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Full text of "A manual of bacteriology for agricultural and general science students"

A MANUAL OF 
BACTERIOLOGY 



REED 







LIBRARY 

NEW YORK STATE VETERINARY COLLEGE 

ITHACA, NEW YORK 




Cornell University Library 
QR 41.R32 

A manual of bacteriology for agricultura 



3 1924 000 225 601 




Cornell University 
Library 



The original of tiiis book is in 
tine Cornell University Library. 

There are no known copyright restrictions in 
the United States on the use of the text. 



http://www.archive.org/details/cu31924000225601 



A 
MANUAL OF BACTEEIOLOGY 



FOR AGEICULTURAL AND GENERAL 
SCIENCE STUDENTS 



BY 



HOWAED S. EEED, Ph.D. 



PEOFESSOK OF MYCOLOGY AND BACTERIOLOGY IN THE 

TIKGINIA POLYTECHNIC INSTITUTE ; PLANT PATHOLOGIST AND BACTERIOLOGIST 

IN THE VIRGINIA AQRICDLTURAL EXPERIMENT STATION 






GINN AND COMPANY 

BOSTON • NEW YORK • CHICAGO • LONDON 
ATLANTA • DALLAS • COLUMBUS • SAN FBANCISCO 






COPYKIGHT, 1914, BT 
HOWARD S. EEED 



ALL EIGHTS RESERVED 




Z5' 



V s;A-n:Gf,.f 



Cte gtjiengeum gre«a 

GINN AND COMPANY • PRO- 
PRIETORS • BOSTON • U.S.A. 



PREFACE 

The study of bacteriology in technical schools of all kinds has 
grown rapidly in the past few years, and especially in the institu- 
tions which teach agriculture and allied subjects. The present 
work is an attempt to outline a profitable course for students 
in such classes. The Manual is the outgrowth of several years' 
experience in teaching bacteriology to students of agriculture 
and engineering, and includes some experiments which have not 
previously appeared in print. 

The writer has tried to outline many experiments calling for 
the simplest kind of equipment, but which should acquaint the 
student with some fundamental facts concerning bacteria. In the 
main, however, the Manual is devoted to more extended experi- 
ments which call for precise results and need precise apparatus 
for their performance. Wherever possible the experiments are 
planned to give quantitative results, to the end that vagueness 
of statement and uncertain thinking may disappear. 

In addition to the outhne for the study of bacteria, a section 
has been added outlining the study of important fermentations 
caused principally by fungi. In most cases the agricultural 
student desires to become acquainted with the more fundamental 
principles of the cultivation of these organisms. 

The appendixes are intended to present in a convenient form 
both new and well-known methods. The section on sterilization is 
designed to aid the advanced worker by supplementing the facts 
given in the body of the Manual. Emphasis is placed upon 
several facts recently brought out in investigations on sterilization. 

References to the literature bearing directly upon the subject 
of the exercise have been introduced wherever possible. The 
conspicuous position of these references is designed to stimulate 
the student to do collateral reading. 



iv A MANUAL OF BACTEEIOLOGY 

The section on soil bacteria has been read by Dr. J, G. Lip- 
man, Director of the New Jersey Experiment Station. That on 
the bacteria of milk has been read by Professor E. G. Hastings, 
of the University of Wisconsin. My colleague, Dr. E. B. Fred, 
has prepared portions of the appendiKCS and given many valuable 
suggestions throughout the work. Mr. C. H. Crabill has aided 
greatly in the preparation of the illustrations. 

In the preparation of the material, frequent use has been made 
of the manuals of Lbhnis, Eyre, Frost, Heinemann, Abel, Russell 
and Hastings, Muir and Ritchie, and others, to which acknowl- 
edgment is due. 

HOWARD S. REED 



CONTENTS 



SECTION I. THE FORM AND OCCURRENCE OF BACTERIA 

EXERCISE PAGE 

1. The Forms op Bacteria 2 

2. Types of Bacteria 2 

3. Spores op Bacteria 2 

4. Motility op Bacteria .... . 4 

5. ZooGLCEA Forms 4 

6. Comparison of the Shapes of Yeasts and Mold Fungi 

WITH Bacteria 5 

SECTION II. THE NUTRITION OF BACTERIA 

7. Preparation op Nutrient Agar 6 

8. Preparation op Bouillon .... .... 9 

9. Preparation op Nutrient Gelatin 10 

10. Preparation of Potatoes for Cultures 11 

SECTION III. STERILIZATION 

11. Sterilization of Culture Media with Moist Heat in the 

Arnold Type op Sterilizer 12 

12. Sterilization of Culture Media with Moist Heat in the 

Autoclave 13 

13. Results op Fractional Sterilization 14 

14. Sterilization of Dishes and Instruments by Dry Heat 15 

SECTION IV. RELATION OF BACTERIA TO FACTORS OF 
THE PHYSICAL ENVIRONMENT 

15. Relation op Bacteria to Oxygen (Growth) 16 

16. Relation of Bacteria to Oxygen (Motility) 16 

17. Effect of Light upon Bacteria 16 

V 



vi A MANUAL OF BACTERIOLOGY 

18. Effect of Light of Different Wave Lengths upon Bac- 

terial Growth 17 

19. Relation of Bacteria to Heat 17 

20. Relation of Bacteria to Concentration of Medium . . 17 

21. Effect op Drying upon Bacteria 18 

22. The Effect of Different Disinfectants . . . .18 

SECTION V. RELATIOiSr OF BACTERIA TO 
BIOLOGICAL FACTORS 

23. Rate of Growth of Bacteria ... 19 

24. Chemotaxis . . .... . . . . . . . 19 

25. Arrangement of Bacteria with Respect to Each Other 20 

26. Production of Enzymes . . . . . 21 

SECTION VI. FUNDAMENTAL METHODS USED IN THE 
CULTURE OF BACTERIA 

27. Inoculation of Cultures on Solid Media .... .21 

28. Inoculation of Cultures on Liquid Media ... . . 23 

29. Preparation of Milk fob Use as a Culture Medium . . 23 

30. Preparation of Litmus Milk . . 23 

31. Preparation of Litmus Whey .... . ... 23 

32. Preparation of Whey Agar ... . . . .23 

33. Preparation of Sugar Bouillon . . ... 24 

34. Preparation of Sugar Gelatin . . 24 

35. Preparation of Litmus-Lactose Gelatin or Agar . 24 

36. Preparation of Glucose-Formate Bouillon . . . 24 

37. Preparation of Dunham's Solution . ... 25 

38. Preparation op Phenol Bouillon .... .... 25 

39. Preparation of Lactose Bile fob Isolation of Intestinal 

Bacteria . . . . ... 25 

40. Preparation of Neutral Red Broth . . 25 

41. Preparation of MacConkey's Bile Salt Broth . . . 26 

42. Preparation of Modified Giltay and Aberson's Fluid 

FOB Denitrifying Organisms . . . . . . . 26 

43. Preparation of Iron Bouillon . . . . 26 

44. Preparation of Lead Bouillon . .... 26 

45. Preparation of Glycerin Bouillon . ... 27 

46. Preparation of van Delden's Solution 27 

47. Preparation of Potato Gelatin ... 27 

48. Preparation of Heyden-Nahrstofp Agar 27 



CONTENTS vii 

49. Prepaeation of Ashby's Solution 28 

50. Preparation op Nitrogen-Free Media for Culture of 

Symbiotic Nitrogen-Fixing Bacteria . . ... 28 

51. Preparation of Winogradsky and Omelianski's Solution 

for Cultivating Nitrite-Forming Organisms ... 29 

52. Preparation op Winogradsky and Omelianski's Solution 

for Cultivating Nitrate-Forming Organisms ... 29 

53. Preparation op N^geli's Solution . . 29 

54. Preparation of Czapek's Nutrient Solution for the Cul- 

tivation op Fungi . . ... .30 

55. Preparation op Sulphindigotate Bouillon fob Detecting 

Anaerobic Organisms ... . . . . . 30 

56. Methods op Cultivating Anaerobic Bacteria . 30 

57. Preparation op Sterile Water Blanks . . . .32 

SECTION VII. FUNDAMENTAL METHODS USED IN THE 
STAINING AND EXAMINATION OF BACTERIA 

58. Preparation of Simple Stains . . . 33 

59. General Method of Making Stained Preparations . 34 

60. Preparation of Contrast Stains . 35 

61. Special Stains .... . . . . ... 36 

62. Differential Staining . . . . .37 

SECTION VIII. FUNDAMENTAL METHODS OF ISOLATION 

63. Isolation op a Pure Culture 39 

SECTION IX. OUTLINE FOR THE ROUTINE CULTIVATION 
OF BACTERIA 

64. Preliminary Examination . . . . .... 41 

65. Study of Bacterial Characters . 41 

SECTION X. BACTERIA OF THE AIR 

66. Relation op Bacteria to Atmospheric Dust 48 

67. Determination of the Number op Bacteria in Air . 48 

68. Study of Micrococcus candicans 50 

69. Study of Sarcina lutea . ... 50 

70. Stddy op Bacillus fluorescens LiQUEFACiErrs 50 

71. Study of Bacillus prodigiosus 50 

72. Study of Bacillus subtilis (Hay Bacillus) 50 



viii A MANUAL OF BACTEEIOLOGY 

SECTION XI. BACTERIA OF WATER AND SEWAGE 

73. Collecting Samples op Water 51 

74. Quantitative Study of Bacteria in Surface Waters . 52 

75. Qualitative Study of Bacteria in Surface Waters . 53 

76. Presumptive Test for Bacillus coli 54 

77. Tests for Fecal Bacteria 54 

78. Quantitative Examination of Sewage 55 

79. Bacterial Content of Snow and of Rain Water ... 55 

80. Study of Streptococci 55 

81. Isolation of Bacillus coli 56 

82. Study of Characters of Bacillus coli 57 

83. Study op Bacillus proteus vulqaris 57 

84. Study of Aerobic Organisms which Decompose Cellulose 58 

85. Studyop Anaerobic Organisms WHICH Decompose Cellulose 58 

86. Cellulose Decomposition with Formation of Hydrogen 59 

SECTION XII. BACTERIA OF THE SOIL 

87. Obtaining Soil Samples 60 

88. Preparing Dilutions and Pouring Plates 61 

89. Determination of the Number of Spores in Soil ... 61 

90. Ammonipication of Peptone by Soil Bacteria .... 62 

91. Ammonipication op Nitrogenous Substances in Soil . . 63 

92. Nitrite Formation in Solution 64 

93. Nitrate Formation in Solution 64 

94. Nitrification in Soil 65 

95. Reduction op Nitrates by Bacteria 66 

96. The Reducing Action of Denitrifying Bacteria upon 

Nitrates and Methylene Blue 66 

97. Nonsymbiotic Bacteria which Fix Atmospheric Nitrogen. 

Isolation and Study of Azotobacter 67 

98. Growth of Azotobacter in Pure Cultures 67 

99. Symbiotic Bacteria which Fix Atmospheric Nitrogen. 

Bacillus radicicola 68 

100. Isolation and Culture of Bacillus radicicola .... 70 

101. The Production of Bacteroids op Bacillus radicicola 

UPON Artificial Media 70 

102. The Formation of Tubercles upon Roots of Legumes . 71 

103. The Reduction of Sulphates by Bacteria 71 

104. The Cultivation op Bacillus amylobacter 72 

105. Study op Bacillus mtcoidbs 72 



CONTENTS ix 

106. Study of Bacillus vulqatus 72 

107. Study of Bacillus denitrifwans 73 

108. Study of Bacillus sadicicola 73 



SECTION XIII. BACTERIA OF MILK 

109. The Contamination of Milk with Bacteria from Various 

Sources 74 

110. Quantitative Examination of the Organisms in Milk . 75 

111. Determination op the Numbers of Bacteria in Milk 

BY Direct Microscopical Examination 76 

112. The Examination of Milk for Body Cells 77 

113. A Direct Microscopical Method for Determining the 

Number of Body Cells in Milk 78 

114. The Germicidal Action of Fresh Milk 78 

115. The Catalase of Milk 79 

116. The Relation of Bacteria to the Normal Souring of 

Milk 80 

117. The Fermentation Test 81 

118. Study of Curds Formed by Different Organisms . . 82 

119. The Reducing Action of Milk of High and Low Germ 

Content 82 

120. The Effect of Pasteurization upon Different Bacteria 84 

121. Study of Bacterium lactis-acidi 84 

122. Study of Bacillus lactis aerooenes 85 

123. Study of Bacillus subtilis and the Type of Fermenta- 

tion IT Produces 85 

124. Study of Bacillus cyahogenus 86 

125. Study of Bacillus lactis viscosus 86 

126. Study of Microspira tyrooena 86 

127. Study of Oidium (Oosfora) lactis ... 87 



SECTION XIV. BACTERIAL DISEASES OF PLANTS 

128. The Blight of Pome Fruits Caused by Bacillus 

amylovorus 88 

129. The Wilt of Sweet Corn Caused by Pseudomonas 

Stewartii 89 

130. The Black Rot of Cabbage Caused by Bacillus campestris 89 

131. The Soft Rot of Vegetables Caused by Bacillus 

carotovorus ... 89 



X A MANUAL OF BACTERIOLOGY 

SECTION XV. BACTERIAL DISEASES OF MAN AND 
ANIMALS 

132. Peepaking a Disinfectant 92 

133. Anthrax (Splenic Fever) 92 

134. Tuberculosis (Consumption, Phthisis) 94 

135. Green Pus 95 

136. Septicaemia, Inflammation, etc. Caused by Streptococcus 

pyooenes 96 



SECTION XVI. SOME ORGANISMS CAUSING IMPORTANT 
FERMENTATIONS 

137. Morphology of Yeast (Saccharomyces) 98 

138. Reproduction of Yeast by Budding 98 

139. Spore Formation in Yeast 99 

140. Preparation of Pure Cultures of Yeast from Single 

Cells 100 

141. Cultivation of Yeasts . . 100 

142. The Invertase of Yeast . . . .... 101 

143. The Zymase op Yeast . . . .102 

144. The Preparation of Yeast Juice 102 

145. The Preparation of an Active Yeast Powder . . . 103 

146. The Estimation of the Chief Products of the Fer- 

mentation OF Sugars . . ... 104 

147. The Fermentation of Bread Dough . . ... 105 

148. The Fermentation of Cider . . .... ... 107 

149. The Fermentation of Wine 107 

150. The Fermentation of Vinegar . 108 

151. Study of Mucor 108 

152. Study op Penicillium .... . . . . 110 

153. Study of Aspergillus ... . Ill 

154. Study op Torula ... . . 112 

155. Study op Dematium pullulans 112 

156. Obtaining Mold Cultures prom Spores in the Air . . 113 

157. Culture op Molds on Liquid Media . 113 

158. The Stimulating Action op Weak Poisons 113 

159. Demonstration of the Presence of Arsenic by Means 

op Peiticillium bbevicaule . 114 

160. The Amylolytic Power of Molds 114 

161. The Peptonizing Action of Molds 114 



CONTENTS 



XI 



APPENDIX A. STERILIZATION 

Sterilization with Chemicals . 

Sterilization with Heat 

Hot-air sterilization 

Steam sterilization at 100° C 

Steam sterilization under pressure ... 

Partial sterilization — Pasteurization ... 

Sterilization by Filtration 

Sterilization by Light . . 

The Technique of Sterilization for Special Purposes 

Glassware, pipettes, and instruments 

Liquid media and other solutions , 

Gelatin and agar media 

Sugar-containing media . . 

Solid vegetables ... 

Milk .... 

Blood serum . . . 

Soil ... 

Seeds . ... . . . 

Growing higher plants under sterile conditions 



115 
117 
117 
118 
119 
120 
121 
124 
125 
125 
125 
126 
127 
127 
127 
123 
128 
131 
1.34 



APPENDIX B. Handling Stock Cultures . . . 
APPENDIX C. Making Permanent Preparations 
APPENDIX D. The Inoculating Chamber 
APPENDIX E. The Titration of Bouillon . . . 
APPENDIX F. The Determination of Ammonia . 
APPENDIX G. The Determination of Nitrate 
APPENDIX H. The Determination of Nitrite 



138 
139 
141 
142 
143 
146 
148 



APPENDIX I. The Determination of Nitrites BY Trommsdorf's 

Method . . . ... 150 

APPENDIX J. Determination op Total Nitrogen .... 151 
APPENDIX K. The Determination of Reducing Sugars . . 154 



xii A MANUAL Or BACTEEIOLOGY 

APPENDIX L. Titanium Trichloride Solution: its Prepa- 
ration AND Standardization 15& 

APPENDIX M. Measures and Weights — Conversion Tables 160 

APPENDIX N. Alcohol Table (Modified from Windisch) . 162 

APPENDIX O. Comparison of Fahrenheit and Centigrade 

Thermometer Scales . . ,-, 164 

APPENDIX P. Chart op the Society of American Bac- 
teriologists 165 

INDEX 177 



A MAIS'UAL OF BACTERIOLOaY 

SECTION I 
THE FORM AND OCCURRENCE OF BACTERIA 

Bacteria are small and simply organized plants. For the most 
part they consist of single cells, but some consist of filaments 
made up of separate individuals and show some tendency toward 
a physiological division of labor. The bacterial cell is supplied 
with a definite cell wall which incloses living protoplasmic sub- 
stance. Bacteria multiply by simple division, usually constricting 
in the middle and forming two new cells from each mother cell. 

The first recorded descriptions of bacteria are those of Leeu- 
wenhoek, a Dutchman, which were written in 1683. Most of 
our knowledge of bacteria, however, has been obtained since the 
year 1869. 

We have learned that bacteria are widely distributed in nature. 
The soil and natural waters contain large numbers of them. The 
dust particles in the air carry numbers of germs. In thickly 
populated regions the number of germs in the air is larger than 
in sparsely settled districts, while the air over the sea and on 
the tops of high mountains is quite poor in bacteria. 

We find that bacterial activities are intimately associated with 
processes of growth and decay, health and disease, life and death. 
Although there are certain bacteria which cause diseases, yet 
the sum total of their activities is beneficial to the human race, 
and it is certain that life as we know it could not long exist on 
the earth were it not for their action. The study of these organ- 
isms has led to the solution of many of the important problems 
of human welfare. 

1 



2 A MANUAL OF BACTERIOLOGY 

Exercise 1. The Forms of Bacteria 

Using prepared laboratory slides, examine bacteria of different 
origin. Note that the orgamsms have been stained artificially. 
You will need to use a microscope having an oil-immersion lens 
and an Abbe condenser vrith an iris diaphragm (Fig. 1).^ 

Exercise 2. T3rpes of Bacteria 

1. Place a small drop of Gram's iodine solution on a glass 
slide. With a needle transfer a bit of scum from a hay infusion 
to the drop of iodine solution. Tease it up and put over it a 
cover glass. Examine with the microscope. Describe the differ- 
ent types of bacteria found. 

2. Following the directions given in Exercise 59, stain bacteria 
from the hay infusion, using methylene blue or carbol-fuchsin. 
Sketch the types of bacteria seen on the stained preparation. 

3. With a clean knife or needle scrape a little of the material 
attached to your teeth and spread it in a very thin layer on a 
cover glass. Dry, fix, and stain as before. Sketch the types of 
bacteria seen on the stained preparation. 

Exercise 3. Spores 

Search the stained preparations made in Exercise 2 for rods 
which contain oval glistening bodies. These bodies are spores 
which, on account of their resistant walls, did not take the 
simple stains employed. How many spores do you find in 
a rod ? Examine prepared slides of Clostridia showing rods 
swollen by the presence of a large spore. Spores are more 
resistant to unfavorable conditions than the vegetative cell. 

1 A suitable microscope for bacteriological purposes would have both coarse and 
fine adjustments and the following fittings: 2 oculars, 1 in. (x 8) and 2 in. (x 4) ; 

3 objectives, f in. (16 mm.), 5- in. (4 mm.), and tV in. (2 mm.) oil-immersion objective, 
mounted on a triple revolving nose piece, together with an Abbe condenser in the 
substage. It is not so important that the image shall be large as that it shall be sharp 
and clear. When you have finished for the time being with the oil-immersion lens, 
remove the oil witli a piece of Japanese lens paper. If the oil has dried upon the lens, 
soften it by the addition of more oil and allow it to stand several minutes, when it 
can usually be removed with the paper. If this method does not succeed, you should 
caU the instructor. 




Fig. 1. Diagram tx) show the parts of the compovmd microscope. (After Russell 

and Hastings) 

Oi, object; Oj, real image in F2, transposed by the collectlye lens to O3, real image 
in eyepiece diaphragm ; O4, virtual image formed at the projection distance C, 250 
mm. from eyepoint EP; CD, condenser diaphragm; L, mechanical tube length 
(160 mm.) ; 1, 2, 3, three pencils of parallel light coming from different points of the 
source of illumination. (After Bausch & Lomb Optical Co.) 



4 



A MANUAL OF BACTEEIOLOGY 



Exercise 4. Motility of Bacteria 

Transfer a small quantity of bacteria from an agar culture 
twenty-four hours old, or a drop from a hay iafusion, to a clean 
cover glass. Take care to use a small drop and place it at the 

center of the cover glass. 
Place a Van Tieghem cell 
on a slide and quickly invert 
the cover glass on the top 
of the cell (Fig. 2). Exam- 
ine this with the ^ objective 
(4 mm.) and with the dia- 
phragm nearly closed. It will be very difficult at first to see the 
bacteria, but by careful focusing they will appear as transparent 
dots or rods. Examine carefully to see whether they are station- 
ary or motile, distinguishing those as motile which actually move 
back and forth across the stage, and not those which simply 
dance back and forth without locomotion (the Brownian move- 
ment, exhibited by inanimate particles also). What causes the 
bacteria to have the power of motion ? Are cocci self -motile ? 



!FiG. 2. Van Tieghem cell for work with 
hanging drop, as seen in section 

The cover glass is supported on the top 
of the cell 



Exercise 5. Zobglcea Forms 

The cell walls of most bacteria have a gelatinous outer sheath. 
Examine stained preparations for chams of bacteria. Do the 




Fig. 3. Optical principles of dry lens and oil-immersion lens compared 

1, path of light in a dry-lens system; 2, path of light in an oil-immersion lens 
system; 0, ohject; C, cover glass ; i, lens of the ohjective 



THE NUTEITION OF BACTEEIA 5 

individual bacteria touch each other ? Is the intervening space 
always equal? Exainine the surface of a hay iofusion for a 
zoijgloea mass. 

Exercise 6. Comparison of the Shapes of Yeasts and Mold Fungi 
with Bacteria 

Put a piece of baker's yeast into a 3 per cent cane-sugar 
solution, and place the dish for a few hours in a warm place 
(25°-30° C). Examine a drop of the solution containing yeast 
cells, comparing their size, form, and structure with that of the 
bacteria studied above. Examiue microscopically such mold 
fungi as Mucor, Aspergillus, or Penicillium. How do these or- 
ganisms compare with bacteria in respect to size and complexity 
of structure ? 

SECTION II 

THE NUTRITION OF BACTERIA 

Bacteria require for their nourishment compounds of carbon, 
nitrogen, hydrogen, sulphur, oxygen, phosphorus, potassium, and 
magnesium. For some organisms iron and calcium compounds 
are also needed. A few bacteria may use carbon dioxide iu 
gaseous form, and several use elementary nitrogen, but most 
bacteria require compounds of the above elements in suitable 
form. In nature, bacteria are found growing upon more or less 
complex organic substances. Unlike the chlorophyll-containing 
plants, most bacteria are unable to lay hold of the atmospheric 
carbon dioxide, but must derive their carbon compounds from 
other plants or animals, or from excreta from these organisms. 

A proper amount of moisture is required in order that certain 
of the compounds may be dissolved. If water is too abundant, 
the food supply will be too dilute to supply the needs of the 
bacteria; on the other hand, if water is scantily supplied, the 
concentration of solutes is too high to permit of absorption, if 
indeed they are dissolved at all. 

In making artificial culture media the general principle halds 
that the medium should approximate as closely as possible that 



6 A MANUAL OF BACTEEIOLOGY 

on which the orgEuaism is found to grow in nature. For the 
study of pathogenic bacteria investigators make use of meat 
extracts, blood serum, etc. Beef extract in some form is the 
basis of nutrient media for many bacteria, since it furnishes car- 
bon and nitrogen in such suitable combinations. By the addition 
of agar-agar or of gelatin we obtain a solid medium which may 
be easily liquefied by heat. 

Agar-agar is a colloidal substance prepared from seaweeds 
growing on islands in the Pacific Ocean. Agar is liquefied at 
about 90° C. and hardens at about 45° C. ; it can therefore be 
incubated at 87.5° C. without danger of liquefaction. Agar media 
are not usually affected by the peptonizing action of bacteria. 
Few instances have been reported in which agar media were 
liquefied by bacteria. 

Gelatin is a colloidal substance of animal origin. It consists 
chiefly, of chondrin and gluten and is prepared from bone, ten- 
don, and hide. Gelatin media are liquefied at 35° C. and harden 
at about 24° C. 

Gelatin media are better suited for the growth of many bac- 
teria than agar media, especially for bacteria which are natives 
of the bodies of warm-blooded animals. 

The peptonizing enzymes produced by many bacteria and 
fungi liquefy gelatin, each in its characteristic manner. The type 
of liquefication caused by bacteria in stab cultures is used as a 
diagnostic character. 

We are indebted to Robert Koch for the introduction of 
gelatin and agar-agar into bacteriological work. 

Exercise 7. Preparation of Nutrient Agar 

1. Place in a clean beaker or granite-ware pitcher the follow- 
ing substances, in the order named. Two students may cooperate 
to make double the quantity named. 

500 cc. . Distilled water 

2 g Beef Extract (Liebig's) 

6 g Peptone (Witte) 

2 g Sodium chloride 

10 g Agar-agar 



THE NUTRITION OF BACTEEIA 



2. Dissolve these ingredients by boiliag them over a gas flame 
with constant stirring. A better method, and one that obviates 
the danger of burniag the material, is to cook it in the autoclave 
at 10 lb. steam pressure (see Exercise 12). After cooking for 
twenty minutes add 50 cc. distilled water to replace that lost 
by evaporation. 

3. Filter immediately through a thin layer of wet absorbent 
cotton in a funnel. Place the filtered medium in a funnel with 
delivery tube and pinchcock, as shown 
in Fig. 5. 

4. Fill test tubes to the depth of 
about 4 cm. with the liquid agar, taking 
care that none is smeared on the inner 
surface' of the tube near the mouth, as 
this will cause the cotton to stick to 
the glass when the plugs are removed. 

5. Plug the mouths of the test tubes 
with cotton. The plugs are rolled from 
strips of cotton about 5 cm. wide, and 
should fit firmly enough to allow the 
tube to be lifted by that part which 
projects; at the same time they should 
be loose enough to permit interchange 
of gases. The plug should extend into 
the tube for about 3 cm., and should be 
in contact with the wall throughout this 
distance. The part of the plug which 
projects from the mouth of the tube 
should overhang the lip of the tube enough to prevent dust 
from lodging there (Fig. 6). 

6. Sterilize in the autoclave according to directions given in 
Exercise 12. 

7. After sterilization lay the tubes in an oblique position, so 
that the agar may solidify in the manner shown in Fig. 7. This 
produces what is known as an agar slant, or agar slope, and is 
much used because it gives a larger surface for the development 
of the bacterial colony. 




Tig. 4. Funnel arranged 
for filtering agar media 

The layer of absorbent cotton 
is supported by a wad of ex- 
celsior or clean straw 




Fig. 5. Method for filling test tubes with liquid media. (After Russell 
and Hastings) 

Hold the tip free from the walls of the tube and avoid smearing the mouth of the 
tube with the medium 



THE NUTKITION OF BACTEKIA 



Exercise 8. Preparation of Bouillon 

Two metliods of preparation are used by bacteriologists, but 
method B is more convenient and hence more generally used. It 
is open to the followiag objec- 
tion: Liebig's Beef Extract 
(the one commonly used) 
often contains very resistant 
bacterial spores. Triple steri- 
lization at 100° C. sometimes 
faUs to kill these organisms; 
however, the bouOlon can 
be sterUized by heating to 
120°C. in the autoclave and 
subsequently used either as pig. g. Cotton plugs 

a culture medium or as a ^, correctly made plug ; 5, shaUow plug 
basis for making up nutrient which will be easily displaced; C, plug 

which does not protect the tube from the 
gelatm, agar, etc. entrance of dust 




A B 

1. Place 500 g. of lean 
chopped beef, free from fat, in 
1000 cc. of distUled water. Stir 
and set in a refrigerator for 
twelve to twenty-four hours. 

2. Strain the meat water 
through a piece of clean cheese- 
cloth. Add distilled water to 
make filtrate up to 1000 cc. 
Place in. a flask or agate-ware 
kettle for cooking. 

3. Add to either of the above 10 g. peptone and 5 g. sodium 
chloride. Weigh the vessel containing the solution on a balance 
and record the weight. 

4. Cook the bouillon on a steam bath or in the Arnold steril- 
izer fifteen to twenty minutes. Add water to restore to the 
original weight. 



1. Add 4 g. of Liebig's Beef 
Extract to 1000 cc. of dis- 
tUled water. 



2. Place the materials in a 
flask or agate-ware kettle for 
cooking. 




10 A MANUAL OP BACTEEIOLOGY 

5. Neutralize. If method B has been used, neutralization is 
often unnecessary, but the reaction should be tested in every 
case. For research work the method described in Appendix E 
is more accurate and should be used. For student work the 

following method is satisfac- 
tory: Add to the hot bouil- 
lon a few cubic centimeters of 
a normal solution of sodium 
hydrate. Stir thoroughly with 

Fig. 7. Method of making an agar slant a glass rod. Test the reaction 

by touching the end of the 

wet stirring rod to a strip of neutral litmus paper. Continue 

the addition of small quantities of sodium hydrate solution until 

the litmus paper shows only a faint pink color. 

6. Boil for five minutes and restore water lost. 

7. Add 0.5-1.5 cc. normal hydrochloric acid solution. For 
ordinary work 1.0 cc. acid is employed. 

8. If the precipitate in the bouillon is mealy, filter through 
folded, moistened filter paper ; if not, the bouillon must be cooked 
again until the precipitate becomes mealy, and then filtered. 

9. Place the bouillon in plugged tubes or flasks and sterilize 
twenty minutes at 120° C. in the autoclave. 

Exercise 9. Preparation of Nutrient Gelatin 

1. Measure 500 cc. sterile bouillon, made according to Exer- 
cise 8, into a clean beaker or pitcher. 

2. Add Gold Label gelatin. In cold weather a quantity equal 
to 10 per cent of the bouillon used is ample, but in warm weather 
15 per cent gelatin should be used. 

3. After weighing the vessel and its contents, place it in the 
Arnold sterilizer or on a steam bath until the gelatin is melted. 

4. Neutralize as in Exercise 8 ; then boil five minutes. 

5. Filter through absorbent cotton while hot. Much clearer 
gelatin may be obtained if the solution, before being filtered, is 
allowed to cool somewhat and the white of an egg added, then 
cooked again until the egg white is completely coagulated before 
filtering. Fill test tubes to a depth of 4-5 cm. 



THE NUTEITION OP BACTERIA 



11 




v_ 



_x 



6. Plug tubes with cotton and sterilize in the Arnold sterilizer 
for twenty-five minutes on three consecutive days. 

Caution. Long exposure to high temperature may alter the 
gelatin so that it vdll not solidify when cool. 

Exercise 10. Preparation of Potatoes for Cultures 

Potatoes have been for a long time used in bacteriological 
work. At the present time they are used chiefly for test-tube 
cultures, the former use of potato 
sHces for isolating bacteria having 
been displaced by the use of gelatin 
or agar media in Petri dishes. 

1. Select large, ripe potatoes and 
scrub them in water. Remove the 
eyes. Cut plugs from the potatoes 
with the special cutter or with a 
large cork-borer. The pieces should 
be dropped at once into cold water 
and washed several times, if they 
are to be kept white. Divide the 
cylinders with a diagonal cut and 
trim if necessary to fit tubes. 

2. The pieces of potato should 
be put into large test tubes con- 
taining a few drops of water. Some 
device is necessary to keep the 
potato plugs from standing in the 
water. Roux test tubes accomphsh 
this by means of a constriction near 
the bottom. Ordinary test tubes 
may be used, however, by first 
putting in glass beads or a wad of absorbent cotton (Fig. 8). 

3. After plugging the tubes with cotton, sterilize them on 
four consecutive days in the Arnold sterilizer, or once in the 
autoclave at 115° C. The potato bacillus (BaciUug mesenterieus 
vulgatus) is often very difiieult to kUl, and the potato media 
should be watched for a week to make sure that they are sterile. 




Fig. 8. Potato tubes 

A^ Rottx tube, with constriction to 

hold the ping out of the "water; 

-B, ordinary tube, "with cotton "wad 

supporting the plug 



12 



A MANUAL OF BACTERIOLOGY 



SECTION III 



STERILIZATION 



The air in few places is free from bacteria. At times the 
number of germs in the air is quite large; if they fall upon suit- 
able media, they soon begin to grow and to cause more or less 
extensive changes in the composition of the media. Therefore 
some method of preserving the media from change is necessary. 
Moreover, for exact work it is generally imperative that we have 
none other than the desired species under cultivation, that is, a 
pure culture. 

Hence the necessity arises that the culture medium should not 
only be cleared of all germ life before using, but that foreign 
germs should be prevented from entering after the sterile condi- 
tion is once attained. The operations of sterilization are directed 
to this end. Heat is the best and most generally applicable method 
we have for sterilizing culture media. Dry heat and moist heat 
are both employed, but these differ somewhat in efficiency. 



Exercise 11. Sterilization of Culture Media with Moist Heat in the 
Arnold Type of Sterilizer 

The most efficient type of sterilizer for work at the tempera- 
ture of boiling water is the Arnold sterilizer, which has super- 
seded the old Koch steam chest. 
The Arnold type of sterilizer 
is so constructed that only the 
water in the false bottom needs 
to be boiled to furnish steam, 
and the steam circulates in the 
sterilizing chamber (Fig. 9). 
The steam which escapes from 
the top of the chamber is con- 
densed between the two walls 
and drips back into the pan.' 
There should be a thermometer 
in the roof of the sterilizing 




Fig. 9. Arnold sterilizer 

The false bottom holds only a thin layer 
of water, which can be quickly boiled 



STEEILIZATIOJSr 



13 



chamber. Plenty of water should always be supplied to the 
lower pan before heat is applied. 

Culture media should be steamed for twenty minutes, begin- 
ning to count the time when the thermometer records 100° C. 
The media should be steamed on 
three successive days. Why? 
Between steamings the media 
should be kept at temperatures 
favorable for spore germination. 
Why? This is kno^vn as Tyn- 
dall's method of fractional steril- 
ization, or as Tyndallization. 

Exercise 12. Sterilization of Cul- 
ture Media with Moist Heat in 
the Autoclave 



Both spores and bacteria are 
killed within half an hour when 
exposed to moist heat at tem- 
peratures of 110°-120° C. In 
practice these temperatures are 
usually obtained by sterilization 
in a strong, tightly closed cham- 
ber. The autoclave is the form 
generally used (Fig. 10). 

The autoclave is first supplied 
with a small quantity of water, 
the culture media are put in, 
and the Hd screwed on tightly. 
Heat is applied to the bottom 
to boU the water supplied. The 
stopcock at the top of the cham- 
ber is kept open untU all the air 

is expelled and steam escapes from the opening. The chamber 
may then be assumed to be at a temperature of 100° C. and 
to be nearly if not entirely free from air. The stopcock is then 
closed and pressure developed up to 15 lb. The autoclave is 




Tig. 10. 



Autoclave fitted with steam 
connections 



This type can also be heated hy a large 
gas burner 



14 



A MANUAL OF BACTERIOLOGY 



usually supplied also with a safety valve. The following table 
gives the temperatures corresponding to the pressure shown 
on the gauge. 

TEMPERATURE CORRESPONDING TO STEAM PRESSURES 



Temperatures 


Steam pressures 


Temperatures 


Steam pressures 


Fahr. 


C. 


Poimds 


Fahr. 


C. 


Pounds 


212 


100.0 





251 


121.5 


15 


228 


109.0 


5 


260 


126.5 


20 


240 


115.5 


10 


287 


141.5 


40 



An exposure of twenty minutes with a temperature of 120°'C. 
is sufficient to kill all germ life. After cutting off the heat the 
stopcock is opened and the steam allowed to escape slowly, else 
the culture media may boil up and force the plugs out. 

Exercise 13. Results of Fractional Sterilization 

Select eight tubes of sterile nutrient agar and give successive 
numbers. Melt the agar and cool until the tubes can be held m 
the palm of the hand. Inoculate all the tubes from a hay infu- 
sion. Pour tubes 1 and 2 immediately into sterile Petri dishes. 
Place the remainder in the Arnold sterilizer and steam them 
twenty-five minutes at 100° C. ; remove and immediately pour 
3 and 4 into sterile Petri dishes, setting away the remainder. The 
following day heat the remaining tubes as before and pour 5 and 6. 
On the following day repeat with 7 and 8. Count the colonies on 
each plate the third day after pouring, and average the duplicates. 
Why were not all germs killed after the first heating ? What 
explanation does this experiment give for the old theory of 
spontaneous generation ? 



Colonies deTeloping in 
forty-eight hours 



Agar not 
heated 



Agar once 
heated 



Agar twice 
heated 



Agar three 
times heated 



STEEILIZATION 



15 



Exercise 14. Sterilization of Dishes and Instruments by Dry Heat 

The hot-air sterilizer is a double-walled oven constructed of 
sheet iron or copper and usually covered with asbestos. It is 
heated by a gas flame. Objects should be sterilized in this 
oven for one hour at a temperature of 140°-150° C. After 



the temperature of the oven 
objects may be taken out of 



has fallen below 40° C. the 
the sterilizer and used. 




Fig. 11. An oven for dry sterilizing 



Objects to be dry sterilized should be clean, dry, and free from 
dust. Volumetric pipettes should be put into a sheet-iron box 
for sterilization. Petri dishes which are to be kept for a time 
before using, may be separately wrapped in manila paper. 




16 A MAJSTUAL OP BACTEEIOLOaY 

SECTION IV 

RELATION OF BACTERIA TO FACTORS OF THE 
PHYSICAL ENVIRONMENT 

Exercise 15. Relation of Bacteria to Oxygen (Growth) 

1. Pour a tube of melted agar into a sterile Petri dish. Keep 
the dish level until the agar has hardened. 

2. Inoculate the plate in tliree parallel lines about 5 mm. 
distant from each other. Use the straight platinum wire charged 

with B. suhtilis. 

3. Sterilize a plate of mica 
or a cover glass in the Bunsen 
flame, wait a few seconds for 
it to cool, then lay it over the 
lines of inoculation. Press it 
Fig. 12. A Petri dish down in firm contact with 

the agar to exclude the air. 

4. After three days examine the plate for bacterial growth. 
Sketch the plate, showing the location of bacterial growth. 

5. Make a duplicate plate, using an anaerobic germ. 

Exercise 16. Relation of Bacteria to Oxygen (Motility) 

Examine water in which seeds have been boiled and allowed 
to putrefy for motile bacteria. Transfer a drop containing bac- 
teria to a slide, add a few filaments of green alga, and seal 
the edge of the cover glass with vaseline. Clamp the slide to the 
microscope stage and set the preparation in the dark until the 
bacteria have ceased to move. Why do they cease to move ? 
Then set the preparation near a window and look for the resto- 
ration of movement. Where does it first take place ? What 
function does the alga filament play ? Why ? 

Exercise 17. Eifect of Light upon Bacteria 

1. Inoculate a tube of melted agar with B. prodigiosus. Mix 
well and transfer three loopfuls to a second melted tube. Pour 
this second tube into a sterile Petri dish. 



BACTERIA AND PHYSICAL ENVIRONMENT 17 

2. When the agar is soHdified, invert the Petri dish and cover 
half the bottom with black paper. 

3. Expose this dish, paper side up, to direct sunlight for half 
a day, or, better still, place it on a table and adjust a 40-candle- 
power incandescent electric Ught about 50 cm. above it while the 
colonies are developing. 

4. Record the differences in the number and size of the col- 
onies on both the illuminated and the darkened side of the dish. 

Exercise 18. Effect of Light of DiSetent Wave Lengths upon 
Bacterial Growth 

To test the effect of primary colors upon bacterial growth : 
1—2. Same as in the preceding exercise. 

3. Coat the exposed half of the dish with photographic collo- 
dion which contains 2 per cent of one of the following aniline 
dyes : Chrysoidine (for red) ; Aurantia (for orange) ; Naples yel- 
low (for yellow) ; Malachite green (for green) ; Eosin, bluish 
(for blue) ; Methyl violet (for violet). Different students may 
employ different dyes. 

4. Expose the plates thus prepared to strong diffused light 
(not direct sunlight) for several days. 

5. Take notes upon the growth of colonies on both sides of 
the dish. Observe the results where lights of different colors are 
used. 

Exercise 19. Relation of Bacteria to Heat 

Make four agar streak cultures of B. coli and four of B. mega- 
therium. Incubate one of each at the following temperatures : 
7° C. (ice chest), 20° C. (room), 28° C. (incubator), 37.5° C. 
(incubator). 

Keep careful notes on the amount and rate of growth of cul- 
tures, and determine the optimum temperature of these organisms. 

Exercise 20. Relation of Bacteria to Concentration of Medium 

Place about 3 cc. of Liebig's Extract of Beef in each of two 
large test tubes. Add to one tube 20 cc. of water. Inoculate 
both with B. fluorescens Kquefaciens. In which tube do bacteria 
develop ? Why ? 



18 A MANUAL OP BACTEEIOLOGY 

Exercise 21. Effect of Drying upon Bacteria 

1. Prepare five clean cover glasses and sterilize by quickly- 
passing them several times through a flame. 

2. Heavily inoculate 10 cc. of distilled water with B. subtilis 
and mix by shaking thoroughly. 

3. With the sterile platinum loop place an approximately 
equal quantity of this bacterial suspension on each cover glass. 

4. Place the cover glasses in a sterile Petri dish and dry them 
in the incubator at a temperature not higher than 28° C. 

5. On the next day and every twenty -four hours later transfer 
one cover glass with sterile pincers to a sterile Petri dish and 
flood it with a tube of melted agar. Rock the dish gently to 
distribute the bacteria before the agar hardens. 

6. Determine the effect of drying upon this organism by 
counting the colonies as they develop on the agar plates. 

7. Duplicate the experiment, using B. coli. 

8. To what difference between these two organisms can you 
refer the difference of results ? 

Exercise 22. The Effect of Different Disinfectants 

Pour beef bouillon into a series of test tubes, filling each about 
one third full. Inoculate all tubes from a hay infusion. To the 
different tubes add the following: a, nothing; b, \ g. NaCl; 
e, 1 g. NaCl ; d, 2g. sugar ; e, 5 g. sugar ; /, 1 drop mercuric 
bichloride solution (1 : 1000) ; g, 6 drops mercuric bichloride 
solution ; h, 1 drop formalin ; i, 3 drops formalin ; j, 1 drop 
carbolic acid solution (1 : 20) ; k, 10 drops carbolic acid solution; 
I, 150 mg. borax ; m, 300 mg. borax. Tubes A, i, j, and k should 
be corked. Why? Place all test tubes in the incubator and 
examine at intervals to see which of them undergo putrefac- 
tion and which are thoroughly disinfected. Note how very 
much more efficient some disinfectants are than others. How 
do you explain the results of h, c, d, and e ? Which proves to 
be the most efficient? For a discussion of the relative value 
of these and other disinfectants the student is referred to the 
Appendix. 



BACTERIA AND BIOLOGICAL FACTOES 19 

SECTION V 

RELATION OF BACTERIA TO BIOLOGICAL FACTORS 

Exercise 23. Rate of Growth of Bacteria 

This question will be studied by noting the rate of growth of 
B. coli in a flask of beef broth inoculated by the instructor. A 
schedule will be made, assigning students to draw and plate 
samples at hourly intervals from the moment of inoculation. 
Students will take samples from the flask, according to direc- 
tions, and put them into sterile Petri dishes, adding immedi- 
ately a tube of melted agar. Mix the contents of the Petri 
dish by tilting it back and forth, allow to cool in a horizontal 
position, and place in the incubator. Each student will com- 
pute the number of organisms per cubic centimeter in his 
samples and report the number to the instructor for the com- 
posite result. Take notes on the entire experiment and draw 
a graph to illustrate the rate of increase. Where does the graph 
rise most rapidly? Why? 

Exercise 24. Chemotaxis 

Free-swimming organisms, such as motile bacteria, show a re- 
sponse to the presence of chemicals commonly serving as nutri- 
ents. This response can be conveniently studied by placing the 
chemicals to be tested in small capillary tubes and observing the 
behavior of organisms near the open ends of the tubes. 

Proceed as follows: 1. Heat a small glass tube in a flame 
until soft, withdraw from the flame, and quickly draw it out to 
a slender filament. Break up the thinnest part of the filament 
into pieces 8-10 mm. long and fuse up one end of each in the 
flame. Place these tubes in a small dish of beef bouillon and 
exhaust the air under the receiver of an air pump. 

2. A culture of motile bacteria may be obtained by boiling a 
few kernels of corn or peas for a minute, to kill them, and 
allowing them to putrefy in an open dish. Mount a drop of this 
culture on a slide ; taking one of the capillary tubes in a pair of 



20 



A MANUAL OF BACTEEIOLOGY 



forceps, rinse it quickly in water and lay the tube with the open 
end near the center of the drop. Put on the cover glass and 
examine with the ^ (4 mm.) objective. 

3. Note the subsequent movements of the free-swimming 
organisms as they enter the area where the bouillon is diffusing 
from the open end of the tube. Study the reactions which ulti- 
mately bring them into the tube. How do you explain them ? 



Exercise 25. Arrangement of Bacteria with Respect to Each Other 

1. Make bouillon cultures of the following organisms : Micro- 
coccus (any species), Sareina lutea, B. prodigiosus, B. subtilis, 
Microspira (any species), Spirillum rubrum. 

2. Make staius and hanging-drop preparations after thirty-six 
hours. 

3. Examine with the oil-immersion objective and classify the 
organisms in the following table : 



Arrangement 


Form 


Name 


Sketch 






^ Spheres 






Isolated 




Eods 
Spirals 

Spheres 






Mlaments 




Kods 
Spirals 






Plane Surfaces .... 


Spheres 






Regular Masses . . . 


Spheres 










Spheres 






Irregular Masses . . . 




Eods 







CULTURE METHODS 21 

Exercise 26. Production of £nz3mies 
Jordan. General Bacteriology, p. 91. Philadelphia, 1908. 

Make two gelatin stab cultures of B. prodigiosus. Incubate 
them below 22° C. until the bacteria have liquefied nearly all of 
the gelatin. Add 1 cc. of toluol or 3 cc. of chloroform, shake 
well, and filter after it has stood for fifteen minutes. Pour one 
culture into a tube of sterile gelatin and the other into a tube 
of milk, and note changes after three to five days. Why is the 
disinfectant used instead of heat to kill the bacteria? Record 
the results of adding these solutions to the gelatin and milk 
tubes. 

SECTION VI 

FUNDAMENTAL METHODS USED IN THE CULTURE 
OF BACTERLAi 

Exercise 27. Inoculation of Cultures on Solid Media 

Platinum needles mounted in glass rods are generally used 
for inoculating cultures of bacteria. A straight needle is used 
for stab and streak cultures, while a needle with a small loop 



o ( ~i 



-C 



Fig. 13. Inoculating needles 
Pieces of platinum wire set in glass rods 

(diameter 2 mm.) at the extremity is used for liquid cultures 
(Fig. 13). These needles are sterilized by puttmg them into 
a gas flame and heating to redness just before and just after 

1 This section is intended to be a summary of methods most likely to he useful to 
the undergraduate student or for a student beginning independent investigation. It 
is not intended to be exhaustive. For more ample treatment of these subjects the 
student is referred to Muir and Ritchie, Manual of Bacteriology ; Abbott, Principles 
of Bacteriology ; Eyre, Bacteriological Technique ; Abel, Bakteriologisehes Taschen- 
buch ; Lafar, Technisclie Mykologle ; and other special works. 



22 A MANUAL OF BACTERIOLOGY 

using. When the cotton plugs are removed from sterile tubes, 
the tubes should be held horizontal or sloping slightly down- 
ward. The plugs should be held between the fingers in such a 
way that the portions entering the tubes will not come in contact 
with each other nor with other objects (Fig. 14). 

Stab cultures are made by thrusting the straight wire down 
the center of the cylinder of the culture medium. Streak cul- 
tures are made by drawing the wire once over the flat surface 
of the medium (agar or potato). 




Fig. 14. Method of inoculating tubes of solid media 

After inoculation, tube cultures should be placed in some re- 
ceptacle which will keep them upright, and set in a place where 
they will not be exposed to strong light. Experimental cultures 
can be stored in incubators at required temperatures. Stock cul- 
tures should be kept in a room of even temperature, preferably 
not too high, and should be transferred to new media once in 
six or eight weeks. Before removing the cotton plugs from old 
cultures it is well to burn over the surface of the cotton, to kill 
germs which may have settled in it. The plugs may be protected 
from dust by caps of paper, tin foil, or rubber. 



CULTUEE METHODS 23 

Exercise 28. Inoculation of Cultures on Liquid Media 

Bouillon, milk, and the various liquid media are inoculated 
by introducing the inoculum upon a platinum needle. Transfers 
from one liquid to another are made with the platinum loop. 
The Hquid media may be used in test tubes or in small flasks 
of about 250 cc. capacity. 

Exercise 29. Preparation of Milk for Use as a Culture Medium 

Strictly fresh milk must be procured. The cream should be 
removed either by a centrifugal separator or by shaking out with 
ether. The milk is placed in test tubes or flasks, as desired, and 
sterilized in the Arnold sterilizer. Prolonged heating should 
be avoided, else the proteins will be wholly coagulated. It is 
well to heat for fifteen minutes on four successive days, then 
discard such tubes as show, after a three-day incubation, any 
sign of bacterial growth. 

Exercise 30. Preparation of Litmus Milk 

This useful medium is prepared by adding aqueous litmus 
solution to fresh milk. Soak 50 g. of dry litmus cubes for twenty- 
four hours in 250 cc. of distilled water. After filtration through 
filter paper, enough of this solution is added to the fresh milk 
to give it a strong lavender color. Sterilize as directed for milk 
cultures. 

Exercise 31. Preparation of Litmus Whey 

1. Precipitate the casein of fresh milk by rennet extract. 
Neutralize the filtered whey with 4 per cent citric acid and heat 
on the steam bath for thirty minutes. 

2. Filter aad add litmus solution until a strong lavender color 
is obtained. Sterilize. 

Exercise 32. Preparation of Whey Agar 

1. Precipitate the casein of fresh milk by adding a few drops 
of acetic acid to boiling milk. FUter. 

2. Neutralize the whey with sodium hydroxide or bring to 
1 per cent acid if desired. 



24 A MANUAL OP BACTEEIOLOGY 

3. Add to the whey 1 per cent peptone, 2 per cent dextrose, 
and 1.5 per cent agar. Cook, filter, and sterilize in the Arnold 
sterilizer. 

Exercise 33. Preparation of Sugar Bouillon 

This is ordinary beef bouillon to which dextrose, lactose, or 
saccharose has been added. It is prepared by adding to the or- 
dinary bouillon (Exercise 8) 1 per cent by weight of one of the 
above sugars. If lactose or saccharose bouillon is prepared, the 
original bouillon should be free from inosite. This can be 
tested by inoculating a fermentation tube containing the plain 
bouillon with a gas-producing organism like B. coli. If no gas 
is formed, the bouillon is free from inosite. Triple sterilization 
in the Arnold sterilizer is best for sugar bouillon, since the heat 
of the autoclave sometimes darkens the sugar. 

Exercise 34. Preparation of Sugar Gelatin 

To the sugar bouillon, prepared as in Exercise 33, add 12 
per cent gelatin. Cook, neutralize, and filter as in Exercise 9. 
Adjust the acidity and sterilize in the Arnold sterilizer. 

Exercise 35. Preparation of Litmus-Lactose Gelatin or Agar 

To ordinary nutrient gelatin, prepared as in Exercise 9, add 
1 per cent of lactose by weight and enough litmus solution to 
give a good blue color (see Exercise 80 for the preparation of 
the litmus solution). It is better to add 15 per cent gelatin 
instead of the usual 12 per cent. Tube the medium as usual 
and sterilize in the Arnold sterilizer. Litmus-lactose agar may 
be prepared by using 2 per cent agar instead of the gelatin 
in the above. 

Exercise 36. Preparation of Glucose-Formate Bouillon (Kitasato) 

Add to a liter of beef bouillon 20 g. glucose and 4 g. sodium 
formate. After they have dissolved, give triple sterilization in 
the Arnold sterilizer. Solid media may be prepared by the 
addition of gelatin or agar. This medium is useful in testing 
the fermentative power of organisms. 



CULTUEE METHODS 



26 



Exercise 37. Preparation of Dunham's Solution (for Indol Tests) 



Peptone . 
Sodium chloride 
Distilled water 



. 1.0 g. 
. 0.5 g. 
100.0 cc. 



r~\ 



Dissolve, place in test tubes, and sterilize as usual. Ten days 
after inoculation, test for indol as 
follows: add 1 cc. of 0.01 per cent 
solution potassium nitrite and a few 
drops of concentrated sulphuric acid. 
Warm gently by putting tubes in 
warm water. A pink color indicates 
the presence of indol. The action of 
an excess of sulphuric acid on peptone 
causes a brown color in the solution. 



Exercise 38. Preparation of Phenol 
Bouillon 



Add 1 g. of phenol (carbolic acid) 
crystals to 1 liter of beef bouillon pre- 
pared as directed in Exercise 8. This 
medium is more frequently used for 
the cultivation or isolation of B. coli. 
A solid medium may be had by the 
addition of gelatin or agar. 




Fig. 15. A fermentation tube 



Exercise 39. Preparation of Lactose Bile for Isolation of 
Intestinal Bacteria 

1. Procure from the slaughterhouse an ox gall bladder. Empty 
the bile into a graduated cylinder and add 1 per cent of lactose. 

2. Fill fermentation tubes and sterilize them in the Arnold 
sterilizer. 

Exercise 40. Preparation of Neutral Red Broth 

Prepare beef bouillon in the regular way, adding 1 per cent 
lactose, and to each 100 cc. of bouillon add 5 cc. of a 1 per cent 
solution of neutral red (Griibler's Neutral Roth nach P. Ehrhch). 

Tube and sterilize in the Arnold sterilizer. 



26 A MANUAL OF BACTEEIOLOGY 

Exercise 41. Preparation of MacConkey's Bile Salt Broth for Isolating 
Intestinal Bacteria 

1. Place 20 g. Witte's peptone in 200 cc. distilled water 
previously warmed to 60° C. Stir until the peptone is in 
suspension. 

2. Weigh out 5 g. sodium taurocholate (commercial) and 5 g. 
glucose and dissolve in the peptone water. 

3. 'Wash the peptone water into a flask with distilled water 
and make the volume up to 1 liter. Cook the solution in the 
steamer for twenty minutes at 100° C. Filter through paper 
into a flask. 

4. Add either (a) sterile litmus solution sufficient to give the 
medium a deep purple or (5) 5 cc. of a freshly prepared 1 per 
cent solution of neutral red. 

5. Fill fermentation tubes, plug, and sterilize in the Arnold 
sterilizer. 

Exercise 42. Preparation of Modified Giltay and Aberson's Fluid for 
Denitrifying Organisms 

Distilled water 1000.00 co. 

Potassium nitrate 2.00 g. 

Magnesium sulphate 2.00 g. 

Citric acid 5.00 g. 

Dipotassium phosphate 2.00 g. 

Calcium chloride 0.20 g. 

Anhydrous sodium carbonate 4.25 g. 

The solution should be neutralized by the addition of potas- 
sium hydrate if, after boiling, it shows any acidity. 

Exercise 43. Preparation of Iron Bouillon for Detecting the Presence of 
Hydrogen Sulphide 

Add 1 g. ferric tartrate (or lactate) to 1 liter beef bouillon. 
Sterilize in Arnold sterilizer. 

Exercise 44. Preparation of Lead Bouillon for Detecting the Presence 
of Hydrogen Sulphide 

Add 1 g. lead acetate to 1 liter beef bouillon. Sterilize in the 
Arnold sterilizer. 



CULTURE METHODS 27 

Exercise 45. Pieparation of Glycerin Bouillon 

Add 6 or 8 per cent glycerin to beef bouillon after filtration. 
This medium is especially used for the cultivation of B. tviereu- 
losis, but may be used for other organisms with good results. A 
sohd medium is usually prepared by the addition of agar. 

Exercise 46. Preparation of van Delden's Solution for Cultivation 
of Sulphate-Reducing Bacteria 

Dipotasslum phosphate . . 0.5 g. 

Sodium lactate . . ... . . 5.0 g. 

Magnesium sulphate 1-0 g. 

Asparagine .... 1.0 g. 

Ferrous sulphate trace 

Tap water 1000.0 cc. 

Exercise 47. Preparation of Pbtato Gelatin (Eisner) 

1. Grate finely about 1 kg. of clean peeled potatoes. Weigh 
the grated potato and add 1 cc. of distilled water for every gram 
of potato. Place the mixture in a 2-hter fiask and let it stand 
in the ice chest for twelve hours. 

2. Strain through cheesecloth; then filter through Swedish 
filter paper into a graduated cylinder. 

3. Add 15 per cent gelatin to the potato decoction and heat 
in the Arnold sterilizer for sixty minutes. 

4. Estimate the reaction and render the final reaction plus 25. 

5. Cool the medium to below 60° C. and clarify with egg- 
white. 

6. Add 1 per cent potassium iodide to the medium. 

7. Filter through papier Chardin. 

8. Tube and give triple sterilization in the Arnold sterilizer. 

Exercise 48. Preparation of Heyden-Nahrstoff Agar 

1. Place 10 g. Heyden-Nahrstoff in a flask containing 300 cc. 
distilled water. Stir the powder until a good suspension is ob- 
tained and allow the flask to stand overnight. Place 20 g. agar 
shreds in 500 cc. water and allow it to stand overnight. 

2. Heat the Nahrstoff suspension in the Arnold sterilizer for 
one to two hours, and filter while hot through paper. Drain the 



28 



A MANUAL OF BACTEEIOLOGY 



agar through a clear cloth, wash briefly, and put into a flask or 
earthenware pitcher. Add to it the filtered Nahrstoff solution, 
and make up to a volume of 1 liter with distilled water. 

3. Melt the agar in the autoclave or over a flame, as described 
in Exercise 7. Filter through absorbent cotton and tube. Steri- 
lize in the autoclave. 



Exercise 49. Preparation of Ashby's Solution for Isolating Azotobacter 

from Soil 



Mannite (or lactose) .... 


. . . 20.0 g. 


Monopotassium phosphate . . 


. . . 0.2 g. 


Magnesium sulphate .... 


. . . 0.2 g. 


Sodium chloride 


. . . 0.2 g. 


Calcium sulphate 


. . . 0.1 g. 


Calcium carbonate 


. . . 5.0 g. 


Distilled water 


. . 1000.0 cc 



Dissolve the monopotassium phosphate separately in a little 
water and neutralize it with potassium hydroxide solution before 
adding it to the other ingredients. 

Exercise 50. Preparation of Nitrogen-Free Media for Culture of 
Symbiotic Nitrogen-Fixing Bacteria 

1. Dextrose solution. 



Potassium biphosphate . . . 


. . . . 1.00 g. 


Magnesium chloride .... 


. . . . 0.20 g. 


Sodium chloride 


. . . . 0.01 g. 


Ferrous sulphate 


. . . . 0.01 g. 


Manganese sulphate .... 


. . . . 0.01 g. 


Dextrose 


.... 20.00 g. 


Distilled water 


. . . 1000.00 cc 



To prepare a solid medium add 20 g. of agar to this solution. 
2. Mannite solution. 



Distilled water . . 
Potassium phosphate 
Calcium chloride 
Magnesium sulphate 
Ferric chloride . . 
"Mannite .... 



1000.00 cc. 
0.20 g. 
0.02 g. 
0.20 g. 
0.01 g. 
15.00 g. 



Neutralize if necessary with NaOH, using phenolphthalein as 
indicator. 



CULTURE METHODS 



29 



3. Soil extract medium Lohnis.^ 

Prepare a soil extract by heating 1 kg. of soil with 1 liter of 
water ia an autoclave for half an hour at 15 lb. pressure, or heat 
1 kg. of soil with 2 hters of water over an open flame for two 
hours. Filter the hot solution through paper and use as directed 
in the f ollowiug formula : 



Soil extract . . . . 
Potassium phosphate 
Mannite or dextrose . 
Agar 



1000 cc. 
• 5g. 
. 10 g. 
. 15 g. 



Exercise 51. Preparation of Winogradsky and Omelianski's Solution for 
Cultivating Nitrite-Forming Organisms 



Ammonium sulphate 
Potassium Iriphosphate 
Magnesium sulphate . . 
Sodium chloride . . . 
Ferrous sulphate . . . 
Basic magnesium carbonate 
Distilled water .... 



1.0 g. 
1.0 g. 
0.5 g. 
2.0 g. 
0.4 g. 
Excess 
1000.0 cc. 



Exercise 52. Preparation of Winogradsky and Omelianski's Solution for 
Cultivating Nitrate-Forming Organisms 



Potassium biphosphate . 
Sodium chloride . . . 
Ferrous sulphate . . . 
Magnesium sulphate 
Fused sodium carbonate 
Sodium nitrite . . . 
Distilled water . . . 



. 0.5 g. 

. 0.5 g. 

■ 0.4 g. 

. 0.3 g. 

. 1.0 g. 

. 1.0 g. 
1000.0 cc. 



Exercise 53. Preparation of Naegeli's Solution 

This solution has been found to be well suited for the culture 
of yeast and of many other fungi. 

Calcium chloride . 0.1 g. 

Magnesium sulphate 0.2 g. 

Dipotassium phosphate 1-0 g. 

Ammonium tartrate 10.0 g. 

Distilled water 1000.0 cc. 

I Centralbl. i. Bakt., 2te Abt., 14 : 590. 1905. 



30 



A MANUAL OF BACTERIOLOGY 



Exercise 54. Preparation of Czapek's Nutrient Solution for the 
Cultivation of Fungi 

Distilled water 1000.00 oc. 

Magnesium sulphate 0.50 g. 

Dipotassium phosphate . . 1.00 g. 

Potassium chloride 0.50 g. 

Ferrous sulphate ... . . . . 0.01 g. 

Sodium nitrate . . . . . . 2.00 g. 

Cane sugar 30.00 g. 



Exercise 55. Preparation of Sulphindigotate Bouillon (Weyl) for 
Detecting Anaerobic Organisms 

To 1 liter of regular beef bouillon add 20 g. of glucose and 
1 g. of sodium sulphindigotate. After sterilization, which should 

be done in the Arnold sterilizer, the 
medium should be blue. The growth 
of anaerobic organisms turns it yellow. 
Solid media may be had by the addi- 
tion of gelatin or agar. 

Exercise 56. Methods of Cultivating 
Anaerobic Bacteria 

These bacteria grow in an atmos- 
phere devoid of oxygen, obtaining 
the oxygen necessary for their meta- 
bolism from carbohydrates and other 
compounds in the medium which they 
break down. Any method of culture 
is successful which either absorbs the 
gaseous oxygen present or replaces 
the air with a nonpoisonous gas. 

1. Test-tube cultures. The cultures 
should be made in dextrose beef agar 
or gelatin, which should be freshly 
prepared and always boiled to expel 
air immediately before being inocu- 
lated. Wright's modification of Buchner's method, given on the 
following page, is very convenient. 




Fig. 16. Novy jar for anaerohic 
cultures 

The jar is sealed by turning the 
stopper in the top 



CULTURE METHODS 



31 



After inoculating the culture medium, burn over the cotton 
stoppers and, with forceps sterilized iu the flame, push the stop- 
pers into the test tubes for a distance of 2 or 3 cm. It is gen- 
erally desirable to cut off part of the 
protruding cotton before doing this. Fill 
the space above the stopper with dry pyro- 
galUc acid. Add with a pipette enough of 
a 5 per cent solution of sodium hydrate 
to dissolve the acid. Close the tubes 
immediately with a tight-fitting rubber 
stopper. Invert the tube if it 
contains a sohd medium, and 
set it aside for development. 




Fig. 17. Kipp apparatus arranged for generating hydrogen 

A, generator ; B, wash bottle containing lead nitrate solution ; C, wash bottle 
containing silver nitrate solution 

2. Plates and miscellaneous cultures. Place the cultures in a 
Novy jar (Fig. 16) connected with a hydrogen generator. Pass 
the hydrogen from the Kipp generator through two wash bottles, 
one of which contains lead nitrate solution, the other a solution 
of sUver nitrate. It is well to add another wash bottle contain- 
ing potassium permanganate. From time to time collect a test 
tube full of gas from the outlet. Test by holding a burning 
match at the mouth of the tube. As long as explosion occurs. 



32 A MANUAL OF BACTEEIOLOGY 

there is air mixed with the hydrogen, and the gas must be kept 
flowing. When the escaping hydrogen is pure, close the Novy 
jar by turning the stopper. Shut off the flow of gas from the 
generator. Disconnect the apparatus and put the Novy jar in 
the culture room. 

Exercise 57. Preparation of Sterile Water Blanks 

Water blanks are used for diluting cultures containing large 
numbers of bacteria. Physiological salt solution (0.6 per cent 
NaCl) or distilled water may be used. 

Water blanks usually contain 10 cc, 100 cc, 200 cc, or 500 ec. 
The liquid should be measured with a burette or accurate pi- 
pette into vessels of approximately twice the volume of the solu- 
tion to be used. Large test tubes or bottles are generally used. 
The mouths of the vessels should be rather tightly plugged with 
cotton and should be sterilized in the autoclave. During steri- 
lization there is slight evaporation, which, however, is usually 
corrected when 1 cc. of the test solution is added. 



SECTION VII 

FUNDAMENTAL METHODS USED IN THE STAINING AND 
EXAMINATION OF BACTERIA 

The general forms of bacteria may be studied in a living con- 
dition, as has already been done ui Section I. However, if the 
exact shape and structure of the bacteria are to be studied, they 
should be stained. In some instances the results of staining are^ 
valuable as diagnostic characters. Most of the stains commonly 
employed in bacteriological laboratories are solutions of aniline 
dyes. 

Solutions of stains should not usually be kept long before 
being used, although in a few cases their staining powers im- 
prove with age. 

By the use of certain chemical agents called mordants the 
staining power of the aniline dyes is increased. Various metallic 
salts and organic acids are used as mordants — for example, 



STAINING METHODS 33 

ferrous sulphate, tannic acid, carbolic acid, etc. These chemical 
agents seem to have great powers of penetration into the cells, 
carrying the stains through the membranes with them. 

Aniline oil water is also used as a mordant. It is prepared by 
shaking a small amount of aniline oil in distilled water for fifteen 
minutes or longer and then filteriag. The aniline oil water so 
obtained is mixed with the dye to be used. It is not stable for a 
long time. 

Exercise 58. Preparation of Simple Stains 

1. Fuchsin (basic). 

a. Saturated aqueous solution. 

Basic fuchsin 1.5 g. 

Distilled water 100.0 cc. 

S.x Saturated alcohoUc solution. 

Basic fuchsin 3.5 g. 

95 per cent alcohol 100.0 cc. 

Place in a stoppered bottle, shake well at frequent intervals, 

and filter after two hours. 

c. Carbol fuchsin (Ziehl). 

Saturated alcoholic solution fuchsin . . . 10 cc. 
Carbolic acid (cryst.) .... . 5 g. 

Distilled water 100 cc. 

Filter before using. This solution improves with age. 

2. Methylene blue. 

a. Saturated aqueous solution. 

Methylene blue .... 1.5 g. 

Distilled water 100.0 cc. 

h. Saturated alcoholic solution. 

Methylene blue 1-5 g. 

95 per cent alcohol . . 100.0 cc. 

Place in a stoppered bottle, shake well at frequent intervals, 

and filter after two hours. 

c. Alkaline methylene blue (Loffier). 

Saturated alcoholic solution methylene blue . 30 cc. 
0.1 per cent aqueous solution KOH . . . 100 cc. 

Filter before using. 



34 A MANUAL OP BACTERIOLOGY 



3. 

a. 

h. 


Gentian violet. 

Saturated aqueous solution. 

Gentiau violet 

Distilled water . ... 

Saturated alcoholic solution. 

Gentian violet 


. . . 2g. 
. 100 cc. 

. . . 5 g. 




95 per cent alcohol 


... 100 cc. 




Place ia a stoppered bottle, shake frequently, and filter after 
two hours. 

Exercise 59. General Method of Making Stained Preparations 

1. Clean a cover glass, holding it only by the edges. Remove 
all greasy material, using alcohol or other solvents for the pur- 
pose. After cleaning the cover glass, place it in the cover-glass 
forceps (Fig. 18) and pass it rapidly several times through a 

Bunsen flame. 
Circular cover 
glasses 18 mm. 
in diameter are 
most suitable 
Fig. 18. Cover-glass forceps for bacterio- 

logical work. 

2. With the platinum loop place a small drop (about the 
size of a pinhead) of distilled water upon the center of the 
cover glass. Transfer the bacteria to be examined with a sterile 
platinum needle to the drop on the cover glass. Mix the bacteria 
thoroughly with the drop and spread it as evenly as possible over 
the cover glass, within 2 mm. of the edge. If the drop does not 
spread well, the cover glass has not been successfully cleaned. 
When the bacteria to be examined are taken from liquid media, 
the platinum loop will be used and a drop of water on the cover 
glass is unnecessary. 

It is difficult, especially for beginners, to get few enough bac- 
teria to make a good preparation. It is well to make a second 
mount from the first by transferring a part of a rather large 
drop to a second cover glass. 



STAINING METHODS 35 

3. Dry the film. If the drop used is sufficiently small, the film 
will dry readily at room temperature. The dryiag process may be 
hastened by holding the cover glass in the fingers, high over the 
flame. If the wet film is overheated, the preparation will be spoiled. 

4. Fix the bacteria to the cover glass by passing it three times 
through the flame, holding the cover glass, film side up, in the 
cover-glass forceps. This process coagulates the albuminous 
substances and causes the bacteria to adhere firmly to the glass 
through subsequent operations. 

5. Stain the preparation, keeping it still in the forceps. Place 
on the film a few drops of the staining solution to be used. 
Allow it to act five or ten minutes, the time depending some- 
what on the species of bacteria and the stain employed. 

Instead of placing the stain upon the film, the cover glass 
may be immersed in a small glass dish containing the stain. 
Special cover-glass forceps are made for this purpose. If heat is 
required to hasten or intensify the process, a watch glass holding 
the stain is placed on the steam bath and in it the cover glass ; 
or the cover glass with the stain upon it may be held over the 
Bunsen flame, the stain being replenished as it is evaporated. 

6. Wash the cover glass in a weak stream of water. 

7. Put the wet cover glass, film side downward, on a slide. 
Blot with filter paper and examine with the microscope. 

8. If it is desired to make a permanent preparation, the cover 
glass should be dried in the air or gently over a flame, and then 
mounted on a slide with Canada balsam. 

9. Label the slide, stating name of organism, stain employed, 
date, and owner's name or initials. 

Exercise 60. Preparation of Contrast Stains 

1. Aqueous eosin. 

Eosin (water sollible) . . . . . 1 g. 

Distilled water 100 cc. 

Dissolve and add absolute alcohol .... 5 co. 

2. Bismarck brown (Vesuvin). 

Bismarck brown . 0.5 g. 

Distilled water 100.0 cc. 



36 A MANUAL OF BACTERIOLOGY 

Exercise 61. Special Stains 

1. Capsule stain (Welch). 

a. Prepare and fix the film in the usual manner. 

h. Flood the slide with 2 per cent acetic acid, leaving it in 
contact for two minutes. This swells and fixes the capsule so 
it will take the stain. 

c. Blow off the acetic acid by the aid of a pipette. 

d. Immerse in aniline-gentian-violet for five to thirty seconds. 

e. Wash in water, drj^ and mount. 

2. Flagella stain. 

a. Make an agar streak of the organism to be stained. 

h. After eighteen to twenty-four hours, by means of the plati- 
num needle remove a portion of the growth (being careful to 
avoid the culture medium) to a large drop of tap water on a 
clean cover glass. Allow this to stand five minutes rather than 
spread, as there is less danger of breaking off the flagella. 

c. Spread carefully two or three loopfuls of this drop on 
each of several clean cover glasses and dry at room temperature. 

d. Fix by passing through the flame, the cover glass being 
held in the hand. 

e. Flood the cover glass thus prepared with the following 
solution (mordant) : liquor ferri sesquichloridi diluted with dis- 
tilled water 1 : 20, one part ; saturated aqueous solution tannic 
acid, three parts. This mixture improves with age but should be 
filtered before using. Allow to act one minute. 

/. Wash the cover glass in water and dry by blotting it be- 
tween strips of filter paper. 

g. Stain with hot carbol-fuchsin for about one minute. 
h. Wash in water, dry, and mount in balsam. 

3. Spore stain. 

a. Prepare and fix film in usual way. ' 

h. Cover the film with hot carbol-fuchsin and hold above a 
small flame or lay the cover glass in a watch glass full of stain 
on a hot steam bath. Replace the stain as it evaporates. Keep 
the film covered with hot carbol-fuchsin for twenty or twenty- 
five muiutes. (Both spores and bacteria are now stained.) 



STAIXIN'G METHODS 3T 

e. Wash with water and decolorize with acetic alcohol (95 per 
cent alcohol, two parts, 1 per cent acetic acid, one part) until only 
a faint pink color remains. Finally wash thoroughly with water. 

d. Mount the cover glass in water and examine microscopi- 
cally with the \ objective. The spores should be red and the 
rest of the film colorless or a very faint pink. If satisfactory, 
pass on to section e ; if not, repeat steps b to d inclusive. 

e. Stain with weak methylene blue. 

/. Wash in water, dry, and examine under the microscope. 
The spores should be red, the cells blue. Young spores are more 
readily decolorized than older ones. 

Exercise 62. Differential Staining 

1. Gram's method. This method depends upon the fact that 
the protoplasm of some bacteria permits aniline-gentian-violet 
and Gram's iodine solution, when consecutively used, to enter 
into chemical combination, resulting in the formation of a blue- 
black pigment which is practically insoluble in alcohol. Such 
organisms are said to be Gram positive ; if the color is not held, 
they are said to be Gram negative. 

a. Prepare aniline-gentian-violet as follows : 

Saturated alcoholic solution of gentian violet 11 cc. 

Absolute alcohol 10 cc. 

Aniline water ^ 100 cc. 

This solution does not keep well. 

h. Prepare Gram's iodine solution as follows : 

Iodine .... . . 1 g. 

Potassium iodide 2 g. 

Distilled water . . . . . . 300 cc. 

e. Prepare a cover-glass film and fix in the usual way. 

d. Stain with aniline-gentian-violet for three to five minutes. 

e. Drain off excess of stain and, without washing, cover the 
film with Gram's iodine solution ; allow it to remain for one 
to three minutes. The cover glass looks black at this point. 

1 Made by shaking together 5-6 cc. of aniline oil in 100 cc. of distilled water, 
with subsequent filtering. 



38 A MANUAL OF BACTERIOLOGY 

/. Wash with 60 per cent alcohol until only a light brown 
shade remains (as if the glass were smeared with dry blood). 

g. Rinse off alcohol with water. Dry and mount, or contrast 
stain with eosin or Bismarck brown. If the bacteria are Gram 
positive, they will appear a deep blue under the microscope. 

2. Ziehl-Neelson method of demonstrating B. tvherculosis and 
other acid-fast organisms. 

a. Spread a thin film on the cover glass ; dry and fix as usual. 

h. Stain with hot carbol-fuchsin five to ten minutes (entire 
film stained). 

c. Decolorize with 25 per cent sulphuric acid or 30 per cent nitric 
acid. (Removes stain from everything but acid-fast organisms.) 

d. Wash thoroughly with water. 

e. Counterstain the film with weak methylene blue. (Stains 
non-acid-fast organisms, leucocytes, epithelial cells, etc.) 

/. Wash in water, dry, and mount. 

SECTION VIII 

FUNDAMENTAL METHODS OF ISOLATION 

In nature single species of bacteria do not often grow alone. 
Even in those cases where they do exist it is not always possible 
to get them into artificial culture without contamination with 
other species. Yet the student of bacteriology must have pure 
cultures of a single species in order to learn anything definite 
about them. This was one of the greatest handicaps from which 
the earlier workers suffered. After the introduction of agar and 
gelatin media the problem of isolating species was greatly simpli- 
fied, since it was then possible to get colonies sufficiently sepa- 
rated from each other, on plates, to obtain bacteria which were 
the descendants of a single organism, and therefore of the same 
species. For special methods of isolation in cases where this 
procedure is not successful, the student is referred to Eyre, 
Bacteriological Technique, Chapter XIII ; Muir and Ritchie, 
Manual of Bacteriology, page 51 ; or Abel, Bacteriologisches 
Taschenbuch. 



FUXDA]\1ENTAL METHODS OF ISOLATIOX 



39 



Exercise 63. Isolation of a Pure Culture 

In order to get a pure culture from a mixture of bacteria such 
as may be had iq water, sewage, or commercial milk, proceed as 
follows : Three tubes of sterile nutrient agar or gelatin are melted 
in hot water and cooled to 
about 42° C. Label the 
tubes A, B, and C. Select a 
platinum inoculating needle 
which has a loop 2 Tmn. in 
diameter at the extremity. 
Sterilize the needle by heat- 
ing to redness in the gas 
flame and, after allowing a 
few seconds for it to cool, 
dip the loop of the wire 
into the hquid containing 
bacteria. A film of hquid 
is held ia the loop at the 
end of the wire. Inoculate 
tube A with three loopf uls of 
the liquid. Sterilize the wire 
and place it in the holder. 
Thoroughly miy the con- 
tents of tube A. This must 
be done without wetting the 
cotton stopper. The result 
can best be accompHshed by 
rolling the tube between the 
pahns of the hands, while 
slanting it alternately right 
and left. Tube A is called the First Dilution. After mixing, trans- 
fer three loopfuls of tube A into tube 5, using the same procedure 
as before. Tube B is the Second Dilution. In the same way trans- 
fer three loopfuls of B into C, constituting the Third Dilution. The 
Third Dilution usually contains few enough bacteria for the pur- 
pose of isolation, but the Second Dilution should also be poured. 




Fig. 19. An inoculating room 

The Interior walls are lined with linoleum and 
can easily be washed with a weak solution 
ol bichloride. If inoculations are made in 
such a room, the danger of contaminations is 
greatly minimized 



40 A MANUAL OP BACTERIOLOGY 

Place two sterile Petri dishes on the table and label them 
Second Dilution and Third Dilution. Remove the plug from the 
mouth of tube B and flame the mouth of the tube ; slightly 
raise the cover of the Second Dilution dish and pour in the 
melted medium (Fig. 20). Take care that none of the liquid 
medium rises over the edge of the Petri dish. If the medium 
fails to cover the entire surface of the plate, gently tilt the dish 
back and forth until the fault is rectified. Keep the Petri dishes 
level until the medium has hardened ; then they may be placed 
in the culture chamber. 

After twenty-four to forty-eight hours, depending upon the 
temperature, the plates will be dotted with colonies, each of 

wliich is supposed 
to consist of the 
descendants of a 
single organism. 
Study the colonies 
by placing the in- 
verted Petri dish 
Fig. 20. Method of pouring melted media into xj^g sta^e of 

Petri dishes " 

the microscope 

and using the | objective with weak light from the substage. 

Sterilize a straight platinum needle in the flame and, after 
allowing it to cool a few seconds, dip the tip of it into one of the 
colonies. Inoculate both an agar slant and a gelatin stab with 
bacteria obtained in this manner. Label the tube, giving it a 
number, and make a record in a notebook stating the kind of 
colony from which the bacteria were taken and the original 
source of the Petri dish. Place the inoculated tubes in the 
culture chamber. 

If the bacteria used for these inoculations were the descendants 
of a single organism, these tubes will contain pure cultures. The 
bacteria may be used for other subcultures, for stained prepara- 
tions, etc. It often happens, however, that the first isolations are 
not pure cultures. In that case another series of plates must be 
poured, using the growth in the test-tube culture as the source. 
In fact, it is always safer to observe this precaution. 




THE CHAEACTEEISTICS OF BACTERIA 41 

SECTION IX 
OUTLINE FOR THE ROUTINE CULTIVATION OF BACTERIA 

In bacteriology the basis for all work is the pure culture. All 
possible care must be taken to obtain pure cultures and to keep 
them from contamination. The characters of most organisms 
can only be studied to a sUght extent with specimens under the 
microscope ; the majority of their characters are determined by 
their behavior in pure cultures on various media. The scheme 
given below calls for cultures sufficient to determine or demon- 
strate the characters of most organisms called for in this manual, 
except where special directions are given. 

The student should consult the Descriptive Chart adopted by 
the Society of American Bacteriologists (Appendix P). 

Exercise 64. Preliminary Examination 

1. Inoculate an agar-slant tube from each pure culture fur- 
nished by the instructor or isolated from mixed infections. 
Place the inoculated tubes in the incubator for twenty-four hours. 

2. After twenty-four hours examine the tubes for growth. 

a. Describe the growth along the stroke of the needle (see 
Exercise 65). 

b. Make a Gram-stained preparation. 

c. Make a simple stain with carbol-fuchsin, methylene blue, 
or gentian violet. 

3. Inoculate the following media from the agar slant : gelatin, 
potato, Utmus milk, beef broth, and agar plate. All cultures 
except gelatin are to be kept in the incubator. 

4. After twenty-four to forty-eight hours describe and make 
sketches of these cultures as directed in Exercise 65. 

Exercise 65. Study of Bacterial Charactere 

A. Morphology. 

1. Form and arrangement : coccus, single and grouped ; 
diplococcus ; streptococcus ; sarcina ; rods, single and in 
chains ; spirals. 



42 



A MANUAL or BACTEEIOLOGY 



2. Size. Measurements in terms of the micron. 

3. Reaction to stains : 

a. Simple stains : stains easily or with difficulty. 
h. Differential stains: Gram's stain, Ziehl-Neelson 
stain, etc. 




B 



r» 




D 




F 



G 




Fig. 21. Types of growth of bacteria in stab cultures 

A to E, nonliquef ying ; F to J, liquefying. A, filiform; B, beaded; C, echinulate; 

D, villous; E, arborescent; F, crateriform; G, napiform; H, iBfundibuliform; 

/, saccate; J, stratiform 

4. Spores : time required for formation, media, position in ceU. 

5. Special characters : 

a. Flagella. 

b. Capsule. 

c. Vacuoles. 

d. Crystals or granules. 

e. Involution forms. 



THE CHAEACTEKISTICS OF BACTERIA 



43 



B. Cultural characters. 
1. Gelatin stab. 
I. NonliquefyiBg. 

a. Line of puncture : filiform, uniform needle-shaped 
growth (Fig. 21, A) ; leaded, succession of small, 
disjointed colonies (Fig. 21, B') ; ecMnulate, prickly 




C 

•A 

I 



I) 







Fig. 22. Types of growth of bacteria on streak cultures 
A, filiform ; B^ echinulate ; C, beaded ; D, effuse ; Ej arborescent 

(Fig. 21, C); villous, beset with unbranched hair- 
Uke extensions (Fig. 21, U) ; plumose, a feathery 
growth; arborescent, beset with rootlike extensions 
(Fig. 21, Ey 
b. Surface growth. Same as for colonies on plate 
cultures. 
II. Liquefying. 

a. Type of liquefaction: crateriform, saucer-shaped 
(Fig. 21, F') ; napiform, turnip-shaped (Fig. 21, G) ; 
infundibuliform, funnel-shaped (Fig. 21, H^ ; saccate, 
sac-shaped (Fig. 21, /); stratiform, the liquefaction 
descending in a horizontal plane (Fig. 21, J"). 

b. Character of the fluid : clear, cloudy, flocculent, 
granular. 

2. Streak cultures (agar or potato). 

a. Growth: invisible, scanty, moderate, abundant, 
h. Form: filiform, a narrow line (Fig. 22, A') ; ecMnulate, 
growth along line of inoculation with toothed or 



44 A MANUAL OF BACTEKIOLOaY 

pointed margins (Fig. 22, 5) ; headed, a succession 
of small, disjointed colonies (Fig, 22, C); effused, 
spreading (Fig. 22, Z)) ; villous, plumose, arborescent 
(Fig. 22, Ey 

c. Luster: glistening, dull, cretaceous. 

d. Optical characters: opaque, translucent, opalescent, 
iridescent. 

e. Odor : absent, decided. 

f. Elevation of growth I „ , ^ ,^ 

„ , ° ^same as for plate cultures. 

g. iopography J 

h. Consistency : slimy ; butyrous, of a consistency like 
butter; viscid, growth follows the needle when 
touched and withdrawn ; coriaceous, growth tough, 
leathery ; brittle, growth dry, friable under the 
needle. 

i. Medium discolored. 

3. Beef-broth cultures. 

a. Condition of fluid : clear, cloudy. 

b. Surface membrane : when formed, color, consistency. 

c. Sediment : amount, compact, flocculent, granular, 
viscid. 

d. Reaction to litmus. 

e. Odor. 

4. Milk cultures. 

I. Curd formed. 

a. Time r^qaired to curdle. 

b. Character of curd : hard ov soft, solid ox perforated, 
changed or not, when boiled. 

c. Whey : amount, clear or turbid. 

d. Reaction to litmus. 

e. Digestion of curd : time required, reaction to litmus, 
solution cloudy or clear. 

f. Gas bubbles. 

g. Odor. 

II. Digestion without formation of curd. 
III. No visible change even after boiling. 



THE CHAEACTEEISTICS OF BACTEEIA 



45 



5. Plate cultures. 
a. Surface colonies. 

I. Naked-eye appearance. 
a. Form: punctiform, too small to be defined by the 
naked eye; circular; oval; spindle-shaped; conglom- 
erate, an aggregate of similar colonies (Fig. 23, A); 
amoeboid, very irregular (Fig. 23, 5) ; rhizoid, branched, 



A 




^3^ 


,fi^,. , -, 


^ 




JC^^^. 




k-) 


|^-~ 


■'■'' At%- 


^. ;. ■■ 




\,t^^l 


\'''- 






Fig. 23. Types of bacterial colonies 
A, conglomerate ; B, amoeboid ; C, rhizoid ; D, curled ; E, myceloid 



rootlike structure (Fig. 23, C) ; curled, filaments in 
strands like curly hair (Fig. 23, D') ; myceloid, fila- 
mentous with the character of a mold (Fig. 23, E). 

h. Size, approximately expressed in millimeters. 

c. Surface elevation : flat (Fig. 24, A) ; spreading ; 
raised (Fig. 24, B') ; convex (Fig. 24, C) ; pulvinate, 
surface a segment of a circle (Fig. 24, D) ; capi- 
tate, surface a semisphere (Fig. 24, .ff) ; umbilicate, 
depressed in the center (Fig. 24, F^ ; umbonate, 
elevated at the center (Fig. 24, G). 



46 



A MANUAL OP BACTEEIOLOGY 



II, 



d. Topography of surface: smooth; contoured, svaoo^hlj 
undulating, like the surface of a relief map ; rugose, 
short, irregular folds due to shrinkage ; verrucose, 
growth wartlike, with wartlike prominences. 

. Microscopic appearance. 

a. Edge of colony: entire (Fig. 25, A) ; undulate, wavy 
(Fig. 25, B); repand(Fig. 25, C); lohate(Fig. 25, D); 



E F O 

Tig. 24. Types of surface elevation of bacterial colonies 

A, flat ; B, raised ; C, convex ; D, pulvinate ; E, capitate ; F, umbilicate ; 
G, umbonate 

aurieulate (Fig. 25, E'); lacerate, irregularly cleft 
(Fig. 25, -F) ; fimhricate, fringed (Fig. 25, (?) ; ciliate 
(Fig. 25, H^ ; erase, irregularly toothed (Fig. 25, 7). 
h. Internal structure : amorphous, no definite structure ; 
finely granular ; coarsely granular ; grumose, appears 








Fig. 25. Types of margin of bacterial colonies 

A, entire ; B, undulate ; C, repand ; D, lobate ; E, aurieulate ; F, lacerate ; 
G, fimbricate ; S, ciliate ; /, arose 

clotted (Fig. 26, B') ; gyrose, showing chinks or 

cracks (Fig. 26, E') ; reticulate, netted (Fig. 26, (?) ; 

filamentous ; floccose. 
/3. Deep colonies. 
a. Color. 
h. Shape : punctiform, lanceolate, oval, circular, spindle 

shaped, irregular, branched, filamentous, 
c. Translucency. 



THE CHAEACTEEISTICS OF BACTEEIA 



47 



C. Physiology. 

1. Relation to oxygen. 

2. Relation to light, desiccation, etc. 

3. Pigment production. 

4. Gas production. 

a. In shake culture. 

6. In fermentation tube : growth in open arm ; growth 

iu closed arm. 
c. Ratio of H : CO.. 




B 










F 


'Si- 


%\ 


^.■■<\ 


■ ■' "-.-O 


%-:. 




iim 


^m 



r^ 


G 




v." 


m 


^ 


^ 


.■Si-> 


^ 



Fig. 26. Types of internal structure of colonies 

A, areolate ; B, gmmose ; C, moruloid ; D, clouded ; JE, gyrose ; F, marmorated ; 

G, reticulate 



5. Acid or alkaU production. 

a. Litmus milk. 

6. Reduction of nitrates to nitrites, to anunonia, or to free 
nitrogen. 

7. Indol production. 

8. Habitat. 



48 A MANUAL OE BACTERIOLOGY 

SECTION X 

BACTERIA OF THE AIR 

In a strict sense there are perhaps no bacteria of the air, since 
in that medium there is small chance for bacterial development, 
yet there are species of bacteria commonly carried in air cur- 
rents. These species are common on dead organic matter, and 
are among those which cause contamination of culture media in 
the laboratory. The greater part of the bacteria in the atmos- 
phere probably are carried on particles of dust. Other things be- 
ing equal, therefore, the more dust in the air, the more bacteria. 

Exercise 66. Relation of Bacteria to Atmospheric Dust 

Melt nine tubes of agar and pour each into a sterile Petri 
dish. Replace the covers and allow the agar to harden. Label 
one dish A and place it in the incubator for a control. Give the 
other dishes consecutive labels and expose one in each of the 
following named locations by removing the cover for exactly ten 
minutes : , B, laboratory ; C, out of doors, at least 50 ft. from 
any building ; D, basement ; U, room before being swept ; F, room 
immediately after being swept with a dry broom ; G, room imme- 
diately after being swept with a damp broom ; H, barn ; I, barn 
after hay has been thrown down. Do not invert the covers while 
making the exposure. Several students may cooperate in this 
exercise. After the results are posted, each student will take 
notes on the entire series. It will be noted that the results 
obtained by this method are comparative, not absolute. 

Exercise 67. Determination of the Number of Bacteria in Air 

Fkankland, p. F. Phil. Trans. Roy. Soc. 178 B. : 113-152. 1887. 

Report of Committee on Standard Methods for the Examination of Air, Amer. 

Jour. Pub. Hyg. 20 : 346. 1910. 
Rettgeb, L. F. Jour. Exp. Med. 22: 461. 1910. 

The following method, while not as exact as some, is simpler 
and more likely to yield good results in the hands of students 
beginning the subject. 



BACTEEIA OF THE AIE 



49 



Two students working together should prepare the following 
simple apparatus: Place 100 cc. of physiological salt solution 
(6 g. XaCl in 1000 cc. water) in a 500-cc. Erlenmeyer flask. 
Close the flask with a tight-fitting rubber stopper through which 
two bent glass tubes are passed. One of the tubes ends about 
2 mm. from the bottom of the flask. The ends of the tubes are 
plugged with cotton and the flask is sterilized in the autoclave. 
Place 5 liters of water in a large bottle and mark the level of 




Fig. 27. Aspirator and flask for determination of bacteria in air 

the water surface. Adjust a siphon with a shut-off and a suction 
tube. After the Erlenmeyer flask and contents have cooled, the 
end of the short tube is connected with the aspirator by means 
of rubber tubing. The cotton plug is removed from the open end 
of the long tube and the aspirator started (Fig. 27). 

When 5 liters of water have run out, a similar volume of air 
has been drawn through the flask. The apparatus is then dis- 
connected and the contents of the flask well shaken. Two 1-ec. 
samples are drawn with a sterile pipette and plated — one in agar 
and one in gelatin. The count multiplied by 100 represents the 
number of organisms in 5 Hters of air. 



50 A MANUAL OF BACTERIOLOGY 

Exercise 68. Study of Micrococcus candicans 

Make cultures oa agar and gelatin. Study the microscopic 
and cultural features. 

Exercise 69. Study of Sarcina lutea 

Make cultures on gelatin, agar, and potato. Study the micro- 
scopic characters and compare with M. candicans. 

Exercise 70. Study of Bacillus fluorescens liquefaciens 

This organism is common in air and in water. It is one of 
those commonly present in decaying albuminous compounds. 

Under the microscope note the small rods. In liquids the rods 
are actively motile. Make cultures on gelatin, agar, and potato. 
Note the fluorescence developed. Is it imparted to the agar or 
is it confined to the colony of bacteria ? 

Exercise 71. Study of Bacillus prodigiosus 

This bacillus was formerly classed as a micrococcus. It is 
quite common and was one of the first to be noticed, because of 
the conspicuous red pigment it produces on starchy media. 

Under the microscope note the small rods. Make cultures on 
gelatin, agar, and potato. 

Exercise 72. Study of Bacillus subttlis (Hay Bacillus) 

This is one of the most widely distributed of bacteria. It not 
only occurs in air but also in water, soil, and all sorts of putre- 
fying liquids if oxygen be present. It is abundant in hay in- 
fusions. It grows rapidly and forms spores abundantly. Under 
the microscope study the individual rods and the chains. Make 
special spore stain, using bacteria from an old culture. One way 
of obtaining cultures of this organism is to cover finely cut hay 
with distilled water and boil for a quarter of an hour. Set 
aside with a loose cover for forty-eight hours. A thick scum 
will show itself on the surface, composed of £. suhtilis whose 
spores have survived the heat. Make cultures on gelatin, agar, 
and potato tubes ; also gelatin and agar plates. 



BACTERIA OF WATEE AND SEWAGE 51 

SECTION XI 
BACTERIA OF WATER AND SEWAGE 

Nearly aU bodies of natural waters contain varying numbers 
of bacteria. ^lany bacteria are introduced from the atmosphere 
or from organic substances comiug iuto the water, but the larger 
number are washed in from the soU. Those bacteria survive and 
multiply which are best suited to the physical environment of 
the water iuto which they are brought. There comes to be a 
characteristic group of organisms in water, which may be re- 
garded as " normal " bacteria. 

The bacteriology of waters is of importance from the stand- 
point of sanitary Hving, and has received much study. A chem- 
ical determination of the chlorides, nitrates, nitrites, albuminoid 
ammonia, and the hardness of the water is required for complete 
investigation of the water sample. 

HoKKOCKS. The Bacteriological Examination of Water. London, 1901. 
KiN-sicuTT, "WixsLOw, and Pratt. Sewage Disposal. Xew York, 1910. 
Pkescott and Wisslow. Elements of Water Bacteriology. New York, 1908. 
Masox. Examination of Water. Xew York, 1909. 

Exercise 73. Collecting Samples of Water 

1. Tie pieces of filter paper or tin foil over five glass-stop- 
pered bottles of 150-2-50 cc. capacity. The bottles, which should 
be perfectly clean, are sterilized in the dry sterilizer at 140° C. 
for two hours. 

2. At the time of taking samples from surface waters, re- 
move the paper cap, then withdraw the stopper, touching only 
the top of it with the fingers. Hold the bottle by the base in 
the other hand and plunge it mouth downward into the water. 
When about 12 in. beneath the surface, turn the bottle mouth 
upwards and allow it to fill. Whenever any current exists, the 
mouth of the bottle should be directed against it, in order to 
carry away any bacteria from the fingers. If there is no current, 
a similar effect can be produced by turning the bottle under 
water and giving it a quick forward motion. Remove the bottle 



52 A MANUAL OF BACTEEIOLOGY 

from the water and replace the stopper, wipe the exterior of the 
bottle dry, and replace the paper cap. Instead of this bottle an 
Esmarch or other type of sampler may be employed. 

3. Samples for bacteriological examination should be used as 
soon as possible. If they are to be kept more than an hour, they 
should be packed in ice. 

4. Samples from pumps should be collected after fifteen min- 
utes of continuous pumping. Samples from a faucet should be 
taken only after the water has run freely for five to ten minutes. 

Exercise 74. Quantitative Study of the Bacteria in Surface Waters 

1. Obtain samples of water from three different sources, fol- 
lowing directions given in Exercise 73. 

2. After shaking the sample at least 25 times, remove 1 cc. of 
water with a sterile pipette and place it in the bottom of a 
sterilized Petri dish. If the sample is suspected of being highly 
infected, the water should be diluted 1 : 10 or 1 : 100, using ster- 
ile water blanks for the purpose. Plates ought not to contain 
over 200 colonies. 

3. Pour into each dish a tube of melted gelatin (not warmer 
than 43° C). Tilt the plate gently to mix the water and gelatin. 
Place on a level until the gelatin has solidified, and incubate at 
a temperature of 22° C. or lower. 

4. In the same way make plates, using beef-peptone agar and 
Heyden-Nahrstoff agar. These plates may be incubated at tem- 
peratures up to 37° C. 

5. Count the colonies on plates incubated at 22° after seventy- 
two hours, and on plates incubated at 37° after forty-eight hours, 
using a counting plate. If possible to do so, all the colonies on 
the plate should be counted. If there are more than 400, it is 
easier and fully as accurate to count a fractional part of the 
plate and estimate from it the total number. It is customary 
in practice to make plates in duplicate or triplicate, thus afford- 
ing a check for one's own work. In the best of hands the limit 
of experimental error is large. With very careful work a varia- 
tion of 10 per cent may be obtained, but a smaller variation is 
not to be expected. 



BACTEEIA OF WATEE AXD SEWAGE 



53 



Exercise 75. Qualitative Study of Bacteria in Surface Waters 

1-2. Same as in the preceding exercise. 

3. Pour plates, using litmus-lactose agar, and incubate them 
at 87° C. for tlnee dajs. 

4. ^lake differential counts of acid-forming colonies (recognized 
by the reddening of the litmus) and the non-acid-f ormiag colonies. 




Fig. 28. Fermentation tube and gasometric chart for reading tlie quantity 

of gas evolved 

The student may copy the chart on cardboard for use as indicated 

The small, compact red colonies usually represent streptococci ; 
verify by making a simple stain. Estimate the proportion of 
streptococci to the total number of colonies. 

5. Examine gelatin plates made in Exercise 74, to determine 
the number of liquefying and nonliquefying colonies. The latter 
are assumed to come from decaying organic matter. 



54 A MANUAL OF BACTERIOLOGY 

Exercise 76. Presumptive Test for Bacillus coli 

The presumptive test is designed to give a fairly accurate 
idea of the existence of pollution without the time-consuming 
operation of isolating B. coli. Several such tests are in use. 
Under diverse conditions diverse results are obtained. 

1. Lactose-bile bouillon. Add 1 per cent of lactose to fresh 
ox bile, place in fermentation tubes, and sterilize in the Arnold 
sterilizer. Inoculate with samples of suspected water or sewage 
and incubate at 37.5° C. for forty-eight to seventy-two hours. 
Tubes showiag 25 per cent or more of gas are regarded as positive. 

2. Dextrose-beef bouillon. Inoculate fermentation tubes of 
sterile dextrose-beef broth with 1 cc. samples of the water to 
be tested. Incubate at 37.5° C. Test for gas after forty-eight 
to seventy-two hours. If the gas is approximately one third 
carbon dioxide and two thirds hydrogen, the test is regarded as 
positive. 

3. Litmus-lactose agar. Although less delicate than the fer- 
mentation tests, this method is of considerable value. With 
suitable dilutions pour Petri dishes, using this agar. Count the 
colonies which redden the agar. A large number of acid-forming 
colonies is regarded with suspicion. 

Exercise 77. Tests for Fecal Bacteria {Bacillus coli and Others) 

The tests here given are those commonly used. None of them 
meets the approval of all bacteriologists. They are to be re- 
garded as presumptive rather than as absolute. 

A. Lactose-bile bouillon. 

1. Inoculate six or more fermentation tubes of sterile lac- 
tose bile with at least 1 cc. of the sample to be investi- 
gated. Incubate at 37° C. 

2. Measure the amount of gas present in the closed arm 
after twenty -four and after forty-eight hours. 

3. Make stains from each tube and use Gram's stain to 
determine the presence of streptococci. 

B. Lactose-beef bouillon. 

Repeat A, using lactose-beef bouillon. 



BACTEEIA OF WATER AXD SEWAGE 55 

C. Neutral Red bouillon. 

Add 1 cc. of the water under examination to tubes contain- 
ing Xeutral Red bouillon. Fecal bacteria transform the 
red color to a canary j'ellow accompanied by green fluo- 
rescence. Make stains for B. coli. 

D. Phenol bouillon. 

Repeat C, using phenol bouillon. 

Exercise 78. Quantitative Examination of Sewage 

1. Procure samples as du-ected for water in Exercise 73. 

2. In making plates and fermentation tube tests use dilutions 
of 1 : 1000, 1 : 10,000, 1 : 100,000, or even greater. 

3. ^lake plates of beef gelatin, beef agar, Heyden-XahrstofE 
agar, and litmus-lactose agar. 

4. Incubate part of the agar plates at 37° C. and others below 
22° C. Count the former after forty-eight hours and the latter 
after sevent^^-two hours. Compare the results. 

Exercise 79. Bacterial Content of Snow and of Rain Water 

If opportunity presents itself, make bacterial counts of snow. 
Collect samples in sterilized glass-stoppered bottles, as in case of 
water. Bring the bottles to the laboratory, allow the snow to 
melt, draw samples with sterile pipettes before all of the snow 
has melted. Collect samples of freshly fallen snow and after 
intervals of twenty -four, forty-eight, and seventy-two hours. 

Collect samples of rain water by uncovering sterile glass jars 
while rain is falling. ]\Iake plate cultures within one hour from 
the time the jars are set out. 

Compare the number of organisms per cubic centimeter with 
that obtained in surface waters. 

Exercise 80. Study of Streptococci 

The term " Sewage Streptococci " is applied to a poorly defined 
group including cocci which may or may not form well-defined 
chains. Under the microscope they appear to occur in pairs, 
short chains, or irregular groups. They grow well on sugar 



56 A MANUAL OF BACTEEIOLOGY 

media, and ferment dextrose and lactose, forming acid but no 
gas. On the litmus-lactose-agar plate, made from a polluted 
water, the streptococcus colonies may be distinguished from 
other acid-forming colonies by their small size, compact structure, 
and permanent deep red color. The organisms grow feebly on 
the surface of beef agar, but flourish better under partial anaerobic 
conditions, such as those prevailing in the lower part of the stab 
canal. 

While it seems entirely reasonable to look upon the strep- 
tococci as evidence of pollution, still there is no such well- 
established connection with sewage as in the case of the colon 
bacteria. 

Houston regards streptococci as indicative of recent and objec- 
tionable pollution. Horrocks, however, found that B. coli gradu- 
ally disappeared from jars of sewage kept in the dark for three 
months, and that those forms which survived were mainly varie- 
ties of streptococci and staphylococci. He believes that strepto- 
cocci indicate pollution with old sewage which is not necessarily 
dangerous. 

1. Transfer from colonies of streptococci to tubes of ster- 
ile media. Cultivate on beef gelatin, beef agar, and dextrose 
bouillon. 

2. Make stained preparations from bouillon cultures. 

Exercise 81. Isolation of Bacillus coli 

Much remains to be desired in the way of methods for iso- 
lating the colon bacillus. With the most careful work it is 
not possible to isolate this germ from all samples of polluted 
waters. 

1. Prepare an " enriching culture " by inoculating fermenta- 
tion tubes of sterile dextrose bouillon each with 1 cc. of the 
sample under investigation. Mix by tilting the tubes. Incubate 
at 37.5° C. for ten to twenty-four hours. Watch the tubes and, 
upon the first appearance of gas, plate out the organisms on 
litmus-lactose agar. 

2. Watch the plates for acid-forming colonies. Transfer from 
them to agar-slope cultures. If the culture resembles B. coli. 



BACTEEIA OF WATEE AXI> SEWAGE 57 

numerous subcultures are made from it. The foUowing gives 
those wliich will be of diagnostic value : 

3. Gelatin. B. coli gro'os well without liquefying the medium. 

4. Milk. In milk it produces acid and forms a curd in twelve 
to twenty -four hours at 37.5° C. 

5. Nitrate solution. When grown on GUtay and Aberson's 
or other nitrate solution it reduces nitrates to nitrites. The 
solutions are kept twelve to twentj^-four hours at 37.5° C. 

6. Sugar bouillon. In sugar bouillon gas is evolved. Analysis 
shows the ratio H : C0„ : : 2 : 1. Incubate twelve to twenty-four 
hours at 37.5° C. 

7. Dunham's solution. Indol is produced when B. coli grows 
on Dunham's solution (Exercise 37). Incubate tubes three or 
four days at 37.5° C. ; then test for indol. 

Exercise 82. Study of Characters of Bacillus coli 

If the preceding exercise has been omitted, make studies of 
pure cultures of B. coli on the various media there described. 
^Make stained preparations with carbol-fuchsin and determine 
its reaction to Gram's stain. 

Exercise 83. Study of Bacillus proteus vulgaris 

Members of the so-called proteus group of bacteria are com- 
monly found in putrefying substances. Their decomposing action 
is marked by a very foul odor and an alkaline reaction. Their 
presence in water does not always indicate sewage pollution, but 
pollution is very probable if they occur in conjunction with the 
colon bacillus. 

^lake cultures on agar slope, gelatin stab, milk, and potato. 
Study the " swarming islands " with a magnification of 60 x in 
colonies in gelatin plates. ]\Iake stained preparations with carbol- 
fuchsin and determine the relation of this organism to Gram's 
stain. 

Study the fermenting activity in shake cultures of dextrose 
agar and in fermentation tubes of dextrose bouUlon. 

Examine cultures on Dunham's solution for indol. 



58 A MANUAL OF BACTERIOLOGY 

Exercise 84. Study of Aerobic Organisms which Decompose Cellulose 

A study of these organisms, even in mixed cultures, is in- 
structive in demonstrating how cellulose substances are liquefied 
in sewage, manure, soil, and other media. 

1. Prepare the following medium and fill Erlenmeyer flasks 
to a depth of 0.5-1 cm. 

Filter paper (in strips) 2.00 g. 

NH^Cl ... 0.10 g. 

K.,HP04 . 0.05 g. 

CaCOj (chalk) 2.00 g. 

Water 100.00 oc. 

2. Inoculate the flasks with fresh horse manure or with slimy 
mud from the bottom of a pond or river. Incubate the flasks 
for three or four weeks at a temperature of 30°-34° C. 

3. Transfers should be made with a platinum loop to other 
flasks of sterile solution after a month. In this way the growth 
of the cellulose-decomposing organisms is increased. It may re- 
quire two or three transfers to get really active decomposition. 

4. The cellulose first becomes brown in color, then perforated 
with holes. A brown color is usually imparted to the solution. 

5. Make cover-glass stains of bacteria taken from a flask in 
which cellulose decomposition is progressing. 

Exercise 85. Study of Anaerobic Organisms which Decompose Cellulose 

1. Proceed to fill Erlenmeyer flasks to the stopper (rubber) 
with the following solution : 

Filter paper . 20.00 g. 

CaCOg (chalk) 2.00 g. 

K2HPO, 0.10 g. 

MgSG^ 0.50 g. 

(NH,)2S0, (or PC,) 1.00 g. 

NaCl trace 

Water 1000.00 cc. 

2. Inoculate with fresh horse manure or slimy mud. If horse 
manure is to be used, the filter paper may be omitted. 

3. Incubate at 30°-34° C. for a month and make transfers as 
in Exercise 84. 



BACTERIA OF THE SOIL 59 

Exercise 86. Cellulose Decomposition with Formation of Hydrogen 

In the foregoing experiments methane (CH^) is the principal 
gas formed from the decomposition of cellulose. If the culture 
made by one of the first transfers be heated after inoculation to 
75° C. for fifteen minutes and then cooled, the production of 
methane will be replaced by that of hydrogen. The organisms 
causing the hydrogenic fermentation of cellulose appear to be 
spore-formers which can withstand the temperature of 75° C. 



SECTION XII 
BACTERIA OF THE SOIL 

Of all the varied activities of bacteria in nature, none com- 
pares in importance with the work of the soil bacteria. They 
not only determine the fertility of the soil, but they serve as the 
connecting link between the world of the Uviug and the world 
of the dead. They are the great scavenging agents which tear 
down the dead bodies of animals and plants and restore the car- 
bon, nitrogen, sulphur, and other elements of the tissue to the 
round of nature. The processes of nature are such that the 
same material is repeatedly used, passing in endless cycle from 
plant to plant or from plant to animal, and back again to plant, 
but always with the intervention of bacteria. Without their 
action dead bodies would accumulate and cover the surface of 
the earth ; the kingdom of the living would be replaced by the 
kingdom of the dead; and the world's supply of carbon and 
nitrogen would be locked up in a form useless to most forms 
of life. 

The soil may be regarded as the greatest field of bacterial 
activity we know. The surface layers of the soil are usually 
inhabited by many thousands of germs per gram. The soil fur- 
nishes many of the germs found in lakes and streams, most 
of those found in air, and some of those concerned in animal 
diseases. Bacteria appear to be most numerous in the surface 
layers of the soil and to diminish at increasing depths. Below 



60 A MANUAL OF BACTEEIOLOGY 

a depth of four to six feet bacteria are rarely found except in. 
places where local drainage currents carry them downward. 
Various factors, such as temperature and moisture, influence the 
number and activities of the soil organisms, but it is probable 
that they are active during the greater part of the year. 

Again, the bacteria of the soil are of various species, and no 
one species has uncontended sway in the field. Those which are 
best suited to the environment thrive ; those which are not so 
well suited are crowded out and diminish in numbers or die. 

In other words, the soil bacteria, like other organisms, have a 
definite problem of existence which must be solved in the main 
under the conditions which they find in arable soils. At times 
one species of bacteria predominates in numbers and activity; 
at other times, under changed conditions, other species predom- 
inate. When the bacteria which set up certain activities pre- 
dominate, the productiveness of the soil is enhanced ; when other 
classes predominate, the reverse is true. 

An enumeration of the number of bacteria in soil does not 
mean much in itself. However, if soil conditions are sufficiently 
well controlled, a quantitative determination may shed light 
on the effects of various factors upon microorganic life in soil. 
Only comparative results can be obtained, even under the best 
conditions, since many soil organisms do not grow upon the 
media available for counting bacterial colonies. 

Exercise 87. Obtaining Soil Samples 

1. Sterilize in the dry oven several trowels or steel spatulas, 
previously wrapped in Manila paper. Also sterilize three wide- 
mouthed, glass-stoppered bottles. 

2. In the field scrape off the top layer of soil ; then, using a 
fresh sterile spatula, loosen about 10 cm. of the soil, stir it thor- 
oughly, and transfer 25-50 g. to a sterile bottle. The surface soil 
varies widely in bacterial content, owing to seasonal extremes 
of moisture, the mhibiting action of light, accidental contamina- 
tion, and other causes. 

3. Carry the samples to the laboratory within an hour after 
collecting them. 



BACTEKIA OF THE SOIL 61 

Exercise 88. Preparing Dilutions and Pouring Plates 

1. Weigh 1-g. samples of soil iato 99-ce. water blanks. Stir 
with a sterile glass rod until all lumps are broken up. 

2. Draw 1 cc. of the muddy water with a sterile pipette and 
transfer to another 99-cc. water blank. These water blanks 
should be contained in narrow-necked flasks or bottles of 200 cc. 
capacity, to permit of agitation without loss of their contents. 
Agitate for two minutes ; then transfer 1 cc. with a sterile pipette 
to a sterile Petri dish and add a tube of melted agar which has 
cooled to 4-3° C. ^lis the contents by tilting the dish. When 
cool, incubate at a temperature between 18° and 23° C 

For making these plates agar media are preferable to gelatin, 
on account of the higher nitrogen content of the latter. Beef- 
peptone agar may be used, although the Heyden-Nahrstoff agar 
will give higher counts. Lipman '■ reports good results from the 
use of a synthetic agar. 

3. Count the colonies as they develop and estimate the num- 
ber of bacteria per gram. 

4. jMany of the bacteria of the soil are anaerobic and can 
only be grown in the absence of oxygen. For method see 
Exercise 56. 

Exercise 89. Determination of the Number of Spores in Soil 

1. ]Make a soil suspension in a dilution of 1 : 1000. Add 1 cc. 
to a tube of sterile melted gelatin. 

2. Heat the tube in a water bath to a temperature of 80° C. 
for ten minutes. 

3. Pour the melted gelatin into a sterile Petri dish. Incubate 
at room temperature and count the colonies after four days. 
Estimate the number of spores in 1 g. of the soil sample. 

1 Lipman and Bro-mi. Centralbl. f . Bakt., 2te Abt., 25 : 4i7. 1910. 

Water ... 1000.00 cc. 

Dextrose ... . . . 10.00 g. 

K„HP04 ... . . 0.50 g. 

MgS04 . . . . 0.20g. 

Peptone . . . 0.05 g. 

Agar-agar .... 20.00 g. 



62 A MANUAL OF BACTERIOLOGY 

SOIL BACTERIA IN RELATION TO NITROGEN COMPOUNDS 

The relations of soil bacteria to compounds of nitrogen have 
been more extensively studied than their relations to other soil 
constituents, although there is reason to beheve that their rela- 
tions to sulphates and phosphates are also of great importance. 
In many soils, however, the amount and form of nitrogen present 
seems to be the chief chemical factor governing plant production. 
Bacteria have been found to play an important part in the trans- 
formations of soil nitrogen, to say nothing of the ability of some 
races of bacteria to add considerable stores of nitrogen to the soil. 

Although there are many unsettled problems, and our knowl- 
edge of these bacteria is far from complete, there is reason to 
believe that we may control to some extent the addition of 
nitrogen to cultivated soils, and its subsequent transformations. 

For a more complete discussion of these and alhed questions 
the student is referred to 

Conn. Agricultural Bacteriology, Second Edition. Philadelphia, 1909. 

LiPMAN. Bacteria in Relation to Country Life. New York, 1908. 

VooRHEES and Lipman. Review of Investigations in Soil Bacteriology, Bulletin 

No. 194. Office of Experiment Stations, U.S. Department of Agriculture, 

1907. 
Marshall. Microbiology. Philadelphia, 1911. 

Lafak. Handbuch der technischen Mykologie, Bd. III. Jena, 1905. 
LoHNis. Handbuch d. landw. Bakteriologie. Berlin, 1911. 
Numerous papers in journals and agricultural literature. 

In many of the experiments which follow, analyses are to be 
made which involve more or less chemical work. The chemical 
methods given in appendixes F, G, H, and I are the simplest 
which will give anything approximating accurate results in the 
hands of undergraduate students. They are mainly qualitative. 
The research worker who has more apparatus and who desires 
more accurate quantitative results, is referred to works on stand- 
ard analysis of waters and soils. 

Exercise 90. Ammonification of Peptone by Soil Bacteria 

1. Prepare 400 cc. of Dunham's solution (see Exercise 37). 
Place 200 cc. in each of two flasks of 500 cc. capacity. 

2. Sterilize the flasks in the autoclave. 



BACTERIA OF THE SOIL 



63 



3. Keep one flask for a control and inoculate the other with 
a few grams of fresh garden soil. Incubate the flasks at 30°-37° C. 
for a week. 

4. Moisten strips of clean filter paper with Nessler's reagent 
and pour upon them a few drops from each of the flasks. A 
reddish-yellow color indicates the presence of ammonia. Another 
method is to remove a portion, say 50 cc, 
make alkaline with NaOH, and boil it in a 
small flask, holding a strip of Nessler paper 
at the mouth of the flask. 

Exercise 91. Ammonification of Nitrogenous 
Substances in Soil 

1. Place 100 g. samples of freshly gathered 
soil in each of four tumblers or beakers. Add 
5 g. of cottonseed meal or dried blood to two 
tumblers, leaving the others for controls. Stir 
with a sterile spatula. 

2. Add water to give optimum moisture 
content as nearly as possible. Cover the tum- 
blers loosely with a glass plate. Incubate at 
room temperature for one week. 

3. At the end of the incubation period 
transfer the soil to a flask, preferably a copper 
flask. Add a little paraffin and some magne- 
sium oxide and distill off the ammonia. 

4. For a quaUtative test a piece of filter 
paper moistened with Nessler reagent may be 
held at the mouth of the flask as in Exercise 90. 
For quantitative work a condenser is attached 
and the distillate is collected in standard acid 
and titrated against standard alkali. 

5. Compare the amount of ammonia liberated in the control 
and in the experimental tumblers. What part do bacteria play 
in this process ? 




Fig. 29. Burettes ar- 
ranged for titration 



64 A MANUAL OF BACTERIOLOGY 

Exercise 92. Nitrite Formation in Solution 

1. Prepare Winogradsky and Omelianski's solution for nitrite- 
forming organisms (see Exercise 51). 

2. Divide the solution equally among five Erlenmeyer flasks 
of 500 cc. capacity. Number the flasks consecutively. 

3. Inoculate flask No. 1 with 1-2 g. of fresh soil collected 
about 10 cm. beneath the surface. Incubate the flasks at 25°- 
30° C. 

4. As soon as growth is evident (usually in four or five days), 
inoculate flask No. 2 from flask No. 1, using the platinum loop. 
Continue the serial inoculation from flask to flask. 

5. Examine the bacteria in a drop of Gram's iodine solution 
and with simple stains. Test the solutions for nitrites with the 
Griess-Ilosvay reagent, or with Trommsdorf reagent. 

Exercise 93. Nitrate Formation in Solution 

1. Prepare 1 liter of Winogradsky and Omelianski's solution 
(see Exercise 52). Divide the solution equally among five Erlen- 
meyer flasks of 500 cc. capacity. Sterilization is of no advan- 
tage in this case, smce none excejit nitrifying bacteria can develop 
in this solution. 

2. Inoculate one flask with 1 g. of a soil sample collected 
about 10 cm. from the surface. 

3. Watch the flask for evidence of bacterial growth. At the 
first indications of growth inoculate the second flask with three 
loopfuls of solution taken from the first. Carry through the 
inoculation in this way from flask to flask, leaving one flask 
uninoculated, to serve as a control. This method of serial trans- 
fers gradually elimiaates all bacteria except those fitted to de- 
velop in this solution, that is, principally nitrifying bacteria. 
At the end one obtains a nearly pure culture of these organisms. 

4. Make cover-slip stains from the flasks and examine the 
organisms microscopically. Stain with warm carbol-fuchsin and 
wash with acidulated alcohol, or stain with Lofiler's alkaline 
methylene blue. At the expiration of ten days test the solutions 
for nitrates with phenol-sulphonic acid. 



BACTERIA OF THE SOIL 65 

Exercise 94. Nitrification in SoU 

For some reasons it is more satisfactory to study the process 
of nitrification in soils than in solutions. Where comparisons 
are desired between the capacity of different soils for nitrifica- 
tion, onlj' soils can be used. ^Moreover, Stevens and Withers 
(Centralbl. f. Bakt., 2te Abt., 23: 355. 1909) have shown that 
soils which show capacity for nitrification often give no result 
when their suspensions are used as inocula hi solutions. The capac- 
ity of a soil for nitrification is not, however, a simple proposition 
depending only upon the number and vigor of the necessary bac- 
teria; it is profoundly influenced by soil conditions — for ex- 
ample, quantity and quality of organic matter, reaction of soil, 
moisture content, porosity, etc. — as well as by other factors 
less readily recognized. 

1. With a sterile trowel procure soil samples about 10 cm. 
below the surface. 

2. Put 100 g. of the freshly collected soils into glass tumblers. 
Reserve one or more tumblers of soil for controls. Add to the 
others 5 ce. of a 2 per cent solution of sterile ammonium sul- 
phate (equivalent to .100 g. (NH^)2S0^). Add 5 cc. of sterile 
distilled water to the control tumblers. Weigh each tumbler and 
record the weight ; cover the tumblers with glass plates. Once 
each week weigh the tumblers and add sterile distilled water to 
bring the weights back to their original value. Incubate the 
tumblers at 80°-35° C. for four to six weeks. 

3. At the end of the experiment transfer the soil from the 
tumblers to agate-ware pitchers or to large, wide-mouthed glass 
bottles. Use 1000 cc. of distilled water for the transfer of each 
sample. Give thorough agitation for five minutes ; then allow 
the soil to settle for thirty minutes. Filter off the supernatant 
extract. Filtration may be accomphshed with a Pasteur-Cham- 
berland bougie, using the apparatus described in Bulletin Ko. 31, 
Bureau of SoUs, U.S. Department of Agriculture (see Appendix), 
or, since only a portion is required for analysis, the extract may 
be decanted through a folded filter paper. The filtration through 
paper may be hastened by adding 5-10 g. of alum (potassium 



66 A MANUAL OF BACTERIOLOGY 

aluminum sulphate) per liter to the soil suspension before filter- 
ing, as directed above. 

4. Draw samples of 50 cc. or larger, as soon as possible, to 
avoid the danger of denitrification. Determiae nitrate qualita- 
tively, or quantitatively by the phenol-sulphonic colorimetric 
method. Each 10 ec. of the extract represents 1 g. of the fresh 
soil sample. By determining the water content of the soil 
sample at the outset of the experiment, it will be possible to 
reduce the nitrate determinations to a moisture-free basis. 

5. Compute the amount of nitrate formed iu the samples to 
which ammonium sulphate was added. If soils from a variety of 
sources under a variety of conditions were included in the plan 
of the experiment, draw conclusions as to the effect of these 
conditions upon nitrification. 

Note. The isolation of nitrite and nitrate organisms from soils involves the 
preparation of silica-jelly media. The work is rather diflBcult for the begin- 
ning student. Directions for procedure will be found in the following works 
and others. 

Heinemann. Laboratory Guide in Bacteriology. 
Lapae. Handbuch der teohnischen Mykologie 3 : 155. 
Smith. Bacteria in Relation to Plant Diseases 1 . 36. 

Exercise 95. Reduction of Nitrates by Bacteria 

1. Prepare Giltay and Aberson's solution and fill several fer- 
mentation tubes and test tubes. Sterilize. 

2. Inoculate some of the tubes with a small quantity of fresh 
horse manure. Inoculate others with B. denitrificans, B. Hartlehii, 
B. pyocyaneiis, or B. fluorescens liquefaciens. Keep the tubes in 
the incubator for ten to fifteen days. 

3. Examine the fermentation tubes for free gases. Examine 
the contents of the test tubes for nitrites and ammonia compounds. 

Exercise 96. The Reducing Action of Denitrifying Bacteria upon Nitrates 
and Methylene Blue 

Fred. Centralbl. f . Bakt., 2te Abt., 32 : 421. 1912. 

Since denitrification is essentially a reducing process in which 
oxygen is removed from its combination with nitrogen, the 
action may well be demonstrated by adding a reducible dyestuff 



BACTERIA OF THE SOIL 67 

to the solutions. ^Methylene blue serves weU because it is non- 
poisonous to bacteria and is easily reduced to the colorless leuco 
compound. 

1. Add 20 ec. of standard methylene blue solution (1 : 1000) 
to each 100 ce. of Giltay and Aberson's solution. Fill several 
test tubes and fermentation tubes. Sterilize. 

2. Inoculate the tubes with B. denitrificans, B. Hartlehii, B. 
pyocyaneus, and B. fluorescens Uquefaeiens. In order to exclude 
oxvgen pour in paraffin oil to a depth of about 2 cm. in the 
test tubes. Keep the tubes in the incubator for three to seven 
days, or until the solutions become colorless. So long as nitrates 
are present the bacteria do not attack the methj-lene blue to any 
great extent. 

3. When a tube has lost its color, test for nitrates ; shake the 
solution or pass air through it. What happens ? Explain. 

Exercise 97. Nonsymbiotic Bacteria which Fix Atmospheric Nitrogen. 
Isolation and Study of Azotobacter 

1. Prepare Ashby's solution (see Exercise 49). 

2. Clean three small flasks or salt-mouthed bottles. Put into 
each 25 cc. of Ashby's solution and sterilize in the Arnold sterilizer. 

3. Inoculate each ■with 1 g. of soil. It will be well to use soil 
samples collected from different places, for Azotobacter is not 
necessarily most abundant in the most productive soils. Incubate 
the flasks at 30° C. for three days ; at the end of that time pick 
out flasks which have a greasy film on the surface of the liquid. 

4. Transfer smaU flecks of this surface fihn to a sHde. Ex- 
amine with the microscope. Add a drop of Gram's iodine solution 
to the preparation. Azotobacter cells are stained golden yellow. 

5. Make a cover-glass preparation and stain with aqueous 
methylene blue. 

Exercise 98. Growth of Azotobacter in Pure Cultures 
Hoffman and Hammek. Centralbl. f. Bakt., 2te Abt., 24 : 181. 1909. 

1. Prepare nutrient agar by adding 2 per cent of agar to 
Ashby's solution. Sterilize in the Arnold sterilizer. 



68 A MANUAL OF BACTERIOLOGY 

2. Plate out Azotobacter from the flasks prepared in Exer- 
cise 97. Examine and transfer from colonies to tubes of 
Ashby's agar. 

3. The most successful method of cultivating Azotobacter is 
upon large surfaces. Put 50 cc. of melted agar into an Erlen- 
meyer flask of 1 liter capacity. After sterilization allow the. 
agar to harden, or pour sterile Ashby's agar into Petri dishes 
with a diameter of 20 cm. or more. Inoculate with a suspension 
of Azotobacter in sterile distilled water. Tilt the culture once 
or twice daily. The result is usually a thick film of growth 
upon the agar. In this way enough material may be collected 
for analytical work, but it should be removed within a week 
from the time of moculation. 

Exercise 99. Symbiotic Bacteria which Fix Atmospheric Nitrogen. 
Bacillus radicicola 

Beijerinck. Botanische Zeitung 46 : 723. 1888. 

Hellriegel. Zeitschr. Riibenzuokerind. deuts. Reichs. 1886. 

Koch. In Lafar's Technische Mykologie, Bd. III. Jena, 1905. 

LoHNis. Handbuch landw. Bakteriologie. Berlin, 1911. 

Marshall. Microbiology. Philadelphia, 1911. 

Peirce. Proo. Calif. Acad. Sci. (3d ser.) 2 : 295-328. 1902. 

Smith. Bacteria in Relation to Plant Diseases, Vol. II. Washington, 1911. 

The student in regular class work can get no more than an 
introduction to the bacterial side of this problem, because most 
experiments in this line require more time than is usually avail- 
able in laboratory classes. Yet it is possible to gain an intelli- 
gent idea of the subject by making a few simple experiments. 
A casual examination of the literature shows that upon some 
important questions uncertainty still prevails. For more ex- 
tended work the student is directed to the researches cited 
above and to others. 

1. Secure the roots of a leguminous plant which shows numer- 
ous tubercles. Sketch a root, showing the shape and arrangement 
of tubercles. Do they seem to have any regular arrangement ? 

2. Secure young roots of peas or vetch plants. Holding a 
root with a young tubercle in pith, cut razor sections embracing 
the tubercle and root tissue at right angles to the long axis of the 



BACTEEIA OF THE SOIL 



69 



root. Stain the sections in 1 per cent acetic acid in which are 
dissolved equal portions of fuchsin and methyl violet. Wash the 
sections briefly with water and make microscopic examination. 




Fis. 30. Increased growth of beans due to inoculation with B. radicicola 

Plant on the left grown in uninoculated soil ; plant on the right grown in inoculated 
soil. (Alter Ferguson) 

Look especially for the " infection threads " extending through 
the cortical cells of the root. The protoplasm of the root cells 
and tubercle cells should appear blue, and the bacteria red. 



70 A MANUAL OP BACTERIOLOGY 

3. Crush a well-washed tubercle between two glass slides. 
Transfer a drop of water from the crushed tubercle to a cover glass 
and stain in the usual manner. Examine the preparation for the 
rod-shaped B. radicicola and for branched rods — bacteroids. 

Exercise 100. Isolation and Culture of Bacillus radicicola 

BccHANAN, R. E. Centralbl. f. Bakt., 2te Abt., 23 : 59. 1909. 
Prazmowski. Landw. Vers.-Stat. 37 ■ 199. 1890. 
Harrison and Barlow. Centralbl. f. Bakt., 2te Abt., 19 : 426. 1907. 
Also works cited under Exercise 99. 

1. Obtain several young tubercles from the roots of clover or 
other legume. Wash thoroughly in tap water. 

2. Immerse the tubercles for several minutes in 1 : 1000 solu- 
tion of mercuric chloride. Transfer with sterile forceps through 
several changes of sterile distilled water. 

3. Crush the tubercles with a sterile spatula in a sterile Petri 
dish and make plates from the contents of the nodule. Use the 
synthetic agar described in Exercise 88. 

4. Watch the plates for small, white, moist colonies. Transfer 
from them to ordinary laboratory media (see Exercise 108). 

5. Pour plates also from commercial cultures of B. radicicola. 

Exercise 101. The Production of Bacteroids of Bacillus radicicola upon 
Artificial Media 

Until recently the branched rod, or bacteroid, of B. radicicola 
has been found only in the nodule of the legume. Zipfel has 
shown, however, that these forms may be produced in cultures 
in the laboratory (Centralbl. f. Bakt., 2te Abt., 32: 97. 1911) 
by the following method: 

1. Extract 100 g. of bean or pea meal with 100 cc. of normal 
KOH and 5 liters of water for twenty-four hours. Siphon off 
the clear liquid and neutralize with phosphoric acid. Make the 
volume up to 5 liters. Prepare a medium by taking 

Legume-seed extract 1000 cc. 

Agar 30 g. 

Dextrose 20 g. 

Normal malic acid 10 cc. 

Caffein 2 g. 



BACTERIA OF THE SOIL 71 

2. Fill tubes, plug, and sterilize in the Arnold sterilizer. 

3. Inoculate with different strains of B. radidcola. At first 
growth will be slow, but between the fourth and tenth days 
many bacteroids may be found. 

Exercise 102. The Formation of Tubercles upon Roots of Legumes 

If time permits, this experiment may be performed by the class, 
different legumes being assigned to different students. 

1. Obtain new flowerpots, or sterilize old pots at 150° C. for 
an hour. Fill the flowerpots with sand which has been baked at 
a temperature of 150° C. for one to two hours. 

2. Plant seeds of various legumes in the sand. Inoculate 
half the pots by adding a suspension of B. radidcola obtained 
either from a pure culture or from soil in which the legume 
has previously been grown. Water the sand as needed with the 
followiug solution : 



Calcium chloride 


1.0 g. 


Magnesium sulphate 


0.5 g. 


Potassium biphosphate .... 


0.5 g. 


Ferric chloride . 


. . . . trace 


Distilled water 


1 liter 



3. Watch the plants for differences in growth. When the 
differences are marked, pull out the plants and examine the roots 
of inoculated and uninoculated plants for tubercles. 

Exercise 103. The Reduction of Sulphates by Bacteria 

When grown under anaerobic conditions, certain bacteria are 
able to reduce sulphates. In water-logged swamps ferrous sul- 
phate may be rediiced and give rise to deposits of bog iron. 

1. Prepare Van Delden's solution for the cultivation of 
sulphate-reducing bacteria (see Exercise 46). 

2. Fill six small flasks nearly full of the solution. Plug and 
sterilize the flasks iu the autoclave. 

3. Set aside two flasks for controls ; inoculate two with a few 
grams of garden soil and two with a few cubic centimeters of 
sewage or ditch water. 

4. Incubate for one to two weeks at room temperature. 



72 A MANUAL OF BACTERIOLOGY 

5. Partial reduction is indicated by the formation of a dark 
color due to the formation of ferrous sulphide. Pour a portion 
of a culture into a test tube and test for sulphates with barium 
chloride. Test the controls for the relative amount of sulphates. 

Exercise 104. The Cultivation of Bacillus amylobacter 

The group of organisrhs represented by B. amylobacter lives 
upon starchy material and effects its decomposition. 

1. Cut slices of beets and place them in wide-mouthed bottles 
or flasks of 50 cc. capacity. Pour in tap water until the flasks 
are full and close them with rubber stoppers. If beets are not 
obtainable, a substitute may be had in thick starch prepared as 
follows: Boil 100 cc. of distilled water until air is expelled; re- 
move the flame and stir in coarsely ground rice until the paste 
is thick enough to support a spoon in a vertical position ; add a 
few grams of wheat starch and stir it in. 

2. Keep the material at 35°- 37° C. for twelve to forty-eight 
hours. 

3. After fermentation is apparent, transfer a drop of paste to 
a drop of Gram's iodine solution. 

4. Examine the preparation for spindle-shaped bacteria contain- 
ing granules. 

Exercise 105. Study of Bacillus mycoides 

This organism represents those soil organisms which play an 
important part in ammonification. 

Make stains of B. mycoides, using carbol-fuchsin and Gram's 
method. Using material from an old culture, make a spore stain. 
Make cultures on agar plates, agar slope, gelatin stab, and potato. 
Grow the organism on ordinary bouillon or on Dunham's solu- 
tion, and test for ammonia. 

Exercise 106. Study of Bacillus vulgatus {Bacillus mesentericus 
vulgatus Fliigge) 

This is the organism commonly known as potato bacillus. It 
is common in soils and hence is a frequent contamination of 
our potato cultures. In incompletely sterilized milk it some- 
times causes slow curdling. Later the curd is digested, with the 
formation of bitter substances. 



BACTERIA OF MILK 73 

Make stains from pure cultures, using carbol-fuchsiii and 
Gram's method. Examine for spores. j\Iake and describe cul- 
tures on agar plate, agar slope, gelatin stab, and potato. 

Exercise 107. Study of Bacillus denitrificans 

This organism shows the ability to reduce nitrates, usually 
with the production of more or less free nitrogen. Studies of 
this organism should be made in pure cultures on gelatin stab, 
agar slope, potato, and Giltay and Aberson's solution in fermen- 
tation tubes. After two weeks the solutions should be tested 
for nitrites and ammonia. 

Comparison of the denitrifying power of B. pyocyaneus, 
B. Hartlebii, and B. fluorescens liquefaciens should also be made 
(compare Exercise 95). 

Exercise 108. Study of Bacillus radicicola 

Continue the study of this organism, begun in exercises 99 
and 100, by making stained preparations from pure cultures. Use 
carbol-fuchsin, methylene blue, and Gram's stain. Study the 
growth of the organisms in pure culture on gelatin stab, agar 
slope, potato, and nitrogen-free media. After three weeks test the 
cultures on nitrogen-free media for nitrogen as nitrites or nitrates. 

SECTION XIII 
BACTERIA OF MILK 

Milk is a favorable medium for the growth of many micro- 
organisms. It contains sugar (a food constituent for many bac- 
teria), proteins represented by casein and lactalbumin. (which 
furnish organic nitrogen in suitable combination), potassium and 
calcium largely as phosphates, and fats wliich may be used by 
various mold fungi. These substances are in solution and at a 
dilution suitable for nourishing bacteria. 

The bacteria in their growth processes bring about more or 
less extensive changes in the character of the milk, and soon 
alter its value for human consumption. These changes are almost 



74 A MANUAL OF BACTERIOLOGY 

inevitable, siiice commercial milk is exposed to more or less con- 
tamination with bacteria before it reaches the consumer. 

One of the methods of determining the quality of milk is by 
bacteriological examination. To determine the exact character 
of the milk, physical and chemical exammations should also be 
made. In general the bacterial content of milk depends upon 
three factors : (a) the number and kinds of organisms gaining 
access to the fresh milk ; (J) the temperature at which the milk 
has been kept ; (c) the age of the milk at the time samples are 
drawn for analysis. 

The student may consult the following works, among others : 

Barthel. Methoden zur TJntersuchung von Milch und Molkereiprodukten, 
2te Aufl. Leipzig, 1911. 

Conn. Bacteria in Millt. New York, 1907. 

Fleischmann. The Book of the Dairy. Transl. by Aikman and Wright. Lon- 
don, 1896. 

Jensen. Essentials of Milk Hygiene. Transl. by Pearson. Philadelphia, 1907. 

Lafae. Handbuoh der technischen Mykologie. Jena, 1905-1908. 

LoHNis. Handbuoh d. landw. Bakteriologie. Berlin, 1911. 

Marshall. Microbiology. Philadelphia, 1911. 

Milk and its Relation to Public Health (by various authors). Bulletin No. 41, 
Hygienic Laboratory, U.S. Public Health and Marine Hospital Service, 1909. 

Russell and Hastings. Experimental Dairy Bacteriology. New York, 1909. 

SwiTHiNBANK and Newman. Bacteriology of Milk. London, 1903. 

Weigmann. Mykologie der Milch. Leipzig, 1911. 

Exercise 109. The Contamination of Milk with Bacteria from 
Various Sources 

1. From atmospheric dust. The relation of bacteria to dust 
has already been demonstrated in Exercise 60. Parts of this 
experiment may, however, be repeated under different conditions 
in the barn or in the milk rooms. 

2. From the coat of the cow. Carry four plates of sterile agar 
to the barn. Wet the inner surface of a milk pail and place an 
uncovered plate of agar in the bottom of the pail. Hold the 
pail in position for milking and manipulate the cow's udder as 
in milking for thirty seconds. 

Expose the other two plates under cows whose udders and 
flanks have been thoroughly moistened with a clean moist cloth. 
Incubate the plates in the laboratory for two days at 30° C. 



BACTERIA OF MILK 75 

Exercise 110. Quantitative Examination of tlie Organisms in Milk 

The enumeration of bacteria in milk, while in many respects 
a simple undertaking, is full of difficulties if one attempts to 
obtain accurate results. In freshly drawn milk the bacteria are 
unevenly distributed, larger numbers being found in the froth, 
on hairs, on fragments of manure, in crevices of the vessels, etc. 
Even when the milk is thoroughly shaken, the distribution of the 
bacteria is not usually uniform enough to give good counts. In 
older milk, numbers of the bacteria are carried to the surface 
layers as the fat globules rise. The lactic-acid-forming bacteria 
begin to predominate as the acid which they produce kills off 
their competitors. Then agaia the temperature employed and 
the media commonly used for making plate cultures are not 
suited for the development of all organisms in milk. 

In spite of these objections the bacteriological examination of 
milk gives a fairly accurate picture of the conditions under 
which the milk has been produced or handled, and continues to 
be used as one of the important standards for the inspection of 
commercial milk samples. 

1. Procure samples of freshly drawn milk, of raw market milk, 
of pasteurized market milk, and of cream, having first thoroughly 
shaken the vessel from which the sample is to be drawn. 

2. Dilute samples about as follows, with sterile physiological 
salt solution, using the method employed for diluting water 
samples (Exercise 74) : 

Freshly drawn milk, 1 : 100. 

Raw market milk, 1 : 1000 and 1 : 10,000. 

Pasteurized milk, 1 : 100. 

Cream, 1:100,000. 

3. Make plates with beef gelatin, Heyden-Nahrstoff agar, 
dextrose-litmus agar, and whey agar. 

4. Determine the total number of colonies and the proportion 
of acid-forming colonies. 

Note. This experiment may be varied ad libitum by counting colonies 
on milk drawn from dlflerent sources, by different methods, and handled in 
various ways. 



76 A MANUAL OF BACTERIOLOGY 

Exercise 111. Determination of the Numbers of Bacteria in Milk by 
Direct Microscopical Examination 

The difficulties of enumerating bacteria by the poured-plate 
method which were mentioned above have been partially over- 
come by the introduction of methods involving a direct micro- 
scopical examination. Such methods are desirable if for no other 
reason, because time is not lost in waiting for colonies to grow 
upon the plates. 

The following procedure is substantially that outlined by 
Breed. See 

"WiNSLOw. Journ. Infect. Dis., Suppl. No. 1., p. 209. 1905. 
Peescott and Breed. Journ. Infect. Dis. 7 : 632-640. 1910. 
Bkeed. Centralbl. f. Bakt., 2te Abt., 30 : 337. 1911. 

1. Shake thoroughly the sample of milk to be examined. Draw 
0.01 cc. by means of a capillary pipette. 

2. Spread the required quantity of milk over an area of 
1 sq. cm. on an ordinary glass slide. 

This can be done by placing the slide on a paper or glass on 
which an area of this size is ruled. Circular areas are better 
than square areas. Make duplicate smears on each shde. 

3. Dry the milk with gentle heat ; treat the film with xylol or 

chloroform to dissolve out 
the fat; when dry, immerse 
the slide in 95 per cent 

Fio. 31. Glass slide with etched circle ^^^O^^l ^^^ *^ee to five min- 
The circle has an area of Isq. cm. ^^^S, tO fix the film on the 

slide. Dry the slide again 
and stain with methylene blue or other simple stain. Avoid 
alkaline stains and all others which would attack the casein 
and loosen the film. 

4. When the slides have dried from washing, they are ready 
to examine. Place the slides under the oil-immersion lens. Move 
the draw tube until the field of the immersion lens is equivalent 
to 0.16 mm. ; then each field of the microscope covers approxi- 
mately one five-thousandth (0.0002) of a square centimeter. 
On this basis each bacterium seen in a field taken at random 






BACTERIA OF MILK 



77 



represents 500,000 per cubic centimeter if the bacteria are 
evenly distributed. As a matter of fact they are not uniformly 
distributed, and it is necessary to count a number of fields for 
accurate results. The total number of bacteria found in ten 
fields, multiplied by 50,000, or the total number in 100 fields, 
multiplied by 5000, gives the total number of bacteria per cubic 
centimeter. 



Exercise 112. The Examination of Milk for Body Cells 

The microscopical examination of milk sediment often reveals 
the presence of leucocytes in greater or less numbers. The 
presence of these cells has 
been regarded as important, 
because it was assumed that 
they showed the presence of 
inflammation and pus forma- 
tion in the cow's udder. Later 
work, however, has cast con- 
siderable doubt upon this as- 
sumption. As a matter of fact 
our methods do not enable us 
to distinguish between leuco- 
cytes and pus cells in the milk. 
In some cities milk may not 
be sold which contains more 
than 500,000 cells per cubic 
centimeter. 

1. Fill 10-cc. centrifuge 
tubes with milk and heat to 70°-75° C. for ten minutes, 
well to break up aggregates of fat globules. 

2. Centrifuge the tubes at high speed for ten minutes. Re- 
move the upper layers of cream and milk with a pipette and 
refill the tubes with distilled water. Centrifuge again for three 
or four minutes. 

3. Draw off all except ^ cc. of Uquid in the point of the 
centrifuge tube. Wipe out the upper part of the tube with a bit 
of absorbent cotton fastened to a glass rod with a rubber band. 




Fig. 32. Field of the Thoma-Zeiss 
blood counter 

The area of all the squares is 1 sq. mm. 
Each ol the 400 squares represents a vol- 
ume of 1^0 eu. mm. 



Shake 



78 A MANUAL OF BACTEKIOLOGY 

Mix thoroughly the remaining sediment and liquid with the 
rounded end of the glass rod. 

4. Obtain a clean Thoma-Zeiss blood corpuscle counting cell. 
The cell represents a volume of 0.1 cu. mm., and each square 
represents ■^■^■^-^ cu. mm. With a rod transfer a drop of thor- 
oughly mixed milk and sediment to the counting cell and cover 
with a clean cover glass. Place the cell under a l objective of 
good working distance. 

If the number of leucocytes is low, the entire area of the, cell 
may be counted, using a mechanical stage to move the slide. If 
their number is large, five or six small squares may be counted 
and averaged. The average number per small square, multiplied 
by 200,000, gives the number of leucocytes per cubic centimeter 
in the original milk. 

Exercise 113. A Direct Microscopical Method for Determining the Number 
of Body Cells in Milk 

Recent work has cast some doubt upon the accuracy of the 
method using the sediment from centrifuge tubes (see Prescott 
and Breed. Jour. Infect. Dis. 7 : 632. 1910). 

The direct method is the same as that employed for counting 
of bacteria described in Exercise 111, except that the film should 
be somewhat overstained with methylene blue and decolorized 
with alcohol. 

Exercise 114. The Germicidal Action of Fresh Milk 

The freshly drawn milk of some, if not of all, cows shows a 
distinct decrease in the total germ content during the first few 
hours. Part of the decrease m numbers may be due to the fact 
that many bacteria fall into milk which are not able to live there, 
and so perish and pass out of sight. Well-controlled inocula- 
tion experiments, however, have shown that milk may have dis- 
tinct germicidal power for seven to eighteen hours after it is 
drawn (see Lohnis, Handbuch d. landw. Bakteriologie, p. 144. 
Berlin, 1911), but this is not always easy to demonstrate. 

1. Procure in sterile flasks samples of freshly drawn milk 
£rom several cows. Keep the milk samples at 20° C. 



BACTERIA OF MILK 



79 



2. ]\Iake plates on gelatin each hour 
up to twelve hours after the milk was 
drawn. 

3. Determine the increase or decrease 
of bacteria as indicated by the number 
of colonies on the plates. 

Exercise 115. The Catalase of Milk 

]\lilk catalase is an enzyme which 
arises in part from the leucocytes, but 
in large part from the microorganisms, 
of the milk. This enzyme may be demon- 
strated by its ability to liberate oxygen 
from hydrogen peroxide, by an action 
which may be represented by the follow- 
ing formula : 

2 HP, = 2 HP + 0,. 

The value of the test is, according to 
Gerber, (a) to determine the health of 
the animals, (5) to reveal inflammation 
of the udder, (c) to detect abnormal 
milk, ((Z) to determine the age of milk 
and the relative number of bacteria pres- 
ent, (e) to detect colostrum. 

Barthel. Jlethoden zur Untersuchung von Milch 

und Molkereiprodukten, 2te Aufl. Leipzig, 1911. 
Jensen. Centralbl. f. Bakt., 2te Aht., 18 : 211. 

1906. 
LoBECK. Molkerei-Zeitung, Hildesheim, Xr. 5, 

1910. 
KnssELi.. Bulletin No. 18, Wisconsin Agr. Exp. 

Sta., 1889. 
Weigmann. Mykologieder Milch. Leipzig, 1911. 

1. The student may use the Gerber- 
Lobeck Reductase apparatus (Fig. 33) 
or the simple arrangement of test tubes 
shown in Fig. 34. 



Fig. 33. Gerber-Lobeck 
Eeductase apparatus 

1, flask ; 2, gas-measuringtube ; 
3, stopper; a, perforation to 
allow escape of air compressed 
by insertion of tbe stopper ; b, 
narrow tube through which 
evolved gases rise and dis- 
place water through the open- 
ings, c, and allow it to rise in 
outer chamber ; d, scale show- 
ing amount of gas evolved 



80 



A MANUAL OF BACTERIOLOGY 



/~\ 



2. Mix in the apparatus 15 cc. of milk and 5 cc. of dilute 
hydrogen peroxide. The dilute hydrogen peroxide should con- 
tain one part H^O^ in 100 parts of 
water. Shake the apparatus to insure 
thorough mixing, and place it in a 
water bath having a temperature of 
22° C. After two to twelve hours 
read off the amount of gas evolved. 

3. Repeat, using separator slime, 
colostral milk, and boiled milk. 

Exercise 116. Relation of Bacteria to the 
Normal Souring of Milk 

1. Secure six samples of milk, three 
of raw milk and three of pasteurized 
milk. Place a sample of each in (a) 
refrigerator, (5) laboratory cupboard 
(near the floor if possible), and (c) in- 
cubator (temperature 37° C). 

2. On the day of installation and on 
each succeeding day pour litmus-lactose- 
agar plates from each of the samples. 
Record differential counts of both acid- 
forming and non-acid-forming colonies 
as far as possible. 

3. As the number of bacteria increases make determinations of 
the increasing acidity of the milk. Remove 5 cc. of milk from 
each sample with a sterile pipette. Add a few drops of phenol- 
phthalein and titrate with N/20 NaOH. Contiuue the determi- 
nations until there is no further increase in acidity. 

4. Express results by plotting curves to show (a) increase in 
acidity, (6) increase in acid-forming bacteria, (<?) total increase 
in bacteria, for each kind of milk used. 

The percentage of acidity may be computed by the following 
formula : 




Fig. 34. Apparatus to dem- 
onstrate catalase 

The corked test tube, containing 

milk and hydrogen peroxide, 

stands in water 



Per cent acidity = 



cc. alkali used x .0045 
cc. of milk tested 



X 100. 



BACTERIA OF MILK 



81 



If N/10 NaOH were used, the factor in the numerator would 
be .009 mstead of .0045. 

5. Does the acidity eventually decline ? Why ? 

6. Procure commercial tablets of lactic-acid-formiug bacteria 
and inoculate pasteurized milk. Carefully compare the odor and 
taste of samples inoculated with different strains. 



Exercise 117. Fermentation Test 

This is a convenient method of showing whether bacteria of 
the putrefying or the Coli-Aerogenes groups are present in milk 
in excess. 




Fig. 35. Constant-temperature water bath for making fermentation 
and other tests 

1. Procure samples of milk from different sources and of dif- 
ferent degrees of purity. Put 25 cc. of each sample into a large 
test tube which has been carefully cleaned and sterilized. Plug 
the tubes with cotton and "put them into a rack which fits a 
water bath (Fig. 35). 



82 A MAJSrUAL OF BAGTEEIOLOGY 

2. The rack full of tubes is then immersed in a water bath 
kept at a temperature between 37° C. and 40° C. 

3. Take out the rack and examiae the tubes at the expiration of 
six hours. Note the appearance, odor, and taste of each sample. 
Replace the rack in the water bath and continue for another six 
hours, when the condition of the milk in each tube is again noted. 

According to Gerber (" Die praktische Milchpriifung ") good 
milk properly handled should not coagulate in less than twelve 
hours when kept under the conditions described. 

Peter interprets the five types of curds as follows : 

I. Fluid (slight coagulation) : germ content low, with cocci 
predominating ; bacteria of the lactic-acid, Coli, and Aerogenes 
types few. 

II. Gelatinous : lactic-acid bacteria predominating ; few Aero- 
genes and Coli varieties. Cocci and Fluorescent bacteria may be 
present. Gas bubbles indicate presence of Coli and Aerogenes 
groups. 

III. Granular : lactic-acid, Coli, and Aerogenes types of bac- 
teria present ; sometimes cocci numerous. 

IV. Cheesy-curdy : lactic-acid bacteria along with cocci, Coli, 
and Aerogenes ; sometimes only the latter are active. 

V. Gassy: Coli and Aerogenes types predominating, along 
with lactic-acid bacteria, cocci, and B. mesentericus. 

Exercise 118. Study of Curds Formed by Different Organisms 

Prepare and fill with milk a number of small bottles or flasks 
holding about 150 cc. Sterilize fractionally and inoculate dif- 
ferent bottles with Bact. lactis-acicli, B. coli, B. lactis aerogenes, 
Streptococcus lacticus, Oidium lactis, a good dairy starter, and 
some separator slime. Incubate at 30° C. and examine the curds 
formed in each case. What does a gassy curd indicate ? 

Exercise 119. The Reducing Action of Milk of High and Low Germ Content 

Milk in which bacterial development has occurred has a greater 
or less reducing action upon methylene blue. This action is due 
to the development of bacteria and not to some substance present 
in the milk at the time it is drawn. Oxygen must be excluded 



BACTEEIA OF MILK 83 

from the milk, otherwise the leuco-methylene blue quickly goes 
back to the methylene blue. 

This action should not be confused with the ability of niUk 
to decompose hydrogen peroxide or to reduce " Schardinger's 
Reagent" (5 cc. sat. ale. solution methylene blue plus 5 cc. 
formalin plus 190 cc. distilled water) at the time of milking. 
This reducing substance is designated aldehyde-reductase, or 
simply reductase. This reductase is destroyed if milk be heated 
to 80° C. or higher — a fact upon which the Storch test for 
heated milk depends. 

The reduction of methylene blue free from formaldehyde is 
due to the development of bacteria and not to the reductase, 
which works only when aldehyde is present. This power is quite 
closely proportionate to the number of bacteria present, and 
hence may be used as a rapid and simple method of determining 
the bacterial content of milk. There are two methods for deter- 
mining the reducing power: the first consists in recording the 
length of time necessary for disappearance of the blue color ; the 
second consists in determining the amount of methylene blue 
remaining unchanged at the end of two to four hours by titrating 
it with titanium chloride. 

Barthel. Zeitschr. Nahr. u. Genussmit. 15 : 385. 1908. 

WicHERN. Zeitschr. physiol. Chem. 57 : 865. 1908. 

Jensen. Rev. gen. du lait 7 : 308. 1909. 

Fred. Centralbl. f. Bakt., 2te Abt., 35 : 391. 1912. 

LoiiNis. Handbuch d. landw. Bakteriologie, p. 170. Berlin, 1911. 

1. Prepare a standard solution of methylene blue by dissolving 
one part of methylene blue in 1000 parts of water. 

2. Procure several milk samples of various ages and of various 
grades of cleanliness. ^lake plates on whey agar for counting 
the number of organisms, as directed in Exercise 110. 

3. Place in test tubes 10 cc. of each sample of milk to be 
tested, and add to each tube 0.5 cc. of standard methylene blue 
solution ; shake until mixed, then cover the Uquid in the tube 
with a layer of paraffin oil 2 cm. deep. The oil layer excludes 
atmospheric oxygen. If whole milk be used, the rising cream 
will exclude atmospheric oxygen, and the oil is unnecessary. 



84 



A MANUAL OF BACTERIOLOGY 



4. Hold the tubes at a temperature of 37° 0. and record 
the length of time required for the tubes to become white in 
a water bath (Fig. 35). Compare the periods required for 
complete reduction with the bacterial count. 

According to Barthel, milk may be classified as follows : 



Time required for reducing 
methylene blue 


Number of bacteria per cubic 
centimeter of milk 


Quality of the milk 


15 minutes .... 


25,000,000 to 250,000,000 


Very sour milk with high 
bacterial content 


1^ hours . . . 


2,000,000 to 15,000,000 


Sour milk with high bac- 
terial content 


7 hours 


20,000 to 2,000,000 


Milk of fair quality 


7-24 hours . . . 


6,000 


Good milk with low bac- 






terial content 



5. Instead of recording the time necessary for reduction, all 
tubes which retain color at the end of three or four hours may 
be titrated with titanium chloride and the amount of reduction 
estimated directly. Directions for this titration may be found in 
Appendix L. 

Exercise 120. Effect of Pasteurization upon Different Bacteria 

1. Inoculate two tubes of sterile milk with each of the follow- 
ing organisms : Baet. lactis-acidi, B. coli. Streptococcus lacticus, 
B. subtilis, B. proteus vulgaris, and B. mycoides. 

2. Immerse one tube of each organism in a water bath. Pas- 
teurize them by raising the temperature to 65° C, or 150° F. 
Hold at this temperature fifteen minutes ; then cool quickly. 

3. Put both sets of tubes in the incubator. Examine at the 
end of three days, and again after five days, for growth. Which 
bacteria are killed by pasteurization ? Why ? 

Exercise 121. Study of Bacterium lactis-acidi 

This is the lactic-acid organism. It occurs widely distributed 
in nature and finds its way into stored milk. At first it 
grows slowly in milk. It breaks up the milk sugar, with the 
formation of lactic acid. As the amount of lactic acid increases, 



BACTERIA OF MILK 85 

other bacteria cease to develop and the lactic-acid organisms 
increase rapidly, causing the milk to become sour. 

Make a study of the characters of the organism on gelatin, 
agar slope, litmus-lactose agar, milk, and litmus milk. Stain 
with carbol-fuchsin and Gram's stam. 

Demonstrate the presence of acid-forming bacteria by means 
of calcium-carbonate agar. Proceed as follows : 

1. Sterilize in the dry oven three Petri dishes and a test tube 
half full of calcium carbonate. 

2. When cool introduce about 1/10 cc. of calcium carbonate 
into each Petri dish. 

3. Melt three tubes of whey agar, and when cooled to 45° C 
inoculate with a very dilute culture of Bact. lactis-acidi or with 
a diluted sample of milk. 

4. Pour the liquid agar into the Petri dishes. The plates 
should appear turbid, but not white, if the right amount of 
calcium carbonate was added. 

5. Set the plates in the incubator at 37° C. As the acid-form- 
ing colonies develop, they dissolve the calcium carbonate in their 
vicinity and appear surrounded by a clear zone in the turbid agar. 

Exercise 122. Study of Bacillus lactis aerogenes 

, This organism forms acid and gas in milk. It is sometimes 
the cause of " gassy " cheese and also injures the cheese by 
producing undesirable flavors. 

Make a study of its cultural characters on agar slope, gelatin, 
htmus-lactose agar, dextrose broth in fermentation tubes, and milk. 

Exercise 123. Study of Bacillus subtilis and the Type of 
Fermentation it Produces 

It has already been shown in Exercise 120 that the spore- 
forming organisms may survive the heat of pasteurization. 
Although such milk may not turn sour, it generally spoils as 
the result of the growth of spore-forming bacteria. In normal 
souring of milk most of this class of bacteria are prevented 
from growing by the lactic acid produced by other bacteria. 



86 A MANUAL OF BACTERIOLOGY 

1. Fill a test tube one-third full of raw fresh milk. Set it for 
fifteen, minutes in boiling water which rises higher than the level 
of the milk in the tube. Cool and place the tube in the incubator. 
Note whether the milk shows curdling. What is the reaction of 
the curd ? Test with litmus paper. 

2. Inoculate tubes of sterile milk and litmus milk with B. suh- 
tilis. Set in the incubator. Note whether the milk curdles. What 
happens to the litmus ? Is it acid ? 

3. Make stains of the bacteria ia the pure cultures of B. subtilis 
and in the heated milk, using carbol-fuchsin and Gram's stain. 
How do the bacteria in the two cultures compare ? 

Exercise 124. Study of Bacillus cyanogenus 

This organism sometimes occurs in the dairy. It is the cause 
of " blue milk." Its growth is quickly inhibited if lactic-acid 
bacteria are also present, since it is quite sensitive to the presence 
of lactic acid. 

Make cultures and study the growth of the organism on 
gelatin stab, agar slope, milk, and litmus milk. Make stains with 
carbol-fuchsin and Gram's stain. 

Exercise 125. Study of Bacillus lactis viscosus 

Ropy milk may be produced by a group of organisms of which' 
this is perhaps the most common. These organisms are so resist- 
ant to heat that they often survive the ordinary methods of 
cleansing dairy utensils. 

Make cultures on agar slope and in flasks of sterile milk. 
Test the viscosity of the milk from time to time with a glass rod. 

Exercise 126. Study of Microspira tyrogena 

This organism, according to Chester, is sometimes found in 
cheese. It has been previously noted as a type of spiral bacteria. 

Make cultures and study the growth on gelatin stab, agar 
slope, potato, milk, and litmus milk. Make cover-glass prepara- 
tions stained with carbol-fuchsin and Gram's stain. 



BACTEEIAL DISEASES OF PLANTS 87 

Exercise 127. Study of Oidium (Oospora) lactis 

This fungus is frequently found in milk or milk products and 
is believed to be of some importance in cheese manufacture. 

It forms chains of barrel-shaped cells which readily break up. 

It may usually be obtained from old flasks of sour milk, 
where -it forms a superficial pelUcle. From these places pure 
cultures may be obtained. 

1. Put raw skun milk to a depth of 3 cm. into each of two 
1-liter flasks. Plug them with cotton. Heat one of the flasks to 
.75° C. for ten to fifteen minutes. 

2. Keep both flasks at room temperature for three or four 
weeks, noting the changes which occur in the milk. Note the 
changes in acid content. Note the sequence of organisms which 
grow in the flasks. 

3. Obtain 0. lactis from the velvety pellicle on the surface of 
the raw milk. 

4. Make cultures on gelatin stab, agar slope, potato, milk, 
and litmus milk. 

5. Make staias with dilute methylene blue and gentian violet. 

SECTION XIV 
BACTERIAL DISEASES OF PLANTS 

A relatively small number of bacteria are known to produce 
diseases in plants. As further investigations are made the number 
will no doubt be increased. Some plant diseases due to bacteria 
are important because of their destructiveness and the great 
difficulty experienced in attempting to control them. 

Several distinct types of disease are produced by organisms 
belonging to this group : The blights are represented by the 
destructive pear blight, caused by B. amylovorus ; galls and 
tumors by the crown gall of nursery trees, caused by Baet. tujne- 
faciens ; leaf spots by the bacterial leaf spot of stone fruits, 
caused by Bad. pruni ; wilts by the wilt of sweet corn, caused 
by Bseudomonas stewartii ; and rots by the black rot of cabbage, 
caused by B. campestris. 



88 A MANUAL OF BACTERIOLOGY 

Remedial measures against bacterial diseases are generally 
unsuccessful. Crop sanitation and the use of immune or resistant 
varieties are the only practices which have as yet afforded any 
relief. The student should consult 

Smith. Bacteria in Relation to Plant Diseases, Publ. 27. Carnegie Inst, of 
Washington, 1905-1911. 

Van Hall. Bijdragen tot de kennis der Baljterieele Plantenziekten. Amster- 
dam, 1902. 

Marshall. Microbiology. Philadelphia, 1911. 

Jordan. General Bacteriology. Philadelphia, 1910. 

DuGGAK. Fungous Diseases of Plants. Boston, 1909. 

Chester. A Manual of Determinative Bacteriology. New York, 1901. 

Exercise 128. The Blight of Pome Fruits Caused by Bacillus amylovorus 

1. Note the general condition of pear or apple trees in various 
stages of blight. What parts of the trees are worst attacked ? 
Look also for cankers on the bark of the trunk and larger 
limbs. How does the appearance of the bark indicate the progress 
of the disease ? If the weather is moist in spring or early summer, 
look for brown, sticky beads on the twigs or blighted fruits. If 
the weather is dry, the only evidence of the beads may be a dry, 
glistening spot. Place such material in a moist chamber with 
the ends of the twigs in water. What is the consistency of these 
beads ? 

2. With a sharp knife cut through diseased twigs. In what 
tissue is the destructive action of bacteria most prominent? 
What evidence is there that bacteria are transferred by pruning 
instruments or by insects ? 

3. Cut some vigorous twigs from the tree and carry them to 
the laboratory. Cut the ends under water and stand them in a 
small jar of water. Prick through the bark of the new growth 
with a steel needle previously sterilized and dipped into a bead 
on a diseased twig (or a pure culture of the organisms). Watch 
subsequently for infection. Collect some immature fruits, inocu- 
late as above, and keep under a bell jar. 

4. Study the growth of the bacillus on agar slope, gelatin 
stab, bouillon, milk, and sterilized pear twigs. Make stains with 
carbol-fuchsin and Gram's stain. 



BACTERIAL DISEASES OF PLANTS 89 

Exercise 129. The Wilt of Sweet Corn Caused by Pseudomonas Stewartii 

In some market-gardening sections this disease may be found, 
but it is not widely distributed. It attacks only sweet corn, not 
field corn nor pop corn. 

1. Note the wilted appearance of an affected plant. 

2. Make razor sections of stalks of diseased plants and examine 
the fibrovascular bundles. Note the slimy yellow material wlxich 
oozes from the fibrovascular bundles. Examine a drop of this 
substance for bacteria. How would you explain the pathological 
symptoms noted in the plants ? 

3. Make a study of the organism growing in gelatin stab, 
agar slope, and potato. Make stains with carbol-fuchsin and 
Gram's stain. 

Exercise 130. The Black Rot of Cabbage Caused by Bacillus campestris 

1. Examine cruciferous plants which may be affected, with 
this disease. Notice particularly the effect on leaf and stem. 

2. Cut stems and leaf petioles transversely. Examine the fibro- 
vascular bundles. Do the organisms ooze from the cut bundles ? 

3. Make razor or paraffin sections and examine minutely with 
the microscope. What effect does the organism exert upon the 
walls of fibrovascular elements ? 

4. Study the growth of the organism on gelatm stab, agar 
slope, litmus milk, potato, and bouillon. Make stains with carbol- 
fuchsin and Gram's stain. 

5. Inoculate young cabbage or turnip plants growing in the 
greenhouse by puncturing their leaves with a needle bearing 
bacteria taken from one of your cultures. JNIake notes and 
sketches illustrating the progress of the disease. 

Exercise 131. The Soft Rot of Vegetables Caused by Bacillus carotovorus 

Jones. Thirteenth Kept. Vermont Agr. Exp. Sta., 1901. 

Potter. Centralbl. f. Bakt., 2te Abt., 7 : 282, 353. 1901. 

Jones, Harding, and Morse. Bulletin No. 147, Vermont Agr. Exp. Sta., 1910. 

1. Examine a carrot or other vegetable infected with the 
organism in question. Note the size and shape of diseased areas. 



90 



A MANUAL OF BACTEEIOLOGY 




Is there a sharply defined line between infected and noninfected 
tissues or do they gradually merge into each other ? 

2. Cut slices about 1 cm. thick from a sound carrot, using a 
flamed knife. Place them on a piece of moist filter paper in a 
Petri dish. Inoculate by scratching them with a needle beariag 
the organism from a pure culture. Scratch other pieces with the 

same needle after sterilization 
in the flame. Keep the Petri 
dish in your desk and make 
examination after three to 
five days. 

3. By reference to the lit- 
erature above cited acquaint 
yourself with the characters of 
pectinase, the cytolytic enzyme 
which this organism produces. 
The following method, de- 
scribed by Jones, is a con- 
venient one for demonstrating 
this- enzyme. 

Pour a tube of melted agar 
into a sterile Petri dish to a 
depth of 3 mm. Allow this 
to become hard, then inocu- 
late an area of 2 or 3 mm. in 
the center of the dish, using 
the platinum loop, with care 
that the layer of agar is not 
punctured. Keep the dish in the incubator until you have a 
surface colony about 1 cm. in diameter. 

With a flamed knife cut a slice from a fresh turnip root 
and place it at once in a sterile Petri dish. Carefully remove 
the agar layer bearing the bacterial colony and transfer with 
sterile instruments to the surface of the turnip slice. Cover at 
once to prevent contamination. Prepare a control by covering 
another turnip shoe with a layer of agar bearing no bacteria 
(Fig. 36). 



d 



^- 



Fig. 36. Method for showing diffusibility 
of enzymes through agar. (After Jones) 

A, surface view ; JB, vertical section along 
dotted line e-e ; a-a, sterile slice of living 
turnip root ; 6-6, layer of nutrient agar bear- 
ing the bacterial colony c-c ; d-d, region of 
most active enzyme action 



BACTEEIAL DISEASES OF MAN AND ANIMALS 91 

After twenty-four to thirty-six hours Uft the layer of agar and 
determine whether the tissues of the turnip have undergone any 
softening. If so, how does the softened area correspond with the 
size and shape of the superimposed bacterial colony ? 

Transfer bits of the softened tissue by means of a sterilized 
needle to tubes of sterile media. If no organisms develop in 
these tubes, what do you conclude as to the diffusibility of the 
enzyme ? Make a microscopic examination of the softened tissue. 

4. Study the characters of the organisms on gelatin stab, agar 
slope, potato, milk, and Dunham's solution. Test the effect of 
desiccation and exposure to strong light. 

SECTION XV 
BACTERIAL DISEASES OF MAN AND ANIMALS 

The first great advance of bacteriology was in the study and 
treatment of disease in the animal body. Its importance to 
medicine and to civilization can hardly be overestimated. The 
proof that a given disease is caused by bacteria depends upon 
the fulfillment of four postulates laid down by Robert Koch. 
They are as follows : (1) the organism must be demonstrated in 
the circulation or tissues of the diseased animal ; (2) this organ- 
ism must be isolated and grown in pure culture for successive 
generations ; (3) the pure culture of the organism, when intro- 
duced into the body of a healthy, susceptible animal, must produce 
the disease in question ; (4) the organism must be found and re- 
isolated from the circulation or tissues of the inoculated animal. 

It is not assumed that the following exercises give an exten- 
sive survey of the subject of animal diseases; they are merely 
intended to be illustrative. An extensive survey of the subject 
is to be had from works on medical or veterinary bacteriology, 
among which the following may be mentioned : 

JoEDAN. General Bacteriology. Philadelphia, 1910. 
Frost. Laboratory Bacteriology. New York, 1903. 
Sternberg. Manual of Bacteriology. New York, 1901. 
Buchanan. Veterinary Bacteriology. Philadelphia, 1911. 
Herzog. Veterinary Micro-organisms. Philadelphia, 1912. 



92 A MANUAL OF BACTERIOLOGY 

Exercise 132. Preparing a Disinfectant 

In all work where pathogenic bacteria are employed great 
care must be used to avoid infection. Apparatus and media should 
be promptly and thoroughly sterilized after having been used. 

The disinfectant of greatest utility is mercuric bichloride, or 
corrosive sublimate, in a solution of 1 : 1000. Prepare 2 liters 
of a 1 : 1000 solution of mercuric bichloride and place it in a 
jar on your table. The solution is readily made by dissolving 
20 g. of mercuric bichloride in 100 cc. of commercial hydrochloric 
acid. 5 cc. of this acid solution in 1 liter of water makes a 
1 : 1000 solution. 

As soon as discarded all cultures should be placed in this jar. 
Be careful to see that the test-tube cultures sink, drop in the 
used cotton plugs, open Petri dishes and drop them carefully in. 
The following day remove the dishes and wash them. 

Carefully wipe up the table top with a towel soaked in 1 : 1000 
solution from the siphon bottle on the general table. If cultures 
or infectious material are spilled on the floor, wipe them up in 
the same way. Wash the hands first with 1 : 1000 bichloride and 
then with soap and water. 

When sterilizing the platinum needles which have infectious 
material upon them hold high above the flame until the material 
is dry before bringing it into the flame. This prevents sputtering, 
which may throw the material off before the germs are killed. 
Some bacteriologists employ metal tubes into which the needles 
are thrust for heating. 

Exercise 133. Anthrax (Splenic Fever) 

This is one of the best-known and longest-studied of bacterial 
diseases. It is pathogenic for man and domestic animals. For 
many years it was known as Wool Sorter's Disease, because of 
its prevalence among those who handle wool. 

The first proof that a disease may be caused solely by a 
bacillus was obtained, in the case of anthrax, by Robert Koch in 
1876, although others had previously seen microscopical rods in 
the blood of diseased animals. 



BACTERIAL DISEASES OF MAN AND ANIMALS 93 

Koch's achievement was due to his success in cultivating the 
anthrax bacillus outside the body in a pure culture, apart from 
any fluids or tissues of the diseased animal, and in being able to 
produce the disease by introducing these germs into the bodies 
of healthy animals. 

The spores, hkewise discovered by Koch, have been the subject 
of much biological study. Spores are only produced in the 
presence of free oxygen and at temperatures between 14° and 
40° C. They are extremely resistant to drying, it having been 
shown experimentally that they may retain their vitality for 
eighteen years, and then produce the disease when inoculated 
into susceptible animals. The spores are not so resistant to heat 
as those of some saprophytic forms, but they are quite resistant 
to disinfectants. 

Vaccination with cultures attenuated by heat is now largely 
used to protect animals against anthrax and is quite successful. 
Immune sera are used on human subjects. 

1. Observe the instructor inoculate a gumea pig with a pure 
culture of B. anthraois. At the next laboratory period make a 
post-mortem examination of the animal. Note the condition of 
the various internal organs. 

2. Inoculate tubes of bouillon from the spleen and tubes of 
agar from the heart blood. 

3. Make smear preparations from the spleen. Hold the tissue 
with sterile forceps and rub it over a clean slide. Dry, fix, and 
stain with Gram's stain. 

4. Place a small drop of blood on a cover glass, add a drop 
of sterile physiological salt solution, mix, and dry. Stain with 
methylene blue. 

5. Examine and draw a field from each slide. 

6. From your observations, how do you explain the death of 
the animal infected with anthrax ? 

7. Make stained preparations from the tubes made in 2. 

8. When spores appear, make a spore stain according to direc- 
tions in Exercise 61. 

9. Make and describe cultures upon gelatin stab and gelatin 
plate. 



94 A MANUAL OF BACTEEIOLOGY 

Exercise 134. Tuberculosis (Consumption, Phthisis) 

This disease is the greatest scourge of the human race. In 
1900 about one ninth of all deaths from known causes in the 
United States were due to this disease. Small nodules, or tuber- 
cles, are so uniformly observed in all advanced stages of the 
disease in animals that their presence has given the name to the 
disease. The tubercle eventually breaks down, the central por- 
tion becomes necrotic, caseation sets in, and then the caseous 
mass softens, probably due to the toxic action of bacterial prod- 
ucts. In many cases a deposit of calcium salts finally takes place 
in the tubercle, and it is converted into a hard, dry, friable body 
which may be, entirely walled off from the neighboring tissue. 

Besides man the disease attacks cattle, swine, birds, and other 
animals. Among these animals the ravages of the disease are at 
times very severe. 

Tuberculosis may attack almost any tissue or organ in the 
body. The lungs constitute the seat of the most common infec- 
tion, but the intestines, the mesenteric glands, the lymph glands 
of the head and neck, the portal glands and liver, the skin, the- 
bones, and the urogenital system are frequently attacked. Be- 
sides the lesions on important organs, the tuberculosis organism 
produces a slow toxemia which works injury to the whole of the 
infected body. 

The ways of infection are usually four : (1) the respiratory 
tract ; (2) the alimentary tract ; (3) inoculation (rare) ; (4) pre- 
natal infection. The first two are more common, so far as we know. 

Tuberculin is a mixture of the toxins produced by B. tuber- 
culosis in artificial cultures. A glycerin-broth culture of the 
organism is sterilized in steam, then concentrated by evaporation 
on a water bath, then filtered through paper and a Berkefeld 
filter. If it is to be kept, a preservative is added. Tuberculin is 
of use in making a diagnosis. Its injection into man or animals is 
followed within two to ten hours by a rise in temperature, which 
continues for a few hours and then disappears. Many modifica- 
tions of the original method of preparing tuberculin are now 
employed. 



BACTERIAL DISEASES OF MAIT AJ^D Al^IMALS 95 

1. Examine and make descriptions of cultures of B. tubercu- 
losis on the general table. Compare cultures of boviue and 
human tuberculosis. 

2. Examine and describe the museum preparations of patho- 
logical material on the general table. 

3. Observe the post-mortem conditions of a tuberculous guinea 
pig. In what parts of the body are tubercles most abundant ? 

4. Select one of the small tubercles or a portion of a diseased 
organ. Grasping it with the dissecting forceps, cut it off. 
Smear the freshly cut surface over the central portion of a 
glass slide. Dry and fix as usual. Stain by the Ziehl-Neelson 
method and counterstain with methylene blue. Do not put a 
cover glass over the smear. Wait until the water has evapo- 
rated ; then put immersion oil on it and examine directly with 
the oil-immersion objective. The tuberculosis organisms should 
appear as slender rods stained red; the body cells are stained 
blue. If the preparation is not satisfactory, repeat until a good 
one is obtained. 

5. Draw a field showing the relation of the B. tuberculosis to 
the cells and tissues. 

6. Read and become familiar with the modem principles of 
treating tuberculosis in man. Has tuberculin any remedial 
value in treating the disease ? 

Exercise 135. Green Pus 

Green or blue-green pus is formed sometimes on surgical 
dressings, due to the development of B. pyocyaneus. In many 
of its characters this organism closely resembles B. fluorescens 
liquefadens, but it has been shown unquestionably to be patho- 
logical both in pure and in mixed infections. 

The deep-blue pigment, pyocyanin, has been isolated and 
studied chemically. It is not toxic to animals. The toxin is 
very resistant to heat. The virulence of the organism may be 
inferred from the statement, made by Jordan, that one tenth of a 
loop of a fresh agar culture will kiU a guinea pig in twenty-four 
hours. 



96 A MANUAL OF BACTERIOLOGY 

1. Shake old broth cultures of B. pyocyaneus with chloroform 
in a separatory funnel until a goodly quantity of pigment is 
extracted. Evaporate the chloroform from a watch glass. 

2. Study the characters of the organisms grown on gelatin 
stab, agar slant, milk, and potato. 

3. Make stains with carbol-fuchsin and Gram's stain. 



Exercise 136. Septicaemia, Inflammation, etc. Caused by 
Streptococcus pyogenes 

This organism may show its pathological effects upon the 
body in various ways. It may cause suppurative inflammation, 
erysipelas, septicaemia, puerperal fever, pneumonia, or other dis- 
eases. It frequently invades organs already attacked by other 
bacteria and causes great injury. In the last stages of pulmonary 
tuberculosis it is likely to invade healthy tissues adjacent to those 
affected with tuberculosis, and thus predispose the patient to 
hemorrhage. " Blood-poisoning " is usually due to an infection 
of streptococci or staphylococci, or both. 

The virulence of different strains of organisms appears to vary 
widely. The toxin may be obtained from cultures killed with 
chloroform or filtered tlirough unglazed porcelain filters. 

Streptococci are always present on the outside of the body, and 
appear to be capable of causing trouble if they gain entrance 
when the bodily resistance is low. This emphasizes the necessity 
of the prompt and thorough disinfection of all wounds. 

1. Make cultures of Streptococcus pyogenes on lactose bouillon, 
gelatin stab, agar slant, "potato, and litmus milk. 

2. After forty-eight to sixty hours shake up the culture in 
lactose bouillon and make a stained preparation from the gran- 
ular sediment formed by the bacteria. Also make a stain from 
the agar slant. Does it stain with Gram's stain ? 

3. Draw and describe the stained preparations with the oil- 
immersion objective. What is the form and arrangement of the 
organisms ? How do staphylococci differ ? 

4. Describe all cultures after three days. On which is there 
no growth ? 



fee:\iextatiox ORGAJSTISMS 97 

SECTIOX XVI 
SOME ORGANISMS CAUSING IMPORTANT FERMENTATIONS 

In the preceding sections the activities of bacteria were the 
chief topic of study. This section will principally treat of the 
activities of another group of plants, namely, the yeasts and mold 
fungi. These organisms, though somewhat more complex, resem- 
ble iQ many ways the bacteria ; they are devoid of chlorophyll, 
grow upon organic substances, and reproduce rapidly by small 
spores, which, when liberated, may be carried long distances in 
the air. They are thus distributed as widely as bacteria. 

The physiological activities of these fungi, on account of their 
great importance ia the arts and industries, have been extensively 
studied. It is as producers of fermentation that they are of 
interest in agricultural and industrial technology. 

In the modern sense the term fermentation is a very broad 
one. As defined by Lafar, " Fermentation is a decomposition 
or transformation of substances of various kinds brought about 
by the vital activity of fungi." In the older and more common 
use of the term fermentation it designates the formation of 
alcohol and carbon dioxide from a carbohydrate — for example, 
the fermentation of wine from must. It is perhaps needless 
to say that these phenomena were observed and used in the 
arts centuries before the scientific basis of the process was 
demonstrated. 

The literature upon fermentation and the organisms which 
cause it is voluminous. References to a few of the more general 
treatises are given herewith : 

Lafar. Handbuch d. teohn. Mykologie. Jena, 1905-1908. 

JoRGEssEN. Micro-organisms and fermentation. Transl. by Davies. Philadel- 
phia, 1901. 

Klocker. Die Garungsorganismen. Stuttgart, 1900. 

Hardex. Alcoholic Fermentation. Xew York, 1911. 

Marshall. Microbiology. Philadelphia, 1911. 

DeBary. Comparative Morphology and Biology of the Fungi, Mycetozoa, 
and Bacteria. Oxford. 

Conn. Agricultural Bacteriology, 2d ed. Philadelphia, 1909. 



98 A MANUAL OF BACTEEIOLOGY 

FowLEB. Introduction to Bacteriological and Enzyme Chemistry. New York 

and London, 1911. 
Const. Bacteria, Yeasts, and Molds in the Home. Boston, 1909. 
Pasteur. Studies on Fermentation. New York, 1879. 
Hansen. Practical Studies on Fermentation. Transl. by Miller. London, 1896. 

Exercise 137. Morphology of Yeast (Saccharomyces) 

Yeast plants are unicellular organisms which reproduce prin- 
cipally by budding. Under certain conditions — for example, 
a sudden diminution in food supply — they produce endo- 
genous spores, usually two to six in each cell. This condition 
is supposed to ally the yeasts with the Ascomycetes, and the 
sporangia are regarded as asci. 

1. Mix a piece of compressed yeast with water, mount a drop 
of the mixture, and examine with both low and high powers. 

2. Note the shape of a single cell. Are all cells the same 
shape ? What is the size of single cells ? Compare with other 
organisms studied. 

3. Study the finer structure of the yeast cell. Note 

a. Color, homogeneity, and transparency. Mount some yeast 
cells in Schultze's iodine solution. This solution turns normal 
cellulose blue, fungus cellulose yellowish-brown. Of which is 
the yeast cell composed ? 

h. The protoplasm, a semitransparent, granular substance fill- 
ing more or less of the cell cavity. The protoplasm often con- 
tains small oil drops, recognizable by their powers of refraction. 
Are they equally abundant in all cells? 

c. Vacuoles, spherical cavities in the protoplasm filled with 
cell sap. How many vacuoles in each cell? 

d. The nucleus, a small spherical body which can only be 
distinguished by the use of special stains. 

Exercise 138. Reproduction of Yeast by Budding 

1. Mount a drop of yeast culture which has grown for twenty- 
four hours in liquid wort medium or on wort agar. 

2. Note the presence of cells which have formed small out- 
growths, or buds. Do cells ever form more than one bud ? Are 
all buds of the same size ? 



FEEMENTATION ORGANISMS 



99 



3. Make hanging-drop cultures of very dilute cultures (one 
or two cells in the hanging drop) and study the process of bud- 
ding. Make observations once or twice daily for three days. 

4. Make a set of drawings to illustrate the stages in the for- 
mation of the bud. 



Exercise 139. Spore Formation in Yeast 

The essential conditions for spore formation are abundance of 
oxygen and paucity of food substances. 

1. Prepare gypsum blocks. Mix gypsum (plaster of Paris) 
with haK its volume of water, stir thoroughly, and pour into a 
paper cylinder mold to harden. The block 
should be about 4 cm. in diameter and 
2 cm. high. When dry, remove the paper 
and place the block in a stender dish cov- 
ered with a loose glass cover or a Hansen 
flask (Fig. 87). Sterilize the block, covered 
as described above, in the dry sterilizer for 
about an hour and a half at a temperature 
no higher than 120° C. 

2. Inoculate a flask of sterilized wort with 
a small quantity of good yeast. Incubate at 
25° C. for twenty-four hours. At the expira- 
tion of that time there should be a good 
layer of yeast at the bottom of the flask. 

3. Draw off the supernatant liquid with 
a pipette. Transfer a small quantity of the yeast sediment to the 
dry surface of a gypsum block. It is essential that the layer 
of yeast on the gjrpsum block be very thin. Pour sterile distilled 
water into the dish so that the gypsum block stands in water 
two thirds of its height. Incubate at 25° C. 

4. Examine yeast from the gypsum block after eight to 
fourteen days. Look for small globular ascospores in the cells. 
How much of the cell do they occupy ? What is their arrange- 
ment ? Does each spore have a cell wall of its ovni ? 

5. Make stained preparations using gentian violet or carbol- 
fuchsin. 




Fig. 37. Gypsum block 
in a Hansen flask 



100 



A MANUAL OF BACTERIOLOGY 



Exercise 140. Preparation of Pure Cultures of Yeast from Single Cells 

1. Sterilize several .01 ec.-capillary pipettes by using 50 per 
cent alcohol and warming them until the alcohol is evaporated. 

2. Draw .01 cc. of a yeast culture from liquid wort and 
examine under the microscope. Dilute the culture with sterile 
wort until .01 cc. contains an average of one cell. (Instead of 
the pipette a 2 mm.-platinum wire loop may be used.) 

3. Inoculate 10-20 tubes of sterile wort with .01 cc. of solu- 
tion containing one yeast cell. Incubate at 25° C. Where 
growth appears, it is a culture developed from one cell. 



Exercise 141. Cultivation of Yeasts 

For much of the student's work yeast may be successfully cul- 
tivated on various carbohydrate media, such as Naegeli's solution 
(Exercise 53), wine must, apple must, and beer wort. These 

media may be sterilized in 
large test tubes or flasks, 
with or without the addition 
of agar or gelatin. In labora- 
tories where cultures are to be 
kept for some time, specially 
constructed flasks are used 
for yeast culture. These flasks 
are provided with narrow 
apertures, and consequently 
evaporation of the culture 
solution is diminished. The 
Freudenreich type has a cap 
which terminates in a narrow, 
cotton-filled tube (Fig. 38). 
The Hansen flask has a cotton stopper in the wide opening, 
over which a cap is fitted which terminates in a narrow tube 
which also contains a tuft of cotton. It also has a side delivery 
tube for transfer of the culture media to other flasks. This side 
tube is closed with a short piece of rubber tubing and a glass 
rod (Fig. 39). 





Fig. 38. Treud- 
enreich flask 



Fig. 39. Hansen 
flask 



FERMENTATION OEGANISMS 101 

Exercise 142. The Invertase of Yeast 

Invertase is one of the important enzymes produced by yeast. 
Its specific action is the hydrolytic splitting of disaccharides to 
monosaccharides, such as dextrose and levulose (invert sugar), 
by an action represented by the equation 

Invertase passes out of the yeast cell by exosmosis into the 
surrounding medium. 

A. A simple and convenient method of demonstrating this is 
the following: 

1. Prepare a 2 per cent solution of pure cane sugar. Make 
certain that it has no reducing action on Fehling's solution. 
Put the solution into several large test tubes or small flasks. 

2. Add to each tube yeast from a yeast cake or from a pure 
culture. Keep the tubes in a warm place (25° C.) for twenty-four 
hours. 

3. Test the contents of the inoculated tubes for invert sugar 
by the use of Fehling's solution. Write the equation for the 
chemical change in the sugar. 

£. By the use of another method the invertase may be obtained 
free from living yeast cells. 

1. Wash 10 g. of brewer's or compressed yeast thoroughly 
with water and filter with suction. 

2. Mix the moist yeast with 100 cc. of distilled water and 
5 cc. of chloroform. The chloroform prevents the yeast from 
growing but does not destroy the enzymic action. Keep the 
solution for three hours at a temperature of 25°-30° C. ; then 
filter. 

3. To test the inverting action of this solution, add 5 cc. to 
25 cc. of a 3 per cent cane-sugar solution and keep at 30° C. for 
an hour or more. Boil the solution to drive off the chloroform, 
which would otherwise interfere with the test with Fehling's 
solution. Add distilled water to restore the boiled solution to 
its original volume and determine the amount of invert sugar 
quantitatively by Fehling's solution or by the polariscope. 



102 A MANUAL OF BACTEEIOLOGY 

Exercise 143. The Zymase of Yeast 

Zymase is the enzyme of yeast which accomplishes the alcoholic 
fermentation of sugars, but it is only capable of fermenting 
hexose sugars, chiefly d-glucose, d-fructose, d-mannose, and 
d-galactose. The action may be represented by the equation 

C,H^A = 2C,HpH + 2CO,. 

Zymase is classed as an endoenzyme, because it occurs only 
in the living cell and does not exosmose into the surrounding 
medium. The isolation of the enzyme from yeast cells is de- 
scribed in exercises 144 and 145, but its action may be demon- 
strated by the following simpler method : 

1. Prepare a 4 per cent solution of dextrose in tap water and 
fiU three flasks about one-third full ; also fill several fermentation 
tubes. Sterilize all in the Arnold sterilizer. 

2. Inoculate two of the flasks and all of the tubes with active 
yeast. Weigh the flasks and record the exact weight of each. 
Keep the cultures in the incubator at 25° C. As fermentation 
progresses, note the changes in weight of the flasks in com- 
parison with the control. To what are the changes due ? After 
a week compare the specific gravity, of the fermented solution 
with that of the control. 

3. Examine the fermentation tubes at the end of twenty-four 
hours. When the closed arm of the tube is about half full of 
gas, test it with 10 per cent NaOH. How much of the gas is 
carbon dioxide ? 

4. For further studies of the action of zymase see exercises 
146, 147, 148, and 149. 

5. State as exactly as possible the characters of invertase 
and zymase. What is the relation of one to another in causing 
fermentations ? 

Exercise 144. The Preparation of Yeast Juice 

In 1897 Buchner succeeded in mechanically breaking down 
the yeast cell and obtaining a yeast-free extract containing zymase 
which would cause alcoholic fermentation in sugar solutions. The 



FERMENTATION OEGANISMS 103 

enzyme was separated from the broken cells by filtration under 
great pressure. The drastic methods which Buchner employed 
are somewhat beyond the facilities of the average student ; 
nevertheless they should be noted and carried out if possible. 

1. Mis a kUo of brewer's yeast with an equal weight of 
clean quartz sand and 250 g. of infusorial earth. Put all into a 
mortar and grind together until plastic and moist. Add 100 cc. 
of distilled water and transfer the material to a press cloth. 

2. Put the material into a filter press and subject to a pres- 
sure of 400 or 500 atmospheres. Remove the cake, grind it, add 
another 100 cc. of water, and press again. Clarify the filtrate 
obtained by shaking it with fresh infusorial earth and filtering. 

3. Mis 25 cc. of the juice with an equal volume of 10 per cent 
dextrose solution. Put the solutions into fermentation tubes, 
with a few drops of toluol added to inliibit bacterial action. Test 
also cane sugar, levulose, maltose, and lactose. Compare with 
the results obtained in Exercise 143. 

Exercise 145. The Preparation of an Active Yeast Powder 
Buchner's yeast juice does not long retain its ability to ferment 
sugar solutions. A proteolytic enzyme appears to digest not only 
the protein present but the zymase also. Owing to this fact, as 
well as to the difficulty of preparation, the yeast juice is not so 
feasible to study in the ordinary laboratory as the permanent 
yeast powder (Bauerhefe), or zymin. The preparation of zymin 
is as follows : 

1. Rub up 500 g. of brewer's yeast, pressed free from most of 
its water. 

2. Put this yeast into 3 liters of acetone and stir for ten 
mkiutes. Filter off the acetone, using suction if necessary. 

3. Mix the material with 1 liter of fresh acetone, stir for two 
minutes, and filter. 

4. Rub up the material with 250 cc. of ether for three minutes, 
and filter. 

5. Spread the residue on filter paper or porous plates to dry. 
Keep it at a temperature of 35°-45° C. for twenty-four hours, to 
drive off ether and acetone. 



104 A MANUAL OF BACTEEIOLOGY 

6. Grind up the dry powder and store it in a clean glass- 
stoppered bottle. 

7. Prepare 2 per cent solutions of cane sugar, dextrose, levu- 
lose, maltose, and lactose. Put them into fermentation tubes 
and add to each as much yeast powder as will lie on the point 
of a penknife. Examine the tubes for gas at the end of ten to 
eighteen hours. Compare the results with those obtained with 
the same sugars in Exercise 143. 

Exercise 146. The Estimation of the Chief Products of the 
Fermentation of Sugars 

The chief products of the fermentation of an invert sugar are 
carbon dioxide and alcohol. In addition, small amounts of glycerin 
and succinic acid are produced, together with smaller quantities 
of other compounds, such as aldehydes, fusel oil, and furfural. 
This exercise demonstrates methods of estimating the carbon 
dioxide and alcohol. 

1. Prepare about a liter of apple- or grape-juice must. Instead 
of must, other saccharine solutions may be used — for example, 
4 per cent dextrose, or molasses diluted with two volumes of 
water. Determine quantitatively the sugar content of the must. 
To do this, make 20 cc. of Fehling's solution by mixing 10 cc. 
of each of the two stock solutions (see Appendix) in a casserole, 
and boil. Above the casserole adjust a burette containing 50 cc. 
of must and add, drop by drop, until the blue color of the 
Fehling's solution disappears. 

1 cc. normal Fehling's solution = 4.94 mg. invert sugar. 
1 cc. normal Fehling's solution = 4.53 mg. dextrose. 

For example, 20 cc. of Fehling's solution required 16.2 cc. of 
must to reduce the copper. 

20 cc. Fehling's solution = .0988 g. invert sugar. 
Then 16.2: .0988: : 100 : a;; 

therefore x = .60988 per cent invert sugar in the must. 

2. Select three Erlenmeyer flasks of 500 cc. capacity. Put 
into each 150 cc. of must and sterilize fractionally in the Arnold 



FEK.ME^'TATIOX OEGAXISMS 105 

sterilizer. After sterilizatioa add 5 cc. of 95 per cent alcohol to 
flask No. 1 ; to flask Xo. 2, enough dextrose to make the sugar 
content up to four per cent. Add to each of the three flasks 
about 3 cc. of a fresh, active yeast culture and fit them with 
fermentation stoppers. Incubate at 25° C. "Weigh the flasks 
ever}- two days until the weight is constant, and estimate the 
grams of carbon dioxide given off. When the fleisks reach con- 
stant weight, attach a condenser and distill off the alcohol from 
each separately. Determine its specific gravity either by a pyc- 
nometer or by a hydrometer. From this compute the percentage 
and the exact amount of alcohol formed from the sugar. Deter- 
mine the amount of sugar remaining in the flask by the method 
given above. 

Write the result, giving formulae with balanced equations. 

Exercise 147. The Fermentation of Bread Dough 

A dough of flour, water, and yeast will rise ff kept in a 
warm place. The flour contains starch and a small amount of 
diastase, an enzyme which forms sugars from starch. The yeast 
ferments the sugars thus formed into alcohol and carbon dioxide. 
The bubbles of gaseous carbon dioxide give the dough a porous 
structure which is not destroyed when baked. The porous struc- 
ture makes the bread easier to masticate and hence easier to digest. 

A dough of flour and water onl}- wiU rise if kept in a warm 
place. The action is due to the production of carbon dioxide 
by organisms occurring naturally in the flour and water. But the 
rising will be much more rapid and certain if at the beginning a 
quantity of yeast (leaven) is incorporated with the dough. The 
leaven may be a piece of dough kept from a previous baking, or 
it may be a yeast cake. The commercial yeast cake contains an 
admixture of more or less bacteria, which are sometimes respon- 
sible for flavors in the bread. Relatively pure cultures of yeast 
are used by bakers who wish a rapid rising with little danger of 
irregularities due to bacterial action. 

1. Soak a. cake of compressed yeast for an hour in distilled 
water and examine the organisms it contains. Both yeasts and 



106 



A MANUAL OF BACTERIOLOGY 



bacteria will be found. Certain of the bacteria have the power to 
transform starch into sugar and to dissolve gluten ; others form 
lactic and acetic acids. The amount of acid formed is not usually 
perceptible unless the dough stands too long before baking or is 
kept too warm, in which case sour bread is obtained. B. mesen- 
tericus vulgatus Fliigge is sometimes the cause of viscid bread. 
2. Soak a dry yeast cake in distilled water which has been 
boiled and cooled to 40° C. After six to eight hours make 




Fig. 40. Bread dough raised with yeast from different sources 

Each beaker (400 cc. capacity) received 75 g. of flour and enough yeast culture to 

make a stiff dough. The yeast in No. 1 came from a compressed yeast cake ; in No. 2, 

from a dried yeast cake ; in No. 3, from a wild yeast culture 

microscopic examination for the organisms present. Is the 
greater number of bacteria m the compressed yeast or in the 
dry yeast cake ? 

3. Make a culture of wild yeast as follows : Cook 200 g. 
of potato in a tin or granite-ware vessel until it can be mashed. 
Add 5 g. of sugar and 5 g. of ammonium tartrate. Stir in enough 
water to make a thin paste. Keep the culture at 25° C- until 
fermentation is evident. Examine microscopically for bacteria 
and yeasts. 

4. Weigh off 100 g. of wheat flour into each of three beakers. 
Stir into each a yeast culture made as directed hi paragraphs 1, 2, 



FERMENTATION ORGANISMS lOT 

and 3, until a firm dough is made. Cover the beakers with a plate 
of glass and place in the incubator. Examine each dough every 
three hours, to determine which rises most rapidly. Note the 
texture, viscosity, and odor in each case. 

Exercise 148. The Fermentation of Cider 

1. Grind a few ripe apples in a meat cutter. Press the pulp 
through a cloth and then filter through paper. 

2. Fill six fermentation tubes with juice. Place four of them 
in the Arnold sterilizer and steam them for half an hour. Inoc- 
ulate two of the sterilized tubes with yeast. Keep the six tubes 
in the incubator for twenty-four hours. 

3. Examine the tubes for fermentation. In which tube is the 
most gas present ? When the arm of the tube is about half full 
of gas, test it with 10 per cent NaOH. How much of the gas 
is carbon dioxide ? '\^^hat caused fermentation in the unsterilized 
tubes ? 

Exercise 149. The Fermentation of Wine 

Wine is usually defined as the fermented juice of some berry 
or grape. The grape is the fruit most commonly used, because 
the juice is rich in sugar and is easily pressed from the crushed 
fruit as a clear liquid. Among the microorganisms which adhere 
to the skin of the grape there are always numerous yeasts. The 
yeasts start fermentation in the saccharine juice pressed from 
the grape. These wild yeasts are sufficient to start the process 
of fermentation, although better results could undoubtedly be 
obtained from the use of cultures of selected yeasts. 

If practically all the sugar has been fermented (only about 0.1 
per cent remaining), the wine is classified as dry ; the wine is 
sweet if the fermentation is checked at an early stage by the 
addition of alcohol or brandy; the wine is still if the carbon 
dioxide produced by the fermentation has been allowed to escape 
before bottling. 

The principles of the fermentation of wine have been demon- 
strated by Exercise 146 and need not be repeated here. 



108 A MANUAL OF BACTERIOLOGY 

Exercise 150. The Fermentation of Vinegar 
This fermentation takes place in some weak alcoholic solution, 
such as cider or wine. The process is carried out by several 
species of bacteria which are common m the air. Inoculation of 
the liquids is not,' therefore, required for vinegar manufacture. 
The brown gelatinous mass found in vinegar barrels, known as 
" mother of vinegar," is the zoogloeum formed by these bacteria. 
The process of fermentation involved is one of oxidation, which 
is most simply expressed by the equation 

CH3 • CH^OH + O, = CH3 • COOH + H^. 

In reality, however, the oxidation process is more complex 
than the above equation would indicate. 

1. Obtain the film from the surface of some old vinegar and 
inoculate several water blanks. 

2. Plate out the organisms in the water blanks, using wort 
agar. Incubate the plates at 25° C. 

3. When colonies appear, transfer organisms from them to 
tubes of wort agar. Incubate tubes at room temperature and at 
37° C. 

4. Prepare liquid cultures in flasks containing 50 cc. of liquid 
wort plus 1 per cent alcohol. Inoculate them from the films 
obtained in 1. Incubate one flask at 15°-20° C, one at 30° C, 
and one at 40° C. Leave two flasks uninoculated for controls. 

5. Examine the flasks daily and note the formation of films. 
Remove organisms, with the sterilized platinum loop, stain with 
carbol-fuchsin, and examine with the microscope. Use the method 
described in Exercise 61 to demonstrate the gelatinous capsule. 
What differences in morphology at different temperatures ? 

6. On every second or third day add a small quantity of 
alcohol. At the end of the experiment, filter the solutions and 
titrate them with yq ^ NaOH to determine acidity. 

Exercise 151. Study of Mucor 

Mucor is one of the molds common everywhere, especially 
upon starchy substances. It is especially common upon bread, 
cooked potatoes, and bananas. It forms a floccose, aerial layer of 



FEEMEXTATION ORGANISMS 109 

sporangiophores 1-5 cm. in height. At first the growth is creamy 
white ; later, as the sporangia mature, it becomes dark brown or 
black. The purely vegetative structure, the mycelium, develops 
mainly in the substratum. 

]Mucor is a fungus belonging to the class Phycomycetes, or 
Alga-like fungi. 3Iucor mucedo may grow in. sacchariae solutions 
and cause therein a weak alcoholic fermentation. 

The sahent features of the fungus are brought out by the 
following outline : 

1. Examine a culture of jSlucor and note 

a. The general appearance of the whole mass. 

b. The position and direction of the threads with reference 

to the supporting substratum. 

c. The aerial hyphse (sporangiophores) upon which the 

black tips (sporangia) have formed. 

2. Examine microscopically a bit of agar from a Petri dish 
inoculated with ]\Iucor at the previous laboratory period. Note 

a. The mycelium, consisting of branched hyphse. 

b. The granular protoplasm in the hyphae may be found in 

motion. Is the movement of the protoplasm uni- 
form ? Is it ever reversed ? Do you find vacuoles 
present? Are cross walls present? Draw a young 
branched hypha. 

3. In suitable material distinguish upright hyphse which have 
a swollen tip containing an abundance of granular protoplasm. 
Is the swollen tip separated from the hypha by a transverse 
wall ? The tip is the beginning of the sporangium. 

4. In older material distinguish the spores forming in the 
sporangia. Find also mature sporangia with ripe spores. Note 

a. The sporangium wall. 

b. The ripe spores. 

c. The swollen tip of the sporangiophore (columella). 

5. Draw a young and a mature sporangium. 

6. Sexually formed spores (zygospores) are rarely found in 
Mucor. For details of the process the student is referred to 
Campbell's " University Textbook of Botany," page 160. 



110 A MANUAL OF BACTEEIOLOGY 

Exercise 152. Study of Penicillium 

The conunon greea mold, Penicillium, is one of the most 
cosmopolitan of the fungi. Its spores are found nearly every- 
where, and it grows upon anythuig from jemons to old boots. 

Penicillium is a fungus genus belonging to the Ascomycetes. 
The perithecia are, however, quite rare in the more common species. 
The spores formed are therefore mostly of the conidial type. 

The green Penicillium is usually abundant in portions of the 
home where food is kept, and causes the loss of much stored 
food. In the fruit busiaess serious losses are caused by Penicillia, 
which attack oranges, lemons, apples, cherries, peaches, etc., 
especially if shipments are made over long distances in warm, 
muggy weather. In breweries Penicillium may cause great 
damage to the malt, as well as to the finished beer. 

Grapes on which this mold is abundant make defective wines. 
The mold not only destroys the necessary sugars, but forms 
waste products which are deleterious to the subsequent growth 
of the yeasts. 

Some species of Penicillium, like P- Camemberti, play a useful 
r61e in the ripening of cheeses. 

1. Examine a culture of Penicillium and note 

a. The general appearance of the whole mass. 

b. The position and direction of the threads with reference 

to the supporting substratum. Remove a small tuft 
of these aerial hyphae, place them in a drop of 50 
per cent alcohol on a slide, tease apart carefully, and 
examine with both low and high powers. 

2. The conidiophores are the straight aerial branches, each of 
which ends in a tuft of spore-bearing branches. Are cross walls 
present? Do all the branches reach to about the same level? 
How many series of branches are present? At the distal end 
note the sterigmata, or club-shaped cells, which bear the spores. 

3. The spores arise in basipetal order from the sterigmata. 
Are spores relatively few or abundant ? "What is their shape ? 
color ? Is the spore wall thickened ? Do the spores contain 
protoplasm? oil? vacuoles? 



FEE:\rEXTATIOX OEGANISMS 111 

4. Draw a conidiophore and spores. 

5. Lift out pieces of agar on which Penicillium has been 
sown for two days. Study the mycelium it contains. Have the 
hyphse cross walls ? Do they branch extensively ? Draw a bit 
of mj'celium. 

Exercise 153. Study of Aspergillus 

This mold fungus is also of wide distribution, usually develop- 
ing at rather high temperatures. 

^Morphologically it is quite similar to PenicHUum, and like- 
wise, the conidial spore form is abundant, while the ascospore is 
comparatively rare. Aspergillus niger is more or less common as 
a black mold on organic substances. It forms oxalic acid in 
considerable quantities when grown upon media containing 
sugars. The fungus produces diastase, maltase, invertase, and 
emulsin. 

Aspergillus oryzae is of considerable interest on account of its 
pronounced diastatic action. It is, in fact, an important source 
of diastase, the takadiastase of commerce. This fungus is used 
in the preparation of rice beer, or sake, a favorite beverage in 
Japan. The Aspergillus is used to convert the rice starch into 
sugar ; then the sugar is fermented by yeasts which find their 
way into the mass. 

1. Examine a culture of Aspergillus and note 

a. The general appearance of the whole mass. 
h. The position and direction of the threads with reference 
to the supporting substratum. 
Remove a small tuft of these aerial hyphse, place them in a 
drop of 50 per cent alcohol on a slide, tease apart carefulh', and 
examine with both high and low powers. 

2. The conidiophores are the aerial branches, each of which 
ends in an enlargement. The surface of this swollen tip gives 
rise to numerous sterigmata, or stalk cells, which bear the spores. 
In some species sterigmata are not formed. 

3. The spores arise in basipetal order from the sterigmata. 
Are the spores relatively few or abundant? What is their 
shape? color? Is the spore wall thickened? Do the spores 
contain protoplasm ? oil ? vacuoles ? 



112 A MANUAL OF BACTERIOLOGY 

4. Draw a conidiophore and spores. 

5. Remove from a Petri dish a bit of agar on which Asper- 
gillus has been sown two days. Study under the microscope 
the mycelium it contains. Have the hyphse cross walls ? Do 
they branch extensively ? Draw a bit of mycelium. 

6. Add some starch to liquid wort and iaoculate with 
Aspergillus oryzae. Test the solution from time to time for 
sugars with Fehliug's solution. 

Exercise 154. Study of Torula 

The genus Torula resembles in most respects that of Saccha- 
romyces, except that it does not produce spores. The Torulae 
are widely distributed ia nature. They have the power of setting 
up alcoholic fermentation, but most of them produce by-products 
which give the solution undesirable odors or flavors. After a 
time they may cause slimy must. 

1. Expose Petri dishes of sterile wort agar to the air. Incu- 
bate them at 25° C. for two days. 

2. Transfer Torulse from pure cultures or from the Petri 
dishes to tubes of wort agar, to fermentation tubes of lactose, 
to tubes of beef broth, and to gypsum blocks. 

3. Make stains and write descriptions of the organisms ; note 
the differences and similarities between Torulse and yeasts. 
Some Torulse produce gas from lactose, but the true yeasts 
do not. 

Exercise 155. Study of Dematium pullulans 

This fungus is widely distributed in nature, especially upon 
fruits, and occurs in damp situations in breweries. It is a dele- 
terious organism in brewing, because it is not only incapable of 
causing fermentation, but also produces films of growth in the 
wort and darkens the wort of light-colored beers. 

1. Inoculate tubes of wort agar and flasks of liquid wort 
with pure cultures of Dematium pullulans. 

2. Find two methods of producing conidia from the hyphse. 
Find gemmse, or chlamydospores, in the older cultures. Do the 
conidia resemble yeast in any way ? 

3. Make drawings to illustrate the different phases found. 



FERMENTATION OKGANISMS 113 

Exercise 156. Obtaining Mold Cultures from Spores in the Air 

1. Put some slices of bread on a plate and steam them for 
ten minutes in the Arnold sterilizer. 

2. Remove and place the plate on a laboratory table for 
fifteen minutes, then cover with a bell jar. 

3. Under another bell jar place sUces of lemon or pieces of 
cheese. 

4. Examine the cultures day by day until they show spores. 
The material may be used for study, or pure cultures may be 
prepared by using the isolation methods described in Exercise 63. 

Exercise 157. Culture of Molds on Liquid Media 

Mold fungi are usually capable of culture upon the surface of 
liquid media. The mycelium of Aspergillus or PenicilUum forms 
a disk which floats upon the surface of the liquid. Spores will 
develop upon the upper surface of the disk of mycelium. 

1. Prepare 100 cc. of the synthetic solution described in 
Exercise 54 ; divide it between two Erlenmeyer flasks of 200 cc. 
capacity. 

2. Make a series of other solutions in which substitutions are 
so made that one essential element is lacking in turn from some 
solution in the set. 

3. Sterilize the flasks in the Arnold sterilizer. Inoculate with 
Aspergillus or Penicillium and make records of the amount of 
mycelium and spore formation in each. 

Exercise 158. The Stunulating Action of Weak Poisons 

The physiological action of small amounts of poison may be 
well demonstrated by the culture of fungi on Uquid media. In 
the weak dilutions there will be stimulation, while at higher 
concentration there is toxic action. 

1. Prepare a hter of the synthetic solution as in the fore- 
going exercise. 

2. Measure off 50 cc. portions which can be put into 200-cc. 
flasks. 



114 A MANUAL OF BACTERIOLOGY 

3. Add to the different flasks amounts of zinc sulphate to 
make the following concentrations of the toxic agent: 1.0, 0.25, 
0.12, 0.06, 0.03, 0.015, 0.007, 0.0035, 0.00 per cent. 

4. Sterilize in the Arnold sterilizer and inoculate with Asper- 
gillus. 

5. Watch the cultures and make records of the amount of 
mycelium and spore development. 

Exercise 159. Demonstration of the Presence of Arsenic by Means of 
Penicillium brevicaule 

1. Mix the materials to be examined with bread crumbs 
(preferably brown or graham bread) and neutralize if necessary. 

2. Sterilize in steam and inoculate when cool with Peni- 
eilUum brevicaule. Close the flasks with tight-fitting plugs. 
Incubate at 37° C. 

3. Twenty-four to forty-eight hours after the mass becomes 
thoroughly overgrown with Penicillium, open the flasks and ex- 
amine for odor. An odor of garlic is indicative of the presence 

of arsenic. 

Exercise 160. The Amylolytic Power of Molds 

1. Measure off 50 cc. of liquid wort into a flask of 200 co. capac- 
ity and add 1 g. of starch. Sterilize in the Arnold sterilizer. 

2. Inoculate with Aspergillus oryzae. After growth is evi- 
dent, test the solution for reducing sugars with Fehling's solution. 

Exercise 161. The Peptonizing Action of Molds 

1. Melt several tubes of beef-peptone gelatin and pour into 
sterile Petri dishes. 

2. When the gelatin has hardened, inoculate the center of 
each plate with spores of Penicillium. 

3. Keep the plates at 20° C. and note the progress of hque- 
faction as the colony spreads over the gelatin. 

4. Another method consists in growing the Penicillium in a 
tube of beef broth until spores are formed, then filtering off the 
medium. Add several drops of toluol and pour the liquid into 
a tube containing sterile gelatin. Follow the hquefaction of the 
solid gelatin from day to day by marks on the wall of the tube. 



APPENDIX A 
STERILIZATION 

Many of the student's difficulties begin and end with the sterili- 
zation of the culture media. Wherever accurate work is to be done, 
much attention must be given to the process of sterilization. Modern 
bacteriological work rests upon the pure culture. So long as con- 
tamination is suspected or known, uncertainty and doubt concerning 
the results will prevail. The first requisite for the vigorous growth 
of a pure culture is, therefore, successful sterilization. 

The principal agents of sterilization are three — chemicals, heat, 
and filtration. Each finds its application for special purposes. 

STERILIZATIOX WITH CHEMICALS 

The efficiency of various chemicals as germicides has been investi- 
gated by numerous biologists. One class of germicides are used in 
surgery and contact disinfection work, while another class of more 
or less volatile compounds are used in laboratories, where toxin and 
enzyme work is carried on. In choosing a germicide for sm^gical 
work it is desii'able to have one which shall be highly toxic to the 
bacteria and shall not at the same time impair its efficiency by form- 
ing insoluble compounds with the body fluids which may be present 
in the wounds. 

Certain salts of the heavy metals have shown strong germicidal 
properties ; among them mercuric bichloride (corrosive sublimate) 
stands first in efficiency and applicability. The concentration of 
bichloride generally used for disinfection and surgical work is one 
part to a thousand of water. Since this substance is slowly soluble in 
water, a hydrochloric acid solution may be first prepared and further 
dilutions made as required. Thus, 200 g. of mercuric bichloride dis- 
solved in a liter of hydrochloric acid makes a 20 per cent stock solu- 
tion. If 5 cc. of this solution be diluted to 1000 cc, it makes a 
1 : 1000 solution of bichloride. In the laboratory the stock solution 
may be conveniently kept in siphon bottles upon the stock table. 

ll5 



116 A MANUAL OF BACTERIOLOGY 

Salts of silver, lead, and copper have also found more or less use 
at times as disinfectants, but their efficiency is not in proportion to 
the cost of materials and the labor of preparation. 

Potassium permanganate is a readily soluble salt which is used 
to some extent in surgery. Its value is impaired by the fact that 
it is easUy reduced by organic substances with which it comes in 
contact. 

Potassium bichromate is less easily reduced than the permanganate 
and is sometimes used for sterilizing seeds. There are no instances, 
however, in which mercuric bichloride would not do the same work 
as well or better. 

Iodine in solution, especially in a solution of potassium iodide, is 
toxic to bacteria and may have limited use as a disinfectant. 

All acids are more or less fatal to microorganisms, although there 
are certain microorganisms that will resist some of them. The more 
highly dissociated acids are the most toxic to bacteria. 

Phenol (carbolic acid) has wide use as a germicide and for contact 
disinfection. It mixes readily with water in all proportions and 
does not readily enter into combination with organic substances. It 
is not as toxic to microorganisms as mercuric bichloride, and is more 
irritating to the skin. It is usually employed in a concentration of 
1 : 20 (5 per cent). 

Lysol and tricresol are cresol products widely used as contact 
disinfectants. They possess efficiency without irritating the skin or 
mucous membrane. Lysol, which contains about 60 per cent cresylic 
acid, is generally used in solutions ranging from 1 to 3 per cent. 
Tricresol has greater germicidal power than phenol, but is not as 
toxic. It does not lose its germicidal power in the presence of albu- 
mins, nor discolor metal instruments. It is used in solutions of 0.5 
to 1.0 per cent. 

Thymol, salicylic acid, and benzoic acid are weak germicides. 
They may be used in special cases, but cannot be relied upon to give 
perfect sterilization. They are to be regarded as agents for retarding 
bacterial activity rather than as sterilizing agents. 

Calcium hypochlorite and chlorine have found extensive use for 
treating water and sewage. At the concentrations used it is seldom 
that complete sterilization is accomplished, but it is possible to 
reduce the number of organisms to a negligible quantity and to 
annihilate bacteria of the colon-typhoid type. Ozone has in some 
cases given complete sterility when applied to milk and waters, but 



APPENDIX A 117 

in other cases it has not proved certain. Hydrogen peroxide is a 
weak germicide but is useful in some cases. 

The volatile group of antiseptics consists of a few organic com- 
pounds, which are more or less toxic to microorganisms. 

Formaldehyde, either as a gas or in a strong solution (known as 
formalin), is an efficient and economical germicide. In a 4 per cent 
solution it quickly kills all organisms with which it is in contact. It 
is quite irritating to the skin, however, and must be employed with 
care. The gas is readUy liberated from the solution by the use either 
of heat or of potassium permanganate. 

Chloroform, acetone, and carbon bisulphide have more or less exten- 
sive use in sterilizing liquid media, where it is not practical to use heat, 
but it will not do to assume too quickly that chloroform or carbon 
bisulphide has sterilized a solution ; the solution should be incubated 
and observed after several days, to make sure that no growth occurs. 

Alcohol and ether have somewhat limited use as sterilizing agents. 
Alcohol of 60 or 70 per cent strength may be used for quick treat- 
ment of glassware when time or location forbids the use of other 
agents. For sterilizing pipettes or tubing used for taking samples in 
the field a brief washing with alcohol just previous to the time of 
using serves very well. After draining off the alcohol, the tubes 
should be rinsed with the solution to be sampled. 

STERILIZATION WITH HEAT 

Heat is the sterilizing agent most useful for general bacteriological 
work. If properly applied, it produces no detrimental conditions in 
the culture media, and at the same time it is efficient in killing 
microorganisms . 

Dry or moist heat may be used, according to the nature of the 
object to be sterilized and the pui'pose for which it is to be used. 

Hot-air sterilization. Dry sterilization is accomplished by placing 
the objects in a hot-air sterilizer, which is a gas-heated oven con- 
structed of sheet iron or other metal. The ovens constructed for this 
purpose have double walls so arranged that the hot air circulates 
through the chamber. The outer walls are covered with asbestos and 
provided with an opening for the insertion of a thermometer. The 
Lautenschlager oven is one of the best for dry sterilization. If a 
simple, cheap oven is to be used, careful tests should be made, to 
ascertain the relative temperature in its various parts. 



118 A MANUAL OF BACTEKIOLOGY 

Experience has shown that objects must be exposed to dry heat' 
for a longer time than to moist heat. In practice it is necessary to 
maintain a temperature of 140°-160° C. for one hour, or of 125° C. 
for two hours. Objects placed in the hot-air sterilizer should be per- 
fectly clean and dry. After the heat is turned ofP, the door should 
be kept closed until the temperature of the oven has fallen to near 
the temperature of the room. 

The Lautenschlager oven is heated by a tubular gas burner extend- 
ing around three sides of the base. If possible, the burner should 
connect directly with the pipe supplying the gas, and entirely avoid 
connections of rubber tubing. The special handling of various objects 
in the hot-air sterilizer is described below. Needles, f orcep tips, and 
scalpel blades may be sterilized directly in the Bunsen flame. 

Steam sterilization at ioo° C. Moist heat in the form of steam is 
the most efficient sterilizing agent for the culture media usually 
employed. 

At the temperature of boiling water most forms of germ life are 
killed, although, as previously stated, numerous spores may success- 
fully resist this temperature. Sterilization may thus be effected in 
a boiler over an ordinary water bath. For economical and effective 
work, however, the Arnold type of sterilizer is preferred and; in 
some form, is in use in most laboratories. 

The type of sterilizer which is heated over a gas burner is pro- 
vided with a double bottom. The lower bottom contains only a thin 
layer of water, which consequently boils quickly and the steam passes 
up through a short chimney into the chamber of the sterilizer. Water 
enters the lower bottom through small holes from the reservoir above, 
which will hold a supply sufficient for several hours. 

The chamber of the Arnold sterilizer has double walls, which are 
so arranged that the condensed steam drips back into the reservoir. 
The chamber of the sterilizer is fitted with one or more shelves upon 
which media may be placed. 

If the laboratory is supplied with steam, it will be found more 
economical and satisfactory to install a sterilizer fitted for steam. 

The Arnold sterilizer and all other steam sterilizers should be 
installed, if possible, in a room separate from the main laboratory, 
and should stand under a hood which will carry off escaping steam. 

The sterilizer should always be provided with a thermometer 
whose bulb extends about 6 cm. into the chamber ; otherwise it is 
difficult to know the exact time at which the contents reach the 



APPENDIX A 119 

temperature of 100° C. The time required for the chamber to heat 
up depends upon the amount of material which it contains. 

When media in test tubes are to be sterilized, the tubes should 
always be held in wire baskets which permit the easy entrance of 
steam. If tin cups or beakers are used, it seems to be almost impos- 
sible to dislodge a layer of cooler air from the bottom of the cup, 
and consequently sterilization is onlj- partial. 

When the steam first enters the chamber of the sterilizer, conden- 
sation takes place, and enough water often drops from the roof to 
soak the cotton plugs. To guard against this, one may wait until the 
interior of the sterilizer has reached a temperature of 100° before 
introducing the objects to be sterilized, or the cotton plugs may be 
covered by tj-ing manila or parchment paper over the top of the 
wire baskets. When flasks of media are being sterilized, small beakers 
may be inverted over the cotton plugs. Such cotton plugs as are 
found water-soaked when the media are rfemoved from the sterilizer 
should be replaced by new plugs and the vessels resterilized. 

Circulating steam at 100° C. will kill the vegetative forms of 
microorganisms in fifteen to twenty minutes, but spores will survive 
much longer heating. The success of discontinuous, or fractional, 
sterilization was demonstrated by Tyndall. In general practice, the 
media are sterilized for twenty to thirty minutes on three successive 
days. In the intervals between sterilizations the media should be 
kept at temperatures favorable for bacterial growth, in order that 
the resting spores may germinate and pass into the vegetative stage, 
and thus be killed at the next sterilization. Even with this treat- 
ment the spores of anaerobic bacteria in the superficial layers of the 
media may not develop, owing to the access of oxygen, and so may 
survive the process of sterilization. 

Steam sterilization under pressure. The autoclave is a strong-walled 
steam chest which maj' be tightly closed. In it the steam is used 
under pressure, and temperatures above 100° C. are obtained. By 
this means both the vegetative forms and the bacterial spores are 
killed, and the necessity for discontinuous sterilization disappears. 

The type of autoclave in most common use is a heavy-walled 
copper cylinder set in a frame above a large gas burner. The upper 
end has a close-fitting lid which clamps on with six turnscrews. 
The cover is provided with a steam-pressure gauge, a thermometer, 
a stopcock, and a safety valve. A rack which fits the inside of the 
autoclave provides one or two shelves for holding the vessels of media. 



120 



A MANUAL OF BACTERIOLOGY 



Before rising the autoclave sujB&cient clean water is poured in to 
cover the bottom to a depth of 5 cm. It is best to use distilled water 
and to renew it frequently. If the water is dirty or contains media 
escaping from broken vessels, it is liable to foam up and wet the 
cotton plugs. 

The lid is securely clamped after the media are put in and the 
gas burners lighted. The stopcock must remain open for the escape 
of air, since the requisite temperature may not be attained unless 
the autoclave contains nothing but steam. When the thermometer 
registers 100° C. and steam issues rapidly from the stopcock, it may 
be assumed that the air has been displaced, and the stopcock may 
then be closed. The gas supply may then be reduced somewhat 
and pressure allowed to run up to 10 or 15 pounds. The contents 
of the sterilizer are held at this pressure for twenty to thirty min- 
utes. The gas is then shut, off and the sterilizer allowed to cool. The 
stopcock may be gradually opened as the pressure falls, but caution 
should be used, because, if the pressure is too suddenly reduced, the 
superheated media may boil over and dislodge the plugs. If direct 
steam is used instead of a gas burner, much time and expense will 
be saved. 

The corresponding pressures and temperatures are shown in the 
following table : 





Steam Pressure 


Temperature 


Steam Pressure 


Temperature 


Founds 


Fahrenheit 


Centigrade 


Foands 


Fahrenheit 


Centigrade 





212 


100 


15 


251 


121.5 


5 


228 


109 


20 


260 


126.5 


10 


240 


115.5 


40 


287 


141.5 





Partial sterilization — Pasteurization. For many industrial purposes 
it is not necessary to obtain complete sterilization, provided certain 
forms can be killed. This is readily accomplished by the use of tem- 
peratures somewhat below the boiling point of water. The process 
is named for Pasteur, who first suggested its use in the treatment of 
grape must to overcome wine faults. 

In this country Pasteurization is extensively used in commercial 
dairy work. In different establishments different temperatures are 
used. As a usual thing the milk is heated to 60°-65° C. for twenty 
minutes. In what is known as the continuous process the milk is sub- 
jected to a momentary heating of 85° C, and is then quickly cooled. 



APPENDIX A 121 

The criterion employed for the Pasteurizing temperature is the 
temperature necessary to kill such pathogenic organisms as the 
typhoid and tuberculosis bacteria, and also the majority, if not all, 
of the lactic-acid bacteria. If the time allowed for heating be short, 
then the temperature must be higher, and vice versa, within certain 
limits. 

The principle of Pasteurization is somewhat similar to that of 
fractional sterilization ; that is, non-spore-forming bacteria are killed 
by the temperatures employed. The process is to be regarded as one 
which retards germ development rather than as one which prevents it. 

Pasteurization is also extensively used in the production of beer. 
The beer, in bottles or jugs, is submerged for half an hour in water 
at a temperature of 60°-65° C. 

STERILIZATION BY MEANS OF FILTRATION 

Liquids which cannot be treated with chemicals or with heat 
may be sterilized by filtration. In the preparation of enzymes and 
toxins this method of treatment is extensively employed. 

The filters used are cylindrical vessels, closed at one end like a 
test tube, which are called bougies. The Chamberland filters are 
made of porous porcelain, hard-burned and unglazed. The Berkefeld 
filters are made of kieselguhr, a fine, diatomaceous earth. The 
Chamberland has the finer pores ; the Berkefeld filters are quicker ; 
with either one, however, it is necessary to use aspiration or pressure 
to hasten the filtration. 

The simplest way to use such a filter, when a small quantity of 
liquid is to be filtered, is to place the liquid inside the filter and force 
it out by means of compressed air. Wrap the nipple end of the 
bougie with a firm band of absorbent cotton about 7-10 cm. wide. 
Invert the bougie in a glass cylinder 5-10 cm. longer than the bougie, 
so that only the nipple end projects from the mouth of the cylinder. 
The cotton wrapping should fit rather firmly in the mouth of the 
cylinder, and the nipple of the bougie should be wired to prevent its 
slipping down into the cylinder. Wrap this apparatus in manila 
paper and sterilize it in the dry oven for two hours at 140° C. 

After the apparatus is cool, insert a small funnel in the nipple of 
the bougie and pour in the liquid to be filtered, taking care not to 
wet the cotton surrounding the upper end of the bougie. Connect 
the bougie nipple with an air pump by means of heavy-walled 



122 



A MANUAL OF BACTERIOLOGY 




Fig. 41. Filtering apparatus 

Air is pumped into the tank and furnishes pressure to force the solution 
through the filters 

rubber tubing, which should be wired on. Turn on the air slowly 
until a pressure of 15-20 pounds is exerted. The liquid should 
drip rapidly from the bougie, but an excess of pressure should be 
avoided, because small organisms might be forced through the wall 
of the filter 



APPENDIX A 



123 



Aside from the limited capacity afforded by the use of the 
above method, it is open to another objection : namely, the residuum 
accumulates upon the inside of the bougie and is hard to remove. 

An extremely satisfactory filtering apparatus is that 
shown in Fig. 41, which was made for the author after C^^^^ 
the filters used in the Bureau of Soils of the United 
States Department of Agricidture and described by ^^s~5 
Schreiner and Failyer in Bulletin No. 31 of that bureau. 
The metal cylinder which contains the bougie is large ^^=2> 
enough to hold 600 ec. or more of solution and to take 
a bougie of any length. This last feature is not with- 
out value, since it is at times impossible to obtain 
bougies short enough to fit the apparatus of European 
manufacturers. The metal filtering cylinders are lined 
with porcelain enamel and are thus entirely nontoxic 
— an advantage over the older brass cylinders or those 
which were silver-plated inside. 

The construction of the metal filtering cylinder is 
evident from the accompanying illustration (Fig. 42). 
Both ends are accurately threaded and receive screw 
caps. The lower end has a rubber gasket which fits 
on the nipple of the bougie. This gasket goes into 
the lower end of the cylinder and is held in place 
by the nipple plate and screw collar. The upper end 
of the cylinder is closed with a cylinder head and a 
screw collar. A good rubber washer lies between the 
cylinder and the cylinder head. The cylinder head 
has a flanged opening at the center, which is threaded 
to fit the pipe from the air tank and usually needs a 
rubber washer. 

The air pressure needed for filtering, which is from 
1 to 2 atmospheres, is supplied by any convenient 
form of pump. The air tank, which should hold from 
10 to 20 gal., serves to store compressed air and to 
equalize the pressure for filtration. The opening on 
the side of the tank is fitted with a stopcock and a bicycle valve, 
so as to hold the pressure in the tank. The bougie is wrapped in 
manUa paper and sterilized in the hot-air sterilizer. A plug of cotton 
should be inserted in the nipple end and the whole carefully wrapped 
ia manila paper. When sterilized and cool, the bougie is put into 



Fig. 42. Cyl- 
inder for the 
Chamberland 
Filter (after 
Schreiner and 
Failyer) 



124 A MANUAL OF BACTERIOLOGY 

place in the filtering cylinder. The solution to be filtered is poured 
into the large end of the cylinder, which is connected with the air 
tank. A piece of glass tubing with a rubber-tubing connection is 
slipped on the nipple end of the bougie. This tubing should pre- 
viously be sterilized in 1 : 1000 bichloride solution or in 70 per cent 
alcohol, and rinsed with sterile distilled water. 

A cotton-plugged Erlenmeyer flask which was previously dry 
sterilized is placed to receive the filtrate. The cotton forms a pack- 
ing around the glass tubing. The time of filtration varies with the 
pressure and the nature of the solution to be filtered. With a pres- 
sure of 1 atmosphere the filtration will usua,lly be rapid. 

The bougie can easily be removed for cleaning by unscrewing the 
cap at the bottom of the cylinder. The scum on the exterior can be 
removed by washing in water, using a small brush with stiff bristles. 
Care should be taken to prevent wash water from entering the mouth 
of the bougie. After washing, rinse in distilled water and dry it for 
resterilization. A Chamberland bougie may be used continuously for 
two or three days before resterilization is necessary. Continued use 
should be avoided, since colonies of microorganisms may grow 
through the wall of the filter bougie. 

When the bougie has been used for some time, its effieiency is 
impaired and the filtration goes very slowly. The dif&culty is due 
to the clogging of the pores of the filter with organic matter. The 
bougie should be dried and heated to redness above a Bunsen flame 
for half an hour. This can be done by hanging the bougie with the 
nipple upward in a cylinder of sheet iron covered with asbestos 
and partially covered at the top and bottom with asbestos rings. A 
single Bunsen burner placed at the bottom will heat the tube to 
redness in a short time. 

After cooling, place the bougie in the filtering cylinder and run 
distilled water through it, to wash out the charred organic matter. 
This treatment of the bougies restores them to their former efficiency. 



STERILIZATION BY MEANS OF LIGHT 

It is almost universally admitted that sunlight is one of the best 
and cheapest of germicides, but for rapid work light is not eco- 
nomical. Eecent work by Henri and his associates shows that the 
ultrarviolet light of a quartz-tube mercury lamp may give complete 
sterilization of water and sewage without producing any chemical 



APPENDIX A 125 

change in the liquids thus treated. In milk the bacteria are likewise 
killed, but there is a complete or partial destruction of the enzymes 
of normal milk. Other workers, like Schwarz and Aumann, have 
found that complete sterilization is frequently not obtained, although 
the bacterial content is greatly reduced. 

The process of sterilization by this means seems promising for 
laboratory work and, when better developed, ought to be of great 
assistance to the bacteriologist. Whether it will be of equal value to 
the engiaeer remains to be seen. 



THE TECHNIQUE OF STERILIZATION FOR 
SPECIAL PURPOSES 

1. Glassware, pipettes, and instruments. The most effective method 
of sterilizing these articles is in the hot-air sterilizer. Objects to be 
sterilized should be perfectly clean, dry, and free from dust. Flasks 
should be plugged with cotton, and the plugs may be covered with 
paper caps. Petri dishes, unless they are to be used immediately, 
should be wrapped in manila paper or inclosed in the sheet-iron 
cases made for the purpose. Pipettes should have the large end 
plugged with absorbent cotton and should be sterilized in sheet-iron 
boxes or wrapped in paper. Knives, scrapers, scalpels, forceps, 
needles, and spatulas may be sterilized, at the time of using them, 
in a gas flame. If they cannot be used while hot, they should be 
sterilized in a sheet-iron box, as pipettes, or in large test tubes 
plugged with cotton. 

Where time or facilities will not permit of hot-air sterilization, 
the articles mentioned above may be sterilized with chemicals. 
Pipettes may be sterilized by drawing up alcohol of 60-70 per cent 
strength and rinsing the pipette with sterile distilled water or with 
the solution to be employed. Flasks may be rinsed with alcohol or 
mercuric bichloride solution and rinsed with sterile water or the test 
solution. Instruments may be washed with a 1 per cent solution of 
lysol or tricresol and dried on sterile surgeon's gauze. Wherever 
possible, however, sterilization by heat is to be preferred. 

2. Liquid media and other solutions. For general work solutions 
are most easily and thoroughly sterilized in the Arnold sterilizer or in 
the autoclave. They may also be sterilized by heat on a steam bath 
or, if organic compounds be absent, by boiling over a flame, although 
in the latter case it is difficult to avoid steaming the cotton plugs. 



126 A MANUAL OF BACTERIOLOGY 

3. Gelatin and agar media. Media whicli contain gelatin should 
not be heated at high temperatures in the autoclave nor for longer 
periods than absolutely necessary in the Arnold sterilizer. An excess 
of heat breaks down the gelatin so that it will not harden when. cool, 
riasks and tubes of gelatin media should be chilled as quickly as 
possible after removal from the stetilizer. After the gelatin has 
hardened, the vessels may be put into the locker at room temperature, 
to await the next sterilization. The temperature at which it is kept 
should be high enough to permit the germination of such resistant 
spores as may have survived heating. When three successive steriliza- 
tions are given at twenty-four-hour intervals, the first heating may be 
twenty minutes, the second fifteen minutes, and the third ten minutes. 
After the last heating the media may be put into the locker and held 
for a few days, during which time they are closely watched for colo- 
nies. Such tubes as show colonies should then be discarded. Gelatin 
media may be sterilized in the autoclave for fifteen minutes, provided 
the pressure does not rise higher than five pounds and the vessels 
containing them, when removed, are plunged at once into cold water. 

One source of difS-Culty in sterilizing media containing gelatin 
comes from the spores of microorganisms which may have developed 
during the preparation of the gelatin itself while in the vats or dry- 
ing house of the manufacturer. Only the best grade of gelatin 
should be used for bacteriological work. Another frequent cause of 
failure in sterilizing media is due to the presence of resistant spores 
in beef extract. These spores develop in the meat juice during the 
process of preparation and evaporation. It is better, when making 
gelatin media, to make the meat extract yourself, according to direc- 
tions given in Exercise 8. If, however, it is necessary to use com- 
mercial beef extract, make the bouillon first and sterilize it in the 
autoclave ; then add the gelatin and carry on subsequent operations 
in the Arnold sterilizer. 

Agar media are not likely to present much difficulty in the way of 
sterilization so far as the agar itself is concerned, although it is not 
advisable to use more heat than necessary. If agar media fail to 
harden after cooling, it is probably due to an excess of sugar or of 
acid. In making media from fruit decoctions this is an important 
precaution. If it is found that the acidity of the agar medium is 
excessive, the medium should be divided into two equal parts. Bring 
one portion to approximate neutrality with sodium-hydrate solution ; 
then recombine the two portions and heat for a short time, with 



APPENDIX A 127 

stirring, on the steam bath. If this fails to reduce the acidity suffi- 
ciently, then the process may be repeated. An excess of alkali, 
however slight, must be avoided, because it will seriously discolor 
media made from plant extracts. 

4. Sugar-containing media. All organic chemists are aware of the 
changes wrought by the action of heat upon sugars. Dry heat con- 
verts part of the sugar into furfuraldehyde. The moist heat of the 
autoclave converts sugars into humuslike bodies and other substances 
unfavorable to the growth of microorganisms. Henderson has found 
that at a temperature so low as that of 37.6° C. a glucose solution 
will, in the course of several days, lose its optical activity. 

An excess of heat should always be avoided in sterilizing culture 
media containing either monosaccharides or disaecharides. The media 
should be intermittently sterilized in the Arnold sterilizer by heating 
for twenty minutes the first day, fifteen minutes the second day, and 
ten minutes the third day. 

5. Solid vegetables. In plant-pathological investigations much 
use is made of sterilized pieces of potatoes, beets, carrots, turnips, 
rice, corn meal, and other vegetable substances. Much difficulty 
is encountered, at times in sterilizing potatoes and beets, because 
they carry spores of very resistant soil organisms. When preparing 
tubers or roots for culture media it is well to scrub them thoroughly 
with a stiff brush, then wash in a 1 : 1000 bichloride solution, and 
finally rinse with distilled water before cutting plugs or slices. 

There is some possibility that the heat of sterilization, acting upon 
these carbohydrate substances, .may produce compounds toxic to 
microorganisms, as has been pointed out by L. ~R. Jones. It is there- 
fore well to use as little heat as possible for sterilization, although 
the autoclave must generally be used in order to successfully sterilize 
potato plugs. 

6. Milk. Sterilization will be much more successful if fresh, clean 
milk is obtained. In cities one shoidd obtain certified milk. Where- 
ever possible, the milk should be obtained directly from the dairy. 
Market milk often contains a large number of organisms, and trouble 
arises from the heat-resistant types. The cream should be removed 
by centrifuging and the milk placed in tubes or flasks for sterilization. 

Here again an excess of heat brings about undesirable changes 
in the medium. Prolonged heating coagulates the casein more or 
less completely and may transform the sugars into compounds less 
favorable for bacterial growth. 



128 A MANUAL OP BACTERIOLOGY 

Milk may be steamed at 100° C. for fifteen minutes on four or five 
consecutive days. It should then be incubated at 30° C. for three 
days, and all tubes -which show signs of microbial activity can be 
thrown away. It is better to discard a few tubes in which growth 
occurs than to lose all on account of overheating. 

7. Blood serum. If the slaughter of the animal can be supervised, 
it is well to have the region of the wound and the knife previously 
sterilized. Collect the blood from the sheep or ox in a sterile glass 
jar in a sterile cylinder. Allow the vessel to stand long enough to 
form a clot before leaving the slaughterhouse. At the laboratory 
detach the clot from the sides of the jar by means of a sterile glass 
rod and place the cylinder in the ice chest for forty-eight hours. The 
clear serum is drawn out with sterile pipettes and placed in sterile, 
cotton-plugged tubes. 

The tubes of sermn are sterilized by heating them to 56° C. in a 
water bath for half an hour on each of three consecutive days. On 
the fourth day the tubes are placed in a sloping position in a serum 
inspissator and heated to 72° C. until coagulation occurs. The tubes 
are then transferred to an incubator at 37° C. for forty-eight hours. 
Those which show indications of infections are then discarded. 

8. Soil. For many experiments with bacteria and phytopathologi- 
cal fungi sterilized soil is required. Unfortunately, successful sterili- 
zation of soil is diificult to accomplish. If all germ life is destroyed, 
so high a degree of heat is required that profound changes in the 
constitution of the soil are brought about. The air held in the soil is 
very difficult to remove and acts as a heat insulator. This condition 
may prevent the soil in the center of a large pot from receiving 
enough heat to kill all germ life. 

Attention may be called to three methods of soil sterilization 
which have been used by investigators : dry heat, moist heat, and 
volatile antiseptics. 

Richter ^ found that exposure to a dry heat of 100° C. for six hours 
on three consecutive days increased its absorptive power for water, 
the amount of nitrogenous material soluble in water or dilute hydro- 
chloric acid, and the amount of water-soluble organic matter. 

Seaver,^ and Seaver and Clark,* have studied the effect of dry 
heat upon soil with relation to the growth of pyrophilous. fungi and 
green plants. They have shown that heat alters the soil beneficially 

1 Landw. Versuchsst. 47 : 269. 1896. 2 Mycologia 1 : 131. 1909. 

8 Mycologia 2 : 109. 1910. Biochemical Bulletin No. 1: 413. 1912. 



APPENDIX A 129 

for the development of the fungus Pyronema. The growth of oats was 
slightly accelerated in soils heated at low temperatures (90°-120° C), 
but above this temperature growth was retarded, the retardation in- 
creasing with the temperature to which the soUs had been heated. 

The water extract of soil heated to 120° C. was yeUow in color, due 
largely to charred organic material. The color was deeper in extracts 
of son which had been heated to stiU higher temperatures. Both the 
inorganic and the organic extractions were increased about ninefold 
by heating soU to 170° C. for two hours. Soils that were heated until 
they were unfavorable for the growth of oats were nevertheless very 
favorable for the growth of Pyronema. 

Moist heat produces much the same effect upon soUs as dry heat, 
provided the temperature goes high enough. 

By steam heating, the physical, chemical, and physiological prop- 
erties of soils are more or less altered. The chemical changes consist 
mainly in an increase in soluble matter in the heated soils. This is 
partly of an inorganic nature, but the largest increase is in the 
organic matter rendered soluble. Ammonia may be formed from the 
reduction of nitrates to nitrites and ammonia, but it is especially 
formed from the decomposition of organic compounds. Large amounts 
of nitrogenous matter are made soluble and apparently more available 
for plant use. Carbon dioxide is also produced in large quantities. 

Pfeiifer and Francke '^ steamed soil at 1 atmosphere pressure for 
three hours and found a consequent increase in dry weight of plant 
growth and nitrogen content over the unheated. Deherain and 
De Moussy'' prevented nitrification in soils by heating them at 
120° C. in an autoclave, although the sterilized soil, when inoculated 
with a portion of the unsterilized soil, produced more nitric nitrogen 
than the unsterilized soil. 

Students of this problem sooner or later find that an improvement 
due to heating soils is often preceded by a marked injury to plant 
growth. The heat, although it increases the quantity of water- 
soluble plant nutrients, seems to produce substances which are poison- 
ous to plants. According to the amount of this poison and the 
sensitiveness of the plants to it, a positive or a negative acceleration 
of plant growth is produced. The results appear to vary in different 
soils. The addition of calcium carbonate to the soil before sterilizar 
tion prevents, almost if not entirely, the formation of poisonous 
substances. 

1 Landw. Versuchsst. 46 : 117. 1896. ^ Ann. Agron. 22 : 305. 1896. 



130 A MANUAL OV BACTERIOLOGY 

The first to notice this unfavorable effect of soil sterilization was 
Dietrich/ who concluded that some poison was generated by the 
action of heat upon the organic matter of the soil. Schulze ^ found 
that the immediate effect of heat was injurious to bacteria as well as 
to the higher plants. 

Koch and Ltlken' found that the application of heat produced 
immediate injurious effects in soils, even though fertilizers were 
applied in each case. The injurious effects were found to be un- 
important if the plants were started in summer instead of early 
spring. 

Lyon and Bizzell,* who also noted the immediate harmful effect of 
heat, found that, upon standing, the quantity of soluble matter de- 
creased, especially the total soluble nitrogen. The soluble organic 
and inorganic matter decreased in about the same ratio. The growth 
of wheat plants on steamed soil seemed to hasten its recovery from 
the injurious effects of heating. The time required for the various 
soils to recover from these effects was, with one exception, in the 
order of their relative productiveness. 

The experiment of Pickering^ dealt with the effects of heated 
soils upon seed germination and upon tree growth. He interpreted 
his results to mean that the heat produced in the soil a nitrogenous 
compound which was inhibitory to the germination of seeds. This 
compound was sufficiently stable in solution for the extract of a 
heated soil to affect a normal soil when added to the latter. 

The first successful attempt to show the chemical nature of the 
changes in organic matter caused by heating soils is that of Schreiner 
and Lathrop.^ Heating the soils in the autoclave for three hours at 
about 135° C. produced an increase in water-soluble constituents and 
an increase in acidity. At the same time ammonia and amines were 
formed. By the process of heating, all the constituents isolated 
from the unheated soil, except nucleic acid, were increased, and, 
when not previously existing, xanthine, hypoxanthine, guanine, 
cytosine, and arginine were formed. These compounds are decom- 
position products of nucleic acid and protein material, and all are 
beneficial to plant growth. 

1 Jahresber. Landw. Versuchs. Marburg, 1901-1902. Bied. Centralbl. 32 : 68. 1903. 

2 Jahresber. angew. Bot. 1 : 37. 1903. Centralbl. f. Bakt., 2te Abt., 2 : 716. 1903. 

3 Jour. Landw. 55 : 161. 1907. 

4 Bulletin JVb. 275. Cornell Exp. Sta., 1910. 

« Jour. Agr. Sci. 2 : 411. 1908. Ibid. 3 : 32. 1908. 

6 Bulletin No. 89. Bur. ol Soils. U. S. Dept. Agr., 1912. 



APPi:]SrDIX A 131 

A substance toxic to plants — dihydroxystearie acid — was also 
formed by the heating process. If already present in the soil, the 
quantity of dihydioxystearic acid was increased ; it appeared, how- 
ever, even when lacking in the unheated soil. 

Sehreiner and Lathrop point out that, although the majority of 
compounds formed when soils were heated must be classed as bene- 
ficial, the harmful compound form.ed at the same time more than 
counteracts their effects. If this harmful compoimd can be elimi- 
nated or diminished, the full beneficial effects of heating will become 
apparent. Such changes in the constitution of harmful substances 
take place if a heated soil is allowed to stand in contact with the 
air, and are probably due to oxidation. 

Soils may also be sterilized by the use of volatile antiseptics like 
chloroform and carbon bisulphide. One or two per cent of the liquid 
antiseptic is usually added to the soil and mixed. After allowing one 
day for the antiseptic to act, the soil is spread out to allow it to pass 
off into the air. This method gives only partial sterilization, yet its 
use is usually followed by a large increment in ammonia and bacterial 
activity, followed by improvement in plant growth. 

There are several theories concerning this beneficial action of 
volatile antiseptics. Hiltner and Stormer'' propose a "Selective" 
action, through which the equilibrium of microorganic life in the soil 
is disturbed in such a way that ammonifying and other beneficial 
bacteria become predominant. 

Kussell and Hutchinson^ hold a somewhat similar view. They 
believe that the effect of partial sterilization is to kill off the larger 
phagocytic organisms which devour the beneficial bacteria. 

Koch" and his associates believe that the small quantities of anti- 
septics remaining in the soil exert a stimulating action upon bacteria 
and green plants alike. 

Grieg-Smith* holds that soils, especially poor soils, contain a 
bacteriotoxine which retards growth, and that the result of partial 
sterilization with carbon bisulphide or by heat is a destruction of 
these bacteriotoxines. 

The subject of partial sterilization by volatile antiseptics is in need 
of thorough investigation from the standpoint of the organic chemist. 

Methods of sterilizing seed. In order to secure vigorous seedlings, free 
from all microorganisms, great care must be exercised in sterilizing 

1 Jahresber. angew. Bot. 5 : 214. 1907. « Centralbl. f . Bakt., 2te Abt. , 31 : 185. 1911. 

2 Jour. Agr. Sci. 3 : 111. 1909. * Centralbl. f . Bakt. , 2te Abt. , 30 : 154. 1910. 



132 A MANUAL OF BACTERIOLOGY 

the seed. Purely physical sterilization is impossible, because the 
bacteria are even more resistant than the seed. Chemical agents 
have been found to give the best results. The ordinary antiseptics, 
such as alcohol, ether, chloroform, and toluol, have proved unsuitable 
for this purpose. The substances commonly employed for sterilizing 
seed are corrosive sublimate, silver nitrate, bichromate of potash, 
copper sulphate, bromine, and many of the mineral acids. The dis- 
infecting power of these substances depends largely upon four factors 
— strength of solution, time of action, temperature, and pressure. 

The ordinary procedure of soaking the seeds in mercuric chloride 
solution does not always give satisfactory results, because of the per- 
sistance with which occasional air bubbles remain on or inside the 
seeds, and thus prevent complete sterilization. 

Very good results have been obtained in sterilizing the seeds of 
corn, wheat, and peas by a modification of the method first described 
by Hutchinson and Miller,'' which consists in sterilizing the seeds in 
a mercuric chloride solution in vacuum. In the first place, well-dried 
seeds of even size are necessary. These should be thoroughly washed 
and cleaned in flowing water and perhaps 60 per cent alcohol before 
beginning sterilization. Transfer the seeds to a sterile bottle and, 
after adding mercuric chloride, remove the air bubbles with a vacuum 
pump. The apparatus for this purpose consists of the following 
(Fig. 43) : 

Five stout-walled glass flasks, wide at the mouth (capacity about 
500-800 cc), carrying steam-proof rubber stoppers with two holes in 
each for glass tubes, which are connected by rubber tubing, as seen 
in the diagram. Flask B is connected to flask A on one side, and on 
the other, by means of a three-way tube, to two glass bottles, C and 
D. ' These, in turn, are connected to flask E. Fill flask C with a 
0.25 per cent solution of mercuric chloride, D with distilled water, 
and E, which is smaller than the other flasks, about half full of 
50 per cent sulphuric acid. This bottle has a drawnout tube passing 
down through the cork almost to the bottom and serves to purify 
the air. 

After connecting the whole apparatus, place screw clamps on- the 
rubber tubing between all flasks, in order to prevent the liquid 
from flowing from one bottle into another; then sterilize in the 
autoclave at 125° C. for half an hour, or three times by fractional 
steam. Cool to 40° C. and connect flask ^ to a vacuum pump ; place 

1 Journal of Agricultural Science 3 : 2, 185. 1908. 



APPENDIX A 133 

the seeds in flask B by means of a funnel, to prevent contact be- 
tween the seeds and the neck of the flask. With the vacuum pump 
draw the mercuric chloride from C into B ; then close with a screw 
clip and exhaust B untn the solution begins to boil. By this means 
the disinfectant comes into direct contact with and is able to act on 
all portions of the seeds. Allow sterilization to go on for four to 
six minutes, then invert B and withdraw, by means of the pump, 
the mercuric chloride ; after this allow sterilized water to flow from 
D into B, and wash the seeds weU two or three times with this water. 
The seeds may then be transferred to Petri dishes and a sterilized 
layer of 1.25 per cent solution of beef-peptone agar poured over them, 
or they may be carried to dextrose-beef bouillon, one seed in each 




Fig. 43. Arrangement of bottles for seed sterilization 
For explanation see text 

tube. If the bouillon remains clear until the eighth day when kept 
in an incubator at 25° C, it indicates that aU bacteria were killed. 
When agar is used, allow the plates to solidify, invert, and place 
in incubator at 20°-25° C. At the end of three or four days the 
majority of the seeds will germinate and form roots 1~1J in. long. If 
these are free from molds and bacteria, they may be carried to 
sterile wide glass test tubes containing about 10 cc. of Tollen's me- 
dium or distUled water, over which cotton plugs have been placed. 
On the cotton plugs the seedlings will grow several inches long, and 
if no subsequent infection is noted, they may be carried over to cul- 
ture bottles. There is less danger of infection, however, when the 
seedlings are carried direct to the culture mediujn or soil in which 
they are intended to grow. 

Schroeder ^ recommends a very simple method for sterilizing seed. 
He found that the seed coats of the Graminese — for example, 

1 Centralbl. f. Batt., 2te Abt., 28 : 492-505. 1910. 



134 A MANUAL OF BACTEEIOLOGY 

barley and wheat — are easily penetrated by such agents as sublimate, 
iodine, alcohol, ether, chloroform, and acetic acid, while silver nitrate, 
copper sulphate, sodium fluoride, and barium chloride penetrate very 
slowly. Taking these results as a basis, he recommends the following 
method for the sterilization of seed : 

Wash the seeds thoroughly and allow them to soak for twelve to 
twenty-four hours in a 5 per cent solution of silver nitrate. Follow 
this with four washings in sodium-chloride water and then allow the 
seeds to stand for twenty-four hours in a dilute solution of sodium 
chloride. After this, drop the seeds into sterile bouillon and test 
them for the presence of molds and bacteria as described above. 

lu sterilizing corn Schulow reports good results from the use of 
1 per cent bromine solution for twenty minutes. 

Methods for growing higher plants under sterile conditions. In order 
to grow plants in a medium free from infection, vessels of careful 
construction are necessary. The weak point with nearly all pots built 
for this purpose is the method of watering and of packing cotton 
around the stem of the plant. Because of the long time required for 
plant growth there is always danger of infecting the culture medium 
if cotton is wrapped around the stem of the plant. To guard against 
this it Is well to conduct this experiment in a clean room, as free as 
possible from all outside contamination. A large variety of vessels 
have been recommended, but many of them are so complicated as to 
be of little practical value. Most of them are prepared either from 
metal or from glass ; the latter have given the best results. Vessels 
of metal — -for example, the Schulze^ pot — have many disadvan- 
tages : first, it is almost impossible to regulate the water supply ; 
second, in sterilization there is danger of the metal's forming com- 
pounds poisonous to plant growth. 

Schulow " claims to have grown corn plants free from all infection 
in glass cylinders thoroughly packed with absorbent cotton. 

The culture vessel (Fig. 44, 1) is a WoulfE's flask with three open- 
ings. The vessel used for water is a 2- or 3-liter flask of Jena glass 
(Fig. 44, 2) which carries a stopper with two openings. All stoppers 
and connecting tubes must be made out of the best Para rubber. The 
entire apparatus is made from glass and is much easier to prepare 
than the Schulze metal pot. The large Woulff flask should hold about 
3-4 liters and should be made from a good quality of glass. 

1 Landw. Jahrbiieher 30 : 219. 1906. 

2 Bericht. d. Deutsch. Bot. Gesell. 29 : 504. 1911. 



APPENDIX A 



135 



U tubes, A and A ', filled with, sulphuric acid, are used to purify the 
air. £ is a long, hard-glass tube enlarged at one end for sterile cot- 
ton and stopper. A and B are for use in the aeration of the soil (if 
liquid culture media are used, these may be omitted). Tubes D, E, 
and A serve for watering ; the sterile air is passed through A, and E 
then carries the water in flask 2 over into culture vessel 1. 

The large opening, C, in flask 1 carries a cylinder constructed as 
shown by Fig. 45. 

A wire net is fastened to the sides of C and passes down to the 
soil in flask 1. An outer glass cylinder 4-5 cm. in diameter and 




Fig. 44. Complete apparatus for growing seedlings under sterile conditions. 

(After Fred) 

1, Wonlft flask containing soil ; 2, Erlenmeyer ilask containing sterile distilled water 

15 cm. in length fits very loosely around C, in order to allow space 
for cotton between C and the cylinder. Within C there is an inner 
tube, about IJ-lJ cm. in diameter and 20 cm. long, which reaches to 
the wire net (see Fig. 45). In the top of this tube there is a cot- 
ton plug. Between these two tubes cotton packing and three glass 
rods about 0.4 cm. in diameter and 15 cm. long are placed. The cotton 
must be packed loosely, with one open space, as seen in Fig. 45, 1. 

If soil is to be used, cover the bottom of flask 1 (Fig. 44) with 
gravel, and fill this vessel with soil. Raise the water content of the 
son to 15 or 20 per cent, according to the type of soil. Now join 
flask 2, which has been previously filled with distilled water. Fasten 
a screw clip between D and E, in order to prevent the water in 



136 



A MANUAL OF BACTERIOLOGY 



flask 2 from flowing over into flask 1 while sterilization is going 
on. Wrap flask 1 with paper and place the entire apparatus, includ- 
ing cylinders for seed, in the autoclave and sterilize for two hours 





Fig. 45. Apparatus for growing seedlings under sterile conditions 
(After Sohulow) 

A, glass cylinder; B, glass tube 1 cm. in diameter; C, glass rods; D, wire net; 1, 

arrangement at start ; 2, arrangement after three to four days ; 3, arrangement 

after six to eight days 

at 2 atmospheres steam pressure. After the vessel is cool, seal all 
stoppers with a wax prepared by melting together equal weights of 
beeswax and rosin. 

The apparatus is now ready for planting. In order to do this 
under conditions as free as possible from all infection, carry the pots 
to inoculation room or to some place where the air is quiet and free 



APPENDIX A 137 

from dust. Remove the cotton plug from the top of the inner glass 
tube (Fig. 46, -B) and, using sterilized forceps, transfer the seeds 
germinated under sterile conditions to the apparatus, allowing them 
to drop down the inner tube on to the wire netting (Fig. 45, D). 

When the young shoot appears after six to eight days, the inner 
tube B should be slightly raised and the rods C, C used to push the 
lower mass of cotton gently but firmly around the base of the shoot, 
as shown in Fig. 45, 2. Finally the tube B, rods C, C, and the 
upper mass of cotton are removed, leaving the seedling as shown 
in Fig. 45, 3. 



APPENDIX B 
HANDLING STOCK CULTURES 

Those in charge of a bacteriological laboratory will have need of 
stock cultures of the organisms commonly used. These cultures may 
be obtained from isolations made at the laboratory or may be obtained 
from other laboratories. In America bacteriologists obtain many of 
their cultures from fellow bacteriologists and also from such institu- 
tions as the laboratory of the American Museum of Natural History, 
and the firm of Parke, Davis and Co. of Detroit. In Europe Krai's 
laboratory in Prag is a convenient and prompt source of supply. 

It should always be remembered that mistakes sometimes occur, 
and that contaminations are liable in transit. Therefore a culture 
received from another source should be cultivated for a time on media 
which will demonstrate its critical characters, before it finds its place 
in the collection of stock cultures. 

The standard beef-peptone agar appears to be best suited for stock 
cultures of most organisms. It is put into rather large test tubes 18 
by 175 mm., which are carefully plugged with deep plugs and slanted 
after sterilization. 

After inoculation the tubes should be kept in a cool, dry place 
where there is not much fluctuation of temperature. A special room 
should be constructed where possible. It should not be built against 
an outside wall and should be entered by one tightly closing door. 
The room should contain plain cupboards which can easily be cleaned 
and are closed by tightly fitting doors. The cupboard shelves should 
be far enough apart so that the cotton plugs will not rub the shelf 
above as they are removed from the cupboard. 

Transfers to newly prepared sterile tubes of media should be made 
every six or eight weeks. The length of time that a culture retains 
its vitality depends largely upon the temperature at which it is kept. 
If it is kept between 10° and 15° C, transfers once in ten to twelve 
weeks may sufSce. 

The old cultures should in every case be kept until one is certain 
that the transfer has grown and is pure. If contaminations appear, 
one may go back to the old culture and pour plates in order to 
reisolate pure cultures. 

138 



APPENDIX C 
MAZING PERMANENT PREPARATIONS 

A supply of demonstration material is very useful for class in- 
struction and for general publicity work. If colonies of organisms 
in pure culture are desired, there is nothing better than the Soyka 
flask. The medium, when hard, can be iaoculated with a needle at the 
center and will produce a good colony. After growth begins and 
the agar has dried somewhat, the cotton plug can be burned off and 
the mouth of the flask sealed with red sealing wax. The flask may 
then be mounted in one of the special holders in which there is dis- 
played a microphotograph or authentic drawing of individual bac- 
teria of the species exhibited. Specimens prepared in this way wiU 
stand much rough handling and will endure for several years. 

The Soyka-flask method is chiefly useful for pure-culture colonies 
or when work can be planned in advance. Teachers are well aware 
of the fact that many instructive examples arise in the progress of 
the daily work, and that such specimens are extremely valuable if 
properly preserved. A method is therefore desirable which will allow 
the preservation of cultures in test tubes and Petri dishes. Hastings ^ 
has described a, very satisfactory method in which glycerin agar is 
used. The method consists in pouring over the surface of the plates 
to be preserved some glycerin agar, which is prepared by mixing 
equal quantities of 2 per cent agar and glycerin. In preparing the 
glycerin agar the ordinary thread agar should be washed in tap water 
for two or three days. A 2 per cent solution of the washed agar 
in distiUed water is made and carefully filtered through paper. An 
equal volume of glycerin is added to this solution. No sterilization 
is necessary, because the medium is dense enough to prevent the 
growth of bacteria and molds. 

When one wishes to preserve an ordinary plate culture containing 
an agar piedium, a small quantity of glycerin agar is melted, cooled 
to about 45° C, and carefully poured over the surface of the plate 
culture. The glycerin agar solidifies and forms a firm protective 
layer over the surface of the plate. The larger part of the water will 
evaporate, but the glycerin, being hygroscopic, holds enough to 

1 Centralbl. f. Bakt., 2te Abt., 34 : 432. 1912. 
139 



140 A MANUAL OF BACTERIOLOGY 

prevent shrinking of the medium. The colonies preserve their orig- 
inal form and appearance. The dishes can be cleaned in the ordinary 
way when their usefulness is over. 

Gelatin plates can be preserved by this method if they are first 
subjected to the vapor of formaldehyde to harden the gelatin and to 
destroy the enzymes which cause liquefication. 

Another method for preserving Petri-dish cultures is by use of a 
sealing mixture. When the agar has become somewhat dried, invert 
the dish and pour melted paraffin into the crevice between the dish 
and the margin of the lid. 



APPENDIX D 
THE INOCDLATING CHAMBER 

In every laboratory where much careful work is to be done an in- 
oculating chamber is necessary. This is usually a closet large enough 
for a person to enter, although chambers are also made into which the 
operator thrusts only his arms. The closet type (Fig. 19) is usually 
preferred. It is 4 or 5 ft. square and at least 7 ft. high. One or 
two windows should be provided, to give plenty of light. The door 
should fit tightly and be provided with a spring which wUl keep it 
closed. The interior of the closet shotdd be lined with some mate- 
rial which can be easily cleansed with a damp cloth ; liaoleum serves 
the purpose well. Near the top of the closet the wall should have an 
opening for ventilation. A hole 15 in. square may be fitted with 
a frame which is covered with a fine wire screen and one or two 
thicknesses of cheesecloth or a thin layer of absorbent cotton. This 
filtering layer of cloth or cotton should be renewed frequently, as 
it becomes filled with dust. 

Before making inoculations in the closet, the walls, floor, eeUing, 
and shelf should be wiped with a cloth wrung out of 1 : 1000 solu- 
tion of mercuric bieloride solution (for method of preparation see 
Exercise 132), or 3 per cent formalin. 

The inoculating closet is not a good place to store stock cultures. 
The only shelf it should contain is the work shelf, on which there 
should be a gas burner for sterilizing the inoculating needles. 



141 



! ' APPENDIX E 

THE TITRATION OF BOUILLON 

After cooking the ingredients, the amount of water lost by evapo- 
ration should be restored. Stir for a moment, then pipette out 5 cc. 
into a porcelain evaporating dish. Add 20 cc. distilled water and boil 
over a flame for three minutes. Add a few drops of phenolphthalein. 

Fill a burette with a one-twentieth normal sodium hydrate solution 
and place the porcelain dish beneath the pinchcock of the burette. 
Run in the alkaline solution, stirring the bouillon with a glass rod, 
until a permanent, faint pink color is obtained. Read off the amount 
of alkali used. Titrate two other 6-cc. samples in the same way. If the 
amounts of alkali used for each of the three samples are reasonably 
close together, average them and record this figure as the amount of 
N/20 alkali necessary to neutralize 5 cc. of bouillon. Compute the 
amount necessary to neutralize the bouillon remaining in the flask. 

The balance of the bouillon is then to be neutralized, but the addi- 
tion of so much weak alkali would dilute the bouillon too much ; there- 
fore it is better to use a normal solution of sodium hydrate, of which 
only one twentieth of the original computation is necessary. 

Test the accuracy of the work by pipetting out a small sample 
and adding a drop or two of phenolphthalein. If a faint pink color 
does not appear, the titration should be repeated. 

For most bacteriological work it is best to have the medium defi- 
nitely acid. This condition is obtained by adding a definite volume 
of normal hydrochloric acid, usually 0.5, 1.0, or 1.5 cc. per liter of 
medium. This is expressed as + 0.5, -f 1.0, or + 1-5. 

The reaction of the medium is usually expressed as plus or minus. 
A reaction expressed as + 1 means that 1 cc. of normal alkali must 
be added to a liter of the culture medium in order to make it exactly 
neutral to phenolphthalein. In the same way, reaction expressed 
as — 1 means that 1 co. of normal acid must be added to bring 
1 liter to the neutral point. It is essential that phenolphthalein 
be used as the indicator for this titration. 

A reaction of + 1.0 is used for work in water and soil bacteriology ; 
+ 0.5 is better for many pathogenic organisms. 

142 



APPENDIX F 
DETERMINATION OF AMMONIA 

For most bacteriological work witli soils the ammonia is best de- 
termined after distillation with magnesium oxide. The analytical 
material is placed in a Kjeldahl or other flask suitable for distilling. 
Magnesium oxide is added to liberate the ammonia, and some shav- 
ings of paraffin to prevent frothing. The distilling flask is connected 
with a suitable condenser, the lower end of which dips into a meas- 
ured amount of deei-normal acid in an Erlenmeyer flask. 

Distillation is continued for a period varying with the amount of 
ammonia present, usually until 50 ce. or more of distillate has been col- 
lected. A few drops of methylrot or of cochineal are added, and the dis- 
tillate is titrated with deci-normal alkali. The difference between the 
amount of alkali required to bring the solution to neutrality and the 
amount of acid taken represents the amount of ammonia distilled over. 
This difference multiplied by .0017 gives the grams of NH^ obtained. 

If the amount of ammonia is small, it is better to use the well- 
known Nessler reaction, as used in water analysis. The solutions 
for analysis must, however, be clear and colorless. Nessler's reagent 
is an alkaline solution of mercuric potassium iodide. The alkali of 
the reagent liberates the ammonia from its salts ; if the concentration 
of the latter is considerable, a precipitate is formed, but if dilute, 
the compound remains in solution, giving a yellow color. The inten- 
sity of the color thus produced is proportional to the amount of 
ammonia present and is compared with that of a dilute standard 
ammonium chloride solution similarly treated. The substituted am- 
monias give a similar precipitate and color with this reagent, and 
in some lines of work it might be necessary to take this fact into 
consideration. 

REAGENTS REQUIRED ' 

1. Ammonia-free water. This may be prepared by redistilling the 
water of the laboratory after acidifying slightly with sulphuric acid. 
For colorimetric purposes it may be quickly prepared by adding 

1 Adapted from Schreiner and Failyer, Bulletin No. 31, Bureau of Soils, U.S. 
Dept. Agr., 1906. 

143 



144 



A MANUAL OF BACTBEIOLOGY 



sodium carbonate to ordinary distilled water until slightly alkaline, and 
boiling until about one fourth, has evaporated. The residual water is 
ready for use when cool. Am- 



A boiled 



nionia/-free water is used in 
the preparation of the follow- 
ing reagents and wherever the 
contamination with ammonia 
would influence the result. 

2. Sodium carbonate solution 
saturated solution. 

3. Nessler's reagent. Prepare a potassium 
iodide solution by dissolving 36 g. in 100 cc. 
of water, and a mercuric chloride solution by 
dissolving 17 g. in 300 cc. of water. Heat 
may be applied, to hasten the solution of the 
mercuric chloride, but the liquid must be 
cooled before it is used. The mercuric 
chloride solution is added to the potassium 
iodide solution until the precipitate of red 
mercuric iodide ceases to redissolve. The 
solution is then diluted to 1 liter with a 
20 per cent solution of sodium hydroxide. 
Then add more of the mercuric chloride 
solution until a slight permanent precipitate 
again forms. Allow this to settle, keeping 
it in a well-stoppered bottle and drawing 
off small quantities into an- 
other bottle from time to 
time as required for the tests. 
The reagent should have a 
light yellow color ; if color- 
less, more mercuric chloride 
must be added. Its sensi- 
tiveness should be tested 
from time to time with a 
very dilute solution of am- 
monium chloride. 



P 






CB 



v 



Fig. 46. The Schreiner Colorimeter 

A, A, immersion tubes ; B, graduated tubes ; 
C, elamgs ; 2), reflector ; E, mirror 



4. Standard ammonium chloride solution. Dissolve 0.7405 g. of 
pure ammonium chloride in ammonia^free water and dilute to 
1 liter. Dilute 10 cc. of this stronger solution to 500 cc. This 



APPENDIX P 145 

constitutes tlie standard ammonium cMoride solution, and each 
cubic centimeter contains 0.005 mg. of NH^. 

5. Standard colorimetric solution. This may be prepared by dilut- 
ing 10 CO. of the standard ammonium chloride solution (4) to about 
90 cc., adding 4 cc. of Xessler's reagent (3) and diluting to 100 cc. 
This standard should be prepared simultaneously with the develop- 
ment of the color ia the solutions to be tested. This colorimetric 
standard contains 0.5 part of NH^ per million. 

AXALTTICAL PROCESS 

If the solution be colorless and free from salts which interfere 
with the reagents, the determinations can be made without previous 
distillation ; otherwise it will be necessary to distiU. a measured 
quantity of the solution after it has been made alkaline with sodium 
carbonate. The flask and condenser should be rinsed with ammonia- 
free water. The condenser must be kept quite cold, with the end 
dipping into a little ammonia^free water at the start. The distUlate 
is made up to a definite volume. The distillation has the advantage 
of concentrating the ammonia in the case of very weak solutions. 

Pirst test a small amount of the solution, to ascertain the dilution 
necessary to get a good color for comparison. This may be done by 
adding some of the Xessler reagent (3) to a few cubic centimeters 
of the solution in a test tube. If a precipitate forms, it wiU be neces- 
sarj- to dilute the solution and again test a small sample. The color 
of the solution should be a light shade of pure yellow. If it has a 
deep yellow or reddish tint, it is too strong to be used direct, and 
further dilution is necessary. 

For the determination of the ammonia the analytical solution is 
diluted to about 45 cc, 2 cc. of Xessler's reagent (3) is added, and 
sufficient water is added to make 50 cc. The standard colorimetric 
solution is prepared at the same time, and after fifteen minutes the 
comparison is made in the colorimeter or ia Nessler tubes. 



APPENDIX G 
DETERMINATION OF NITRATE ^ 

The nitrates, if present in small quantities, are best determined 
by means of the color produced by the action of phenoldisulphonic 
acid and making alkaline with ammonia. 

Chlorides, when present in considerable quantities, interfere quite 
markedly with the determination of nitrates and must be previously 
removed. This is best accomplished by means of silver sulphate free 
from nitrates. This can be added in the solid form, thus avoiding 
dilution of the original solution. 

REAGENTS REQUIRED 

1. Phenoldisulphonic acid reagent. This is prepared by mixing 30 g. 
of pure crystallized phenol with 37 g. (20.1 cc.) of concentrated sul- 
phuric acid (sp. gr. 1.84) and heating for six hours at 100° C. by 
setting the lightly stoppered flask in boiling water. 

The acid thus prepared may crystallize out on standing, especially 
during the cold season. It may be brought into solution by heat, but 
the addition of water to affect solution is to be avoided. 

2. Ammonium hydroxide. Dilute strong ammonium hydroxide so- 
lution (sp. gr. 0.9) with an equal volume of water. 

3. Standard nitrate solution. Dissolve 0.1631 g. of pure, dry potas- 
simn nitrate in water and make up to 1 liter. Of this stronger solu- 
tion 100 cc. are diluted to 1 liter. This constitutes the standard 
nitrate solution and contains 0.01 of a milligram of NO^ in each 
cubic centimeter. 

4. Standard colorimetric solution. Evaporate 10 cc. of the standard 
nitrate solution (3) to dryness in a porcelain dish on a water or steam 
bath and treat as described under Analytical Process below, finally 
diluting the solution to 100 cc. This standard colorimetric solution 
has the strength of one part of NO, per million. 

1 Adapted from Schreiner and Failyer, Bulletin No. 31, Bureau of Soils, U.S. 
Dept. Agr., 1906. 

146 



APPENDIX G 147 

ANALYTICAL PROCESS 

Evaporate 60 ce. or other convenient quantity, depending upon the 
amount of nitrate present, to dryness in a porcelain dish on a water 
bath, removing the dish as soon as it is completely dry. Add 1 cc. 
of the phenoldisulphonic acid reagent (1) and stir thoroughly with 
the rounded end of a glass rod so as to loosen the residue and bring 
the acid well in contact with every portion of it. The time of action 
on the nitrate should be about ten minutes. At the end of this time 
the acid is diluted with about 15 cc. of water and made alkaliae 
with ammonium hydroxide (2), a yellow color being developed when 
the solution becomes alkaline. This is then diluted to 50 cc. or 
100 cc. and compared in the colorimeter with the standard colori- 
metric solution (4). If the color is too intense for direct compari- 
son with this standard, an aliquot portion may be taken and diluted 
to definite volume and the strength of this determined. 



APPENDIX H 
DETERMINATION OF NITRITE 

The nitrite is best determined by means of Ilosvay's modification 
of Griess's test, which has the advantage that the color is more rapidly 
developed and the reagents are less liable to change. The red color 
produced is dne to the action of the liberated nitrous acid on the sul- 
phanilic acid, the resulting diazo-compound being in turn converted 
by the naphthylamine into azo-amidonaphthalenebenzenesulphonic 
acid. 

REAGENTS REQUIRED 

1. Sulphanilic acid solution. Dissolve 0.5 g. of pure sulphanUic 
acid in 160 cc. of dilute acetic acid (sp. gr. 1.04). 

2. Naphthylamine acetate solution. Boil 0.1 g. of a-naphthylamine 
in 20 cc. of water and strain through a well-washed plug of absorb- 
ent cotton into 180 cc. of acetic acid (sp. gr. 1.04). 

3. Nitrite reagent. Mix equal volumes of the sulphanilic acid so- 
lution (1) and the naphthylamine acetate solution (2). This reagent 
is prepared in small quantities from time to time. If a reddish tint 
is developed on mixing the solutions, it indicates the presence of 
nitrites. In this case the reagent is treated with zinc dust, which 
destroys the color, and after removing the excess of zinc, it is ready 
for use. 

4. Standard sodium nitrite solution. Dissolve 0.0836 g. of pure silver 
nitrite in water. Add a solution of pure sodium chloride until silver 
chloride ceases to be precipitated. The volume is then made up to 
260 cc. and, after thorough shaking, allowed to stand in the dark 
until the precipitate has completely settled. Dilute 10 cc. of the 
supernatant liquid to 100 cc. with nitrite-free water. This standard 
must be kept in a well-stoppered bottle in the dark. Each cubic cen- 
timeter of this standard solution contains 0.01 mg. of NO^. The pure 
silver nitrite is prepared by adding to a hot concentrated solution 
of sixteen parts of silver nitrate a hot concentrated solution of ten 
parts of potassium nitrite. Allow to cool, and separate the mother 

148 



APPENDIX H 149 

liquor by filtration witli a filter pump. The silver nitrite is redis- 
solved in the smallest possible quantity of hot water, allowed to cool, 
and the crystal mass agam separated by means of suction. The 
crystals are then quickly dried iu a water bath and preserved in a 
tightly stoppered bottle in the dark. 

5. Standard colorimetric solution. This may be prepared by dilut- 
ing 10 ec. of the above standard sodium nitrite solution (4) to about 
80 CO., adding 16 cc. of the nitrite reagent (3) and making up to 
100 cc. It must be prepared at the same time as the test solutions. 
This colorimetric standard has the strength of one part NO^ per mil- 
lion, and solutions of other strengths may be similarly prepared when 
necessary, using the same quantity of reagent. 

ANALYTICAL PROCESS 

Dilute a measured volume of the solution, if necessary, to about 
40 cc, add 8 cc. of the nitrite reagent (3), and dilute to 50 cc. Develop 
the color of the standard solution at the same time. Allow the solu- 
tions to stand fifteen minutes and then determine their relative 
strengths in the colorimeter or by direct comparison by any of the 
other colorimetric procedures. 



APPENDIX I 
DETERMINATION OF NITRITES BY TROMMSDORF'S METHOD 

This method has certain advantages, although it is not strictly 
quantitative. Some organic compounds interfere with the test. 

REAGENTS REQUIRED 

1. Starch-iodide solution. Triturate 4 g. of starch with 150 cc. of 
water in a porcelain mortar. Cook the starch water in a steam steri- 
lizer or on a water bath. Dissolve 20 g. of zinc chloride in 100 cc. of 
water and add to the boiling starch paste. Kemove from the steam 
bath and allow the solution to stand until it becomes clear. Add 2 g. 
of zinc iodide and dilute the solution to 1 liter. Filter. Keep in a 
glass-stoppered bottle in the dark. 

ANALYTICAL PROCESS 

Take 100 cc. of the solution to be analyzed, 3 cc. of the starch-iodide 
solution, and 1 cc. of dilute (1 : 3) sulphuric acid. After a few minutes 
a blue color appears. Develop the color at the same time in the 
standard nitrite solution as used for the Griess-Ilosvay method (see 
above). 



1.50 



APPENDIX J 

DETERMINATION OF TOTAL NITROGEN BY A MODIFICATION 
OF KJELDAHL'S METHOD, TO INCLUDE THE NITROGEN OF 

NITRATES 

This method in some form is used by most chemists for the de- 
termination of the total nitrogen, and is useful iu many ways in bac- 
teriological work. 

REAGENTS REQUIRED 

1. Standard hydrochloric acid solution. For careful work the abso- 
lute strength of the solution may be determined by precipitation 
with silver nitrate and by weighing the silver chloride. 

2. Standard sulphuric acid solution. For ordinary work half-normal 
acid is used, but for work where small amounts of nitrogen are 
involved, decinormal acid is better. 

3. Sxilphuric acid. This should be the strong acid, sp. g. 1.84, and 
should be free from nitrates and ammonium sulphate. 

4. Zinc dust. This should be an impalpable powder. Granulated 
zinc or zinc filings will not answer. 

5. Sodium thiosulphate. 

6. Commercial salicylic acid. 

7. Metallic mercury or mercuric oxide. If mercuric oxide is used 
it should be prepared in the wet way, but not from mercuric nitrate. 

8. Potassium permanganate. This is used as a fine powder. 

9. Granulated zinc or pumice stone. One of these substances should 
be added to the contents of the distillation flasks to prevent bumping. 

10. Potassium sulphide solution. A 4 per cent solution of com- 
mercial potassium sulphide. 

11. Sodium hydroxide solution. A saturated solution of sodium 
hydroxide free from nitrates. 

12. An indicator. A solution of cochineal prepared by digesting and 
frequently agitating 3 g. of pulverized cochineal in a mixture of 50 cc. 
of strong alcohol and 200 cc. of distilled water for a day or two at 
room temperature. The filtered solution is employed as an indicator. 

151 



152 . A MANUAL OF BACTEEIOLOaY 

APPARATUS REQUIRED 

1. Kjeldahl flasks for both digestion and distillation. These are 
flasks having a capacity of about 550 cc, made of hard, moderately 
thick, and well-annealed glass. When used for the distillation, the 
flasks are fitted with rubber stoppers and bulb tubes, as described 
under distillation flasks. 

2. Kjeldahl digestion flasks. These are pear-shape, round-bottom 
flasks, made of hard, moderately thick, well-annealed glass and having 
a total capacity of about 250 ce. They are 22 cm. long and have 
a maximum diameter of 6 cm., tapering gradually to a long neck, 
which is 2 cm. in diameter at the narrowest part and flared a little 
at the edge. 

3. Distillation flasks. For distillation a flask, of ordinary shape, of 
about 550 cc. capacity may be used. It is fitted with a rubber stopper 
carrying a bulb tube to prevent the possibility of sodium hydrate 
being carried over mechanically during distillation. The bulbs may 
be about 3 cm. in diameter, but the tubes are the same diameter as 
the condenser, -^^ith which they are connected by a rubber tube. 

ANALYTICAL PROCESS 

Place from 0.7 g. to 3.5 g. of the substance to be analyzed in a 
Kjeldahl digestion flask ; if soils are being analyzed, a 10-15 g. sample 
is necessary. Add 30 cc. of sulphuric acid containing 1 g. of salicylic 
acid and shake until thoroughly mixed ; then add 5 g. of crystallized 
sodium thiosulphate ; or add to the substance 30 cc. of sulphuric acid 
containing 2 g. of salicylic acid, then add gradually 2 g. of zinc dust, 
shaking the contents of the flask at the same time. Finally, place the 
flask on the stand for holding the digestion flasks, where it is heated 
over a low flame until all danger from frothing has passed. The heat 
is then raised until the acid boils briskly, and the boiling continued 
untU white fumes no longer escape from the flask. This requires 
about five or ten minutes. Add approximately 0.7 g. of mercuric 
oxide, or its equivalent in metallic mercury, and continue the boil- 
ing until the liquid in the flask is colorless or nearly so. In case the 
contents of the flask are likely to become solid before this point is 
reached, add 10 cc. more of sulphuric acid. Complete the oxidation 
by adding small amounts of potassium permanganate until, after 
shaking, the liquid has a permanent green or purple color. 



APPENDIX J 153 

After cooling, dilute with about 200 cc. of water, add a few pieces 
of granulated zinc or pumice stone, if necessary in order to keep 
the contents of the flask from bumping, and add 25 cc. of potas- 
sium sulphide solution with shaking. Next add 50 cc. of the soda 
solution, or suf&cient to make the reaction strongly alkaline, pouring 
it down the side of the flask so that it does not mix at once with the 
acid solution. Connect the flask with the condenser, mix the con- 
tents by shaking, and distill until all ammonia has passed over into 
the standard acid. The tip of the condenser should dip into a 300-cc. 
Erlenmeyer flask containing 20-40 cc. of standard acid and enough 
distilled water to make a depth of 1-2 cm. The first 150 cc. of 
the distillate will generally contain all the ammonia. This operation 
usually requires from forty minutes to one hour and a half. The dis- 
tillate is then titrated with standard alkali, using cochineal as the in- 
dicator. Each cubic centimeter of decinormal ammonia is equivalent 
to 1.4 mg. of nitrogen. 



APPENDIX K 
DETERMINATION OF REDUCING SUGARS 

REAGENTS. REQUIRED 

1. Copper sulphate solution. Dissolve 34.639 g. of crystallized cop- 
per sulphate in water and dilute to 600 cc. 

2. Alkaline tartrate solution. Dissolve 173 g. of Eochelle salts and 
126 g. of potassium hydroxide in water and dilute to 600 cc. 

GRAVIMETRIC METHOD FOR THE DETERMINATION OF 
DEXTROSE (ALLIHN) 

Place 30 cc. of the copper solution, 30 cc. of the alkaline tartrate 
solution, and 60 cc. of water in a beaker and heat to boiling. Add 
25 cc. of the solution of the material to be examined (which must be 
so prepared as not to contain more than 0.250 g. of dextrose) and boil 
for two minutes. 

Prepare an asbestos felt at least half a centimeter thick in the 
bottom of a Gooch crucible. Wash the asbestos thoroughly with 
water to remove small particles, then with 10 cc. of alcohol and 10 cc. 
of ether. Dry the crucible and contents thirty minutes in a water 
oven at the temperature of boiling water ; cool and weigh. 

Filter the precipitated cuprous oxide through the asbestos felt and 
wash thoroughly with hot water, then with 10 cc. of alcohol and 
finally with 10 cc. of ether. Dry the crucible and contents thirty 
minutes in a water oven at the temperature of boiling water ; cool 
and weigh. 

The weight of cuprous oxide multiplied by 0.8883 gives the weight 
of metallic copper. From either figure the corresponding weight of 
dextrose is found from the following table : 



154 



APPEKDIX K 



155 



ALLIHN'S TABLE FOR THE DETERMINATION OF DEXTROSE 



MiUi- 


MiUi- 


MiUi- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


MiUi- 


Milli- 


MUU- 


Milli- 


giams 


gramsof 


grams 


grams 


grams of 


grams 


grams 


grams of 


grams 


grams 


grams of 


grams 


of cop- 


cnprouB 


of dex- 


of cop- 


cuprons 


of dex- 


of cop- 


cuprous 


of dex- 


of cop- 


cuprous 


of dex- 


per 


oxide 


trose 


per 


oxide 


trose 


per 


oxide 


trose 


per 


oxide 


trose 


11 


12.4 


6.6 


51 


57.4 


26.4 


91 


102.4 


46.4 


131 


147.5 


66.7 


12 


13.5 


7.1 


52 


58.5 


26.9 


92 


103.6 


46.9 


132 


148.6 


67.2 


13 


14.6 


7.6 


53 


59.7 


27.4 


93 


104.7 


47.4 


133 


149.7 


67.7 


14 


15.8 


8.1 


54 


60.8 


27.9 


94 


105.8 


47.9 


134 


150.9 


68.2 


15 


16.9 


8.6 


55 


61.9 


28.4 


95 


107.0 


48.4 


135 


152.0 


68.8 


16 


18.0 


9.0 


56 


63.0 


28.8 


96 


108.1 


48.9 


136 


153.1 


69.3 


17 


19.1 


9.5 


57 


64.2 


29.3 


97 


109.2 


49.4 


137 


154.2 


69.8 


18 


20.3 


10.0 


58 


65.3 


29.8 


98 


110.3 


49.9 


138 


155.4 


70.3 


19 


21.4 


10.5 


59 


66.4 


30.3 


99 


111.5 


50.4 


139 


156.5 


70.8 


20 


22.5 


11.0 


60 


67.6 


30.8 


100 


112.6 


50.9 


140 


157.6 


71.3 


21 


23.6 


11.5 


61 


68.7 


31.3 


101 


113.7 


51.4 


141 


158.7 


71.8 


22 


24.8 


12.0 


62 


69.8 


31.8 


102 


114.8 


51.9 


142 


159.9 


72.3 


23 


25.9 


12.5 


63 


70.9 


32.3 


103 


116.0 


52.4 


143 


161.0 


72.9 


24 


27.0 


13.0 


64 


72.1 


32.8 


104 


117.1 


52.9 


144 


162.1 


73.4 


25 


28.1 


13.5 


65 


73.2 


33.3 


105 


118.2 


53.5 


145 


163.2 


73.9 


26 


29.3 


14.0 


66 


74.3 


.33.8 


106 


119.3 


54.0 


146 


164.4 


74.4 


27 


30.4 


14.5 


67 


7.5.4 


34.3 


107 


120.5 


54.5 


147 


165.5 


74.9 


28 


31.5 


15.0 


68 


76.6 


34.8 


108 


121.6 


55.0 


148 


166.6 


75.5 


29 


32.7 


15.5 


69 


77.7 


35.3 


109 


122.7 


55.5 


149 


167.7 


76.0 


30 


33.8 


16.0 


70 


78.8 


35.8 


110 


123.8 


56.0 


150 


168.9 


76.5 


31 


34.9 


16.5 


71 


79.9 


36.3 


111 


125.0 


56.5 


151 


170.0 


77.0 


32 


36.0 


17.0 


72 


81.1 


36.8 


112 


126.1 


57.0 


152 


171.1 


77.5 


33 


37.2 


17.5 


73 


82.2 


37.3 


113 


127.2 


57.5 


153 


172.3 


78.1 


34 


38.3 


18.0 


74 


83.3 


37.8 


114 


128.3 


58.0 


154 


173.4 


78.6 


35 


39.4 


18.5 


75 


84.4 


38.3 


115 


129.6 


58.6 


155 


174.5 


79.1 


36 


40.5 


18.9 


76 


85.6 


38.8 


116 


130.6 


59.1 


156 


175.6 


79.6 


37 


41.7 


19.4 


77 


86.7 


39.3 


117 


131.7 


59.6 


157 


176.8 


80.1 


38 


42.8 


19.9 


78 


87.8 


39.8 


118 


132.8 


60.1 


158 


177.9 


80.7 


39 


43.9 


20.4 


79 


88.9 


40.3 


119 


134.0 


60.6 


159 


179.0 


81.2 


40 


45.0 


20.9 


80 


90.1 


40.8 


120 


135.1 


61.1 


160 


180.1 


81.7 


41 


46.2 


21.4 


81 


91.2 


41.3 


121 


136.2 


61.6 


161 


181.3 


82.2 


42 


47.3 


21.9 


82 


92.3 


41.8 


122 


137.4 


62.1 


162 


182.4 


82.7 


43 


48.4 


22.4 


83 


93.4 


42.3 


123 


138.5 


62.6 


163 


183.5 


83.3 


44 


49.5 


22.9 


84 


94.6 


42.8 


124 


139.6 


63.1 


164 


184.6 


83.8 


45 


50.7 


23.4 


85 


95.7 


43.4 


125 


140.7 


63.7 


165 


185.8 


84.3 


46 


51.8 


23.9 


86 


96.8 


43.9 


126 


141.9 


64.2 


166 


186.9 


84.8 


47 


62.9 


24.4 


87 


97.9 


44.4 


127 


143.0 


64.7 


167 


188.0 


85.3 


48 


54.0 


24.9 


88 


99.1 


44.9 


128 


144.1 


65.2 


168 


189.1 


85.9 


49 


55.2 


25.4 


89 


100.2 


45.4 


129 


145.2 


65.7 


169 


190.3 


86.4 


50 


56.3 


25.9 


90 


101.3 


45.9 


130 


146.4 


66.2 


170 


191.4 


86.9 



156 



A MANUAL OE BACTEEIOLOGY 



ALLIHN'S TABLE FOR THE DETERMINATION OF 
DEXTROSE (Continued) 



Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


Milli- 


grams 


gramB of 


grams 


grams 


grams of 


grams 


grams 


grams oi 


grams 


grams 


grams of 


grams 


of cop- 


cuprous 


of dex- 


of cop- 


cuprous 


of dex- 


of cop- 


cuprous 


of dex- 


of cop- 


cuprous 


of dex- 


per 


oxide 


trose 


per 


oxide 


trose 


per 


oxide 


trose 


per 


oxide 


trose 


171 


192.5 


87.4 


211 


237.6 


108.4 


251 


282.6 


129.7 


291 


327.4 


151.6 


172 


193.6 


87.9 


212 


238.7 


109.0 


252 


283.7 


130.3 


292 


328.7 


152.1 


173 


194.8 


88.5 


213 


239.8 


109.5 


253 


284.8 


130.8 


293 


329.9 


152.7 


174 


195.9 


89.0 


214 


240.9 


110.0 


254 


286.0 


131.4 


294 


331.0 


153.2 


175 


197.0 


89.5 


215 


242.1 


110.6 


255 


287.1 


131.9 


295 


332.1 


153.8 


176 


198.1 


90.0 


216 


243.2 


111.1 


256 


288.2 


132.4 


296 


333.3 


154.3 


177 


199.3 


90.5 


217 


244.3 


111.6 


257 


289.3 


133.0 


297 


334.4 


154.9 


178 


200.4 


91.1 


218 


245.4 


112.1 


258 


290.5 


133.5 


298 


335.5 


155.4 


179 


201.5 


91.6 


219 


246.6 


112.7 


259 


291.6 


134.1 


299 


336.6 


156.0 


180 


202.6 


92.1 


220 


247.7 


113.2 


260 


292.7 


134.6 


300 


337.8 


156.5 


181 


203.8 


92.6 


221 


248.7 


113.7 


261 


293.8 


135.1 


301 


338.9 


157.1 


182 


204.9 


93.1 


222 


249.9 


114.3 


262 


295.0 


135.7 


302 


340.0 


157.6 


183 


206.0 


93.7 


223 


251.0 


114.8 


263 


296.1 


136.2 


303 


341.1 


158.2 


184 


207.1 


94.2 


224 


252.4 


115.3 


264 


297.2 


136.8 


304 


342.3 


158.7 


185 


208.3 


94.7 


225 


253.3 


115.9 


265 


298.3 


137.3 


305 


343.4 


159.3 


186 


209.4 


95.2 


226 


254.4 


116.4 


266 


299.5 


137.8 


306 


344.5 


159.8 


187 


210.5 


95.7 


22T 


255.6 


116.9 


267 


300.6 


138.4 


307 


345.6 


160.4 


188 


211.7 


96.3 


228 


256.7 


117.4 


268 


301.7 


138.9 


308 


346.8 


160.9 


189 


212.8 


96.8 


229 


257.8 


118.0 


269 


302.8 


139.5 


309 


347.9 


161.5 


190 


213.9 


97.3 


230 


258.9 


118.5 


270 


304.0 


140.0 


310 


349.0 


162.0 


191 


215.0 


97.8 


231 


260.1 


119.0 


271 


305.1 


140.6 


311 


350.1 


162.6 


192 


216.2 


98.4 


232 


261.2 


119.6 


272 


306.2 


141.1 


312 


351.3 


163.1 


193 


217.3 


98.9 


233 


262.3 


120.1 


273 


307.3 


141.7 


313 


352.4 


163.7 


194 


218.4 


99.4 


234 


263.4 


120.7 


274 


308.5 


142.2 


314 


353.5 


164.2 


195 


219.5 


100.0 


235 


264.6 


121.2 


275 


309.6 


142.8 


315 


354.6 


164.8 


196 


220.7 


100.5 


236 


265.7 


121.7 


276 


310.7 


143.3 


316 


355.8 


165.3 


197 


221.8 


101.0 


237 


266.8 


122.3 


277 


311.9 


143.9 


317 


356.9 


165.9 


198 


222.9 


101.5 


238 


268.0 


122.8 


278 


313.0 


144.4 


318 


358.0 


166.4 


199 


224.0 


102.0 


239 


269.1 


123.4 


279 


314.1 


145.0 


319 


359.1 


167.0 


200 


225.2 


102.6 


240 


270.2 


123.9 


280 


315.2 


145.5 


320 


360.3 


167.5 


201 


226.3 


103.1 


241 


271.3 


124.4 


281 


316.4 


146.1 


321 


361.4 


168.1 


202 


227.4 


103.7 


242 


272.5 


125.0 


282 


317.5 


146.6 


322 


362.5 


168.6 


203 


228.5 


104.2 


243 


273.6 


125.5 


283 


318.6 


147.2 


323 


363.7 


169.2 


204 


229.7 


104.7 


244 


274.7 


126.0 


284 


319.7 


147.7 


324 


364.8 


169.7 


205 


230.8 


105.3 


245 


275.8 


126.6 


285 


320.9 


148.3 


825 


365.9 


170.3 


206 


231.9 


105.8 


246 


277.0 


127.1 


286 


322.0 


148.8 


326 


367.0 


170.9 


207 


233.0 


106.3 


247 


278.1 


127.6 


287 


323.1 


149.4 


327 


368.2 


171.4 


208 


234.2 


106.8 


248 


279.2 


128.1 


288 


824.2 


149.9 


328 


369.3 


172.0 


209 


235.3 


107.4 


249 


280.3 


128.7 


289 


325.4 


150.5 


329 


370.4 


172.6 


210 


236.4 


107.9 


250 


281:5. 


129.2 


290 


326.5 


151.0 


330 


371.5 


173.1 



APPENDIX K 



157 



ALLIHN'S TABLE FOR THE DETERMINATION OF 
BEXTROSE (Continued) 



MiUi- 


MQli- 


MUli- 


MUli- 


Milli- 


Milli- 


Milli- 


MiUi- 


Milli- 


Milli- 


Milli- 


Milli- 


giamB 


giamaof 


grams 


grams 


grame of 


grams 


grams 


grams of 


grams 


gnime 


grams of 


grams 


of cop- 


cuprouB 


of dex- 


of cop- 


cuproUB 


of dex- 


of cop- 


cuprous 


of dex- 


of cop- 


cuprous 


of dex- 


per 


oxide 


trose 


per 


oxide 


trose 


per 


oxide 


trose 


per 


oxide 


trose 


331 


372.7 


173.7 


866 


412.1 


193.4 


401 


451.5 


213.5 


436 


490.9 


233.9 


832 


373.8 


174.2 


367 


413.2 


194.0 


402 


452.6 


214.1 


487 


492.0 


284.5 


333 


374.9 


174.8 


368 


414.3 


194.6 


403 


453.7 


214.6 


438 


493.1 


235.1 


334 


376.0 


175.3 


869 


415.4 


195.1 


404 


454.8 


215.2 


439 


494.3 


285.7 


335 


377.2 


175.9 


370 


416.6 


195.7 


405 


456.0 


215.8 


440 


495.4 


236.3 


336 


378.3 


176.5 


871 


417.7 


196.3 


406 


457.1 


216.4 


441 


496.5 


236.9 


387 


379.4 


177.0 


372 


418.8 


196.8 


407 


458.2 


217.0 


442 


497.6 


237.5 


838 


380.5 


177.6 


373 


420.0 


197.4 


408 


459.4 


217.5 


443 


498.8 


238.1 


339 


381.7 


178.1 


374 


421.1 


198.0 


409 


460.5 


218.1 


444 


499.9 


238.7 


340 


382.8 


178.7 


875 


422.2 


198.6 


410 


461.6 


218.7 


445 


501.0 


289.3 


341 


883.9 


179.3 


876 


423.3 


199.1 


411 


462.7 


219.3 


446 


502.1 


239.8 


342 


385.0 


179.8 


377 


424.5 


199.7 


412 


463.8 


219.9 


447 


503.2 


240.4 


343 


386.2 


180.4 


378 


425.6 


200.3 


413 


465.0 


220.4 


448 


504.4 


241.0 


344 


387.8 


180.9 


379 


426.7 


200.8 


414 


466.1 


221.0 


449 


505.5 


241.6 


345 


388.4 


181.5 


380 


427.8 


201.4 


415 


467.2 


221.6 


450 


506.6 


242.2 


346 


389.6 


182.1 


381 


429.0 


202.0 


416 


468.4 


222.2 


451 


507.8 


242.8 


347 


390.7 


182.6 


382 


480.1 


202.5 


417 


469.5 


222.8 


452 


508.9 


243.4 


848 


391.8 


183.2 


383 


431.2 


203.1 


418 


470.6 


228.3 


453 


510.0 


244.0 


349 


392.9 


183.7 


384 


432.8 


208.7 


419 


471.8 


223.9 


454 


511.1 


244.6 


350 


394.0 


184.3 


385 


433.5 


204.3 


420 


472.9 


224.5 


455 


512.3 


245.2 


351 


395.2 


184.9 


386 


434.6 


204.8 


421 


474.0 


225.1 


456 


513.4 


245.7 


352 


396.3 


185.4 


887 


435.7 


205.4 


422 


475.6 


225.7 


457 


514.6 


246.3 


353 


397.4 


186.0 


888 


436.8 


206.0 


423 


476.2 


226.8 


458 


515.6 


246.9 


354 


398.6 


186.6 


889 


438.0 


206.5 


424 


477.4 


226.9 


459 


516.8 


247.5 


355 


399.7 


187.2 


390 


439.1 


207.1 


425 


478.5 


227.5 


460 


517.9 


248.1 


356 


400.8 


187.7 


391 


440.2 


207.7 


426 


479.6 


228.0 


461 


519.0 


248.7 


357 


401.9 


188.3 


892 


441.3 


208.3 


427 


480.7 


228.6 


462 


520.1 


249.3 


358 


408.1 


188.9 


398 


442.4 


208.8 


428 


481.9 


229.2 


468 


521.8 


249.9 


359 


404.2 


189.4 


394 


448.6 


209.4 


429 


483.0 


229.8 








860 


405.3 


190.0 


395 


444.7 


210.0 


480 


484.1 


280.4 








361 


406.4 


190.6 


396 


445.9 


210.6 


431 


485.3 


231.0 








362 


407.6 


191.1 


397 


447.0 


211.2 


432 


486.4 


231.6 








363 


408.7 


191.7 


398 


448.1 


211.7 


433 


487.5 


282.2 








864 


409.8 


192.3 


399 


449.2 


212.3 


434 


488.6 


232.8 








365 


410.9 


192.9 


400 


450.8 


212.9 


435 


489.7 


288.4 









158 A MANUAL OF BACTERIOLOGY 

GRAVIMETRIC METHOD OF DETERMINATION 
(DEFREN-O'SULLIVAN) 

For the determination of reducing sugars many prefer the method 
of Defren and 0' Sullivan, because it is applicable to the determi- 
nation not only of dextrose but also of maltose and lactose.^ Mix 
15 CO. of the copper solution with 16 cc. of the alkaline tartrate in 
an Erlenmeyer flask, and add 50 cc. of distilled water. Place the 
flask and its contents in a boiling-water bath and allow them to re- 
main five minutes. Then run rapidly from a burette into the hot 
liquor in the flask 25 cc. of the sugar solution to be tested (which 
should not contain more than J per cent of reducing sugar). Allow 
the flask to remain in the boiling water just fifteen minutes after 
the addition of the sugar solution, remove, and, with the aid of a 
vacuum, filter the contents rapidly in a platinum or porcelain Gooch 
crucible containing a layer of prepared asbestos fiber about 1 cm. thick, 
the Gooch, with the asbestos, having been previously ignited and 
weighed. The cuprous oxide precipitate is thoroughly washed with 
boiling distilled water till the water ceases to be alkaline. 

Dry the Gooch with its contents in the oven, and finally heat to 
dull redness for fifteen minutes, during which time the red cuprous 
oxide is converted into the black cupric oxide. If a platinum Gooch 
is used (and this variety is preferable), it may be heated over the 
low flame of the burner. If the Gooch is of porcelain, considerable 
care must be taken to avoid cracking the crucible, the heat being 
increased cautiously. After oxidation as above, the crucible is trans- 
ferred to a desiccator, cooled, and quickly weighed. From the mil- 
ligrams of cupric oxide the milligrams of reducing sugar may be 
calculated (for a convenient table see Leach, loc. cit. p. 490). 

1 See Leach, Food Inspection and Analysis, New York, 1907. 



APPENDIX L 

TITANIUM TRICHLORIDE SOLUTION: ITS PREPARATION 
AND STANDARDIZATION 

TMs reagent is useful for titrating methylene blue solutions. To 
prepare it, take : 

Titanium trichloride (Merck) 15 per cent solution . . . . 9 cc. 
Hydrochloric acid, concentrated . . .... . . 9 cc. 

Boil this mixture on a steam bath until aU the H.^S is expelled. 
Dilute with oxygen-free water to 3 liters, adding hydrochloric acid 
to slight excess. Keep the solution in a flask under carbon dioxide 
or hydrogen. When estimations are to be made, the burette should 
be filled quickly with the reagent and a little oil added to prevent 
contact at the top with atmospheric oxygen. The tip of the bu- 
rette should dip a few centimeters below the surface of the liquid 
under titration. 

Owing to almost unavoidable changes in the strength of the 
titanium solution, frequent standardization is necessary. For this 
purpose a solution of ferric chloride is used with potassium sulpho- 
cyanate as iadicator. The titanium solution is added slowly until 
the test shows that the ferric chloride is reduced to ferrous. A 
standard iron solution is made by dissolving 2.9 g. FeClj in 100 cc. 
water. This corresponds to 1 g. Fe, and 1 cc. corresponds to .01 g. Fe. 
Not more than 10 cc. of this solution should be used for purposes of 
titration ia estimating titanium samples. 



159 



APPENDIX M 

MEASURES AND WEIGHTS— CONVERSION TABLES 

A. METRIC TO ENGLISH 
Length 



Metric Name 


Abbrevi- 
ation 


Relation to 
Standard 


English Equivalent 


Approximately 
in English 


Kilometer . . 


km. 


1000 meters 


fl093.61yd. I 
\. 62138 mi. J 
r 39.37 in. ] 


fmi. 


Meter .... 


m. 


Standard 


J. 3.28 ft. I 
[ 1.094 yd. J 


39 in. 


Decimeter . . 


dm. 


y\j^ meter 


3.937 in. 


4 in. 


Centimeter . . 


cm. 


tU meter 


.3937 in. 


|in. 


Millimeter . . 


mm. 


ttjVw meter 


.03937 in. 


sVim 


Micron or micro- 




tttVct of ^ 






millimeter 




millimeter 


.00003937 in. 


^zisv m- 



Capacity 



Liter .... 

Cubic centimeter 
(Milliliter) 



Standard 



liter 



r 1.057 U.S. qt. \ 
\61.03cu. in. J 

.001057 XJ.S.qt. 

.06103 cu. in. 

.034 fluid ounce 



1 qt. U.S. 
measure 



Weight 





kg. or 
kilo. 




r 2.205 lb. •] 




Kilogram . . . 


1000 grams 


J 2 lb. 3 oz. 4| dr. . 


2|lb. 






avoirdupois 










r 15.43 gr. 1 










(avoir.) 




Gram .... 


g- 


Standard 


- .0353 oz. (avoir.) • 
.643 pennyweight 
. (troy) 


jV oz. 


Milligram . . . 


mg. 


rauTS of a 
gram 


.01543 gr. (avoir, or 
troy) 





160 



APPENDIX M 



161 



B. ENGLISH TO METRIC 
Length 



English Name 


Metric Equivalent 


Mile 


1.609 km. 


Yard 

Foot 

Inch 


r .914 m. 

\91.44 cm. 

30.48 cm. 

f 2.54 cm. 




\ 25.40 mm. 



Capacity 



English Name 



Metric Equivalent 



Quart (U.S.) 

Pint (U.S.) 
Gill (U.S.) . 
Fluid ounce 
Cubic inch . 



f .946 liters 

|946.36cu. cm. 

473.18 ou. cm. 

118.29 cu. cm. 

29.57 ou. cm. 

16.39 cu. cm. 



"Weight 



English Name 


Metric Equivalent 


Pound (avoir.) 

Ounce (avoir.) 

Grain (avoir.) 

Ounce (troy) 


f .4536 kg. 
1453.59 g. 

28.35 g. 
f .0648 g. 
\ 64.79 mg. 

31.103 g. 
1.555 g. 
r .0648 g. 
[ 64.80 mg. 


Grain (troy) (= avoir, gr.) .... ... 



APPENDIX N 
ALCOHOL TABLE MODIFIED FROM WINDISCH 



Specific 


Per Cent of 


Per Cent of 


Specific 


Per Cent of 


Per Cent of 


Gravity of 


Alcohol by 


Alcohol by 


Gravity of 


Alcohol hy 


Alcohol by 


Distillate 


Weight 


"Volume 


Distillate 


Weight 


Volume 


1.0000 


0.00 


0.00 


0.9908 


6.20 


6.55 


0.9998 


0.11 


0.13 


6 


5.32 


6.71 


6 


0.21 


0.27 


4 


5.46 


6.86 


4 


0.32 


0.40 


2 


6.67 


7.02 


2 


0.42 


0.53 





5.70 


7.18 





0.63 


0.67 


0.9898 


5.83 


7.33 


0.9988 


0.64 


0.80 


6 


5.96 


7.50 


6 


0.74 


0.93 


4 


6.08 


7.66 


4 


0.85 


1.07 


2 


6.21 


7.82 


2 


0.96 


1.20 





6.34 


7.99 





1.06 


1.34 


0.9888 


6.47 


8.15 


0.9978 


1.17 


1.48 


6 


6.59 


8.31 


6 


1.28 


1.61 


4 


6.73 


8.48 


4 


1.39 


1.75 


2 


6.88 


8.64 


2 


1.50 


1.88 





6.99 


8.81 





1.60 


2.02 


0.9878 


7.12 


8.98 


0.9968 


1.71 


2.16 


6 


7.26 


9.15 


6 


1.82 


2.30 


4 


7.39 


9.32 


4 


1.93 


2.44 


2 


7.53 


9.48 


2 


2.04 


2.58 





7.66 


9.66 





2.16 


2.72 


0.9868 


7.80 


9.83 


0.9958 


2.27 


2.86 


6 


7.94 


10.00 


6 


2.38 


3.00 


4 


8.07 


10.17 


4 


2.49 


3.14 


2 


8.21 


10.36 


2 


2.60 


3.28 





8.35 


10.52 





2.72 


3.42 


0.9858 


8.49 


10.70 


0.9948 


2.82 


3.56 


6 


8.63 


10.88 


6 


2.94 


3.71 


4 


8.77 


11.05 


4 


8.06 


3.85 


2 


8.91 


11.23 


2 


3.17 


4.00 





9.06 


11.41 





3.29 


4.14 


0.9848 


9.20 


11.59 


0.9938 


3.40 


4.29 


6 


9.34 


11.77 


6 


3.52 


4.43 


4 


9.49 


11.95 


4 


3.64 


4.58 


2 


9.63 


12.14 


2 


3.75 


4.73 





9.79 


12.32 





3.87 


4.88 


0.9838 


9.92 


12.60 


0.9928 


3.99 


5.03 


6 


10.07 


12.69 


6 


4.11 


5.18 


4 


10.22 


12.88 


4 


4.23 


5.33 


2 


10.36 


13.06 


2 


4.35 


5.48 





10.52 


13.25 





4.47 


6.53 


0.9828 


10.66 


13.44 


0.9918 


4.59 


5.78 


6 


10.81 


13.63 


6 


4.71 


5.93 


4 


10.96 


13.82 


4 


4.83 


6.09 


2 


11.12 


14.01 


2 


4.95 


6.24 





11.27 


14.20 





5.08 


6.40 









162 



APPENDIX N 



163 



ALCOHOL TABLE MODIFIED FROM WINDISCH (Continued) 



Specific 


Per Cent of 


Per Cent of 


Specific 


Per Cent of 


Per Cent of 


Gravity of 


Alcohol by 


Alcohol by 


Gravity of 


Alcohol by 


Alcohol by 


Distillate 


Weight 


Volume 


Distillate 


Weight 


Volume 


0.9818 


11.42 


14.39 


0.9718 


19.30 


24.32 


6 


11.57 


14.58 


6 


19.45 


24.51 


4 


11.72 


14.77 


4 


19.60 


24.70 


2 


11.88 


14.97 


2 


19.76 


24.89 





12.03 


15.16 





19.91 


25.08 


0.9808 


12.19 


15.36 


0.9708 


20.06 


25.27 


6 


12.34 


15.55 


6 


20.21 


25.47 


4 


12.50 


15.75 


4 


20.36 


25.66 


2 


12.65 


15.95 


2 


20.51 


25.84 





12.81 


16.14 





20.66 


26.03 


0.9798 


12.97 


16.34 


0.9698 


20.81 


26.22 


6 


13.13 


16.54 


6 


20.96 


26.41 


4 


13.28 


16.74 


4 


21.10 


26.59 


2 


18.44 


16.94 


2 


21.25 


26.78 





13.60 


17.14 





21.40 


26.96 


0.9788 


13.76 


17.34 


0.9688 


21.54 


27.14 


6 


13.92 


17.54 


6 


21.69 


27.33 


4 


14.08 


17.74 


4 


21.83 


27.51 


2 


14.23 


17.94 


2 


21.97 


27.69 





14.39 


18.14 





22.12 


27.87 


0.9778 


14.55 


18.34 


0.9678 


22.26 


28.05 


6 


14.71 


18.54 


6 


22.40 


28.28 


4 


14.87 


18.74 


4 


22.54 


28.41 


2 


15.03 


18.94 


2 


22.68 


28.59 





15.19 


19.14 





22.82 


28.76 


0.9768 


15.35 


19.34 


0.9668 


22.96 


28.94 


6 


15.51 


19.55 


6 


23.10 


29.11 


4 


15.67 


19.75 


4 


23.24 


29.29 


2 


15.83 


19.95 


2 


23.38 


29.46 





15.99 


20.15 





23.52 


29.64 


0.9758 


16.15 


20.35 


0.9658 


23.65 


29.81 


6 


16.31 


20.55 


6 


23.79 


29.98 


■4 


16.47 


20.75 


4 


23.93 


80.15 


2 


16.63 


20.96 


2 


24.06 


30.32 





16.79 


21.16 





24.19 


80.49 


0.9748 


16.95 


21.36 


0.9648 


24.33 


30.66 


6 


17.11 


21.56 


6 


24.46 


30.82 


4 


17.27 


21.76 


4 


24.59 


80.99 


2 


17.42 


21.96 


2 


24.78 


31.16 





17.58 


22.16 





24.85 


31.32 


0.9738 


17.74 


22.35 


0.9638 


24.99 


31.49 


6 


17.90 


22.55 


6 


25.12 


31.65 


4 


18.05 


22.75 


4 


25.25 


81.81 


2 


18.21 


22.95 


2 


25.37 


31.98 





18.37 


23.14 





25.50 


32.14 


0.9728 


18.52 


23.34 


0.9628 


25.63 


32.30 


6 


18.68 


23.54 


6 


25.76 


82.46 


4 


18.84 


23.73 


4 


25.88 


82.62 


2 


18.99 


23.93 


2 


26.01 


32.78 





19.14 


24.12 





26.13 


32.93 



APPENDIX O 

COMPARISON OF FAHRENHEIT AND CENTIGRADE 
THERMOMETER SCALES 



Fahr. 


Cent. 


Fahr. 


Cent. 


Fahr. 


Cent. 


Fahr. 


Cent. 


212 


100 


144 


62.2 


76 


24.4 


8 


-13.3 


210 


98.9 


142 


61.1 


74 


23.3 


6 


-14.4 


208 


97.8 


140 


60.0 


72 


22.2 


4 


-15.6 


206 


96.7 


138 


58.9 


70 


21.1 


2 


-16.7 


204 


95.6 


136 


57.8 


68 


20.0 





-17.8 


202 


94.4 


134 


56.7 


66 


18.9 


-2 


-18.9 


200 


93.3 


132 


55.6 


64 


17.8 


-4 


-20.0 


198 


92.2 


130 


54.4 


62 


16.7 


-6 


-21.1 


196 


91.1 


128 


53.3 


60 


15.6 


-8 


-22.2 


194 


90.0 


126 


52.2 


58 


14.4 


-10 


-23.3 


192 


88.9 


124 


51.1 


56 


13.3 


-12 


-24.4 


190 


87.8 


122 


50.0 


54 


12.2 


-14 


-25.6 


188 


86.7^ 


120 


48.9 


52 


11.1 


-16 


-26.7 


186 


85.6 


118 


47.8 


50 


10.0 


-18 


-27.8 


184 


84.4 


116 


46.7 


48 


8.9 


-20 


-28.9 


182 


83.3 


114 


45.6 


46 


7.8 


-22 


-30.0 


180 


82.2 


112 


44.4 


44 


6.7 


-24 


-31.1 


178 


81.1 


110 


43.3 


42 


5.6 


-26 


-32.2 


176 


80.0 


108 


42.2 


40 


4.4 


-28 


-33.3 


174 


78.9 


106 


41.1 


38 


3.3 


-30 


-34.4 


172 


77.8 


104 


40.0 


36 


2.2 


-32 


-35.6 


170 


76.7 


102 


38.9 


34 


1.1 


-34 


-36.7 


168 


75.6 


100 


37.8 


32 


0.0 


-36 


-37.8 


166 


74.4 


98 


36.7 


30 


-1.1 


-38 


-38.9 


164 


73.3 


96 


35.6 


28 


-2.2 


-40 


-40.0 


162 


72.2 


94 


34.4 


26 


-3.3 


-42 


-41.1 


160 


71.1 


92 


33.3 


24 


-4.4 


-44 


-42.2 


158 


70.0 


90 


32.2 


22 


-5.6 


-46 


-43.3 


156 


68.9 


88 


31.1 


20 


-6.7 


-48 


-44.4 


154 


67.8 


86 


30.0 


18 


-7.8 


-50 


-45.6 


152 


66.7 


84 


28.9 


16 


-8.9 


-52 


-46.7 


150 


65.6 


82 


27.8 


14 


-10.0 


-54 


-47.8 


148 


64.4 


80 


26.7 


12 


-11.1 


-56 


-48.9 


146 


63.3 


78 


25.6 


10 


- 12.2 







To change Centigrade to Fahrenheit : (C. x f ) + 32 = F. For example, to 
find the eqmvalent of 10° C, (10° x f ) + 32 = 50° F. 

To change Fahrenheit to Centigrade : (F. — 32°) x ^ = C. For example, to 
reduce 50° F. to Centigrade, (50° - 32°) x f = 10°C ; and - 40° F. to Centigrade, 
(- 40° - 32°) = - 72°, whence - 72° x | = - 40° C. 

164 



APPENDIX P 

THE DESCRIPTIVE CHART OF THE SOCIETY OF AMERICAN 
BACTERIOLOGISTS 

The identification of species of bacteria is rendered much more 
certain if one uses the chart prepared by the above-mentioned 
society. This chart enumerates practically all characters of bacteria 
■which have a differential value when the organisms are grown upon 
media prepared according to the standard methods of the American 
Public Health Association as adopted in 1905. 

Copies of this chart, in form suitable for use in the laboratory, 
are supplied at a nominal price by the Secretary of the Society 
of American Bacteriologists. 



DESCRIPTIVE CHAET — SOCIETY OP AMERICAN 
BACTERIOLOGISTS 

Prepared by F. D. Chester, F. P. Gorham, Erwin F. Smith, Committee 

ON Methods of Identification of Bacterial Species. Indorsed by the 

Society for General Use at the Annual Meeting, Dec. 31, 1907 

DETAILED FEATURES 

(Note. Underscore required terms) 

I. Morphology (1) 

1. Vegetative cells, medium used , 

temp , age days. 

Perm, round, short rods, long rods, short chains, long chains, filaments, 

commas, short spirals, long spirals, Clostridium, euneate, clavate, curved. 

Limits of size Size of majority 

Ends, rounded, truncate, concave. 

(Orientation (grouping) 
Chains (number of elements) 
Short chains, long chains. 
Orientation of chains, parallel, irregular. 

2. Sporangia, medium used , 

temp , age days. 

Form, elliptical, short rods, spindled, clavate, drumsticks. 

165 



166 A MANUAL OF BACTERIOLOGY 

Limits of size Size of majority 

r Orientation (grouping) 

Agar hanging block i Chains (number of elements) 

[ Orientation of chains, parallel, irregular. 
Location of endospores, cerdral, polar 

3. Endospores 

Form, round, elliptical, elongated. 

Limits of size Size of majority 

Wall, thick, thin. 

Sporangium wall, adherent, not adherent. 

Germination, equatorial, oblique, polar, bipolar, by stretching. 

4. Flagella.No Atta.chjiient,polar,bipolar,peritrichiate. Howstained 

6. Capsules, present on 

6. Zoogloea, Pseudozoogloea. 

7. Involution fonns, on In days at °C. 

8. Staining reactions 

1:10 watery fuchsin, gentian violet, carbol-fuchsin, Loffler's alkaline 

methylene blue. 
Special stains 

Gram Glycogen 

Fat Acid-fast 

Neisser 

II. Cultural Features (2) 

1. Agar stroke 

Growth, invisible, scanty, moderate, abundant. 

Form of growth,^K/on?i, echinulate, beaded, spreading, plumose, arborescent, 

rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Luster, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, vemicose. 
Optical characters, opaque, translucent, opalescent, iridescent. 

Chromogenesis (7), 

Odor, al>sent, decided, resembling 

Consistency, slimy, butyrous, viscid, membranous, coriaceous, brittle. 
Medium, grayed, browned, reddened, blued, greened, 

2. Potato 

Growth, scanty, moderate, abundant, transient, persistent. 

Form of growth, filiform, echinulate, beaded, spreading, plumx)se, arborescent, 

rhizoid. 
Elevation of growth, ^ai, effuse, raised, convex. 
Luster, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Chromogenesis (7), Pigment in water, insoluble, soluble ; other 

solvents, 

Odor, absent, decided, resembling 

Consistency, slimy, butyrous, viscid, membranous, coriaceous, brittle. 
Medium, grayed, browned, reddened, blued, greened. 



APPENDIX P 167 

3. Loffler*s blood sermn 

Stroke, inmsible, scanty, moderate, abundant. 

Form of growth, JUiform, echinulate, beaded, spreading, plumose, arbores- 
cent, rhizoid. 
Elevation of growth, .^oi, effuse, raised, convex. 
Luster, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 

Chromogenesis (7), 

Medium, grayed, browned, reddened, blued, greened. 
Liquefaction begins in days, complete in days. 

4. Agar stab 

Growth, uniform, best at top, best at bottom ; surface growth scanty, abun- 
dant; restricted, widespread. 

Line of puncture, filiform, beaded, papillate, mlkms, plumose, arborescent ; 
liquefaction. 

5. Gelatin stab 

Growth, uniform, best at top, best at bottom. 

Line of puncture, ^Ji/orm, beaded, papiUate, villous, plumose, arborescent. 

Liquefaction, crateriform, napiform, infundibuliform, saccate, stratiform; 

begins in days, complete in days. 

Medium, fluorescent, browned 

6. Hntrient broth 

Surface growth, ring, pellicle, flocculent, membranous, none. 

Clouding, slight, moderate, strong ; transient, persistent ; none ; fluid turbid. 

Odor, absent, decided, resembling 

Sediment, compact, flocculent, granular, flaky, viscid on agitation, abun- 
dant, scant. 

7. Milk 

Clearing without coagulation. 
Coagulation, yrompi, delayed, absent. 

Extrusion of whey begins in days. 

Coagulum, slowly peptonized, rapidly peptonized. 

Peptonization begins on day, complete on day. 

Reaction, 1 day , 2 days , 4 days , 10 days , 

20 days 

Consistency, slimy, viscid, unchanged. 
Medium, browned, reddened, blued, greened. 
Lab ferment, present, absent. 

8. Litmus milk 

Acid, alkaline, acid tfien alkaline, no change. 
Prompt reduction, no reduction, partial slow reduction. 

9. Gelatin colonies 

Growth, slow, rapid. 

Form, punctiform, round, irregular, amoeboid, mycelioid, filamentous, rhizoid. 
Elevation,.^, ^ffiise, raised, convex, pulvinate, crateriform (liquefying). 
Edge, entire, undulate, lobate, erase, lacerate, fimbriate, fHamentous, floccose, 

curled. 
Liquefaction, cup, saucer, spreading. 



168 A MANUAL OF BACTEKIOLOGY 

10, Agar colonies 

Growth, slow, rapid, (temperature ). 

¥oTm,punctiform, round, irregular, amoeboid, mycelioid, filamentous, rhizoid. 
Surface, smooth, rough, concenirically ringed, radiate, striate. 
Elevation, flat, effuse, raised, convex, pulvinate, umbonate. 
Edge, entire, undulate, lobate, erose, lacerate, fiml»iate,floccose, curled. 
Internal structure, amorphous, finely granular, coarsely granular, grumose, 
filamentous, floccose, curled. 

11. Starch jeUy 

Growth, scanty, copious. 

Diastasic action, absent, feMe, profound. 

Medium stained, 

13. Silicate jelly (Fermi's solution) 

Growth, copious, scanty, absent. 

Medium stained, 

IS. Cohn's solution 

Growth, copious, scanty, absent. 
Medium, fluorescent, nonfluorescent. 

14. Uschinsky's solution 

Growth, copious, scanty, absent. 
Fluid, viscid, not viscid. 

15. Sodium chloride in tiouillon 

Per cent inhibiting growth 

16. Growth in bouillon over chloroform, unrestrained, feeble, absent. 

17. Nitrogen 

Obtained from^epione, asparagine, glycocoll, urea, ammonia salts, nitrogen. 

18. Best media for long-continaed growth 

19. Quick tests for differential purposes 

III. Physical and Biochemical Features (5) 



1. Fermentation tubes contain- 
ing peptone water or sugar- 
free bouillon and 


1 

ID 




1 




1 


a 

■a 

I 
3 












Gas production, in per cent 






















Vcoj 






















Growth in closed arm 






















Amount of acid produced in 1 day 






















Amount of acid produced in 2 days 






















Amount of acid produced in 4 days 























APPENDIX P 



169 



2. Ammonia production, fed}le, moderate, strong, absent, masked by acids. 

3. Nitrates in nitrate broth 

Reduced, not reduced. 

Presence of nitrites ammonia 

Presence of nitrates free nitrogen 

4. Indol piodaction, feeble, moderate, strong. 

5. Toleration of Acids, great, medium,, slight. 

Acids tested 

6. Toleration of NaOH, great, medium, slight. 

7. Optimum reaction for growth in bouillon, stated in terms of Fuller's scale 

8. Vitality on culture media, brief, moderate, long. 

9. Temperature relations 

Thermal death point (ten minutes exposure in nutrient broth when this 

is adapted to growth of organism), C. 

Optimum temperature for growth C. ; or best growth at 15° C, 

20° C, 25° C, 30° C, 37° C, 40° C, 50° C, 60° C. 

Maximum temperature for growth, C. 

Minimiun temperature for growth, C. 

10. Killed readily by drying ; resistant to drying. 

11. Per cent killed by freezing (salt and crushed ice or liquid air) 

12. Sunlight, exposure on ice in thinly sown agar plates, one-half plate covered 

(time fifteen minutes), sensitive, not sensitive. 
Per cent killed 

13. Acids produced 

14. Allsalies produced 

15. Alcohols 

16. Ferments, pepsin, trypsin, diastase, invertase, pectase, cytase, tyrosinase, 

oxidase, peroxidase, lipase, catalase, glucose, galactose, lab, etc 



17. Crystals formed 

18. Effect of germicides 



Substance 


Method used 


1 


£ 
1 


1 

s 


O 















































































































170 A MANUAL OF BACTEEIOLOGY 

IV. Pathogenicity 

1. Pathogenic to animals 

Insects, crustaceans, fishes, reptiles, birds, mice, rats, guinea pigs, rabbits, 
dogs, cats, sheep, goats, cattle, horses, monkeys, man 

2. Pathogenic to plants 

3. Toxins, soluble, endotoxins. 

4. Non-toxin-forming. 

5. Immunity bactericidal. 

6. Immunity nonbactericidal. 

7. Loss of virulence on culture media, prompt, gradual, not observed in 

months. 

(1) The morphological characters shall be determined and described from growths 
obtained upon at least one solid medium (nutrient agar) and in at least one liquid 
medium (nutrient broth) . Growths at 37° C shall be in general not older than twenty- 
four to forty-eight hours, and growths at 20° C not older than forty-eight to seventy- 
two hours. To secure uniformity in cultures, in all cases preliminary cultivation 
shall be practiced as described in the revised Report of the Committee on Standard 
Methods of the Laboratory Section of the American Public Health Association, 1905. 

(2) The observation of cultural and biochemical features shall cover a period of at 
least fifteen days and frequently longer, and shall be made according to the revised 
Standard Methods above referred to. AU media shall be made according to the same 
Standard Methods. 

(3) Gelatin-stab cultures shall be held for six weeks, to determine liquefaction. 

(4) Ammonia and indol tests shall be made at end of tenth day, nitrite tests at end 
of fifth day. 

(5) Titrate with N/20 NaOH, using phenolphthalein as an indicator ; make titra- 
tions at same times from blank. The difference gives the amount of acid produced. 

The titration should be done after boiling, to drive off any CO2 present in the 
culture. 

(6) Generic nomenclature shall begin with the year 1872 (Cohn's first important 
paper) . 

Species nomenclature shall begin with the year 1880 (Koch's discovery of the 
poured-plate method for the separation of organisms). 

(7) Chromogenesis shall be recorded in standard color terms. 



APPENDIX P 



171 



BRIEF CHARACTERIZATION 

Mark + or 0, and when two terms occur on a line, erase the one which does not 
supply, unless both apply. 



o 

a 

e 
1 


Diameter over 1 /i, | | 


Chains, filaments 




Endospores 




Capsules 




Zoogloea, Pseudozoogloea 




Motile 




Involution forms 


Gram's stain 




03 

o 
a 

a 

D 


5 


Cloudy, turbid 




Ring 




Pellicle 




Sediment 






Shining 




DuU 




Wrinkled 




Chromogenic 






Round 




Proteuslike 




Rhizoid 




Filamentous 




Curled | 


OS 


Surface growth | 


Needle growth | 


2 


Moderate, absent 




Abundant 




Discolored 




Starch destroyed 




Grows at 370 c, 




Grows in Cohn's Sol. 




Grows in Uschinsky's Sol. 




m 

a 
a 
o 

< 
.J 

D 

s 

§ 
s 


§1 


Gelatin (3) 




Blood serum 




Casein 




Agar, mannan 




1 


Acid curd 




Rennet curd 1 { 


Casein peptonized 




Indol (4) 




Hydrogen sulphide 




Ammonia (4) 




Nitrates reduced (4) 




Fluorescent 




Luminous 




1 

n 


Animal pathogen, epizoon 




Plant pathogen, epiphyte 




Soil 




Milk 




Fresh water 




Salt water 




Sewage 




Iron bacterium 




Sulphur bacterium 













172 A MANUAL OF BACTEEIOLOGY 

GLOSSARY OF TERMS 

Agar hanging block, a small block of nutrient agar cut from a poured plate 
and placed on a cover glass, the surface next the glass having been first touched 
with a loop from a young fluid culture or with a dilution from the same. It is 
examined upside down, the same as a hanging drop. 

Amoeboid, assuming various shapes, like an amceba. 

Amorphous, without visible differentiation in structure. 

Arborescent, a branched, treelike growth. 

Beaded, in stab or stroke, disjointed or semiconfluent colonies along the line 
of inoculation. 

Brief, a few days, a week. 

Brittle, growth dry, friable under the platinum needle. 

BuUate, growth rising in convex prominences, like a blistered surface. 

Butyrous, growth of a butterlike consistency. 

Chains, short chains, composed of 2 to 8 elements ; long chains, composed of 
more than 8 elements. 

Ciliate, having fine, hairlike extensions, like cilia. 

Cloudy, said of fiuid cultures which do not contain pseudozooglcEse. 

Coagulation, the separation of casein from whey in milk. This may take 
place quickly or slowly, and as the result either of the formation of an acid 
or of a lab ferment. 

Contoured, an irregular, smoothly undulating surface, like that of a relief map. 

Convex, surface the segment of a circle, but flattened. 

Coprophil, dung bacteria. 

Coriaceous, growth tough, leathery, not yielding to the platinum needle. 

Crateriform, round, depressed, due to the liquefaction of the medium. 

Cretaceous, growth opaque and white, chalky. 

Curled, composed of parallel chains in wavy strands, as in anthrax colonies. 

Diastasic action (same as diastatic), conversion of starch into water-soluble 
substances by diastase. 

Echinnlate, in agar stroke, agrowth along line of inoculation, with toothed or 
pointed margins ; in stab cultures, growth beset with pointed outgrowths. 

Effuse, growth thin, veily, unusually spreading. 

Entire', smooth, having a margin destitute of teeth or notches. 

Erose, border irregularly toothed. 

Filamentous, growth composed of long, irregularly placed or interwoven 
filaments. 

Filiform, in stroke or stab cultures a uniform growth along line of inoculation. 

Fimbriate, border fringed with slender processes, larger than filaments. 

Floccose, growth composed of short, curved chains, variously oriented. 

Flocculent, said of fluids which contain pseudozoogloese, that is, small adherent 
masses of bacteria of various shapes and floating in the culture fluid. 

Fluorescent, having one color by transmitted light and another by reflected 
light. 

Gram's stain, a method of differential bleaching after gentian violet, methyl 
violet, etc. The + mark is to be given only when the bacteria are deep blue or 
remain blue after counterstaining with Bismarck brovm. 



APPENDIX P 173 

Grumose, clotted. 

Infundibaliform, form of a funnel or inverted cone. 

Iridescent, like mother-of-pearl. Tlie effect of very thin films. 

Lacerate, having the margin cut into irregular segments, as if torn. 

Lobate, border deeply undulate, producing lobes (see Undulate). 

Long, many weeks or months. 

Mairimiini temperature, temperature above which grovrth does not take place. 

Medium, several weeks. 

Membranous, growth thin, coherent, like a membrane. 

lyrininiiini temperature, temperature below which growth does not take place. 

Mycelioid, colonies having the radiately filamentous appearance of mold 
colonies. 

Hapifoim, liquefaction with the form of a turnip. 

Nitrogen requirements, the necessary nitrogenous food. This is determined by 
adding to nitrogen-free media the nitrogen compound to be tested. 

Opalescent, resembling the color of an opal. 

Optimum temperature, temperature at which growth is most rapid. 

Pellicle, in fluid, bacterial growth either forming a continuous or an inter- 
rupted sheet over the fluid. 

Peptonized, said of curds dissolved by trypsin. 

Persistent, many weeks or months. 

Plumose, a fleecy or feathery growth. 

Pseudozooglss, clumps of bacteria, not dissolving readily in water, arising 
from imperfect separation or more or less fusion of the components, but not 
having the degree of compactness and gelatinization seen in zoogloeae. 

Pulvinate, in the form of a cushion, decidedly convex. 

Punctifonn, very minute colonies, at the limit of natural vision. 

Raised, growth thick, with abrupt or terraced edges. 

Rapid, developing in twenty-four to forty-eight hours. 

Repand, wrinkled. 

Rhizoid, growth of an irregular-branched, or rootlike character, as in 
B. mycoides. 

Ring (same as rim), growth at the upper margin of a liquid culture, adhering 
more or less closely to the glass. 

Saccate, liquefaction the shape of an elongated sack, tubular, cylindrical. 

Scum, floating islands of bacteria ; an interrupted pellicle or bacterial mem- 
brane. 

Short, applied to time, a few days, a week. 

Slow, requiring five or six days or more for development. 

Sporsmgia, cells containing endospores. 

Spreading, growth extending much beyond the line of iuoculation, that is, 
several millimeters or more. 

Stratiform, liquefying to the walls of the tube at the top and then proceeding 
downward horizontally. 

Thermal death point, the degree of heat required to kill young fluid cultures 
of an organism exjxised for ten minutes (in thin-walled test tubes of a diameter 
not exceeding 20 mm.) in the thermal water bath. The water must be kept 
agitated, so that the temperature shall be uniform during the exposure. 



174 



A MANUAL OF BACTEEIOLOGY 



Transient, a few days. 

Turbid, cloudy, with flocculent particles ; cloudy plus flocculenoe. 

Umbonate, having a buttonlike, raised center. 

Undulate, border wavy, with shallow sinuses. 

Vermiform contoured, growth like a mass of worms or intestinal coils. 

Verrucose, growth wartlike, with wartlike prominences. 

Villous, growth beset with hairlike extensions. 

Viscid, growth follows the needle when touched and withdrawn ; sediment, on 
shaking, rises as a coherent swirl. 

Zoogloeae, firm gelatinous masses of bacteria, one of the most typical examples 
of which is the Streptococcus mesenterioides of sugar vats {Leuconostoc mesenteri- 
oides), the bacterial chains being surrounded by an enormously thickened firm 
covering, inside of which there may be one or many groups of the bacteria. 

TABLE 



A Numerical System of Rkcording the Salient Characters of 

AN Organism. (Group Number)^ 

100. Endospores produced 

200. Endospores not produced 

10. Aerobic (strict) 

20. Eacijltative anaerobic 

30. Anaerobic (strict) 

1. Gelatin liquefied 

2. Gelatin not liquefied 

0.1 Acid and gas from dextrose 

0.2 Acid without gas from dextrose 

0.3 No acid from dextrose 

0.4 No growth with dextrose 

.01 Acid and gas from lactose 

.02 Acid without gas from lactose 

.03 No acid from lactose 

.04 ■ No growth with lactose 

.001 Acid and gas from saccharose 

.002 Acid without gas from saccharose 

.003 No acid from saccharose 

.004 No growth with saccharose 

.0001 Nitrates reduced with evolution of gas 

.0002 Nitrates not reduced 

.0003 Nitrates reduced without gas formation 

.00001 Fluorescent 

.00002 Violet chromogens 

.00003 Blue chromogens 

.00004 Green chromogens 

.00005 Yellow chromogens 

1 This system will be found useful as a quick method of showing close relationships 
inside the genua, but is not a sufficient characterization of any organism. 



APPENDIX P 



175 



.00006 Orange chromogens 

.00007 Red chromogens 

.00008 Brown chromogens 

.00009 Pink chromogens 

.00000 Nonchromogenlc 

.000001 Diastasic action on potato starch, strong 

.000002 Diastasic action on potato starch, feeble 

.000003 Diastasic action on potato starch, absent 

.0000001 Acid and gas from glycerin 

.0000002 Acid without gas from glycerin 

.0000003 No acid from glycerin 

.0000004 No growth with glycerin 
The genus according to the system of Migula is given its proper symbol, 
which precedes the number thus (6) : 

Bach-lus coli (Esch.) Mig. becomes B. 222.111102 

Bacillus alcaligenes Petr. becomes B. 212.333102 

PsEUDOMONAs cAMPESTRis (Pam.) Sm. becomes Ps. 211.333151 
Bacteeium suicida Mig. 



becomes Bact. 222.232203 



INDEX 



Agar, nutrient, 6 ; sterilization of, 126 ; 

synthetic, 61 
Agar-agar, 6 
Agar slant, 10 
Air, bacteria of, 48 
Alcohol table, 162 
Aldehyde-reductase, 83 
Allihu's method for determination of 

sugars, 154 
Ammonia, determination of, 143 
Ammonification by soil bacteria, 62, 63 
Amylolytic action, 114 
Anaerobic bacteria, 30, 58 
Anthrax, 92 

Arsenic, detection of, 114 
Ashby's solution, 28 
Aspergillus, 5, 111, 114 
Atmospheric dust, 48 
Autoclave, 13 ; temperature of, 14, 119 
Azotobacter, 67, 68 ; medium for, 28 

Bacillus amylobacter, 72 

Bacillus amylovorus, 88 

Bacillus anthracis, 93 

Bacillus campestris, 89 

Bacillus carotoTorus, 89 

Bacillus coli, 54, 56, 57 

Bacillus cyanogenus, 86 

Bacillus denitrificans, 66, 67, 73 

Bacillus fluorescens liquefaciens, 50, 

66, 67 
Bacillus Hartlebii, 66, 67 
Bacillus lactis aerogenes, 82, 85 
Bacillus lactis viscosus, 86 
Bacillus mycoides, 72 
Bacillus prodigiosus, 50 
Bacillus proteus vulgaris, 57 
Bacillus pyocyaneus, 66, 67, 95 
Bacillus radicicola, 68, 70, 71, 73 
Bacillus subtilis, 50, 85 
Bacillus tuberculosis, 95 
Bacillus vulgatus, 72 
Bacterium lactis-acidi, 84 
Bacteroids, production of, 70 
Beef extract, 6 
Berkefeld filters, 121 
Bile medium, 25, 26 
Black rot of cabbage, 89 



Blight of pome fruits, 88 

Blood poisoning, 96 

Blood serum, 128 

Blue milk, 86 

Body cells in milk, 77, 78 

Bouillon, 9; glucose-formate, 24; glyc- 
erin, 27 ; iron, 26 ; lead, 26 ; neutral 
red, 25, 55 ; phenol, 25 ; sugar, 24 ; 
sulphindigotate, 30 ; titration of, 142 

Bread dough, 105 

Brownian movement, 4 

Carbol fuchsin (Ziehl), 33 

Catalase in milk, "79 

Cellulose decomposition by bacteria, 

58, 59 
Chamberland filter, 121 
Chart, descriptive, 166 
Cider, 107 
Clostridia, 2 
Colonies, 45, 47 
Colorimeter, Schreiner's, 144 
Colostrum, 79 
Cotton plugs, 9 
Cover-glass forceps, 34 
Cultural characters, 43 
Cultural features, descriptive terms, 

166-168 
Czapek's solution, 30 

Defren-O'SuUivan method for deter- 
mination of sugar, 158 

Dematium, 112 

Descriptive terms, for culturalfeatures, 
166 ; for morphology, 165 

Differential staining, 37 

Diseases of man and animals, 91 

Diseases of plants, 87 

Disinfectants, 18 ; preparation of, 92 

Drying, effect upon bacteria, 18 

Dunham's solution, 25, 57 

Enriching cultures, 56 

Enzymes, 21 

Fecal bacteria, 54 

Eermentation, 97 ; of bread dough, 
105 ; of cider, 107 ; organisms of, 
97 ; of vinegar, 108 ; of wine, 107 



177 



178 



A MANUAL OF BACTERIOLOGY 



Fermentation test for milk, 81 
Fermentation tube, 25 
Filtering apparatus, 122 
Filtration, 121 

Fractional sterilization, 13, 14 
Fuchsin, 33 

Gelatin, 6 ; nutrient, 10 ; sterilization 

of, 126 
Gentian violet, 34 
Giltay and Aberson's solution, 26 
Glossary of terms, 172-174 
Glucose-formate bouillon, 24 
Gram's iodine solution, 37, 67 
Gram's stain, 37 
Green pus, 95 

Heat, effect upon growth, 17 
Heyden-Nahrstoft agar, 27, 61 
Hot-air sterilizer, 15 

Infection threads, 69 
Inflammation, 96 
Inoculating chamber, 141 
Inoculating needles, 21 
Inoculating room, 39 
Invertase, 101 
Isolation methods, 38 

Kjeldahl method, 151 

Koch, Robert, 6 ; postulates of, 91 

Leeuwenhoek, 1 

Light, effect upon growth, 16 

Litmus milk, 23 

Litmus whey, 23 

Litmus-lactose agar, 24 

Litmus-lactose gelatin, 24 

Measures, conversion tables, 160 

Methylene blue, 33, 66, 83 

Micrococcus candicans, 50 

Microscope, 3 

Microspira tyrogena, 86 

Milk, acidity of, 80 ; bacteria of, 73 ; 
contamination of, 74 ; as a culture 
medium, 23 ; germicidal action of, 
78 ; organisms in, 75 ; reducing ac- 
tion of, 82 ; sterilization of, 127 

Mold cultures, 113 

Molds, amylolytic action of, 114 ; pep- 
tonizing action of, 114 

Mordants, 32 

Morphology, 41 ; descriptive terms, 
165-166 

Motility, 4 

Mucor, 108 



Nsegeli's solution, 29 

Nessler's reagent, 144 

Neutral red broth, 25, 55 

Neutralization of media, 10 

Nitrate, determination of, 146 

Nitrate formation, 64, 65 

Nitrate-forming organisms, medium 
for, 29 

Nitrification, 65 

Nitrite, determination of (Griess- 
Ilosvay), 148 ; (Trommsdorf), 150 

Nitrite formation, 64 

Nitrite-forming organisms, medium 
for, 29 

Nitrite reagent, 148 

Nitrogen, determination of, 151 ; fixa- 
tion by nonsymbiotic organisms, 67 ; 
fixation by symbiotic organisms, 68 ; 
relation of soil bacteria to, 62 

Nitrogen-free media, 28 

Novy jar, 30 

Numerical nomenclature, 174 

Nutrition, 5 

Occurrence of bacteria, 1 

Oidium lactis, 87 

Oxygen, relation to aerobes, 16 

Pasteurization, 84, 120 
Pectinase, 90 
Penicillium, 110, 114 
Peptonizing action of molds, 114 
Permanent preparations, 139 
Petri dishes, 16, 125 
Phenol bouillon, 25 
Phenoldisulphonic acid reagent, 146 
Physical and biochemical features, 

descriptive terms, 168 
Physiological salt solution, 32 
Poisons, stimulation by, 113 
Potato gelatin, 27 
Potatoes used for cultures, 11 
Pseudomonas Stewartii, 89 



Reductase, 83 
Root tubercles. 
Ropy milk, 86 
Roux tube, 11 



71 



Saccharomyces, 98 
Sake, 111 
Sarcina lutea, 50 
Seeds, sterilization of, 131 
Septicaemia, 96 
Sewage bacteria, 55 
Sewage streptococci, 55 
Snow, bacteria in, 55 
Soft rot of vegetables, 89 



INDEX 



179 



Soil, ammonification in, 63 ; bacteria 

of, 59 ; changes due to heating, 129 ; 

nitrification in, 65 ; sampling, 60 ; 

sterilization of, 128 
Sporangium, 109 
Spores, 2 
Stab cultures, 22 
Staining methods, 34 
Stains, capsule, 36 ; contrast, 35 ; 

flagella, 36 ; Gram's, 37 ; spore, 36 
Starch-iodide solution, 150 
Sterilization, 12, 115 ; by chemicals, 

115; by filtration, 121; by heat, 117; 

by steam, 118, 119; by ultra-violet 

light, 124 ; technique of, 125 
Stimulation, 113 
Stock cultures, 138 
Streptococcus pyogenes, 96 
Sugar bouillion, 24 
Sugar gelatin, 24 

Sugars, determination of, 154, 158 
Sulphates, reduction by bacteria, 71 

Titanium chloride, standardization of, 

159 
Torula, 112 



Total nitrogen, determination of, 151 
Tuberculin, 94 
Tuberculosis, 94 
Tyndallization, 13 
Tyndall's method, 13 

Van Delden's solution, 27 
Vinegar, fermentation of, 108 

Water, bacteria of, 51, 55 ; collecting 
samples, 51 

Water blanks, 32 

Water cultures under sterile condi- 
tions, 134 

Weights, conversion tables, 160 

Whey agar, 23 

WUt of sweet com, 89 

Wine, 107 

Yeast, 98 ; spore formation in, 99 

Ziehl-Neelson stain, 38 
Zoogloea, 4 
Zymase, 102 
Zymin, 103 










Each of the large^ 
40 and 60 represe^ 
by the respei 





mm 







mm 





« 




rter. The figures 
circles bounded 
of its circle