 


SOURCES OF ENERGY FOR THE AZOTOBACTER 263 


naturally occurring glucosides and many benzin derivatives are 
unsuitable as sources of energy for Azotobacter. Molasses, which 
should serve as a useful source of energy, often results in a loss of 
nitrogen when applied to the soil. This may be due to the time of 
applying, for Peck maintains that molasses applied to a land lying 
fallow at an interval of several weeks before planting of the crop 
may produce beneficial results by increasing nitrogen-fixation. 

Beijerinck early recognized that certain decomposition products 
of cellulose can also serve as sources of energy for Azotobacter, and 
Pringsheim found that Clostridiwm americanum does not fix atmos- 
pheric nitrogen on sterilized cellulose unless other carbohydrates 
like dextrose, lactose, mannitol, or sucrose are present. However, 
in the presence of cellulose, Clostridium will fix nitrogen and this 
more efficiently than it will in the regular carbohydrate medium. 
The same holds for agar. Just how completely cellulose must be 
broken down before it can be utilized by Azotobacter is not definitely 
known, but it is known that Azotobacter cannot utilize cellobiose 
except when grown in conjunction with Aspergillus niger or other 
organisms. It is, therefore, certain that the products which are 
utilized by the Azotobacter are comparatively simple. 

Cellulose when applied to the soil may serve as a valuable source 
of energy, provided sufficient time is allowed for its decomposition. 
The cellulose ferment is probably the most efficient organism in the 
soil in bringing about this decomposition. But the number of soil 
fungi which possess this power is large. 

Hoppe Seyler thinks that cellulose is decomposed according to the 
following formula: (a) the hydration of the cellulose with the 


formation of hexose, 
(CsH105+ H20 =CceH120e. 


the destruction of the carbohydrate with the formation of equal 
quantities of carbon dioxid and methane. 


CeH2rOs—3CO2+ 3C Ha 


None of the cellulose ferments studied by McBeth, however, yielded 
gaseous products when acting on cellulose or sugar; hence the 
Azotobacter probably gets from the cellulose ferments, pentoses 
and hexoses, and similar products upon which they can readily fix 
nitrogen. 

At times in fermenting straw and manure, the thermophilic 
anaérobic bacteria play a major part, in which case fatty acids 
probably make up the greater part of the end products. 

It is claimed by Dvarak that substances with low carbon and high 
oxygen content are usually the best sources of energy for A. chrod- 
coccum, which assimilated 5.73 mgm. of free nitrogen per 100 gm. 
of carbon in pine leaves as compared with 1237.9 mgm. per 100 gm. 


264 AZOFICATION 


of carbon in red clover. He obtained for other substances the 
following results: 

1456.5 mgm. of nitrogen per 100 gm. as glucose. 

280.4 mgm. of nitrogen per 100 gm. as cornstalks. 

596.8 mgm. of nitrogen per 100 gm. in stalks and root residues of 
corn. | 

325.4 mgm. of nitrogen per 100 gm. in wheat straw. 

The carbon—nitrogen ratio in compounds is no indication of their 
value to nitrogen-fixing organisms, for non-leguminous hays and 
straws are utilized just as effectively as are the legumes. Mockeridge 
found that the ratios of nitrogen fixed to the heat of combustion with 
the four lower fatty acids is almost constant. The same holds true 
with starch, dextrin, and gum arabic, when allowance is made for 
experimental error, which is greater with these compounds than 
with the simpler compounds. This close relationship is not, how- 
ever, graduated and no such uniformity is observed with the series 
of monohydrie alcohols. 

The quantity of nitrogen fixed per gram of carbohydrate varies 
greatly with the species. Winogradsky found Clostridium pasteuria- 
num to assimilate 2 to 3 mgms. of nitrogen for each gram of sugar. 
But this like other anaérobic organisms is very wasteful of energy, 
leaving much of it in the butyric acid, acetic acid, and butyl] alcohol 
formed. In the experiments of Bredemann with B. amylobacter and 
of Pringsheim with Clostridium americanum, the amounts fixed were 
at times much larger. Much greater fixations have been reported 
with Azotobacter, and Lipman has obtained as high as 15 to 20 mgms. 
of nitrogen per gram of mannite assimilated by A. vinelandii. This 
quantity is considerably greater than that fixed by any of the other 
members of the group. 

Koch and Seydel claim that the usual method of estimating the 
nitrogen-fixing powers of Azotobacter is erroneous, as it does not 
represent accurately the intensity of the process. In a series of 
experiments made by them, the amounts of nitrogen fixed per gram 
of dextrose used were 53, 70 to 80, 20 to 30, and 5 to 8 mgms. on the 
first, second, third, seventh, and eighth days, respectively. 

Krainsky considers that there should be sufficient organic matter 
in the soil to permit that for 1 part of nitrogen tormed there will be 
90 parts of carbon for the use of the organism. The organisms, how- 
ever, utilize the carbohydrates more economically when only small 
quantities are present. Walton finds with Indian soil that highest 
fixation is obtained per gram of mannite when 10 grams are used in 
1 liter of nutritive solution. Young, vigorously growing cultures 
usually fix more nitrogen than the older ones. The nitrogen fixed 
is greatest in the first stages of the growth of the organisms, as is 
seen from Fig. 33 from the work of Omelianski. 

The efficiency of these organisms is, therefore, greatest when they 


35 


J,0 


5) 


20 


13) 


SOURCES OF ENERGY FOR THE AZOTOBACTER 265 


are rapidly multiplying, and it decreases as their metabolic products 
accumulate. Hoffmann and Hammer claim this to be due in impure 
cultures to a loss of nitrogen or free ammonia occasioned by the 
decomposition of the cells of Azotobacter. This explanation would 
hardly hold in the presence of pure cultures, unless we ascribe the 
breaking down to an autolytic ferment secreted by the Azotobacter 
cell. According to Koch and Seydel this indicates that in the latter 
stages of fixation, when there occurs an accumulation of nitrogenous 
material in the medium, the organisms employ the carbohydrates 


10 


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|_| alas eae rah ore Ty ca | | | fsa | | eta 
AGS eiciaeoet ee eaace ee eee 
Be a | a | | A a aa | ae | | |p| | | 9 |e at | 
aia slelsig|siaie Aabea nisi eae ieee ea 
BSE eID ATs 62a 
SEN a 0 a a ha 
BIRMG eee eicialelats alsin aie ele ic Ie ae a 
Se) a dS 
micemisiida ee eee Cee et eee ee 
ea a a a a | a | | || Oe | Os || ys a ge |] |g 
RO SCGVeRSe <n ne see ee) @ cee 
nba icici ee tat piesa 
pant aa | HEROS aeee 
Biciguissiecistaiatet Fisch e 
0 = eg ao k es eGo SS FO 


Fie. 33.—Graph showing the fixation of nitrogen and decomposition of sugar in 
mixed cultures of Azotobacter chroécoccum and Clostridium pasteurianum. 


for other purposes than for nitrogen-fixation. Under natural con- 
ditions in the soil this accumulation and concentration of nitrogenous 
material by the Azotobacter is not likely to occur; hence, they assume 
that the organism will continue fixing nitrogen at the high ratio 
noted in the early part of laboratory experiments. 

The quantity of nitrogen fixed, however, is dependent upon factors 
other than the source of energy; e. g., Krzemieniewski found with 
A. chroécoccum that the addition of humates to the cultural solutions 
increased the nitrogen fixed from a maximum of 2.4 mgm. to 14.9 


266 AZOFICATION 


mgm. Moreover, Krainsky found Azotobacter to utilize from 100 
to 200 gm. of sugar in the assimilation of 1 gm. nitrogen when 
grown in tees but when grown on sand it réqnired only 11 to 
30 gm. for the same fixation. 

They utilize their energy more economically in the presence of a 
liberal supply of phosphorus than when the quantity of available 
phosphorus is limited. This accounts, in a measure, for the high 
fixation noted in most Utah soils. 

Manure.—It has been known for a long time that humus exerts 
a highly favorable effect on nitrogen-fixation. The great question, 
however, has been as to the manner of action. Humus, being such 
a complex, variable substance, varies greatly in action, depending 
upon its source. Remy considered that some of the products from 
humus are favorable sources of organic matter for Azotobacter. 
Definite and valuable information is furnished by the work of 
Lohnis and Green. They worked with mixed cultures of A. chroé- 
coccum, A. beijerinckii, A. vinelandiu, and A. vitrium in Beijerinck’s 
mannite solution with various forms of organic matter. 


Nitrogen fixed in 
100 ce. of solution 


Material. after 3 weeks. 
mgm. 
Fresh straw . . ee eee is Soh el el ioe Peete aN eS teen ar ee TINS 
Fresh stable manure Diaby Bek Matte sk Sep edl tty ta lias Whe oem PX Ramat yale 9.8 
Fresh peat Se ia Ra Me Oe ear Fr Ne: aS Se ene 9.3 
Green manure... preg ae ee. Oa SU Ree ae 8.0 
Beijerinck’s mannite Solution 5.6 


After humification, these substances were even more readily 
assimilated and the nitrogen-fixation was greater than when the 
unhumified substance was used. 

The same year Hanzawa published results which show that stable 
manure even up to 3 per cent. greatly stimulated bacterial activities. 
Green manure humus was found by him to be injurious. From this 
it is certain that humus can act as a source of energy and usually 
stimulates bacteria, but the extent is governed largely by its com- 
position and by the quantity of available combined nitrogen which 
is being supplied with it to the organism. In addition to this, corn 
roots, cornstalks, oak leaves, lupine, alfalfa, maple leaves, and pine 
needles may all serve as a useful source of energy to the nitrogen- 
fixing organisms. Apparently, the tissues from the non-legume 
give a greater gain than do those from the legumes. Fulmer has 
recently confirmed these results. 

The influence of stable manure upon the nitrogen-fixing powers 
of the soil under field conditions is seen from the following table in 
which the quantity of nitrogen fixed in the unmanured soil has been 
taken as 100 per cent. 


METABOLISM OF AZOTOBACTER 267 


Treatment. Nitrogen fixed. 
per cent. 
No manure : ; Cm a TRL tn yt. Put utales hy tite a: Me estes NOU: 
Rinna on mene per LCC ta ae RIE ai Tee ae el Pon aes; Fee eet Le 
Ottonsofemanune: Der aCrenm suey eee ey ie re Nee d wcom sh) hme IO) 
(otons Onmantire-DenAChe, Muae) on eae eu fs ny oe a se LOD 
POonsoOtmanune  DeracCre nes fhe eet fen let 2 le tore OS 
PO OELOnS OLMManUew pel: Che). caret Mime ein lu cele beck ry eel efyu scp ne MALO 


These results indicate clearly that stable manure has a beneficial 
effect upon the nitrogen-fixing powers of the soil, but if used in 
large quantities the benefit is not so pronounced as if used in smaller 
quantities. 

This decrease in nitrogen-fixation with increased additions of 
manure must be considered as due to its physical effect upon the 
soil, for Richards found that Azotobacter grow and fix nitrogen in 
horse manure when it is well aérated and contains sufficient moisture 
and calcium carbonate. There is, too, a close connection between 
the diet and the effect. Horses fed on oats gave feces which induced 
the greatest fixation; horses on grass next; cattle receiving oatmeal 
cake third; but the feces from cattle fed on grass proved unsuitable. 

Manures often contain nitrogen-fixing organisms of considerable 
activity. Their activity appears to be greatest in fermenting ma- 
nures mixed with straw which serves as a source of energy. 

Although Fulmer and Fred were unable to find Azotobacter in any 
of the samples of manure examined, they did obtain many nitrogen- 
fixing bacteria from it. One of these organisms, for which they 
suggested the name of B. azophile, is as efficient in fixing nitrogen 
as is Azotobacter. This would make it appear that manure may often 
carry to the soil nitrogen-fixing organisms. 

Metabolism of Azotobacter.— Much time has been given to a study 
of the metabolism of Azotobacter, yet our knowledge of this subject 
is far from satisfactory. It is well known that the organisms oxidize 
the various carbohydrates and with the energy thus obtained build 
up complex nitrogen compounds. Berthelot early recognized that 
the nitrogen so fixed is insoluble in water, thus indicating its protein 
nature. Lipman found that there was a small but ‘appreciable 
quantity of nitrogen in both young and old cultures of A. vinelandii 
not precipitated by lead acetate and a large proportion not precipi- 
tated by phosphotungstic or by tannic acid. Further work indicated 
that the substances were either amino-acids or comparatively simple 
peptids. He considered that one of the early substances synthesized 
by these organisms is alanin. An analysis of the Azotobacter mem- 
brane gave the following results: 


Nitrogen as ; ‘ Non-Basic Nitrogen in MgO Total per cent. 
Ammonia Basic Nitrogen Nitrogen Precipitate Nitrogen 
per cent. per cent. per cent. per cent. per cent. 


0.98 2.76 6.39 0.42 10.45 


268 AZOFICATION 


This he finds corresponds remarkably closely with that of legumin. 
Experiments with plants indicate that the nitrogen of the Azoto- 
bacter cells is not readily assimilated. 

Stoklasa found the Azotobacter cells to contain 10.2 per cent. ot 
total nitrogen and 8.6 per cent. of ash. The ash contained from 58 
to 62.35 per cent. of phosphoric acid. Nitrogen and phosphorus 
were mainly in the form of nucleo-proteins and lecithin. The per- 
centage of both nitrogen and phosphorus in the cell increase with 
age. 

The most complete analyses of the Azotobacter cells, so far reported, 
show them to contain when grown on dextrin agar and rapidly 
dried at 30° C., 6.63 per cent. of water, 4.12 per cent. of ash, and 
12.92 per cent. of protein. The protein is similar to other plant 
proteins. It contains 10 per cent. of ammonia nitrogen, 26.5 per 
cent of diamino-nitrogen, and 60 per cent. of monoamino-nitrogen. 
The quantity of lysin present is very high, but the histidin is present 
only in traces. 

Krzemieniewski states that Azotobacter produces no hydrogen or 
other combustible gases in its metabolism, but according to Stoklasa 
it does, and in the presence of nitrates it produces ammonia and 
nitrites. Moler claims that during its life, 4. chrodcoccum separates 
no soluble compounds, and it is only after death that it furnishes 
nitrogen to higher plants. Nor are their bodies readily broken 
down by proteolytic enzymes. Both A. agilis and A. vinelandu 
separate a soluble compound. ‘The protein compounds so formed 
in soil are quickly broken down by other bacteria. Remy considers 
the nitrogen fixed by Azotobacter in a readily available form for 
plant assimilation. Beijerinck found that 50 per cent. of the total 
nitrogen in Azotobacter cells when supplied to the soil is nitrified in 
about seven weeks. None of the Azotobacter so far studied produce 
nitrates in the media. 

Turning now to the breaking down of the carbohydrates, we find 
that the organisms produce ethyl alcohol, glycocoll, acetic acid, 
butyric acid, lactic acid, carbon dioxid and hydrogen. The quantity 
and quality of the different products vary with the species and with 
the carbohydrate used. 

It is likely that many of the end-products have not been deter- 
mined, for Stoklasa starting with 15.9 gm. of dextrose recovered 
7.9 as carbon dioxid, 0.3 as ethyl alcohol, 0.2 as formic acid, 0.7 as 
acetic acid, 0.2 as lactic acid, but could not trace the remaining 6.6 
gm. The organisms are extremely active when growing under 
appropriate conditions, for 1 gm. weight of Azotobacter has evolved 
no less than 1.3 gm. of carbon dioxid in twenty-four hours. A great 
distinction between the Azotobacter and the other species is that the 
former decompose their sugar with carbon dioxid as the chief 


METABOLISM OF AZOTOBACTER 269 


product, whereas the other species produce large quantities of butyric 
acid. Some of these products may be accounted for as follows: 


CHO CHO CHO CHO HCOOH — CO; 
Intakes beset uA us Ise fees CHO > CH.OH 
CHOH — iis 5 cae La CH; ue 
vee CHOH see ZGHO! te} CHOW... CHO | 


| | 
CHOH CHOH CHOH CletOMEr ==] (C=——Osl = ew 


| | | | | 
CH.OH CH:0OH CHLOH CH.OH CH: CH; 


It is known that when sugars, such as glucose, levulose and man- 
nose are acted upon by alkalies, there are produced a great many 
products, some of which are formic, carbonic, oxalic, lactic, pyruvic 
tartronic, malic, malonic, tartaric, ribonic, saccharic, and gluconic 
acids in addition to many other either more or less complex com- 
pounds. We can readily conceive that the Azotobacter brings about 
a somewhat similar reaction, the stages, however, being more nicely 
governed, because of enzymes. Many of the products would be 
oxidized to carbon dioxid and water with the liberation of energy 
necessary for the endothermic nitrogen reaction; others readily 
react with the resulting nitrogen compounds. We are completely in 
the dark as to what this first nitrogen compound is, but we know that 
the Azotobacter possess the power of changing nitrates or nitrites 
under appropriate conditions into ammonia. Up to date it has been 
impossible to detect nitrate formation; it is not impossible that 
nitrates are formed and utilized by intracellular enzymes. By using 
nitrates, nitrites or ammonia, we can offer a rough explanation of 
protein anabolism. 

The endothermic reaction, 

2N-+2H:0 = NH:NO:, 
may take place and the ammonia thus formed may react with the 
decomposition products of the sugars, pyruvic acid for instance, 


with the formation of alanin which Lipman considered as one of 
the first products: 


CH;—CO—COOH + NH;s = CH;s—CHN—COOH + H:0 
CH;—CHN—COOH + Hz = CH;—CHNH:—COOH 


or with glyoxylic acid forming glycocoll: 


HCO—COOH + NH: 
CHINHCOOH? He 


HCNH—COOH + H:0 
CH»NH:»—COOH 


“il il 


By similar reactions other amino-acids may be formed. More- 
over, Windas and Knoop have shown that methylimadazol may be 


270 AZOFICATION 


produced from glucose and ammonia, presumably through the 
formation of pyruvic aldehyd and formaldehyd: 


ae pay wee 

rt + 2NH; + HCHO = HC | + 3H:20 
|| 

CHO N—C— Ei 


which is nearly related to the amino-acid, histidin: 


FE ENE Se H—N—C—CH:—CHN H:2COOH 
| | 

+ CHNH»COOH —~H—C | 
| 


| 
| {| 
N—C—H N—C 


| 
| 
| 
=i 


The various amino-acids may, through the intervention of pro- 
teinases, condense with the formation of dipeptids, thus: 


CH;—CHNH2COOH + CH:CHNH»COOH = 
CH;CHNH:»,CONHCHCH:COOH + H20 


By the continuation of this process and by condensing with phos- 
phorus and sulphur-bearing compounds, probably through the 
intervention of other enzymes, there may result the complex protein 
of the Azotobacter cell. | 

Pigment Production by Azotobacter.— Most species of Azotobacter 
produce pigments. These vary in color from brown to black of the 
A. chrodcoccum to a yellow or orange of the A. vinelandu. The 
pigmented film usually develops on the culture media in from three 
to seven days. It is formed by A. chrodcoccum earlier and in more 
abundance where old brownish cultures are used as the inoculating 
material. The pigment is produced and retained within the bacterial 
cell; it occurs in neither the capsule nor the medium. The pigment 
produced by A. chrodcoccum is most pronounced when a dextrin 
agar medium to which calcium carbonate is added is kept at a tem- 
perature of 30° C. under well aérated conditions. According to 
Jones, it is produced only when there is a lack of suitable available 
nutrient material and when organisms in the pigmented area have 
ceased to multiply. The color of the pigment is intensified if 
nitrates are added to the medium in which the organism is growing. 
The non-pigmented strains apparently fix nitrogen just as readily 
as do those which have not lost the power of forming pigments. 

The pigment from Azotobacter chrcdcoccum is insoluble in water, 
alcohol, ether, chloroform, benzol, and carbon bisulphid. It dis- 
solves in alkalies, undergoing decomposition with the formation of 
a dark brown solution. Sackett maintains that the peculiar brown- 
ish color which is characteristic of certain “ nitre spots” of some soils 
is due to the pigment produced by Azotobacter. Such soils are high 


MORPHOLOGY OF THE NITROGEN-FIXING ORGANISMS 271 


in nitrates and alkalies which would dissolve the pigments from the 
body of the organism. But Omelianski and Sswewrowa are of the 
opinion that althought in some cases the dark color of vegetable 
soil may be due in a measure to the action of these microdrganisms, 
it would be a mistake to attribute it to this factor alone. Further- 
more, it has recently been proved that the brown color of the 
“nitre spots’ is due to solvent and decomposing action of the 
nitrates on the colored organic compounds of the soil, for they may 
be produced at will in a rich greenhouse soil with an excess of sodium 
nitrate, and this too in soils which have been rendered sterile with a 
saturated solution of mercuric chlorid. 

Morphology of the Nitrogen-fixing Organisms.—Of the many 
different bacteria which have been isolated and proved to have the 
ability to assimilate free nitrogen, Clostridium pasteurianum may be 
taken as a type of the anaérobic and Azotobacter chrodcoccum as a 
type of the aérobiec. 

Clostridium pasteurianum is a short thick rod from 1.2 to 1.3 
in diameter and 1.5 to 2u long in the young cells; the older spore- 
bearing cells take on a spindle shape. The bacteria stain a violet 
brown with iodin. The spores when ripe are 1.6u long and 1.3y 
broad and often lie in a roughly triangular covering. The ripe 
spore escapes through the wall of the mother in a longitudial 
direction. ‘Their germination is polar. 

Azotobacter chrodcoceum occurs ordinarily as diplococci or short 
rounded rods 1 to 2u thick and 1.5 to 3u long, and according to 
Prazmowski the microérganism first presents itself in its vegetative 
stage as a bacterium, in the fruiting stage as a micrococcus, and 
possesses a nucleus which functions in the same way as that of higher 
animals. In the resting stage the nucleus assumes a globular form, 
having a strongly refractive nucleolus with clearly differentiated 
boundary layers. The individuality of the nucleus appears to be 
practically lost at times, because of its relation to the cytoplasm. 
The division of the nucleus marks the first stage of cell division. 
According to Bonazzi the organism shows peculiar granulations 
apparently not related to reproduction. These take the basic dyes 
and are neither fats, glycogen, starch nor chromatin, but appear to 
be of metachromatic nature and seem to have their genesis in the 
nucleus. Their disposition in the cells is not constant but changes 
in different individuals. Involution forms occur and cell division 
is preceded by a simple form of mitosis. Some, but not all, varieties 
have been observed to form spores. The volutin bodies within the 
organism increase in number and size when the organisms are grown 
on media rich in nitrates. Hills suggests that they may have some 
relation to nitrogen-fixation, but his results appear to oppose this 
view; whereas the addition of nitrates to a medium greatly increased 
the reproduction, it very materially decreased the physiological 


O72 - AZOFICATION 


efficiency of the organism. It seems, therefore, more likely that - 
they are reserve protein material. * 

Lohnis and Smith have recently observed that Azotobacter, in 
common with many other bacteria, pass through a life cycle which 
is not less complicated than those of other microérganisms. Under 
certain conditions they pass over into an amorphous or “symplastic’’ 
stage, appearing under the microscope either as an unstainable or a 
readily stainable mass without any easily distinguishable organiza- 
tion, which, if not discarded as dead, later gives rise to new regenera- 
tiveforms. They multiple not only by fission, but by the formation 
of gonidia. 

Methods.— Clostridium pasteurranum grows readily in a vacuum on 
carrots. ‘The organism also grows on sliced potatoes, but ordinarily 
is grown in an aqueous solution containing 1 gm. K;PQO,, 0.5 gm. 
MgsSO,, 0.1 to 0.02 gm. NaCl, FeSO, and MnSO,, and 1.0 gm. 
CaCOs;, and 10 to 15 gms. of a suitable carbohydrate in 1 liter of 
water. One method used by Winogradsky in isolating B. Clostridiwm 
pasteurianum was to add garden soil to a non-nitrogenous solution 
and to allow a stream of nitrogen gas to pass through the solutions, 
after which several successive transfers were made into similar 
media. The final culture, after B. Clostridium pasteurtanum had 
formed spores, Was heated to 80° C. 

The organism ferments certain carbohydrates with the formation 
of butyric acid, acetic acid, carbon dioxid, and water. When grown 
in nutritive solution devoid of combined nitrogen, it assimilates 
atmospheric nitrogen. Although in pure cultures it is an anaérobe, 
in impure cultures it may fix nitrogen under aérobic conditions. In 
nature it occurs In connection with two other bacteria which do not 
possess the power of fixing nitrogen, and their nitrogen requirements 
are small. When in conjunction with these organisms, Clostridiwm 
pasteurianum has the ability of growing in the upper layers or soil 
and of assimilating free nitrogen. 

Azotobacter chroécocceum grows readily on solid or liquid media, 
one of the best being: 


Per Cent. 
Monopotassium eh he aa to peu uees by 
Sodium hydroxid 5 . F FeO 
Marnesium:sulphate.- % hv 5 a ee ee OO 
Sodnugm) chlorid)“4.05.. aL. oo ewe 1 per ee pie a ee O02 
Calcium sulphate . . 0.01 


Ferric chlorid (1 per cent. solution); 2 drops per 100 C.c, mannite 1.00 


The organism is readily isolated by seeding this medium with 
soil. After the characteristic membrane forms, it is transferred by 
dilution to a similar medium containing agar in which the charac- 
teristic brownish black colonies form readily. 

On mannite agar the colonies first appear as milk-white glistening 
drops, round and convex, which under a low magnification show 


METHODS 273 


a coarsely granular structure extending to the margin. The colonies 
rapidly increase in size, and after a week or more become brown at 
the center with concentric rings alternating dark and white to the 
circumference and darker streaks radiating from the center outward. 
In old cultures, where the agar has partly dried up, the cells are often 
united in sarcina-like packets; the cell walls are much swollen and 
the contents are aggregated to a small ball at the center. At the 
same time giant cells, both round and elongated and filled with oil 
drops, can be seen. Often a number of involution forms are seen, 
drawn out with long threads and false septa. By successive 
dilutions and transfers, it may be obtained in pure culture, although 
at times considerable difficulty is experienced in freeing it from a 
small organism— B. radiobacter. 

Several different methods have been used for studying its nitro- 
gen-fixing powers: 

(a) Seeding into 100 c.c. of the medium given above and after a 
certain time determining the nitrogen. 

(b) The use of the same medium, but the addition of sufficient 
sand for the formation of sand slopes on which the organism can 
grow. 

(c) The addition of a definite quantity of a carbohydrate to a 
soil and the incubation of this. 

Each of these methods has its value. The first is much more 
readily handled in the final Kjeldahl determination, but the others 
give much higher results. 

Freudenreich found that when Azotobacter are grown upon gyp- 
sum, the gain in nitrogen is considerably in excess of that assimilated 
in the liquid media. Srainsky found Azotobacter to utilize from 100 
to 200 gm. of sugar in the assimilation of 1 gm. of nitrogen when 
grown in solution, but when grown on sand it required only 11 to 
30 gm. for the same fixation. Many other workers have noted 
similar variation when grown in the soil. Where the organisms 
have been grown on gypsum or soil, we may attribute the stimula- 
tion to certain soluble constituents, yet this explanation scarcely 
seems plausible when considered in relation to sand cultures. Three 
strains of Azotobacter were grown in Ashby’s mannite solution 
and sand (nearly pure silicon dioxid) to which Ashby’s solution was 
added, with the following results: 


Nitrogen fixedin Nitrogen fixed 


Ashby’s solution, in sand, 
mgm. mgm. 
PAOLO DOAGCLERCA SS ot tr IPO ME FD a Rg AL Form 6.86 22.61 
PA OLODUCLEN sxe x a: Sobre eae te eee ate eee hee acd 5.00 12.60 
VA ZOLODACLCTZ Co ON setae elt rane morte Bae Tod As 6.44 16.80 


Moreover, arsenic is very toxic in the solution, whereas when 
added to the soil or to pure quartz, in small quantities, it stimulates. 
Although the total quantities of nitrogen fixed under the two 

18 


274 AZOFICATION 


methods differ greatly, the relative efficiency of the organisms is 
about the same in both cases. In testing soils similar results are 
obtained, as may be seen from the following results, which are the 
average for several hundred determinations made on different soils 
by the two methods. 


Nitrogen fixed in: 
100 c.c. of Ashby’s 


_— 


100 gm. of soil containing 1.5 gm. 
Depth of Sample. 1.5 gm. of mannite, of mannite 
mgm. mgm. 
ITS SOOD elas a to ne ce ye Tat, ee en Se 2 1A 
Second footwec (oko ee rie ee ae aD 0.77 
SD hard TOOb Mw c-0 et Ae crf See ae ane elec 0.58 


Although the greater aération in the sand and soil culture probably 
play a great part, there is little doubt that the colloids also assist. 

Relation of Azotobacter to other Organisms.—In the early study of 
nitrogen-fixation, the view was held that alge growing on or near 
the surface of soil are able to fix nitrogen. Frank in 1888 had 
observed such a growth on sand exposed to light and found that the 
soil showed a considerable increase in nitrogen. In 1892 Schlésing 
and Laurent proved, both by determining the nitrogen fixed by a 
soil in a closed vessel and by observing the diminution of the nitrogen 
gas in the enclosed air, that a soil exposed to light gains in nitrogen 
if algee are allowed to grow on the surface, and that the gain is 
confined to the upper few millimeters. They did not, however, 
employ a pure soil or pure cultures of algee. Kossowitsch, working 
with pure cultures of two green alge, found no fixation, but observed 
a considerable increase of soil nitrogen when they were grown with 
soil bacteria. Later, Kruger and Schneidewind, employing pure 
cultures of many other chlorophycez, obtained no nitrogen-fixation, 
Hellriegel and Dehérain had found a large increase in the nitrogen 
content of sand in pots when exposed to the light, which was always 
accompanied by a development of alge. In the light of such results, 
the conclusion has been reached that alge alone cannot assimilate 
free nitrogen, but only in concurrence with soil bacteria, the former 
producing carbohydrates which are used by the latter as a source 
of energy for the nitrogen-fixation. Heinze actually observed rapid 
fixation of nitrogen when cultures of algze were inoculated with Azoto- 
bacter or other nitrogen-fixing organisms. Stoklasa found that Azoto- 
bacter are especially abundant in soils having a vigorous growth of 
blue-green algee. Azotobacter are often absent from virgin soils, 
but are always found in such soils when there is a vigorous growth 
of alge. Bottomley claims that both Azotobacter and Pseudomonas 
live in true symbiosis with cycas. It, therefore, appears certain 
that the nitrogen-fixing powers of Azotobacter are greatly enhanced 
when growing with alge, but the exact réle played by each is yet to 
be explained. This offers a rich and inviting field for research. 

Nor is it alone in combination with alge that these organisms 


AZOTOBACTER RELATION TO OTHER ORGANISMS 275 


may grow and thus be benefited. Beijerinck and van Delden early 
recognized that an apparent symbiosis exists between Azotobacter 
and other bacteria, and that the nitrogen fixed is considerably 
greater in the mixed than in the pure cultures. This symbiosis, | 
though in many cases beneficial to Azotobacter, is not essential for 
nitrogen-fixation. Radiobacter, with which the Azotobacter are 
usually associated, have only slight nitrogen-fixing powers, yet they 
increase the nitrogen-fixing powers of Azotobacter. The carbo- 
hydrates disappear more rapidly from mixed than from pure cultures 
and with a greater fixation per gram of carbohydrate utilized. 
There is also a greater fixation when two strains of Azotobacter are 
grown in conjunction with each other. This is especially marked in 
an aqueous solution of mannite. Results have been reported where 
Azotobacter fixed twice as much in the presence of Psewdomonas as 
when grown alone. 

The manner in which this mutual benefit is exerted is not clear. 
In some cases it may be due to the associated organism rendering 
more available the carbonaceous material. 

Omelianski and Salunskov offer the following explanation con- 
cerning the association of aérobic and anaérobic nitrogen-fixers: 

“The synergetic activity of nitrogen-fixing and accompanying 
microbes, is both in laboratory experiments and under natural 
conditions (cultivable stratum of the soil) of a different character 
according to the properties of the species taking part in the process 
and their environment; in both cases the function of the satellite 
organism seems to consist in fixing the oxygen of the air and creating 
the anaérobic environment for Clostridium pasteurranum. The 
species added to the cultures of nitrogen-fixing microbes sometimes 
supply the compounds of carbon needed for the process of fixing 
nitrogen as energetic substance. In the case of the combination: 
Azotobacter and Clostridium pasteurranum, the function of the former 
is not confined to fixing the oxygen of the air only, and consequently 
to creating an anaérobic environment for the Clostridium. But this 
combination is also useful inasmuch as it destroys the injurious 
products of disassimilation created by the second (chiefly butyric 
acid) and maintains the action of the environment. (Azotobacter 
is alkaligenic and the Clostridium acidogenic.) 

“The satellite species may also unfavorably affect the nitrogen- 
fixing microbe, either through products of assimilation or by con- 
sumption of the carbon compounds needed by this microbe for 
nitrogen-fixing. The energetic fixation of oxygen by the satellite 
aérobic species creates conditions favorable to the development of 
Clostridium pasteurianum, but at the same time hinders the growth 
of the Azotobacter, which is necessarily aérobic. 

“The form endowed with the maximum vitality and at the same 
time the most common form in which combination of the nitrogen- 


276 AZOFICATION 


fixing organisms takes place in the upper soil strata is that of sym- 
biosis between the aérobic and anaérobic nitrogen fixers, principally 
between Azotobacter and Clostridium pasteurianum. In spite of the 
opposite properties of the two species, their synergetic activity in 
the upper strata of the soil results in a harmonious mutual develop- 
‘ment producing the maximum economy in consumption of energetic 
substances.” 

So far, little has been done to determine the relationship of 
Azotobacter to the higher plants, but it is interesting to note that 
Beijerinck has observed a distinct relationship between the distribu- 
tion of the organism and leguminous plants. Fischer suggests that 
some nitrogen-fixing bacteria presumably exist first as saprophytes, 
then as exoparasites in loose combination with green plants, then as 
endoparasites. Finally they develop the true symbiosis or root 
nodule bacilli. Hopkins has questioned whether there may not be a 
relationship between the legume bacteria and Azotobacter. 

The Influence of Water.— Azotobacter are very resistant to drying; 
they may be dried for a considerable time in a desiccator over sul- 
phuric acid. Pure cultures are just as resistant to drying as are mixed 
cultures. This would vary some with. the media in which the 
bacteria are dried, for the survival of non-sporebearing bacteria in 
air-dry soil is due, in part, to the retention by the soil of moisture 
in the hygroscopic form. ‘This, however, is not the only factor, for 
the longevity of bacteria in a solid is not directly proportional to its 
grain size and hygroscopic moisture. Guiltner and Langworth found 
that bacteria resisted desiccation longer in a rich clay loam than in 
sand. Furthermore, if bacteria are suspended in the extract from 
a rich clay loam before being subjected to desiccation in sand, they 
live longer than if subjected to dessiccation after suspension in a 
physiological salt solution. Because of this, they consider that soils 
contain substances which have a protective influence upon bacteria, 

subject to desiccation. 

- Lipman and Burgess have found that many soils manifest a vigor- 
ous nitrogen-fixing power even after being air-dried and kept in 
stoppered museum bottles for periods varying from five to twenty 
years. In some cases the fixation was equally as high as in freshly- 
collected samples. ‘The organisms from such soils are more easily 
attenuated than are other organisms which have not been so dried. 
The tendency is for soils gradually to decline in nitrogen-fixing 
power or drying. This may manifest itself as early as the second 
week. 

During the periods of drying, the organisms are inactive, as they 
require moisture for growth and reproduction. For maximum 
nitrogen-fixation a definite moisture content is needed, Warmbold 
found the optimum moisture content to be 20 per cent, When it was 
below 10 per cent. there was no nitrogen fixed, and in some cases 
there was a decided loss of nitrogen. Krainsky allowed soil with 


THE tNFLUENCE OF WATER 977 


varying moisture content to stand for some time and then inoculated 
it into mannite solutions and obtained maximum fixation in the soils 
containing fairly small quantities of water. Later, however, he 
decided that soil should be damp—but not wet—and well aérated 
for maximum nitrogen-fixation. ‘The water requirements vary with 
different soils. As a general rule, the higher the humus content of 
the soil, the more water will be required for optimum nitrogen- 
fixation. The quantity of water present may, however, become so 
great that it may kill all Azotobacter in addition to stopping nitrogen- 
fixation. 

An insufficient supply of moisture checks both nitrification and 
nitrogen-fixation. ‘This occurs in some soils when the water content 
has been reduced to 16.5 per cent. This again varies with the soil, 
for Schlésing found bacterial activity less in fine-grained soils than 
in lighter, coarse-grained soils. A difference in moisture content 
of 1 per cent. according to Dafert and Bollinger, is sufficient to pro- 
duce a marked change in the oxidation going on in the soil. 

The moisture requirement of the nitrogen-fixing bacteria, accord- 
ing to Lipman and Sharp, is more nearly that of the ammonifying 
than of nitrifying organisms. Ina sandy loam it was found to vary 
between 20 and 24 per cent. the anaérobic nitrogen-fixers are most 
active, but the action of the aérobes is slightly depressed. Thus, 
in many soils two maxima of nitrogen-fixation occur, depending upon 
whether the conditions are favorable for the anaérobic or aérobic 
organisms. 

Traaen’s results differ from Lipman’s in showing only the one 
maximum, as 1s seen from the following, which gives the milligrams 
of nitrogen fixed in 100 gm. of soil. 


5 percent. 10 percent. 17.5 percent. 25 percent. 30 per cent. 
H:0. H:0 O. 


Temperature. 20. 2! 20. 20. 
TLIO a is on eee ee Oey 15 11.2 13.4 5.4 
PAA Ce C : 1.9 alee!) 13.2 16.6 15.5 


He used a loam soil with a maximum water capacity of 27.4 per 
cent. It is quite evident from his statement that anaérobic organ- 
isms played a prominent part in the fixation at the higher moisture 
contents. 

Since the carbohydrates disappeared much more rapidly in the 
soils containing the greater quantities of water, it is quite possible 
that greater quantities of nitrogen per gram of carbohydrate con- 
sumed are fixed where the smaller quantities of water are applied. 
This, together with the different methods used by the several 
investigators, would explain the apparent discrepancy in their 
results. 

Ina series of pot experiments in which a calcareous loam receiving 
various amounts of water was used, the author found the moisture 
content for maximum nitrogen-fixation to lie between 15 and 22 per 
cent. ‘These results also bring out the two maxima which were first 


08 (Coa meal?) 


OL 


O€ On OS 


O7 


278 AZOFICATION 


noted by Lipman. ‘These soils were kept at the various moisture 
contents for four months. All were then incubated at 28° C. for 
twenty-one days with a moisture content of 20 per cent. 


Treatment. Nitrogen fixed. 
Per cent. Per cent. 
12.5 100 
15.0 108 
17.5 102 
20.5 104 
22.5 108 


In this soil the optimum for the aérobes would appear to be at 
17.5 per cent. and that for the anaérobes 22.5 per cent. or higher. 


O 10 20 JO 40 JO 60 70 80 


Fic. 34.—Average percentages of ammonia ————— and nitric nitrogen +-+-+-+ 
formed and nitrogen fixed — —— -— in soil receiving varying quantities of water. On 
the ordinate is given the per cent. increase of the respective substances and on the 
abscissa the quantity of water applied as per cent. of water-holding capacity. 


/00 


TEMPERATURE 279 


When too large a quantity of water is applied there is a tendency 
to depress the total nitrogen fixed, as is illustrated by the following 
results in which various quantities of water were applied to a soil 
throughout the year under field conditions: 


Inches of Nitrogen fixed 
water applied in 100 grams 

during summer. soil. 
mgm. 

37.5 ee ek ee eee a AT fodkial te i 
PAT LUE AS ane ee ea) Oe eh ee AS ee ane a le rs Peis i 
15.0 PM eh ed ee te reer 8.5 
None . ih 


The maximum for anaérobic conditions does not appear in these 
results probably because the soil did not become filled with water 
and because under field conditions the water rapidly drains away 
or is evaporated. There would seem to be a correlation between 
the water content of a soil as measured in terms of its water-holding 
capacity irrespective of physical composition and its nitrogen-fixing 
powers. This is brought out in Fig. 34 in which water requirements 
for ammonification, nitrification, and nitrogen-fixation are compared. 

Temperature. — Berthelot early recognized that the biological gain 
of nitrogen in soils is dependent upon a suitable temperature. 
He found nitrogen-fixation to occur best at summer temperatures 
between 50° and 104 F°. The process was immediately stopped on 
heating to 230° F. Later Thiele maintained that although Azoto- 
bacter possess the ability to fix small quantities of nitrogen under 
laboratory conditions, the temperature would be unfavorable under 
field conditions. Heinze, however, found that although the nitro- 
gen-assimilating organisms are most active at a temperature between 
20° C. and 30° C., they nevertheless fix appreciable quantities at 
temperatures as low as 8 to 10°C. Still more recent work has shown 
the optimum temperature to be 28° C. and the limits of activity of 
Azotobacter chrodcoccum to lie between 9° C. and 33°C. The actual 
quantitative variation in nitrogen fixed is seen from the results 
reported by Lohnis. He inoculated 100 c.c. of a 1 per cent. mannite 
soil extract with 10 gm. of soil and obtained the following fixation 
at the various temperatures: 


Nitrogen. 
Mgm. 


EPIL BO ne cna ie Sle RN Coc at IM A De 3.15 

DO one oC ae ahent yeaa RL ORY S Dre x sal yrot ise. 1 a BD 

30° to 32°C. 4.27 
Better fixation at a lower temperature is noted when the soil is 
incubated and the gain in nitrogen determined directly. Koch 


obtained fixations of 3 mgm., 11 mgm., and 15.5 mgm. of nitrogen 
in 100 gm. of soil when incubated with a carbohydrate at 7° C., 


280 AZOFICATION 


15° C., and 24° C., respectively.. Traaen, using a loam soil with a 
maximum water-holding capacity of 27.4 per cent., obtained nearly 
as great a fixation at 13° C. as at 25° C. when the optimum moisture 
content was maintained. This is seen from the following: 


Nitrogen fixed in 100 gm. of soil. 


Temperature. rina 
5percent. 10percent. 17.5 percent. 25 per cent. 30 per cent. 
H:0. HO. H20. BS 20. 
Mgm. Mgm. Mgm. Megm.: Mem. 
La Ce ac as rae DOM 1.9 11:2 13.4 5.4 
DOO ee Meee hm LEG 1.9 AS 2 16.6 15.5 


A temperature, favorable even though not ideal for nitrogen- 
fixation, would occur in soils under natural conditions. The 
temperature of soil in Utah during the months if September averaged 
14° C., with a minimum of 10° C. and a maximum of 17°C. During 
June, July and August the mean temperatures would be much 
higher. 

The mean daily temperatures of the soil for Bismarck, North 
Dakota; Key West, Florida; and New Brunswick, New Jersey; for 
the months of June, July, August and September were 18° C., 28° C., 
and 24.5° C., respectively. From this it is evident that during a 
considerable period of each year an arable soil has a temperature high 
enough for moderately rapid nitrogen-fixation. 

Although it is generally maintained that there is no nitrogen- 
fixation in soils during the winter months, cold or even freezing does 
not injure the organism; for the cooling of a soil, even to the freezing 
point, increases its nitrogen-fixing powers. This is probably due to 
the suppression of competing species and to the establishment of a 
new flora. ‘The same is true when the soil is heated, as may be seen 
from the results given below. 


Temperature Nitrogen fixed, 
deg. C. per cent. 
Normal i hal 
50 9.00 
55 14.14 
60 ' 16.38 
65 14.42 
70 13.02 
75 11.34 
80 12.66 
85 10.36 


This soil had been autoclaved and then inoculated with a soil 
extract which had been heated to the temperature indicated. The 
stimulation could not, therefore, have been due to the heat rendering 
more of the plant-food in the soil available. The results indicate 
that many of the organisms which take part in nitrogen-fixation 
are highly resistant to heat. It is significant that the greatest stimu- 
lation is exerted in a soil which had been inoculated with solutions 


SEASON 981 


heated just above the temperature which Cunningham and Lohnis 
found to be the thermal death-point of soil protozoa. 

Light and other Rays.—As a class, bacteria are sensitive to light, 
but the extent to which they can withstand it varies, among other 
things, with the conditions of exposure and the specific organism. 
Unfortunately, we have but fragmentary information concerning the 
effect of light upon azofiers, but what we do know would lead us to 
believe they are more resistant than many microérganisms—prob- 
ably more so than many other soil bacteria. Berthelot recognized 
that nitrogen-fixation in the soil occurred both in daylight and in 
darkness, though more freely in the light. Jones found many 
Azotobacter to be alive in a small Petri dish of dried soil that had 
stood in the laboratory in front of a south window for two years. 
They can withstand the direct action of the violet and of the longer 
ultraviolet rays for five minutes, but are killed in much less time by 
the shorter ultraviolet rays. They are more resistant even to these 
than are many other species. 

The fixation of elementary nitrogen by A. chroécoccwm is distinctly 
increased when the air is activated by pitchblende. Somewhat 
better results are obtained with weak than with stronger radio- 
active intensity. 

Aération.— Under field conditions there is a mixed flora consisting 
of the anaérobic ands aérobic nitrogen-fixing microérganisms. A 

soil condition which would be ideal for one species might be unfavor- 

able for the other. It has already been pointed out that there are 
two maxima of nitrogen-fixation in soils, depending upon the 
moisture content., This is illustrated in Figure 32. 

Although it is usually conceded that nitrogen-fixation is most 
rapid when soils are well aérated, this may not always be the case. 
Concerning this Murray reports the following results: 


Nitrogen fixed 


re nS 
hind of soil. Aérobic Anaérobic 
conditions. condition. 
mgm. mgm. 
Greenhoucessollers «eee ee hee to toe eee ee, OL S4 8.50 
NO AIMASOUS Leda pers mera Pe eta lar Ge eee lt OOS 5.29 
Clayscolltcs t.co ee ren pet gciat bomen rt Nex er yicn MOE A 4.69 


This condition must be attributed to a great difference in the 
physiological efficiency of the two groups found in these particular 
soils and not to a lack of aérobic nitrogen-fixing organisms, for more 
than ten times the number of organisms developed on nitrogen- 
poor media from these soils under aérobic as under anaérobic condi- 
tions. 

Season.— Berthelot was unable to show any gain in nitrogen of 
his soils during the winter, but Koch found a considerable increase 
during this season in soils which were kept in a heap and shovelled 


282 AZOFICATION 


over from time to time. Loéhnis observed that Azotobacter mem- 
branes are more readily obtained in winter than in summer. He 
later found that the nitrogen-fixing power of soil varies from month 
to month throughout the year, there being two maxima—one in 
spring and another in autumn. The extent of the variation noted 
may be seen from the following: 


1903=1904:7 ‘Marches Fie ee Oe, Laie eee ee LOE se eee ee OO 
bia Misi 3: pres, ese A Duk 22g SR ee Spee Re ae ene an ale nmr mene oat 

. Sully ie ee ee a ee yey Fg ae ane eee Ce ee ae 50 

<< September saw sic ea ote han ee Se ee eee OU 
TOO Ss Atpral iy a le ee ea En coe telecine eh ae Orme OLD 
C= ANT = UMC es pe oP ne Mca kee ee eee ds ee Li 
(SFO TL ATIP USE Heres Are ans Pcs UM ne oes a ne emer 
“~~ October-November : 122 


The relative numbers are based on the spring months as 100. 


Green found nitrogen-fixation in 1 per cent. mannite solution to be 
low during August, September and April. In other months he 
noted a fairly constant fixation of about 10 mgm. of nitrogen per 
gram of mannite. He also noted a marked yearly variation in the 
nitrogen fixed during July and August. 

Walton found nitrogen-fixation lowest in Indian soil between 
October and January and highest between June and September. 
This corresponds with moisture and temperature changes. Peterson 
has found that although the nitrogen-fixation of Utah soils is highest 
from June to September, the number of types of Azotobacter occur- 
ring in the soil was greatest in May. Moll goes so far as to maintain 
from his work that the season of the year is the principal factor in 
determining the biochemical transformation in soils. This would 
appear to be especially true as regards nitrogen-fixation. 

Crop.— Heinze called attention to the fact that the fallowing of the 
soil increased its nitrogen-fixing power. This could be due to better 
aération, moisture, temperature, etc., and not to any depressing 
influence exerted directly by the plant. Most experiments which 
deal with plant and bacterial activity could be interpreted in this 
light. Hiltner maintained that. the free nitrogen-fixing bacteria 
are stimulated in their activities by the growing plant roots. ‘There 
may be considerable truth in this, for here the higher plants are 
rapidly removing from the solution the soluble nitrogen compounds. 
In this case, the nitrogen-fixing organisms would be forced either to 
compete with the higher plant for the soil nitrogen or else to make 
use of their ability to live upon the atmospheric nitrogen. It is 
certain that different cultural methods vary sufficiently with crops 
to influence profoundly a soil’s nitrogen-assimilating properties, 
for the Azotobacter occur more widely distributed in cultivated than 
in virgin soil. The analyses of hundreds of samples of cultivated 
and virgin soils in Utah have in nearly every case shown the virgin 
soil to have a low nitrogen-fixing power as compared with the culti- 


CLIMATE 283 


vated soil. This was the case even where the soil was incubated 
without carbohydrates and the nitrogen determined directly. The 
average results for many determinations were as follows: 


Mem. of 
nitrogen fluid. 
Watroncctoll 5 bs Sa a Pe es lene ea el 6.99 
Cultivated se. wae IS aS RO Sr ee 14.28 
Wheat . er eRe Cn ee eres etn OSE ye! eS 
AUER, 0 FS Rts OP Rg A eae eet ed Se er es Mn cme en Ltr 
Huai lO Wann s A Pa ita Ee Oy hrs er ee oe 2eeOd 


The fallow soil had received considerable manure, hence these 
results are undoubtedly high. It would, however, be possible to 
fallow or crop soils so continuously that extremely small quantities 
of plant residues would be returned to the soil, under which condi- 
tions there might be a decrease in nitrogen-fixation. ‘The conditions 
of moisture and aération are much more nearly ideal in a fallow soil 
than in a cropped soil. It is just possible that the high fixation 
noted where wheat is grown continuously may be due to the method 
in vogue in the arid districts of leaving the greater part of the straw 
on the soil. This would act as readily assimilable carbonaceous 
material for the Azotobacter. Welbel and Winkler have found that 
fallowing not only increases the assimilable nitrogen but also the 
available phosphorus of the soil, a liberal supply of which causes the 
Azotobacter to utilize its energy more economically. That the 
increased nitrogen-fixation noted when soils are cultivated is not 
confined to the arid soil, is seen from the recent work of Reed 
and Williams. Brown’s work indicates that crop rotation increases 
the nitrogen-fixing powers of a soil. 

Climate.—It has been maintained for a long time that there is a 
close correlation between the chemical, physical, and biological! 
transformations going on in a soil and the climatic conditions, but 
there was nothing definite on this subject until the highly interesting 
work of Lipman and Waynick appeared. They found a definite 
relationship between climate and the nitrogen-fixing powers of a soil. 
Removal of California soil to Kansas increased the vigor of the 
Azotobacter flora and especially that of A. chroécoccum. It increased 
the nitrogen-fixation by 50 per cent. over that attained by the same 
soil in California. Similar results were obtained in California soils 
removed from Maryland. Kansas soil taken to California lost its 
power to produce a membrane in mannite solution, the Azotobacter 
flora became rather feeble, and the nitrogen-fixing powers of the soil 
were greatly reduced. The removal of the Kansas soil to Maryland 
increased the vigor of the Azotobacter and induced a higher fixation 
of nitrogen. The Maryland soil in California diminishes in nitrogen- 
fixing powers, but not in so great a degree as does the Kansas soil. 
This also happened when the Maryland soil was taken to Kansas. 


984 AZOFICATION 


The bacterial flora of a soil, therefore, is dependent upon climatic 
conditions which affect many of the other properties of a soil. 

Relationship of Azotobacter to Nitrate Accumulations.—The fact 
that certain spots in western cultivated soils were very rich in 
nitrates was first observed by Hilgard This he attributed to the 
rapid nitrification of the organic matter of the soil in the warm arid 
climate of the West when ethe moisture limit was removed by 
irrigation. 

A number of years later Headden noted these “nitre spots” in a 
number of Colorado soils, but he attributed it to the fixation of 
atmospheric nitrogen by the non-symbiotic bacteria which find in 
the western soils ideal conditions for growth and rapid nitrogen 
fixation. This conception has been further amplified by Headden 
and also Sackett. In the early work by Headden it is assumed 
that the Azotobacter not only fix the nitrogen but also produce the 
nitrates. Itis known, however, that these organisms do not produce 
nitrates. 

Moreover, there are a number of other vital objections to this 
theory. (1) Lipman has shown that for the fixation of the quantity 
of nitrogen which Headden maintains to have occurred, it would 
require from 1000 to 2000 tons of carbohydrates. ‘There is no such 
visible supply of energy in these soils. ‘True, many of these soils 
have a rich alge flora, but it has not been proved that this will 
furnish a sufficient supply of available energy. (2) The average 
amount of nitrogen fixed in thirty-two samples collected in the 
nitrate region was 7.4 mgms. and the average nitrogen fixed in 
thirty-one samples of dry-farm alkali-free soil in Utah was 12.2 
mgms. Yet there is no accumulation of nitrates in these latter 
soils. (3) The quantity of soluble salts occurring is often sufficient 
to stop the activity of all nitrogen-fixing organisms, if not to kill 
them. (4) The quantity of nitric nitrogen and of chlorin in any 
given “nitre spot” varies in the same spot from year to year or from 
period to period within a year. (5) The country rock adjacent to 
the nitrate accumulations and which has contributed to the soil 
formation contains abundance of nitrates to account for the accumu- 
lations noted. (6) Soils having a similar physical appearance may 
be produced in the laboratory in the absence of bacteria. Because 
of this, we must conclude that the accumulation of nitrates in spots 
in western soils have their origin as do other accumulations of 
soluble salts found in the soil and not in the fixation in place by 
bacterial activity. 

Soil Inoculation.— High hope was entertained that the nitrogen 
problem in agriculture had been solved, when Caron announced that 
he had prepared a culture of bacteria which would enable non- 
leguminous plants to utilize free atmospheric nitrogen, provided 
certain precautions were observed. Many of the results which he 


SOIL INOCULATION 285 


reported on pot experiments were clearly in favor of the inoculated 
plant. Stoklasa was one of the first to study in detail the commercial 
preparation “alinit” which was placed on the market as a result of 
Caron’s work. His findings were fully as favorable as Caron’s, 
but the work of others soon demonstrated that “alinit” neither in 
the laboratory nor in the field had the ability to fix nitrogen. When 
Beijerinck discovered the free-living aérobic nitrogen-fixers, the hope 
that soil inoculation may be so perfected that it would be beneficial 
to crops was revived, and since that time many investigators have 
attempted to inoculate soil in order to increase its crop-producing 
powers, but usually with negative results. Stoklasa has made 
great claims for soil inoculation. He found that soils, inoculated 
with Azotobacter chrodcoccum and adequately supplied with carbo- 
hydrates and lime, showed an increase in the number of nitrogen- 
fixing organisms, and also an increased yield both in quantity and 
quality of the crop. Stranak also obtained a pronounced increase 
in the production of beets, grain, and potatoes on inoculating with 
Azotobacter. 

There may be a decrease in the crop during the first year when 
carbohydrates and Azotobacter are added to the soil with a marked 
increase In crop during the second and third year. Even then, the 
soil may be left richer in nitrogen than it was at first. 

The effect of dextrose and sucrose on the productiveness and nitro- 
gen content of the soil is shown below: 


Crops obtained. Total 

La are ae || eeotal nitrogen Nitrogen 

Carbohydrate added per Oats, 1905. Sugar beets, 1906. | Mtrogen left in ., a8 
100 gms. of soil. remained soil nitrates, 
, in crop, | spring of | pts. per 

Dry |Yield of | Dry | Yieldof | &™- 1906, mil. 

matter.| nitrogen. | matter.) nitrogen. per cent. 

INGTe wayne e cen 10020) ee OOLOEs e100; 0) set 0050 0.9514 0.093 10 

2 per cent. dextrose .| 32.8 62.5 | 186.0 | 190.0 | 0.6814 0.105 17 

2 per cent. sucrose .| 33.3 58.7 | 179.0) 195.0 0.6800 0.105 15 

4 per cent. sucrose .| 37.7 78.1 | 283.0 | 339.0 1.0092 0.119 37 

| 


It is often the case that the addition of starch to a soil during the 
first year retards plant growth. This injurious action may be due 
to the augmented bacterial activity in the soil brought about by the 
carbohydrates which injure the roots of the plant by withdrawing 
oxygen and by forming hydrogen sulphid in the deoxygenated 
atmosphere of the soil through the reduction of sulphates by the 
bacteria. 

The effect produced by the carbohydrate applications also varies 
with the season. If applied to the soil in the spring when the soil 
temperature is low and when other bacteria are more active than 


286 AZOFICATION 


Azotobacter, the results are that they rapidly multiply and compete 
with the higher plants for the limited available plant-food. If, 
however, the carbohydrates are applied in the autumn directly 
after the removal of the crop, when the soil is warm, Azotobacter 
are active, with the result that sufficient nitrogen is fixed to produce 
an increased crop the following season. 

If the same quantity of carbohydrates per unit of nitrogen fixed 
be required by the organism under natural conditions, as are found 
necessary in laboratory experiments, enormous quantities would be 
required for the fixation of any considerable quantity of nitrogen; 
but it is possible that in the soil they are more economical with their 
energy or they may live in symbiosis with other organisms which 
furnish them part of their carbon. 

Many workers have noted either no effect or even a detrimental 
influence when soils are treated with the carbohydrates and then 
inoculated with Azotobacter. This may be due in a great measure to 
any or all of the following factors: (a) Absence of a suitable 
environment, as temperature, moisture, aération, food and alkalinity; 
(b) absence of a suitable host from which Azotobacter may obtain 
part of its carbon; (c) injurious effects due to the decomposition 
products of the carbohydrate added. 

There is considerable interest in the work of Bottomley who 
uses bacterized peat, or humogen. ‘The bacterizing process consists 
of three stages: (a) Treatment of peat with a culture solution of the 
special ‘““humating”’ bacteria and an incubation of it at constant 
temperature for a week or ten days, during which period soluble 
humates are formed; (b) destruction of the humating bacteria by 
sterilization with live steam; (c) treatment of this sterilized peat 
with mixed cultures of nitrogen-fixing organisms— Azotobacter 
chroécoccum and Bacillus radicicola—and an incubation at 20° C. for 
a few days, after which it is ready for use. 

Theoretically, there is much in this process which recommends it, 
for there is no abrupt change in environmental conditions for the 
organism added, as would be the case when added from laboratory 
culture. Moreover, they are added in enormous quantities and 
with a source of carbon which is not far different from that found in 
the soil. Russell, however, after carefully reviewing all of the 
experimental evidence on the subject, concludes: “There is no 
evidence that humogen possesses any special agricultural value. 
There is not the least indication that it is fifty times as effective as 
farmyard manure, to quote an often repeated statement, and there 
is nothing to show that it is any better than any other organic 
manure with the same nitrogen content.’ Furthermore, he con- 
cludes that there is no definite evidence that “bacterization” really 
adds to the value of peat. 

The conclusion is evident that soil inoculation, in order to be 


SOLL INOCULATION 287 


successful, must be accompanied by the rendering of the physical 
and chemical properties of the soil ideal for the growth of the specific 
organisms to be added. A few organisms placed in a new environ- 
ment already containing millions can never hope to gain the ascend- 
ency over the organisms naturally occurring in the soil, for they have 
been struggling for countless generations to adapt themselves to the 
environment and only those which are fitted have survived. The 
problem becomes even more complicated when we recall the findings 
of Lipman that the bacterial flora of a soil is in many cases entirely 
changed by climatic conditions. On this account, it would appear 
that ever to make soil inoculation a success the chemical, physical, 
and even the biological condition must be made suitable for the 
growth of the specific organism added. Furthermore, strains of the 
organism must be used which have been evolved under similar 
climatic conditions. 

Soil Gains in Nitrogen.—It is well established that many forms of 
microscopic organisms possess the power of fixing nitrogen either 
when grown alone or in combination with other organisms of the 
soil. Many of these have been obtained in pure culture and their 
morphology and physiology carefully studied. The most favorable 
conditions for their maximum nitrogen-fixation in pure cultures in 
liquid solutions have been accurately determined. Some of the 
conditions requisite for their activity in soils are known, but on this 
phase of the subject there are many gaps in our knowledge and much 
work must yet be done before we can state definitely the part which 
they play in the economy of nature and before we can say which 
are the very bestmethods for increasing their usefulness. Never- 
theless, it is interesting to consider the results obtained by a see 
workers. 

Berthelot’s early laboratory experiments led him to believe that 
sands and clays may fix in a year from 75 to 100 pounds of nitrogen 
to the acre. In two exceptional instances he noted that nitrogen 
was fixed by sands at the rate of 525 pounds and 980 pounds amacre, 
but soils which contained fairly large quantities of nitrogen never 
made markedly rapid gains. 

Thiele, on the other hand, maintained that while there is no doubt 
that Azotobacter possessed the power of fixing free nitrogen, under 
laboratory conditions, yet it is not certain that conditions would be 
such in soils for any gain of nitrogen due to the activity of these 
organisms. We have already seen, however, that the Azotobacter 
do not require as high a temperature for nitrogen-fixation in soil as 
he thought necessary. It is also certain that in most arable soil the 
temperature is sufficient during a large part of the year for a fairly 
rapid nitrogen-fixation by bacteria. 

Krainsky thinks that even better results should be obtained in 
soils than in pure culture, for there the nitrogen-fixers grow in sym- 
biosis with autotrophic organisms which make organic compounds 


288 AZOFICATION 


available to the Azotobacter. In soils the nitrogen fixed is rapidly 
removed by other plants, because of which the slowing-up process 
that becomes perceptible so early in laboratory experiments should 
not occur. 

In addition to an optimum temperature and moisture content of 
the soil, the Azotobacter are dependent upon a supply of carbon sfor 
energy and inorganic nutrients for the building of cell protoplasm. 
Unfortunately, it is too often the case that under natural conditions 
those soils which are deficient in nitrogen are also lacking in available 
carbon, and especially in phosphorus, which are so essential for rapid 
nitrogen-fixation. Then there are the technical difficulties which 
the chemist encounters in determining the gain or loss of nitrogen 
which occurs in soils under natural conditions and which may be 
attributed to non-symbiotic nitrogen-fixation. 

There are, however, several cases in which the gain has been 
measured with a fair degree of accuracy. 

Lipman, in pot experiments carried on with a soil containing about 
5000 pounds of nitrogen per acre-foot of soil, found a gain of more 
than one-third this amount in two short seasons. Much of this 
must be attributed to non-symbiotic nitrogen-fixation. To these 
soils had been applied solid and liquid manure, which furnished to 
the organisms readily-available supplies of energy and vacious 
necessary inorganic constituents. This fixation was not nearly so 
rapid where legumes were turned under as green manures. 

Koch found a gain of from 0.019 to 0.093 per cent. in soil nitrogen 
during two seasons which must be attributed to non-symbiotic 
nitrogen-fixation. In addition to this there was a threefold gain in 
the nitrogen content of the crops—oats, buckwheat, and sugar-beets 
—which must also be attributed to the action of Azotobacter. 

Hall noted an annual gain of 100 pounds of nitrogen on Broadbalk 
field at Rothamsted and 25 pounds on Grescroft field. He feels that 
much of this gain must be due to the action of non-symbiotic 
bacteria. Lipman points out that the actual gains of nitrogen are 
even greater, for this does not take into consideration the various 
losses which are sure to occur even under the best of conditions. 
Hopkins takes the stand that the apparent gain is due in a large 
measure to drifting dust and plant residues coupled with the diffi- 
culty of obtaining representative samples of soil at the two different 
periods. Even when all of these factors are considered the evidence 
points to a gain of nitrogen through bacterial activity. 

The analysis of a great number of soils in Utah showed that the 
average nitrogen content of the soil which had grown wheat and 
other non-leguminous plants for from twenty to fifty years was 
0.2009 per cent., whereas adjoining virgin soil on the average showed -° 
only 0.1984 per cent. of total nitrogen. The evidence is very strong 
that considerable nitrogen has been added to these soils by micro- 
scopic organisms, for: 


SOIL INOCULATION 289 


(a) In nearly every case the cultivated soil fixed much more 
nitrogen in the laboratory than did the virgin soil. This was the 
case when the soil was incubated with or without the addition of 
carbonaceous material. 

(b) There is a richer nitrogen-fixing bacterial flora in the cultivated 
than in the virgin soil. 

(c) The conditions of moisture, alkalinity and food constituents 
in the soil were ideal for rapid nitrogen-fixation, and the temperature 
of the soil was high enough during a considerable part of the year 
for the growth of Azotobacter. 

(d) The cultivation of the soil would increase aération and avail- 
able phosphorus in the soil. 

(e) The large quantity of plant residues would act as a supply of 
carbon which is readily rendered available by the soil’s rich flora of 
cellulose ferments. If these soils had produced a wheat crop every 
alternate year and all of the nitrogen which had been added to the 
soil without loss from leaching or bacterial activity taken by the 
crop, it would have necessitated the addition of 25 pounds an acre 
yearly, which is evidently the very minimum which can be attributed 
in these soils to non-symbiotic nitrogen-fixation. 

Eighty different samples of these soils were incubated in the 
laboratory for twenty-one days and the gains in nitrogen determined 
by comparing with sterile checks. The soils were incubated without 
the addition of anything except sterile distilled water. At the end 
of the period the average gain per acre for the cultivated soils was 
202 pounds and that for the virgin soil was 92. 

True, fixation would not continue long at this rate, for when the 
nitrogen content of the soil passed beyond a certain limit decay 
bacteria would increase rapidly, and in the struggle for existence 
they are able, with the advantage at their disposal, to suppress the 
more slowly growing Azotobacter, which would gain the ascendency 
again only when the nitrogen of the soil became low. 

Thus, there is an upper as well as a lower limit to the nitrogen 
content of the soil as far as bacterial activity is concerned, but by 
making the conditions for nitrogen-fixation as nearly ideal as possible 
we may maintain in a soil the upper and not the lower nitrogen 
content. 

In conclusion, it may be stated that although the part played by 
Azotobacter in maintaining the nitrogen of the soil has not been 
definitely measured, it is nevertheless an important factor. Hall 
found it to be at least 25 pounds, Léhnis 35.7 pounds, and the author 
25 pounds per acre annually. It is, therefore, conservative to state, 
as has Lipman, that these organisms, under favorable conditions, 
add from 15 to 40 pounds of available nitrogen to each acre of soil 
yearly. 

REFERENCE. 
Greaves, J. E.: Azofication, Soil Science, 1918, vi, 163-217. 
19 


GHAPTE RR CATY. 
SYMBIOTIC NITROGEN FIXATION. 


From the earliest day of agricultural practice it has been the 
experience of practical men that legumes under appropriate condi- 
tions render the soil more productive. It was the practice of the 
Roman farmers to plow under lupines in order to enrich their soil. 
This practice has persisted through all the succeeding ages by the 
farmers of Europe and Asia. But it is only within the memory of 
men now living that we have been able to state the cause of the 
increased fertility. 

Early Theories.— Liebig, by applying the exact methods of chemis- 
try to agriculture, was able to demonstrate that plants get their 
carbon from the carbon dioxid of the air and not from the carbon 
compounds of the soil. He came to regard the ammonia of the air 
as analogous to the carbon dioxid and taught the doctrine that the 
plants are able to derive their nitrogenous food from the atmosphere. 
He wrote: ‘If the soil be suitable, if it contains a sufficient quan- 
tity of alkalies, phosphates, and sulphates, nothing will be wanting. 
The plants will derive their ammonia from the atmosphere as they 
do carbonic acid.” Liebig considered all crops capable of securing 
the nitrogen from the air, but the legumes and other broad-leafed 
plants were especially fitted for this task, as is witnessed by the 
fact that,they benefit the succeeding cereal crops and do not respond 
as readily to nitrogenous fertilizers. 

It was soon proved that the ammonia and other nitrogen com- 
pounds of the air which were brought down by snow and rain were 
very small and would account for only a small fraction of the nitro- 
gen removed by the crops. 

Lawes and Gilbert (1855) reached the conclusion that non- 
leguminous plants require a supply of some nitrogenous compound, 
nitrates and ammonium salts being about equally effective. ‘The 
amount of ammonia obtainable from the atmosphere is insufficient 
for the need of crops. Leguminous plants behave abnormally. 

They took the precaution of calcining the soil and removing all 
of the ammonia from the air before it was admitted to the vessel in 
which the plants were grown. Their results and those of Boussin- 
gault agree fully in pointing to the conclusion that free nitrogen of 
the air was not available to the plants. ‘These conclusions were 
accepted as decisive for a number of years, although much evidence 


EARLY OBSERVATIONS ON ROOT TUBERCLES 291 


pointed in the other direction. Pot and field experiments carried 
out in England, France, Germany, and the United States during the 
early eighties furnished unmistakable evidence that the legumes 
possessed the power of utilizing atmospheric nitrogen. Atwater’s 
experiments (1883-84) fully demonstrated this. In some of his 
trials the nitrogen gained was 50 per cent. or more of the total 
quantity harvested. However, the mystery was not solved until 
1886 when Hellriegel and Wilfarth announced that the fixation of 
free nitrogen is a property possessed by the legumes and is due to 
the bacteria associated with them in the root tubercles. 

Early Observations on Root Tubercles.—The presence of tubercle 
on the roots of leguminous plants had long before been noted by 
Malpighi. He regarded them as root galls. Later they were 
regarded as buds of incomplete plants, or as rudimentary roots. 
In 1866 Woronin found in them numerous minute bodies which bore 
some resemblance to bacteria. ‘They were rod-shaped but often. 
slightly forked to “'T’’- or “ Y’’-shaped bodies. On account of this 
irregularity in shape the discoverer was unable to say whether they 
were true bacteria or not. He, therefore, called them bacteroids, 
and regarded them as the cause of the tubercles. In 1874 Erickson 
found that in the early stages of the development of the tubercle it 
was filled with long, branching threads resembling the mycelium of 
fungi, and to these hyphe he attributed the formation of the tuber- 
cles. In later stages of the growth of the tubercles he found bac- 
teroids, but was unable to determine whether they had any connec- 
tion with the hyphe or not. 

Frank (1879) not only showed that tubercles are almost invariably 
present on the roots of legumes but that their formation may be 
prevented by the sterilization of the soil. He was thus in possession 
of facts which might have revealed to him the true nature of the root 
tubercles. However, he accepted the interpretation of his pupil, 
Brunchhorst, who claimed the bacteria-like bodies, were merely 
reserve food materials. 

Marshall Ward not only proved that tubercle formation is due to 
outside infection but that such infection may be brought about by 
placing pieces of old tubercles in contact with the roots of growing 
leguminous plants. . 

Hellriegel found, as the result of a long series of experiments, that 
when pea plants were grown in sterilized soils as a rule no tubercles 
were formed, but when the plants were watered with soil infusions 
made by allowing water to act upon soil in which peas had been 
grown, the tubercles appeared in abundance. If the soil infusion 
was sterilized by boiling before it was put upon the plants no tuber- 
cles appeared. These experiments were thought to prove that the 
tubercles were really caused by living organisms in the soil infusion, 
which were killed by heat. The tubercles could not, therefore, be 


292 SYMBIOTIC NiTROGEN FIXATION 


regarded as normal products of the roots, but were certainly infec- 
tions from the soil. In a series of researches, undertaken with the 
assistance of Wilfarth these results were thoroughly confirmed. 
They showed that in sterilized soil the legume behaves the same as 
the non-legume and dies of nitrogen hunger if not supplied with 
suitable forms of nitrogen. When the sterilized soil was inoculated 
with fresh soil on which legumes had made a normal growth they 
then made a vigorous growth in sterilized soil. Under similar 
conditions non-legumes did not recover. The recovery of the 
starving legume was found to coincide with the formation of root 
tubercles. 

Wigand (1887) found that the tubercles contained true bacteria 
and the following year these were obtained in pure cultures by 
Beijerinck. He found further that there were bacteria associated 
with all tubercles, and although the bacteria differed somewhat in 
the tubercles of different species of plants, still there were certain 
constant characteristics to be seen in them all. He, therefore, 
regarded the tubercles as the result of the action of bacteria and gave 
to the organism producing the tubercles the name of Bacillus 
radicicola. Beijerinck regarded the so-called bacteroids of Woronin 
as degenerate forms of the bacteria-involution forms, which appeared 
only after the bacteria had lost their vigor. Ina later investigation, 
after isolating the bacteria and keeping them in pure cultures for 
many months, he was able to produce the tubercles at will by iocu- 
lating soils in which his plants were grown with the pure cultures of 
the organisms. 

Prazmowski (1890) published researches which confirmed all of 
Hellriegel’s results, showing conclusively that if sufficient precau- 
tions were taken to sterilize the soil in which leguminous plants were 
grown no tubercles were ever produced. He further showed that 
the tubercles grow on plants developing both in the light and in the 
dark, but are larger on plants growing in the light; that they only 
appear on healthy plants; that there are very few on plants growing 
in well-washed sand; that if plants growing in sterilized soil be 
watered with brook or river water, tubercles occasionally develop 
but never in abundance; and that the infection of the roots occurs 
early in the germination of the plant and cannot take place in the 
older roots. 

‘Two years later Schlésing and Laurent demonstrated the fixation 
of atmospheric nitrogen through the joint activities of leguminous 
plants and Pseudomonas radicicola by the actual diminution of the 
amount of elementary nitrogen in the inclosed atmosphere sur- 
rounding the plants. 

Species.— Whether the different varieties of legume bacteria are 
distinct species is a perplexing question which today cannot be 
definitely answered. It is known that certain legumes are readily 


SPECIES 293 


infected by one variety, whereas with another variety—infection is 
accomplished with difficulty or not at all. Moreover, the sero- 
logical test yielded by different varieties is specific. These facts have 
led some observers to consider the types as quite distinct, whereas 
others consider them as simply physiological varieties of the same 
general species. On the whole, the consensus of opinion at the 
present time seems to be decidedly in favor of this latter view. 

Some of Hellriegel’s experiments indicated that bacteria from 
clover could not produce tubercles on lupines and serradella. Simi- 
lar results were obtained by Nobbe and his associates, yet they were 
finally led to conclude that the root invasion of legumes is caused by 
a single species. Long-continued growth of the organism on a 
legume adapts it to that legume so it no longer invades the roots of 
other legumes. But Petermann (1893) considered it probable that 
every genus of plant has its specific bacteria. Buhlert considered 
that all of the organisms are forms of B. radicicola but that the 
bacteria best adapted to a given species of leguminous plant are those 
naturally found upon that plant. However, cross inoculation is 
possible within certain limits. From the root tubercles of some 
leguminous plants he obtained bacteria which seemed to be very 
highly specialized, but he considers that this specialization does not 
extend to differences that may be regarded as specific. 

As a result of a large number of experiments'with different kinds 
of legumes, Maassen and Miiller (1907) reached the conclusion that: 
(1) The organisms of Pisum’ satiwm will inoculate Vicia faba, V. 
sativa, V. villosa, Lens esculenta, Lathyrus sativus, L. odoratus, 
and L. silvestris; (2) that of Trifolium incarnatum will inoculate 
T. pratense; (3) that of Medicago sativa will inoculate M. lupulina 
and Melilotus officinalis; and (4) that of Lupinus lutens will inoculate 
L. angustifolius and Ornithopus sativus. ‘The organisms of Phaseolus 
vulgaris, Soja hispida, and Robinia pseudacacia, according to 
Maassen and Miiller, will apparently not inoculate any other plant. 
Similar conclusions were reached regarding the organisms of Coronilla 
varia, Onobrychis satava, Anthyllis vulneraria, Sarcthamnus scoparius, 
Amorpha fruticosa, Caragana frutescens, and Acacia lophanta. 

De Rossi (1907) described a specific organism derived by him from 
root tubercles of V. faba which produces root tubercles and which 
he claimed is morphologically, biologically, and culturally widely 
different from Bacillus radicicola Beijerinck. 

Nobbe and coworkers (1908) showed that pure cultures of 
bacteria from tubercles of one member of a genus are effective on 
other members of the same, and, as a rule, only of the same genus. 
They found, however, complete interchangeability in case of peas 
and vetches and partial in case of lupines and serradella. 

Zipfel (1912), with the hope of throwing light upon the kinship 
among the various nodule bacteria, made use of the agglutination 


SYMBIOTIC NITROGEN FIXATION 


294 


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SPECIES 295 


method. From his results he concluded that the nodule bacteria 
were not varieties of the same species, but that distinct species 
existed. 

Klimmer and Kriiger two years later used serological tests to 
distinguish species. They used the agglutination method princi- 
pally and complement bindingand precipitation for confirmation and 
control. Working with organisms from eighteen legume species, 
they divided the oe ae according to thea methods, into nine 
species which they asserted differed sharply from one another. 

Simon (1914) tested various cultures upon seedlings of several 
legume species and compared the results with those obtained by 
using Zipfel’s agglutination method. He found that the results ot 
both methods agree substantially. His grouping of the organisms 
is in general agreement with that of Klimmer and Kriiger. He 
concluded, however, that “the root bacteria of legumes are rather 
to be conceived as more or less constant adaptations of the species 
Bacillus radicicola.”’ 

Burrill and Hansen (1917), after an extensive study of various 
legume bacteria both with the pot-culture method and the agar 
test-tube method of Garman, divided the nodule organisms into the 
following eleven groups according as they are interchangeable for 
the purpose of inoculation: 


Group I. 


Mammoth red clover, 7'rifoliwm pratense perenne. 
Alsike, or Swedish clover, Trifoliwm hybridum. 
Crimson clover, T'rifoliwm incarnatum. 

Berseem, or Egyptian clover, Trifoliwm alexandrianum. 
White clover, T'rifolium repens. 

Zigzag, or cow clover, Trifoliwm medium. 


Group II. 


White sweet clover, Melilotus alba. 

Yellow sweet clover, Melilotus officinalis. 

Wild yellow sweet clover, Melilotus indica. 

Alfalfa, Medicago sativa. 

Alfalfa, Medicago falcata. 

Bur clover, Medicago hispida. 

Black medick, or yellow trefoil, Medicago lupalina. 
Funugreek, T7'rigonella foenum-graecum. 


Group III. 
Cowpea, Vigna sinensis. 
Partridge pea, Cassica chamaecrista. 
Peanut, Archis hypogoea. 
Japan clover, Lespedeza striata. 


296 SYMBIOTIC NITROGEN FIXATION 


Slender bush clover, Lespedeza virginica. 
Velvet bean, Mucuna utilis. 

Wild indigo, Baptisia tinctoria. 
Tick trefoil, Desmodium canescens. 
Tick trefoil, Desmodium illinoense. 
Acacia, Acacia armata. 

Acacia, Acacia floribunda. 

Acacia, Acacia longifolia. 

Acacia, Acacia melanozylon. 

Acacia, Acacia semperflora. 

Acacia, Acacia from California. 
Dyer’s greenweed, Genista tinctoria. 


Group IV. 


Common garden pea, Pipsum sativum. 

Field pea, or Canada field pea, Pisum sativum arvense. 
Hairy vetch, Vicia villosa. 

Spring vetch, Vicia sativa. 

Broad bean, Vicia faba. 

Narrow-leaved vetch, Vicia angustifolia. 

Vetch, Vicia daysiecarpa. 

Lentil, Lens esculenta. 

Sweet pea, Lathyrus odoratus. 

Perennial pea, Lathyrus latifolius. 


Group V. 
Soybean, Glycine hispida. 


Group VI. 


Garden bean, Phaseolus vulgaris. 
Garden bean, Phaseolus angustifolia. 
Searlet runner bean, Phaseolus multiflorus. 


Group VII. 


Lupine, Lupinus perennis. 
Serradella, Ornithopus sativus. 


Group VIII. 


Hog peanut, Amphicarpa monoica. 


Group IX. 


Lead plant, Amorpha canescens. 


CULTURAL CHARACTERISTICS 297 


Group X. 
Trailing wild bean, Strophostyles helvola. 


Group XI. 


Black, or common locust, Robinia pseudo-acacia. 


Hiltner and Stormer (1903), however, arranged the tubercle 
bacteria into two groups possessing, according to them, well-defined 
morphological and physiological differences. One of these groups 
is included under the species Rhizobium radicicola and the other 
under Rhizobium beijerincku. The former comprises the organisms 
from lupines, serradella, and soybeans, whereas the latter comprises 
all of the others. 

Grieg-Smith (1902) reports having found three races of the 
nodule bacteria in the same nodule, while Gino de Rossi (1907) 
reported the finding of two organisms which differ in that one forms 
a large hyaline colony not developing well on beef and peptone 
gelatin, while the other forms white non-transparent colonies on beef 
gelatin. He believes that the one is morphologically, biologically, 
and culturally widely different from Bacillus radicicola (Beijerinck). 

Cultural Characteristics.—'The nodule bacteria grow well on a great 
variety of cultural media, perhaps best on a medium of ash-maltose- 
agar or one of legume extract to which has been added a sugar, 
dextrose, sucrose, or maltose, and dipotassium phosphate. 

In an agar stab typical drop-form colonies are produced at the 
surface, while a thin gray growth follows the line of the needle. In 
standard beef broth the growth of the organism is slow. The liquid 
becomes cloudy, a gray-white ring is formed, and a thin membrane 
covers the surface. Later a flocculent precipitate settles to the 
bottom of the tube. In standard beef broth gelatin the growth of the 
organism is at first funnel-shaped and then stratiform. Gelatin is 
slowly liquefied, the process sometimes requiring two or three months 
for completion. In gelatin stabs the growth sometimes seals over 
the stab with a drop-form growth and liquefaction does not occur. 
On the ordinary cultural media the organisms do not show any very 
characteristic growth. The most noticeable difference between 
various strains is the rapidity of development. Slight alkalinity to 
+20° to +25° acid (Fuller’s scale) with phenolphthalein is tolerated ; 
neutral to + 10° is best. 

The results obtained by Fred and Davenport clearly indicated 
that the nodule bacteria from different plants behave differently 
toward acid. They divided the legume bacteria into five groups 
depending upon their sensitiveness to acid. 


1. Critical px 4.9 ae) ee ao eAlfalfia andi sweet clover: 

2. Critical pu 4.7 - . . . . . Garden pea, field pea and vetch. 
3. Critical pH 4.2 . . . . . . Red cloverand common beans. 
4. Critical pH 3.3 - . . . . . Soybeans and velvet beans. 

5 


eaGriticalpHsslow ess) 2.)  ..  Jpines! 


298 SYMBIOTIC NITROGEN FIXATION 


The alfalfa organism is the most sensitive of the legume bacteria 
to acidity and conversely the lupine organism is the most resistant to 
acidity. 


Fie. 36 


Fics. 36 and 37.—Ash-agar plate from bean (Phaseolus vulgaris), showing giant 
colonies in a thickly seated plate. Ash-agar plate from perennial pea (Dathyrus 
latifolius); the clear spaces are due to sterilized fluid carried over with pieces of 
nodule tissue. (After Burrill and Hansen). 


MORPHOLOGY OF THE COLONIES 299 


The zone of optimum temperature (Zipfel) is between 18° and 20° 
C.; the limits of growth are 3° and 45° C.; and the upper limits of 
life are from 60° to 62° C. Burrill gives a considerably higher 
optimum temperature—25° to 28° C. Grieg-Smith found the best 
temperature for the production of slime to be 22° C. with most organ- 
isms and 26° C. for one obtained from Robinia. The organism is 
aérobic, and he found that diffused sunlight of the laboratory is not 
harmful; even exposure to direct sunlight for several months with- 
out transferring did not kill the organisms when grown upon favor- 
able media with precautions to prevent evaporation. 

Ball found that the organism endures at least two years in dry 
soil. Harrison and Barlow found that the limit of viability on ash- 
maltose-agar varied somewhat, but in the majority of cases it was 
about two years. How long the organism will exist in a soil under 
field conditions is not yet known, but practical observations indicate 
that it must be many years. 


Fig. 38.—Young nodule magnified, showing affected root hair and same root hair 
more highly magnified. (After Atkinson.) 


Morphology of the Colonies.— Two types of colonies appear on agar 
plates—buried and surface colonies—and are thus described by 
Burrill and Hansen: 


5300 SYMBIOTIC NITROGEN FIXATION 


‘Buried colonies are small and submerged, most frequently lens, 
or spindle-shaped, with smooth and even edges. ‘They are rather 
opaque, granular in structure, and in color are cream to a chalk 
white. They increase slowly in size, eventually appearing on the 


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Fic. 39.—Young nodule, showing the beginning of the differentiation of its tissues. 
(After Prazmowski.) 


surface of the agar as surface colonies, when the growth becomes 
rapid. The lens colonies, however, remain visible for many days in 
the center of the new growth. 
“Surface coloni igi I f f tl : 
Surface colonies originate at or near the surfaces of the agar or 
develop from buried colonies. ‘They are drop-form, watery, muci- 


301 


MORPHOLOGY OF THE COLONIES 


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302 SYMBIOTIC NITROGEN FIXATION 


laginous (in appearance, though not always to the touch), gray- 
white to pearly white in color, glistening, and semitranslucent 
to opaque. The edges are smooth and even. Under the low power 
the interior is granular. They frequently attain considerable size, 
a centimeter or more in diameter. 

“Plates made direct from the nodule lack uniformity to a marked 
degree. The undiluted plate (first plate) begins to show a few 
colonies in two to four days. These colonies become extremely 
large in a very short time, their rapid growth being due to small 
pieces of nodule tissue or to clumps of bacteria carried over into the 
agar. In five or six days numerous colonies begin to make their 
appearance, most of them as submerged colonies, which later grow 
to the surface. 

“The dilution-plate (second plate) colonies are always extremely 
slow in growth. Generally colonies are large enough for transfer 
in six to fourteen days, the plates should not be discarded for two or 
even three weeks. 

“The rate of growth of colonies also varies with the organisms of 
different nodules. Among the fast growers are the organisms from 
pea (Pisum), vetch (Vicia), lentil (Lens), sweet pea (Lathyrus), 
bean (Phaseolus), lupine: (Lupinus), wild bean (Strophostyles), 
clover (Trifolium), sweet clover (Melilotus), alfalfa (Medicago), 
and fenugreek (7'rigonella). The organisms appreciably slower in 
growth are those from the cowpea (Vigna), Japan clover (Lespedeza), 
tick trefoil ( Desmodium), acacia (Acacia), partridge pea (Cassia), 
false indigo ( Baptisia), dyer’s greenweed ( Genista), peanut (Arachis), 
soybean (Glycine), and hog peanut (Amphicarpa).” 

Morphology of the Bacteria.— They are bacilli and when full-grown 
vary in length from 1 to 4 or 5u. It is not uncommon to find them 
from 0.5 to 0.64 wide and from 2 to 3u long and some have been 
found to measure only 0.184 wide and 0.9u long. The bacilli prevail 
in the young nodule, whereas the branched forms or bacteroids 
predominate in the older structure. In the cowpea nodules Burrill 
frequently found large club-shaped bacteroids, though the branched 
forms were not so numerous. The bacteroids are best demon- 
strated when the young nodule is just beginning to show a reddish 
interior. At this stage the characteristic 2 and y forms’ occur in 
great number and show considerable vacuolation and unevenness in 
staining, especially when stained with carbol-fuchsin. 

“Tn the old, decomposing nodule the bacteroids are extremely 
vacuolated and ghost-like, showing small, oval, deep-staining bodies 
within. ‘The inference is that these bodies are motile swarmers 
which later free themselves from the ghost-like capsules, rather than 
bud off, as has been described by some writers. Frequently the 
swollen rods have a beaded appearance with unstained bands or 
areas, A few motile rods may sometimes be seen in hanging drops 


BACTEROIDS 303 


in this stage, and sometimes a bacteroid is seen to oscillate as though 
swung about by some propelling force in one end. Division of the 
bacteroids into bacilli, as represented by Dawson, may also occur. 

“When first plated out, the young colonies consist of small rods 
which show considerable variation in length. No bacteriods are 
present, though the rods are sometimes slightly club-shaped and 
sometimes show vacuolation. However, they never attain the size 
of bacteroids. With frequent transfers the rods become quite 
uniform in size and stain deeply and evenly, especially with anilin- 
gentian violet. 

“Tn very old cultures (three months on ash agar, without transfer) 
the small, oval swarmers and the normal rods predominate, though 
a few club-shaped and a few branched bacteroids are found. The 
bacteroids produced upon artificial media are never so large nor so 
numerous as those seen in mounts direct from a young nodule. 

“Staining.—The organisms do not stain well with ordinary 
aniline stains. Carbol-fuchsin and aniline-gentian-violet (used 
steaming) are the most satisfactory stains. Though carbol- 
fuchsin was preferred, anilin-gentian-violet stains were always 
used as checks, because the former stain accents the vacuolated 
appearance, particularly in bacteroids. Carbol-fuchsin 1s especially 
useful in staining bacteroids, direct from the nodule and also old 
agar cultures. Kiskalt’s amyl-Gram stain, described by Harrison 
and Barlow, is useful since the amyl alcohol clears up the field, 
leaving the bacteria stained, though not so intensely. This stain, 
however, should not be considered a means of identifying Ps. 
radicicola. 


c= 
LOT wh 
rE se @ 
b, 
Fic. 41.—Bacteroids, showing shape and occurrence of vacuoles. (After Whiting). 


“Bacteroids.— While Ps. radicicola produces no spores, it produces 
bacteroids which are very evidently more resistant than the normal 
rods. Unfavorable conditions, such as unsuitable media, infrequent 
transfer, or addition of caffein to the medium, cause their appear- 
ance. ‘Thisis inaccord with what takes place in the nodule. In the 
growing nodule, when development is most rapid, the bacteroids are 
at their maximum; they enable the organisms to multiply rapidly 
in spite of the resistance offered by the plant cells. ‘Transferred to 
favorable media from this stage the normal uniform bacilli are 
produced. ‘The bacteroid then must be regarded as a normal and 


304 SYMBIOTIC NITROGEN FIXATION 


a very necessary stage in the life of the organism. Its significance 
in the actual fixation of nitrogen, however, is pure speculation.” 

The organisms are actively motile and when viewed under the 
microscope may be seen darting about with amazing rapidity, now 
tumbling end over end, now spinning violently on the short axis 
and then sweeping across the field in a darting, jerking course. 
They contain from 6 to 20 flagella. The number and distribution 
of the flagella are variously given by the different investigators due 
probably to either variation in organisms or to the difficulty with 
which flagella is demonstrated owing to the gum or slime produced 
by the organism. 

Mode of Entrance into Host.—The method of inoculation and the 
growth of the nodule is described as follows by Whiting: 

“As the tip of the root hair of the legume pushes itself out into the 
soil, it chances to come into intimate contact with the organism, 
B. radicicola. Some scientists have exploited the view that the 
organism is attracted to the plant by chemotaxis, believing that the 
plant excretes a substance, probably a carbohydrate, which diffuses 
into the soil solution and attracts the motile organism. While it 
has been rather definitely shown that this organism progresses in 
the soil at a rapid rate, nevertheless the number of root hairs infected 
is too small to lend support to a chemotactic theory. However the 
case may be, the organisms cluster at the tip of the hair and by 
means of an enzyme (or otherwise) rapidly dissolve the cellulose of 
the cell wall, thus enabling the organism to enter the root hair. As 
a result, there is a decided bending of the tip, causing it to resemble 
a shepherd’s crook. ‘This was early observed as a sign of complete 
infection. It is claimed that other root hairs which form after infec- 
tion are immune to the attack of other leguminous bacteria. 

“The organisms, by rapid division and growth, advance through 
the center of the infected root hair. Prazmowski found organisms 
in the'cell sap and even in the epidermis only two days after inocula- 
tion. In this advance an infection strand (Infektion-schlauche) is 
formed, which consists of gelatinous material, and in the earlier 
stages of development this strand may be traced from the root hair 
into the inner tissue of the root and from cell to cell throughout the 
nodule. ‘This infecting strand is not supposed to constitute a 
portion of the living tissue, nor is it a well-defined tube; but, as Fred 
has recently shown, it consists of a large number of zodglea occurring 
adjacent to one another, in which separate bacteria can be distin- 
guished. ‘The infecting strand branches profusely and it was this 
habit of growth which caused the early investigators to consider it 
the mycelium of a fungous growth. 

“Growth of the Nodule.—The presence of B. radicicola in the 
tissues of the root causes a rapid cell division in the pericycle. 
These cells become larger and contain more protoplasm than the 


RELATIONSHIP TO HOST 305 


surrounding cells, and as growth takes place the cortical parenchyma 
and epidermis are forced outward, thus forming a nodule. The 
growth of the nodule is apical. The various tissues common to the 
plant are present. In the central portion of the nodule is the so- 
called bacteroidal tissue, which is ochre, flesh, or gray in color, 
according to the age of the nodule, and in this portion the infecting 
strand (Infektion-schlauche) is distinguished in the young nodule. 
It ramifies throughout the cells, causing those which it enters to 
lose their power of cell division but not of growth. Later, or in 
older nodules, the infecting strand is not visible, and the bacteroidal 
tissue loses its firmness. At the period when seed formation is at 
its height, most of the nodules are soft, and the internal tissues slough 
off, leaving the more resistant epidermal tissue a mere shell, which 
later decays. ‘The endurance of the nodule depends upon several 
factors,—chiefly, however, upon the kind of legume plant on which 
it is produced and the need of nitrogen by that plant. 

“Pierce considers the nodules as originating endogenously from 
the same layer of cells as the lateral roots, and as being morpho- 
logically similar to them; however, as the lateral roots rupture the 
epidermis the above statement is not entirely in accord with what 
actually takes place. 

“The nodules are largest and most numerous where “aération is 
best in the soil. In saturated soils they occur at the surface and 
are often found colored green, very similar to sunburned potatoes. 
Nodules form in solutions, and exceptionally well in certain nutrient 
solutions. Several interesting instances have been brought to the 
attention of the Experiment Station, in which the observers believed 
that the nodules had grown above the ground. These peculiarities 
were undoubtedly caused by unobserved phy sical conditions occur- 
ring at the time of infection or afterward.” 

Relationship to Host.—Even today the relationship between Ps. 
radicicola and its host is a mooted question. Some authors claim that 
they are true parasites and that the relationship between the 
tubercle organisms and their host plants is that of two contending 
parties and the bacteria draw on the nitrogen of the air in their 
endeavor to make up the deficiency of nitrogenous substances which 
have been taken from them by the plant. Moreover, inoculation 
experiments have demonstrated that Ps. radicicola causes a certain 
resistance similar to that produced by an organism in combating a 
true parasite. 

Hiltner has given the six following conditions as instances in which 
immunity demonstrates itself: 

1. The organisms cannot get into the plant. 

2. The organisms gain admission into the plant, but do not pro- 
duce nodules because the plant, by its greater resistance, absorbs 
the bacteria. 

20 


306 SYMBIOTIC NITROGEN FIXATION 


3. The organisms enter the plant and produce nodules, but no 
fixation of nitrogen occurs. 

4. The organisms enter, produce nodules, and nitrogen is fixed 
and assimilated by the plant. 

5. The organisms are so efficient in comparison with the plant 
that the latter is injured. 

6. The organisms are parasitic and the plant is actually killed. 

Certain products which are produced by the invading organism in 
connection with the host have been taken as evidence of the parasitic 
nature of the bacteria, whereas others consider the nodule which 
forms on the legume root a result of irritation due to a parasite. 
Grieg-Smith, however, considers the formation of root tubercles not 
as a result of irritating parasitic action but rather as the consequence 
of the production of nutrients at that place resulting in better 
nourishment and growth of the cells than in other parts of the roots. 

Fuhrmann considers that the fixation of atmospheric nitrogen 
by the root-tubercle organisms begins when the bacteroids have 
reached a stage when they are colored brown-red by addition of 
tincture of iodine. This occurs only when the organisms are feeding 
almost exclusively upon carbohydrates and the available nitrogen 
compounds have been almost completely exhausted. Many 
workers prefer to call the relationship up until this stage a true 
parasitic and later a true mutual symbiosis. 

By careful staining Fred was able to demonstrate the entering of 
the bacteria through the root hairs, immediately after which a 
tubercle started to form. A series of sections showed that mitosis 
goes on in the nodules much the same as it does in diseased tissue of 
animals. The mitotic figures are larger, very irregular, and not well 
marked and have an uneven number of chromosomes. In the 
normal roots the mitotic figures are about one-sixth as large, very 
clear, and the chromosomes in numerous pairs. This he considers 
bears out the theory that the legume bacteria are symbiotic parasites 
of the plant. 

If we accept Whiting’s definition of mutual symbiosis “as the 
contiguous association of two or more morphologically distinct 
organisms not of the same kind, resulting in an acquisition of 
assimilated food substances which implies that the organisms con- 
cerned have the power of independent existence, but that both are 
benefited by the close association,’’ we must conclude that all the 
evidence bears out the idea that the relationship existing between 
Ps. radicicola and legumes is one of mutual symbiosis. 

Mechanism of Fixation (Metabolism).—Ior a long time it was 
believed that the nitrogen fixed by legume bacteria and assimilated 
by the plant was obtained through the leaves. ‘The organisms on 
the roots were considered to in some way stimulate the plant so that 
it possessed the power to assimilate nitrogen. Stoklasa considered 


MECHANISM OF FIXATION ' 307 


that amids were first formed and that these migrated to the nodules, 
reacted with glucose and produced protein which served as the 
nutrient medium for the bacteria. In this connection he advanced 
the idea that the bacteria produced an enzyme which enabled the 
plant to fix the nitrogen. This theory, however, was shown to be 
untenable by Whiting who grew soybeans and cowpeas under careful 
control conditions. One lot received a definite proportion of oxygen, 
and carbon dioxid, a second oxygen and carbon dioxid, while a third 
received ordinary air. He found that these plants utilize atmos- 
pheric nitrogen through their roots and not through their leaves. 

Nobbe and Hiltner (1893) considered the root tubercles to be the 
parts of the leguminous plants where the free nitrogen is assimilated 
and that the direct agents of the assimilation are the bacteroids and 
not the bacteria themselves. As to the metabolism of the nitrogen 
by these bacteroids the ideas at present are very indefinite. Loew 
and Aso (1908) suggested that ammonium nitrite was the first 
compound produced, the nitrous acid being readily reduced to 
ammonia. 

Gautier and Drouin considered that the nitrogen is oxidized to 
nitrous and nitric acids, whereas Winogradsky has advanced the idea 
that the free nitrogen in the plasma of the organism may unite with 
nascent hydrogen and form ammonia which by oxidation would 
become assimilable. 

Gerlach and Vogel concluded that there is a direct union of free 
nitrogen with some organic compound inside the bacterial cell. 
Heinze thinks it probable that nitrogen is at once brought into 
combination with a carbohydrate (glycogen) and suggests that a 
salt of carbonic acid may be formed first, or that carbonic acid may 
be produced from cyanamid. All of these theories, however, are 
purely speculative as there is little experimental evidence on the 
subject. 

It is in keeping with our knowledge of bacteria to assume that 
the changes are catalyzed by enzymes produced by the bacteria, 
and Hiltner reported the findings of a substance which is produced 
by the legume bacteria which can dissolve the cell wall and root 
hairs. Yet Beijerinck claims that no enzyme has been found which 
attacks starch, cellulose, or saccharose. No true proteolytic enzyme 
has been reported, but Benjamin has reported the presence of urease 
in the nodules of various legumes. This enzyme is, however, found 
quite generally in plants and may have come from the host and not 
the bacteria. Fred, although unable to detect a proteolytic enzyme, 
has obtained evidence of the presence of oxidases in the slime of 
various legume bacteria. 

There are two main suppositions regarding the assimilation of the 
nitrogen by the plant as follows: (1) That the bacteroids are bodily 
absorbed by the plant fluids; and (2) that the bacteroids, by some 


308 SYMBIOTIC NITROGEN FIXATION 


sort of change, produce the substance containing the assimilable 
nitrogen w hich the plant used. 

There appears to be considerable evidence in favor of this second 
theory. Stefan thinks that the transfer of the assimilable nitrogen 
from the organism to the host plants follows the ordinary physical 
laws of osmosis, and Golding has conducted some very interesting 
experiments on the removal of the products of growth in the assimila- 
tion by nitrogen by legume bacteria. He reasoned that the plant 
played an important réle in the removal of the products produced by 
bacteria in the nodules aside from the mere furnishing of suitable 
food. He used a porous Chamberland filter candle placed in a 
culture vessel to serve to imitate natural conditions. The parts of 
the plants used in some of his experiments were sterilized in order to 
avoid the possibility of plant enzyme action. As a result of his 
method of experimentation he obtained a much greater fixation of 
nitrogen than other experimenters had obtained. He concluded that 
the plant plays a part in the removal of soluble products of growth, 
thus permitting a more rapid reaction than where the products 
accumulate. 

The results of Golding’s most extensive experiment are sum- 
marized as follows: 

Nitrogen in 


Grams. 

500.0 gms. of stems and leaves. . atl nT ee RROD 
26.2 gms. of roots and nodules (quite freak) VT Aa dee Pl lye eee OOS 
3000.0 c.c. ammonia-free distilled water . . . . . . . . 0.000 
Totalinitrozen'toistart with) 90-0 le) en) oe es nee 009 

28/02 0lecahltrates and <drainingses) en) ee eee ee eer Ol sen 
566.2 gms. wet residue. . en SPR Bie 2 ee oro ao 
Total nitrogen after Pope oe CREO 2 i Or oU 

Total gain of nitrogen during experiment aot hen he yi ()) Be 


Attempts have been made to obtain an insight into the transforma- 
tion going on in the nodules by various analyses. These have been 
summarized by Whiting as follows: 

“Chemical—The chemical composition of legumes from the 
standpoint of their nitrogenous constituents has been investigated 
to some extent, but the studies closely related to this point are 
relatively few. The following data are very general in character 
and relate to studies concerning the total nitrogen content of the 
different parts of legumes at different periods of growth. Studies 
upon some of the various nitrogenous compounds are also included. 

“Tn 1895 Stoklasa, working with lupines (Lupinus luteus and L. 
angustifolius), found that the nodules were richest in the element 
nitrogen at the time of blooming, while the roots appeared to be 
richest in that element at the fruiting period. His results are given 
in Table I. The figures for the nodules indicate the nitrogen is 
either taken up by the plant for seed production or diffused into the 
soil. 


CHEMICAL 309 


TABLE I.—TOTAL NITROGEN IN LUPINUS LUTEUS: RESULTS OBTAINED 
BY STOKLASA. (PERCENTAGE ON DRY BASIS). 


Period. Roots. Nodules. 
Blooming ewes ts wee este Sn ean pad ll. 64 Dawe 
Bil ee ee ee eee Ae i ee Se Daal 
Wittintt yee ee oee aad ae a eee eee be La? rhs? 


“Stoklasa also determined protein, amids, and asparagin in 
lupine nodules. The protein was obtained by the Stutzer method, 
the amids by the Kjeldahl method, and the asparagin by calcula- 
tion from the ammonia obtained by distillation with magnesium oxid. 
Table II shows his results. 


TABLE II.—NITROGEN COMPOUNDS IN LUPINE NODULES: RESULTS 
OBTAINED BY STOKLASA. (PERCENTAGE ON DRY BASIS). 


Period. Protein. Amids. Asparagin. 
Blossoming SL ee eotiat, 2) Bena t99, 0.35 0.34 
Miatinttvan were eke ere. BO mT b4 0.15 Trace 


The presence of asparagin in the nodule is important, as it is 
thought to be intimately related with the formation of protein. 

“In 1901 Wassilieff studied the nitrogen compounds in white 
lupine (Lupinus alba) seeds and seedlings. He found that the seeds 
contained 7.68 per cent. of total nitrogen; and that of this, 6.89 per 
cent. was in the form of protein and 0.53 per cent. was precipitated by 
phosphotungstic acid, leaving a difference of 0.26 per cent., aspara- 
gin. The occurrence of asparagin in large amounts in the seed- 
lings is shown by the data given in Table ITI. 


TABLE III.—NITROGEN COMPOUNDS IN FOURTEEN—DAY—OLD GREEN 
SEEDLINGS OF WHITE LUPINES: RESULTS OBTAINED BY ‘° 
WASSILIEFF (EXPRESSED IN PERCENTAGE ON DRY BASIS). 


pep eAGt Total 

Parts. nitrogen. Asparagin. Protein. nitrogen. 
MECRVES yom ethers ee 0.53 1.45 4.11 6.57 
Cotyledons Souls ee 72 0.63 3.83 2.44 7.83 
SUCIIS® Pameece 24 0 vat oat 0.42 4.57 1.56 6.77 
FOOTSIE) Mies ce fee a 0.46 2.20 1.87 5.40 


Wassilieff also demonstrated the presence of leucin and tyrosin 
in the cotyledons of one-week-old seedlings of white lupines. These 
and other amino-acids would be expected to be present when the 
protein of the seed is breaking down for the nutrition of the seedling. 

“Knisely analyzed the leaves, pods, stems, roots, and nodules 
of lupine plants for total nitrogen at three distinct periods of develop- 
ment. His results show better than the others presented where the 
nitrogen accumulates as the plant matures. 


1 Phosphotungstie acid. 


310 SYMBIOTIC NITROGEN FIXATION 


TABLE IV.—TOTAL NITROGEN IN LUPINES: RESULTS OBTAINED BY 
KNISELY. (EXPRESSED IN PERCENTAGE ON DRY BASIS). 


Period. Leaves. Pods. Stems. Roots. Nodules. 
Full bloom os et Oe 3.07 ealip 0.92 Denline 
Pods wellformed . . 38.70 3.38 0.88 0.83 3.29 
Podsverylarge . . 3.41 3.68 0.90 0.66 3.70 


“Schulze and Barbieri examined lupine and soybean seeds and 
seedlings for nitrogen and obtained the results shown in Table V. 


TABLE V.—NITROGEN IN LUPINE AND SOYBEAN SEEDS AND SEEDLINGS: 
RESULTS OBTAINED BY SCHULZE AND BARBIERI (EXPRESSED 
IN PERCENTAGE ON DRY BASIS). 


Total Pep eAS Filtrates 
Material. nitrogen. Protein. nitrogen. from P.T.A. 

Lupine seeds . . . . 8.63 8.17 0.24 0.22 
Soy beansve. aoevisee 8 6.73 6.32 (Meals 0.28 
Lupine dark seedlings, 

eleven to twelve daysold 10.64 3.40 1.60 5.64 
Lupine dark seedlings 

twelve days old Seen vil Oo PAB) yedi les 6.01 
Soybean seedlings fifteen 

days old lad iids ro EE 7.42 3.86 0.56 3.00 


“They also found a large amount of asparagin in both the lupine 
and the soybean seedlings. 

“Schulze has made a careful study of the compounds in plants, 
and has formulated the hypothesis that the same decomposition 
products arise from protein in the plant as outside it, but that in the 
plant the compounds are further altered, thereby affecting in varying 
degree the individual products of the hydrolytic decomposition. 
A comparison of the analyses of pea seedlings one week old and those 
three weeks old show the following differences: 


Leucin. Tyrosin. Arginin. Asparagin. _ 
lweek . . . . Abundant Little Present Absent. 
3weeks. . . . Muchless Absent Almost absent Very abundant. 


Arginin and amido-acids were shown to be present in the lupine 
cotyledons, but asparagin was absent, although the latter substance 
was found in the stem of the seedling. It has been suggested that 
the occurrence of asparagin is associated with the disappearance 
of amido-acids and not of protein. Phenylalanin, tyrosine, and 
tryptophane have been reported in the white lupine (Lupinus alba), 
tyrosin and tryptophane in vetch (Vicia sativa), and tryptophane 
in the garden pea (Pisum satiwum). 

“Smith and Robinson found 4.19 per cent. of nitrogen in soybean 
nodules and 3.90 per cent. in cowpea nodules. They observed that 
inoculation increased the protein content of soybean plants without 
increasing the yield of beans. This has been noted by other 
experimenters. 


CHEMICAL 511 


_“ Hopkins has reported the analyses of cowpea plants for total 
nitrogen with and without inoculation. ‘The nodules, root, and tops 
were analyzed separately, as will be seen by reference to Table VI. 


TABLE VI.—NITROGEN FIXATION BY COWPEAS: RESULTS OBTAINED 
BY HOPKINS. (EXPRESSED IN MGS.) 


Nitrogen 

Treatment. Tops. Roots. Nodules. fixed. 
Ten plants with bacteria . . . 146 9 ita 125 
Ten plants without bacteria . . 38 3 
Ten plants with bacteria . . . 171 10 18 140 
Ten plants without bacteria .. 55 4 
Ten plants with bacteria . . . 1438 8 ily 124 
Ten plants without bacteria .. 40 4 


The inoculated plants contained a much greater percentage of 
nitrogen than the uninoculated, the average content of the inoculated 
being 4.24 per cent. in the tops, 1.48 per cent. in the roots, and 5.92 
per cent. in the nodules, while the average content of the uninoculated 
was 2.48 per cent. in the tops and 0.88 per cent. in the roots. 

“The ash and the ash constituents of the nodules and the roots 
of lupines have been determined by Stoklasa, as presented in 
Table VII. The total ash of the nodules was found to be 6.32 per 
cent., while that of the roots was found to be 4.55 per cent. 


TABLE VII.—ASH CONSTITUENTS IN LUPINE NODULES AND ROOTS: 
RESULTS OBTAINED BY STOKLASA. (EXPRESSED IN PERCENTAGE.) 


Constituents. Nodules. Roots. 
Si = es A ee Bs i a ee ree oe 1.59 1.90 
(Sis) aye, hal Spe Opt entice perce Se ees ee 4.90 6.38 
eee ee er Wie ee ee IS Cid) 6.51 4.28 
K ay ON ee ray EA wat ee eM ah re eee! Lets 12.05 
Nair ete ae ee ee Pen Be? SG SOA: 19-94 
Vier ae as eee he tS er een mena PERM gs ON eee 7.41 W105 
(GaROert es fh. eee aa. panel) oe eB i iat are, 2 7.64 12.04 
HOMME TET te et Pi Unc ie ge ns ofl ibe ay Sears 0.83 Oar 5) 


“The analyses of red-clover nodules show a potassium content 
of 2.63 per cent. in the dry matter. The nodules, therefore, are 
relatively rich in mineral elements as well as nitrogen compounds; 
and Stoklasa’s results (See Table VII) show that the chief differences 
between the roots and the nodules in the composition of the ash 
constituents are in phosphorus, potassium, calcium, and sodium. 
The nodules are richer in the first two elements and the roots in the 
latter two. 

“Tn brief, the chemical data which have been considered, although 
small in amount, show the relative richness in nitrogen of the 
nodule as compared with other parts of the plant. ‘They point to 
the accumulation of nitrogen in the seeds, at the expense of the other 
parts, as the plant matures. That the nitrogen exists in the form 


312 SYMBIOTIC NITROGEN FIXATION 


of protein, asparagin, and other soluble forms, is also clear. The 
presence of various aliphatic and carbocyclic amino-acids has been 
mentioned.” 

Sources of Energy.— Under natural conditions the legume bacteria 
undoubtedly obtain the energy required for the endothermic reaction 
which they catalyze from the plant carbohydrates. It has long been 
known that decoctions of the legumes makes the best media on which 
to grow these organisms. Temple found that the presence of 
ground alfalfa caused a rapid multiplication of the organisms either 
in solution or in soil. Grieg-Smith found dextrose, levulose, sac- 
charose, maltose, and mannite to furnish a good source of carbon for 
the organisms, but lactose was a very poor nutrient. Temple found 
saccharose and dextrose superior to lactose, whereas he found levulose 
wholly unsuited to their needs. 

No one so far has attempted to measure their energy requirements 
when growing under their natural symbiotic condition. Fred has 
studied their growth apart from the host plant and found that when 
considered per unit of carbohydrate consumed the legume bacteria 
fix as much or more nitrogen than Azotobacter. 

“ Aération.— The legume bacteria are all aérobic and the nodules 
on the roots of the plants are usually near the surface. Although 
nodules will form on plants grown in water cultures, yet they are 
not as large and active as when grown in a well aérated soil. The 
addition of oil to a soil or water culture in which legumes are growing 
prevents the formation of the nodules. Moreover, as shown by 
Whiting, the legumes get their nitrogen through the root and not 
the leaves. The result of cultivation of legumes is, therefore, 
threefold: (1) The loosening up of the soil, thus making available 
to the nodule bacteria atmospheric nitrogen and oxygen; (2) the 
working of the soil increases other bacterial activity which in turn 
renders soluble potassium, phosphorus, and other essential elements 
in the soil; (3) the loose aérated surface tends to conserve the 
moisture of the lower layers which can be drawn on by the plant, 
thus making more nearly optimum moisture conditions. 

Moisture.—The root systems of plants vary greatly with the 
moisture content of the soil. Gain found that legumes grown in 
moist soil spread widely, were full of water, became covered with 
root hairs, and presented a large surface of young tissues. In the 
dry soil the roots were less spreading and the epidermis was greatly 
thickened. 

In moist soil the tubercles of the peas were scattered all over the 
roots, were five or six times as abundant as in the dry soil, and were 
about four times as large and ovoid in shape; while in the dry soil 
no tubercles were produced on the superficial roots. At a depth of 
about 20 centimeters some tubercles were found of a hemispherical 
shape and much smaller than those grown in moist soil. 


INFLUENCE OF FERTILIZERS 315 


On beans about twenty times as many tubercles were found in 
the moist soil and microscopic examinations showed important 
differences in the number of bacteria present and the structure of the 
tubercles. Similar results were obtained with lupines and other 
plants. This is what is to be expected, for when the root system is 
not actively functioning the nodules are slowly destroyed by the 
nodule-forming bacteria within and the saprophytic organisms 
without. The nitrogen fixed by the plant is proportional to the 
number and size of the nodules. Hence, the gains made in combined 
nitrogen are dependent upon the water applied to the legume. 
This optimum will vary with different soils. Kalantarov found in 
a loam soil that nodule bacteria require for their growth a minimum 
moisture content of about 30 per cent., whereas Prucha found in a 
sandy soil the optimum moisture content was from 20 to 40 per cent. 
Wilson found that an increase in moisture content from 35 to 45 
per cent. more than doubled the production of nodules, while with 
an. increase from 45 to 55 per cent. it was nearly doubled. There- 
fore, water is necessary for the normal functioning of the plant and 
bacteria, and it tends to leach out the soluble nitrogen and thus 
stimulates to greater action the legume bacteria, for it is known that 
the legume feeds first on the combined nitrogen of the soil and turns 
to the atmosphere only when this is greatly reduced. 

Excessive quantities of water may exclude the nitrogen from the 
roots and also favor anaérobic action, both of which would be 
detrimental to the legume bacteria. 

Temperature.—It is a well-known fact that the temperature of a 
soil varies with its moisture content and a water-logged soil is slow 
to heat up. Gain found the temperature of moist soil at midday 
to be 7 degrees cooler than dry soil. This difference in temperature 
persists throughout the day and night and is noted in a diminished 
degree even to a depth of four feet. This difference may be suffi- 
cient in some soils to greatly decrease or increase the metabolism of 
the organism which Zipfel finds is at its optimum at a temperature 
between 18 and 20° C. 

Influence of Fertilizers.—The legume bacteria require the same 
elements for their growth as do other plants, and the application 
of fertilizers to a soil which increases the available potassium and 
phosphorus is attended by an increased bacterial activity. How- 
ever, it has long been known that nitrates inhibit nodule formation. 
Wilson found that nodule development was prevented by the 
presence of nitrates, sulphates, and ammonium salts, although the 
organisms retain their vitality in the presence of these salts. It is 
thought by some that the addition of soluble nitrates to the soil 
decreases by a kind of compensatory action the formation of root 
tubercles by legumes. Legumes growing on soil rich in nitrates 
may actually be immune to the nodule bacteria and prevent their 


314 SYMBIOTIC NITROGEN FIXATION 


entrance into the roots. Small quantities of nitrates tend to 
stimulate. 

Legumes Associated with Non-legumes.— or centuries it has been 
the practice in China, Japan, Western Asia, Northern Africa, as well 
as ancient Rome, to grow legumes and non-legumes in combination 
and there is no doubt that time and again practical farmers have 
noted the more vigorous growth and darker green of non-legumes 
when so grown, long before the investigations of Hellriegel and his 
associates established the fact that legumes are capable of utilizing 
atmospheric nitrogen. His discovery gave the key to the mystery, 


Fic. 42.—Double pots used in showing influence of Legume on non-legume. 
(After Lipman). 


—the non-legume was getting combined nitrogen from the legume. 
This was strikingly demonstrated by a very ingenious experiment 
conducted by Lipman. He grew non-leguminous plants in soil in 
a porous pot surrounded by earth in a larger glazed earthenware pot 
in which leguminous plants were growing and found that under 
favorable conditions non-legumes associated with legumes may 
secure large amounts of nitrogen from the latter. This power of 
supplying nitrogen to non-legumes varies wth different legumes. 

At times it may appear as an increased yield, whereas at others it 
may appear as an increased proportion of nitrogen in the dry matter 
of the non-legume or both. The following table gives the per- 


SOIL GAINS IN NITROGEN 315 


centages of increase in the protein content and length of leaf in the 
grasses grown with clover over grasses grown alone (Evans). 


Protein, Length of 
Kind of grass. n X 6.25. leaf. 
Timothyecrowninlawn plat... 4 . = §. 218289 POA 
Rimothysorowmindeld. 9s ee 6 ke 28. a 7.68 19.41 


This, however, varies with the soil and there may be conditions 
in which not only the protein content of the non-legume is slightly 
reduced by the association with the legume, but that the percentage 
of nitrogen in the legzume may decrease as the proportion of non- 
legume in the mixture increases as noted by Westgate. Even in 
these cases the total nitrogen of the combined crops is usually 
increased, provided the legumes are properly inoculated. 

Soil Gains in Nitrogen.—The gains made by soil in nitrogen are 
dependent upon a number of factors. 

(1) It is self-evident that the soil must be in good physical condi- 
tion for maximum nitrogen gains. (2) The soil must contain the 
elements essential to plant growth, with the exception of nitrogen. 
The law of minimum holds rigidly in this case and the gains in nitro- 
gen are limited by the limiting element of plant production other 
than nitrogen. (3) Soils which contain sufficient available nitrogen 
for the production of good crops gain little, if any, nitrogen from the 
growth of legumes, for the plants together with the bacteria feed 
first upon the combined nitrogen of the soil and only upon atmos- 
pheric nitrogen when the soil nitrogen is exhausted. Soils may 
contain an abundance of combined organic nitrogen which for some 
reason is not rendered available and still gain from the turning under 
of properly inoculated legumes. (4) The legume must be properly 
inoculated; otherwise it obtains its nitrogen as do other plants. 
(5) The soil must be a suitable home for the legume and bacteria, 
that is, it must have a correct reaction, moisture, temperature, and 
aération for maximum nitrogen fixation. Hence, we can expect 
to find a wide divergence in the results reported by investigators. 

Frank in 1891 found that soil which had been green-manured with 
legumes showed an appreciable gain of nitrogen. And it is a well- 
known fact that, in sand culture experiments in which the nitrogen 
of the soil is very low much more nitrogen may be removed in the 
legume crop than was found at first in the soil, and after the removal 
of the crop the soil may have gained in nitrogen. But what would 
happen in normally productive soil? The most reliable data now 
existing are contributed by the Illinois Experiment Station and 
indicate that two-thirds of the nitrogen in legumes grown on soils of 
normal productive power is obtainedfrom the air. These figures were 
obtained from the analysis of inoculated and uninoculated legumes 
from like areas of normal soils and as a result of pot experiments. 
Computed by these data a four-ton alfalfa crop adds 132 pounds, a 


316 SYMBIOTIC NITROGEN FIXATION 


four-ton crop of clover adds 107 pounds, a four-ton crop of cowpea 
hay adds 115 pounds. 

These are the quantities of nitrogen which reach the soil under 
ideal conditions, but some may be lost under natural conditions 
with the drainage, and possibly by other means. The New Jersey 
Experiment Station has reported a gain of 200 pounds per acre where 
crimson clover has been grown, whereas the Rhode Island Experi- 
ment Station, as a result of pot culture experiments, reports a gain of 
400 pounds per acre yearly. This experiment extended over five 
years, and legumes were grown both in the summer and in the 
winter. The tops of the summer legumes (cowpeas and soybeans) 
were removed from the soil, while the winter legumes (vetch) were 
turned back into the soil. 

Shutt, in pot and plat experiments extending over two years in 
which mammoth red clover was grown on soil and turned under, 
showed a gain of 179 pounds of nitrogen per acre to a depth of 9 
inches in the pot experiments and 175 pounds to a depth of 4 inches 
in the plat experiments. A light sandy loam with a sandy subsoil, 
when planted to clover continuously and reseeded every two years, 
doubled in nitrogen in ten years. ‘This was a yearly gain of nitrogen 
of 50 pounds per acre. 

Soil Inoculation.—The early experiments demonstrated that 
legumes assimilate atmospheric nitrogen only when properly inocu- 
lated. Since that time much has been written on soil inoculation. 
However, it is being found that in the majority of cases where the 
physical and chemical conditions of the soil are optimum, the 
ordinary legume bacteria are already present. This is especially 
true where that particular legume has been grown in that district 
for some time. The legume organism may have been in the virgin 
soil, having come from the native legume, or carried into the soil 
with manure or dust. When a new legume is being introduced into 
a district, one should see that the soil is properly inoculated for the 
members of that group. Successful seed inoculation can be per- 
formed with fresh properly prepared artificial cultures, but in many 
cases this has not proved successful and in the majority of cases 
inoculation with soil known to be infected is to be preferred. ‘The 
method suggested by the Illinois Experiment Station for large seeds, 
such as soybeans, is very satisfactory. ‘The seeds are thoroughly 
moistened by a 10 per cent. solution of glue and sufficient dry 
pulverized infected soil sifted on to absorb all of the moisture. ‘The 
seed is shovelled over a few times. Such infected seed should be 
planted very soon or else spread out to dry to prevent mould action. 
Neither infected seed nor soil should be long exposed to bright sun- 
shine, as this is very destructive to the bacteria. 

Where the old plants are to be inoculated a few hundred pounds 
of soil may be obtained from an old infected field spread on the new 
field and harrowed. 


ALTERNATIVE METHOD 317 


Dr. C. B. Lipman gives the following method for inoculating 
beans, and in a modified form it can be used for other legumes: 

“Method Involving the Use of One Commercial Culture.— Prepare 
one-half barrel full of good loam soil (150 pounds) with sufficient 
water to make about optimum moisture conditions. This soil can 
be kept in a shallow vat about a foot in depth or in some other 
convenient receptacle where it can be well aérated. Purchase one 
commercial culture from any of the commercial firms selling legume 
bacterial cultures, choosing a culture for beans of the variety desired. 
The amount usually sold in a culture for one acre is sufficient. 
Shake this up with a few quarts of boiled water. The shaking should 
be continued for about ten minutes to get all the bacteria in sus- 
pension. Pour this suspension all over the surface of the soil in the 
vat and add to the solution about one-quarter of a pound of ordinary 
sugar per one hundred pounds of soil used in the vat. This should 
be distributed as evenly as possible through the soil and the latter 
thoroughly mixed with a spade or hoe, thus distributing both the 
sugar solution and the culture. After that the inoculated soil is to 
be kept in a warm place like a kitchen or a warm stable and the 
moisture content maintained at optimum until you are ready to use 
it for the inoculation of beans when they are planted. It is well to 
allow a period of two or three months for such incubation. 

“At the end of the incubation period or when getting ready to 
plant, shake up some of the inoculated soil with clean water for 
a few minutes as before to get a good suspension of the bacteria. 
Pour enough of this suspension over the bean seed in large tanks 
or similar receptacles to wet the seed thoroughly, but not enough 
to allow any excess of water. ‘Then spread the bean seed out on a 
canvas in a thin layer in the shade. As soon as the seed is air-dry and 
will not stick, place in a planter and plant immediately. In cases 
in which only small quantities of seed are to be planted, the suspen- 
sion need not be made, but the inoculated soil in small quantities 
can be mixed with the seed in the planter and allowed to drop with 
the beans as they are dropped from the machine, thus introducing 
the bacteria into the soil with every seed, or nearly so. 

“ Alternative Method.— Where it is not desired even to purchase 
one commercial culture, inoculation can also be carried out entirely 
successfully by obtaining soil from a garden in which beans have 
grown successfully for some years and using that soil for making up 
the soil suspension or for mixing with the seed as above described. 
In other words, this garden soil, which contains the necessary bac- 
teria, will serve fully as well as the inoculated and incubated soil 
just described above. This is of course the simpler method to those 
who have access to garden soil which has produced beans successfully. 
Soils like this may also be obtained from old and more extensive 
bean fields, where successful bean-growing has been carried out. 


318 SYMBIOTIC NITROGEN FIXATION 


For small plots, such soil can be directly harrowed into the soil to 
be inoculated after being spread (about one bushel per acre) in moist 
condition on a cloudy or rainy day.” 

Commercial Cultures.—Because inoculation by means of soil from 
old fields may transmit fungus diseases, weed seed, and necessi- 
tates the transference of large quantities of soil numerous workers 
have endeavored to inoculate with pure cultures. The first 
attempt was made by Nobbe and Hiltner who placed on the market 
a preparation called “nitrogin.”” Eight kinds were prepared suit- 
able for the different legumes and were sent out on gelatin. Some 
of the results were satisfactory, but on the whole the percentage of 
failures was so great that the method was largely discredited. 

Later the subject was investigated by Moore who considered the 
failures of Nobbe due to the fact that he had grown his cultures on 
gelatin. This contained combined nitrogen in abundance and the 
bacteria lost their virulence and no longer possessed the power of 
forcing their way into the roots of leguminous plants and producing 
nodules. Moore used a nitrogen-free medium for the growth of 
his cultures, thus increasing their nitrogen-fixing power. ‘They 
were distributed on cotton. Later liquid cultures were used and 
since that time many different media, including even soil humus 
have been used by different workers with varying degrees of 
success. Asa result, at the present time some commercial cultures 
are being put upon the market which are probably just as efficient 
as are the natural-occurring soil organisms. However, some work- 
ers claim to have developed organisms possessing a high physio- 
logical efficiency. But after taking all the tacts into consideration, 
one must conclude that at the present time the pure cultures have 
little advantage over the natural-occurring organisms. 


REFERENCES. 


Lohnis: Handbuch der Landwirtschaftlichen Bakteriologie. 

Lafar: Handbuch der Technischen Mykologie. 

Voorhees and Lipman: A Review of Investigations in Soil Bacteriology, U. 8. 
Dept. Agr., Off. Exp. Sta. Bull. 194. 

Whiting: A Biochemic Study of Nitrogen in Certain Legumes, Illinois Exp. 
Sta. Bull. 179. 

Burrill and Hansen: Is Symbiosis Possible between Legume Bacteria and Non- 
legume Plants, Illinois Exp. Sta. Bull. 202. 


CHAPTER XX.YV. 


CROP ROTATION. 


From time immemorial it has been considered a self-evident fact 
that where crop rotation is practised there is a bigger and better 
yield. The farmers of ancient Rome understood that crops fol- 
lowing beans, peas and vetches were usually better than those 
following wheat or barley, but it was not until the last quarter 
of the nineteenth century that it was learned that the leguminous 
plants, with the aid of associated bacteria, have the power of feeding 
on the free nitrogen of the air, whereas the non-leguminous plants 
have not this power and require a supply of combined nitrogen. 
Today the best farmers practise some system of crop rotation. 
They have learned from experience that where crop rotation is 
practised the crops are bigger and better than under the single 
crop system. ‘This is usually interpreted as indicating that crop 
rotation has increased the fertility of the soil. Many farmers 
plant legumes for a number of years on run-down soil, remove the 
entire crop and feel confident that their soil is becoming richer in 
plant-food. Let us examine some of the results obtained in care- 
fully planned experiments to see if this conclusion is warranted by 
the experimental evidence. 

Essential Elements.— Plants are composed of ten elements, each 
one of which is absolutely essential to growth and seed formation. 
Only two—carbon and oxygen—are secured from the air by all 
plants; only one—hydrogen—from the water; the other seven are 
secured by all plants from the soil. One class of plants—the 
legumes—may, under appropriate conditions, obtain their nitrogen 
from the air. Six elements—phosphorus, potassium, magnesium, 
calcium, iron and sulphur—are obtained from the soil by the growing 
plant. 

Element Added by Legumes.—'The great majority of agricultural 
soils contain large quantities of all these elements, with the excep- 
tion of nitrogen, phosphorus and potassium. These are used by 
the growing plant in larger quantities than are any of the other 
elements which are obtained directly from the soil. In the great 
majority of soils nitrogen, phosphorus, or potassium is the limiting 
element in crop production. Therefore, our problem resolves 
itself into the question: Can crop rotation maintain these elements 
in the soil in quantities sufficient for maximum yields? Phosphorus 


320 CROP ROTATION 


and potassium are obtained by the growing plant only from the 
soil; it is, therefore, self-evident that no simple system of crop 
rotation can maintain the phosphorus and potassium, since the 
quantity within the soil must of necessity be reduced with each 
crop removed, the extent depending upon the specific crop grown. 
Hence, nitrogen is the only element which we can hope to maintain 
by crop rotation. But this is the element which is found in the 
soil in smallest quantity and removed by most plants in larger 
quantities than the phosphorus or potassium. Moreover, large 
quantities of this element are at times lost from the soil by leach- 
ing, while the loss of the others is comparatively small. It is of 
the greatest importance, therefore, that this nitrogen be supplied 
to the soils in sufficient quantities for crop production and in the 
cheapest manner possible. The total quantity of these three ele- 
ments found in an acre-foot section of two Utah agricultural soils, 
assuming one acre-foot to weight 3,600,000 pounds, is given below: 


Greenville Farm (Utah), Nephi Farm (Utah), 


pounds per acre. pounds per acre. 
Nitrogen Pre Ne ess eR ee 4,904 3,744 
Phosphorisen sh he ae ae 2,700 8,388 
IPGtASSIUIEO Re ee aye, anes Le ee On) OU) 87,840 


Both soils contain an abundance of potassium, but the supply 
of phosphorus and nitrogen is much lower. A study of these 
results shows that a 50-bushel crop of wheat each year for forty- 
nine years would remove the equivalent of the total quantity of 
nitrogen in the Greenville soil to a depth of one foot, while a similar 
crop on the Nephi farm would accomplish this in just thirty-seven 
years. It would, however, require a 50-bushel crop one hundred 
and seventy years to remove the phosphorus from the Greenville 
soil and five hundred and twenty-five years to remove it from: the 
Nephi soil. Of course a crop would never remove all the nitrogen 
or phosphorus from a soil, but in actual practice the elements are 
slowly removed, the crop yields beng reduced each year until 
a certain minimum is reached. When crops can no longer be 
produced economically then the owner abandons his soil, moves 
on to virgin soils, or if it be in an old district he resorts to the 
expensive commercial fertilizer. The illustration is, however, sufh- 
ciently accurate to make it clear that the limiting factor, in so far 
as soil fertility is concerned in both of these soils, is the nitrogen. 
And it is true of the great majority of all soils that an increased 
nitrogen supply means an increased yield. This principle is one 
of the fundamentals of soil fertility. 

Nitrogen.— Nitrogen exists in the atmosphere in inexhaustible 
quantities, every square yard of land having seven tons of nitrogen 
lying over it, or if the quantity covering one acre could be combined 
into the nitrate it would be worth as a fertilizer $125,000,000. 


ROTHAMSTED ROTATION 321 


Now it has been demonstrated that the legumes—peas, beans, 
alfalfa, ete.—when properly infected have the power of feeding on 
this limitless supply of atmospheric nitrogen, while the non-legumes 
—barley, wheat, oats, ete.—must depend upon the supply within 
the soil, and the farmer must take advantage of this fact to supply 
nitrogen for his crops, as the commercial fertilizer cannot be eco- 
nomically used for the production of most crops, as is seen from the 
fact that the nitrogen in a 50-bushel wheat crop would cost $14.40, 
or 20 tons of sugar-beets $15.00, or 1 ton of alfalfa hay $7.50, if 
bought as a commercial fertilizer. But will the legume draw nitro- 
gen from the atmosphere while there is a supply in the soil, or will 
it follow the line of least resistance and turn only to the atmosphere 
when nitrogen is lacking in the soil? If it does, it must first drain 
the soil of its valuable nitrogen and thus leave it no richer than it 
was before the legume was grown upon the soil. This is the prob- 
lem which this chapter is to answer. 

Rothamsted Rotation.—Crop rotation has been practised for 
centuries, but the oldest system on which we have accurate infor- 
mation is the one on Agdell Field at the Rothamsted Experiment 
Station. This system was inaugurated in 1848 and is still being 
carefully followed. It consists of a four-year rotation, as follows: 

First year: Swede turnips (rutabagas). 
Second year: Barley. 

Third year: Clover or beans. 

Fourth year: Wheat. 

Still another system has been running parallel and similar to 
this, except that fallow cultivation is practised in the third year 
instead of growing a legume. The average yields for twenty-year 
periods are given below. ‘These systems are of special interest to 
western farmers, for when we substitute sugar-beets for the turnips 
and alfalfa or peas for the clover or beans, we have nearly an ideal 
rotation for our soils. 


Legumes. Fallow. 
Crop. ve 
| Yield 1st | Yield 2d | Yield 3d | Yield 1st Yield 2d | Yield 3d 
| 20 years, | 20 years, | 20 years, | 20 years, | 20 years, | 20 years, 
| 1848-68. | 1868-88. | 1888-08. | 1848-68. | 1868-88. | 1888-08. 
| | | 
| = | 
Turnips: | | | 
Roots (bso es le o2040F 1 723510 967.0 | 5785.0 | 3067.0 | 2502.0 
Leaves (lbs.) ae ee Oe 447.0 | 242.0 629.0} 538.0 458.0 
Barley: | | ; 
Grainy (bu) er Ae 2 38.0 22.5 | Lend BCU Peete: F597 
Straw (lbs.) . . . «| 2373.0) 1496.0 1726 02244" 0) 14890) ee 72e0 
Wheat: | 
Grains) tae 29.6 201 DET SS 3415 ll emo es PaO 
Straw (Ibs.) . . . ./| 3169.0 | 2082.0 | 2445.0 | 3761.0 | 2420.0 | 2412.0 
| | | 


21 


o22 CROP ROTATION 


Even where the legume was used in the system there had been 
a decline in the yield. The yield of the turnips during the first 
twenty years was 5264 pounds, the second 1723, and the third 
only 967 pounds, thus showing a decrease of about five-sixths the 
original in sixty years. 

The results with the barley are no better, for there is a drop 
from the fair yield of 38 bushels per acre during the first period to 
only 13.7 during the third. The wheat which followed the legume 
in the rotation, and hence occupied the most favored place in the 
system, shows a decrease of 5.3 bushels. Not even a good yield 
has been maintained for the clover, for from 1850 to 1874 the 
average yield was 4165 pounds, while from 1882 to 1906 the yield 
was only 1246 pounds. In reality we find no greater decline in 
the yields where fallow cultivation is practised. But both sys- 
tems strongly testify to the fact that rotation is not maintaining 
the productive powers of this soil. And the evidence is strong 
that the legume gets no more nitrogen from the air than that 
which is removed with the plant. Otherwise, we should expect 
better results in the legume system than in the fallow system. 

Nitrogen Obtained from Atmosphere by Legumes.—'That the 
alfalfa, when grown on fertile soil and the crop removed, does not 
increase the nitrogen of the soil is seen from experiments con- 
ducted by Dr. Hopkins at the University of Illinois. The experi- 
ments were made possible by the fact that many of the Illinois 
soils do not normally contain the symbiotic bacteria which make 
it possible for the alfalfa to obtain nitrogen from the air. This 
being the case, a field was taken which had not grown alfalfa and 
which did not contain the symbiotic nitrogen-gathering bacteria. 
This was planted to alfalfa, only one-half of it being inoculated 
with the legume organism. ‘To some of the plots were added lime 
and phosphorus to make sure that these were not the limiting fac- 
tors. The results thus obtained are given below: 


Pounds in crop: 


——_—_——————-._ Pounds, nitrogen 
Plot No. Treatment applied. Dry matter. Nitrogen. fixed by bacteria. 

la Nones ti 5 eee be kc ee ESO) PA el 40.23 
1b Bacteria.) co Soa ee pee oO 62.04 ee 

2a TSIIMNIG © Scie ta ey eetce coy gin dee O0 26.20 41.82 
2b lime bacteria... : . . 2570 68.02 ne 
3a Lime phosphorus. . . . 1740 35.40 \ 53.65 
3b Lime phosphorus bacteria . 3290 89.05 jer 


It is evident from these results that the alfalfa had obtained 
from 40 to 53 pounds of nitrogen fromthe air, depending upon 
the treatment. There was slightly more than one-third as much 
nitrogen in the alfalfa crop from the uninoculated asin the inoculated. 
Therefore, it is quite evident that the alfalfa in these plats had 
obtained one-third of its nitrogen from the soil and two-thirds from 


LEGUMES FEED ON NITRATES 329 


the air. Now, nitrogen is required by the root for its growth as 
well as for the growth above the ground, and we have every reason 
for believing that the root also would obtain it in the same pro- 
portion from air and soil as did the hay crop. 

Distribution of Nitrogen in Legumes.—If we examine dry matter 
and total nitrogen occurring in the roots and stalks of alfalfa, we 
should be able to decide whether more nitrogen is being returned 
to the soil in the roots and residues than is removed by the growing 
plants. 

The results for this comparison have been obtained from Illinois 
and Delaware experiments and are tabulated below: 


Dry matter Nitrogen Per cent. of 
per acre, per acre, total nitrogen, 
Legume, pounds. pounds. in tops. 
Sweet clover: 
SLOPE EPO eee are), Se es le 410 9029 174 \ 76 
Rootsandresidues . . . . . 3748 54 f 
Crimson clover: 
INE Ss es Ee Sha Oe ae oe a 103 | 70 
Rotate rune a. Py fee 2022 41 f 
Alfalfa: 
Boone ee eee. oe ey OSD 40.4 f 


With the clover, three-fourths of the total nitrogen is found 
in the plant above ground and only one-fourth in the roots, while 
alfalfa shows a greater proportion in the roots—40 per cent. This 
represents the proportion for the first-year growth for alfalfa and 
it is not likely that in the older plant this proportion of the total 
nitrogen would be maintained in the roots. Hence, it is quite 
certain that if only two-thirds of the total nitrogen of the plant 
is obtained from the air the quantity returned to the soil with the 
roots and plant residues does not exceed that removed from the 
soil by the growing plant, which would give no increase in soil 
nitrogen from the growing of a legume where the entire crop is 
removed, and this even where the roots are allowed to remain 
and decay. Yet we find some farmers who remove the roots from 
the soil and even then expect an increase in their soil fertility. 

Legumes Feed on Nitrates.—It is, therefore, rather certain that 
the legume, where the crop is harvested, does not increase the 
soil nitrogen of the fertile soil of Illinois and other soil fairly rich 
in nitrogen. But what will happen on the arid and semi-arid soil 
where nitrogen in many cases is the limiting element and is present 
in much smaller quantities than it is in the soils on which the 
experiments considered have been conducted. Experiments which 
have been conducted at the Utah Experiment Station during the 
last twelve years have demonstrated that even on soils poor in 
nitrogen the legume first feeds upon the combined nitrogen of the 
soil. It is known that plant residues and other complex nitrogen 


324 CROP ROTATION 


compounds found in the soil are transformed by bacteria into 
ammonia, and this in turn by another class of bacteria into nitric 
nitrogen, and it is mainly on this nitrogen that the growing plant 
feeds. The quantity of this found in the soil at different periods 
under different plants has been measured at the Utah Experiment 
Station and the average results for twelve years are given in tabular 
form below, stated as pounds of nitric nitrogen per acre to a depth 
of six feet. 


Season. 
Crop. == —— Average. 
Spring. Midsummer. Fall. 
Alfialic 2G 2s" ei else eee a Ua 15.8 32.8 23.6 
Oats aN) Se Ae oe OT 14.1 20.6 23.5 
Cornuns ce ec in tees Ler ee ee eS 18.9 22.0 21.9 
IPOtRTOES!>~tant yee. ao ee ee Se 60.8 54.2 65.3 
Fallow Scath ter Ota Sal ge Mma ee tr lief Leta) 53.6 62.6 65.9 


The legume, alfalfa, removes the nitric nitrogen from the soil 
equally as fast as do the non-legumes. Yet this soil was well- 
inoculated with the symbiotic bacteria which undoubtedly assisted 
- the alfalfa in obtaining free nitrogen from the air when needed, but 
not until the soluble nitrogen had been drained from the soil to 
its full extent, as shown by the fact that alfalfa soil never contains 
more than does oat and corn land, and is very poor as compared 
with potato and fallow soil. 

Nitrification in Soils——It may be argued that the small quantity 
of nitric nitrogen in the alfalfa soil is due to a lack of its formation, 
as it is not needed by the legume, and hence not formed. This 
conclusion, however, is not warranted by the facts in the case, as 
may be seen from the results obtained where nitrification was 
measured. ‘These also are the average results extending over a 
number of years and obtained at the Utah Experiment Station. 


Milligrams, nitric nitrogen produced 


P in 100 gms. of soil in twenty-one days. 
Crop. —_——_—_s——— -__ Average. 


Spring. Midsummer. Fall. 
Alfalfa Tale the al Nee rt ke Sree icoalat ie to the! Ha 7.48 3.08 4.56 
Oats. eS AeA Baebes 40 4.00 3.00 3.138 
(Orn HALAS Les Pe Sica a eS 3.50 1.48 2.38 
Potatoes . . gt ee RAI 15.55 5.60 8.04 
: Fallow Mpa) igvtes oat ee ee LOO D000 2.48 3.09 


Here the quantity of soluble nitrogen produced in the alfalfa 
soil is greater than that produced in either the oat, corn, or fallow 
soil. There is no doubt that this is one reason why an increased 
yield is obtained the year following the plowing up of legumes for this 
increased action also occurs the next year after an alfalfa field is 
planted to some other crop. This is due to the stimulation of 
bacterial organisms of the soil by the alfalfa plant so that they 
make available faster the nitrogen of the soil, but this only depletes 
the soil of its nitrogen more readily than the non-legume, as it is 
the nitrogen already combined in the soil on which the nitrifying 


HOW TO MAINTAIN SOIL NITROGEN 325 


organisms act. Hence, we must conclude that alfalfa not only 
feeds closer on the soluble nitrates of the soil, but it also makes a 
greater drain upon the insoluble nitrogen of the soil by increasing 
its nitrifying powers and would therefore deplete the soil if the 
entire crop be removed, more readily than would otl 
conclusion which is borne out by the direct analysis of the soil. 

The analysis of a great number of Utah soils which have grown 
various crops for a number of years—some of them having been 
into alfalfa or wheat for upward of thirty years—revealed the 
fact that almost invariably the alfalfa soil contained less total 
nitrogen than did the wheat soil. The average for a great number 
of determinations made from alfalfa soils was 7232 pounds per 
acre of total nitrogen, while the average for a great number of 
wheat soils was 7398 pounds. These are average results from a great 
number of determinations made on adjoining alfalfa and wheat soil 
and they clearly indicate that in ordinary farm practice the alfalfa 
is making just as heavy a drain upon the soil nitrogen as is the 
wheat. 

Hence, from a consideration of the yields obtained in crop rota- 
tion, the relative quantities of nitrogen obtained from the atmos- 
phere and the soil by the alfalfa, the feeding and stimulating effect 
of the alfalfa upon nitrification, and finally the actual quantity of 
total nitrogen remaining in the soil after wheat and legumes, we 
must conclude that the legume does not increase the nitrogen of 
a common agricultural soil—even in the arid region where the 
nitrogen is low—when the entire crop is removed. 

This conclusion does not, however, mean that crop rotation 
should not be practised, for there are many reasons why crop rota- 
tion commends itself to the careful farmer, but it must not be used 
and the legume removed with the intention of maintaining soil 
fertility. This may appear to be an unfortunate conclusion, but 
it is just the reverse, and if its teachings be heeded it means a 
fertile soil and an economic gain to the farmer from the system of 
farming which it requires him to adopt. 

How to Maintain Soil Nitrogen.—There are two practicable 
methods of maintaining the nitrogen content of the soil. (1) 
Planning systems of crop rotations with legumes, the legumes 
being plowed under and allowed to decay, thus furnishing nitrogen 
to the succeeding crop; (2) practising a combined system of crop 
rotation and livestock farming. 

Three tons of alfalfa contain 150 pounds of nitrogen, all of 
which we could assume came from the atmosphere. Assuming 
the quantity found in the roots as coming from the soil, this is the 
equivalent of the nitrogen found in the grain and straw of 75 
bushels of wheat. If the alfalfa is plowed under some of the 
nitrogen would be lost to the growing plant in the processes of 
decay and leaching, but that the total nitrogen of the soil may 


326 CROP ROTATION 


actually be increased by the turning under of the legume is certain 
from field experiments. 

The Dominion of Canada Experiment Stations grew mammoth 
clover for two successive seasons on a soil very low in nitrogen. 
The two cuttings of mammoth clover with all the residues were 
turned under each year with the result that the soil gained as an 
average 177 pounds per acre of total nitrogen which is the quantity 
ot nitrogen found in three 40-bushel crops of wheat, provided the 
straw was returned to the soil, as two tons of this contains 20 
pounds of nitrogen. On the other hand, work on the soil of the 
Utah Nephi Experiment Farm, with a rotation of wheat and peas 
where the peas were plowed under, showed a gain in total nitrogen 
of 240 pounds in four years. That is, in addition to furnishing the 
small quantity of nitrogen required by the wheat crop, the peas 
had added to the soil an average of 60 pounds of nitrogen per year. 

The second method of maintaining the nitrogen and organic 
matter of the soil—the combined rotation and livestock method— 
is the more practical, and if systematically practised will not only 
maintain the nitrogen of the soil but will prove of great economic 
value to the individual following it. For it consists of a rotation 
in which the legume plays a prominent part. The legume to be 
fed and all the manure returned to the soil, which would mean the 
selling from the farm of the hay crop in the form of butter, milk or 
beef which carries from the soil only a fraction of the nitrogen 
stored up by the legume. Moreover, it brings for the producer 
much greater returns than does the system in which the legume is 
plowed under. 

It must, however, be remembered in this system that only three- 
fourths of the total nitrogen of the feed is recovered in the dung 
and urine. So that in place of three tons of alfalfa adding 150 
pounds of nitrogen to the soil from the air, it would add only 120 
pounds, and this is where all of the liquid and solid excrements are 
collected and returned to the soil. But where the alfalfa is to be 
fed and the manure returned to the soil, the legume can occupy 
a much longer period in the rotation and that with greater economy 
than where the legume is to be plowed under directly. 

Hence, we find that if these principles which have been estab- 
lished for soils even low in nitrogen be systematically applied to 
the soil, it will result in greater revenue from an increased live- 
stock industry and will maintain the soil rich in nitrogen and 
organic matter in place of depleting it of its stored-up nitrogen, 
as is so often the case with the present methods. 


REFERENCES. 


Hopkins: Soil Fertility and Permanent Agriculture. 
Greaves, Stewart and Hirst: Influence of Crop, Season and Water on the Bace- 
terial Activities of the Soil, Jour. Agr. Research, ix, 293-341. 


CHAP THR XOX VE. 
CELLULOSE-DECOMPOSING ORGANISMS. 


Tuer plant,residues which find their way into the soil contain, 
in addition to protein, non-protein compounds. ‘These are decom- 
posed by microérganisms, thus liberating the energy and returning 
the carbon to the atmosphere so that it is again available to plants. 
The reactions occurring in this process are probably the reverse 
of those occurring in the fixation of carbon by the plant. 


CO: + HO CO. + HO 
sugars ana sugars eneney 
Plant Ss absorbed Microérganisms é ie liberated 
cellulose cellulose 


Cellulose.—'‘The term cellulose does not designate a single indi- 
vidual compound but undoubtedly a whole series of compounds. 
All of these are extremely complex and pass gradually from the 
tender hemi- or pseudo-cellulose of the young plant, which is 
comparatively soluble in acids and alkalies, to the more complex 
and very resistant lignocelluloses. All are forms of cellulose, but 
their properties are exceedingly different. The first may serve as 
sfood even to man, but the latter is highly resistant to all the common 
solvents. It is, however, dissolved by a few special solvents, such 
as ammoniacal solutions of copper oxid, carbon bisulphid in sodium 
hydroxid, and a few others. Cellulose is nitrogen-free and is 
made up of carbon, hydrogen, and oxygen having the empirical 
formula, (CsHiwOs;)n- On hydrolysis, it yields various sugars, 
depending upon its source, as glucose, mannose or xylose. In 
the process of hydrolysis, there results certain intermediate 
dextrin bodies, a study of which has shown cellulose to be ex- 
tremely complex. Besides these there are certain gums, pectins, 
lignins and similar compounds, which are nearly related to cellu- 
lose and which have not been differentiated from the true cellulose 
by many investigators. The results are that the power of decom- 
posing cellulose has been attributed to certain organisms but a 
careful study of the subject has revealed later that the organism 
decomposed some of the related compounds but left cellulose 
unaltered. 

Early Observations.—'That carbon passes through a definite cycle 
from the solid organic tissues of plants to the gaseous form of the 


328 CELLULOSE-DECOMPOSING ORGANISMS 


atmosphere has been known for a long time, but it was usually 
thought of as passing from the solid complexes to the gaseous 
compounds through its direct combination with oxygen at a high 
temperature. In fact, this was considered as being the only 
method until Pasteur pointed out that there were other means. 
He considered it as being brought about by molds. Later Mit- 
scherlich (1850) observed that when moist potatoes decay the cell 
wall is dissolved and the starch gradually passes out. This he 
thought to be due to a group of organisms, but nothing was done 
to show that it was the work of any species until about fifteen 
years later when Trectl isolated an organism which had the power 
of decomposing young plant tissues and which was stained blue 
by iodine. To this organism he gave the name amylobacter. 
The organism he claimed had the power of decomposing cellulose 
with the formation of butyric acid, carbon dioxid, and hydrogen. 
As all of his work, however, was carried on with plant tissues, it 
leaves. a question as to whether the amylobacter had actually 
decomposed cellulose or only some of the nearly related compounds. 

The decomposition of cellulose in manure was studied by Dehé- 
rain, Gayon, Herbert and Popoff. The last investigator was the 
first to recognize the similarity between the method of production 
of methane in sewage and the intestines of animals. He studied 
the action which took place when a medium containing Swedish 
filter paper was seeded with sewage, and obtained a large volume 
of gas, an analysis of which showed it to consist of carbon dioxid, 
methane and hydrogen. ‘The first two he thought to be due to 
a cellulose ferment, but the latter to a butyric acid ferment. At 
the end of the incubation period, there was a gummy mass in the 
fermentation flasks. 

For a long time after this the attention of the investigators 
seemed to be directed mainly to a quantitative study of the result- 
ing products of fermentation. This is especially true with the 
work of Tappeiner and Hoppe-Seyler. The former, with the idea 
of determining the bacterial changes which take place normally in 
the intestinal canal, introduced finely divided cotton or paper into 
flasks containing a 1 per cent. neutral solution of beef extract. 
The flasks and contents were sterilized and then inoculated with 
small quantities of pancreatic juice and incubated at 35° C. They 
were so arranged that the gases could be collected and analyzed. 
The resulting product consisted of acetic acid, isobutyric acid, 
acetaldehyd, methane and carbon dioxid. ‘The last two were 
in the ratio of 1 to 7.2 at the beginning of the process and 1 to 3.4 
at the close. In another set of experiments he used an alkaline 
medium and obtained the same qualitative but different quanti- 
tative results, there being a large amount of hydrogen evolved in 
the alkaline medium. 


EARLY OBSERVATIONS 329 


From his work, he concluded that cellulose undergoes a fer- 
mentation in the first stomach of ruminants and in the alimentary 
canal of all herbivora. In later work he tried to decide whether 
this fermentation was due to an organized or to an unorganized 
ferment. This he did by inoculating suitable flasks with the 
contents of the alimentary canal of oxen. The flasks were divided 
into three sets and treated as follows: (1) Heated, (2) treated 
with antiseptics (thymol and the like) and (3) untreated. Fer- 
mentation occurred only in the last set from which he concluded 
that it was due to bacterial action. From his work in general 
he decided that bacteria have the power of decomposing cellulose 
with the formation of carbon dioxid and methane and that this 
process plays a large part in the digestive processes of herbivorous 
animals. ; 

Hoppe-Seyler, who considered the fermentation process mainly 
from the changes which take place when cellulose is decomposed 
in soil or beneath water, commenced his experiments by collecting 
and analyzing the gases given off from soils and swamps. These 
he found to consist mainly of carbon dioxid and methane. Later 
he carried out laboratory determinations by placing 25.773 grams 
of filter paper into 1000 c.c. flasks containing 700 ec.c. of water and 
inoculated with mud. ‘They were so arranged that the gaseous 
products were collected over mercury. He incubated them at 
room temperature for four years. During the first year there was 
considerable gas evolved, but the evolution gradually became 
slower until at the end of four years the evolution of gas had practi- 
cally ceased. An analysis showed that 15 grams of the cellulose had 
been decomposed with the formation mainly of carbon dioxid 
and methane. He was unable to find any of the true sugars, 
although he thought it possible that there were some of the dextrin 
compounds in the solution. When air was excluded he found 
that there was a greater production of methane and a smaller one 
of carbon dioxid. From his work he considered the reaction pro- 
ceeded in two stages: First, a hydration of the cellulose with 
the formation of a hexose according to the equation, CsHjO;+ 
H.O0 — C,H»O;. From the hexose, carbon dioxid and methane 
was formed (CsHi20O, — 3CO, + 3CHs,), or perhaps acetic acid 
was an intermediate product and then carbon dioxid and methane 
were formed according to the equation, CH;COOH — CO, + CHg. 

In 1889 Schlésing published his quantitative results of the 
investigation on the decay of manure. He collected the gases 
given off in the course of two months in the decay of manure and 
analyzed them. He concluded that the change was similar to 
alcoholic fermentation. 

Three years later the work of Herbert appeared. He inoculated 
5 per cent. solutions of potassium carbonate or ammonium car- 


330 CELLULOSE-DECOMPOSING ORGANISMS 


bonate containing finely divided straw with manure. At the end 
of three months, when the evolution of carbon dioxid and methane 
had nearly ceased, he examined the residue with the following 
results: 


At beginning At end 
Substance in the straw. of experiment. of process. Loss, 
Cellulose J iras pe ee Se CRS gene as 1D 6.18 56.2 
Wioodigumice Oye te So ee ey ent oe 0200 4.67 GSi083 
Viasctlosen4: Dita o Life ctae 14s01 Beye) 16.1 


Dehérain studied the substances given off in the decomposition 
of manure with the following results: 


Top layer of Middle of Bottom of 
manure heap, manure heap, manure heap, 
per cent. per cent. per cent. 
Carbon dioxids 26m ae Le ee eo 31.0 ote 
Oxy cents tess bo cia ae © Megane 0.0 0.0 OZR 
Methane Se Me WA, Tp cer ae Mew Oe 0.0 eeiey 58.0 
Nitrogen ON ON ees cles Pr eed fees iad, 4.9 


In the layers in which there was considerable oxygen, as may 
be seen, the amount of combustible gas given off was zero, but in 
the middle and lower layers of the manure heap the resulting 
methane was over half of the gaseous product. Similar results 
were obtained by Gayon, who studied the changes resulting with 
a limited and free access of air and found that methane was obtained 
in much larger quantities when the air had been excluded. From 
this, he concluded that methane fermentation is due to an anaérobic 
organism. 

Preceding this work was that of van Senus, who found that 
cotton and plant tissues were decomposed by microérganisms with 
the formation of carbon dioxid, methane, hydrogen, butyric acid, 
acetic acid, alcohol, aldehyd and a trace of the higher fatty acids. 
He thought the methane was formed through the reduction of 
acetic acid by means of hydrogen. He considered the action as 
being brought about by two organisms—orie the amylobacter of 
Trecil, and another very small bacillus which he had isolated from 
the alimentary canal of a rabbit. He considered that they acted 
by means of an excreted enzyme, which he precipitated by means 
of aleohol and found an aqueous solution of the same had the 
power of decomposing cellulose. 

Work of Omelianski.— As may be seen from the preceding brief 
summary of the work, practically all that had been done on the 
subject prior to 1895 was directed at a study of the chemistry of 
the process and little had been done in trying to isolate in 
pure cultures the specific organism or organisms which had the 
property of decomposing cellulose. It was at this point that the 
work was taken up by Omelianski, who studied very carefully the 
chemical and bacteriological phases of cellulose fermentation. 


MORPHOLOGY AND PHYSIOLOGY dol 


In his work, the following medium was used: 


Potassium phosphate 5 1"gm. 
Miaionesrimi sulphate sus enee carmen ee Rate et Ry ns 1 
Ammonium sulphate . Cg Th OL i apis Cae] Fee 1 
Sodimmpchloniclawis-as itera aie wi OF hee ks ee nr ee wr trace 
Distiledswatcram ry kettle eee ay Sete... cee SLOOOcc: 


In some cases he replaced the ammonium salt with 0.5 per cent. 
asparagin, 1 per cent. peptone, or 0.5 per cent. beef extract. The 
solutions were placed in flasks containing filter paper and then 
inoculated. Inasmuch as the incubation period of cellulose fer- 
mentation is long and variable, he found it best to seed with con- 
siderable of the organism. Ordinarily, this was done by taking 
a small piece of the decomposing paper from an old culture. 

Soon after inoculation there was observed a slight turbidity 
in the flasks. ‘Then the paper thickened and assumed a decayed 
appearance. It was covered with little specked places where it 
had been decomposed by the organisms. ‘This latter appearance 
varied; at times the holes were large and few, and at other times 
they were small and very numerous. At still other times the 
paper seemed to thicken and then to fall to pieces. At the end of 
the process there remained a residue entirely different from the 
original paper. In old cultures the white appearance of the paper 
had disappeared ‘and it had taken on a yellowish brown color, 
which often appeared even in the surrounding solution. The 
gases given off had the odor of decayed cheese. At the height of 
the process particles of filter paper were carried to the surface of 
the liquid by the gas. ‘The above description applies to the process 
as brought about by both the hydrogen and methane organ- 
isms which Omelianski succeeded in isolating in pure cultures by 
the method of repeated heating (75° C. for fifteen minutes), which 
is based on a difference in the life history of the two organisms. 
The methane fermentation organism develops more rapidly than 
the other variety and gains the supremacy in the early stages of 
the process. If heat be applied at this stage the more slowly germi- 
nating spores of the hydrogen-fermenting organism, being in a 
resistant stage, survive. 

Morphology and Physiology.—A microscopic examination of the 
hydrogen ferment reveals the following: In the young culture the 
bacillus is about 0.5 « in width and from 4 to 8 uw in length. In 
old cultures they are from 10 to 15 win length. They never occur 
linked together in chains but appear as slightly bent rods. In 
still older cultures they take the drumstick form which gradually 
develops into a round spore about 1.5 w in diameter. They are 
readily stained by the anilin dyes. In old cultures they give the 
characteristic colors for the spore and surrounding sheath with 
carbol-fuchsin and methylene blue. They are not stained blue 


302 CELLULOSE-DECOMPOSING ORGANISMS 


with iodin, and consequently are different from the amylobacter 
of Trecil. No growth occurs usually in the ordinary cultural 
media, though Omelianski has observed on some occasions very 
minute translucent colonies on potatoes. 

This investigator carried out quantitative determinations of the 
substances yielded by the organism. It was done in flasks con- 
taining 300 ¢.c. of a mineral salt solution containing calcium car- 
bonate and Swedish filter paper. The flasks were inoculated 
with the organism and incubated for thirteen months. On analysis 
the following results were obtained: 


Resulting products. 


Cellulose at beginning of process 3.4743 Fatty acids 2.2402 
Cellulose at end of period . . 0.1272 Carbon dioxid 0.9722 
Decomposedi 3.) hp a. i ae a oad Hydrogen 0.01388 


Chief among the fatty acids yielded were acetic, butyric, and 
valeric acid. Besides these there were traces of the higher acids 
found. . 

The methane fermentation, according to Omelianski, takes 
place if a flask containing filter paper, lime and a mineral neutral 
solution be inoculated with mud or fresh horse manure and kept 
under anaérobic conditions at a temperature of from 35° to 37° C. 
After a short time a careful examination of the filter paper revealed 
numerous bacilli. By successive subculturing, while the methane 
fermentation was at its height, the hydrogen ferment was soon 
eliminated. The methane organism. is rod-shaped, slightly more 
bent than the hydrogen ferment. It never develops in chains, 
but in old cultures assumes the typical drumstick form with a spore 
in the end. The organism is 0.4 w in width and 5 win length. It 
is not stained blue by iodine and hence is different from the amylo- 
bacter of Trecil. From this it may be seen that both the vegetative 
cell and spore are slightly smaller than the hydrogen ferment. 
Though morphologically very similar, physiologically they are 
very different, since one yielded hydrogen and the other methane. 
This is shown by the quantitative studies of Omelianski. They 
were conducted in 500 ¢.c. flasks containing 2.0685 gms. of Swedish 
filter paper, 4.9482 gms. of calcium carbonate, and a 0.1 per cent. 
solution of ammonium sulphate. They were inoculated with 0.013 
em. of filter paper from an old culture. Over one month elapsed 
before fermentation became perceptible and even then it was very 
slow as is shown by the fact that the gaseous material evolved 
never exceeded 1.1 ¢.c. in twenty-four hours, and at the end of 
four and a half months had dropped to 0.01 c.c. for twenty-four 
hours. The resulting gas was mainly carbon dioxid and methane, 
0.7146 gm. of the carbon dioxid and 0.1372 gm. of the methane. 
In the flask there remained only a small amount of cellulose but 


FUNCTION 333 


a large amount of acetic and butyric acids. In fact over one-halt 
of the decomposed cellulose had been transformed into these acids. 

Later Work on Cellulose Fermentation.—Later work which has 
been carried out by van Iterson has shown that there are certain 
of the non-sporeforming, denitrifying organisms which have the 
power of decomposing cellulose. In the presence of nitrates, the 
chief products are nitrogen and carbon dioxid, According to this 
investigator, the decomposition is brought about by Bacillus ferru- 
gineus, Which is the chief cause of the brown color of humus due 
to a pigment formed from cellulose by this organism. 

Recently Kellermann and McBeth have questioned the work of 
Omelianski. While they admit the great importance of these 
organisms in the formation of humus in agricultural soils, they 
claim that the organisms described by Omelianski were not pure 
cultures and furthermore that cellulose is decomposed under 
aérobic conditions. These investigators have isolated thirty-six 
species from various sources. ‘These were found to be much more 
active than the ones described by Omelianski. They were all rod- 
shaped organisms varying in length from 0.8 to 3.5 uw. Involution 
forms have been observed for only three species. Five species 
have been found to produce spores. Twenty-seven species are 
motile; of these, seven are pseudomonas and twenty are bacilli. 
A few are facultative anaérobes. The optimum temperature lies 
between 28° and 33° C., but they grow well from 20° to 37.5° C. 
They grow readily on solid media such as beef agar, gelatin, starch 
and potato. Nineteen species liquefy gelatin. They rapidly 
decompose cellulose and other carbohydrates with the production 
of acids, but none of the organisms so far studied produce a gas. 

Function.—It may be well to call attention to the great part 
taken by this class of organisms in returning carbon to the atmos- 
phere. This is especially the case with the material which passes 
off in the sewage. In septic tanks there are millions of these 
organisms busy changing the most resistant organic matter into 
gaseous products, and many large cities today depend upon this 
for the disposal of their sewage. Organisms also take a great part 
in the purification of a-city’s water supply. They also take part 
in the formation of soil humus, as was pointed out by Omelianski 
when he gave the general reaction, 2C5H190; — 5CO, + 5CH4 + 2C, 
and he says, “It is possible that a general reaction of this sort 
torms the basis of the universal processes of humification, that 1s, 
the gradual transformation of organic substances into a mixture 
of brown and black substances with a high content of carbon, such 
as is characteristic of fossil coals. But whatever the mechanism 
of these transformations, the active participation of microérgan- 
isms in the latter cannot be denied.” 

The cellulose ferments break the plant residues into less com- 


304 CELLULOSE-DECOMPOSING ORGANISMS 


plex organic compounds which are fermented by other organisms 
with the generation of large quantities of organic acids. These 
would react with the minerals of the soil rendering them available. 
This is very likely the cause of the good results obtained from raw 
rock phosphate and stable manure on phosphorus-poor soil. The 
fermentation of the cellulose yields acids which render soluble 
the phosphorus. This formation of acids may at times, however, 
become excessive, giving rise to the sour humus of moors and 
heaths. 

It is well known that the fermentation processes in the soil 
resulting in the decomposition of organic matter may give rise to 
large quantities of carbon dioxid, methane and hydrogen. The 
hydrogen and methane do not all pass into the atmosphere, but 
according to the researches of recent investigators furnish energy 
to numerous soil organisms, the importance of which remains 
almost wholly for future workers to develop. The first work on 
this subject was done by Immendorff, who in 1892 found that 
hydrogen and oxygen may be made to unite under the influence of 
soil. He found that the oxidation of hydrogen was brought about 
only by normal soil and not by soil previously treated with chloro- 
form vapor. This observation remained unnoticed until recently 
when two papers appeared—one by Kaserer and the other by 
Solmgen—which throw considerable light cn this phase of carbon 
and hydrogen transformation. They used an inorganic solution 
containing dipotassium phosphate, ammonium chlorid, magnesium 
sulphate, sodium bicarbonate, and a trace of ferric chlorid. This, 
they inoculated with a small quantity of soil and confined in 
an atmosphere consisting of a mixture of hydrogen, oxygen and 
carbon dioxid. Growth took place and the hydrogen disappeared. 
The presence of a small quantity of carbon dioxid seemed to be 
necessary for the development of the organisms, and it would 
appear that like the nitrifying bacteria they can produce bacterial 
protein in inorganic solutions, deriving their carbon from carbon 
dioxid. This reaction, according to Lipman, is of great significance 
in agriculture, for a great loss of energy is prevented by the bacterial 
oxidation of hydrogen formed. in the deeper layers of the soil by 
anaérobic ferments. It also partly counteracts the rapid minerali- 
zation of organic materials, in that it leads to the formation of com- 
plex compounds from carbon dioxid, hydrogen and oxygen. 

Kaserer and Solmgen also obtained organisms capable of utilizing 
methane as the sole source of energy in their life process. The 
latter investigator secured pure culture of an organism which he 
named Bacillus methanicus. When grown in inorganic solutions 
confined in an atmosphere of one-third methane and two-thirds 
air, it caused the disappearance of the methane with the production, 
of considerable quantities of organic material. 


FUNCTION 300 


The cellulose ferments also perform other direct functions in 
the soil, as for instance, the liberating of plant food which is bound 
up in plant residues. Heinze has very recently ascribed to bacterial 
activities much of the benefits obtained from summer fallowing. 
In quantitative studies he found them to be more numerous in 
fallow soil than in cropped soil, and he thinks it to be due to their 
activities in rendering the cellulose more suitable as a carbon 
supply for the Azotobacter that causes the increase of soil nitrogen 
in fallow land noted by a number of recent workers. One of the 
’ more important problems of today in soil bacteriology is the rela- 
tionship between this class of organisms and other important soil 
organisms, especially the nitrifiers and the nitrogen fixers. 


REFERENCES. 


Lafar: Handbuch der Technischen Mykologie. 
McBeth: Studies on the Decomposition of Cellulose in Soils, Soil Science, i, 
437-488. 


CPLA TE Reece Lr: 
BACTERIA IN AIR. 


BaAcrERIA require for their growth moisture, food, a suitable 
temperature and usually the absence of direct sunlight. The 
moisture conditions of the atmosphere at times may be optimum 
for the growth and reproduction of bacteria, but none of the other 
conditions are. Hence, bacteria do not multiply and grow in the 
atmosphere as they do in water, soil and milk. These substances 
may and do have a natural bacterial flora, but we cannot consider 
the air as having a definite one, for the number and kind continually 
vary with many factors and there are scarcely two places having 
the same number and species of microérganisms. 

How Bacteria Enter Air.—The earth is surrounded by the atmos- 
phere, which when “looked at as a whole, its calms are exceptional, 
and its movements are the rule. We may find the gentle breeze, 
the cyclonic wind or the restless tornado, but always active. These 
movements do not confine themselves to horizontal paths, but the 
gases rise and plunge, pursue broad curves and narrow spirals, and 
would present—to an eye that could see them from above—a 
tumult like the sea in storm.” In all this aetivity it is picking 
up bits of sand, organic matter and oftentimes even water. These 
contain microdrganisms which are carried into the air and may 
subside with the particle to which they adhere or become free and 
float about for a period. 

As the waters of the ocean, lakes, rivers and smaller streams beat 
against some barrier the fine spray so formed carries into the air 
bacteria, as do also the hurrying feet and rattling wheels in a 
crowded street. Furthermore, individuals speaking or coughing 
force from the mouth numerous bacteria which for a time help to 
make up the microbial inhabitants of the atmosphere. 

Number and Kind.—The number and kind of organisms found 
in the air are governed largely by the locality. They are most 
plentiful in densely: populated areas and within buildings such as 
churches, theaters and other places where a large number of people 
congregate. Miquel found thatas an average 1 cubic meter of airfrom 
the streets of Paris contained 3480 bacteria, laboratory air, 7420, 
the air of old houses 36,000, whereas the air of the Paris Hospital 
contained 79,000 bacteria in 1 cubic meter. It is quite evident from 
these figures that air of inhabited districts contains many bacteria. 
These are carried on the dust particles. It does not, however, 
always follow that the number of bacteria in the air is an exact 


FACTORS GOVERNING NUMBER AND KIND Oa0 


measure of the number of dust particles. An examination of 
the air of the London streets showed it to contain between 300,000 
and 500,000 dust particles per cubic centimeter, but there was 
only one microérganism to every 38,300,000 dust particles. The 
number of species present will vary with the locality, but probably 
in the majority of cases it is not great. Fischer states that an 
examination of the street dust in the city of Freiburg showed it 
to contain from five to seventeen different species of bacteria in 
1 gram of dust which contained from 24,000 to 2,000,000 organisms 
per gram. Graham Smith found at the top of the Clock Tower of 
the House of Parliament in London only one-third the number 
of bacteria that were found at ground level. Whipple found 
1330 bacteria per cubic foot in air at street level, while at the tenth 
story of the John Hancock Building in Boston the air contained 
330. 

Factors Governing Number and Kind.—The sprinkling of the 
streets greatly increases the number of bacteria in the dust, but 
it decreases the number in the air. This is due to the fact that 
the moist particles are not dislodged’ and carried into the air as 
freely as are the dry. 

The air of the country contains fewer bacteria than does the air 
of the city. Miquel found as an average 300 organisms per cubic 
meter of air taken outside the city of Paris and 5445 bacteria per 
cubic meter of air taken within the city. 

The number of bacteria in air over oceans is low and varies 
with the nearness to land. Close to shore there are often very 
many, while at great distances from land the air may be free from 
bacteria. 

On mountain tops, in deserts, and in other uninhabited regions 
the air is nearly free from bacteria. The classical illustration of 
this fact is found in the experiments carried on by Pasteur to 
refute the doctrine of spontaneous generation. He exposed flasks 
containing organic infusions in various localities. Of 20 such 
flasks exposed to the air of Mer de Glace 19 showed no contamina- 
tion. In similar experiments Tyndall exposed 27 flasks containing 
beef infusion to the air of the Aletsch Glacier (8000). None 
showed contamination, whereas 90 per cent. of a similar number 
opened in a hayloft did. 

The number of organisms in air decreases with the altitude as 

well as locality. Jean Binot did not find a single microédrganism 
in 100 liters of air taken on the summit of Mount Blane. The 
number rapidly increased on descending. 


On the summit . Se Sct a el Si i a eee ie 0 
At the Grand Pieteaul RT Le a Aiea wend se i. Pek! Ln gee a ae 6 
ACT heEGranGelVial ets eo meet aa eRe Gu bn Fae eee, ule 8 
At the Place de l'aiguille Ree hee ee MIE A Wat Rg one ee I om NE ewe atl A, 
PACA CPV TEIN Ce KGa COn Mame Ihrer ne Mer Meee hi tS. van te SOM Hehe eer eS 
PAT PVLOMICAI eT Glas tiike Meteo itd ta nae area uty ek SN ON ce ag ere YA 


22 


338 BACTERIA IN AIR 


The number of bacteria in the air varies with the season, increas- 
ing from winter to summer and decreasing from summer-to wanter. 
There is also a marked decrease in the number of bacteria in the 
air after a rainstorm. ‘The rain carries them to the ground and 
also moistens the surface so that particles of dust are not carried 
into the air by every breeze. But the added moisture of the soil 
greatly increases the speed of multiplication so that later as the 
surface soil dries out more dust and with it a greater number of 
bacteria are carried into the air. It is also true that the number 
of microdrganisms in the air decreases in the winter months not 
because cold is inimical to the life of the microérganisms—for just 
the reverse is true—but the conditions are not as good for them 
to find their way into the atmosphere. This is due to the fact that 
the soil is covered with snow or the greater moisture prevents 
the dust from being carried into the atmosphere. 

It is quite evident that there would be a relationship between 
the number of bacteria in the atmosphere and the climate of that 
region. Bacteria would multiply rapidly in the soil of a warm, 
humid district and these in turn may be carried into the atmosphere, 
but the rains would quickly wash them out. Hence, there would 
be a great variation in a short time, whereas in an arid region the 
number in the air may be smaller but will not vary as greatly as 
in the humid region. 

The stay of the bacteria within the atmosphere will vary, depend- 
ing upon a number of factors: 

1. The hardy spore-forming saprophytes may remain suspended 
in the air for days or even weeks, whereas the frail non-spore- 
forming pathogens soon perish due to either drying or the steriliz- 
ing action of the sun’s rays. 

2. Small particles settle out more slowly than do large ones, 
for as the size of an object is decreased the surface area decreases 
less rapidly proportionately than does the volume. Hence, those 
bacteria which are floating free in the atmosphere would subside 
more slowly than those attached to dust particles. 

3. The time of suspension is also determined by the velocity 
of the air current. Organisms settle out of a still atmosphere 
more readily than from one in motion, whereas it may require an 
air current of considerable velocity to dislodge microdrganisms 
and bring them in suspension a slight current will sustain them. 

4. Moisture in the atmosphere tends to cause particles to adhere 
together and as they grow in size the tendency for them to settle 
out is increased proportionately. 

5. Although the air of London and many Cee cities contains’ 
numerous particles of dust, the number of living organisms is com- 
paratively small as the various gases thrown into the atmosphere 
have a slight germicidal effect upon the bacteria. 


AIR-BORNE INFECTION 309 


Bacteria in Inspired and Expired Air.— Inasmuch as the atmosphere 
contains numerous bacteria it is to be expected that many will be 
inhaled with the inspired air. It is estimated that a person living 
in London breathes about 300,000 bacteria each day and individuals 
living in other districts may take many times this number. Most 
of these are harmless and are caught on the moist mucous mem- 
branes of the upper respiratory passages, very few finding their 
way into the deeper alveoli. 

The expired air, during normal respiration, is practically free 
from bacteria. But during the acts of coughing, sneezing. and 
speaking the air is forced out and with it bacteria, some of which 
may be pathogens and if inhaled by a second individual may give 
rise to the specific disease. 

Air-borne Infection.—The air has long been considered as the 
chief vehicle for the spread of communicable diseases. ‘This 
was but natural, for until recently the virus of these diseases was 
believed to be gaseous or at least readily diffusible and borne by 
air currents. After the bacterial nature of disease was discovered 
and it was found that the discharges from the nose and mouth of the 
diseased body often contains the causative organisms, and hence 
could readily find their way into the air, this was a favorite 
method for explaining infection. Recent work, however, has 
demonstrated that the pathogens do not long retain their vitality 
when free in air, and where infection i is conveyed by air it is due to 
dust or droplet infection. 

Dust infection occurs only in the case of those diseases caused 
. by organisms which can survive considerable periods of drying. 
The most important is that of tuberculosis and is here confined to 
rooms and dusty places which have been occupied by careless 
consumptives. The extent to which dust is a factor in the trans- 
mitting of disease is not well known, but it probably is not great. 

Flugge and his students were the first to demonstrate that 
minute droplets may be emitted from the mouth during talking, 
coughing and sneezing. The droplets may be carried in a quiet 
room as far as twenty or thirty feet. The large ones soon settle 
out, whereas in the smaller ones there is a great tendency for many 
pathogens to perish. Hence, droplet infection is conveyed only a 
few feet. 


CHAPTER XXVITL 
WATER BACTERIOLOGY. 


CoMmMON things are often little prized, and this is true of water. 
Yet there is no other compound which plays so many and such 
vital parts as does this substance. It composes two-thirds of the 
body weight, entering into the make-up of every tissue. The 
muscles which do our work contain 75 per cent. water; the liver 
which acts as the body protector against poisons consists of 75 
per cent.; the bones, which* possess a tensile strength of 25,000 
pounds per square inch and are one and one-fourth times as strong 
as cast-iron, consist of 40 per cent.; the brain, the most complicated 
and wonderful organ of the body, consists of 85 to 90 per cent.; 
the blood, that cosmopolitan fluid which visits every tissue of the 
body bearing to it nutrients and from it waste products, contains 
over 90 per cent. water. All the secretions of the digestive glands 
consist mainly of water, and it is not there merely as a vehicle in 
which are conveyed the active principles, for it enters into practi- 
cally every chemical reaction through which carbohydrates, fats 
and proteins pass in the process of digestion and metabolism. 
It is the fluid in which are held the mineral nutrients which play 
such a vital part in the life phenomena. Water gives to the tissues 
their plumpness, carries off waste products, regulates the body 
temperature and acts as a catalyzer in most reactions. Hence, a 
substance which is of such vital importance and so often polluted 
or infected must receive more than passing notice by the bacteri- 
ologists. 

Classification of Waters.—From a_ bacteriological viewpoint, 
natural waters are best classified according to their relation to the 
rich layers of bacterial growth upon the surface of the earth. ‘There 
are four distinct classes: (1) Atmospheric water, (2) surface waters, 
(3) stored waters and (4) ground waters. 

1. Atmospheric water consists of rain and snow. It is really 
water which has been vaporized and then condensed. It contains 
none of the non-volatile substances and should, therefore, more 
nearly approach pure water than any of the other natural sources. 
But even this is far from pure, for as it falls through the atmosphere 
it absorbs gases and collects large amounts of floating dirt. Every 
one has observed how a shower will wash the air so that it becomes 
beautifully clean and clear. The minute the water reaches the 


CLASSIFICATION OF WATERS 341 


earth further contamination occurs and it is a well-known fact 
that some of the filthiest water used for domestic purposes comes 
from rainwater tanks. This is due both to the methods of collect- 
ing and of storing which pollutes but usually does not infect it. 

2. Surface waters include rivers, creeks and smaller streams 
and are immediately exposed to contamination. They vary 
greatly in composition, depending upon the nature of the catch- 
ment basin. Waters flowing through rock, gravel or sand forma- 
tion are better than are those which flow over or drain loam or 
swamps. But even the waters from sand and gravel regions may 
be polluted or even infected, depending upon the relationship borne 
by the drainage basin to animal life, and especially to human beings. 
In the thickly settled portions of the country and as the new dis- 
tricts build up these waters must be more carefully protected. 
Sanitary workers are being forced to the conclusion that it is 
impossible to protect such waters against contamination, and as 
far as possible such waters should be purified before they are used. 

3. Stored waters include lakes and large ponds. ‘These, when 
fresh and kept free from the pollution with the wastes of human 
life and industry make admirable sources of water. On account 
of the limited area of the drainage basins they are more easily 
protected than large streams. Moreover, the natural agencies 
for purification—time, sedimentation and enormous dilution—play 
a great part in freeing the water from any accidental foreign material 
which may find its way into the water. 

4. Ground waters. are of two classes: (a) Deep springs and 
wells, from which most bacteria and other suspensoids have been 
removed by filtration. Such waters in passing through the soil 
take up large quantities of carbon dioxid which has been set free 
by the decay of organic matter. Water heavily charged with 
carbon dioxid has a great solvent action for lime and other inor- 
ganic constituents. Hence, while such waters are usually safe they 
are hard and carry large quantities of organic material. (b) Shal- 
low springs and wells correspond more nearly to surface waters 
and are often polluted and at times infected. 

Waters are also classified as polluted and infected. A good 
water is one of high standard quality, as determined by physical 
inspection, sanitary survey of the watersheds, clinical experience, 
bacteriological and chemical analysis. 

A polluted water is one containing organic waste of either animal 
or plant origin. A polluted water is not*necessarily a dangerous 
water but is always looked on by the bacteriologist with suspicion. 

An infected water is one which contains the specific micro- 
organism which causes disease and is always dangerous. The 
bacteriologist in examining seldom proves that a water is infected, 
but draws his conclusions from indirect evidence. 


342 WATER BACTERIOLOGY 


Numbers of Bacteria in Waters.—The bacterial content of the 
several waters varies greatly. Atmospheric waters after a long- 
continued storm may be free from bacteria, whereas rain after a 
long drought may contain many. ‘There is also a variation in the 
number, depending upon whether the rain is collected in the country 
or city. Miquel obtained for the period 1883-1886 an average 
of 4.3 bacteria per cubic centimeter in the country and 19 per 
cubic centimeter in Paris. Snow contains rather higher numbers 
than does rain. Janowski found in freshly fallen snow from 34 
to 463 bacteria per cubic centimeter of snow-water. 

Surface waters are never free from bacteria, but the numbers 
vary greatly from a few hundred, in the case of clear mountain 
streams, to millions, in the case of the sewage polluted rivers. 

The number varies with the turbidity of the stream. ‘The 
Thames River carries 277 bacteria per cubic centimeter in April, 
whereas the Illinois carries between 6000 and 8000 per cubic centi- 
meter. The number also varies with the season of the year. In 
May the Potomac River carries about 750, while in March it 
earries 11,500 per cubic centimeter. The number is increased 
when the drainage basin is manured with the various animal 
manures, as it is also by the entrance of sewage into the streams. 

The bacterial content of lakes is usually lower than that of 
streams, but shows wide variations. Lake Michigan near Chicago 
gives count for from 68 to 2000 per cubic centimeter, while Lake 
Lucerne’s variation is from 8 to 51 per cubic centimeter. 

The same wide variation is shown in ground waters. Shallow 
wells and springs often contain as many and just as dangerous 
organisms as do surface waters. But deep wells and springs contain 
few organisms, and it is not an uncommon experience to find some 
which are sterile. 

The seasonal variation of bacteria in deep wells and springs 
is zero, and where we have seasonal variation in these sources of 
water it indicates surface contamination, and with shallow wells 
and springs it is often enormous. 

Surface waters are subject to marked variations in bacterial 
contents, especially during spring and fall, due to melting snow 
and rains of these seasons. A heavy shower is likely to increase 
contamination by introducing fresh material from the surface 
of the ground. Prolonged moderate rains may have the opposite 
effect and after the main impurities have been washed away may 
dilute the stream with a better water than itself. The net effect, 
therefore, depends upon the character of the stream as well as the 
catchment basin. A stream highly polluted with sewage may 
actually contain fewer bacteria after a heavy storm than before, 
but a normal stream contains more, as emphasized by the following 
data compiled by Prescott and Winslow: 


LIGHT 043 


MONTHLY VARIATION OF BACTERIA IN A NORMAL AND POLLUTED 


STREAM. 
Bacteria per c.c. Bacteria per c.c. 
Date, 1904. l ae 
Lahn Wieseck Date, | Wieseck 
(normal). (polluted). 1904-05. | eee | (polluted). 
| ee a 
July See eee 4 = 318 | 104,000 December!) 1220 21,200 
Ful veh sateen eas soe sont 132 | 156,800 | January! | 3668 29,920 
UP Hakeem cae ee oe 840 | 98,000 | February'; 5380 | 11,900 
October. = ene) : 235 | 28,400 | March! | 1210 8,250 
October scars ay) beh? 2 420 | 58,000 | April! \@ 4905 “1-9 55,910 
(Noyembern naan ane 2340 | 39,200 | May 570 | 14,800 
INevermbersen ae ee 1740 | 52,000 | June | 686 50,180 
December . . . «| 780 4 28,600 | 


Sedimentation.—Bacteria disappear more rapidly from still or 
slow-flowing streams than from rapid-flowing streams, due to the 
fact that the transporting power of a stream varies as the sixth 
power of its velocity. A current moving six inches a second will 
carry fine sand; one moving twelve inches a second will carry 
gravel; four feet a second, stones of about two pounds’ weight; 
and thirty feet a second, blocks of three hundred and twenty tons. 

The sedimentation of bacteria themselves takes place very 
slowly even in still water, for the difference in numbers between 
the top layer and the bottom layer of water in tall jars in laboratory 
experiments of a few days’ duration is very slight, being quite 
within the limits of experimental error. In the natural streams 
however, the bacteria are, to a great extent, attached to larger 
solid particles, and upon these the action of gravity is more import- 
ant. Sedimentation is one of the most important factors, according 
to Jordan, in purifying waters. He states that “it is noteworthy 
that all the instances recorded in the literature where a marked 
bacterial purification has been observed are precisely those where 
the conditions have been most favorable for sedimentation.” 

Light.—Light is one of the best germicides, for when it plays 
upon the naked protoplasm of the bacterial cell it kills both vege- 
tative and spore forms in a short time. Opinions vary, however, 
as to the part played by light in destroying bacteria in natural 
waters. Buchner found that plates containing B. tuberculosis 
were sterilized in four and one-half hours at a depth of five feet, 
but were unharmed at a depth of ten feet. Plates exposed at 
various depths and containing various saprophytes gave the fol- 
lowing counts after three hours: 


1 Rain or high water due to previous thaw. 


344 WATER BACTERIOLOGY 


Before exposure. Sunshine. Darkness. 
At surface of water (pere.c.) . . 2100 9 3103 
Under 20 inches of water (per c.c.) 2103 10 3021 


Under 40 inches of water (per c.c.) 2140 2115 3463 


Few studies have been made of the effect of light on bacteria in 
flowing water. Jordan, after an investigation of several Illinois 
streams, concluded that at least in eight moderately turbid waters 
the sun’s rays are virtually without action. Much, therefore, 
depends on the turbidity and speed of the current, the maximum 
germicidal effect being produced in shallow, clear, slow-moving 
water. 

Temperature. —'The action of temperature upon the bacteria varies 
with the food and specific organism. When they are in a medium 
in which they can grow and multiply, warmth within reasonable 
limits favors their development. ‘This is true of the natural bac- 
terial flora and may, as was found to be the case at Harrisburg, 
Pennsylvania, hold for B. coli. But this does not hold for the 
pathogens which in the majority of cases do not multiply in water, 
and, as pointed out by Prescott and Winslow, “when a bacterium 
cannot multiply, the only vital activity which can take place is 
a katabolic wasting away, which soon proves destructive, and the 
higher the temperature the more rapidly the fatal result is reached. 
A frog in winter lives at the bottom of a pond breathing only 
through its skin and eating not at all, but as soon as the temperature 
rises it must eat and breath through its lungs or perish.” The 
typhoid bacilli will survive longer in ice than in water. The 
speed with which they perish varies inversely with the temperature, 
as was found by Houston. 


Percentage of typhoid Period of final 
bacilli surviving disappearance of 
Temperature. after one week. bacilli, weeks. 
Oger Week... bey tok oi ce ee eee ee LOMO) 9 
ee op eee AG Smee eae emi ebm. 4 temeonl fy (111) 7 
IN Uke ete ete Ce See he ohh enire re (es saan oe 0.07 5 
1 fee ce ee GS eS POE Worn ory) 0.04 4 


In the natural-occurring waters probably many factors play a 
part; sometimes it is the inhibiting action of microérganisms and 
their products on one another; at other times protozoa which feed 
upon bacteria and the development of which is directly proportional 
to the temperature of the medium in which they are growing. 

Hinds found that in pure, natural and distilled water B. coli 
and B. typhosus die from starvation at a regular rate. The rate 
of death increases with the temperature and is similar to the rate 
of a chemical reaction, thus following the mono-molecular law. 

Food.— Bacteria are dependent upon food and respond quickly 
to comparatively slight changes in their food supply. Wheeler 
found that typhoid bacilli would persist in almost undiminished 


CLASSES OF BACTERIA 345 


numbers in sterilized water from a polluted well containing con- 
siderable organic matter and kept in the dark at 20 degrees, while 
in purer water or in the light they died out in from two to six 
weeks. In unsterilized water the results may be just the opposite, 
for in the presence of an abundant supply the saprophytes may 
multiply at the expense of the pathogens. 

Whipple and Mayer find that the presence of oxygen is essential 
to the existence of typhoid and colon bacilli in water, and even 
small quantities of acid and alkali are fatal. It is for this reason 
that we find few organisms in acid and alkali water of various 
regions. The factors, therefore, which are at work on the puri- 
fication of water are numerous, and “although it is hard to estimate 
the exact importance of each factor, the general phenomena of the 
self-purification of streams are easy to comprehend. A small 
brook, immediately after the entrance of polluting material from 
the surface of the ground, contains many bacteria from a diversity 
of sources. 

“Gradually those organisms adapted to life in the earth or in the 
bodies of plants and animals die out, and the forms for which water 
furnishes ideal conditions survive and multiply. It is no single 
agent which brings this about, but that complexity of little-under- 
stood conditions which we call the environment.” 

Classes of Bacteria.— The bacteria found in water may be roughly 
classed as: (1) Natural-water bacteria, (2) soil bacteria and (3) 
sewage or intestinal bacteria. There is no hard and fast line 
between these classes, for organisms belonging to the water flora 
are found in the soil and water draining from manured soil will 
contain intestinal organisms. The classification, however, is valu- 
able; for the first two groups usually contain the saprophytes, 
whereas the third contains the pathogens. 

A number of attempts have been made to classify water bacteria. 
Ward, in his study of the bacterial flora of the Thames River, 
arranged them into twenty-one groups. But the work is beset 
with certain difficulties which were recognized by Ward, for he 
made the following statement: “My work goes to show that 
species cannot be made out, but that the limits of the species are, 
in most cases, far wider than is assumed in descriptions—in other 
words, that many so-called species in books are merely variation 
forms, whose characters, as given, are not constant but depend on 
treatment. How far this is true for any given case will have to 
be tested on the particular form in question.” 

Fuller and Johnson, from a study of the bacteria in the rivers 
of America, suggested a classification containing thirteen groups. 
Their system was based mainly on morphological data, and hence 


they experienced considerable difficulty in differentiating short 
bacilli from cocci. 


f 


346 WATER BACTERIOLOGY 


Jordan studied 548 strains of bacteria from the Illinois, Missouri 
and Mississippi Rivers and grouped them into the following classes, 
depending upon their biochemical properties: 

I. B. coli communis. 
Il. B. lactis aerogenes. 
Ill. B. proteus. 
IV. B. enteritidis. 
V. B. fluorescens liquefaciens. 
VI. B. fluorescens non-liquefaciens. 
VII. B. subtilis. 
VIII. Non-gas formers, non-fluorescent, non-sporeforming bac- 
teria which liquefy gelatin and acidify milk. 
IX. Similar to Group VIII, save that milk is rendered alkaline. 
X. Similar to Group VIII, save that gelatin is liquefied. 
XI. Similar to Group IX, save that gelatin is not liquefied. 
XII. Similar to Group XI, save that the reaction of milk is not 
altered. 
XIII. Chromogenic bacteria not included above. 
XIV. Chromogenic staphylococci. 
XV. Non-chromogenic staphylococci. 
XVI. Sarcine. 

XVII. Streptococci. 

The natural water flora are saprophytes and the most important 
members found were: 

Group V (B. fluorescens liquefaciens) is probably more often 
found in water than any other species. It liquefies gelatin and 
produces a green fluorescence. 

Group VI (B. fluorescens non-liquefaciens) produces colonies with 
a fluorescent shimmer and does not liquefy gelatin. They are 
often very abundant in river water. 

Group VIII: Organisms which liquefy gelatin and acidify milk. 
These are closely related to the proteus group and some of them 
are B. liquefaciens, B. punctatus, B. circulans. These are found 
more commonly at some seasons than at others. 

Groups XIII and XIV: Chromogenic bacilli and cocci. The 
red-pigmented B. prodigiosus belongs to this type, as does also B. 
ruber, B. indicus, B. rubescens and B. rubefaciens. Those pro- 
ducing a yellow or orange pigment and belonging to this group are 
B. aquatilis, B. ochraceus, B. aurantiacus, B. fulvus. At times 
there occur organisms which produce violet-pigment— B. violaceus. 
The chromogenic cocci occurring in water are not so numerous; 
of these, Sarcina lutea is the most common species. The non- 
chromogenic cocci, which Jordan classes as Group XV, are more 
numerous. 

Soil Bacteria.—'The flood waters are continually carrying to the 
surface waters soil organisms, so we may at times find any of 


INTESTINAL BACTERIA 347 


the bacteria which occur in soil also in water. Many of these 
find this an unsuitable medium for growth and multiplication and 
soon perish. But some species, among which are 6. mycoides, B. 
subtilis, B.megatertum and B. Mesentericus vulgatus persist for a 
considerable time. 


Intestinal Bacteria.—'These are usually of sewage origin. To 


this class belongs a heterogeneous group of microérganisms which 
find their way into water from sewage. Many of them are true 
saprophytes and of themselves are not injurious, but their presence 
in a water constitutes a danger signal to the bacteriologist. This 
is especially true of the B. coli group of organisms, the natural 
habitat of which is the intestinal tract of the higher animal—man. 
Hence, whenever there is opportunity for these organisms to find 
their way into waters there may also be opportunity for the patho- 
gens which cause typhoid fever, cholera and dysenteria to reach 
the water. It is, therefore, certain that even a little sewage may 
cause much damage if it enters a water supply for only a few hours 
at rare intervals, but it is the slight continuous infections which 
can give rise to a prolonged outbreak of disease. It is well estab- 
lished that typhoid bacteria die quite rapidly in ordinary waters, 
and so far as known never multiply in such waters, as is seen from 
the following (Mills): “To prove whether typhoid-fever germs 
would survive in the Merrimac River water, when at the low 
temperature of the month of November, long enough to pass from 
the Lowell sewers to the service-pipes in Lawrence, a series of 
experiments was made by the Board by inoculating water from the 
service-pipes with typhoid-fever germs, and keeping the water 
in a bottle surrounded by ice, at as near freezing as practicabie, 
for a month and each day taking out one cubic centimeter and 
determining the number of typhoid germs. The number continu- 
ally decreased, ‘but some survived twenty-four days. 
“On the first day there were 6120 germs. 

On the fifth day there were 3100 germs. 

On the tenth day there were 490 germs. 

On the fifteenth day there were 100 germs. 

On the twentieth day there were 17 germs. 

On the twenty-fifth day there were 0 germs.” 

At a higher temperature the life of the organism would have 
been of even shorter duration. 

Our information in regard to the cholera vibrio is not quite as 
definite, but experiments indicate that it may multiply to some 
extent in sterilized river or well water, and that it maintains its 
vitality in such water for several weeks or even months. 


Natural Purification of Water.—Nature’s methods of puriying. i 


water are mainly: 


ae 


re 4, 


348 WATER BACTERIOLOGY 


1. Evaporation and condensation which gives the purest of 
natural waters. Millions of gallons of water are annually evapo- 
rated from the surface of the globe. Thus, we have an enormous 
natural still by which water is constantly being purified in Nature. 

2. The self-purification of running streams which although . 
important is often hard to estimate quantitatively. It is due to 
many factors, chief among which are: (a) Chemical—the oxidation 
and reduction of organic and inorganic constituents of the water 
with the formation of simple substances which are not well suited 
to the maintenance of life and growth of many forms of bacteria, 
and the germicidal influence of sunlight which is an important but 
very variable factor. (b) Biological—the death of microérgan- 
isms through various not well-understood conditions grouped under 
the heads of symbiosis, antibiosis, time and various other means. 
(c) Physical—of which dilution and sedimentation are the more 
important. 

3. The storage in lakes and ponds which through the prolonging 
of the time of action greatly intensifies those factors at work in 
the natural purification of running streams. 

4. The combined physical, chemical and biological action of 
soil upon water which filters through the soil. This is one of 
Nature’s greatest purifying agents and stands second to evapora- 
tion and condensation in effectiveness. 

Artificial Purification.—Those methods which are so effective in 
the purification of water under natural conditions are usually’ the 
methods which are made use of in the artificial purification of 
water. Only a few of the best known can be briefly considered 
here. The student who is more deeply interested in the subject 
is referred to any of the many comprehensive works on this subject. 

The slow sand filter frees water from impurities through the 
interaction of sedimentation, filtration, and the biological destruc- 
tion of organic matter and bacteria. It has been extensively used 
for over one hundred years, but a great impetus was given to this 
measure when Koch, in 1893, showed that the proper filtration of 
the water from the Elbe River saved Altona from an epidemic ot 
cholera which devastated Hamburg which was using unfiltered 
water. 

The method consists in causing water to pass through a layer . 
of sand of such fineness and thickness that the requisite removal 
of suspended substances is accomplished. The filter as usually 
constructed is a basin having a water-tight concrete base on the 
surface of which are laid perforated tiles or pipes. These are 
covered with about a foot of gravel graded in size from 25 to 3 mm. 
in diameter from bottom to top. Over this is placed three or four 
feet of sand which acts as the real filter. The water passes through 
this and is conveyed to the mains by the underlying pipes. The 


CHEMICAL METHOD 049 


suspended material, including bacteria, is removed by the sand 
which becomes more efficient as used, due to the rapid formation 
of a mat of finely divided sediment, in which protozoa often multi- 
ply, and assist biologically in removing many bacteria. In time the 
mat becomes very thick and the filtration although effective is 
unduly slow. The water is then allowed to subside below the 
surface and about half an inch of the sand removed, after which 
filtration is resumed. The sand removed is washed to free it from 
collected impurities and is later replaced on the bed after succes- 
sive scrapings have reduced the filter to about one foot in thickness. 

The filters are usually divided into units of convenient size, 
about half an acre, so that one unit may be cleaned without inter- 
ruption of the system. The slow sand filter removes about 99 
per cent. of the bacteria, about one-third of the coloring matter 
and its long effective use has established the fact that it has a favor- 
able effect upon the health of the community where used. 

Chemical Method.—The chemical disinfection of water on a 
large scale is now almost exclusively effected with substances 
yielding chlorin, chief of which are bleaching powder (chlorid of 
lime), sodium hypochlorite and free chlorin. The action of these 
substances is essentially similar and dependent upon the quantitative 
active chlorin which they contain. They are usually added in 
quantities sufficient to give from 0.5 to 1 part of active chlorin 
per million parts of water. 

The use of bleaching powder in the purification of waters is 
cheap, reliable, harmless and easy of application, which makes it 
an attractive method, but when used on impure waters containing 
organic matter it gives rise to amins, chloramins and other com- 
pounds of unknown composition which impart to the water unpleas- 
ant flavors. 

Alum is often used either alone or in connection with the mechan- 
ical sand filter, and if used under controlled conditions is very 
effective and leaves no undesirable constituents in the water. 
The quantity should be accurately determined for each water as 
it varies with the turbidity and quantity of calcium carbonate 
contained in the water. 

Potassium permanganate is often used in the disinfecting of 
small quantities of waters, but its effectiveness cannot be depended 
upon except against the cholera spirillum. Moreover, the disagree- 
able taste and the color imparted to the water are a serious drawback. 

Chlorazene, the new disinfectant suggested by Dakin, has much 
which commends itself for use in the disinfection of small quantities 
of water, as in the concentration of 1 : 300,000 it will sterilize ordi- 
narily heavily contaminated water in thirty minutes. Such a 
concentration imparts a very slight taste to the water but is per- 
fectly palatable. It is non-toxic and if used for only short intervals 


350 WATER BACTERIOLOGY 


would probably be without effect upon the health of the individual. 
The compound, chlorazene (p-sulphondichloraminobenzoic acid— 
Cl,NO.SC,HiCOOH), is excreted in the urine as p-sulphonamido- 
benzoic acid. 

Ice.—It is often the case that water which one would not con- 
sider fit for drinking is used in the manufacture of ice. This 
should not be the case as the freezing of water reduces only slowly 
the number of organisms present. In fact Keith considers that 
low temperatures alone do not destroy bacteria. On the contrary, 
cold appears to favor longevity doubtless by diminishing destructive 
metabolism. i 

Probably the decrease in number is due to mechanical rupturing 
of the cell, lack of oxygen, food and moisture which are due to 
the low temperature. Although there is a decrease of bacteria, 
yet experiments have demonstrated that even the pathogen Bacillus 
typhosus may persist in ice for one hundred days. The cholera 
vibrio perish much sooner. Hence, the evidence is conclusive 
that just as pure a water should be used in the manufacture of 
ice as is required in domestic supplies. 


CHAPTER XXIX. 
WATER AND DISEASE. 


History is replete with facts indicating that early in the history 
of the race there was a general conception that water might cause 
disease. [arly tribes sought out those streams and springs which 
yielded a generous supply of cool, clear water. They followed 
them on their course to the sea and learned that some furnished 
water which promoted health, whereas the user of other waters 
suffered certain plagues. Centers of population sprang up in 
ancient times around those points where water was readily avail- 
able and great expenditures of labor and treasure were made to 
protect and carry it to places where it was needed. About 400 
B.c. Hippocrates pointed out the danger from polluted water and 
advised the filtering and boiling of such water. But apparently 
during the following centuries no relationship was observed between 
the character of the drinking water and the epidemics of typhoid, 
cholera and other intestinal diseases which swept over Europe. 
During the Dark Ages the belief that water caused diseases of the 
human race became very popular. But the attributing factor was 
thought to be witches who by some occult magic poisoned pure 
wells, springs and streams. 

The statements in the literature during the beginning of ihe 
nineteenth century became more definite, “Seana that the rela- 
tionship-between the character of the drinking water and the 
prevalence of intestinal diseases was being recognized. By the 
middle of the century Michel had collected such a mass of statis- 
tics as to warrant the conclusion that there is a direct relationship 
between the purity of a drinking water and typhoid fever. 

Disease First Definitely Proved as Due to Water.—'The first clear- 
cut demonstration that disease is caused by infected water was 
that of the now famous Broad Street well (1854) so ably studied 
by Snow. During this outbreak of cholera in London there was 
an enormous concentration of cases in a very limited area just 
east of Regent Street. There were during a period of about six 
weeks over 600 fatal cases. A careful study of the site, soil, sub- 
soil, streets, density and character of population, dwellings, yards, 
closets, cesspools, vaults, drains, conditions of cleanliness and 
atmospheric conditions revealed nothing of importance. A study 
of the water supply revealed the following facts; 


352 WATER AND DISEASE 


1. Nearly all of the cases were nearer a certain public pump 
in Broad Street than any other source of water and most of them 
gave a definite history of getting water from the pump. 

2. Of the few cases which developed outside of the area sup- 
plied by the pump most of them were known to have drunk water 
from the Broad Street well. 

3. The few scattered cases in distant parts of London were 
individuals who had used water from the well. 

4. Right in the midst of the district was a workhouse with 235 
inmates and a brewery with 70 employees, each having its own 
well, and there were only 5 deaths in the workhouse and none in 
the brewery. 

5. It was shown that a privy vault and cesspool in an adjoining 
house discharged through a leaky drain which ran within two 
<8 of the Broad Street well. 

. There were 4 fatal cases of cholera in the hous at the time 
a ate outbreak and earlier cases which were probably cholera. 

It was not until 1880 that the typhoid bacillus was isolated 
by Eberth and studied in detail by Gafky in 1884 that we had 
definite information concerning the causative agent of typhoid 
fever, the way in which it leaves the body, and the routes by which 
it may reach drinking water. This same year Koch isolated the 
cholera vibrio from stools of patients suffering with the disease. 
He also isolated the organism from tankwater in India. We now 
know that water is a vehicle for a number of infections such as 
typhoid fever, cholera, dysentery and other intestinal diseases. 
It may be the medium for conveying infections not now generally 
regarded as water-borne. It may carry inorganic poisons such as 
lead, or may be of such a nature as to bring about derangements of 
‘metabolism resulting in goiter, or may lower resistance, so as to 
favor infections not water-borne. It occasionally conveys animal 
parasites, amebee and worms. 

Amount of Sickness due to Water.— Water is probably responsible 
for more sickness and death than any other article of diet except 
milk. This is due to the facts: (1) That it is used raw, while 
many other substances are rendered sterile by cooking; (2) water 
comes in contact with numerous substances upon the earth’s surface 
and is a universal solvent; (3) it is used as the great vehicle for the 
removal of waste, much of which may contain pathogenic organisms. 

It is difficult to obtain statistics to indicate accurately the mor- 
bidity and mortality due to impure water, but Whipple states 
that the average typhoid death-rate in American cities is about 
35 per 100,000, while cities with a good water supply average 20. 
He, therefore, attributes 40 per cent. of the typhoid fever of the 
United States to infected water. Chapin, however, considers it 
would be more conservative to place it at 15 per cent. for the whole 
country rather than at 40. But even these figures show a large 


THE MILLS-REINCKE PHENOMENON 309 


unnecessary mortality and morbidity when we remember there 
were 25,000 deaths in the United States in 1910, representing at 
least 250,000 cases. 

Dysentery and diarrhea, although not as fatal as typhoid fever 
or cholera, are not to be neglected, for when we consider the sick- 
ness and economic loss resulting each year in the United States 
from these causes, much of which is due to infected water, we 
find that they are not negligible. Moreover, the better care of 
drinking water has resulted in a marked decrease in the ravages 
of dysentery, for it is estimated that the mortality from dysentery 
in England toward the end of the last century was but a fraction 
of a per cent. of what it was in the middle of the century. More- 
over, the reduction of dysentery in the United States has kept pace 
with the advancement made in water protection and purification, 
as seen by the fact that the death-rate from dysentery in this 
country in 1850 was 6.32 per cent.; of the total mortality in 1860, 
2.65 per cent.; 1870, 1.6 per cent.; and in 1880, less than 1.5 per cent. 

The Mills-Reincke Phenomenon.— Mills, of Lawrence, Massa- 
chusetts, and Reincke, of Hamburg, Germany, in 1893 noted 
that the purification of the water supplies of their respective 
towns was followed by a decline in the general death-rate which 
was more rapid than could possibly be accounted for by the death 
from typhoid fever. This condition was later searchingly studied 
by Sedgwick and MacNutt who gave to it the name of the “ Muills- 
Reincke Phenomenon.” Later (1904) Hazen, a sanitary engineer 
formulated a numerical expression for the comparative effect of 
purified water upon the typhoid fever and total mortality as fol- 
lows: ‘Where one death from typhoid fever has been avoided 
by the use of a better water, a certain number of deaths, probably 
two or three, from other causes have been avoided.” This propor- 
tion varies greatly in different instances. It was 1 to 16 in Ham- 
burg, in Lawrence 1 to 4.4, Lowell 1 to 6, Albany 1 to 4.4 and 1 
to 1.5 in Binghamton. Hence, in all of the cases studied by Sedg- 
wick and MacNutt it appears to be sound and conservative, but 
in some of the American cities more recently studied it does not 
appear so exact. 

The cause of this decline in mortality is not clearly understood. 
It may be due to the exclusion of specific pathogenic organisms, 
to increased vital resistance resulting from the use of a better 
water, or in some cases the appearance and taste of the water 
may be improved with the result that greater quantities are used, 
and hence a better condition of the body in general. Probably 
many factors are at work and these studies have revealed a remark- 
able relationship between polluted water and infant mortality. 
Rosenau considers that it bids fair to assume a causal importance 
in gastro-intestinal disturbance of children second only to that of 
contaminated milk. 

23 


O04 WATER AND DISEASE 


Cholera.— Water has been proved to be the causative agent in 
the conveying of cholera in a number of instances. The two best 
known cases are that of the Broad Street well, which has 
already been considered, and the epidemic of 1892 in Hamburg. 
This latter will ever remain classic on account of the clearness of 
the circumstances and the fact that there is no missing link in 
the chain of evidence, as the cholera vibrio was isolated from the 
Elbe River water. 

The Hamburg epidemic occurred in 1892, and in a little over 
two months there were 17,000 cases with 8605 deaths, whereas 
Altona, which in reality forms with Hamburg one large city, was 
practically free. The two cities are built on the same soil, pro- 
vided with the same sewage system, and have the same climatic 
conditions. They have the same social customs and were sepa- 
rated only by a political boundary line. The boundary runs 
through a street on one side of which is Altona and on the other 
Hamburg. They have separate water supplies, but both derive 
their water from the Elbe River which is a grossly polluted stream. 
However, the water supply for the city of Altona was purified by 
filtration, while that of Hamburg was not. The boundary of 
the epidemic was just as clear as was that of the water system, or 
in the words of Koch, “cholera in Hamburg went right up to the 
boundary of Altona and there stopped. In one street, which for 
a long way forms the boundary there was cholera on the Hamburg 
side, whereas the Altona side was free from it.” 

Typhoid.— Contaminated water was the first recognized and 
probably the most significant vehicle of typhoid infection. ‘The 
improvement in water supplies during recent years has been respon- 
sible for the reduction in typhoid morbidity. The results compiled 
by Kober clearly show the effect of improved water supplies on 
typhoid mortality in American cities. 


EFFECT OF WATER PURIFICATION ON GENERAL AND TYPHOID 
DEATH-RATE. 
_ General Typo? 
Genuante Same | Percentage) death-rate | Same Percentage 
City. uheneeol after. reduction. | before | after. reduction. 
inert, | change of | 
| supply. ee 
pply. 
Providence, R. I. . | 19.3 19.0 = eeG a as 1327 +37.2 
St. Louis, Mo. . | 18.0 16.1 +10.6 39.2 19.1 +51.3 
Youngstown, O. 15.6 Mays It + 3.2 96.1 39.1 +59.4 
Tthaca, ania see 16.4 Toei + 7.9 108.8 25.3 +76.8 
Paducah, Ky. . .| 23.4 cas eso Oe Sze 78.7 + 4.2 
Watertown, N.Y. . ey 1 4 —11.1 100.6 38.2 +62.1 
Paterson, N. Je. |. NFAY 16.5 4.1 28.2 | Wiha; +57.8 
Binghamton, N. Y. 17.6 Lie 6 | 0 1 FE OLS Pal eltes see + 67.2 


TYPHOID 305 


Water still remains the most important single channel by which 
the typhoid bacilli reach the human body. Estimates vary as to 
the actual percentage of typhoid cases which are referable to water 
infection. It is placed by various authors at from 10 to 40 per 
cent. According to Gay, Schuder found that of 640 typhoid 
epidemics 22 per cent. were due to water. Schegehdahl found 
that of 682 cases about 33 per cent. were water-borne. Typhoid 
is, therefore, the most important water-borne disease. 

The proof that a typhoid epidemic is due to water infection 
is usually indirect, for the actual isolation of the offending organ- 
ism is effected with considerable difficulty and has been accom- 
plished in only seven or eight cases. However, in those cases 
where it is found it is not always possible to prove that it was 
present at the time the infection occurred. Strong presumptive 
evidence is given whenever waters are proved through the presence 
of colon bacillus to have been infected by sewage. 

The best evidence, however, obtainable that a specific typhoid 
outbreak is due to polluted water is that obtained by the epidemi- 
ologist. He knows that the important characteristics of water- 
borne epidemics are: 

1. They may be preceded by a period of dysentery. 

2. The epidemic usually has a sharp onset, the curve rising to a 
peak and the decline being rapid. 

3. The cases are quite evenly divided over the city, that is, 
provided the city is served by a municipal supply. 

4. They nearly always occur in the spring, fall, or winter. 

5. The pollution is usually nearby and the epidemic is of short 
duration unless there be a continuous source of new infecting 
material. 

The work of the epidemiologist is vividly portrayed by Hill 
as follows: 

“To illustrate the general principles, let us suppose notification 
be received that a typhoid fever outbreak exists in a far-off com- 
munity. The public health detective packs his grip and _ goes. 
He knows no details; he has never heard of this particular com- 
munity before; he has not even any general information about the 
character of the country; he enters the community with no pre- 
conceived ideas. But he does know how typhoid fever originates 
and how it spreads. Water, milk, food, flies and fingers are the 
routes—typhoid cases or typhoid carriers, the source. His duties 
are to find both; and to find them, not as a scientific amusement, 
or as a matter of record; not to furnish food for speculation—above 
all not to make a show of doing something—but to stop the outbreak, 
and then to advise measures to prevent recurrence. 

“The public health detective on entering the community affected 
by typhoid fever does not first examine the water-supply, the 


306 WATER AND DISEASE 


milk supply, the sewage disposal system, the markets, the back 
alleys, the dairies, or anything else. He goes directly to the bedsides 
of the patients. Of course he must obtain the names and addresses 
of the patients from someone—from the local health officer, if he 
has them; from the attending physician, if the health officer has 
no list; from the lay citizens themselves, if no one else is immediately 
available. The more complete the list, the faster he can work, 
because then he is not compelled to hunt up the cases personally. 
But if there be no list, he begins making one himself. His inten- 
tion vs to see just as many patients as he can, for each furnishes evi- 
dence and he wants it all. But he knows that it is not always 
necessary at this stage to see absolutely all the patients, so long 
as he sees the majority. ; 

“Reaching the patient’s bedside, his investigation begins. 
Automatically, almost mechanically, he decides whether or not 
the patient has typhoid fever or not. Satisfied on that point, 
his first question is not, “Tell me all the different water supplies 
you have used, or all the sources of milk you have used.’ The 
first question is, ‘When did you first show the earliest symptoms of 
the disease?’ Why? Because this date once fixed, at which infec- 
tion entered the patient’s mouth is fixed also, 7. e., a date between 
one and three weeks previous to the date of the earliest symptoms. 
Remember that at that stage the detective may not have even an 
inkling as to which of the usual factors—water, milk, food, flies 
or fingers—is involved. Still less can he guess which particular 
water supply, milk supply, ete., of the many possible ones, may be 
the guilty one. But the answer to this question reduces possi- 
ble routes to those used by this patient—not at any time—but during 
a specific period, i. e., from one to three weeks preceding his date 
of earliest symptoms. 

“Not yet, however, are the milk and water questions offered. 
The second question is ‘Where were you during that period?’ 
Why? Because if the patient were not in the community during 
that period, he could not have contracted his infection within it, 
and does not belong to the outbreak under examination at all but 
to some other. He is in brief an ‘imported case,’ and while, of 
course, he is to be supervised lest he spread his infection to others, 
he cannot help to locate the source of the main outbreak—unless 
perchance he be himself that source, 7. ¢., the introducer to the 
community of the original infection. If he be an imported case 
he is noted for further reference and the detective goes to another 
patient. If not, the questions continue. But not yet is water 
or milk or flies mentioned. The third question is, ‘Were you asso- 
ciated during your period of infection with any then known typhoid 
case?’ Why? Because such association, especially if intimate, 
makes it more probable that the case under examination received 


TYPHOID 307 


his infection from the preceding case, rather than from any general 
route and that he is, therefore, a ‘secondary case.’ If he had 
such associations, this is noted for further reference and the investi- 
gator passes on to another bedside. If not, the questions continue, 
and now at last take up milk, water, food, ete., but of course only 
so far as to determine those used by the patient during his infec- 
tion period. 

“Then the investigator passes to the next patient. What has 
he learned so far? Nothing much yet. But he has narrowed the 
possible routes of infection to certain water supplies, certain milk 
supplies, certain food supplies, ete., 2. e., those used by the first 
patient during a certain period, and he has done this in thirty minutes 
—in searcely the time it takes for the old-style investigator to get 
his bottles ready to collect his first water sample. 

“At the bedside of the second patient, the same inquiries in 
the same order are made. If this second patient be an imported 
case, or a secondary case, he also is merely noted for future refer- 
ence. If he be a primary, however, the origin of his drinking water, 
milk, food, ete., during his infection period are also ascertained. 
Perhaps he coincides with the first patient in every detail of aliment- 
ary supplies, in history and associations. If so, nothing much has 
been added to the detective’s knowledge. But more than likely, 
dissimilarities have developed. Since the responsible water supply, 
milk supply, etc., must be one of those water supplies, milk supplies, 
etc., used in common by primary cases, all those not common to 
both of these primary cases may be dropped from consideration 
(except in rare instances of multiple routes). Thus, if both have 
used the same water, water from that origin remains as a possi- 
bility. But if the water supplies have been different, water is 
eliminated from the question entirely. If the milk supplies are 
identical, milk remains as a possible route of infection; if not, 
milk is eliminated from the question entirely. 

In brief, provided the information obtained be reliable, and it 
is part of the public health detective’s training to distinguish at 
a glance truth from falsehood, the honestly mistaken, or forgetful, 
or stupid replies from the reliable ones—and above all never to 
believe anything (to the extent of recording it) unless it is checked, 
confirmed and established as a fact, the modern investigator has 
in one hour narrowed his investigation to a point which the old- 
style investigator often would not reach for weeks. 

“And so from patient to patient the inquiry proceeds. In the 
course of the day the investigator has seen perhaps 30 patients. 
The tabulation (probably already made in his own mind) shows, 
say, 3 imported cases, 5 secondaries, 2 uncertain or indefinite. 
The remaining primary cases show in common, say, 1 water supply 
only, the milk, ete., varying; or 1 milk supply only, the water, 


308 WATER AND DISEASE 


etc., varying; or no connection except attendance at some one 
social function. 

“Going straight to the route thus indicated, the public health 
detective quic ely confirms the indications of his results. He knows 
that the route indicated must be the guilty one, for only that 
route can account for all the cases. He concentrates on that 
route until the evidence is complete—when and how that route 
became infected, when and by what sub-routes the infection was 
distributed, why it infected the patients found and not others, ete. 

“Tn this illustration I have assumed complete ignorance on the 
part of the epidemiologist as to everything connected with the 
community he is investigating, except what he finds by cross-exam- 
ining the patients. As a matter of fact, every epidemiologist, 
however much a stranger to the particular community he enters, 
begins to learn about it from the moment he enters it. 

“Thus, almost unconsciously he notes the size of the town and 
compares it with the number of cases reported as existing; if it 
is summer time he almost automatically notes the presence or 
absence of open toilets in the backyards, of manure piles and of 
garbage cans—all bearing upon fly infection. If it is winter time 
or the community be ll sewered, he does not even consider flies. 
If the cases are grouped in one quarter of the town, while the public 
water supply extends all over it, he tentatively eliminates the 
water supply before he asks a question. If good surface drainage 
and a sandy soil exist, or driven wells are chiefly in vogue, he 
tentatively eliminates well water—even before he registers at the 
hotel. 

“This is not and cannot be a complete synopsis of all the com- 
binations of circumstances which the epidemiologist meets. It is 
intended to illustrate his methods and to show why they are incred- 
ibly rapid and incredibly accurate—how they eliminate specula- 
tion and guarantee a correct solution—which means, of course, the 
achievement of the great end, the finding of proper measures for 
suppression. 

“As soon as the route is indicated, he must go to that route, and 
establish beyond peradventure that it was in truth responsible. 
A water supply cannot convey typhoid if typhoid fever discharges 
have not entered it. There is no object in attributing an outbreak 
to fly infection from toilets into which typhoid feces have not 
been discharged at such a time as to account for the cases. A 
milk supply, not handled at some point by an infected person, nor 
adulterated at some time with infected extraneous matter cannot 
convey typhoid. Whatever his results, they cannot be true unless 
they are consistent—they should not be accepted unless they are 
provable—and proved. 

“Tf the public health detective is familiar with the community 


TYPHOID 309 


where the outbreak occurs, including its water supplies, its milk 
supplies, the sociological relationships of its people, etc., he can 
often tentatively determine the cause of the outbreak by a mere 
inspection of the names and addresses of primary cases, especially 
if plotted on a map of the community, taking into account also 
the time of year, and other general points. But such deductions, 
while often wonderfully reliable, can never be as conclusive and 
satisfactory as are the results of an investigation by even a total 
stranger, if the investigation be conducted as above described.” 


REFERENCES. 


Savage, W. G.: The Bacteriological Examination of Water Supplies. 

Harracks, W. H.: An Introduction to the Bacteriological Examination of Water. 
Prescott and Winslow: Elements of Water Bacteriology. 

Thresh: The Examination of Water and Water Supplies. 

Mason: Water Supplies. 

Rosenau: Preventative Medicine and Hygiene. 

Don and Chisholm: Modern Methods of Water Purification. 


CHAPTER XXX. 
SEWAGE AND SEWAGE DISPOSAL. 


Man early learned that both esthetic and sanitary reasons 
demand that sewage be properly treated. In the early history 
of the race and also of a district the pit or trench was used for the 
disposal of the refuse. Later this was lined with stone, brick 
or cement to partly prevent diffusion into the surrounding soil, 
and hence the contamination of the well. This, when properly 
covered, became the cesspool which is largely in use in the rural 
districts today. As population increased with its constantly 
growing volume of human waste the old methods became inade- 
quate, and hence there has developed the modern sewage system. 

Source, Composition and Quantity of Sewage.—A city’s sewage 
consists of the public water supply carrying human and animal 
excreta, refuse from the kitchen, laundry, manufacturing estab- 
lishments and the dust and dirt of the streets. Its quantity is 
directly proportional to the consumption of water in the district. 
In small cities it may be as low as forty or fifty gallons per capita 
daily, whereas in larger cities it may reach from 100 to 200 gallons 
or over. 

Its composition depends upon the density of population, the 
number and kinds of manufacturing establishments, and whether 
there is a separate or combined system. Where the combined 
system is used the composition and quantity of the sewage varies 
with the rainfall and street washing. There is also a diminution 
in quantity and composition at night. 

Fuller gives the estimated amount of dry suspended solids in 
the New York City sewage per 1000 inhabitants annually as 
follows: 


Tons per 1000 
Inhabitants 


Material. annually. 
Feces oS Pda oe de. [NT DONS ee > ce ah ge oe OC 
Toletipsaperiand newspaper’ erence te be ce ees eS 8 
Soap and washings dy cole se aim var eae pie oe eer 
Street wabtes* =-.0>.or cee ee a ee cae ee 8 
Miscellaneous, “205 Pt) en he ee Se SA Oe ee 4 

Total By. 7 ey ae tcg tia Whaat San alae le a aS aR et ECL 


From the viewpoint of purification sewage contains proteins, 
carbohydrates, fats, soaps, urea and other organic substances. 


BACTERIA IN SEWAGE 361 


The important elements present are nitrogen and sulphur. ‘The 
quantity of these present determines the nature and repulsiveness 
of the resulting products. 

Bacteria in Sewage.— The number and kind of bacteria in sewage 
varies widely with its composition and origin. According to Fuller 
it contains 320 billion for each person connected with the sewer 
system. Johnson found B. coli to average about 500,000 per c.c. 
He isolated the following species from the crude sewage of Columbus: 


: Number of times 
Species. found. 


UG DORIA G OS putin ek OG Sa an) bag eed Coan Io Cera enemy 41 
MICOLIRCOTILIILILTLUSM IRL ew? alts Mae et tin ae ten ORAL ee, be ae AG 
COUT OLS eee 

. mesentericus vulgatus 
bruneus 

hyalinus . 

Suscus 

delicatulus 

pyocyaneus 

fluorescens 

circulans . 

nibilus 

weichselbaumii 

sporogenes 

stellatus 

. helvolus 

. Cereus 

. cloace 

. proteus zenkeri 

. monadiformis 

. aeris muintissimus b 
. tetragenus mobilis ventriculi 
. CASEL . Se aae 

. albicans amplus 

. fervidosus 

Str. coli gracilis 

Str. enteritis 

Sarcina alba 

Ps. turcosa 

Ps. nebulosa 

Ps. ochracea 


BBS SSB BB By Dy Oy Dy yy te te 
ee i RE ee RB BPN NNN NWwWwwen co 


In addition to these, many of the pathogens may find their way 
into sewage and survive for various lengths of time. 

However, the interest centers more in the changes produced by 
the various bacteria found in sewage than in the specific classes. 
Most of them are not only harmless, but of genuine importance 
in the economy of Nature through the scavengering work which 
they accomplish. A few of them are dangerous on account of 
their causing certain infectious diseases. Many of them play 
an important ‘rdle in decomposing sewage with the formation of 
malodorous gases and products “associated with putrefactive 
nuisances. 

The modern tendency is, therefore, to classify sewage bacteria 


362 SEWAGE AND SEWAGE DISPOSAL 


from a physiological viewpoint. They may roughly be divided 
into four classes—hydrolyzing, oxidizing, reducing and pathogenic. 

Fuller gives the estimated constituents of average sewage as 
follows: 


| Grams per capita | Parts per 
| daily. million. 
Oxygen consumed: 
‘wo wmMinutes + boline.) 24) 5 4. Gene 15.0 39.6 
Five minutes’ boiling . . . . Sle || 22.0 58.0 
Nitrogen: 
Free ammonia Nop eke a ae ON ee | 7.0 18.5 
Albuminoid ammonia ; 225 6.6 
Organic Ere | 8.0 Dikesl 
—oeed | "— 
Meshal tee wee 5 eee 17.5 46.2 
= at fe ——— 
GEhlorint ee ose hee LE cee eee ce eee a 19.0 50.2 
Sora aes Ei reared aot nner coe ea Sah 19.0 50.2 
Dissolved matter: 
IMEIN CTH Saeki ee eee nae 58.0 140.0 
Organic and volatile ; Le. 40.0 | 106.0 
Total BSE | Weel ce Seon me ES hs 98.0 246.0 
Total solids: 
IManerally 225 te pk Loree ten pe eon Sedat 152.0 402.0 
Organic and volatile ; See ete Le ite 77.0 203.0 
Total ee A ee ee hy LA es 229.0 605.0 
Bacteria, 322 billion per capita daily. 


Hydrolyzing Bacteria.— Probably most of the early changes which 
occur are hydrolytic, that is, the substance is caused to take up 
water, becomes unstable, and for some reason falls into fragments, 
thus often passing from a non-soluble compound of complex con- 
stitution to a simple soluble substance. 

Protein liquefaction belongs to this type of changes and is brought 
about by a great variety of bacteria working in conjunction with 
each other. The proteins are hydrolyzed by successive stages to 
proteoses, peptones, peptids, amino-acids and finally to ammonia, 
carbon dioxid, methane, hydrogen, ete. It probably corresponds 
in the main with the changes which have been considered under 
ammonification. The final products vary widely, depending upon 
whether the process is being carried on under aérobic or anaérobic 
conditions. The tendency is for it to partake more of putrefaction 
in the septic tank and decay in soil. 


REDUCING BACTERIA 363 


Cellulose fermentation, next to protein hydrolysis, is the most 
important work of bacteria in sewage purification. Paper, cotton 
fabric, wood and other cellulose-containing substances are rapidly 
attacked by various organisms with the production of soluble 
substances—starches, sugars, acids and finally carbon dioxid, 
methane and hydrogen. 

Probably fewer organisms possess the power of saponifying fat 
than of liquefying proteins or hydrolyzing cellulose. For this 
reason and also due to the fact that the fat tends to rise to the 
surface out of the sphere of bacterial action, there is a great ten- 
dency for the fat to accumulate. At times this may accumulate 
around some solid and give rise to “grease balls’ which cause 
clogging of pipes. The fat which is acted upon by bacteria is 
broken into fatty acids and glycerin. The fatty acids are quite 
resistant to further bacterial activity, but the glycerin is rapidly 
broken into simpler products. 

Oxidizing Bacteria.—'The complex microflora of the sewage must 
have energy. This they get in a great degree from the oxidation 
of the comparatively simple products yielded through the hydrolysis 
of the proteins, carbohydrates and fats. These are changed prob- 
ably similarly to: the acetic acid fermentation with the production 
of acids and finally carbon dioxid and water. 

The ammonia liberated through the deaminization of the amino- 
acids is oxidized by the Nitrosomonas to nitrous acid and by the 
Nitromonas to nitric acid. 

Reducing Bacteria.—'The nitrites and nitrates formed by the 
nitrifying bacteria are in a great measure reduced to free nitro- 
gen through denitrification. The sulphur in the protein molecule is 
liberated as sulphates, sulphur dioxid and hydrogen sulphid. The 
sulphate formed is reduced to hydrogen sulphid. This reacts with 
the small amounts of iron and other metals present with the result- 
ing black residue of metallic sulphids always found on the bottoms 
of tanks and streams in which sewage 1s decomposing. 

Each of these processes is going on simultaneously in sewage 
and the one is dependent upon the other, there being a true _bio- 
logical cycle, as is pointed out by Whipple. 

“The decomposition and oxidation of the organic matter in 
sewage are brought about by bacteria, and the bacteria serve as 
food for protozoa and other forms of microscopic animal life. The 
dissolved organic matter in sewage serves as food for alge. These 
alge and protozoa are, in turn, consumed by rotifers and crustacea, 
while the latter form the basis of food supply for various aquatic 
animals and fishes. Thus, there is a continuous biological cycle. 
Again, animal forms require oxygen and produce carbonic acid, 
while plants consume carbonic acid and produce oxygen. Where 
these processes occur normally and with a proper equilibrium main- 


364 SEWAGE AND SEWAGE DISPOSAL 


tained between animal and plant life, offensive conditions do not 
result, but where abnormal conditions are produced, as, for example, 
by the discharge of excessive quantities of sewage or trade wastes 
into a stream, a depletion of the dissolved oxygen may follow, or 
there may be an overproduction of alge so that the conditions 
become offensive. It is coming to be realized that in order to 
properly determine the dilution required in any particular case 
the conditions required to bring about this condition of biological 
equilibrium must be determined.” 

Pathogenic Bacteria.— Owing to the origin and nature of sewage 
it may at any time contain pathogenic bacteria. The ones with 
which the sanitarian is most concerned are the typhoid, cholera 
and dysenteria, but it is always possible for many other species 
to find their way into sewage and from it cause infection. This 
is especially true of B. tuberculosis which is quite resistant to putre- 
faction. With the exception of the cholera and dysentery organ- 
isms, there is no evidence that they ever multiply in sewage, and 
they produce no appreciable change in its composition. 

The majority of the pathogens soon die in sewage. The results 
as reported by Whipple for typhoid bacilli are given in Fig. 44. 

The speed with which the typhoid bacilli disappear from water 
varies with the vitality of the organism, the temperature of the 
water, the organic matter, and the bacterial antagonism exerted 
by other organisms. ‘Typhoid bacilli seem to die more quickly in 
sewage than in fairly pure water, probably because of the great 
bacterial antagonism existing. Furthermore, the absence of 
oxygen probably plays an important part, as Whipple found oxy gen 
necessary for longevity of typhoid bacilli. Jordan thus summarizes 
our present knowledge of the longevity of typhoid bacilli: 

“Laboratory experiments have shown that the typhoid bacillus 
can survive in sterile water in glass vessels for upward of three 
months, and for possibly two or three weeks in unsterilized ground 
or surface water. Other evidence indicates that the bacillus is 
able to travel in water a distance of at least 140 kms., and to retain 
its vitality in natural bodies of water for at least four or five days. 
It is possible that water may continue to be the vehicle of infection 
during a much longer period, but the available data point to a 
comparatively short duration of life of the specific germ in the 
water of flowing streams. Under ordinary conditions no multi- 
plication of the typhoid bacillus takes place in water, even when 
a considerable amount of organic matter is present, but, on the 
contrary, a steady decline in numbers goes on. The history of 
typhoid epidemics tends to show that sewage pollution is to be 
feared chiefly when the sewage is fresh, and that the danger of 
infection diminishes progressively with the lapse of time. 

“Tn soil in the fecal matter of privy vaults the duration of life 


PATHOGENIC BACTERIA 365 


of the typhoid bacillus is much longer than in water. Levy and 
Kayser found typhoid bacilli in soil that had been manured fourteen 
days previously with the five-months-old contents of a vault. The 
evidence that any genuine multiplication can take place ing the 


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soil is not convincing, but it has been proved that the bacillus 
may be carried by water-currents to a considerable distance from 
the point where it was first introduced. Infection of wells and 
small water-courses is thus brought about sometimes by the wash- 


366 SEWAGE AND SEWAGE DISPOSAL 


ing of bacilli out of soil in which they may have lain dormant for 
many months. The persistence of typhoid fever around certain 
habitations may be plausibly explained on the supposition of an 
extensive soil infection. There is no doubt that the practice of 
using human excrement for manuring vegetable gardens entails 
a danger no less real because often unrecognized.” 

Necessity of Sewage Disposal.—Sewage is obnoxious to the senses 
because of its appearance, and especially because on decomposing 
it yields malodorous compounds. It is usually considered that 
hydrogen sulphid is the main offender, but indol, skatol, cadaverin, 
mercaptan and some other compounds are considerably more 
repulsive and exist in sewage. 

More important still is the fact that sewage contains bacteria 
which have come from persons sick with typhoid fever, dysentery, 
tuberculosis and other diseases, as these may reach the food or 
water of healthy individuals and thus give rise to epidemics. Statis- 
tics show that the abandonment of privies and the substitution 
of a good sewerage system have greatly reduced the general death- 
rate In many a city. 

What Should be Accomplished in Sewage Disposal—he sanitary 
engineer attempts to dispose of sewage as rapidly as possible, with 
the least nuisance to the smallest number of people, with the least 
damage to health or property, and at the smallest cost. Sewage 
can be made entirely harmless only by the complete destruction 
of its organic matter and bacteria. A complete purification is 
not attempted normally as the plant required for such would be 
so elaborate and too expensive. Moreover, practical experience 
has shown this to be unnecessary. 

Methods of Disposal.—The method selected for sewage disposal 
will vary with the district, location and means at the disposal of 
the sanitary engineer. However, in all cases he must keep in 
mind convenience and public health. In rural districts the well- 
constructed cesspool may of necessity be used, whereas in the 
urban district this may not be tolerated. One of the readiest 
methods, and the one which until the last few years has been uni- 
versally used in this country, is to allow the sewage to flow without 
treatment into the nearest stream, lake or harbor. This is very 
successful as long as the quantity is not excessive, the dilution 
great, and the receiving water is not used by other communities 
for drinking and culinary purposes. Where this method is used 
the dilution should be great. The Chicago drainage canal was 
designed on the basis of 3.3 cubic feet per second for 1000 people. 
The efficiency of purification, however, varies with the nature of 
the sewage. The presence of trade wastes, especially those of an 
oily nature, which float on the surface, may form scums which 
interfere with the absorption of oxygen from the air. Rapidly 


METHODS OF DISPOSAL 367 


flowing streams, on account of their absorption of oxygen, tend to 
purify themselves more rapidly than do slower ones. Cold water 
holds more oxygen than does warm, and fresh than salt water; 
hence, there is a greater tendency for oxidation in cold fresh waters 
than in warm or salty waters. 

There is, however, a growing demand that sewage be treated 
before it is thrown into streams or lakes. This may be done by 
various methods, such as sedimentation, sub-surface irrigation, 
broad irrigation and other means. For a description of each 
together with its relative value the student is referred to any 
of the standard works on sewage. 


REFERENCES. 


Whipple, George C.: Sewage Disposal. 
Rosenau: Preventative Medicine and Hygiene. 
Phelps, Earle B.: Microbiology of Sewage. 
Marshall: Microbiology. 

Folwell, A. Prescott: Sewerage. 

Samtee, E. M.: Farm Sewage. 

Whipple, George C.: Typhoid Fever. 

Fuller, George W.: Sewage Disposal. 


CHAPTER XXXL 
MILK BACTERIOLOGY. 


Axout ten billion gallons of milk are produced annually in the 
United States, one-fourth of which is consumed as milk and the 
other three-fourths as butter and cheese. The quantity of milk 
consumed varies in different localities, being greater in the North 
than in the South and greater in the country districts than in the 
city. It also varies with different classes, as seen from a survey made 
by Williams of fifteen sections of Rochester, New York. He found 
that the average consumption of milk by 21,600 individuals was 
little more than 0.24 pint per capita. Furthermore, he found that 
the poor not only used less milk and bought it in smaller quantities 
than the well-to-do, but the use of store milk and of condensed milk 
was largely confined to the laboring classes. In other words, the 
people who most needed to be careful in their buying used smaller 
quantities of the cheapest food which they bought in the most 
expensive manner. It is usually stated that about 16 per cent. of 
the average dietary in the United States consists of milk and milk 
products, yet the average daily consumption per capita of milk as 
such is only 0.6 pint, which is about half what it should be. 

Milk as Food.— Milk has been regarded from the earliest times as a 
most important article of food, and although little was known as to 
its chemical composition previous to the eighteenth century, the 
ancients attributed many and peculiar hidden virtues to it. 

Good whole milk or skimmed milk are among the best and cheap- 
est of foods. Good fresh milk is all but essential to the welfare of 
young children, and to the babe that for any reason is deprived 
of its mother’s milk, cows’ milk is practically indispensable. The 
reason is due to its composition. The composition of human and 
cow’s milk is as follows: 


Fat, Lactose, Protein, Ash, 


per cent. per cent. per cent. per cent. 
laholenvshoyrlllie 5 wa Sy | 2a 6.0-7.5 OV 7=155 0.15-0.30 
Cows’ milk a), eae o=O 3.5-5.0 2.5-4.0 0.66-0.77 


Besides these substances both cows’ and mothers’ milk carry 
organic substances which contain little or no nitrogen, one of which 
is soluble in ether and alcohol, the other in water. The true chemical 
nature of these is unknown. The amount of these substances in 
human milk at the beginning of lactation is about 1 per cent.; in 


the middle period of lactation about 0.5 per cent. 
the middle of lactation contains about 0.3 per cent. 


MILK AS FOOD 


369 


Cows’ milk at 


It is usually stated that one quart of milk is about equal in food 
value to any one of the following: 


Salt codfish 
Fresh fish 
Chicken 
Beets 
Turnips. 
Butter . : 
Wheat flour 
Cheese . 
Lean round beef 
Potatoes 
Spinach 
Lettuce 
Cabbage 
Eggs 


Fic. 44. —Composition of cow’s milk, 
investigation, Boston Chamber of Commerce, 


Milk Problem.) 


TOTAL 
SOLIDS 


WATER 


at hid TL 


Fat 


Su MN 
ny NLU 
fi ' Hy a hth 


SOLID: 
NOT 
FAT 


1915.) 


— | 
L. 3 fo h=5/2 7, 
AN eS ' 


showing variations. 
(MacNutt, The Modern 


2 pounds 
3 pounds 
2 pounds 
4 pounds 
5 pounds 
pound 

pound 

pound 

pound 

pounds 
pounds 
pounds 
pounds 
pounds 


CO He ST Db FIO OR OH OI 


(Report on milk 


This, however, considers milk only from the total calories yielded. 
Digestibility and assimilation must be considered as well as chemical 
composition and caloric value; when this is done milk ranks even 


higher than suggested by the foregoing. 


24 


Moreover, milk has other 


370 MILK BACTERIOLOGY 


functions and furnishes essential constituents to the growing animal 
which is not furnished by many other foods and which cannot be 
measured in heat units. It contains “vitamins” or “accessories,”’ 
substances belonging to a group of agents which are widely dis- 
tributed in nature and which are now regarded as essential factors 
in diet. 

Hopkins clearly demonstrated that the feeding of very small 
quantities of milk to rats which had been living on a diet inadequate 
for normal growth brought about a rapid growth in the animals. 
Moreover, Osborne and Mendel in their extensive experiments on 
the growth of animals have for several years been employing 
“protein-free milk’? as an indispensable ingredient of their basic 
diet to which certain isolated food substances are added. They find 
that no artificial imitation of this natural mixture has been devised 
to replace it satisfactorily for considerable periods of time. ‘The 
weight and health of adult rats can be maintained for many months 
on a ration consisting of protein, starch, sugar, protein-free milk, 
and lard. Young animals kept on this mixture decline after a time. 
If, however, butter is substituted for the lard growth is resumed. 
The active constituent is the fat soluble vitamin of the butter in 
contrast to the water soluble accessories present in the protein-free 
milk. Ordinary skimmed milk contains both the fat-soluble and 
water-soluble accessories. The influence of milk and sour milk upon 
the growth of chicks is seen from the following summary of a 


great many tests made by Rettger: 
Gain per chick, Pounds. 


Wed-sour milk, ;.9 1) one st ake ee BS ie Bee ices ee en 
Bed sweetimnillle*. (Oeil Joke Poe eee oe ond Ae wae e a ear oe eee 
Given no Millet ss, pcs ee ie ew OED OMe ee Pasi ohn ee oe ROTO S, 


Moreover, individuals who have lived to extreme old age have 
used milk in some of its forms. Several French laborers whose diet 
consisted largely of milk lived to be one hundred and ten years or 
over. There are also authentic records of a number of individuals 
in the Balkans, Persia, Arabia, and in the Caucasus Mountains who 
have reached extreme old age, whose main diet was milk. 

Scientists have long studied the habits of these centenarians and 
their diet was found to contain large quantities of sour milk. Metch- 
nikoff attributes their long life as due to specific bacteria taken into 
the alimentary tract with the sour milk, and the organism, Bacillus 
bulgaricus, is sometimes known as “the bacillus of long life” and is 
often used by the physician in combating certain digestive disturb- 
ances—sometimes with good effects and at other times without. 
The cause of these failures is only at the present time being fully 
understood. 

Even the acid-forming bacteria cannot gain the ascendency when 
growing on a protein-rich diet, but if grown on a carbohydrate diet 


CLASSES OF MILK 371 


soon produce sufficient acid to check, if not kill, the putrefiers which 
give rise to ptomaines. 

Milk undoubtedly owes its beneficial action to its lactose which 
is slowly absorbed and hence regulates the biochemical changes 
which take place in the lumen of the intestines. Hull and Rettger 
have conclusively demonstrated that a high lactose diet markedly 
influences the intestinal flora of man. 

Hence, nothing should be done or said to decrease the consump- 
tion of milk, but much should be done to see that the milk consumed 
is pure, clean, and free from disease-producing bacteria. For although 
milk is one of the cheapest and best of foods it is responsible for 
more sickness and deaths than perhaps all other foods combined. 

Classes of Milk.— Milk is often roughly divided into three classes, 
depending upon the care exercised in its production and handling— 
certified milk, inspected, or guaranteed milk, and common milk. 

Certified milk has no unusual properties other than those of 
exceptional cleanliness and purity. It is milk which has been pro- 
duced according to the regulations and under the supervision of a 
medical milk commission. The cows from which the milk is pro- 
duced are tuberculin-tested. The stable and cows are kept extremely 
clean and no dust is allowed in the stable at the time of milking. 
Small-top sterilized pails are used. The cows are carefully groomed 
long enough before milking to let the dust settle. The cow’s udder 
and flanks are washed just before the milking. The milker wears a 
white suit and washes his hands before milking each cow. The milk ~ 
is cooled either before or after bottling. The caps are so constructed 
that they completely cover the top of the bottle, and many dairies 
use a double cap. The caps are sterilized before use and the milk 
is kept cool during transit. The number of bacteria should not 
exceed 10,000 per c.c. of milk. Moak gives the average count of 
321 samples of certified milk delivered in Brooklyn during 1910 as 
4095 bacteria per c.c. . 

Such milk is as near pure as it is possible to produce it on a com- 
mercial scale, and although it is required that it be delivered to. the 
consumer within thirty hours after production, yet it will keep for a 
great length of time. At the Paris Exposition in 1900 certified milk 
from the United States, to the astonishment of the judges, was 
placed on exhibition in perfectly sweet condition after a journey of 
fourteen to eighteen days, or 3000 to 4000 miles, in midsummer. 

It is probable that in none of our large cities does the production 
of certified milk exceed 1 per cent. of the total supply. This is due 
to the greater price which must be charged for such milk, and the 
tendency at the present time is to produce a high grade of milk 
under less ideal conditions which can be sold at a more moderate 
price. 

This is being met in the selected, inspected or guaranteed milk 


372 MILK BACTERIOLOGY 


which is being placed on the market. This is milk produced from 
herds free from tuberculosis and which are housed and cared for 
under good sanitary conditions. Nearly as great care is taken in 
its production as in that of certified milk. Some milk so produced 
compares favorably with certified milk. 

Common milk is all milk not classified under the preceding 
heads and may vary in microbial content from a few thousand to 
many millions. The number and kind vary with the different 
dairies which produce the milk and often with the city or state in 
which it is produced, depending upon the nature of the law and the 
strictness with which it is enforced. 

The number of bacteria reported by Hill and Slack for Boston 
milk is given below: 


Per cent. 
Below 100; 000:bacteria per cielo ce acy det bea te oe See ee OD 
‘Between 100,000 and’ 500;000'per' cies. 9 ae eee ei 
Between 500,000 and 1,000,000 pere.c. . . . . . . . . 9.75 
Between 1,000;000'and..5,000;000 per cic)... Se Se ee Leo 
Above 5,000,000 pere.c..  . FRIST TAPER yh oe oe (He 5.00 
Unecountablespiates: “7 e.eeccmete oak oe ae tee ere 0.75 


Bacteria in Milk.— Milk is one of the best foods for man. Itisalsoan 

excellent food for bacteria, as is seen from the facts that millions are 
often found in a few drops, and in many cases the bacteriologist finds 
it one of the best mediums on which to grow his laboratory cultures. 
Therefore, milk should be protected from substances which contain 
bacteria, especially the disease-producing ones. It is the methods 
by which they enter and the speed with which they multiply that 
we want to consider. But it should be stated at the outset that large 
numbers of bacteria in milk indicate dirt, lack of refrigeration, or 
age. It may or may not contain the germs of disease, but there is the 
possibility. So milk with a high bacterial content is not necessarily 
harmful, but when used as a food—particularly for children—is a 
hazard too great to be countenanced, or, as stated by Conn: “Good, 
clean, fresh milk will have a low bacterial count, and a high bacterial 
count means dirt, age, disease, or temperature. A high bacterial 
count is, therefore, a danger signal and justifies the health officer in 
putting a source with a persistently high bacterial count among the 
class of unwholesome milk.”’ 

The number of bacteria occurring in milk varies with age, initial 
contamination, the care with which it is handled and kept, tempera- 
ture, and age. Milk may contain only a few or millions in each drop, 
or some market milks at times contain as many, but not as danger- 
ous, organisms as sewage. 

Initial Contamination.—The source of bacteria in milk are: (1) 
Intramammary, (2) introduced during milking process, (3) from 
milk utensils, (4) from the use of special milk apparatus, (5) con- 


INITIAL CONTAMINATION 373 


tamination in transit, (6) contamination on sellers’ or consumers’ 
premises. 

Milk as it is secreted is a sterile fluid, but it is fairly well estab- 
lished that as it is excreted from the udder it is not sterile. Harding 
and Wilson examined 1230 samples from the udders of 78 cows 
which showed an average of 428 bacteria per c.c. The ae vary 
widely with different cows, some yielding milk with as few as 25 per 
c.c., Whereas others yielded milk with bacterial contents up to 100,- 
000. The organisms obtained from the healthy udder are non- 
pathogenic and are almost invariably staphylococci, streptococci, 
and other forms of cocci. It is regarded as certain that the origin 
of these bacteria is from the outside of the teat. They find their 
way in through the orifice of the teat and extend up the milk column, 
thus infecting the milk cistern and ultimately the ramifications of the 
milk tubes through the udder. The work of Savage makes it appear 
that the number found in freshly drawn milk is determined by the 
numbers entering the teat, and the selective action of the specific 
animal. 

The bacteria introduced during the milking process are derived 
from (a) the coat, udder, and teats of the cows, (b) from the milking 
shed and clothes of the milker, and (c) from fhe hands of the milker. 
It is impossible to produce clean milk from cows, the color of which 
cannot be distinguished even a few rods away because of the filthy 
condition of their coat. Even where the animal is in a fairly clean 
condition the wiping of the udder just before milking greatly reduces 
the number of bacteria in the milk. An average of thirteen experi- 
ments at the Storrs Experiment Station yielded the following 


results: 
Bacteria in milk 


per ¢.c. 

Unwipedmiddense:- Mest twee ta ee eS ea eek ka wn bo, HUDS 
Witpedtuddersoin sec el. al arcri te os stain : 716 
DEGKreaSexchllehio awaloin oe eee ee eee ene Pore wie et er Ooo 


Numerous investigators have shown the presence of bacteria in 
large numbers in cowsheds, and many individuals have seen stables 
or milk houses in which each beam of light passing through the 
crevices seems to be filled with myriads of dancing specks. ‘These 
dust particles carry bacteria and will increase the bacteria content 
of milk. However, recent work at the New York and Illinois 
Experiment Stations has demonstrated that under fair conditions 
this is a negligible factor. 

Then the hands of the milker may not be quite clean, or perchance 
they have come in contact with disease germs from his own or some 
one’s else body, and these may find their way into the milk and at 
times multiply with an enormous rapidity. 

The influence of the milker in adding bacteria is clearly illustrated 


O74 MILK BACTERIOLOGY 


by the following experiment reported by Stocking. The average of 
19 tests with two milkers, one who had had no training in dairy sanita- 
tion, and one who had, showed 17,105 bacteria per c.c. for the 
untrained man and 2455 for the trained man. The only difference 
was the knowledge possessed by the trained man. 

Even more important than the surroundings in contaminating 
milk are the utensils. Many buckets are wrongly constructed or 
not scalded each time so that every seam contains hidden away 
millions of bacteria. These immediately grow on reaching the 
fresh, warm milk. Then the strainer may contain a good seeding 
of bacteria. It would be a great step in advance could the strainer 
by some means be done away with, for then greater care would be 
taken in the production of milk; otherwise, it would be unsalable. 
The condition is somewhat similar to that which existed when it was 
first suggested that bread be wrapped. There was a baker’s con- 
vention and the subject had come up for consideration and the 
members had practically agreed that all bread offered by them should 
be wrapped, when an old veteran arose and said, “If we wrap our 
bread in white paper and handle it as we do now the paper will be 
so dirty that when it reaches the consumer he will refuse to buy.” 
So it is with milk; if it had to be sold in the condition in which it 
comes at times from the barn, it would be refused. Not that the 
strainer reduces the number of bacteria in the milk, for it does not. 
It only removes the particles which are visible to the naked eye after 
they have been washed nearly free from bacteria. 

Prucha and coworkers studied the influence of all the utensils 
that normally come into contact with the milk both at the barn 
and at the dairy. They found that when they were all carefully 
steamed the germ content of the milk in the bottles was about 4566 
bacteria per c.c. When similar conditions obtained, except that the 
steaming of the utensils was omitted, the germ content of the milk 
approximated 257,240 bacteria per c.c. 

Of all the various utensils coming into contact with the milk at 
the barn and at the dairy, it was found that the clarifier and the 
bottle-filler, when unsteamed, proved to be the most prolific sources 
of contamination. 

It would, therefore, seem that the most important factor in 
producing good milk is the scrupulous cleanliness of the milk uten- 
silsand not so much surroundings, as has been so much taught in the 
past. 

It is difficult to accurately measure the contamination in transit 
and on the sellers’ premises, but it is quite evident that at times it is 
large. Orr reported average increases as high as 22.7 per cent., 
whereas it should be zero under ideal conditions. 

Growth of Bacteria in Milk.—Saprophytic and many pathogenic 
bacteria multiply in milk so that the number found in milk is 


CHANGES PRODUCED IN MILK BY BACTERIA 375 


governed, in addition to the factors considered above, by age and 
temperature. The influence of temperature is illustrated by the 
following: 


Temperature 
maintained for Bacteria per c.c. at Hours to curdle 
twelve hours F. end of twelve hours. at 70° F. 
40 “4G iS whe Boa ee eee ee 4,000 75 
45 Cee lett = be IE ale Se 9,000 75 
50 esa Pk ee Ty ea tS 18,000 72 
55 2 Pc A ee Lee 38,000 49 
60 Ree ead ee eh nese We alk thay ds 453,000 43 
70 Re eee Pep tis he) ye eee TR 8,800,000 32 
80 PROP Se Mien Re a fae MEE oe “OO OOOOO0 28 


All of these samples at first contained the same number of bacteria 
but were kept for twelve hours at the different temperatures and 
then all maintained at the high temperature. We find over ten 
thousand times as many bacteria at the end of twelve hours in the 
sample kept at a high temperature as the one kept at a low. 
Although the difference in temperature was maintained for only 
twelve hours, the milk at 40° kept three times as long as did that at 
80°. 

Changes Produced in Milk by Bacteria.— The changes occurring 
in milk are governed by the specific bacterial flora which it contains 
and the temperature at which it is kept. Normal clean milk, if 
kept at a temperature of between 10° and 21° C., passes through a 
sequence of changes which can be divided into four stages. 

First Stage.—The first of these is known as the germicidal stage, 
and lasts a few hours after the milk has been drawn from the udder. 
During this stage there is a decrease in the number of organisms, 
as shown by the plate method. The extent of this decrease varies 
with the milk of different cows and the temperature at which the 
milk is kept. The higher the temperature, the more marked the 
decrease, the sooner the end of the germicidal period is reached. 
There is a great difference in opinion among bacteriologists con- 
cerning the nature of the phenomenon. Some would account for 
it on the grounds that milk is a favorable cultural media for many 
bacteria, but not all. The ones for which it is unsuited rapidly 
die off. Others consider that the milk, like the blood and many 
other body fluids, possesses bactericidal power which is very weak 
and soon lost. Rosenau and McCoy, however, consider that the 
bacteria are agglutinated and not killed. On plating, the clump 
gives rise to the colony in place of each individual organism, as is nor- 
mally the case. 

This germicidal power is lost on boiling the milk or heating to a 
temperature of 80° C., and some have urged this as an objection 
against pasteurization, but in the “holder” process this is not a 
warranted objection. 


376 MILK BACTERIOLOGY 


Second Stage.—'This stage extends from the end of the germicidal 
period to the time of curdling. There may be a gradual increase 
during this time of many species, but the predominating types are 
the Bact. lactis acidi. These rapidly produce lactic acid which 
exerts a suppressing influence on many species. When the milk 
reaches an acidity of .75 to .80 per cent. it usually curdles. The lactic 
acid organisms seldom produce more than 1.25 per cent. acid. 

Third Stage.—This stage extends from the time of curdling until 
the neutralization of the acid. The acidity becomes so great that 
the action of the lactic acid bacteria is checked and their number, 
which at first may be as high as 100,000,000 per c.c., rapidly 
decreases. The predominating species become Oidiwm lactis, certain 
species of molds and yeasts. The proteins are broken down with 
the formation of ammonia which neutralizes the acidity. 

Fourth Stage.—The liquefying and peptonizing bacteria which 
remained inactive in the sour milk find suitable conditions in the 
alkaline media for their growth. They rapidly decompose the 
casein. 

Abnormal Changes in Milk.— At times foreign undesirable organ- 
isms find their way into milk and produceabnormal and objectionable 
changes. The B. coli communis and the Bact. lactic aerogenes types 
produce considerable gas and disagreeable odors and flavors in the 
milk. B. lactis viscosus produces a slimy or ropy condition of the 
milk. The slimy condition is supposed to be due to the mucin con- 
taining capsule which surrounds these bacteria. Milk may be normal 
in color when first produced, but on standing may turn blue due to 
B. cyanogenes or red due to B. erythrogenus or B. prodigiosus. At 
times a bitter taste develops in milk some time after it has been 
drawn from the udder. This, according to Conn, is due to a micro- 
coccus. 

Although these changes are very objectionable when considered 
from the standpoint of the dairymen, they are not known to be the 
cause of illness. However, when milk putrefies with the production 
of a bitter alkaline milk illness often does result from its use. This 
may be due to the poisonous action of the ptomaines which it con- 
tains, or probably more often to the bacterial infection. 

Classes of Bacteria.—'The bacteria found in milk are a hetero- 
geneous lot but, according to Hastings, may be roughly divided 
into five classes, as follows: 

1. Acid-forming Bacteria.—There are constantly present in milk 
many acid-forming bacteria. ‘These vary in morphology, cultural 
characteristics, and products of fermentation. They may be divided 
into five groups. The number and kind vary greatly in milk, 
depending upon the methods of handling. 

(a) The most important organism of this group is Bact. lactis 
acidi. The group, however, includes a number of organisms. They 
produce no gas, a mild acid flavor, and are desirable. 


ABNORMAL CHANGES IN MILK BYMs 


(b) The best known representatives of this group are B. coli 
communis and Bact. lactis aerogenes. These organisms give to milk 
a sharp tang and are the particular enemies of the cheese maker as 
they are the cause of gassy curd. They are especially numerous in 
milk which is produced and handled under unsanitary conditions 
and in such milk outnumber those of Group a, but the rapid growth 
and acid production of Group a soon checks them. 

(c) This is represented by Bacillus bulgaricus and the rod-shaped 
organisms which have been especially studied by de Freudenreich. 
They produce a curd which is easily broken by shaking and shows no 
tendency to express whey. They give to the milk a pleasant acid 
flavor and are desirable. 

(d) Acid-forming Udder Bacteria.—These are the characteristic 
bacterial flora of the healthy udder and consist mainly of cocci with 
few bacilli. They are slow growers and may curdle milk, but the 
curd so formed resembles that formed by rennet. They produce 
acetic, propionic, butyric, and caproic acid but no lactic acid. 
They are an unimportant ou of organisms, so far as the milk 
is concerned. 

2. Peptonizing Bacteria.—These organisms digest the casein 
either with or without coagulation at times with the formation of 
an alkaline reaction. Most of these are bacilli of various shapes 
and sizes, some of them being the largest organism found in milk. 
There are both motile and non-motile varieties. Many develop 
very strong putrefactive odors. Barny or cowy odors are caused by 
this type of bacteria. They are all undesirable and their presence 
in milk indicates unsanitary condition of production and handling. 

3. Bacteria Producing Milk of Unusual Character.—Occasionally 
bacteria which produce abnormal changes or so-called “diseases” 
of milk find their way into milk from unclean surroundings. They 
produce various queer milks, for example, red, blue, and green. 
Sometimes milk develops a bitterness after it is drawn. This is due 
to the products from a number of bacteria and.yeast. At other times 
milk is changed to a slimy or ropy consistency and may at times 
result in considerable economic losses. These organisms are quite 
resistant to heat and frequently pass uninjured through the ordinary 
methods of cleaning and scaldings. Because of this, dirty utensils 
once infected become a constant source of infection. 

4. Inert Organisms.—These are mostly cocci which produce no 
appreciable change in milk and are unimportant. 

5. Pathogenic Bacteria.—This class consists of the pathogenic 
bacteria, B. dysenterie shiga, B. dysenterie fleener, B. typhosus, B. 
paratyphosus a. and B. paratyphosus B., V. cholerw, Bact. diphtherve, 
Bact. tuberculosis, B. lactimorbimic melitensis. These organisms 
produce no perceptible change in the milk in which they grow but 
are dangerous and may give rise to epidemics. 


CHAPTER -XoCxTA: 
MILK AND DISEASE. 


ALTHOUGH milk is one of the cheapest and best of foods, yet it is 
responsible for more sickness and death than perhaps all other foods 
combined. The reasons for this have been summarized by Rosenau 
as follows: . 

“1. Bacteria grow well in milk; therefore, a very slight infection 
may produce widespread and serious results. (2) Of all foodstuffs, 
milk is the most difficult to obtain, handle, transport, and deliver 
in a clean, fresh, and satisfactory condition. (3) It is the: most 
readily decomposable of all our foods. (4) Finally, milk is the only 
standard article of diet obtained from animal sources consumed in 
its raw state.” 

Diseases conveyed through milk are of two classes: (1) Definite 
diseases of animal origin—tuberculosis, foot-and-mouth disease, 
malta fever, and anthrax, and indefinite ailments as diarrheal infec- 
tions and probably contagious abortion. (2) Diseases of human 
origin—typhoid fever, paratyphoid fever, diphtheria, scarlet fever, 
tuberculosis, septic sore throat, and possibly others. 

Sources of Infection.—Infection of bovine origin is very common, 
especially in the case of tuberculosis wherein the animal is suffering 
with open cases of this disease and the organism gets into the sur- 
roundings from the respiratory or alimentary tract. Extreme care 
in the milking process may decrease the infection from this source, 
but not so in the case of tuberculosis of the udder, which probably 
accounts for the main cases where the tubercle bacilli find their way 
into milk. 

As a rule milk becomes infected from human sources. This may 
be either direct or indirect human infection. 

Direct human infection may come from a person either suffering 
with the disease or carrying the infective organism. ‘The more 
common are the following: 

1. The most common method is where the milkers or other 
handlers of milk are suffering with a communicable disease in a mild° 
unrecognized condition. 

2. A second common source of infection is where the milker or 
vender of milk has been brought in contact with sufferers of com- 
municable diseases and still attends to his regular work in the hand- 
Jing of milk. 


CHARACTER OF MILK-BORNE DISEASE 379 


3. A third and probably very important source of infection comes 
from carriers who work on the farms, in dairies, or other places 
where milk is handled. 

Indirect human infection comes largely from the use of infected 
water which is used in the washing of buckets, bottles, and other 
milking utensils. Cows often have access to polluted water and 
infection from this source may find its way into the milk from being 
on the body of the animal. 

Character of Milk-borne Diseases.—Milk-borne diseases have 
characteristics which greatly assist the epidemiologist in his work. 
The most important are the following: 

1. The cases usually follow the route of the milkman and it is 
often possible to plot his route from the cases of the specific disease. 
There would thus be the inhabitants of homes where the infected 
milk is used suffering with the disease, while neighbors who use 
other milk escape. There may be many purchasers of the infected 
milk who may escape, but when careful inquiry is made it is found 
consumers of the implicated supply furnish a much higher percentage 
of eases than does the rest of the community. The smallest per- 
centage invasion of households is met with in scarlet fever outbreaks. 
But this is easily explained when one considers the number of missed 
cases in this disease. 

2. The outbreaks from infected milk are usually explosive. 
Sometimes the majority of the cases occur within a few, days of each 
other. Usually there is little secondary infection and the decline is 
rapid on removal of the source of infection. The epidemic at Stam- 
ford, Connecticut, in 1895, is a good example. There were 586 
cases of typhoid fever and 22 deaths in the period from April 15 to 
May 28. There were 176 persons stricken during the first week. 

Although the explosive type of epidemic is usually characteristic 
of milk-borne outbreaks, yet Parker points out that the smoldering 
kind may be very commonly due to infected milk. He cites as an 
example the experience of Hill of North Branch, Minnesota, where 
one of the physicians pointed out that in his seventeen years of 
practice during the first twelve there was no typhoid fever, but in the 
last five years native cases of unknown origin had been frequent. 
Acting on this information, a list of 21 cases of typhoid fever that 
had appeared in the town in the last five years was made, and 
inquiry showed that seventeen of the patients were regular custo- 
mers of a dairyman who came to town five years before, two others 
were irregular customers, and two others may have used milk from 
his dairy. It was learned that the wife of the dairyman, who washed 
the cans, had suffered with typhoid fever twenty-two years before 
and gave a positive Widal reaction, but typhoid bacilli were not 
isolated from her stools. She was forbidden to have anything more 
to do with the dairy and the proprietor was told that if another 


380 MILK AND DISEASE 


primary case developed among his customers his dairy would be 
closed. Rumors of the affair spread through the town and his 
customers left him and the family moved away, after which there 
was no more typhoid fever. 

3. It frequently happens that the better class suffer more than 
do the poor, as they can afford more milk and use it more freely. 

4. There is a special incidence among milk drinkers, there fre- 
quently being an individual who dislikes milk escaping, whereas 
the remainder of the family is attacked. 

5. Women and children are more often victims in milk-borne 
typhoid than are the adult male population due to their use of raw 
milk. 

6. There is some evidence which indicates that the incubation 
period is shorter and the mortality lower in milk-borne epidemics 
than in others. 

The mild character of the disease is usually attributed to the 
attentuation of the pathogenic properties of the microdrganisms 
through their growth in milk. 

7. Milk epidemics of typhoid spread over a rather short milk 
route, whereas when milk is brought from a considerable distance 
there is not the likelihood of infection, thus indicating that typhoid 
germs tend to disappear from milk under certain conditions. 

Extent of Milk-borne Disease.—The extent of milk-borne epidemics 
cannot be accurately measured, as even at the present day many 
cases go undetermined or perhaps attributed to other causes. But 
the experience of Boston, Massachusetts, which has a fair milk 
supply indicates the gravity of the subject. 


Year. Epidemics. Cases. 
1907 Diphtherta’ vyos- elo sae Is pecicehne Bee Ay ar ane means 72 
1907 Scarletiiever i unis ey, eet ee Ee PA ene ee thili7¢ 
1908 Ty phOrdHlGver a ay a sckc tQhnin, ORS ee ey Ler ae Oe 400 
LOLO. we Searlettever: co sarees ee, ae ee tes eee 842 
1911 Septic sore’ throatie ... ofa soe tLe ee ae ee ee Oe 

Total Ree Ss hae hee eater e mare fe ee OS 


This indicates that scarlet fever and septic sore throat may be 
conveyed even more often than typhoid fever. 

Tuberculosis is the most important of all milk-borne diseases, 
both because of the frequency with which it is conveyed and the 
seriousness of the disease. It may be either bovine or human in 
origin. Human infection is rarer than bovine, but it is certain that a 
tubercular patient may infect milk, and Hess in 1918 actually isolated 
the human tuberculosis bacillus from a sample of market milk. 

Koch in 1901 announced that there was practically no danger of 
man’s contracting tuberculosis from cattle, but his statement was 
immediately challenged by many bacteriologists who have since 
brought forth conclusive evidence of the falseness of Koch’s dictum. 


EXTENT OF MILK-BORNE DISEASE os 


The summarized English, German, and American findings in 
1511 cases are given below: 


COMBINED TABULATION SHOWING ORIGIN OF CASES OF 


TUBERCULOSIS. 
2 ran : neler =F | ee 
| Adults sixteen | Children five to | Children under 
Diagnosis of cases years and over. | sixteen years. | five years. 
examined. 
Human.) Bovine. Human.) Bovine. | Human.| Bovine. 
Pulmonary tuberculosis . . err kes: 3 14 as 35 1 
Tuberculosis adenitis, axillary or 
inguinal : 3 4 2 mts 
Tuberculosis adenitis, cervical : SOM mera 36 PPP A AN | 24 
Abdominal tuberculosis . . . “| Gs | 4 8 elo 14 
Generalized tuberculosis, aliment- | | | 
ary origin ahs! ee | 6 1 3 4 Pili 15 
Generalized tuberculosis aaa ZO Seal 5 1 (ae 7 
Generalized tuberculosis, including | 
meninge, alimentary origin . | 5 rae 11 as 81 11 
Tubercular meningitis. . . ya eee 3 $8 28 | 4 
Tuberculosis of bones and joints | By 4 1 At OP e wu) 
Genito-urinary tuberculosis . . | 22 | 1 Pe IM he ae He ee 
Ruberculastnat sities eens le LD Bh Aes Ie Go| Qo 
Miscellaneous cases: | | | 
Tuberculosis of tonsils . . ae | AEN Te Leia 
Tuberculosis of mouth and cer- | 
vicalmodes, 95). eh ites os aly ll 
Tuberculosis sinus or SRSERE Si 2 Sots bee xan Roe 
Sepsis, latent bacilli . . . aa Ke: ig! ee 1 
OLAS peer ae are LS 940 115) 11 i AG PALA AG 


The percentage incidence of bovine infection would, therefore, 
be as follows: 
Adults sixteen Children five Children under 


years and over. to sixteen years. five years. 
Pulmonary tuberculosis . . . . 0.4 0 2.8 


Tuberculosis adenitis, cervical . . Det 38.0 61.0 
Abdominal tuberculosis 5 20.0 53.0 58.0 
Generalized tuberculosis, Aline tang 

origin ; See etd ise te ASO Gif 0) 47.0 
Generalized piberenlonts ; Mt 0 16.0 8.6 
Generalized tuberculosis, including 


meningitis, alimentary origin . 0 0 66.0 
Tubercular mentingitis . . , 0 0 4.6 
Tuberculosis of bones and foints 3.0 6.8 0 
Quberculosisiot-skiny =. ee.) =) =) 2370 60.0 0 


It is probable that the majority ‘of all cases of bovine tuberculosis 
in man are due to infected milk, as there is little danger from meat 
since it is usually cooked and tuberculosis of the muscles is very 
rare. 

Tuberculosis is quite prevalent among cows, varying in different 
places from a few to as high as 50 per cent. Savage gives from 


382 MILK AND DISEASE 


Dallar the following as being the percentage found in various places: 
England, 20 per cent.; Denmark, 31 per cent.; Sweden, 42 per cent.; 
Norway, 8.4 per cent.; Belgium, 60 per cent.; Massachusetts (1897), 
58.9 per cent. 

It is a general conception that tubercle bacilli occur only in milk 
obtained from animals suffering with tuberculosis of the udder; this 
is not strictly true as is seen from the following conclusions by 
Mohler: 

“The tubercle bacillus may be demonstrated in milk from tuber- 
cular cows when the udders show no perceptible evidence of the 
disease either macroscopically or microscopically. 

“The bacilli of tuberculosis may be excreted from such udder in 
sufficient numbers to produce infection in experimental] animals 
both by ingestion and inoculation. 

“The presence of the tubercle bacilli in the milk of tubercular 
cows is not constant, but varies from day to day. 

“Cows secreting virulent milk may be affected with tuberculosis 
to a degree that can be detected only by the tuberculin test. 

“The physical examination or general appearance of the cow 
cannot foretell the infectiveness of the milk. 

“The milk of all cows which have reacted to the tuberculin test 
should be considered as suspicious and should be subjected to sterili- 
zation before using.” 

Shroeder, however, concluded that only 40 per cent. of the cows 
which react to the tuberculin test actively expel tubercle bacilli. 

Market milk often contains the tubercle bacilli, as may be seen 
from the following table compiled by Parker. 


TUBERCLE BACILLI IN MARKET MILK. 


Samples Number _ Percentage 
Date. Place. Investigator. examined. positive. positive. 
1899 England Macfayden tial 17 Jo 
1904 ' Germany Miller 1596 97 6.2 
1904 Germany Beatty 272 27 10.0 
1898 Liverpool Delepine 12 22 Mea} 
1897 Liverpool Hope 228 12 5.2 
1900 London Klein 100 7 7.0 
1893 St. Petersburg Scharbekow 80 4 Sy) 
1900 Kiew Pawlowsky 51 1 2.0 
1900 Krakow Bujwid 60 2 3.3 
1900 Naples Marconi 14 7 50.0 
1898 Berlin Petri 64 9 14.0 
1900 Berlin Beik 56 17 30.3 
1898 Schev Ott 27 27 Vibe a 
1898 K6nigsburg Jaeger. 100 7 TW 
1908 Leipzic Eber 210 22 10.5 
1905 Rotterdam Smit 567 14 DT | 
1906 Rotterdam Smit 1584 45 2.8 
1908 Washington Anderson 223 15 Grd 
1909 Louisville Field 119 46 29.5 
1909 New York Hess 105 17 16.2 
1909 Philadelphia Campbell a 810) 18 13.8 
1910 Chicago Tonney 144 15 10.5 
1910 Rochester Goler 237 30 12.6 


THE TUBERCULIN TEST 383 


It has been shown that man is susceptible to the bovine type of 
tuberculosis and that the organism often is found in market milk, 
and Rosenau estimates that probably 7 per cent. of the cases of 
tuberculosis thus have their origin. The significance of these 
figures becomes apparent when we realize that 160,000 individuals 
die each year in the United States of this disease, and 11,200 would 
get their infection from milk. 

This is a needless loss of human life; for the information now 
available is sufficient to prevent every one of these cases if milk be 
obtained only from cows which have given negative tuberculin 
tests. 

The Tuberculin Test.— This reaction should be applied to all cows 
and is carried out as follows: 

“Tnspections should be carried on while the herd is stabled. If it 
is necessary to stable animals under unusual conditions or among 
surroundings that make them uneasy and excited, the tuberculin 
test should be postponed until the cattle have become accustomed 
to the new conditions. The inspection should begin with careful 
physical examination of each animal. This is essential, because in 
some severe cases of tuberculosis no reaction follows the injection 
of tuberculin on account of saturation with toxins, but experience 
has shown that these cases can be discovered by physical examina- 
tion. The latter should include a careful examination of the udder 
and of the superficial lymphatic glands and auscultation of the 
lungs. 

“Each animal should be numbered or described in such a way 
that it can be recognized without difficulty. It is well to number 
the stalls with chalk and transfer these numbers to the transfer 
sheet, so that the temperature of each animal can be recorded in 
its appropriate place without danger of confusion. The following 
procedure has been used extensively and has given excellent results: 

(a) “Take the temperature of each animal to be tested at least 
twice at intervals of three hours before tuberculin is injected. 

(b) “Inject the tuberculin in the evening, preferably between 
the hours of 6 and 9 p.m. The injection should be made with care- 
fully sterilized hypodermic syringes. The most convenient point 
for injecting is back of the left scapula. Prior to the injection the 
skin should be washed carefully with a 5 per cent. solution of car- 
bolic acid or other antiseptic. 

(c) “The temperature should be taken nine hours after the injec- 
tion, and temperature measurements repeated at regular intervals 
of two to three hours until the sixteenth hour after the injection. 

(d) “When there is no elevation of temperature at this time 
(sixteen hours after injection) the examination may be discontinued, 
but if the temperature shows an upward tendency, measurements 
must be continued until a distinct reaction is recognized or until 
the temperature begins to fall. 


o84 MILK AND DISEASE 


(e) “If the reaction is detected prior to the sixteenth hour the 
measurements should be continued until the expiration of this 
period. 

(f) “If there is an unusual change of temperature of the stable 
or a sudden change in the weather, this fact should be recorded on 
the report blank. 

(g) “If a cow is in a febrile condition tuberculin should not be — 
used, because it would be impossible to determine whether, if a rise 
in temperature occurred, it was due to the tuberculin or to some 
transitory illness. 

(h) “Cows should not be tested within a few days after or before 
calving, for experience has shown that the results at this time may 
be misleading. 


INSIDE 
SECTION 
SHOWING 
GRACKET 
FOR 
TRAY 


Fig. 45.—Straus’s home pasteurizer. (From Rosenau’s Preventative Medicine.) 


(1) “The tuberculin test is not recommended for calves under 
three months old. 

(7) “In old, emaciated animals and in retests use twice the usual 
dose, for these animals are less sensitive. 

(k) “Condemned cattle must be removed from the herd and kept 
away from those that are healthy. 

1. “In making postmortems the carcasses should be thoroughly 
inspected and all the organs should be examined. 

“No animal whose temperature exceeds 39.5° C. (103° F.) is a 
fit subject for the tuberculin test. 

“A rise of temperature to above 40° C, (104° F.) in any animal 


PASTEURIZATION 389 


whose temperature at the moment of injection was below 39.5° C. 
(103° F.) is to be regarded as a positive reaction. 

“Any rise in temperature between 39.5° C. (108° F.) and 40° C. 
(104° IF.) must be regarded as of doubtful significance; animals 
exhibiting such require special study.” 

Milk-conveyed typhoid fever can be handled nearly as effectively 
as can tuberculosis by excluding typhoid carriers as producers and 
handlers of milk. This can be very easily and efficiently done by 
requiring the blood test for all dairymen and their workers. 


Milk Pasteurized for. 30 Minutes at 


; : 3 
siete 82.2°C. 87.8°C. 93.3 °C. 
(170° F.) (180° F.) = (190° F.) (200° F) 
-— 
-— 
— 
= 
— 
HF — 
Acid — ; 
3 
a —— 
Coaguldting E> = = 
Group FS Ny Sis 
— ow “2 
p ; z 
— Z zz = 
-— 2 os ue 
= Ga $a “ 
: sz wo 3 
a ie 
Acid 28 ew $0 20 
2= 3 350 <a 
Group oe Zw 29 $9 
ac ae 25 ee 
St <3 95 oS 
ba <> ou Ou 
ra 22 >> zr 
tA So zz oF 
y te 9° 50 420 
a « 
aw ° 
2 ur o% $2 eS 
= oa oz oa 3 
2 ao 3° az ot 
= cw <> Ox B59? 
re) zS : oo Zu ~ 
= FO ets Zo zz ae 
2 we 5 - to yu 8 
= Zz, or az vz = 
w Ors we 3 2 3 = 
= KE I” <t aS ‘ 
u 3a rw se ga 4 oe 
* <9 - 23 = g Fy 
Inert 2 ~w ua ox rz = = 
a Se 62 << < 2 2 
Group 2 z< 32 So = = 2 
a =" as : 3 = = 
= <5 Fe La za < = 
S 53 wu? . AO we » ® 
9 = w 7, w 
oo a3 0 AS = = = 
3 3 Js Ao = = = 
2 ee or zc ae b - 
3 a z ag Az > 5 
i 3] {ile!]_ 7722 3 3 
5 $6 25 Zo Az? 2 = 
u oF re F $3 
oO oo no Ee a< 
< oo oe dz ox 
s ee az 25 ow 
a2 ae <u Az 
z< es oo AR 
8s O5 Za Se 
< oo <> AA 
ax z< zo Aol 
32 58 ro 32 
. od = 93 
Alkali Su Zo so Ace 
Grou ee Eo = o< 
p rn a< tS 38 
3 ey 28 5° 
c Of = Az 
< z Ca 
4 <w <u 
Se eB 
=x a uw 
=3 5x 
z is 
ee = 
Peptonizing . 
Are 


Group 


Fic. 46.—The hypothetical relation of the bacterial groups in raw and pasteurized 
milk. (Tanner, after Ayers and Johnson.) 


Pasteurization.—There are two methods of pasteurization—the 
“flash” and “continuous” processes. In the flash method the milk 
is heated to 80° or 90° C. for from one to five minutes and then 
cooled to 50° C. or below. This method is rapid, cheap, and much 

25 


386 MILK AND DISEASE 


in vogue but does not give uniform results, is not entirely reliable, 
and does not meet the approval of the sanitarian. 

The continuous method is much to be preferred and consists in 
heating the milk to a temperature of 65° C. for thirty minutes and 
then cooling to 50° C. or below. 

The ideal method for home pasteurization of milk is outlined by 
Rosenau as follows: 

“Pasteurization in the bottle is the perfection of the art. It is 
the ideal method, because the danger, however slight, of recontami- 
nation is entirely eliminated. In order to pasteurize milk in bottles 
the bottles must be well sealed with a tight cork and cap, or equally 
effective stopper. The bottles containing the milk may either be 
immersed in a water bath, brought to the proper temperature, held 
there a sufficient length of time and then chilled; or the method used 
in beer pasteurization, such as the Loew pasteurizers, may be used. 
In this case the bottles are subjected to a spray or shower of heated 
water. 

“ After the bottles have been thoroughly cleaned they are placed 
in the tray (A) and filled with the milk or mixture used for one 
feeding. Then put on the corks or patent stoppers without fasten- 
ing them tightly. 

“The pot (B) is now placed on the wooden surface of the table 
or floor and filled to the support (C) with boiling water. Place the 
tray (A) with the filled bottles into the pot (B) so that the bottom 
of the tray rests on the support (C), and put cover (D) on quickly. 

“After the bottles have been warmed up by the steam for five 
minutes, remove the cover quickly, turn the tray so that it drops 
into the water, replace the cover immediately. This manipulation 
is to be made as rapidly as possible to avoid loss of heat. Thus it 
remains for twenty-five minutes.” 


REFERENCES. 


Rosenau: The Milk Question. 

McNutt: The Modern Milk Problem. 

Savage: Milk and the Public Health. 

Parker: City Milk Supply. 

Lane, Clayton: Milk and Its Hygienic Relations. 
Heineman: Milk. 

Douglass: The Bacillus of Long Life. 


CHAPTER XX XLT 
BACTERIA IN OTHER FOODS. 


Aux foods except those cooked just previous to eating contain 
bacteria, the number and kind varying with the specific product and 
especially the method of handling. The greater part of such 
bacteria are without special significance. Some are beneficial and 
play an important réle in the ripening or other changes through 
which the food passes. These are considered in a later chapter, the 
present one being reserved for a consideration of those bacteria 
which cause spoilage of food though not necessarily injurious to health, 
but of importance from an economic standpoint, and the pathogenic 
organisms which find their way into food and may infect the con- 
sumer. 

Bacteria in Butter.— Many of the bacteria which occur in unclean 
milk multiply and give bad flavors to the butter produced from 
such milk. This is true to such a degree that most dairies first 
sterilize their cream and then add to it a pure culture for the ripen- 
ing of the cream. 

Any pathogenic bacteria which find their way into the milk may 
persist in the butter. While the typhoid organism grows well in 
fresh milk the increased acid production tends to check their mul- 
tiplication and may actually kill many. They are, however, fairly 
resistant to lactic acid, as seen from the following results (Krum- 
wiede and Noble): 


Reaction. Number of typhoid 

Days. Per cent. bacilli. 
PEEL A SORE Si Reracee a el ae Rea ee EL IN WAS [er 392,000 
Uf aah AA GR Cees Eth dee aan Ol eR er eee ats eS A 65,000,000 
ST a ga Se PR ee ere tt es cA Psy tO) 300,000,000 
Le ee serrata | 3. St Viasat met ane 113,000,000 
UR ae el orale © abies epee teh) AA aor ny hye heen 181,000 
| ee ee ek Ce oS eek eS pe ge Be bee (SO 400 


Hence, lactic acid cannot be depended upon to free butter from 
the typhoid bacilli. Moreover, numerous investigators have found 
typhoid bacilli in butter after varying lengths of time—Bolley, 
five to ten days; Heim, twenty-one days; Pfulel, twenty-four days; 
Buck, twenty-seven days. Washburn obtained the organism after 
one hundred and fifty days from butter which had been experimen- 
tally infected with typhoid bacilli, and Boyd reports an epidemic 
of typhoid fever which resulted from butter. Probably the longevity 
of BL. typhosus in butter would vary greatly with the temperature 
and other factors, but it is quite evident that butter should not be 
produced from infected milk. 

Tubercle bacilli multiply only slowly—if at all—in milk; hence, 
it is the initial contamination which counts. But we have seen in 


388 


BACTERIA IN OTHER FOODS 


the preceding chapter that these organisms often find their way into _ 
milk, and the following table after Briscoe and MacNeal indicates 
that they often occur in butter. 


Author. 


Brusaferro 
Roth : 
Obermiiller 
Schuchardt 
Obermiiller 
Gréning 
Himesch 
Rabinowitsch. 
Rabinowitsch. 
Petri 
Hormon and 
Morgenroth | 
Rabinowitsch. 
Rabinowitsch. 
Rabinowitsch. | 
Rabinowitsch. 
Obermiiller 
Korn 
Ascher. 
Jiger . 
(CS 
Weissenfield . 
Grassberger 
Herbert 
Herbert 


Herbert 
Herbert 


Abenhausen 
Hellstr6m: 
Bomhoff 
Pawlowsky 
Tobler 
Lorenz 
Markl 
Herr and 

Beninde . | 
Aujeszky .. | 
ahh eed ae, Blea 
Teichert | 
Reitz 


Eber 

Briscoe and 
MacNeal 

Riper” 7 Ses cal 

Rosenau et al | 


Marchiotti 


| Date. 


1890 
1894 
1895 
1896 
1897 
1897 
1897 
1897 
1897 
1897 


1897 
1899 
1899 
1899 
1899 
1899 
1899 
1899 
1899 
1899 
1899 
1899 
1899 
1899 


1899 
1899 


1900 
1900 
1900 
1900 
1901 
1901 
1901 


1901 
1902 
1902 
1904 
1906 


1908 
1911 


1912 
1914 


1917 | 


Samples 


Place. | exam- 
ined. 
Turin 9 
Zurich 20 
Berlin 13 
Marburg 42 
Berlin 14 
Hamburg 17 
Wien ? 
Berlin 30 
Philadelphia 50 
Berlin 102 
Berlin 10 
Berlin 15 
Berlin ? 
| Berlin 15 
| Berlin 19 
Berlin 10 
Freiburg 17 
K6nigsburg 27 
K6onigsburg 3 
Milan 100 
Bonn 32 
Wien 10 
| Tibingen 43 
Wirttem- 58 
burg 
Berlin 20 
|Minchen | 5 
Marburg 39 
Helsingfors 8 
Marburg 28 
Kiew 23 
Zurich 12 
Dorpat 30 
Wien 43 
Breslau 52 
Budapest 17 
Christiania | 16 
Rosen | 40 
Stuttgart 94 
| Leipzic 150 
| Urbana, II. 6 
| Boston eel 


bo 
o 


ee oo _ 
bo Ge wocorocnrarownds = 


—y 


4 
CCoCWNrFrNHKHOGM 


o 


o 


coonNroroco 


Samples |Per cent. 
positive. positive. 


INCIDENCE OF TUBERCLE BACILLI IN MARKET BUTTER. 


Remarks. 


oooccor 


w 


onwo 


Bow Ff OO 


Microscopic method. 


16 tested, 2 lost. 


First series. 
Second series. 
Third series. 
Fourth series. 


Pseudotuberculosis, 
5 per. 

Pseudotuberculosis, 
8 per. 

Pseudotuberculosis, 
4 per. 


.5 | 12 samples, 4 lost. 


39 samples, 11 lost. 


Two were doubtful. 


.5 | Butter from eighty- 


eight dairies. 


9 
.6 | Creamery butter. 
4 


| 52 per cent. of the 
samples contained 
acid-fast bacilli. 


BACTERIA IN BUTTER 389 


Tubercle bacilli have also been found in oleomargarine. Briscoe 
and MacNeal tabulated the analysis of 209 samples, 4.5 per cent. 
of which were found to contain tubercle bacilli. 

The longevity of tubercle bacilli in butter is even greater than that 
of B. typhosus, for Schroeder and Colton demonstrated their pres- 
ence in butter which had been kept for one hundred and sixty days; 


130 

WD ees Commercial score 
——__.. Bacteria content 

110 

100 


= co iS) 
i=) i=) i) 
~ 

4 

/ 


Millions of Bacteria per Gram of Cheese 
$ 


50 60 70 80 ¥0 100 
Days 


10 20 30 40 


Fic. 47.—Bacteria content and commercial score of Cheddar cheese. (After Harding 
and Prucha, 1903.) 


and contrary to prevailing opinion Mohler, Washburn, and Rogers 
found tubercle bacilli were not devitalized by cold storage, and in 
salted butter they were found to retain their virulence for six 


months. 
Diphtheria bacteria have been found in butter, and an outbreak 
at Lewiston, Minnesota, was believed to have been caused by eating 


infected butter. There had been no diphtheria in the place until a 


390 BACTERIA IN OTHER FOODS 

boy returned from the “Twin Cities” after an attack of diphtheria. 
The milk from the farm where he lived was sent to a creamery and 
every family in the place, in which there was diphtheria, was found 
to have used butter from this creamery. Experiments have shown 
that the diphtheria bacilli can live in butter for a month. 

Bacteria in Cheese.—If undesirable species of microérganisms are 
present in the milk they will pass into the cheese and there produce 
their harmful effect. This is more important in cheese-making 
than in butter-making since it has not been found possible to 
make many of the important varieties of cheese from pasteurized 
milk. The bacteria most dreaded by the cheese manufacturer are 
those of the B. coli aerogens group. These give rise to the formation 
of large holes which can be often taken to indicate bad flavor as the 
organisms produce in addition to hydrogen and carbon dioxid 
offensive smelling and tasting compounds. 

The bacteria of cheese increase during the first few weeks (Fig. 
45), after which there is a decrease, but even in ripened cheese there 
are millions of bacteria. 

Pathogenic bacteria which are in the milk will find their way into 
the cheese, as Schroeder and Brett purchased 256 samples of cheese 
on the Washington market and examined them for tubercle bacilli 
by means of guinea-pig inoculation; 7.42 per cent. contained tubercle 
bacilli. Probably many of the tubercle bacilli die during the ripening 
process, but this cannot be entirely depended upon, as Washburn 
and Done prepared a cheese from infected milk and after 220 days 
produced generalized tuberculosis by injection into guinea-pigs, 
and even after 260 days injection of emulsions caused slight lesions. 
According to the findings of Rowland, B. typhosus and M. cholere 
soon perish in cheese. 

Bacteria in Ice Cream.—The number of bacteria in ice cream may 
at times be enormous. There is only a slight decrease on storage, 
as is seen from the following results of Hammer and Goss: 


SHOWING THE EFFECT OF STORAGE ON THE NUMBER OF BACTERIA IN 


ICE CREAM. 
Mixed 236,000 32,800,000 120,000 172,500,000 110,000,000 120,000,000 
Frozen 735,000 30,850,000 146,000 271,000,000 170,000,000 140,000,000 
Days old: 
1. . 360,000 7,750,000 137,000 157,000,000 194,000,000 70,000,000 
2 310,000 4,450,000 216,000, 128,000,000 216,000,000 71,000,000 
3 260,000 2,435,000 dig 52,000,000 102,000,000 
4, i 1,150,000 152,000 
De, 310,000 “fs 300,000 34,000,000 39,000,000 41,000,000 
6 me 139,000 
7 156,000 31,000,000 54,000,000 
8 ae a Bs 61,000,000 
Oe 36,000,000 
10 
11 15,000,000 


BACTERIA IN CONDENSED MILK og 


Freezing does not kill the bacteria in milk or cream; hence, ice 
cream may and does convey all of the milk-borne diseases. [urther- 
more, the ice cream is often made in unsanitary places and handled 
in an unsanitary manner; so that even though the milk be safe 
there are numerous opportunities for infection, especially where ice 
cream is vended on the street or from the little ice-cream stands on 
the corner. 

Bolton reported experiments on the freezing of B. typhosus in 
cream. One-twentieth of the organisms were alive after one month 
and even after forty-five days some of the organisms were alive. 
Furthermore, epidemics of typhoid have actually been traced to 
ice cream. Cumming reported 23 cases which developed among 
twenty-nine persons who partook of ice cream at a school picnic 
at Helm, California, in 1916. Ice cream was the only food partaken 
of by all, and as chocolate ice crream was the favorite flavor this 
was determined to be the source of the infection. This was because 
(1) those not partaking of it did not become ill, (2) those partaking 
of it but no other food were taken ill, (3) those eating chocolate ice 
cream were taken with acute intestinal symptoms, and (4) those 
eating the largest quantity of chocolate ice cream were the most 
seriously ill. 

Dysentery is also often spread by means of ice cream. Smillie 
studied 75 cases and found the etiology of them to be as follows: 


Cases. 
Gontactiawithiantacucercase: pen 9s. 6h) A os. cl oe) Seen od om all 
Contact with a carrier s 
Contact with house cases . Be eed ek Wer Dey 
@ondensedumiliaepidemic: ile) siren salt ee aot ae ie 
Ice-cream cones 5 
Flies . 
Milk . 
Water 
Fruit 3 Neh gs yoda) Ect Ge SIA cP ee Seen ae ee 
Wnknownspices wit we eee Pee ee A ee Oey Teed 


oe em oO © Ore bo 


The dysentery bacillus of Flexner was actually isolated from the 
ice-cream cones. 

Hamilton has pointed out that ice-cream epidemics can be pre- 
vented by (1) the use of ingredients with a clean sanitary history, 
(2) the use of properly cleaned utensils and a clean factory, and (3) 
the proper handling of materials by individuals with a clean bill 
of health. The first of these is to be controlled by the pasteurization 
of the milk and cream; the second by frequent inspection; and the 
last requires regular and careful inspection of all workers for com- 
municable diseases. 

Bacteria in Condensed Milk.—Sweetened condensed milk is not 
intended to be sterile. The large quantity of sugar added prevents 
the growth of microérganisms. But the unsweetened or evaporated 


392 BACTERIA IN OTHER FOODS 


milk is sterilized after sealing, and hence when properly done the 
finished product should contain no bacteria. The organisms which 
at times survive pasteurization and later may cause spoilage are 
B. subtilis, B. mesentericus, and B. coagulans. 

The degree of heat to which all the condensed, concentrated, and 
powdered milk are subjected is probably sufficient to kill all tubercle 
bacilli and typhoid bacilli, but Andrews’ work would indicate that 
at times condensed milk may act as a differential medium for 
Staphylococci. He reports instances where at time of condensation 
afew Staphylococer Pyogenes aureus were present, but later when the 
cans were opened many were present. 

Bacteria in Bread.— The interior of the loaf reaches a temperature 
of 101° to 103° C. and the crust 125° to 140.5° C. in the baking 
process; hence only the more resistant sporebearing organisms 
would survive the baking process. Disease germs, therefore, seldom 
—if ever— occur in the freshly baked bread. However, B. mesen- 
tericus vulgatus, and probably other organisms which cause slimy 
or ropy bread, may survive and cause considerable economic loss. 

When they have found their way into a bakery the ease with 
which they are overcome depends upon whether they are in the 
yeast, on the utensils, or in the flour. If they are in the yeast, a new 
start must be obtained, and Kayser suggests the use of acidulated 
water for washing all of the apparatus and even states that some of 
the apparatus may have to be discarded. Great economic loss, 
however, results when the flour is the infected material. 

Although bread is free from pathogenic bacteria when it leaves 
the oven, this is often not true when it reaches the consumer, for 
unwrapped bread must ever remain a constant danger. But when 
once wrapped, the danger is not as great, for the wrapper acts asa 
protection and if carelessly handled tells the tale to such an extent 
that it may be refused by the consumer. 

Bacteria in Eggs.—All investigators have found more bacteria 
in the egg yolk than in the egg white, and many of those who have 
found no bacteria in egg white have assumed that this part of the 
egg possesses a bactericidal action. Rettger considers that the 
contents of normal fresh eggs are as a rule sterile, although he con- 
siders it quite probable that an egg yolk may become invaded before 
it is expelled from the ovary. But this he considers an uncommon 
occurrence, except when the ovary is infected with the organism 
of bacillary white diarrhea. 

The percentage of infected eggs found by different investigators 
as reported by Tanner is given below: 


Number Per cent. 
Author. examined. infected. 
Rettger: 8. iusto elite: Tike aD ee Sen ee OG 9.5 
RetigerGuoleie samples) — 150 ssacs sere oats 647 3.86 
Bushnelliand Wiauer. ete Gee een ae ne 23.70 
Mauer. PONS O Med © i NRE oe 600 18.10 


Fiewdiley andi Caldwellesa thee at ee ae Rumored 8.70 


BACTERIA IN MEAT 393 


The kind of organism and the number of times found by Rettger 
are listed below: 


Fresh eggs. Number of times 
; found. 
Staphylococcus, usually aureus and albus owe SAL lec NA 
Subtilis group, usually B. mesentericus and B.ramusus  . . . . 60 
B. coli and closely related organisms Pip apie Asch Azul) ke ite pe ee OT 
IBTOUCIAEETOUL Re MER: Riria” WM Soc ie sey tate he cm Fe ae me () 
Streptococcus . . en ere URE). Tree chek Pak ee Bee 
Micrococcus (tetragenus, big Se 9 
Streptothrix 6 
Diphtheroid bacillus 5 
Putrefactive anaérobes 5 
B. fluorescens 2 
Mold 4 
B. mucosus . 3 
Mixed 2 


Total 


bo 
on 
x 


Hadley and Caldwell studied forty different strains isolated from 
eggs. They were divided as follows: rods, 28; cocci, 11; spirillum, 1. 
They found no member of the hemorrhagic septicemia, intestinal, 
proteus colon, enteritidis, typhoid, dysentery, nor diphtheria group. 

Rettger considers that under normal conditions the shell is bac- 
terium-proof. Moisture lessens its impervious character, however, 
and when combined with dirt or filth makes it possible for micro- 
organisms to enter and bring about decomposition. Hence, eggs 
should be stored under sanitary conditions. 

Cold storage, frozen, and dried eggs often contain millions of 
bacteria, yet of all food, so far as known, eggs are less liable to con- 
tain harmful products or to convey disease than any other single 
food of animal origin. They have an exceptionally clean health 
record. There is no known infection of the hen transmissible through 
the eggs to man. The literature is exceptionally free from instances 
where sickness has been attributed to eggs except in the case of 
anaphylaxis which is undoubtedly due to an idiosyncrasy of the 
individual and not to any inherent injurious constituent of the egg. 

Bacteria in Meat.—It is usually considered that the tissues of 
healthy animals are free from bacteria, but Haagland states that 
certain bacteria (chiefly micrococci) may be normally present in the 
carcass of healthy animals slaughtered for beef. These bacteria he 
considers possess no pathological significance and do not appear to 
multiply in the cold-stored carcasses, provided the cold storage 
room is maintained at the proper temperature. 

Meat kept for some time may contain many bacteria. Weinzirl 
and Newton found that four out of ten samples of meat which had 
been stored at —10° C. for one year, contained over 10,000,000. 

Chopped meats invariably contain many bacteria for the reason 
that meat used for that purpose is often that which has been dis- 


394 BACTERIA IN OTHER FOODS 


carded for other purposes and then the hashing carries the bacteria 
throughout the mass which is an excellent medium for their multi- 
plication. This is recognized by the fact that Weinzirl and Newton 
proposed a bacterial standard of not over 10,000,000 per gram for 
hamburger and then found that 50 per cent. of the samples examined 
by them had to be condemned. 

Sausage always contains numerous bacteria, but as pointed out 
by Carey the kind of organism present is more important than the 
number. He isolated the following organisms from 34 samples of 
sausage purchased on the market in Chicago: 


Bacillus colt sgl ath ene RNa ey ete ce ipl kad Oey ak wn Bt De ee a (D 
Proteus vulgaris SaMery ey aa eee eae IE cn ry gee Nine tb Od ota SK] 
B. paracolon efommaret at Wise t dagger tee tis, Om, Maekawa aE eae 9 
B. fecalis ea tae RS Re ite Sata ete ee 8 
BV CASE me Poder en Were oho wer Nencseece yh Map Leer ML We Hs ST x eo 8 
ISIFEDLOCOCCUS™ 2 ose uc) Be,s = 5 
Staphylococcus aureus . 2; 


Bacteria in Canned Foods.—The majority of the canned meats 
and fruits are free from bacteria, but in the case of swelled and 
spoiled products numerous organisms are found. 

Some of the organisms identified by Donk as causing spoilage of 
canned goods were as follows: 

M. acidi in cheese. 

. candicans in roast beef, sardines, and bulk granulated sugar. 
. candidus in baked beans. 

. cereus in baked beans. 

. lactis in cheese. 

M. luteus in corn. 

M. pyogenes in two samples of Maine style corn and one sample 
of canned corn on cob. 

M. stellatus in canned roast beef. 

B. cloace in canned roast beef. 

B. detrudens in cheese. 

B. licheniformis in stringbeans. 

B. megathervum in sauerkraut brine (not canned) and cheese. 

B. mesentericus in cheese. 

B. pammellit in cheese. 

B. subtilis in corn. 

B. tenus in cheese. 

B. viscosus in cheese. 

B. vilatis in spinach and bulk granulated sugar. 

B. welchii in corn. 

B. vulgatus in two samples of corn. 


SSS 


REFERENCES. 


Rosenau: Preventative Medicine and Hygiene. 
Tanner: Bacteriology and Mycology of Foods. 


CHAPTER XXXIV. 
BACTERIA AND FOOD-POISONING. 


Tue term “food-poisoning,” or ptomain poisoning, in the past 
has been used to cover a multitude of physiological disturbances. 
As Jordan points out, “that convenient refuge from etiological 
uncertainty ‘ptomain poisoning’ is a diagnosis that unquestion- 
ably has been made to cover a great variety of diverse conditions 
from appendicitis and pain caused by gall-stones to the simple 
abdominal distention resulting from reckless gorging.’’ But even 
when account is taken of this, its toll of human life and suffering is 
great. Food-poisoning is also a great cause of inefficiency, depres- 
sion, sluggish mental processes, dissatisfaction, or abnormal irrita- 
bility which are often overlooked or attributed to other causes. 

Classes of Food-poisoning. — Present knowledge permits the follow- 
ing rough classification of food-poisoning: 

1. Poisoning due to the eating of foods which naturally contain 
poisonous products. 

2. Poisoning due to the eating of foods containing mineral poisons 
added either intentionally or accidentally. 

3. The eating of foods which are normally non-poisonous but 
which have been obtained from animals suffering from disease. 

4. The eating of food which has been accidentally infected with 
pathogenic bacteria in handling or preparation. 

5. The eating of foods which contain poisonous products of 
bacterial katabolism-toxins. 

6. The eating of a normal food by an individual who possesses 
peculiar idiosyncrasies toward a specific food. 

Poisonous Foods. —The first group consists of naturally-occurring 
plants and animals which are always poisonous or become so during 
certain seasons of the year. According to Chestnut there are 
16,673 leaf-bearing plants included in Heller’s Catalog of North 
American Plants. Of these nearly 500 have been alleged to be 
poisonous, but fortunately only a few are ever accidentally par- 
taken of by man. Chestnut lists about thirty important poisonous 
plants occurring in the United States and some of these are not 
known to be poisonous except to domestic animals. Some of the 
more common are as follows: 

American false heltebore (Veratrum virid?)—mistaken for marsh- 
marigold. 


396 BACTERIA AND FOOD-POISONING 


Kkentucky coffee tree (Gymmnocladus dioica) —mistaken for honey- 
locust. 

Broad leaf laurel ( Kalmia litifolia)—mistaken for wintergreen. 

Water hemlock (Cicuta maculata)—roots mistaken for horse- 
radish, artichoke, parsnip, etc. 

By far the most common of these cases of plant-poisoning is due 
to the eating of the poisonous mushrooms or “toadstools” (Amaneta 
muscaria), (A. phalloides), (A. verna). 

The symptoms of poisoning with A. phalloides is thus described 
by Ford: 

“Following the consumption of the fungi there is a period of six 
to fifteen hours during which no symptoms of poisoning are shown 
by the victims. This corresponds to the period of incubation of 
other intoxications or infections. The first sign of trouble is sudden 
pain of the greatest intensity localized in the abdomen, accompanied 
by vomiting, thirst, and choleraic diarrhea with mucous and bloody 
stools. The latter symptom is by no means constant. The pain 
continues in paroxysms often so severe as. to cause the peculiar 
Hippocratic facies, la face vulteuse of the French, and though some- 
times ameliorated in character, it usually recurs with greater 
severity. The patients rapidly lose strength and flesh, their 
complexion assuming a peculiar yellow tone. After three to four 
days in children and six to eight in adults the victims sink into a 
profound coma from which they cannot be roused and death soon 
ends the fearful and useless tragedy. Convulsions rarely if ever 
occur and when present indicate, I am inclined to believe, a mixed 
intoxication, specimens of Amanita muscaria being eaten with the 
phalloides. The majority of individuals poisoned by the ‘deadly 
Amanita’ die, the mortality varying from 60 to 100 per cent. in 
various accidents, but recovery is not impossible when small 
amounts of the fungus are eaten, especially if the stomach be very 
promptly emptied, either naturally or artificially.” 

Metallic Poisons.— Various canned goods have been repeatedly 
accused of causing poisoning, but the cases in which this has occurred 
when the foods have been sterilized by the pressure method are 
extremely rare. Where it has caused trouble it is usually due to 
some metallic poison found in the cans and not to poisons developed 
in the food due to bacterial activity. 

Asparagus is often looked upon as one of the canned products 
most likely to cause poisoning. Thisis due in a large measure to the 
fact that asparagus takes up large quantities of tin, and some indi- 
viduals are especially susceptible to this substance. The quantity 
of tin, and especially copper, which is taken up in most cases varies 
with the amount and kind of acid found in the fruit or vegetables. 
Moreover, when a low or poor grade of copper is used, it is more 
readily attacked by the fruits than are the pure compounds. 


TYPICAL PARATYPHOID OUTBREAKS 397 


Fairly large quantities of copper have to be eaten before death 
results, and it is doubtful whether many foods would dissolve 
sufficient to result fatally. Whereas a small small quantity of one 
of the metallic poisons taken once may cause no ill effects, their — 
constant use would, for their action is cumulative. Moreover, 
sanitarians insist that chemical substances likely to be irritating to 
the human tissues in assimilation or elimination should not be 
employed in food. Each new irritant, even in small quantities, 
may add to the burden of organs already weakened by age or 
previous harsh treatment. 

Animals Suffering from Disease. —The milk and flesh of animals 
suffering with certain diseases are continually being used without 
adequate cooking, the result being that thousands die each year from 
this cause. The majority of these cases come from the use of milk, 
which has been considered’ in an earlier chapter. A typical out- 
break of paratyphoid due to the eating of diseased meat is thus 
given by Jordan: 

“The most characteristic examples of ‘food poisoning,’ popularly 
speaking, are those in which the symptoms appear shortly after 
eating and in which gastro-intestinal disturbances predominate. 
In the typical group-outbreaks of this sort all grades of severity 
are manifested, but as a rule recovery takes place. The great 
majority of such cases that have been investigated by modern 
bacteriological methods show the presence of bacilli belonging to the 
so-called paratyphoid group (B. paratyphosus or B. enteritidis). 
Especially is it true of meat-poisoning epidemics that paratyphoid 
bacilli are found in causal relation with them. Hiibener enumerates 
forty-two meat-poisoning outbreaks in Germany in which bacilli of 
this group were shown to be implicated, and Savage gives a list of 
twenty-seven similar outbreaks in Great Britain. In the United 
States relatively few outbreaks of this character have been placed 
on record, but it cannot be assumed that this is due to their rarity, 
since no adequate investigation of food-poisoning cases is generally 
carried out in our American communities. 

“Typical Paratyphoid Outbreaks. — Kaensche describes an outbreak 
at Breslau involving over eighty persons in which chopped beef was 
apparently the bearer of infection. The animal from which the meat 
came had been ill with sever diarrhea and high fever and was 
slaughtered as an emergency measure (notgeschlachtet). On exami- 
nation a pathological condition of the liver and other organs was 
noted by a veterinarian who declared the meat unfit for use and 
ordered it destroyed. It was, however, stolen, carried secretly 
to Breslau, and portions of it were distributed to different sausage- 
makers, who sold it for the most part as hamburger steak (Hack- 
fleisch). The meat itself presented nothing abnormal in color, odor, 
or consistency. Nevertheless, illness followed in some cases after 


398 BACTERIA AND FOOD-POISONING 


the use of very small portions. With some of those affected the 
symptoms were very severe, but there were no deaths. Bacilli of 
the Bacillus enterttidis type were isolated from the meat. 

“A large and unusually severe outbreak reported by McWeeney 
occurred in November, 1908, among the inmates of an industrial 
school for girls at Limerick, Ireland. There were 73 cases with 
9 deaths out of the total number of 197 pupils. The brunt of the 
attack fell on the first or Senior class comprising 67 girls between 
the ages of thirteen and seventeen. Out of 55 girls belonging to this 
class who partook of beef stew for dinner 53 sickened, and 8 of these 
died. One of the two who were not affected ate the gravy and 
potatoes but not the beef. Some of the implicated beef was also 
eaten as cold meat by girls in some of the other classes, and also 
caused illness. Part of the meat had been eaten previously without 
producing any ill effects. ‘The escape of those who partook of 
portions of the same carcass on October 27 and 29 (five days earlier) 
may be accounted for either by unequal distribution of the virus or 
by thorough cooking which destroyed it. Some of the infective ma- 
terial must, however, have escaped the roasting of the 29th, and 
multiplying rapidly, have rendered the whole piece intensely toxic 
and infective during the five days that elapsed before the fatal Tues- 
day when it was finally consumed.’ Theanimal from which the fore- 
quarter of the beef was taken had been privately slaughtered by alocal 
butcher. No reliable information could be obtained about the 
condition of the calf at, or slightly prior to, slaughter. The meat, 
however, was sold at so low a price that it was evidently not regarded 
as of prime quality. In this outbreak the agglutination reactions 
of the blood of the patients and the characteristics of the bacilli 
isolated showed the infection to be due to a typical strain of Bacillus 
enteritidis.’ 

Offending Foods.— Meat is so often the cause of poisoning that 
the terms “meat-poisoning”’ and food-poisoning”’ have come to 
be used almost synonymously. Of meats, chicken and pork are 
more likely to cause poisoning than are meats from other animals, 
while the internal organs—liver and kidney—are more likely to 
contain disease-producing bacteria than are the muscular tissues. 
Sausages, hamburger steaks, meat pies, puddings, and jellies are 
especially likely to cause food-poisoning. This is probably due to 
the products from which they are made, the methods of treating, and 
the fact that the heat used in cooking such foods is not sufficient to 
kill the bacteria in the food. While there are a few cases on record 
where individuals have been poisoned by the eating of freshly well- 
cooked meats they are so rare as to be of little importance: so the 
thorough cooking of meat greatly diminishes the likelihood of 
trouble, 


HUMAN INFECTION 399 


Human Infection.—It is necessary that food be protected from 
contamination during the whole process of preparation and serving 
to prevent its infection with pathogenic bacteria, as is illustrated by 
the remarkable instance reported by Sawyer where ninety-three 
typhoid cases were caused by eating Spanish spaghetti served at a 
public dinner. Investigation showed that the dish had been 
prepared by a woman typhoid-carrier who was harboring living 
typhoid bacilli at the time she prepared the dish. The dish was 
baked after it was infected, but the baking was‘shown by laboratory 
experiments to have incubated the bacteria instead of sterilizing 
the food. 

Then there is the celebrated case of Typhoid Mary, investigated 
by Saper. In the pursuit of her work as a cook in and about New 
York City she is known to have caused at least seven typhoid out- 
breaks in various families and one extensive hospital epidemic. 

The danger from this source is voiced by Chapin as follows: 
“There are doubtless 200,000 cases of this disease (typhoid fever) 
in the United States each year. If only 3 per cent. of these become 
chronic carriers, and if a carrier remains such only three years, we 
should have a carrier population of 18,000 persons, practically 
unknown and taking no precautions against infecting others. If we 
add to these the 25 per cent. of convalescents, who for some weeks 
are excreting the bacilli in their urine, it appears that there is a very 
respectable army of unrecognized sources of typhoid infection.” 

This is a situation which will be solved only when all handlers 
of food in public places are examined for various diseases which are 
transmissible through food. A move in the right direction has been 
made by the California State Board of Health which enters into the 
following agreement with all carriers discovered: 

“T have this day been informed that my excreta contain typhoid 
bacilli and that, unless unusual precautions are taken, persons will 
contract the infection from me. Realizing this danger I agree to 
observe the precautions stated below, and request that I be permitted 
to remain in free communication with other persons. 

“1. I will take no part in the preparation dr handling of food 
which will be consumed by persons outside of my immediate family, 
and I will not participate in the management of a boarding house, 
restaurant, food store, or in any other occupation involving the 
preparation or handling of food. 

“2. I will not dispose of my excretions in a toilet to which flies 
have access without first exposing such excretions to either a 5 per 
cent dilution of liquor formaldehyd or 5 per cent. phenol (carbolic 
acid). 

“3. I will notify the local health officer of any cases of typhoid 
among persons with whom I come in contact. 

“4. I will inform the local health officer of any contemplated 


400 BACTERIA AND FOOD-POISONING 


change of residence so that he can notify the State Board of Health 
and obtain its approval. 

“5. I will submit specimens for examination when requested by 
the State Board of Health. 

“6. I will fill out the following report blank when submitted to me 
semi-annually, and return the same to the California State Board 
of Health: 

‘I have, during the last six months, complied to the best of my 
knowledge with the five separate agreements entered into between 
myself and the California State Board of Health. Precautions 
involved in these separate agreements are for the purpose of prevent- 
ing typhoid infection.’ ” 

Ptomain Poisoning.— Plant and animal tissues under appropriate 
temperature, moisture, and aération putrefy. The proteins are 
broken down with the formation of basic, often highly toxic, sub- 
stances spoken of as “animal alkaloids’ or ptomains. These 
compounds are not poisonous in every case. The presence of oxygen 
in the compound seems to be necessary for the development of strong 
toxicity. In putrefying mixtures these toxic bodies appear on or 
about the fifth to seventh day after putrefaction sets in and disappear 
through further cleavage more or less rapidly yielding less complex 
nitrogenous substances that are non-toxic. It was formerly thought 
that they played a very important part in food-poisoning. But 
recent work has indicated that they are seldom the causative agent. 
Vaughan and Navy, who have made an extensive study of ptomains, 
have proposed a very elaborate nomenclature for supposed food- 
poisoning due to their ingestion. Some of them are as follows: 


Gheese-poisoning®y.) 0 ss se ees ee ee te ee OLOxismgs 
Righ-poisoning seta ee ee ee ee ee we Chih voto xisimuls 
Hood=poisoning 4). ae ee ee eer ee oe oe,  romototexemnus 
Meat-poisoning 4 Se a Pe ee ee ren tosiaminls 
Mallk-poisoning yy) .20 t8 ee oe eae ee Galactotoxiamus 


Botulism.— Botulism results from the eating of food in which the 
Bacillus botuiinus has grown and elaborated a poison. The organism 
is a large bacillus 4 to 6u by 0.9 to 1.2u, having slightly rounded ends 
and they may arrange themselves in pairs, end to end, or in an 
unfavorable environment in long chains. It is a strict anaérobe, but 
may grow under imperfect anaérobic conditions if in symbiosis with 
certain aérobic bacteria. The optimum temperature for the growth 
of the bacillus and for the elaboration of the poison is between 20° 
and 30° C. The vegetative cells are easily destroyed by heat, but 
the spores are quite resistant (according to Van Ermengen, 85° C. 
for fifteen minutes), but Dickson considers them even more resistant. 
Thom and associates isolated strains from asparagus which survived 
steaming under 10 pounds’ pressure for fifteen minutes, or a tempera- 
ture of 100° C. for one hour. They remain visable, according to 


BOTULISM 401 


Dickson, for months or even years if protected from the action of 
light and air, even though the medium in which they are immersed 
is of acid reaction. The toxin which they produce is quickly de- 
stroyed by exposure to the action of light and air, but will maintain 
its virulence for six months or more if kept in the dark and sealed. 
According to Van Ermengen, the toxicity is diminished by heating 
at 56° C. for three hours and destroyed by heating to 80° C. for half 
an hour or by boiling. Dickson found that the toxin may develop 
in mediums such as green corn, artichokes, asparagus, apricots, and 
peaches to which no traces of animal proteins have been added in 
addition to the various meats. 


Fic. 48.—Bacillus Botulinus. (After Dickson.) 


The pronounced symptoms which develop on the ingestion of the 
toxin are thus described by Wilbur and Ophiils: 

“Girl, aged twenty-three, Tuesday evening, November 23, 1913, 
ate the dinner including the canned stringbeans of the light green 
color together with a little rare roast beef. The following day she 
felt perfectly normal except that at 10:00 in the evening the eyes 
felt strained after some sewing. Thursday morning, thirty-six 
hours after the meal, when the patient awoke, the eyes were out of 
focus, appetite was not food, and she felt very tired. At night she 
had still no appetite, was nauseated, and vomited the noon meal 
apparently undigested. Friday morning, two and one-half days 
after the meal, the eyes were worse, objects being seen double on 
quick movement, and it was noticed that they had a tendency to be 
crossed. A peculiar mistiness of vision was also complained of. 
She was in bed until late in the afternoon, when she visited Dr. 
Black. She had had some disturbance in swallowing previous to 

26 


402 BACTERIA AND FOOD-POISONING 


this time and stated that it felt as if “something came up from below’ 
that interfered with deglutition. The fourth day she remained in 
bed, was much constipated, and noticed a marked decrease in the 
amount of urine voided. There was at no time pain except for 
occasional mild abdominal cramps, no headache, subnormal tem- 
perature, and anormal pulse. The fourth and fifth days the breath- 
ing became difficult at times and swallowing was almost impossible. 
The patient complained of a dry throat with annoying thirst. The 
sixth day there were periods of a sense of suffocation with a vague 
feeling of unrest and as if there might be difficulty in getting the 
next breath. The upper lids had begun to droop. The voice was 
nasal. When the attempt was made to swallow liquids they passed 
back through the nose. The patient felt markedly weak. 

“Physical examination at this time showed ptosis of both upper 
eyelids, dilatation of the right pupil, sluggish reaction to light of 
both pupils, apparent paralysis of the internal rectus of the left 
eye, normal retina, inability to raise the head, control apparently 
having been lost of the muscles of the neck, inability to swallow, 
absence of taste. The tongue was heavily coated and the throat 
was covered with a viscid whitish mucus clinging to the mucous 
membrane. The soft palate could be raised but was sluggish, 
particularly on the right side. ‘The exudate on the right tonsil was 
so marked that it resembled somewhat a diphtheritic membrane. 
The seventh day there was some change in the condition; occasional 
periods occurred when swallowing was more effective, and there was 
less tendency to strangle. On the eleventh day there was some 
improvement of the eyes, still strangling on swallowing, sensation 
of taste was keener, and the general condition improved. The 
twelfth day the patient was able to move her head, but was unable 
to lift it except when she took hold of the braids of her hair, and 
pulled the head forward. The eyes could be opened slightly, speech 
was less nasal and more distinct, and improvement in swallowing 
was marked. At the end of two weeks the patient was able to take 
soft diet freely, and at four weeks she was up in a chair for a couple 
of hours complaining only of general weakness and inability to use 
her eyes. At the end of five weeks she was able to leave the hospital 
and return to her home and later to resume her regular work.” 

Prevention.—The prevention of food-poisoning from canned foods 
consists of processing the material according to the best experience 
available, the selection of good, sound material and the rejection of 
any infected material. Dirty, wilted, and partly rotted food carries 
many more organisms into the canning process than does fresh, 
sound, clean fruits and vegetables. Dirty tables, dirty jars, lids, 
and sewage-polluted water and flies are sources of contamination 
which should be eliminated. 

When a can presents a convex appearance (technically called a 


PREVENTION 403 


“blown can”), or on opening a can a foul smelling gas escapes, it is 
a warning to the consumer that the contents should be destroyed, 
not salvaged, fixed up into salads, mincemeat, or made-over dishes 
for human consumption, nor should it be fed to lower animals as 
there are many cases in which chickens and other animals have been 
killed by such products. This probably distributes the organism 
on the premises. 

At other times the products have a peculiar rancid odor resembling 
spoiled butter which becomes more pronounced on standing. Such 
vegetables should not be tasted, but destroyed. All vegetables 
which have been put up by any other than standard methods should — 
be boiled before being eaten or even tasted, and no such products 
should be served as salads unless they: have been cooked after remov- 


ing from the container. 
REFERENCES. 
Boldnau: Food Poisoning. 
Jordan: Food Poisoning. 
Tanner: Bacteriology and Mycology of Food. 
Dickson: Botulism Monographs of the Rockefeller Institute for Medical Research. 


CHAPTER XXXYV. 
PRESERVATION OF FOOD. 


WHERE a race is dependent upon a local or seasonal supply of 
food it occurs that in one district or during one season there is an 
abundance, whereas in another locality or at another season there 
may be a scarcity which at times amounts to a famine. This state 
of affairs was very common in the earlier history of the race, but 
modern methods of transportation and food preservation has made 
it possible for the modern individual to have a sufficient and varied 
diet at allseasons. A varied diet is more certain to contain all those 
constituents which are essential to the body needs than is a restricted 
or monotonous diet. Moreover, it is well recognized today that 
nutritional disorders are more likely to occur on a restricted than on 
a varied diet. Hence, the general result of the expansion in the 
kinds of food consumed is good, but the food should be preserved 
in a manner such that as little as possible of the nutrient constituents 
are lost; so there is little change in appearance and taste and nothing 
is added nor developed in the food which is deleterious to health. 

Heat, cold, drying, and the use of some chemicals have long been 
in use for the preservation of food, but it is only recently that the 
art has been developed to its highest perfection. ‘This is due to the 
fact that the art of food-preserving depends upon the science of 
bacteriology, and today it is possible to preserve some foods indefi- 
nitely without injuring their nutritive value or seriously interfering 
with their taste and appearance, and all such methods are legitimate. 

But “the chief harm has come from the blind use of chemical 
germicides without regard for their harmful properties. The 
simplest and cheapest way to preserve food is by adding one of these 
chemicals and the method was, therefore, seized upon by alert men 
whose chief interest was of the pecuniary kind. The question was: 
to find the smallest percentage of a chemical which would prevent the 
decay of some particular food product, trusting to luck that the 
preservative used would prove harmless to the consumer. Often 
these chemicals were added with a liberal hand; further, it was soon 
found that chemical preservatives could be used to preserve food 
products for the market from materials already so decayed as to be 
unsalable in their original condition.” 

Methods of Preserving Foods.— Methods of preserving foods may 
be roughly classified as follows: 


METHODS OF PRESERVING FOODS 405 


1. Physical —The more important methods of this class are heat, 
cold, and drying. The last two are regarded by the sanitarian as 
antiseptic rather than germicidal, as they usually arrest the growth 
of the organisms, but do not kill them. 

2. Chemical.—Under this class we have two groups. 

(a) Those chemicals which preserve through their influence 
upon the medium in which they are placed. The majority of these 
act by increasing the osmotic pressure of the solution, and the 
important constituents used are salt and sugar. These are without 
objection from the hygienic standpoint. 

(b) Those substances which bring about a chemical change within 
the medium in which they are placed or which combine chemically 
with the living protoplasmic substance, or inhibiting their natural 
functions. Their function is then to prevent bacterial digestion. 
They are for the most part injurious, for if bacteria have attempted 
to digest food and failed, man need not try. 

Cold.—Cold has come in recent times to be of inestimable value in 
food preservation, and such food usually commands a higher market 
value than food preserved by other means. This is due to the fact 
that refrigerated foods in most cases very closely resemble in 
appearance, taste, and nutritive value the fresh article. Food 
can be kept in a satisfactory condition in cold storage for a very 
long time. The time varies with the specific article, its condition 
when placed in cold storage, and the temperature at which it is kept. 
Moreover, some foods such as meats pass through a stage of ripening 
while in cold storage so that when removed they have developed a 
tenderness and even more delicate flavor than that of the fresh 
product. 

The temperature at which foodstuffs must be kept varies with 
different articles. Fish are usually frozen, dipped in water, and 
refrozen. The formation of a coating of ice acts as a protection 
against bacteria and also prevents dessication. They are then 
stored at 16° C. Smith has shown that fish may be so kept for as 
long as two years without depreciating in nutritive or sanitary value. 

Meats are usually surface-dried before they are placed in cold 
storage. This prevents the formation of a film of water on the 
surface which under some conditions may act as a good cultural 
medium. Eggs and milk are materially injured by freezing; hence, 
they are ordinarily kept at a temperature just above 0° C. 

Cold is a disinfectant and not a germicide, and although a tempera- 
ture of 0° C. will prevent the growth of pathogens, some saprophytes 
may actually multiply at this temperature, but as the temperature 
is decreased the pathogens slowly succumb. Nevertheless, cold 
alone should. not be relied upon as a protection against pathogenic 
bacteria. | 

Most animal parasites die if kept in cold storage long. Rosenau 


406 PRESERVATION OF FOOD 


gives the following periods for some: Trichine die at or below 5° C. 
in twenty days; Taenia saginata, the beef tapeworm, dies in twenty- 
one days; but Taenia soliwm, the pork tapeworm, may live more 
than twenty-nine days. 

Food spoilage in cold storage is usually due to wrong temperature. 
This is often true of the home icebox which is usually placed so as 
to be convenient, not considering practicability, and a survey of 
such refrigerators revealed the fact that the temperature is often 
15° C. or higher. Such a temperature is ideal for rapid bacterial 
growth. 

Foods taken from cold storage spoil rapidly as the bacteria in and 
on the food have not been killed and the freezing has loosened up the 
texture so the micro6drganisms can gain entrance. Moreover, the 
enzymatic change which proceeds in the cold-storage product gives 
rise to substances which accelerate bacterial activity. 

Drying.—Nature’s method of preserving foods is by drying, 
for this is the universal principle used in preserving seeds. Bacteria 
must have moisture to grow and multiply, and if the dessication be 
great enough they die. Pathogens die quite rapidly when dried. 
Furthermore, fruits, vegetables, and meats when preserved by this 
method are usually cooked before eating; hence, the process has a 
decided sanitary as well as economical significance. Although 
nothing is added to the dried food and only water is lost, yet some 
dried food loses its savor and probably at times decreases in digesti- 
bility. 

The effectiveness of drying as a means of food preservation 
depends upon the completeness of dessication and the specific food. 
Those foods which are rich in soluble constituents are easily pre- 
served by this method, for while the moisture present may be 
considerable yet the osmotic pressure in the solution is too great for 
bacterial growth. This is the reason grapes, apples, and prunes are 
so easily preserved by drying, whereas meats and some fruits are 
preserved with difficulty. 

A great variety of foods, such as meat, fruit, eggs, and even milk, 
can be successfully kept by drying. According to Rohn’s classifica- 
tion the following groups of foods can be kept by this method: 


Groupe ie =) ee a STOLEN Hoods 

Group IIT-=: . . : 2) .~ Carbohydrate Foods 

Group III, . . \ . = .»*Proteins’ ---\ ‘Carbohydrates 

Group IV. . .... . . Acids -+ Proteins -+ Carbohydrates 


Pressure.—The use of pressure for the preservation of foods 
is yet in the experimental stage. Hite and coworkers found that the 
bacteria which cause spoilage in many fruits can be killed by press- 
ure. Apple juice kept for five years after being subjected to a 
pressure of from 90,000 to 120,000 pounds. Peaches and pears 
exposed to a pressure of 60,000 pounds for thirty minutes never 


METHODS OF PRESERVING FOODS 407 


spoiled. Inconsistent results were obtained with blackberries, 
raspberries, and tomatoes, thus indicating that more work is neces- 
sary before it can be used on a commercial scale. Larson, Hartzell 
and Diehl found that a direct pressure of 6000 atmospheres kills 
non-spore-forming bacteria in fourteen hours. A pressure of 
-12,000 atmospheres for the same length of time is required to kill 
spores. They think sterilization by means of pressure may prove 
valuable from a medical viewpoint as cultures so killed were found 
very effective in immunization. They are disposed to attribute the 
sterilization to the sudden change in the osmotic tension of the fluid 
in which the bacteria were suspended. However, Bridgman’s 
results indicate that it may be due to the coagulation of the bacterial 
protoplasm. 

Canning.—This process in most cases leaves the food sterile. 
It is, therefore, a sanitary safeguard, and can be used with most 
meats, fruits, and vegetables, and if properly conducted yields very 
satisfactory products. 

The method used and the success met with varies with the 
different products and their condition at the time of canning. Acid 
foods or those containing large quantities of soluble constituents 
are canned with considerable ease as compared with the neutral 
substances (corn, peas, and beans). 

The various methods used may be arranged under three groups: 

1. The heating of the products under pressure for a sufficient 
time to sterilize. This method although it requires the use of an 
autoclave is more efficient and requires less time than the other 
methods. It is used very extensively in large canneries, whereas 
the intermittent and continuous methods are used to a greater extent 
in the home. 

2. The intermittent method consists of heating the products 
on three successive days, maintaining the food at a temperature 
between heating such that spores will vegetate. The objection to 
this method is the time necessary in the preparation of the finished 
product, and anaérobic organisms may not germinate in the intervals 
between heating but may later with the production of toxins. 

3. The continuous or cold-pack method is being extensively 
used of late, but Dickson and later Thom and coworkers have shown 
that the temperature is not always sufficient to insure the death of 
all injurious organisms. 

Sugar and Salt.—Sugar and salt preserve by increasing osmotic 
pressure and are very extensively used as they are without injury 
upon the health of the consumer. 

Sugar is largely used in the manufacture of jellies and preserves. 
These substances are cooked in the preparation, and this together 
with the high osmotic pressure of the solution renders them free from 
pathogens. 


408 PRESERVATION OF FOOD 


Salt is extensively used in the preservation of meats and pickles, 
and our knowledge concerning the action of salt is more exact than 
it is concerning sugar. 

Tanner lists the various reasons which have been ascribed for the 
keeping powers of salt as follows: 

I. Exerts a poisonous action. 

II. Renders the moisture unavailable for microérganisms. 

III. Destroys the cells by plasmolysis. 

Salt does not render the medium sterile but exerts a_ selective 
action upon the bacterial flora. A 7 to 10 per cent. solution of salt, 
according to Stadler, inhibited the growth of the following organisms: 
B. Colt commune, B. morbificcius bows, B. enteriditis, B. (proteus) 
vulgaris, and B. botulinus. 

De Freytag and Stadler found that a saturated salt solution had 
the following effect upon bacteria: 


INFLUENCE OF CONCENTRATED SALT SOLUTION ON BACTERIA. 


Author, Organism. Observation. 
Freytag B. anthracis Not killed after a number of hours. 
Freytag B. anthracis spores Not killed in six months. 
Freytag : B. typhosus Killed after five months. 
Stadler... B. typhosus Not killed in six weeks. 
Freytag B. diphtheria Not killed in three weeks. 
Stadler . B. diphtherie Not killed in four and a half weeks. 
Freytag B: tuberculosis Not killed in three months. 
Stadler . B. pestis ' Not killed in sixteen weeks. 


Homer found that B. botulinus does not develop in media con- 
taining over 6 per cent. of salt, and he considers meat which is 
properly covered with brine safe. But much higher concentrations 
—12 to 19 per cent. acting for seventy-five days—are necessary to 
destroy the bacteria, and even then ptomains which had previously 
been formed in the food would not be rendered harmless. 

It is quite evident from these results that salt is an efficient food 
preservative. It does not, however, destroy pathogens, and in 
dilute solutions the organisms involved in food-poisoning may 
develop. 

Chemical Preservatives.—All authorities are agreed that the 
preservation of food by drying, refrigeration, heating, canning, 
salting, and preserving with sugar is justified on theoretical grounds 
as well as practical experience, whereas the use of sulphites in sausage 
and chopped meat, the addition of formaldehyd to milk, or of boric 
acid or sodium flourid to butter are objectionable from the stand- 
point of public health. The addition of sulphites to meat is especially 
objectionable, as it places in the hands of the unscrupulous dealer a 
method of concealing the signs of decomposition in meat, in addition 
to being injurious to the health. 

The use of other preservatives such as benzoic acid and sodium 


CHEMICAL PRESERVATIVES 409 


benzoate is defended by some authors, while others argue that any 
chemical which is poisonous in large quantities should be considered 
as poisonous in small quantities until the contrary is proved. This 
can be determined only by tests extending over long periods, for 
whereas one dose may not be injurious the continuous use may. 
‘So it is best to exclude as far as practical the use of chemical pre- 
servatives from food. The subject is well summarized by Jordan 
as follows: 

“The remedy is obvious and has been frequently suggested— 
namely, laws prohibiting the addition of any chemical to food except 
in certain definitely specified cases. The presumption then would 
be—as in truth it is—that such chemicals are more or less dangerous, 
and proof of innocuousness must be brought forward before any one 
substance can be listed as an exception to the general rule. Such 
laws would include not only the use of chemicals or preservatives, 
but the employment of substances to ‘improve the appearance’ of 
foodstuffs. As already pointed out, the childish practice of arti- 
ficially coloring foods involves waste and sometimes danger. It 
rests on no deep-seated human need; food that is natural and 
untampered with may be made the fashion just as easily as the color 
and cut of clothing are altered by the fashionmonger. The incor- 
poration of any chemical substance into food for preservative or 
cosmetic purposes could wisely be subject to a general prohibition, 
and the necessary list of exceptions (substances such as sugar and 
salt) should be passed on by a national board of experts or by some 
authoritative organization like the American Public Health Associa- 
tion.” 

An advance in the right direction was made by the passage of the 
National Food and Drug Law in 1906. This is being rapidly 
incorporated in the statutes of the various states. According to 
this law a food is adulterated: 

1. “If any substance has been mixed and packed with it so as to 
reduce or lower or injuriously affect its quality or strength. 

2. “Tf any substance has been substituted wholly or in part for 
the article. 

_ 3. “If any valuable constituent of the article has been wholly or 
in part abstracted. 

4. “Tf it is mixed, colored, powdered, coated, or stained in any 
manner whereby damage or inferiority is concealed. 

5. “If it contains any poisonous or other added deleterious 
ingredient which may render such article injurious to health. 

6. ‘If it consists in whole or in part of a filthy, decomposed or 
putrid animal or vegetable substance or any portion of an animal 
unfit for food, whether manufactured or not, or if it is the product 
of a diseased animal or one that has died otherwise than by 
slaughter.” 


410 PRESERVATION OF FOOD 


Those chemical preservatives concerning which there is a question 
as to their influence upon the health need only be listed on the article 
leaving the consumer to decide for himself as to whether he cares to 
use it. 

REFERENCES. 

Rosenau: Preventative Medicine and Hygiene. 


Tanner: Bacteriology and Mycology of Foods. 
Wiley: Foods and Their Adulteration. 


CHAPTER XXXVI. 
BACTERIA IN THE ARTS AND INDUSTRIES. 


Bacteria play an ever-increasing part in the arts and industries 
and man is learning that the majority of them are his friends and not 
his enemies. In addition to the processes considered in the preced- 
ing pages bacteria play a leading réle in many important industries, 
a few of which are briefly considered below. 

Alcoholic Fermentation.—The development of bacteriology as a 
science is intimately associated with the history of fermentation. 
Some of Pasteur’s classic studies dealt with this subject and ever 
since it has commanded considerable attention. 

Although from a commercial viewpoint the yeasts are of first 
importance in alcoholic fermentation, yet there are many bacteria 
which produce alcohol, for instance: 

B. fitzianus, ferments glycerin with the formation of ethyl alcohol. 

B. ethaceticus ferments glycerin, starch, sucrose, lactose, glucose, 
mannite, and arabinose with the formation of ethyl alcohol and 
acetic acid. 

A number of bacteria, chief among which are B. butylicus, B. 
B. orthobutylicus, B. amylozyme, and Beijerinck’s genus Granulo- 
bacter, ferment carbohydrates with the production of butyric acid. 

Recently Northrop and coworkers have outlined a method of 
producing acetone on a commercial scale, ethyl alcohol being a by- 
product. The organism used is B. aceto-ethylicum which acts on a 
solution of beet molasses. The fermentation yielded from 8 to 8.5 
per cent. of the sugar as acetone and 20 to 21 per cent. as ethyl 
alcohol. 

Milk usually undergoes lactic acid fermentation, yet Koumiss, 
Matzoon, Keffir, and Leben all contain alcohol and bacteria play an 
important role in their fermentation. 

Vinegar.—Many species of bacteria have been described which 
produce acetic acid. They are all closely related but differ slightly 
in morphology and fermentative power. It is believed that the 
oxidation of the alcohol is due to an intracellular enzyme. All of 
the organisms are bacilli and a few of the most common species 
are as follows: 

Bactervum pasteurtanum—non-motile rods, lu x 2u, that do not 
form spores. Their optimum temperature is about 34° C. They 
develop best in solutions not over 9.5 per cent. of alcohol and 
produce under favorable conditions about 6 per cent. of acetic acid. 


412 BACTERIA IN THE ARTS AND INDUSTRIES 


Bacterium schiitzenbachi and several related species are the main 
factors in the production of vinegar by the quick-vinegar process. 
The organisms vary considerably in size—0.3—0.4u x 3—6yu. Their 
optimum temperature is 25°—30° C. They produce as high as 
11.5 per cent. acetic acid. In the-absence of sufficient alcohol the 
acetic acid may be oxidized by them to carbon dioxid and water. 

Two methods of preparing vinegar are in general use—(1) the 
Orleans and (2) the Quick, or German Method. 

1. The Orleans Method is the oldest commercial method and 
produces vinegar of the highest quality. There are many modifica- 
tions of this method but they all contain essentially the same 
principles. The filtered wine is placed in barrels or covered vats 
furnished with openings so the entrance of air is facilitated and can 
be controlled. The receptacle is filled about two-thirds full of a 
mixture of four parts of good, new vinegar and six parts of wine, 
preferably that which has been pasteurized at 55° C. At times 
there is placed a light wooden grating which floats and helps to 
support .the bacterial film. A small quantity of a good bacterial 
film is placed in asa starter. Periodically a portion of the contents 
is drawn off and replaced by wine, and so the process continues. 

2. In the Quick or German Method the liquid to be acetified 
is allowed to trickle through barrels filled with beech chips, the 
pressed pomace of red wines, rattan shavings, corn cobs, or charcoal. 
Although the main function of these is to increase aération, yet the 
best results are obtained with the beech shavings or pomace. 

Sauerkraut.—The cabbage is cleaned, cut into pieces of con- 
venient size, and tightly packed with from 1 to 3 per cent. of salt 
into wooden or earthen vessels on the top of which is placed a 
weighted perforated cover. ‘This, together with the osmotic press- 
ure of the salt, draws from the vegetable considerable water. The 
respiration of the cells of the leaf and the yeast soon remove all 
oxygen.’ The mass undergoes lactic acid fermentation which in 
time reaches from 0.5 to 1 per cent. The brine is then drawn off 
and replaced by 4 to 8 per cent. salt solution. In this the vegetable 
will keep for a considerable time. Many substances are produced 
in the process, the chief of which are lactic acid, alcohol, succinic 
acid, volatile acids, mannite, amid bodies, carbon dioxid, hydrogen, 
methane, and various aromatic esters. 

The bacteria responsible for the process come from the vegetable. 
Although Weiss has isolated 65 different species of bacteria from 
sauerkraut, probably the principal changes are due to a few species. 
The lactic acid is usually produced by Streptococcus lacticus and 
Bacterium lactus acide. 

By similar means other vegetables—stringbeans, cucumbers, 
etc.—may be preserved. 


ENSILAGE 413 


Ensilage.—The changes through which ensilage passes during its 
curing was looked upon a few decades ago as being entirely microbic 
in origin, but due to the work of Babcock and Russell (1906-10) 
opinion swung in the opposite direction to such an extent that 
microdrganisms were generally considered as of little if any signifi- 
cance in the normal fermentation of silage. Later (1912) Esten 
and Moson considered the process entirely bacteriological. Three 
chief fermentations were thought to take place: the lactic acid, 
alcoholic, and acetic acid fermentation. The lactic acid fermenta- 
tion was thought to be due to organisms similar to those concerned 
in the souring of milk. It was also believed by these workers that 
yeasts cause an alcoholic fermentation and that acetic acid bacteria 
then oxidize the alcohol so formed to acetic acid. Samarani con- 
cludes that the acetic acid fermentation in silage is due to the res- 
piration of the plant cells, while the lactic acid fermentation is due 
to bacterial action. The organisms responsible for the latter proc- 
ess were identified by him as a bacillus and a coccus which occurred 
in about equal proportions. The former he designated as the B. 
acidi lactict of Hulppe, and the latter was considered identical with 
the common streptococcus of milk. 

Counts made by Sherman on silage juice showed the presence of 
from 1,500,000,000 to 4,800,000,000 per cubic centimeter, most of 
which were slender rods, and he considered the organisms con- 
cerned to be nearly related to the B. bulgaricus group of milk and the 
B. acidophilus groups. 

However, the temperature, kind of silage, and other factors 
would govern the bacterial flora, and Gorini distinguishes four types 
of grass-silage prepared in pits, depending upon the predominating 
type of bacteria as follows: (1) Butyric, (2) lactic, (3) putrefactive, 
and (4) sterile or atypical. If the silage stage reaches a temperature 
of 60° C. butyric organisms predominate; if 50° C. lactic organisms 
prevail; putrefaction occurs at lower temperatures, and sterile or 
atypical when the mass becomes superheated. Butyric silage is 
objectionable because of the odor and taste which it is apt to impart 
to the milk and the bacteria which enter from the surroundings 
render the milk unsuitable for cheese-making. 

The acid-producing bacteria of silage are found constantly on 
corn fodder, so that silage made from corn is always amply seeded 
with the organisms, but Gorini achieved considerable success by 
inoculating fresh grass-silage with lactic acid bacteria, and Crolhois 
found that the inoculating beet silage with lactic acid organisms 
preserves it better, furnishes a more nutritive product, and sup- 
presses the diseases to which the cattle fed on non-inoculated pulp 
are subject. 

At least from a theoretical basis this would seem quite probable, 
for it is known that beets contain in addition to many other products 


414 BACTERIA IN THE ARTS AND INDUSTRIES 


cholin and betain. The quantity of this last product in the ripe 
beet is 0.1 per cent., in the unripe beet 0.25 per cent., and in the beet 
molasses as high as3 per cent. The quantity may be even greater 
in the leaves and upper part of the beet than in the main beet. 

Now certain organic reactions are known which relate these 
products to a toxic substance. Cholin on oxidation and the subse- 
quent elimination of a molecule of water passes into betain: 


CH:CH:OH CHe 
Pig eh 
(CH3)3N + 20 = (CHs3)3N C=O + 2H:20 
SNe cae 
OH Oo 
Cholin. Betain. 


This is a typical oxidation and dehydration reaction which could 
be brought about by mold or bacteria under aérobic conditions, 
whereas betain can be converted into muscarin through being made 
to take up water and reduced thus: 


CH» CH2:—CHO.H20 
Wce os 
(CH3)3N C=O + He + H:20 = (CH3)3N 
see aS 
O OH 


Betain. Muscarin. 


This is a reaction which theoretically could be catalyzed by bac- 
teria or molds and would probably occur under anaérobic conditions. 

It, therefore, appears plausible that under-appropriate tempera- 
ture, moisture, aération, and microflora there may develop in beet 
silage toxic compounds. 

Retting.— The separation of the fibers of flax and hemp is brought 
about by a complex fermentation in which bacteria dissolve certain 
pectin bodies which cement the fibers together. The reaction 
occurs best at a temperature of 30° to 32° C. and is due to many 
species .of bacteria. In the water-retting of hemp, the anaérobic 
butyric acid bacteria (Clostridia) play a leading réle, and the water- 
retting of flax is ascribed to a specific anaérobic bacillus (Granu- 
lobacter pectinovorum). 

Tanning.—Animal skins are tanned in order to increase their 
resistance to decomposition and also to increase their adaptability 
to the various purposes to which leather is put. In tanning bacteria 
play important parts. When the skin is soaked in baths rich in 
organic matter an energetic bacterial flora soon develops which 
quickly softens the hide. Bacteria cause the depilation ar 
of the hair by which the dermis is separated from the epidermis and 
the hair which accompanies it. This is true in the sweating and lime 
methods, whereas the alkaline sulphid and arsenic sulphid are both 
chemical methods. 

The third step in the process is conducted in the excrement or 


VACCINES 415 


bathing tubs which contain the droppings of hens, pigeons, and dogs. 
Here a true digestion of the hide is carried on by bacteria. Wood 
has isolated 90 species of bacteria from such a tube, no one of which 
possessed the power of bringing about the desired change, but all 
acting conjointly gave the desired product. The heated hides are 
next placed in a tan pit or in bark liquor. Numerous bacteria 
yeast and molds occur in the bark liquor and play a part in the 
finishing of the product. 

Vaccines.—A vaccine is a killed or weakened (attenuated) sus- 
pension of organisms to be inoculated into the body for the purpose 
of causing the development of an active immunity. 

The vaccine is usually | prepared from a fresh twenty-four-hour 
growth of the microdrganism on agar. The surface growth only is 
taken, thus avoiding secondary metabolic products which may be 
formed. ‘The cultures are usually killed by exposure to heat at 
from 538° to 60° C. for one hour. High heat, while certain to kill 
the virus, is undesirable for the reason that it coagulates the protein 
substances in the bacterial cell and otherwise alters its chemical 
structure. The closer the vaccine approaches the virus the better 
the result and higher the resulting immunity. For this reason many 
workers prefer to kill the bacteria with chemicals, carbolic acid, 
chloroform, or some other suitable germicide. 

The attenuated virus is obtained by passing the microérganism 
through the body of some animal, as smallpox through the heifer 
by which its virulence for man is reduced. At other times it is 
grown under adverse conditions—high temperatures, artificial media, 
or in the presence of antiseptics, after which it becomes less virulent. 
Drying is used in the case of the virus of rabies. 

In the preparation of antitoxins the bacterial cell or some of its 
products are injected into a suitable animal, and after sufficient 
time has elapsed blood is drawn and after appropriate treatment is 
used for the cure or prevention of disease. 


REFERENCES. 


In addition to the references listed at the end of the several chapters, the fol- 
lowing have been freely consulted and to these, the students are referred for further 
information. The date given is that in which the first volume appeared. 


Abstracts of Bacteriology, 1917, vol. i. 

Agricultural Index, 1916, vol. i. 

Biedermann’s Ceniralblatt fiir Agrikulturchemie, 1872, Bd. i. 
Botanical Gazette, 1876, vol. i. 

Centralblatt fiir Bakteriologie, 1887, Abt. I, Bd. i. 
Centralblatt fiir Bakteriologie, 1892, Abt. II, Bd. i. 
Chemical Abstract, 1907, vol. i. 

Experiment Station Record, 1888, vol. i. 

Comptes Rendus Académie des Sciences, 1835, vol. i. 
Experiment Station Bulletins. 

International Catalogue of Scientific Literature, 1911. 
Jahresbericht titber die Fortschritte der Agrikulturchemie, 1858, Bd. i. 


416 BACTERIA IN THE ARTS AND INDUSTRIES 


Jahresbericht iiber die Landwirtschaft, 1886, Bd. i. 
Journal of Agricultural Science, 1906, vol. i. 

Journal of the American Chemical Society, 1875, vol. i. 
Journal of the American Medical Association. 

Journal of Agricultural Research, 1913, vol. i. 

Journal of the Infectious Diseases, 1904, vol. i. 

Journal of American Society of Agronomy, 1910, vol. i. 
Journal of Bacteriology, 1916, vol. i. 

Journal of Biological Chemistry, 1906, vol. i. 

Journal of Industrial and Engineering Chemistry, 1909, vol. i. 
Journal fiir Landwirtschaft, 1853, Bd. i. 
Landwirtschaftliches Jahrbuch der Schweiz, 1887, Bd. i. 
Landwirtschaftliche Versuchs-Stationen, 1859, Bd. i. 
Phytopathology, 1911, vol. 1. 

Soil Science, 1916, vol. i. 

United States Department of Agriculture Bulletins. 


INDEX OF 


A 


ABENHAUSEN, 388 

Adler, 180 

Allen, 141 

Allison, 262 

Ampola, 245 

Ampola and Garino, 241 
Anderson, 382 

Andre, 234 

Andrews, 392 

Armsby, 249 

Armstrong, 75 

Arrhenius, 95, 99, 106, 111 
Ascher, 388 

Ashby, 219, 221, 253, 273 
Atkinson, 299 

Atwater, 291 

Aubin, 217 

Aujeszky, 388 

Avery, 249 

Ayers, 385 


B 


Bascock and Russe, 4138 

Ball, 299 

Barbieri, 310 

Barlow, 299, 303 

Barthel, 153 

Bassler, 156 

Baumann, 210 

Bayliss, 73, 81 

Bazarewski, 230, 240, 245 

Beatty, 382 

Beckwith, 123 

Beddies, 226, 230 

Beijerinck, 27, 57, 93, 144, 251, 263, 
266, 268, 276, 292, 293, 297, 307, 411 | 

Beijerinck and Van Delden, 53, 251, 
254, 275 

Beik, 382 

Beninde, 388 

Benjamin, 307 

Bergonzini, 56 

Berman, 70 

Bernard, 65 

Berthelot, 60, 249, 250, 261, 267, 278, 
281, 287 


27 


AUTHORS. 


Berzeluis, 71 

Biedermann, 415 

Bienstock, 190 

Binot, 337 

Birner, 249 

Bizzell, 131, 140, 219, 233, 234 
Black, 401 

Bodenstein, 74 

Boldnau, 403 

Bolley, 387 

Bollinger, 227, 277 

Bolton, 391 

Bomhoff, 388 

Bonazzi, 271 

Borisson, 78 

Bornemann, 153 

Bottomley, 138, 235, 286, 274, 286 
Bouilhac, 122 

Boullanger, 221, 225 
Boullanger and Massol, 221, 225 
Bouonami, 18 

Boussingault, 209, 218, 227, 248 
Boyd, 387 

Breal, 218, 240 

Bredemann, 157, 251, 264 


| Brenchley, 122 


Brett, 390 

Briailles, 130 

Bridgman, 106, 407 

Brieger, 189 

Briggs, 201 

Briscoe, 152, 388, 389 

Briscoe and MacNeal, 388, 389 

Brown, 41, 96, 129, 185, 140, 141, 142, 
144, 152, 158, 162, 174, 199, 200, 203, 
233, 283 

Brown and Allison, 262 


| Brown and Hitchcock, 146 


Brunchorst, 291 
Brusaferro, 388 
Buchanan, 30, 94, 106 


| Biichner, 72, 79, 88, 101, 343 
| Buck, 387 


Buffon, 19 

Buhlert, 292 

Bujwid, 382 

Burgess, 122, 124, 140, 195, 252, 255, 
276 

Burri, 216, 226, 240 

Burri and Stutzer, 216, 226, 240 


418 


Burrill 27, 295, 298, 300, 302, 318 

Burrill and Hansen, 295, 298, 299, 300, | 
318 

Bushnell and Mauer, 392 

Busley, 224 

Bychiklin, 175, 232 

Bychiklin and Skalski, 232 


C 


CALDWELL, 392, 393 

Campbell, 382 

Carbone, 202 

Carey, 394 

Caron, 151, 241, 242, 250, 284, 285 
Carter, 159, 180, 199, 201 
Causemann, 157 

Celli-Zuco and Heraeus, 210 
Chamberlain, 182 

Chapin, 352, 399 


Chester, 51, 141, 161, 163, 164, 196, 


Dili ave Paley aay 
Chestnut, 395 
Chick, 111, 113, 117 
Chick and Martin, 113 
Chisholm, 359 
Christen, 98 
Christensen, 254, 258, 259 
Christensen and Larson, 254 
Cienkowski, 55 
Clark and Gage, 123 
Clausen, 95 
Clayton, 386 
Coggi, 388 
Cohn, 21, 46, 55, 57, 190 
Coleman, 136, 138, 221, 222, 227, 256 
Colton, 389 


Conn, 64, 96,°162, 164, 165, 166, 167, 


168, t70, 197, 372, 376 
Conn and Brown, 96 
Cook, 182 
Coudon, 194 
Cramer, 61 
Crochetelle, 218, 220 
Crolhois, 413 
Crooks, 183 
Cumming, 391 
Cunningham, 126, 128, 173 
Cunningham and Léhnis, 126, 281 
Czapek, 118 


D 


Darert and BoLLInGER, 227, 277 
Dakin, 113, 114, 115, 117, 349 
Dakin and Dunham, 113, 117 
Dallar, 382 

Darbishire and Russell, 130 
Davenport, 297 

Davis, 252 

Davy, 239 


INDEX OF AUTHORS 


| Dawson, 303 

De Bary, 165 

Dehérain, 145, 151, 227, 230, 231, 232, 
239, 243, 249, O74, 328, 330 

| Dehérain and Demoussy, esi Py 

Delepine, 382 

| Delwiche, 156 

| Demolon, 1s 

| Demoussy, 151, 224, 227, 

Depetit, 240, 244 

| DeSchweinitz and Dorset, 59 

| Dezani, 142 

| Dickson, 400, 401, 403, 407 

Diehl, 407 

Doleris and Pasteur, 190 

Don, 359 

Done, 390 

Donk, 394 

Dorset, 59 

Douglass, 386 

| Drouin, 307 

DuBois and Raymond, 105 

Duclaux, 98, 241 

Duggar and Davis, 252 

Dumas, 208 

Dumont, 145, 218, 220 

| Dumont and Crochetelle, 218, 220 

Dunean, 181 

Dunham, 113, 117 

Dusch, 20 

Duschechkin, 153, 158 

Dvarak, 263 

| Dzierzbicki, 199, 202, 203, 258 


231 


E 


EBER, 382, 388 
Ebermayer, 141 

Eberth, 352 

Effront, 190, 193, 206 
Egorou, 128 

Ehrenberg, 54, 56, 123, 141 
Ehrlich, 84 

Einecke, 152 

Ellis, 180 

Elwell, 218 

Emerson, 252 

Emmerich, 152 
Emmerling, 197 
Engberding, 140, 143, 144, 151, 158 
| Engelmann, 109 

| Krickson, 291 

Escherich, 56, 190 

Esten and Moson, 413 
Euler, 81 

Evans, 315 


F 


Fapricrus and von FrinitzEn, 151 
Feilitzen von, 151, 225 
| Fernan and Pauli, 108 


INDEX OF 


Field, 382 

Fischer, 29, 77, 97, 98, 140, 151, 153, 
158, 188, 200, 203, 219, 222, 254, 276 

Fletcher, 132 

Flexner, 391 

Fliigge, 38, 57, 165, 166, 168, 190 

Folwell, 367 

Ford, 396 

Forster, 96 

Fraenkel, 216 

Frangois, 118 

Frank, 53, 127, 210, 274, 291, 315 

Frankfurt and Duschechkin, 153, 158 

Frankland, 165, 167, 210 

Fraps, 139, 227 

Fred, 129, 131, 133, 136, 137, 244, 267, 
304, 306, 307 

Fred and Davenport, 297 

Fred and Gainey, 136 

Fred and Hart, 145 

Fresenius, 198 

Freudenreich, 251, 273, 377 

Freytag, 408 

Friedliinder, 83 

Froehde, 239 

Fromberz, 206 

Frost, 54 

Fuhrmann, 79, 306 

Fuller, 193, 345, 360, 361, 362, 367 

Fuller and Johnson, 345 

Fulmer, 266, 267 

Fulmer and Fred, 267 


G 
Garry, 352 
Gage, 123, 196 . 
Gain, 312, 313 
Gainey, 136, 203, 228 
Gandechon, 225 
Garino, 241 
Garman, 295 
Gartner, 216 
Gasperini, 169 
Gay, 355 
Gay-Lussac, 77 
Gayon, 240, 244, 328, 330 
Gayon and Depetit, 240, 244 
Gerlack and Vogel, 69, 154, 251, 257, 
307 
Gibbs, 212, 213, 238 
Gilbert, 191, 209, 235, 240, 290 
Giltner and Langworth, 276 
Girard, 127 
Godlewski, 221 
Golding, 308 
Goler, 382 
Goodsir, 55 
Gorini, 413 
Goss, 390 
Gottheil, 165, 166 
Grassberger, 388 


AUTHORS 419 


Grazia, 151, 175 

Greaves, 126, 146, 149, 159, 162, 164, 
180, 199, 201, 218, 232, 234, 237, 238, 
244, 255, 277, 289, 326 

Greaves and Carter, 159, 180, 199, 201 

Greaves, Stewart and Hirst, 238 

Green, 200, 203, 234, 260, 266, 282 

Gregory, 28 

Greig-Smith, 138, 235, 236, 297, 299, 
306, 312 


|Greig-Smith and Bottomley, 235, 236 


Groenewege, 252 
Groning, 388 

Grove, 74 . 

Grunner, 118 

Guatier and Drouin, 307 
Guffroy, 1438 

Guignard, 249 


HAAGLAND, 393 

Haas, 99 

Hadley and Caldwell, 392, 393 

Haeckel, 29 

Hall, 172, 288, 289 

Hamilton, 391 

Hammer, 265, 390 

Hammer and Goss, 390 

Hansen, 27, 295, 298, 299, 300, 318 

Hanzawa, 266 

Harden, 83, 86 

Harding, 164, 373, 389 

Harding and Wilson, 373 

Harracks, 359 

Harrison and Barlow, 299, 303 

Hart, 145 

Hartleb, 216, 217 

Hartwell, 141 

Hartz, 51, 169 

Hartzell, 407 

Hastings, 376 

Hauser, 56, 190 

Hazen, 353 

Headden, 118, 284 

Hecker, 151 

Heim, 387 

Heineman, 386 

Heimze, 128, 135, 153, 157, 231, 234, 
256, 274, 278, 282, 307 

Heller, 395 

Hellriegel, 27, 248, 274, 291, 293, 314 

Hellriegel and Wilfarth, 248, 291 

Hellstrom, 152, 388 

Helmholtz, 21 

Heraeus, 210 

Herbert, 328, 329, 388 

Herr and Berninde, 388 

Herzfield and Lange, 118 

Herzog, 41 

Hess, 382 

Hilgard, 141, 191, 201, 230, 235, 284 


420 


Hill, 44, 80, 157, 355, 372, 379 

Hill and Slack, 372 

Hills, 256, 257, 271 

Hiltner, 232, 282, 305, 307, 318 

Hiltner and Stormer, 129, 133, 
160, 164, 257, 297 

Himesch, 388 

Hinds, 344 

Hinterberger, 197 

Hippocrates, 351 

Hirschler, 188 

Hirst, 238, 326 

Hiss and Zinsser, 99 

Hitchcock, 146 

Hite, 406 

Hoelling, 52 

Hoffmann and Hammer, 265 

Homer, 408 s 

Hope, 382 

Hopkins, 63, 183, 237, 276, 288, 311, 
322, 326, 370 

Hopkins and Whiting, 175 

Hoppe-Seyler, 263, 328, 329 

Hormon and Morgenroth, 388 

Houston, 344 

Hull and Rettger, 371 

Hulme, 244 

Hulppe, 413 

Hutchinson, 121, 123, 130, 135, 187, 
138, 230, 231, 235, 236, 253 


151, 


IMMENDOREF, 334 
Itano, 204 


JACKSON, 123 

Jaeger, 382, 388 

Janowski, 342 

Jeannert, 188 

Jenner, 24 

Jennings, 108 

Jensen, 52, 53, 67, 234, 242 

Jodidi, 206 

Johnson, 132, 138, 239, 345, 361, 385 

Jones, 281 

Jordan, 46, 82, 343, 344, 346, 364, 395, 
397, 403, 409 

Joshi, 226 


KAENSCHE, 397 

Kalantarov, 313 

Karg and Schmori, 42 

Kaserer, 52, 216, 225, 259, 260, 334 
Kayser, 194, 365, 392 

Keith, 350 


INDEX OF AUTHORS 


Kellermann and Beckwith, 123 

Kellermann and McBeth, 333 

Kellermann and Robinson, 140, 143 

Kelley, 141, 176, 199 

Kellner, 249 

Kellogg, 141 : 

Kendall, 37, 44, 62, 70, 88, 94, 188 

Kern, 57 

Kiesow, 249 

King, 151, 230, 231, 232, 234 

King and Whitson, 232, 234 

Klebs, 59 - 

Klein, 382 

Klimmer and Kruger, 295 

Knisely, 309, 310 

Knoop, 269 

Kober, 354 

Koch, 24, 54, 99, 127, 131, 183, 157, 
160, 251, 264, 265, 279, 281, 288, 
348, 352, 354, 380i, 

Koch and Seydel, 264, 265 

Kochenavsk, 219 

Kolle and Zetnow, 41 

Kopeloff and Coleman, 136, 138 

Korn, 388 

Kossowiez, 180, 187, 238 

Kossowitsch, 274 

Krainsky, 255, 264, 266, 273, 277, 287 

Kresslig, 59 

Krober, 175 

Krogh, 182 

Kronig, 112, 249 

Kruger, 140, 245, 274, 295 

Kruger and Heinze, 128 

Krumwiede and Noble, 387 

Kruse, 62, 70 

Krzemieniewski, 255, 260, 265, 268 

Kuhlmann, 239 

Kuhlmann and Dumas, 208 

Kuhne, 72, 215 

Kunkel, 118 

Kunnemann, 240 

Kurth, 56 


LABAVIUS, 71 

Ladd, 218 

Lafar, 94, 178, 179, 180, 187, 207, 223, 
225, 238, 318, 335 

Landolt, 210 

Lange, 118 

Langworth, 276 

Larson, 254, 407 

Latham, 123 

Laurent, 158, 241, 274, 292 

Laws and Gilbert, 191, 235, 240, 290 

Lazear, 27 

Leach, 62 

Leeuwenhoek, 17, 18, 42 

Lemmermann, 141, 162, 198, 244 

Lemmermann and Ernecke, 152 


INDEX OF AUTHORS 


Lemmermann and Fresenius, 198 

Leoneini, 144 

Lery and Kayser, 365 

Libby, 28 

Tiebig, 21, 23, 71, 189, 290 

Lipman, C. B., 142, 143, 145, 146, 164, 
196, 199, 200, 218, 235, 252, 255, 260, 
277, 278, 287, 31¢ 

Lipman, J. G., 27, 60, 135, 138, 140, 
141, 152, 159, 191, 193, 197, 202, 203, 
204, 207, 238, 239, 246, 247, 251, 256, 
264, 267, 269, 288, 289, 314, 318, 334 

Lipman and Brown, 129, 140, 199, 200 

Lipman, Brown and Owen, 140 

Lipman and Burgess, 122, 124, 140, 
143, 195, 252, 255, 276 

Lipman and Green, 203 

Lipman and Sharp, 146, 277 

Lipman and Waynick, 200, 283 

Lipman and Wilson, 123 

Lister, 26 

Locy, 28 

Loeb, 108, 117 

Loew, 386 

Lohnis, 38, 126, 138, 160, 161, 198, 207, 
221, 224, 238, 251, 252, 260, 262, 266, 
272, 279, 281, 282, 289, 318 

Lohnis and Green, 260, 266 

Loéhnis and Smith, 272 

Lohnis and Pillai, 262 

Lorenz, 388 

Lumia, 145 

Lyon and Buzzell, 131, 140, 219, 233, 
234 


M 


MaassEn and M@LiER, 293 
Macaulay, 24 

Macfayden, 382 

MacNeal, 64, 388, 389 
MacNutt, 353, 369, 386 
Makrinov, 143 

Malpighi, 291 

Marchal, 194, 195, 196, 203, 204 
Marchiotti, 388 

Marcille, 221, 235 

Marconi, 382 

Markl, 388 

Marshall, 70, 94, 367 
Martelly, 190 

Martin, 113 

Mason, 359 

Massol, 221, 225 

Mauer, 392 

Mayer, 345 

McBeth, 145, 228, 231, 263, 333, 335 
McBeth and Smith, 228, 231 
McBeth and Wright, 145 
McClendon, 117 

McCoy, 375 

McLean and Wilson, 197, 204 
MecWeeney, 398 


421 


Mendel, 370 
Metchnikoff, 32, 370 
Meyer, 44, 59, 231 
Michel, 351 

Migula, 38, 46, 168 
Millard, 153 

Mills, 347, 353 

Miquel, 96, 336, 337, 342 
Mitscherlich, 328 
Miyaka, 204 

Moak, 371 

Mockeridge, 255, 258, 264 
Mohler, 382, 389 
Molisch, 180 

Moll, 153, 234, 282 
Montanan, 144 

Moore, 73, 251, 318 
Morgan, 131 
Morgenroth, 388 
Morrey, 18 

Moson, 413 

Mueller, 54 

Mulder, 208 

Mulford, 224, 225 
Miiller, 209, 293, 382 
Miinter, 170, 231 
Miinter and Robson, 228 
Miintz, 157, 209, 210, 217, 222 
Miintz and Aubin, 217 
Miintz and Coudon, 194 
Murray, 281 

Myer, 196 


N 


Napson, 173, 196 

Nathan, 70 

Navy, 400 

Neale, 156 

Needham, 19 

Neish, 232 

Nencki, 188 

Neubauer and Fromberz, 206 
Newton, 393 

Niklewski, 52, 153, 222 
Nishimwia, 60 

Nobbe, 293, 307, 318 

Nobbe and Hiltner, 307, 318 
Noble, 387 

Northrop, 411 


OBERLIN, 127 

Obermuller, 388 

Olaru, 144, 259 

Omelianski, 159, 224, 252, 264, 271, 
275, 330, 331, 332, 333 

Omelianski and Salunskov, 275 

Omelianski and Sswewrowa, 271 

Ophiils, 401 


422 


Orr, 374 

Osborne and Mendel, 370 
Ostwald, 75, 77 

Ott, 382 

Owen, 140, 201, 203, 219, 221 


P 
PaGET, 28 


Pagnoul, 198 
Parker, 379, 382, 386 


Pasteur, 20, 21, 23, 24, 25, 26, 28, 72, 


182, 190, 209, 328, 387, 411 
Paterson, 141, 142, 143 
Paterson and Scott, 227 
Paul and Kronig, 112 
Pauli, 103, 105 
Pawlowsky, 382, 388 
Peck, 140, 144, 263 
Perotti, 175 
Peterson, 141, 282 
Peterson and Wollny, 141 
Petit, 151 
Petri, 382 
Pettenkofer, 182 
Pfeiffer, 57, 108, 156, 198, 242 
Pfulel, 387 
Phelps, 367 
Pichard, 142 
Pierce, 305 
Pillai, 262 
Platt, 210 
Plenge, 174 
Plummer, 231 
Popoff, 328 
Potter and Synder, 206 
Prazmowski, 57, 271, 292, 300, 304 
Prescott and Winslow, 344, 359 
Pringsheim, 252, 263, 264 
Prucha, 313 374, 389 
Pugh, 240 
Putnam, 240 


Qur1RoGA, 232 


R 


RABINOWITSCH, 388 

Radot, 28 

Rahn, 80, 199 

Raymond, 105 

Redi, 19 

Reed, 232, 259, 283 

Reed and Williams, 232, 283 
Reincke, 353 

Reitz, 388 

Remy, 141, 151, 161, 255, 266, 268 
Remy and Fischer, 151 


INDEX OF AUTHORS 


Renault, 145, 223 

Rettger, 56, 67, 70, 204, 370, 371, 392, 
393 

Richards, 242, 267 

Richards and Rolfo, 242 

Richardson, 232 

Riviere and Bouilhac, 122 

Robertson, 81 

Robinson, 140, 148, 310 

Robson, 228 

Rogers, 389 

Rohn, 406 

Rolfs, 242 

Rosenau, 111, 353, 359, 367, 375, 378, 
383, 384, 386, 388, 394, 405 

Rosenbach, 51, 55, 190 

Rosing, 259, 260 

Rossi, 293, 297 

Roth, 388 

Rowland, 390 

Ruppel, 59 

Russell, 92, 121, 123, 130, 131, 136, 
137, 188, 231, 234, 236, 247, 286, 413 

Russell and Hutchinson, 121, 123, 130, 
131, 136, 1387, 138, 236 


Ss 


Sackert, 270, 284 

Samtee, 367 

Sanarani, 413 

Sanfelice, 169 

Saper, 399 

Savage, 359, 373, 381, 386, 397 

Sawyer, 399 

Scharbekow, 382 

Schegehdahl, 355 

Schettenhelm, 174 

Schlésing, 145, 209, 210, 214, 227, 230, 
234, 274, 292, 329 

Schlésing and Laurent, 274, 292 

Schlésing and Miintz, 210 

Schmori, 42 

Schneider, 152 

Schneidewind, 231, 274 

Schénbein, 208 

Schreiner, 193 

Schroeder and Brett, 390 

Schroeder and Dusch, 20 

Schroeter, 55, 174 

Schuchardt, 388 

Schuder, 355 

Schultz, 156 

Schultz and Borisson, 78 

Schulze, 20, 310 

Schulze and Barbieri, 310 

Schwann, 20 

Scott, 143, 227 

Sedgewick and MacNutt, 353 

Senus, 330 

Setchell, 96 

Severin, 142, 177, 178, 241, 250 


INDEX OF AUTHORS 


Seydel, 264, 265 
Sharp, 146, 201, 277 
Shedd, 123 
Sherman, 413 
Shroeder, 382, 389 
Shutt, 234, 316 
Silberberg, 123 
Simon, 295 

Sirker, 135 

Skalski, 232 
Skinner and Sullivan, 143 
Slack, 372 

Smillie, 391 
Smirnoy, 222 

Smit, 382 


Smith, 138, 228, 231, 235, 236, 239, 272, 


297, 299, 306, 310, 312, 337, 405 
Smith and Robinson, 310 
Snow, 351 
Snyder, 191 
Solmgen, 334 
Sérenson, 204 
Spallanzani, 19 
Sswewrowa, 271 
Stadler, 408 
Stefan, 308 
Stevens, 153 
Stevens and Withers, 158 
Stewart, 232, 234, 238, 326 
Stewart and Greaves, 232, 234 
Stigell, 151 
Stocking, 374 


Stoklasa, 60, 122, 131, 135, 152, 177, 


425 


'Trecol and Fremy, 23 
Treciil, 328, 332 

Trevisan, 54, 57 

Troop, 151 

Truog, 178 

Tsiklinsky, 96 

Tyndall, 23, 24, 26, 72, 337 


Vv 


| Van DELDEN, 53, 178, 251, 254, 275 
Van Ermengen, 400, 401 

Van Helmont, 18, 71 

Van’t Hoff and Arrhenius, 95, 99, 106 
| Van Tieghem, 55, 252 

| Vaughan, 62, 91 

' Vaughan and Novy, 400 

Veillon and Zuber, 190 

Vitek, 242, 243, 244 

| Voelcker, 123 

Vogel, 69, 123, 154, 234, 251, 257, 307 
| Vollery, 28 

Voltaire, 19 

| Vorhees, 138, 141, 193, 203, 207, 238, 
246, 247, 318 

'Vorhees and Lipman, 138, 193, 207, 
238, 246, 247, 318 


Ww 


WaGner, 131, 151, 245 


241, 242, 243, 244, 256, 258, 262, 268, | Waksman, 170 


274, 285, 306, 308, 309, 311 
Stoklasa and Vitek, 242, 243, 244 
Stormer, 129, 133, 151, 160, 257, 297 
Stranak, 256, 262 
Straus, 384 
Stutzer, 216, 217, 226, 240, 242, 309 
Stutzer and Hartleb, 216, 217 
Sullivan, 143 
Sulunskov, 275 
Synder, 206 


i 


TANNER, 385, 392, 394, 403, 408; 410 
Tappeiner and Hoppe-Seyler, 328 
Taylor, 94, 204 

Teichert, 388 

Temple, 152, 312 

Thaysen, 138 

Thorn, 407 

Thomasen, 217 

Thomson, 75 

Thresh, 359 

Thu, 388 

Tissier and Martelly, 190 

Tobler, 388 

Tonney, 382 

Traaen, 277 


| Walton, 259, 264, 282 

Ward, 291, 345 

Warington, 130, 145, 153, 210, 215, 218, 

220, 230, 231, 238, 245 , 

Warnbold, 276 

Washburn, 387, 389, 390 

Washburn and Done, 390 

Wassilieff, 309 

Waynick, 200, 283 

Weichselbaum, 55 

Weinzierl and Newton, 393, 394 
Weiss, 412 

Weissenfield, 388 

Welbel, 153, 218, 231, 232, 234, 283 
Welbel and Winkler, 232, 283 
Wenner, 56 

Westgate, 315 

Wheeler, 62, 344 

Whipple, 337, 345, 363, 364, 365, 367 
Whipple and Mayer, 345 

White, 219 

Whiting, 294, 303, 304, 306, 307, 308, 

312, 318 

Whitson, 232, 234 

Wigand, 292 

Wilbur and Ophiils, 401 

Wiley, 218, 410 

Wiley and Elwell, 218 

Wilfarth, 27, 248, 291, 292 


424 INDEX OF 


Wilhemy, 197 

Williams, 44, 232, 268, 283 

Wilson, 123, 197, 204, 313, 373 

Windas and Knoop, 269 

Winkler, 232, 283 

Winogradsky, 27, 53, 153, 159, 179, 
180, 211, 214, 215, 216, 220, 221, 222, 
225, 250, 252, 264, 272, 307 

Winogradsky and Omelianski, 159 

Winslow, 344, 359 

Withers, 139, 158 

Withers and Fraps, 139 

Wohl, 84 

Wohltmann, 153 

Wojtkiewiez, 235 


AUTHORS 


Wollny, 128, 141, 153 
Wood, 415 

Woronin, 291, 292 
Wright, 145 


Zeit, 103, 104 

Zetnow, 41, 43 

Zinsser, 91, 99 

Zipfel, 298, 295, 299, 313 
Zopf, 55 

Zuber, 190 

Zuco, 210 


. 


SUBJECT INDEX. 


A 


ABIOGENESIS, 19 
Acid, acetic, production of, by bacteria, | 
85, 411, 412 
butyric, production of, by bacteria, 
86 


carbonic, influence on calcium car- 


bonate, 172 
citric, production of, by mold, 87 


formic, produced by bacteria, 85, 87 | 


gluconic, from dextrose, 86 

hippuric, action of bacteria on, 92 

lactic, mechanism of formation, 86 
production of, by bacteria, 85 

oxalic, produced by molds, 87 


phosphates, action on calcium car- | 


bonate, 172 


propionic, from propyl alcohol, 86, 87. 


sulphuric, produced by sulphur bac- 
teria, 179 
uric, action of bacteria on, 91 
valeric, produced by molds, 87 
Acid-forming bacteria in milk, 376 
Acids, amino-, from proteins, 86 
influence on denitrification, 241 
on potassium of soil, 180 
produced by Azotobacter, 269 
by bacteria, 84 
in soil, 172 
in cellulose fermentation, 328, 330. 
in milk, 376 
by nitrifying organisms, 176, 178 
Actinomyces in soil, 169 


Aération, influence on ammonia _ pro- | 


duction, 202, 203 
on Azotobacter, 281 
on denitrification, 242, 246 
on nitrification, 230 


on number of bacteria in soil, 162) 


on Ps. radicicola, 312 


on toxicity of arsenic in soil, 124 | 


Aérobacter, 251 
Aérobes, 69, 109 
Air, bacteria in, 336, 339 


inspired and expired, 339 
factors governing number and kind | 
of bacteria in, 336, 337, 338 
how bacteria enter, 336 
Air-borne infection, 339 
Aleohol as disinfectant, 111, 112 | 


| Aleoholic fermentation, 83, 411 


reactions of, 83, 84 

| Algee, blue-green, 30, 31 

relationship to Azotobacter, 274 
Alinit, 250, 251, 285 
Alkali, influence on nitrification, 220 
Amaneta muscaria poisoning, 396 
_Amidases, production of ammonia by, 

2 


Amino-acids, alcohol from, 84 
ammonification of, 206 
in Azotobacter, 268 
formation of, by Azotobacter, 270 
in legume nodules, 309, 310 


‘ Amins, formation of, by bacteria, 90 
_Ammonification, 27, 34, 92, 194-207 


by actinomyces, 170 
chemistry of process, 204—207 
fungi-producing, 197 
influence of aération on, 202, 203 
of antiseptics on, 130, 135 
of arsenic on, 119 
of calcium carbonate on, 139 
chloride on, 142 
of carbohydrates on, 199 
of climate on, 200 
of crop on, 200 
of green manure on, 156-159 
of gypsum on, 142 
of iron sulphate on, 142 
of lime on, 141 
and magnesia on, 203 
of manganese salts on, 143, 144 
of manure on, 153, 154, 155, 159 
of moisture on, 200, 201 
of phosphorus on, 203, 204 
of potassium salts on, 144, 145 
of season on, 200 
of sodium salts on, 145 
of soil on, 200 
of water on, 154, 155 
methods of studying, 198 
organisms concerned in, 194, 195 
reactions, 192, 193 
stimulation by salts, 147 
toxicity of salts on, 149 
variation with substrate, 199 


_Ammonifiers, distribution, 196, 197 


| Amyedalin, 80 


| Amylobacter, 328, 330, 332 
Anabolism of Azotobacter, 269 


426 


Anabolism, bacterial, 71 
Anaérobes, 69 
Animals and plants, differences in, 29 
Anthrax, organism discovered, 24 
vaccine, 25 
Antiseptic action on ammonifiers, 130 
on bacterial activities of soil, 127, 
128 
on oxidation in soil, 130 
on plants, 128 
on soil, 127 
theories concerning, 133-138 
Grieg Smith’s, 138 
Hiltner’s and Stormer’s, 
133 
Koch’s, 138 
Russell’s and Hutchinson’s, 
135, 136 
definition of, 110 
Antitoxins, 415 
Arsenic, action in soil, 120 
influence of aération of 
toxicity of, 125 
on ammonification, 119 
on azofication, 120 
on Azotobacter, 261 
on bacterial activities, 118-126 
on nitrification, 119 
on soil phosphorus, 122 
occurrence in soils, 118 
solubility of, 118 
Ash of bacteria, 69 
Aspergillus, influence of salts on, 67 
Azofication, 248-289 
definition of, 249 
history of, 248-252 
influence of aération on, 281, 282 
of climate on, 283-284 
of colloids on, 260, 261 
of crop on, 282, 283 
of light on, 281 
of manure on, 266, 267 
of reaction on, 254-256 
of season on, 281, 282 
of temperature on, 278-281 
of water on, 276-278 
methods of studying, 272-274 
Azofiers, distribution of, 252-254 
food requirements of, 256-259 
influence of organic substances on, 
259-261 
metabolism of, 267-271 
morphology of, 271, 272 
pigment production by, 270, 271 


soil on 


relationship to nitrate accumulation, | 


284 

to other micro6rganisms, 274-276 
soil gain in nitrogen from, 287-289 

inoculation with, 284-287 
source of energy for, 261—266 

Azotobacter agilis, 251 

occurrence in water, 254 

ash of, 268 


SUBJECT INDEX 


Azotobacter beijerinckil, 251 
influence of manure on, 266 
calcium requirements of, 259 
carbonic acid generated by, 177 
chroécoccum, 251 
calcium carbonate on, 255 
formation of calcium carbonate 
by, 173 
humates on, 265 
manure on, 266 
methods of growing, 272 
morphology of, 271 
pigments of, 270, 271 
physiology of, 272 
radium rays on, 103 
soil inoculation with, 284-287 
composition of, 59, 60 
distribution, 252, 253 
food of, 256-259 
index of lime requirements, 254 
of phosphorus requirements, 258 
influence of aération on, 281 
of aluminum on, 259 
of antiseptic on, 137 ; 
of arsenic on, 121, 122, 261 
of carbon bisulphid on, 128 
of carbonates on, 254-257 
of climate on, 283 
of colloids on, 259 
of drying on, 106, 276 
of green manures on, 157 
of humus on, 266 
of light on, 281 
of manganese on, 259 
of nitrates on, 256, 257 
of phosphorus on, 176, 177 
of season on, 281, 282 
of sodium on, 256 
of temperature on, 278, 279, 280 
of water, 276, 277 
in fallow soil, 335 
iron requirements of, 259 
isolation of, 251 
life cycle of, 272 
metabolism of, 267—270 
nitrogen of, 268 : 
fixed in different media by, 273, 
274 
phosphorus requirements of, 257-259 
potassium requirements of, 257 
products of, 86 
relationship to cellulose ferments 
257 
to higher plants, 276 
to other organisms, 274-276 
soil gains in nitrogen due to, 289 
sulphur requirements of, 258, 259 
symbiosis amongst, 276 
utilization of cellulose by, 263 
vinelandii, 251 
composition of, 60 
forms of nitrogen in, 267, 268 
nitrogen fixed by, 264 


SUBJECT INDEX 


Azotobacter vinelandii, pigments of, 
270, 271 
vitrium, 251 
influence of manure on, 266 
woodstownli, 251 


B 


“Bases Ernst” granules, 43 
Bacilli, 37 
Bacillus, aceto-ethylicum, 
of acetone by, 411 
acidophilus, in ensilage, 413 
aéris munitissimus, in sewage, 361 


formation 


amylobacter, nitrogen fixation by, 
251 

amylozyme, formation of butyric 
acid by, 411 


anthracis, action of salt on, 67, 408 
influence of pressure on, 107 
temperature relations, 97, 98 

aquatilis, in water, 346 

asteroporus, nitrogen fixer, 251 

aurantiacus, 346 

azophile, 267 

bifermentans sporogens, 190 

botulinus, 400, 401 
acid production by, 86 
influence of salt on, 408 
morphology of, 400 

bulgaricus, 370, 377 
in silage, 413 


centropunctatum, as denitrifier, 241 
243 

cereus, 167, 168 
cultural characteristics, 167, 168 


morphology of, 167 
physiology of, 168 
in soil, 165 
choleree, action of salt on, 67 
circulans in sewage, 361 
in water, 346 
cloacee in canned beef, 394 
in sewage, 361 
coagulans, survive pasteurization, 
392 
coli, 248 
action of copper on, 123 
on phosphates, 174 
aérogens in cheese, 390 
communis in milk, 376, 377 
in eggs, 393 
indol and skatol production by, 89 
products formed by, 85 
as putrefier, 190 
in sausage, 394 
in water, 346 
weight of, 64 
cyanogenes in milk, 376 
pigment production of, 93 
delicatulus in sewage, 361 
denitrificans, 240, 241 


427 


Bacillus denitrificans, enzymes of, 244 
longevity of, 245 
detrudens in cheese, 394 
diphtheriz in butter, 389 
influence of salt on, 408 
in milk, 377 
temperature relations, 98 
diplococcus griseus in putrefaction, 
190 
dysenteriz, Flexner, in milk, 377 
Shiga, in milk, 377 
ellenbachensis, 250 
action on eyanamid, 221 
enteritidis in food, 397 
in water, 346 
erythrogenes i in milk, 376 
ethaceticus, alcohol produced by, 83, 
411 
fecalis in sausage, 394 
ferrugineus, 333 
filefaciens, as denitrifier, 241 
fitizianus, aleohol-producing, 83, 411 
flitrovorum, as denitrifier, 248 
fluorescens, action on cyanamid, 221 
in eggs, 393 
liquefaciens, carbon requirements, 


242 
denitrifier, 241, 243 
in water, 346 


non-liquefaciens, in water, 346 

pigment production by, 93 

in sewage, 361 
fulvus in water, 346 
fuscus, 361 
hartlebi, denitrifier, 241, 242, 243 
helvolus in sewage, 361 
hyalinus in sewage, 361 
icteroides, ammonia-producing, 196 
janthinus, pigment production by, 93 
eigen action on cyanamid, 221 
lactimorbimic melitensis, in milk, 377 
lactis acidi, in milk, 376 

aérogenes in water, 346 

viscosus in milk, 376 

nitrogen fixer, 251 

leprae, nitrogen requirements, 68 
levaniformus, nitrogen fixer, 251 
licheniformis in string beans, 394 
liquefaciens in sewage, 361 
liquidus in sewage, 361 
megatherium, 165, 166 

action on cyanamid, 221 

ammonia produced by, 196 

cultural characteristics of, 165 

denitrifier, 241 

in foods, 394 

morphology of, 165 

physiology of, 165, 166 

in soil, 165 
mesentericus, in bread, 392 

in cheese, 394 

in eggs, 393 

nitrogen fixer, 251 


428 SUBJECT INDEX 


Bacillus mesentericus, survive pasteur- | Bacillus simplex in soil, 165 
ization, 392 sporogenes in sewage, 361 
vulgatus, 196 stillatus in sewage, 361 
in sewage, 361 stutzeri, as denitrifier, 241 
methanicus, 334 subtilis, action on cyanamid, 221 
monadiformis in sewage, 361 on phosphates, 174 
mucosus in eggs, 393 in corn, 394 
mycoides, 166, 167 | as denitrifier, 241, 243 
action on cyanamid, 221 physiological solution for, 142 
on phosphates, 174 in soil, 165 
ammonia produced by, 194-196 survives pasteurization, 392 
cultural characteristics of, 167 temperature relationship of, 98 
denitrifier, 241, 242 in water, 346 
morphology of, 166 tenus in cheese, 394 
optimum conditions for, 195 tetani, acid produced by, 86 
physiology of, 167 thermophilis, temperature relations 
in soil, 165 of, 96, 98 
nibilus in sewage, 361 tumescens, ammonia-producing, 196 
nitrator, 225 typhosus, acid produced by, 85 
nitrovorum, denitrifier, 241 action of copper on, 123 
ochracens in water, 346 in cheese, 390 
orthobutylicus, formation of butyric in cream, 391 
acid by, 411 as denitrifier, 241 
pammelli in cheese, 394 in ice, 350 
paratyphosus, in food, 397 influence of freezing on, 100 
in milk, 377 of salt on, 408 
perfringens in putrefaction, 190 longevity of, in butter, 387 
pestis, influence of salt on, 408 in milk, 377 
phosphorescens, influence of temper- in sewage, 364 
ature on, 96, 98 in water, 344 
pheumone, nitrogen fixer, 251 vilatis in spinach, 394 
prodigiosus, denitrifier, 241, 245 violaceus, pigment production by, 93 
in milk, 376 in water, 346: 
nitrogen fixer, 251 viscosus in cheese, 394 
pigment produced by, 93 vulgaris, action on cyanamid, 221 
in water, 346 ammonia-producing, 196 
proteus, indol and skatol formation in putrefaction, 190 
‘ vulgatus, ammonia-producing, 196 
in putrefaction, 190 in corn, 394 
vulgaris, action on phosphates, 174| weichselbaumii in sewage, 361 
ammonia-producing, 196 welchil in corn, 394 
denitrifier, 241 zopfi lepsiense, action of, on cyana- 
in water, 346 mid, 221 
zenkeri, as denitrifier, 241 in putrefaction, 190 
in sewage, 361 Bacteria, acid production by, 84 
pseudodiphtheriz, influence of press-| action of drying on, 105 
ure on, 107 on fats, 87 
punctatus in water, 346 on hippuric acid, 92 
putidum, action on cyanamid, 221 on minerals, 92 
putidus in putrefaction, 190 on proteins, 87, 88, 89 
putrificus in putrefaction, 190 on sulphur, 175, 178 
pyocyaneus, carbon requirements of, on urea, 91 
42 ! on uric acid, 91 
denitrifier, 241 | in air, 336-339 . 
pigment produced by, 93 expired, 339 
radicicola (See Ps. radicicola) inspired, 339 
nitrogen fixer, 251 methods of entering, 336 
ramosus, ammonia-producing, 196 number and kind of, 336, 337 
in eggs, 393 amins, formation of, 90 
non-liquefaciens, as denitrifier, 241} in arts and industries, 411, 416 
rubefaciens in water, 346 autotrophic, 67 
ruber in water, 346 in body, 31 32 
rubescens in water, 346 in bread, 392 


SUBJECT INDEX 


Bacteria, Brownian movement of, 40, 
Al 


in butter, 387 
in canned foods, 394 
capsules of, 43 
carbohydrates in, 58 
carbon requirements of, 68 
cellulose in, 42 
changes produced in milk by, 375, 
376 
in cheese, 289, 390 
chitin in, 43 
classes of, in milk, 376 
in water, 345, 346 
classification of, 46-57 
composition of, 58-62 
cytoplasm of, 43 
definition of, 29 
discovery of, 17 
in eggs, 393 
in ensilage, 413 
energy for, 65 
extractives in, 58 
factors influencing number and kind 
in soil, 162, 164, 337, 338 
food requirements of, 63-70 
gradation of, 38 
hemicellulose in, 42 
hydrolyzing in sewage, 362 
hydrogen requirements of, 68 
‘in ice cream, 390, 391 
indol, production by, 89 
influence of antiseptics on, 129-138 
of calcium carbonate on, 139 
chloride on, 142 
of chemicals on, 108-117 
of cold on, 100 
of electricity on, 103 
of gypsum on, 142 
of heat on, 95 
of iron sulphate on, 142 
of light on, 95, 101, 105 
of lime on, 141 
of magnesium salt on, 143 
of manganese salts dn, 143, 144 
of manure in soil, 151, 153, 154 
of moist heat on, 99 
of moisture in soil on, 154, 155 
of osmotic pressure on, 106 
of oxygen on, 105 
of potassium salts in soilon, 144, 
145 
of radium rays on, 103 
of Réntgen rays on, 103 
of saltsion, 139 
of shaking on, 107 
of sodium salts in soil on, 145 


of temperature and light on, 95-102 | 


involution forms, 38 

as liberators of phosphorus, 174 
longevity of, 45 

in lungs, 32 

in meat, 393 


Bacteria, metachromatic granules, 43 
method of determining number of, 
160 
im milk, 31) 372, 373 
certified, 371 
common, 372 
condensed, 391, 392 
sources of, 372, 373 
speed of growth in, 374, 375 
moisture in, 65 
content of, 58 
morphology of, 37-45 
motility of, 17, 42 
nitrogen requirements of, 69 
occurrence of, 31 
oxygen requirements of, 69 
phosphorus requirements of, 69 
pigment production of, 93 
in plants, 32 
pleomorphism of, 38 
potassium requirements of, 69 
production of heat by, 94 
light by, 94 
ptomains produced by, 91 
role in nature, 32 
in sewage, 361 
oxidizing in, 363 
pathogens in, 364, 365, 366 
reducing in, 363 
sheath of, 48 
skatol formation by, 89 
soil formers, 33, 171, 172 
in soils, kinds of, 164, 165 
number of, 161, 162 
spores of, 44 
stimulation by salts, 147 
in stomach, 32 
sulphur requirements of, 69 
toxicity of salts for, 149 
vital movement of, 41 
vitamine requirements of, 70 
in water, 342 
classes of, 345, 346 
influence of light on, 343 
of sedimentation on, 343 
of temperature on, 344 
weight of, 39 
zoogloea, 43 


Bacterial activities, influence of 
arsenic on, 118-126 
of enzymes on, 71-81 
of manure on, 150-159 
of salts on, 139-149 
counts, value of, 161 
metabolism, 71—94 
products of, 81-94 
toxins, 91 
Bacteriology, agricultural, 27, 36 
dairy, 36 


definition of, 29 
development of, 17-28 
industrial, 36 
pathological, 36 


429 


430 


Bacterium, acetic acid produced by, 
85 


chrysogloea, nitrogen fixer, 252 
diphtheriz in milk, 377 
lactis aérogenes in milk, 376, 377 
lactus acidi in sauerkraut, 412 
lipsiense, nitrogen fixer, 252 
pasteurianum, acid produced, 85, 86 

morphology of, 411 
pneumoniz, alcohol-forming, 83 
schutzenbachi, morphology of, 412 
tartaricus, nitrogen fixed by, 252 
termo, in putrefaction, 190 
tuberculosis, in butter, 388 

composition of, 59 

influence of salt on, 408 

in milk, 377, 382 

in oleomargarine, 389 

in sewage, 364 

temperature relation of, 96, 98 

Bacteroids, 291, 302, 303 


C 


Catcium carbonate formed in soil, 173 
losses in soil, 172, 173 
on soil flora, 139 
transformation in soil, 172, 173 
Capsules, 43 
composition of, 61 
Carbohydrates, action of, in soil, 246 
in bacteria, 58 
influence on ammonia production in 
soil, 199 
on denitrification in soil, 242, 243 
on nitrification in soil, 222 
on nitrogen fixation in soil, 262 
products formed from, by Azoto- 
bacter, 268 
as source of carbon, 68 
Carbon bisulphid, action of, on soil, 127, 
8 


on Azotobacter, 128 
on plants, 127 
compounds, influence of, on denitrifi- 
cation, 243 
cycle, 182, 183 
nitrogen ratio influence on nitrogen 
fixation by Azotobacter, 263 
264 
in soil, 191 
sources of, for bacteria, 68 
for nitrifiers, 220, 221 
for plants, 290 
Catalyzers, definition of, 75 
as ferments, 71, 73 
Canned food, bacteria in, 394 
Canning, 407 
Cellulose, 327 
in bacteria, 42 
decomposing ferments, 327-335 
early observations on, 327-330 


SUBJECT INDEX 


Cellulose decomposition by actinomy- 
cetes, 170 
ferments, aérobic, 333 
function, 333, 334, 335 
involution forms of, 333 
isolation of, 330, 331 
morphology of, 331-333 
products formed by, 332 
recent work.on, 333 
in sewage, 363 
temperature relations of, 333 
sources of energy for Azotobacter, 


263 
Cell wall of bacteria, 42 
Cheese, bacteria in, 389, 390 
Chemical preservation of foods, 408, 
409 
Chemicals, influence of, on bacteria, 
108-117 
Chemotaxis, 108, 109, 304 
Chitin in bacteria, 43 
Chlorinated lime as disinfectant, 115 
Chlorine compounds as disinfectants, 
114 
Cholera due to water, 351, 352, 354 
vibrio, composition of, 61 
Classification of bacteria, 46—57 
bacterial products, 82 
difficulties of, 46, 48 
from food requirements, 67 
Jensen’s, 67 
Migula’s, 46 
in regard to heat, 96 
Soc. Am. Bact., 50 
of bacterial enzymes, 79 
pigments, 93 
Linnaean system of, for plants, 48 
of waters, 340, 341 
Climate, influence of, on nitrogen fixa- 
tion, 283, 284 
Clostridium, 86 
americanum, nitrogen fixed by, 264 
utilization of cellulose by, 263 
gelatinosum, 241 
nitrogen liberated by, 248 
pasteurianum, discovery of, 250 
method of growing, 272 
morphology of, 252, 271 
nitrogen fixed by, 264 
occurrence of, in water, 254 
physiology of, 272 
symbiosis with Azotobacter, 276 
Cocci, 37 
Cold, influence of, on pathogens, 406 
preservation of food by, 405 
Colloids, influence on Azotobacter, 260, 
261 
Composition of bacteria, 58-61 
of water bacillus, 60 
Copper, action of, on soil bacteria, 122, 
123 


in food, 397 
Crenothrix polyspora, 180 


SUBJECT INDEX 


Crop, influence of, on bacterial flora, 
250 
on nitrates of soil, 236 
on nitrification, 230, 231 
on nitrogen fixation, 282, 283 
production, essential elements in, 319 
rotation, 319-326 
Cycle, biological, in sewage, 363 
of carbon, 182, 183 
of elements, 181-187 
of nitrogen, 183, 184 
of phosphorus, 184, 187 
of sulphur, 184 
Cytoplasm, composition of, 61 


D 


. Decay, 188-193 
definition of, 188 
products of, 190, 191 
Denitrification, 239-247 
by actinomyces, 170 
early theories.on, 239, 240 
enzymes concerned with, 81 
influence of media reaction on, 241, 
242 
of temperature on, 245 
of water on, 244, 245 
losses of nitrogen in, 245, 246, 247 
organisms concerned in, 240, 241 
reactions of, 92 
Denitrifiers, food requirements of, 242, 
243 
function of, 247 
metabolism of, 243, 244: 
Deodorants, 110 
Desiccation, influence on Azotobacter, 
276 
Diplocoeci, 37 
Disease due to meat, 397 
to milk, 378 
to water, 351, 352 
milk-borne, character of, 379, 380 
extent of, 380-383 
Disinfectant, chlorinated lime as, 115 
definition of, 110 
formaldehyd as, 116 
hydrocyanic acid as, 117 
mercuric chlorid as, 117 
sulphur dioxid as, 116 
Disinfectants, chlorin compounds as, 
114 
classes of, 111 
emulsions as, 113, 114 
influence of medium on, 113 
of moisture on, 111 
of temperature on, 111 
laws governing action of, 111 
mode of action, 111 
Drying, preservation of food by, 406 
Dysentery due to water, 353 


431 


E 
Ecropiasm, 42 
composition of, 61 
Eggs, bacteria in, 392, 393 
Electricity, germicidal influence of, on 
bacteria, 104 
influence of, on bacteria, 103 
on medium, 104 


| Emulsion, 80 


Energy, liberation by enzymes, 80 
sources of, 64 
for Azotobacter, 261-266 
for denitrifiers, 243 
for nitrifiers, 223 
Ps. radicicola, 312 
Ensilage, 418, 414 
Enzymes, 71-81 
action of, on phosphates, 175 
classification of, 79 
concerned in ammonification, 204, 
206 
in cellulose fermentation, 330 
in denitrification, 243 
in nitrification, 224 
in Ps. radicicola metabolism, 305 
definition of, 72, 73 
factors governing action of, 76-79 
hydrolytic, 79 
influence of poisons on, 79 
of temperature on, 77, 79 
of time on, 78 
oxidizing, 81 
properties of, 76 
reversible action of, 78 
specificity of, 77 
terminology of, 75, 76 


_Epidemiologist’s method of working, 


355-358 
Extractives in bacteria, 58, 59 


F 


Fatiow, influence of, on nitrification, 
231-235 
loss of nitrates from, 231 
Fats, action of bacteria on, 87 
Fermentation, 188-193 
of alcohol, 83, 84 
alcoholic, 411 
definition of, 188 
early theories of, 21, 71, 72° 
enzymes of, 79 
Ferments, extracellular, 72 
intracellular, 72 
organized, 72 
unorganized, 72 
Fertilizers, influence of, on legumes, 
318, 314 
Food, bacteria in, 387-394 
function of, for bacteria, 64 
milk as, 368-370 
preservation of, 404-410 


432 


Food, preservation of, by canning, 407 
by chemicals, 408, 409 
by cold, 405 
by drying, 406 
importance of, 404 
methods of, 404, 405 
pressure, 406, 407 
by sugar and salt, 407, 408 
pure, law, 409, 410 
requirements of bacteria, 63-70 
maximum, 64 
minimum, 63 
Food-poisoning, 395-403 
botulism, 400, 401 
classes of, 305 
diseased animals causing, 397 
foods causing, 398, 399 
metallic, 396, 397 
paratyphoid causing, 397, 398 
prevention of, 402, 403 
ptomain, 400 
Formaldehyd as disinfectant, 116 
Freezing, influence of, on bacteria, 100 
in soil, 162, 163 
Fruits, preservation of, by pressure, 107 
Fungi, ammonia production by, 197 
filamentous, nitrogen fixers, 252 
Fusel oil, produced in alcoholic fer- 
mentation, 84 
Future work in bacteriology, 27 


G 


Gasgs, in cellulose fermentation, 329, 
332, 333, 334 
Germicidal action of moist heat, 99 
Germicide, 110 
Glucose, alcohol from, 83, 84 
energy from, 65 
lactic acid from, 86 
succinic acid from, 86 
Glycogen in bacteria, 62 
Gonococcus, 37 
Gradation of bacteria, 38 
Granules, “‘ Babes-Ernst,”’ 43 
metachromatic, 43 
Granulobacter, 251 


H 


Heat, influence of moist, on bacteria, 
98, 99 
on nitrifiers, 226 
production of, by bacteria, 94 
relationship of bacteria to, 96 
Hemicellulose in bacteria, 42 


Humates, influence of, on nitrogen fixa- | 


tion, 265 
Humus, chemistry of formation of, 192, 


formation of, 190 


SUBJECT INDEX 


Humus, formation of, by cellulose fer- 
ments, 333 
influence of, on Azotobacter, 266 
on nitrification, 227 
on water requirements in nitrifi- 
cation, 227 
value of, in soil, 191, 192 
Hydrocyanic acid, 117 
Hydrogen requirements of bacteria, 68 
Hydrolytic enzymes, 79 


I 


Icn, bacteria in, 350 
cream, bacteria in, 390 
disease due to, 399 
Indican, formation of, 90 
Indol, formation of, 89 
Infection, air-borne, 339 
caused by food, 399 
sources of, in milk, 378, 379 
Inorganic constituents in bacteria, 61 
Insecticide, hydrocyanic acid as, 117 
sulphur dioxid as, 116 
Involution forms of bacteria, 38 
Iron, action of bacteria on, 92 
bacteria, 180 
influence of, on nitrification, 219 
requirements of Azotobacter, 259 


K 


Karasorism, bacterial, 71 
Keffir, 411 

Kinase, 76 

Kumiss, 411 


L 


Leap, influence of, on soil bacteria, 122 
Leben, 411 
Lecithin, hydrolysis of, 174 
Legumebacter, species of, 295, 296, 297 
Legumes, chemical composition of, 
308-3 12 
elements added to soil by, 319, 320 
feed on nitrates, 323, 324 
influence of, on non-legumes, 314, 315 
immunity to Ps. radicicola, 305 
methods of assimilating nitrogen by, 
307 
nitrogen in, 323 
power to fix nitrogen, 291 
sources of nitrogen for, 322, 323 
Life cycle of Azotobacter, 272 


| Light, influence of, on Azotobacter, 281 


on bacteria, 101, 105 
on denitrifiers, 245 
production of, by bacteria, 94 
Lime, influence of, on ammonification, 
203 


SUBJECT INDEX 433 


Lime, influence of, on nitrification, 219 
on nitrogen fixation, 255 
requirements of soil, Azotobacter 
as an indicator of, 254 
Lipases, 80 
Listerism, 26 « 
Longevity of bacteria, 45 


M 


MaGnesium carbonate, influence of, on 
nitrification, 219 
salts, influence of, on bacteria, 143 
Maltose, 79, 80 
Manganese salts, influence of, on bac- 
teria, 148, 144 
Manure, denitrifying organisms in, 240 
green, ill effects from use of, 158 
influence of, on soil, 152, 156, 159 
influence of, on ammonification, 153 
on Azotobacter, 266, 267 
on bacteria, 151-153 
on bacterial activities, 150-159 
on moisture of soil, 151 
on nitrification, 153 
on nitrogen fixation, 153, 154 
on temperature of soil, 151 
products of decomposition, 330 
Mass and enzyme action, 78 
Matzoon, 411 
Micrococcus acidi in cheese, 394 
albicans amplus, 361 
candicans in beef, 394 
candidus in baked beans, 394 
casel in sewage, 361 
cereus in beans, 394 
fervidosus amplus in sewage, 361 
aa liquefaciens in putrefaction, 
1 
gonorrhee nitrogen requirements, 68 
temperature relationship of, 98 
lactis in cheese, 394 
luteus in corn, 394 
meningitidis, vitamine requirements 
of, 70 
pyogenes in corn, 394 
stellatus in beef; 394. ' 
tetragenus mobilis ventriculi, 361 
ures, 81 
Micron, 39 
Micro6rganisms, kinds of, in soil, 164 
Microspira eestuarii, action of, on sul- 
phates, 179 
desulphuricans, action of, on Sale 
phates, 179 
Mills Reincke phenomenon, 353 
Milk, abnormal changes in, 376 
acid-forming bacteria in, 376, 377 
bacteria in, 372 
bacteriology of, 368-377 
bitter, 376 
certified, 371 


28 


Milk, changes produced in, by bacteria, 
375-376 
classes of, 371 
of bacteria in, 376, 377 
common, 371 
composition of, 368 
disease and, 378-386 
factors influencing number of bac- 
teria in, 372, 373 
as food, 368, 369, 370 
growth of bacteria in, 374, 375 
influence of, on intestinal microflora, 
oil 
pasteurization of, 385, 386 
pathogenic bacteria in, 377, 378 
peptonizing bacteria in, 377 
quantity consumed, 368 
sources of infection in, 378, 379 
tubercle bacilli in, 382 
tuberculosis due to, 381 
Milk-borne disease, character of, 379. 
380 
extent of, 380, 381 
Mineralization of soil constituents, 
171-180 
Moisture in bacteria, 58, 65 
function of, in organism, 66 
influence of manure on soil, 151 
requirements of nitrifiers, 226-230 
Molds, acid produced by, 87 
as denitrifiers, 241 
difference of, from bacteria, 30 
in eggs, 393 
Morphology of Azotobacter, 271 
of bacteria, 37-45 
of cellulose ferments, 331 
of clostridium pasteurianum, 271 
of nitrifiers, 225, 226 
Motility, organs of, 42 
Mustard, as green manure, 157 


N 


Nitrate accumulations, relationship 
of, to Azotobacter, 284 
influence on loss from soil, 247 
losses of, from soil, 236, 237 
prevention of, 238 
quantities formed in soil, 153, 158, 
235, 236 
used by legumes, 323, 324 
Nitrification, 28, 34, 208, 238 
of calclum cyanamid, 221 
chemistry of process, 223, 224 
discovery of organisms concerned, 
209, 210 
early knowledge of, 208 
theories of, 208, 209 
energy transformations in, 223 
influence of aération, 230, 231 
of antiseptics on, 1384 
of arsenic on, 119 


434 


Nitrification, influence of calcium on, 
142 


carbonate on, 139 
of chloroform on, 210 
of crop on, 231, 232, 233, 324, 325 
of cultivation on, 230, 231 
of fallow on, 231-233 
of green manure on, 156-159 
of gypsum on, 142 
of iron sulphate on, 142 
of light on, 230 
of lime on, 141 
of magnesium salts on, 143 
of manganese salts on, 148, 144 
of manure on, 153, 154, 155 
of moisture on, 154, 155, 226-230 
of organic matter on, 211, 222 
of potassium salts on, 144, 145 
of reaction on, 218, 219 
of season on, 234 
of sodium salts on, 144, 145 
of temperature on, 230 
of water-holding capacity of soil 
on; 228, 229 
isolation of organisms concerned, 
211-215 
stimulation of, by salts, 147 
toxicity of salts on, 149 
Nitrifying ferments, distribution of, 
217, 218 
function of, in soil, 215 
influence of depth of soil on, 218 
of heat on, 226 
isolation of, 211-215 
metabolism of, 223, 224 
Nitrite nitrogen in soil, 228 
Nitrobacter, 214, 216 
morphology of, 226 
sources of energy for, 214, 215 
Nitrogen in crops, 320, 321 
cycle, 183, 184 
fixation, chemical theory of, 249, 250 
early theories of, 290, 291 
energy for, 312 
influence of aération on, 312 
of arsenic on, 120 
of combined nitrogen on, 250 
of cultivation on, 283 
of fertilizers on, 313 
of green manures on, 156-159 
of heated soil on, 131 
of manure on, 151 
of moisture on, 312 
of season on, 282 
of temperature on, 279, 280, 313 
mechanism of, 306-312 
in soils, 34, 35 
fixed by Azotobacter, 264, 265 
in legumes, 323, 324 
liberation of, by bacteria, 243 
losses in nitrification, 225 
in nodules, 309 
soil gains in, 287, 315, 316, 352 


SUBJECT INDEX 


Nitrogen, soil losses of, 239 
sources of, for bacteria, 68 
in Utah soils, 320, 325 
“Nitrogen,” 318 
Nitrosococcus, morphology of, 226 
Nitrosomonas, influence of, on phos- 
phates, 175 
media for, 220 
morphology of, 214, 225, 226 
sources of nitrogen for, 221 
Nodule, ash of, 311 
bacterial growth of, 304, 305 
composition of, 309 
Nuclein, action of bacteria on, 174 


O 
O1prum lactis, action of, on lactic acid, 
87 


influence of pressure on, 107 
in milk, 376 
Oleomargarine, tubercle bacilli in, 389 
Organic compounds, action of, on 
Azotobacter, 259, 260 
on denitrification, 243 
in bacteria, 58 
as energy for Azotobacter, 262, 263 
bacteria, 159 
of soil, 193 
manures, speed of decomposition, 
199 
Osmotic pressure, 67 
on bacteria, 106 
influence of, in soil, 149 
Oxidation, of acids, 87 
incomplete, 82 
Oxidizing enzymes, 79, 81 
Oxygen, influence of, on bacteria, 105 
requirements of bacteria, 69 


P 


PATHOGENIC bacteria in cheese, 390 
Peat, bacterized, 286 
denitrification in, 241 
Penicillium, influence of salt on, 67 
Peroxidases, 81 
Phosphate, action of bacteria on, 176 
reaction in soil, 175 
required by zymase, 83 
Phosphoproteins, action of bacteria 
on, 174 
Phosphorus, action of cellulose fer- 
ments on, 334 
cycle, 184—187 
requirements of Azotobacter, 257- 
25 


of bacteria, 64 
in soil, 173 r 
in Utah soil, 320 


SUBJECT INDEX 


Pigment, production of, by actino- 
mycetes, 169, 170 
by Azotobacter, 94, 270, 271 
Pigments, classes of, 93 
production of, 93 
Plants, classes of, 30, 31 
elements in, 181 
poisonous, 395, 396 
Plasmolysis, 67 
Pleomorphism, 38 
Pneumococcus, 37 
Poison, metallic, 396, 397 
Poisoning, 395-403 
classes of, 395 
food causing, 398 
from diseased meat, 397, 398 
from ensilage, 414 
prevention of, 402, 403 
ptomain, 400 
symptoms, 397, 398 
Poisonous foods, 395, 396 
Polar bodies, 43 
Potassium carbonate, influence of, on 
nitrification, 218, 219 
liberation of, by bacteria, 180 
requirements of Azotobacter, 257 
of bacteria, 69 
salts, influence of, on bacteria, 144, 
145 
in Utah soils, 320 
Preservative, definition of, 110 
Pressure on bacteria, 106, 107 
for preserving food, 107 
preservation of food by, 406, 407 
Proteases, 80 
Protein, action of bacteria on, 88 
hydrolysis, 204, 205 
liquefaction in sewage, 362 
as source of carbon, 68 
Protista, 29 
Protozoa, action of antiseptic on, 131 
of arsenic on, 121 
of heat on, 131 
Protozoan theory, 135 
Pseudomonas fluorescens, 168 
ammonia-producing, 196 
morphology of, 168 
physiology of, 168 
in soils, 165 
nebulosa in sewage, 361 
ochracea in sewage, 361 
radicicola, 292 
bacteroids of, 303, 304 
commercial cultures of, 318 
cultural characteristics of, 297-299 
entrance into host, 304 
influence of acids on, 297 
of aération on, 312 
of drying on, 299 
of fertilizers, 313, 314 
of moisture on, 312 
of phosphorus in soil, 178 


435 


Pseudomonas radicicola, influence of 
temperature on, 299, 313 
metabolism of, 306-308 
morphology of, 299-303 
relationship to host, 305, 306 
sources of energy for, 312 
species, 292-299 
staining of, 303 
turcosa in sewage, 361 
Psychrophelic bacteria, 96, 97 
Pteridophytes, 30 
Ptomain poisoning, 400 
Ptomains, 90, 91, 189 
Putrefaction, 188-193 
definition of, 188 
organisms concerned with, 190 
products of, 190, 191 


R 


Rasies, 26 
Radiobacter, 251 
Radium rays, action of, on bacteria, 103 
Rain, influences of, on soil nitrates, 236 
Rays, ultraviolet, as catalyzers, 225 
Reducing enzymes, 79, 81 
Reductase, 81 
Retting of flax, 414 
Rhizobium beijerinckii, 297 
radicicola, 297 
Rontgen rays, influence of, on bac- 
teria, 103 
Rotation and soil fertility, 325 


| Rothamsted, 321, 322 


Ss 


SALT, influence of, on bacteria, 67 


Salts, influence of, on bacteria, 139, 147 
cespactert et activities of soil, 139- 
49 
toxicity of, 145, 146, 149 
Sarcina alba in sewage, 361 
aurantiaca, pigments of, 93 
lutea, pigment of, 93 
production of ammonia by, 196 
in water, 346 


| Sauerkraut, 412 


| 


Schizases, 79 

Schutz-Borissow law, 78 

Season, influence of, on Azotobacter, 
281, 282 


|Sewage, bacteria in, 361, 362 


composition of, 360 

disposal of, 366, 367 

hydrolyzing bacteria in, 362, 363 

oxidizing bacteria in, 363 

pathogenic bacteria in, 364, 365, 366 

reducing bacteria in, 363, 364 
Shaking, influence of, on bacteria, 107 
Sheath, 43 


436 


Silage, temperature of, 94 
Silkworm disease, 23 
Skatol, formation of, by bacteria, 89 
Smallpox, 24 
Sodium salts, influence of, on bacteria, 
145 
Soil actinomycetes, 169 
arsenic in, 118 
bacteria, action of, on proteins, 88 
as formers, 32, 33, 171, 172 
number of, 161, 162, 163, 164 
in water, 346, 347 
biological changes produced in, by 
bacteria, 150 
definition of, 171 
flora, 160, 170 
gain in nitrogen, 248, 287, 289, 315, 
316 


influence of antiseptics on, 127 
of carbon bisulphid on, 128, 129, 
131 at 
of freezing on, 100, 101 
of green manure on, 156-159 
of heat on, 127, 131, 132 
of manure on, 150 
inoculation, 284-287 
methods of, 316-317 
loss of nitrates from, 236, 238 
organic constituents of, 193 
phosphorus, liberation of, 173-175 
plant food in Utah, 320 
potassium, liberation of, 180 
psychrophilic bacteria in, 96, 97 
temperature, 151, 154, 280, 318 
Spirillum cholere asiaticz, 97, 98 
desulphuricans, 178 
Spirophyllum ferrigineum, 180 
Spontaneous generation, 18, 19 
Spores, 44, 45 
germination, 45 
resistance, 44 
Staphylococci in eggs, 393 
Staphylococcus aureus, pigment of, 93 
survives pasteurization, 392 
pyogenes albus in putrefaction, 190 
Streptococcus coligracilis in sewage, 
361 
enteritis in sewage, 361 
lacticus, absence.of katalase in, 81 
acid produced by, 85 
in sauerkraut, 412 
pyogenes in putrefaction, 190 
Substrata, 75 
Sugar and salt, as preservatives, 407 
Sulphates, action of bacteria on, 93 
reduction of, by enzymes, 81 
Sulphur, action of bacteria on, 175, 


bacteria, 179 

cycle, 184 

dioxid as a disinfectant, 116 

requirement of Azotobacter, 259 
of bacteria, 69 


SUBJECT INDEX 


Sulphuric acid, catalytic production of, 
76 
Symbiosis, 306 
among Azotobacter, 276 
T 


TANNING, 414, 415 
Temperature, and coagulation of bac- 


terial protoplasm, 96 
fatal, 98 
influence of, on bacteria, 95, 100, 101 
on enzymes, 79 
on denitrification, 245 
on legume bacteria, 313 
on manure in soil, 151, 154 
on nitrification, 230 
on nitrogen fixed, 279, 280 
on reactions, 95 
on soil, 154 
on water bacteria, 344 
relationships of bacteria, 97 
Thallophytes, 30, 31 
Thermal death point, 99 
Thermophilic bacteria, 96, 97 : 
organisms as nitrogen fixers, 279, 
280 
Thiothrix, 179 
Toluene, action of, on soil, 136 
Toxic compounds in ensilage, 414 


_| Toxicity of salt for bacteria, 149 


Toxins, 91 
bacterial, in soil, 138, 236 
formation of, in soil, 235 
influence of heat on, 132 
Tryptophan, action of bacteria on, 89 
Tubercles, root, early observations on, 
291, 292 
Tuberculin test, 383, 384, 385 
Tuberculosis among cattle, 381, 382 
due to milk, 380, 381 
Typhoid bacilli, resistance of, to lactic 
acid, 387 
carriers, 399 
cause of, how determined, 355-359 
character of outbreak of, 355 
due to milk, 379, 380 
to water, 351, 352, 354 
para, outbreaks, 397, 398 


U 
UREA, action of bacteria on, 91 
Urease, 80 
Uric acid, products from, 91 
Uricarse, 92 

vV 


Vaccine, for anthrax, 25 


| Vaccines, 415 


SUBJECT INDEX 


Vinegar, bacteriology of, 411, 412 
oxidase, 81 

Vital movement, 41 

Vitamin requirement of bacteria, 70 

Vitamins in milk, 370 


Ww 


Water, catalytic action of, 66 
chemical purification of, 349 
cholera due to, 352, 353 
classes of bacteria in, 345, 346 
classification of, 340, 341 
disease and, 351-359 
diseases transmitted by, 352 
drainage, nitrogen in, 237 
dysentery due to, 353 
ground, 341 
importance of, 340 
influence of, on ammonification, 200, 
201 
on Azotobacter, 276, 277 
on denitrification, 244 
of food on number of bacteria in, 
344, 345 
of light on number of bacteria in, 
343, 344 
on nitrification, 226-230 
on nitrogen fixation, 276-278 
on Ps. radicicola, 312, 318 
of penn eaoe on bacteria in, 
34 


437 


Water, influence of temperature on 
number of bacteria in, 344 
intestinal bacteria in, 347 
natural purification of, 347, 348 
purification of, by alum, 349 
by chlorazine, 349 
by potassium permanganate, 349 
soil bacteria in, 346, 347 
solvent, 66 
stored, 341 
surface, 341 
typhoid due to, 354, 355 
Water-holding capacity of soil, re- 
pe ee of, to ammonification, 
201 


x 


Yeast, action of, on phosphates, 175 
as denitrifier, 240 
difference of, from bacteria, 30 
nitrogen fixed by, 252 
production of ammonia by, 194 
Yellow fever, 26 


Z 


Zinc, action of, on bacteria, 123, 124 

Zodgloea, 43 

ate phosphorus requirements of, 
8 

Zymases, 80 

Zymo-excitor, 76 

Zymogen, 76 


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