Saunders'  (juestion  Compends 


Essentials  of 
Bacteriology 

M.V.BALL.  M.D, 


Seventh  Edition 


New  (9th)  Edition 


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ESSENTIALS 


OF 


BACTERIOLOGY 


Since  the  issue  of  the  first  volume  of  the 
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BACTERIOLOGIC    MICROSCOPE. 


SAUNDERS'   QUESTION-COMPENDS.    No.  20 


ESSENTIALS 


BACTERIOLOGY 


CONCISE  AND  SYSTEMATIC  INTRODUCTION  TO  THE 
STUDY  OF  BACTERIA  AND  ALLIED  MICROORGANISMS 


t  *   a  *  ■ 


professor'   of'    pathology,      NBW     YORK      MEDICAL     COLLEGE     AND     HOSPITAL      FOR 

WOMEN,      NEW     YORK     CITY  ;       MEMBER     OF     THE       ACADEMY      OF      NATURAL 

SCIHKCBS        OF        PHILADELPHIA;         FORMERLY       INSTRUCTOR       IN 

BACTERIOLOGY  AT  THE  PHILADELPHIA  POLYCLINIC. 


Assisted  By 

PAUL   G,    WESTON,    M.  D. 

PATHOLOGIST   STATE    HOSPITAL    FOR    INSANE    AT    WARREN,    FA. 

SEVENTH   EDITION,  THOROUGHLY   REVISED 

With  118  lUustrations,  some  in  Colors 
PHILADELPHIA   AND   LONDON 

W.   B*    SAUNDERS    COMPANY 

J9I8 


Copyright,  1891,  by  W.  B.  Saunders.     RepW.jted  October,  1892.     Revised,  reprinted, 
and  recopyrighted  May,  1893.      Reprinted  June,  1894,    Revised,  reprinted,  and  re- 
copyrighted  November,  1896.     Reprinted  October,   1898.     Revised,  reprinted, 
and  recopyrighted  March,  1900.    Reprinted  May,  1903.    Revised,  reprinted, 
and    recopyrighted    August,    1904.      Reprinted    October,    1905,    and 
August,  1905:.  -  Re<'ised,  entirely  resgt.  reprinted,  §nd  rec9p_yrighted 
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reset,  rv-'prtoteii,  a«id<.iecopy-ig4i{cd   De'?em|)e'-,  1919. 
c      ,         ,     ,Repryite4  January,  1914  ^ 


Copyright*  i9i3,*by  W.  ET.  launders  Company. 


Reprinted  July,  1916 


Reprinted  May,  191 8 


PRINTED    IN    AMERICA 


PRESS   OF 
SAUNDERS    COMPANY 
PHILADELPHIA 


(^/O- 


PREFACE  TO  THE  SEVENTH  EDITION 


This  book  has  undergone  a  complete  revision  and  many 
of  the  chapters  have  been  rewritten  in  their  entirety.  Those 
which  relate  to  immunity  and  infection  have  been  carefully 
edited  by  Dr.  Paul  G.  Weston,  Pathologist  at  the  State 
Hospital,  Warren,  Pa.,  who  has  also  furnished  the  article 
on  the  Wassermann  reaction.  The  author  is  likewise  in- 
debted to  him  for  valuable  aid  in  other  portions  of  the  re- 
vision. 

The  author  realizes  that  compends  of  this  nature  must 
necessarily  suffer  in  comparison  with  the  larger  and  more 
elaborate  works,  and  he  trusts  that  the  reviewers  will  bear 
this  in  mind  in  their  criticisms. 

When  this  book  first  appeared  in  1891  it  was  one  of  the 
first  American  publications  on  the  subject,  and  only  a  few 
text-books  had  been  issued  in  other  countries.  Although 
since  then  a  great  many  excellent  treatises  have  appeared, 
there  still  remains  a  place  for  this  compend,  and  hence  this 
new  edition.  The  author  hopes  that  he  has  succeeded  in  incor- 
porating all  the  newer  established  facts  in  bacteriology  and 
in  eliminating  all  that  is  obsolete  and  no  longer  in  use. 

M.  V.  Ball. 


3577<^ 


PREFACE  TO    THE  FIRST   EDITION 


Feeling  the  need  of  a  Compendium  on  the  subject  of 
this  work,  it  has  been  our  aim  to  produce  a  concise  treatise 
upon  the  Practical  Bacteriology  of  to-day,  chiefly  for  the 
medical  student,  which  he  may  use  in  his  laboratory. 

It  is  the  result  of  experience  gained  in  the  Laboratory 
of  the  Hygienical  Institute,  Berlin,  under  the  guidance  of 
Koch  and  Frankel;  and  of  information  gathered  from  the 
original  works  of  other  German,  as  well  as  of  French,  bac- 
teriologists. 

Theory  and  obsolete  methods  have  been  slightly  touched 
upon.  The  scope  of  the  work  and  want  of  space  forbade 
adequate  consideration  of  them.  The  exact  measurements 
of  bacteria  have  not  been  given.  The  same  bacterium  varies 
often  much  in  size,  owing  to  differences  in  the  media,  staining, 
etc. 

We  have  received  special  help  from  the  following  books, 
which  we  recommend  to  students  for  further  reference: 

Mace:  Traite  pratique  de  Bacteriologie. 
Frankel:  Grundriss  der  Bakterienkunde. 
Eisenberg:  Bakteriologische  Diagnostik. 
Crookshank,  E.  M.:  Manual  of  Bacteriology. 
Gunther:  Einfiihring  in  das  Studium  der  Bacteriologie,  etc. 
WooDHEAD  and  Hare:  Pathological  Mycology. 
Salmonsen:  Bacteriological    Technique    (English    transla- 
tion) . 

M.  V.  BAUL. 
11 


CONTENTS 


PACK 

Introduction 17 


PART  I 

GENERAL  BACTERIOLOGY 

Chapter         I — Structure  and  Development  of  Bacteria  ... .  21 

Chapter        II — Biologic  and  Chemic  Activities 26 

Chapter      III — Infection 30 

Chapter       IV — Immunity 34 

Chapter         V — Methods  of  Studying  Bacteria — Microscope  43 

Chapter       VI — Methods  of  Studying  Bacteria  (Continued), 

Solutions  and  Formulas  for  Staining 47 

Chapter     VII— General  Method  of  Staining  Specimens 54 

Chapter   VIII — Special  Methods  of  Staining  and  Modifica- 
tions   „ 58 

Chapter      IX — Cultivation  of  Bacteria 62 

Chapter        X — Preparation  of  Nutrient  Culture-media  ....  67 

Chapter      XI — Inoculation  of  Culture-media 78 

Chapter     XII — Cultivation  of  Anaerobic  Bacteria 82 

Chapter  XIII — The  Growth  and  Appearances  of  Colonies  ...  87 

Chapter    XIV — Animal  Inoculation 91 

Chapter     XV — Bacterins  (Vaccines) 95 

PART  II 

SPECIAL  BACTERIOLOGY 

Chapter    XVI — Some  Common  Bacteria  Slightly  Pathogenic.  97 

Bacterium  Prodigiosum 97 

Bacillus  Mesentericus  Vulgatus 98 

Bacillus  Megaterium 99 

Bacillus  Ramosus .  .  .  .• 99 

Bacterium  Zopfii 100 

Bacillus  Subtilis  (Hay  Bacillus) icx> 

13 


14  CONTENTS 

PAGE 

Boas-Oppler  Bacillus loi 

Bacillus  Violaceus 102 

Microorganisms  Found  in  Urine 102 

Micrococcus  Ureae 102 

Spirilla 103 

Spirillum  Rubrum 103 

Sarcina 103 

Sarcina  Lutea 103 

Sarcina  Aurantiaca 104 

Sarcina  Ventriculi 104 

Chapter  XVII — Bacillus  of  Anthrax 105 

Chapter  XVIII — Bacillus  Tuberculosis  and  Allied  Organ- 
isms    no 

Other  Acid-fast  Bacteria 116 

Bovine  and  Human  Tuberculosis 119 

Products  of  Tubercle  Bacilli 1 20 

Lepra  Bacillus 122 

Smegma  Bacillus  of  Alvarey  and  Tavel 1 23 

Bacillus  of  Glanders  (Bacillus  Mallei;  Rotz-Bacillus) 124 

Chapter  XIX— Diphtheria  Bacillus 126 

Chapter   XX — The  Colon-typhoid  Group 133 

Bacillus  Coli 134 

Bacillus  of  Typhoid  or  Enteric  Fever 135 

Antityphoid  Bacterins  (Vaccines) 139 

Differentiation  Between  Colon  and  Typhoid 142 

Typhoid  Bacilli  from  Blood 143 

Paracolon  or  Paratyphoid  Bacilli 143 

Bacillus  Botulinus 144 

Bacillus  Dysenteriae 145 

Bacterium  Termo 147 

Bacillus  Proteus  Vulgaris 147 

Proteus  Mirabilis 147 

Proteus  Zenkeri 148 

Chapter  XXI — Cholera  Bacteria 148 

Spirillum  Cholerae  (Comma  Bacillus  of  Cholera) 148 

Chapter  XXII — Bacteria  in  Pneumonia 155 

Diplococcus  Pneumoniae 157 

Bacillus  Pneumoniae 159 

Bacillus  of  Rhinoscleroma 159 

Bacillus  of  Influenza 160 

Koch-Weeks  Bacillus 161 

Bacillus  of  Pertussis  (Whooping-cough) 161 

Bacillus  Melitensis 162 

Chapter  XXIII — Pyogenic  Cocci 163 

Streptococcus  Pyogenes;  Streptococcus  Erysipelatis 164 

Staphylococcus  Pyogenes  Aureus 166 


CONTENTS  1 5 

PACK 

Staphylococcus  Pyogenes  Albus 168 

Micrococcus  Pyogenes  Citreus 169 

Micrococcus  Cereus  Albus 169 

Micrococcus  Cereus  Flavus 169 

Micrococcus  Pyogenes  Tenuis 169 

Micrococcus  Tetragenus 170 

Morax-Axenfeld  Diplobacillus  of  Conjunctivitis 171 

Bacillus  Pyocyaneus 171 

Chapter  XXIV — Gonococcus — Meningococcus 174 

Micrococcus  Gonorrhoeae 174 

Allied  Varieties 176 

Diplococcus  Intracellularis  Meningitidis 177 

Bacillus  of  Soft  Chancre,  Chancroid 178 

Chapter  XXV — Anaerobic   Bacteria   (Bacillus  of  Tetanus; 

Bacillus  of  Malignant  Edema,  Etc.) 180 

Bacillus  of  Tetanus 180 

Bacillus  (Edematis  Maligni;  Vibrion  Septique 184 

Bacillus  Aerogenes  Capsulatus 186 

Bacillus  Enteritidis  Sporogenes 187 

Bacillus  Chauvei 187 

Chapter  XXVI — Hemorrhagic  Septicemia  Group 190 

Bacteria  of  Hemorrhagic  Septicemia 192 

Bacillus  of  Chicken  Cholera 193 

Bacillus  of  Erysipelas  of  Swine 194 

Bacillus  Murisepticus;  Mouse  Septicemia 195 

Micrococcus  of  Mai  de  Pis 196 

Chapter  XXVII — Protozoa 197 

Entamoeba  Histolytica;  Amoeba  Dysenteriae 198 

Life  Cycle  of  Malarial  Sporozoa 199 

Three  Forrns  of  Malarial  Protozoa 201 

Methods  of  Examination  for  Malarial  Organisms 203 

Trypanosomata 204 

Trypanosoma  Lewisi 205 

Trypanosoma  Brucei 206 

Sleeping  Sickness;  Trypanosoma  Ugandense  Gambiense 207 

Trypanosoma  Evansi 208 

Herpetomonas  (Leishman-Donovan  Bodies) 208 

Piroplasma  Boyis  (Piroplasma  Bigeminum) 208 

Rabies  or  Hydrophobia — Negri  Bodies 209 

Chapter  XXVIII — The  Micro-organism  of  Syphilis  and  Al- 
lied Organisms 209 

Spirochaeta  Pallida 209 

Wassermann  Reaction 211 

Noguchi  Modification  of  Wassermann  Reaction 214 

Luetin  Reaction 216 

Yaws ; 217 

Spirillum  of  Relapsing  Fever 217 

African  Tick  Fever 218 


l6  CONTENTS 

PAGE 

Chapter  XXIX— Filterable  Organisms 218 

Filterable  or  Ultra-microscopic  Organisms 218 

Small-pox  and  Vaccinia 218 

Yellow  Fever 219 

Measles .  219 

Typhus  Fever 219 

Acute  Poliomyelitis 219 

Chapter  XXX— Yeasts  and  Molds 220 

Blastomycetes 220 

Saccharomyces  Cerevisiae  (Torula  Cerevisiae) 220 

Saccharomyces  Rosaceus;  S.  Niger;  S.  Albicans 221 

, Saccharomyces  Mycoderma 221 

Oidium 221 

Oidium  Lactis 222 

Oidium  Albicans  (Soor;  Thrush  Fungus  ) 222 

Pathogenic  Yeasts 222 

Blastomycetic  Dermatitis  or  Oidiomycosis 223 

Hyphomycetes  (True  Molds) 223 

Penicillium  Glaucum 223 

Mucor  Mucedo 224 

Achorion  Schonleinii 224 

Trichophyton  Tonsurans  (Ring- worm) 225 

Microsporon  Furfur ' 226 

Aspergillus  Glaucus 226 

Aspergillus  Fumigatus 226 

Examination  of  Yeasts  and  Molds 226 

Cladothrices  and  Streptothrices 227 

Crenothrix  Kiihniana 227 

Cladothrix  Dichotoma 227 

Leptothrix  Buccalis 228 

Beggiatoa  Alba 228 

Streptothrix  or  Cladothrix  Actinomyces  (Ray  Fungus) 228 

Streptothrix  Madurae 230 

Nocardia  (Streptothrix)  Farcinica;  Bovine  Farcin  du  Boeuf .  .  .  231 

Plant  Diseases  Due  to  Bacteria 23 1 

Chapter     XXXI — Examination  of  Air,  Soil,  antj  Water 232 

Chapter   XXXII— Bacteria  in  Milk  and  Food 246 

Chapter  XXXIII — Bacteriologic  Examination  of  the  Organs 

AND  Cavities  of  the  Human  Body 257 

Chapter  XXXIV — Germicides,  Antiseptics,  and  Antisepsis  .  .  .  261 

Tables  of  Chief  Characteristics  of  Principal  Bacteria 268 

Non-pathogenic 268 

Pathogenic 286 

Index 30^ 


Essentials  of  Bacteriology 


INTRODUCTION 


History. — The  microscope  was  invented  about  the  latter 
part  of  the  sixteenth  century,  and  soon  after,  by  its  aid, 
minute  organisms  were  found  in  decomposing  substances. 
Kircher,  in  1646,  suggested  that  diseases  might  be  due  to 
similar  organisms,  but  the  means  at  his  disposal  were  insuffi- 
cient to  enable  him  to  prove  his  theories.  Anthony  van 
Leeuwenhoek,  of  Delft,  Holland  (1680  to  1723),  so  improved 
the  instrument  that  he  was  enabled  thereby  to  discover 
micro-organisms  in  vegetable  infusion,  saliva,  fecal  matter, 
and  scrapings  from  the  teeth.  He  distinguished  several 
varieties,  showed  them  to  have  the  power  of  locomotion,  and 
compared  them  in  size  with  various  grains  of  definite  measure- 
ment. It  was  a  great  service  that  this  "Dutch  naturalist" 
rendered  the  world;  and  he  can  rightly  be  called  the  "father 
of  microscopy." 

Various  theories  were  then  formulated  by  physicians  to 
connect  the  origin  of  different  diseases  with  bacteria;  but  no 
proofs  of  the  connection  could  be  obtained.  Andry,  in  1701, 
called  bacteria  worms.  Mliller,  of  Copenhagen,  in  1786, 
made  a  classification  composed  of  two  main  divisions — monas 
and  vibrio;  and  with  the  aid  of  the  compound  microscope  was 
better  able  to  describe  them.  Ehrenberg,  in  1833,  with  still 
better  instruments,  divided  bacteria  into  four  orders:  bac- 
terium, vibrio,  spirillum,  and  spirochaete.  It  was  not  until 
2  17 


1 8  ESSENTIALS   OF  BACTERIOLOGY 

1863  that  any  positive  advance  was  made  in  connecting  bac- 
teria with  disease.  Rayer  and  Davaine  had,  in  1850,  found 
a  rod-shaped  bacterium  in  the  blood  of  animals  suffering 
from  splenic  fever  (sang  de  rate),  but  they  attached  no  special 
significance  to  their  discovery  until  Pasteur  made  public  his 
grand  researches  in  regard  to  fermentation  and  the  role 
bacteria  played  in  the  economy.  Then  Davaine  resumed  his 
studies,  and  in  1863  established  by  experiments  the  bacterial 
nature  of  splenic  fever  or  anthrax. 

But  the  first  complete  study  of  a  contagious  affection  was 
made  by  Pasteur  in  1869,  in  the  diseases  affecting  silk- worms, 
— pebrine  and  flacherie, — which  he  showed  to  be  due  to 
micro-organisms . 

Then  Koch,  in  1875,  described  more  fully  the  anthrax 
bacillus,  gave  a  description  of  its  spores  and  the  properties 
of  the  same,  and  was  enabled  to  cultivate  the  germ  on  arti- 
ficial media;  and,  to  complete  the  chain  of  evidence,  Pasteur 
and  his  pupils  supplied  the  last  link  by  reproducing  the  same 
disease  in  animals  by  artificial  inoculation  from  pure  cultures. 
The  study  of  the  bacterial  nature  of  anthrax  has  been  the 
basis  of  our  knowledge  of  all  contagious  maladies,  and  most 
advances  have  been  made  first  with  the  bacterium  of  that 
disease. 

Up  to  1875  most  medical  men  believed  that  bacteria 
originated  in  pus  and  did  not  associate  them  with  the  cause 
of  suppuration.  Lister  then  began  the  practice  of  treating 
wounds  and  operating  antiseptically,  having  formed  the 
theory  that  inflammation  and  suppuration  were  due  to  the 
contamination  of  wounds  by  germs  from  the  air,  instruments, 
etc.  From  1880  to  1890  the  most  important  organisms  were 
discovered  and  associated  with  disease. 

In  1890  the  discovery  of  the  blood-serum  therapy,  the 
antitoxin  of  Behring,  established  a  new  field  of  research,  and 
much  work  was  undertaken  with  a  view  to  curing  disease. 

The  researches  of  Ehrlich  and  the  endeavors  of  Metch- 
nikoff,  Hankin  and  Ehrlich,  to  account  for  the  phenomena  of 
immunity,  brought  forth  a  great  mass  of  literature  and  es~ 


INTRODUCTION  1 9 

tablished  the  "lateral-chain"  theory  and  theory  of  phago- 
cytosis. These  theoretic  problems  occupied  the  attention  of 
the  workers  from  1890  to  1905  and  are  by  no  means  ended. 
Laveran,  in  1881,  had  discovered  the  protozoa  of  malaria, 
and  in  1903  Button  had  associated  trypanosomes  with 
sleeping  sickness.  In  1905  Schaudinn,  by  demonstrating 
the  cause  of  syphilis  to  be  a  protozoon,  gave  added  im- 
portance to  this  particular  group  of  micro-organisms,  and 
today  investigators  are  looking  in  this  branch  of  microbiology 
for  the  cause  of  cancer. 

The  serum  reactions  of  Wassermann  and  Noguchi,  the 
tuberculins  and  other  products  of  bacterial  growth  useful  in 
diagnosis  and  treatment,  have  interested  the  whole  medical 
world,  and  every  physician  must  of  necessity  be  familiar  with 
some  part  of  this  knowledge. 

There  is  hope  that  the  technic  and  the  microscope  will 
receive  more  attention  in  the  next  few  years,  so  that  the  so- 
called  ultramicroscopic  and  filterable  organisms  that  are 
believed  to  exist  will  be  definitely  determined,  and  also  the 
cause  of  such  epidemic  diseases  as  smallpox  and  scarlet  fever 
be  ascertained. 


PART  I 
GENERAL  BACTERIOLOGY 


CHAPTER  I 
STRUCTURE  AND  DEVELOPMENT  OF  BACTERU 

The  bacteria  occupy  the  lowest  plane  of  plant  life  known 
to  us,  though  they  are  by  no  means  as  primitive  in  their 
biology  as  was  formerly  supposed,  and  it  is  quite  possible 
that  still  simpler  forms  may  be  discovered.  The  ultra- 
microscope  gives  promise  of  such  minute  organisms,  and 
has  made  visible  particles  of  matter  yj--^-  the  size  of  our 
smallest  known  bacteria. 

The  numerous  unicellular  vegetable  organisms  which  form 
the  lower  limit  of  plant   life   multiply  by  fission  and   are 


*^'l^fe>  -^ 


Fig.  I. — ^Types  of  bacteria:  a,  Micrococcus;  b,  spirillum;  c,  bacillus. 

hence  called  the  Schizophyta,  or  splitting  plants.  This 
group  is  subdivided  into  two  classes — (a)  the  Schizophycece, 
or  fission  algae,  and  (b)  the  Schizomycetes^  or  fission  fungi, 

jor  bacteria,  as  we  usually  call  them. 

TV  Bacteria  are  unicellular  masses  of  protoplasm  of  microscopic 
size,  multiplying  by  fission  and  existing  without  chlorophyl, 

I  Three  main  types  are  found:  (i)  Globular  forms,  called 
cocci;  (2)  straight  rod-shaped  forms,  called  bacilli;  (3)  curved 
or  spiral  rods,  called  spirilla.     (See  Fig.  i.) 


22  ESSENTIALS    OF   BACTERIOLOGY 

Classifications. — Various  ones  have  been  proposed:  Mor- 
phologic, as  micrococci,  spirilla,  and  bacilli.  Physiologic,  ac- 
cording to  their  activities  and  functions,  as  acid  bacteria, 
alkah  and  indol  bacteria;  then  subdivisions,  according  to 
motiHty  or  need  for  oxygen,  but  none  are  satisfactory. 
The  tendency  to  place  bacteria  similar  in  their  disease- 
producing  manifestations  in  one  group  is  growing,  as,  for  in- 
stance, the  colon  group,  the  pus-producers,  the  pneumonic 
group,  etc. 

Structure. — Bacteria  are  cells;  they  appear  as  round  or 
cylindric,  of  an  average  diameter  on  transverse  section  of 
o.ooi  mm.  (=1  micromillimeter),  written  /x  =  ^5o0o  inch. 
The  cell,  as  other  plant-cells,  is  composed  of  a  membranous 
cell-wall  and  cell-contents  or  cytoplasm. 

Cell-wall. — The  cell- wall  is  composed  either  of  hemi- 
cellulose,  or  a  form  of  albumin,  since  it  is  less  permeable  than 
cellulose  membrane.  The  membrane  is  firm,  and  can  be 
brought  plainly  into  view  by  the  action  of  iodin  upon  the 
cell-contents,  which  contract  them. 

Cell-contents. — ^The  contents  of  the  cell  consist  mainly  of 
protoplasm,  usually  homogeneous,  but  in  some  varieties 
finely  granular,  or  holding  pigment,  chlorophyl,  fat-droplets, 
and  sulphur  in  its  structure.  The  protoplasm  permits  osmo- 
sis, and  is  like  that  of  other  plant-cells  in  its  structure. 

Chemic  Composition  of  Bacteria. — The  ash  is  mostly 
phosphoric  acid;  potassium,  chlorin,  and  calcium  are  present 
to  a  small  extent;  80  to  90  per  cent,  is  water.  The  bacteria 
resemble  the  lower  animals,  rather  than  plants,  in  chemic 
composition. 

Nuclein,  hypoxanthin,  and  other  nitrogen  compounds  are 
found  in  most  bacteria.  Varies  with  media  in  which  grown; 
the.  proteids  are  about  10  per  cent.;  fats,  i  per  cent.;  ash, 
0.75  per  cent. 

Gelatinous  Membrane. — ^The  outer  layer  of  the  cell- 
membrane  can  absorb  water  and  become  gelatinoid,  forming 
either  a  little  envelop  or  capsule  around  the  bacterium  or 
preventing  the  separation  of  the  newly  branched  germs, 


STRUCTURE  AND  DEVELOPMENT   OF   BACTERIA  23 

forming  chains  and  bunches,  as  streptococci  and  staphylococci. 
Long  filaments  are  also  formed. 

Zooglea. — When  this  gelatinous  membrane  is  very  thick, 
irregular  masses  of  bacteria  will  be  formed,  the  whole  growth 
being  in  one  jelly-like  lump.  This  is  termed  a  zooglea  {^Coov, 
animal,  yKoios,  glue). 

Locomotion. — Many  bacteria  possess  the  faculty  of  self- 
movement,  carrying  themselves  in  all  manner  of  ways  across 
the  microscopic  field — some  very  quickly,  others  leisurely. 

Vibratory  Movements. — Some  bacteria  vibrate  in  them- 
selves, appearing  to  move,  but  they  do  not  change  their 


/ 


/5  a 


Fig.  2. — XyP^s  of  flagelia:  a.  Vibrio  choleras,  one  flagellum  at  the  end 
— monotrichla  txpe;  &,  Bacterium  syncyaneum,  tuft  of  flagella  at  the 
encl^  rarely  atlfie  side — lophgtrichia  type;  c,  Bacterium  vulgare,  flagella 
arranged  all  about — peritrichia~type  (Lehmann  and  Neumann). 


place;  these  movements  are  denoted  as  molecular  or  ''Brown- 
ian,"  and  are  due  to  purely  physical  causes,  such  as  may  be 
obtained  by  suspending  fine  grains  of  carmin  in  water. 

Flagella. — Little  threads  or  lashes  are  found  attached  to 
many  of  the  motile  bacteria,  either  at  the  poles  or  along  the 
sides — sometimes  only  one,  and  on  some  several,  forming  a 
tuft. 

These  flagella  are  in  constant  motion,  and  can  probably  be 
considered  as  the  organs  of  locomotion;  they  have  not  been 
discovered  upon  all  the  motile  bacteria,  owing,  no  doubt,  to 
our  imperfect  methods  of  observation.  They  can  be  stained 
and  have  been  photographed.     (See  Fig.  2.)     Flagella  serve 


24  ESSENTIALS    OF   BACTERIOLOGY 

sometimes  to  increase  food-supply,  and  have  been  found  on 
some  species  whicJi  are  non-motile. 

Reproduction. — Bacteria  multiply  through  simple  divi- 
sion or  fission,  as  it  is  called.  Spore  formation  is  simply  a 
resting  stagehand  hot  a  means  of  multiplication.  To  accom- 
plish division  the  cell  elongates,  and  at  one  portion,  usually 
the  middle,  the  cell-wall  indents  itself  gradually,  forming  a 
septum  and  dividing  the  cell  into  two  equal  parts,  just  as 
occurs  in  the  higher  plant  and  animal  cells.     (See  Fig.  3.) 

Successive  divisions  take  place,  the  new  members  either 
existing  as  separate  cells  or  forming  part  of  a  community  or 


Fig.  3. — Division  of  bacteria:  a,  Division  of  a  micrococcus;  h,  division  of 
a  bacillus  (after  Mace). 

group.  It  has  been  computed  that  if  division  takes  place 
every  hour,  as  it  often  does,  one  individual  in  twenty-four 
hours  will  have  7,000,000  descendants. 

Spore  Formations. — Two  forms  of  sporulation,  endo- 
sporous  and  arthrosporous. 

Endosporous. — First,  a  small  granule  develops  in  the  pro- 
toplasm of  a  bacterium;  this  increases  in  size,  or  several  little 
granules  coalesce  to  form  an  elongated,  highly  refractive, 
and  clearly  defined  object,  rapidly  attaining  its  real  size,  and 
this  is  the  spore.  The  remainder  of  the  cell-contents  has  now 
disappeared,  leaving  the  spore  in  a  dark,  very  resistant  mem- 
brane or  capsule,  and  beyond  this  the  weak  cell-wall.     The 


STRUCTURE   AND   DEVELOPMENT   OF   BACTERIA 


25 


cell-wall  dissolves  gradually  or  stretches  and  allows  the  spore 
to  be  set  free. 

Each  bacterium  gives  rise  to  but  one  spore.  It  may  be  at 
either  end  or  in  the  middle  (Fig.  4).  Some  rods  take  on  a 
peculiar  shape  at  the  site  of  the  spore,  making  the  rod  look 
like  a  drum-stick  or  spindle — Clostridium  (Fig.  5). 

Spore  Contents. — What  the  real  contents  of  spores  are  is 
not  known.  In  the  mother-cell  at  the  site  of  the  spore  little 
granules  have  been  found  which  stain  differently  from  the  rest 


Fig.  4. — Sporulation  (after  De  Bary). 


Fig.  5. — Clostridium. 


of  the  cell,  and  these  are  supposed  to  be  the  beginnings — the 
sporogenic  bodies.  The  most  important  part  of  the  spore  is 
its  capsule;  to  this  it  owes  its  resisting  properties.  It  con- 
sists of  two  separate  layers — a  thin  membrane  aroimd  the 
cell,  and  a  firm  outer  gelatinous  envelop. 

Gennination. — When  brought  into  favorable  conditions, 
the  spore  begins  to  lose  its  shining  appearance,  the  outer  firm 
membrane  begins  to  swell,  and  it  now  assumes  the  shape  and 
size  of  the  cell  from  which  it  sprang,  the  capsule  having  burst, 
so  as  to  allow  the  young  bacillus  to  be  set  free. 

Requisites  for  Spore  Formation. — It  was  formerly 
thought  that  when  the  substratum  could  no  longer  maintain 


26  ESSENTIALS    OF   BACTERIOLOGY 

it,  or  had  become  infiltrated  with  detrimental  products,  the 
bacterium-cell  produced  spores,  or  rather  turned  itself  into  a 
spore  to  escape  annihilation;  but  we  believe  now  that  only 
when  conditions  are  the  most  favorable  to  the  well-being  of 
the  cell  does  it  produce  fruit,  just  as  with  every  other  type  of 
plant  or  animal  life,  a  certain  amount  of  oxygen  and  heat 
being  necessary  for  good  spore  formation.  The  question  is 
still  unsettled,  however. 

Asporogenic  Bacteria. — Bacteria  can  be  so  damaged  that 
they  will  remain  sterile — not  produce  any  spores.  This  con- 
dition can  be  temporary  only  or  permanent. 

Arthrosporous. — In  the  other  group,  called  arthrospores, 
individual  members  of  a  colony  or  aggregation  leave  the  same, 
and  become  the  originators  of  new  colonies,  thus  assuming  the 
character  of  spores. 
'The  micrococci  furnish  examples  of  this  form. 

Some  authorities  have  denied  the  existence  of  the  arthro- 
sporous formation. 

Resistance  of  Spores. — Because  of  the  very  tenacious  en- 
velop, the  spore  is  not  easily  influenced  by  external  measures. 
It  is  said  to  be  the  most  resisting  object  of  the  organic  world. 

Chemic  and  physical  agents  that  easily  destroy  other  life 
have  very  little  effect  upon  it. 

Many  spores  require  a  temperature  of  140°  C.  dry  heat  for 
several  hours  to  destroy  them.  The  spores  of  a  variety  of 
potato  bacillus  (Bacillus  mesentericus)  can  withstand  the 
application  of  steam  at  ioo°  C.  for  four  hours. 


CHAPTER  II 

BIOLOGIC  AND  CHEMIC  ACTIVITIES 

Origin  of  Bacteria. — As  Pasteur  has  shown,  all  bacteria 
develop  from  preexisting  bacteria  or  the  spores  of  the  same. 
They  cannot  arise  out  of  nothing. 

Distribution. — The  wide  and  almost  universal  diffusion 
of  bacteria  is  due  to  the  minuteness  of  the  cells  and  the 


BIOLOGIC   AND   CHEMIC   ACTIVITIES  27 

few  requirements  for  their  existence.  In  a  drop  of  water 
1,700,000,000  cocci  can  find  space. 

Very  few  places  are  free  from  germs;  the  air  on  the  high  seas 
and  on  the  mountain-tops  is  said  to  be  free  from  bacteria, 
but  this  is  questionable. 

Specific  Nature. — One  kind  of  bacterium  will  not  pro- 
duce another  kind.  A  bacillus  does  not  arise  from  a  micro- 
coccus, or  the  typhoid  fever  bacillus  produce  the  bacillus  of 
tetanus. 

Saprophytes  and  Parasites. — Saprophytes:  caTrpos, put- 
rid; (f)VT6v,  plant.  Parasites:  irapa,  aside  of;  alros,  food. 
Those  bacteria  which  live  on  the  dead  remains  of  organic  life 
are  known  as  saprophytic  bacteria,  and  those  which  choose  the 
living  bodies  of  their  fellow-creatures  for  their  habitat  are 
called  parasitic  bacteria.  Some,  however,  develop  equally 
well  as  saprophytes  and  parasites.  They  are  called  faculta- 
tive parasites.  All  pathogenic  (disease-producing)  bacteria 
are  parasites. 

Conditions  of  Life  and  Growth  of  Bacteria. — Influence 
of  Temperature. — In  general,  a  temperature  ranging  from  io° 
C.  to  40°  C.  is  necessary  to  the  life  and  growth  of  bacteria. 

Saprophytes  take  the  lower  temperatures;  parasites,  the 
temperature  more  nearly  approaching  the  animal  heat  of  the 
warm  blooded.  Some  forms  require  a  nearly  constant  heat, 
growing  within  very  small  limits,  as  the  bacillus  of  tubercu- 
losis. 

Some  forms  can  be  arrested  in  their  development  by  a 
warmer  or  colder  temperature,  and  then  restored  to  activity 
by  a  return  to  the  natural  heat. 

A  few  varieties  exist  only  at  freezing-point  of  water,  and 
others  again  will  not  live  under  a  temperature  of  60°  C.  and 
thrive  in  hot  springs  at  a  temperature  of  89°  C. 

For  the  majority  of  bacteria  a  temperature  of  60°  C.  will 
prevent  development,  but  steam  under  pressure  at  125°  C.  is 
necessary  to  destroy  spores.  Ice  may  contain  active  bac- 
teria; frozen  milk  permits  the  growth  of  bacteria. 

Influence  of  Oxygen. — Two  varieties  of  bacteria  in  relation 


28  ESSENTIALS    OF   BACTERIOLOGY 

to  oxygen — the  one  aerobic,  growing  in  air;  the  other,  anae- 
robic, Hving  without  air. 

Obligate  aerobes,  those  which  exist  only  when  free  or 
atmospheric  oxygen  is  present. 

Facultative  aerobes,  those  that  live  best  when  atmos- 
pheric oxygen  is  present,  but  can  live  without  it. 

Obligate  or  true  anaerobes,  those  w^hich  cannot  exist  in 
the  presence  of  free  oxygen;  facultative  anaerobes,  those 
which  exist  better  where  there  is  no  free  oxygen,  but 
can  live  in  its  presence. 

Some  derive  the  oxygen  which  they  require  out  of  their 
nutriment,  so  that  a  bacterium  may  be  aerobic  and  yet  not 
require  the  presence  of  free  oxygen. 

Aerobes  may  consume  the  free  oxygen  of  a  region  and  thus 
allow  the  anaerobes  to  develop.  By  improved  methods  of 
culture  many  varieties  of  anaerobes  have  been  discovered. 

Influence  of  Light. — Sunlight  is  very  destructive  to  bacteria. 
A  few  hours'  exposure  to  the  sun  has  been  fatal  to  anthrax 
bacilli  and  the  cultures  of  Bacillus  tuberculosis.  The  sun's 
rays,  however,  must  come  in  direct  contact  with  the  germs, 
and  are  usually  active  only  on  the  surface  cultures.  The 
rays  at  the  violet  end  of  the  spectrum  are  the  most  active. 
The  electric  arc-light  has  much  the  same  effect  as  sunlight  on 
bacteria;  the  effect  of  sunlight  is  not  due  to  heat-rays. 

Effects  of  Electricity. — Electricity  arrests  growth. 

Effects  of  Rontgen  Rays. — Have  little  or  no  effect  on  arti- 
ficial cultures,  but  in  the  living  tissues  a  pronounced  bacteri- 
cidal effect  is  produced,  perhaps  through  the  stimulation  of 
the  body-cells. 

Moisture. — Water  is  necessary  for  the  development  of  most 
bacteria;  complete  drying  is  usually  destructive  after  a  few 
days. 

Heat. — Dry  heat  is  much  less  destructive  than  moist  heat, 
steam  under  pressure  most  destructive. 

Biologic  Activities. — Bacteria  feeding  upon  organic  com- 
pounds produce  chemic  changes  in  them,  not  only  by  the 
withdrawal  of  certain  elements,  but  also  by  the  excretion 
of  these  elements  changed  by  digestion.     Sometimes  such 


BIOLOGIC    AND    CHEMIC    ACTIVITIES  29 

changes  are  destructive  to  the  bacteria  themselves,  as  when 
lactic  and  butyric  acids  are  formed  in  the  media. 

Oxidation  and  reduction  are  carried  on  by  some  bacteria. 
Ammonia,  hydrogen  sulphid,  and  trimethylamin  are  a  few  of 
the  chemic  products  produced  by  bacteria.  Nitrites  in  the 
soil  are  reduced  to  ammonia. 

Nitrification. — Albuminoids  changed  into  indol,  skatol, 
leucin,  etc.;  then  these  into  ammonia,  am.monia  into  nitrites, 
nitrites  into  nitrates. 

Ptomains. — Brieger  found  a  number  of  complex  alkaloids 
closely  resembling  those  found  in  ordinary  plants,  and  which 
he  named  ptomains,  from  Trrwjua,  corpse,  because  obtained 
from  putrefying  objects.  These  were  at  one  time  held  to  be 
the  chief  causes  of  bacterial  disease,  but  are  no  longer  con- 
sidered of  much  importance. 

Chemic  Products. — Secretions,  as,  for  instance,  enzymes, 
toxins.     Excretions,  pigments,  indol,  cell  proteins,  bacterins. 

Proteins. — The  protein  contents  of  the  bacterial  cell  may 
cause  inflammation  and  fever. 

Producers  of  Disease. — Various  pathologic  processes 
are  caused  by  bacteria,  the  name  given  to  such  diseases  being 
infectious  diseases,  and  the  germs  themselves  called  disease- 
producing  or  pathogenic  bacteria.  Those  which  do  not  form 
any  pathologic  process  are  called  non- pathogenic  bacteria. 

Fermentation. — This  is  an  important  property  of  bac- 
terial activity. 

Enzymes. — An  enzyme  or  ferment  is  a  substance  capable 
of  inaugurating  a  chemic  reaction  without  entering  into  the 
reaction,  and  is  a  product  of  living  cells. 

Bacterial  enzymes  are  closely  related  to  the  ferments  of 
special  cells  of  higher  animals  and  plants,  like  ptyalin  and 
diastase. 

Ferments  may  be  diastatic,  changing  starch  into  sugar,  or 
proteolytic,  transforming  albumins  into  more  soluble  sub- 
stances, of  which  gelatin  liquefaction  is  an  example.  Invert- 
ing, changing  a  sugar  from  one  that  does  not  undergo  fermen- 
tation into  one  that  does. 


30  ESSENTIALS    OF   BACTERIOLOGY 

Coagulating,  fat-splitting,  hydrolytic  ferments  are  some  of 
the  other  varieties. 

Toxins  and  toxalbvimins  are  various  albuminoids  pro- 
duced in  the  animal  organism  and  in  culture-media  which  are 
very  poisonous,  and  are  considered  the  prime  cause  of  disease. 

Putrefaction. — When  fermentation  is  accompanied  by 
development  of  offensive  gases,  a  decomposition  occurs  which 
is  called  putrefaction,  and  this,  in  organic  substances,  is  due 
entirely  to  bacteria. 

Pigmentation. — Some  bacteria  are  endowed  with  the 
property  of  forming  pigments  either  in  themselves,  or  pro- 
ducing a  chromogenic  body  which,  when  set  free,  gives  rise  to 
the  pigment.  In  some  cases  the  pigments  have  been  isolated 
and  many  of  the  properties  of  the  anilin  dyes  discovered  in 
them. 

Phosphorescence. — Many  bacteria  have  the  power  to 
form  light,  giving  to  various  objects  which  they  inhabit  a 
characteristic  glow  or  phosphorescence. 

Fluorescence. — An  iridescence,  or  play  of  colors,  devel- 
ops in  some  of  the  bacterial  cultures. 

Gas-formation. — Many  bacteria,  anaerobic  ones  especi- 
ally, produce  gases,  noxious  and  odorless;  in  the  culture- 
media  the  bubbles  which  arise  soon  displace  the  media. 

Odors. — Some  germs  form  odors  characteristic  of  them: 
some  are  pleasant  and  even  fragrant;  others,  foul  and  nause- 
ous. 

Effect  of  Age. — With  age,  bacteria  lose  their  strength  and 
die. 


CHAPTER  III 
INFECTION 


How  Bacteria  Cause  Disease. — Many  theories  have 
been  advanced  to  explain  the  action  of  bacteria  in  causing 
disease,  but  only  a  few  of  the  more  important  ones  can  be 
discussed.     Nearly  all  the  changes  found  in  the  organs  of  the 


INFECTION 


31 


body  are  similar  to  those  produced  by  drugs  and  can  be 
reproduced  by  the  injection  of  bacterial  poisons. 

Infection  is  the  successful  invasion  of  an  organism  by 
microparasites,  and  implies  an  abnormal  state  resulting  from 
the  deleterious  action  of  the  parasite  upon  the  host. 

Sources  of  infection  may  be  exogenous  or  endogenous. 
Exogenous  infections  result  from  the  successful  invasion  of 
the  body  by  microparasites  from  sources  entirely  apart  from 
the  individual  infected.  Infection  by  the  typhoid  bacillus 
from  water  or  milk,  by  the  Spirochaeta  pallidum  from  dental 
instruments  or  drinking-cups,  contraction  of  smallpox  from 
fomites,  and  contraction  of  malaria  from  the  bites  of  mosqui- 
tos  are  examples  of  exogenous  infection. 

Endogenous  infections  result  from  the  successful  invasion 
of  the  body  by  microorganisms  normally  present  on  the 
body.  The  skin  and  mucous  membranes  furnish  lodgment 
for  a  great  variety  of  virulent  pathogenic  organisms  which, 
when  the  resistance  of  the  body  is  lowered,  immediately 
become  invasive.  The  pneumococcus  is  a  normal  inhabitant 
of  the  mouth  and  pharynx,  but  causes  no  infection  until  the 
body  resistance  is  lowered.  When  this  occurs,  tonsillitis, 
pharyngitis,  or  lobar  pneumonia  may  follow. 

Pathogenesis. — The  ability  of  a  microorganism  to  do 
harm  depends  on  its  invasive  powers  and  its  ability  to  gener- 
ate toxins  or  both. 

Toxins. — Little  is  known  of  the  chemic  nature  of  toxins. 
Undoubtedly  some  are  related  to  albumins.  Others  give  no 
reactions  common  to  compounds  of  this  group. 

(A)  Intracellular  or  Insoluble  Toxins. — These  are  chiefly 
within  the  bodies  of  the  bacteria,  and  are  set  free  by  disin- 
tegration of  the  organism.  This  group  comprises  most  of 
the  pathogenic  bacteria. 

(B)  Extracellular  or  Soluble  Toxins. — These  toxins  are  ap- 
parently excreted  by  the  bacteria,  and  are  foimd  in  the 
surrounding  medium.  This  group  includes  the  diphtheria 
and  tetanus  bacilli. 

It  has  been  shown  that  bacteria  which  apparently  do  not 


32  ESSENTIALS   OF   BACTERIOLOGY 

produce  toxins  in  artificial  media  may  do  so  in  the  human 
body.  These  toxic  substances  are  formed  by  the  bacteria  to 
combat  the  body  defenses,  and  have  been  called  by  Bail 
aggressins.  They  have  a  paralytic  action  on  phagocytes. 
A  sublethal  dose  of  bacteria,  if  injected  along  with  aggressin, 
will  cause  death. 

Toxins  are  not  stable,  though  tetanus  toxin  has  been  kept 
in  powdered  form  for  a  number  of  years.  They  are  soluble 
in  water,  destroyed  by  heat  (thermolabile),  and  precipitated 
by  ammonium  sulphate. 

The  Cardinal  Conditions  for  Infection. — (i)  The 
microorganism  must  be  sufficiently  virulent;  (2)  it  must  enter 
in  sufficient  numbers  and  by  appropriate  channels;  and  (3}  the 
host  must  be  susceptible. 

Virulence  is  a  very  variable  quality,  and  depends  on  the 
ability  of  the  micro-organism  to  invade  or  produce  toxin  or 
both.  The  virulence  may  be  decreased  by  repeated  trans- 
planting on  artificial  culture-media  or  by.  the  action  of  heat. 
It  may  be  increased  by  adding  animal  juices  to  the  culture- 
medium,  by  inclosing  the  micro-organism  in  a  collodion  sac, 
and  placing  the  sac  in  the  abdominal  cavity  of  an  animal,  and 
by  repeatedly  passing  it  through  animals. 

Infection  Depends  on  Quantity  of  Bacteria. — Unless 
a  sufficient  number  of  bacteria  enter  the  tissues  no  infection 
follows,  because  the  body  defenses  immediately  destroy  the 
bacteria.  The  number  necessary  to  cause  infection  depends 
on  their  virulence  and  the  susceptibility  of  the  host.  Strep- 
tococci may  become  so  virulent  that  a  single  coccus  will  cause 
death  in  a  rabbit.  It  has  been  found  that  820  tubercle  bacilli 
are  necessary  to  kill  a  guinea-pig,  and  1,000,000  staphylo- 
cocci to  kill  a  rabbit.  The  period  of  incubation  can  be  ex- 
plained on  the  supposition  that  the  organism  requires  a 
definite  time  to  generate  the  amount  of  toxin  necessary  to 
produce  symptoms. 

Avenues  of  Infection.— The  organism  must  gain  en- 
trance into  the  tissue  or  find  lodgment  on  some  part  of  the  body 
that  has  been  injured.     Even  when  several  avenues  of  infec- 


INFECTION  33 

tion  are  open,  the  parasite  most  commonly  invades  through 
one  that  may,  therefore,  be  regarded  as  the  most  appropriate 
for  entrance;  this  channel  furnishes  the  typical  picture  of 
the  infection. 

Susceptibility  of  the  Host. — Susceptibility  varies  in 
different  species  of  animals,  in  different  members  of  the  same 
species,  in  the  same  individual  at  different  times,  and  in  the 
same  individual  to  different  organisms. 

Susceptibility  may  he  natural,  as  in  smallpox;  acquired,  as 
from  exposure  to  conditions  which  lower  the  vitality,  such 
as  hunger,  cold,  intoxication,  fatigue,  inhalation  of  noxious 
vapors,  and  traumatic  shock.  Inherited  susceptibility  also 
occurs.  The  transmission  of  certain  inherited  character- 
istics, as  narrow  chest,  predisposes  to  infection  of  the  lungs. 

Mixed  infections  are  the  result  of  two  or  more  micro- 
organisms successfully  invading  and  intoxicating  the  host 
at  the  same  time. 

Local  Effects  of  Bacteria. — By  mechanical  obstruction 
from  rapid  growth  of  the  bacteria,  thrombosis,  w4th  its  con- 
sequences, may  occur.  Destruction  of  a  part  of  the  cells  of  a 
tissue  with  necrosis  can  arise  from  irritation,  the  bacteria 
acting  as  a  foreign  body. 

General  Effects. — Bacteremia  or  septicemia  occurs  when 
bacteria  proliferate  and  enter  the  whole  system,  as  when 
anthrax  and  typhoid  cause  general  disease. 

Toxemia. — When  the  poisons  become  widely  distributed, 
though  the  bacteria  remain  few  and  localized,  and  never  or 
seldom  enter  the  circulation,  as  diphtheria  and  tetanus. 

Pyemia,  a  form  of  bacteremia,  in  which  secondary  or 
metastatic  foci  of  suppuration  occur  throughout  the  body. 

Suppurative  bacteria  are  those  which  give  rise  to  inflam- 
mation and  suppuration  locally  at  the  point  of  entrance,  and 
secondarily  through  metastasis.  Any  organism  may  cause 
suppuration,  but  certain  ones  are  peculiarly  inclined  to  give 
rise  to  pus,  and  are  known  as  pyogenic  organisms. 

Specific  Bacteria. — Infective  bacteria  are,  as  a  rule, 
specific,  the  particular  toxin  having  a  specific  action  and  caus- 
3 


34  ESSENTIALS   OF   BACTERIOLOGY 

ing  a  disease  peculiar  to  the  micro-organism.  Thus  typhoid 
fever  is  a  disease  distinctly  different  from  tuberculosis;  the 
infective  organisms  are  distinct  and  the  poisons  they  produce 
have  specific  characteristics. 

The  Nature  of  Toxins. — Very  similar  to  the  venom  of 
serpents;  highly  poisonous  in  minute  doses  (tto  o"  gram  of 
tetanus  toxin  will  kill  a  horse  weighing  600  kilos — 1200 
pounds).  At  first  toxins  were  called  ptomains,  or  cadaveric 
alkaloids;  but  this  term  is  applied  now  to  such  poisons  as 
have  a  basic  nature  and  arise  in  decomposing  meat,  cheese, 
and  cream  as  a  result  of  chemical  change  in  the  material,  the 
bacteria  causing  the  change.  Then  they  were  called  toxal- 
bumins,  and  were  supposed  to  belong  to  an  albumin  series; 
but  when  the  bacteria  are  grown  in  non-albuminous  media, 
the  toxins  correspond  more  in  their  chemical  composition  to 
a  ferment,  and  therefore  it  is  supposed  that  the  albumin  part 
of  the  toxin  is  furnished  by  the  blood  or  albuminous  media  in 
which  it  is  formed.  The  term  toxin  is  to  be  preferred  in 
speaking  of  bacterial  poisons. 


CHAPTER  IV 
IMMUNITY 

Ordinary  Defenses  to  Bacterial  Invasion. — The  un- 
broken skin  and  the  connective  tissue  underneath  prevent 
the  passage  of  bacteria.  The  unbroken  mucous  surface  of 
eye,  nose,  and  mouth,  because  of  the  continuous  washing, 
prevents  the  numerous  bacteria  that  are  constantly  present 
in  the  discharge  from  finding  suitable  lodgment.  The  hairs 
and  ciliated  epithelium  in  upper  respiratory  tract  retain 
many  a  dust  particle  and  pathogenic  cell  on  its  way  to  the 
lungs.  The  acid  gastric  juice  is  destructive  to  most  bacteria, 
and  protects  not  only  the  stomach,  but  the  intestines  as  well. 


IMMUNITY  35 

The  intestinal  secretions  are  but  mildly  preventive  of  bac- 
terial growth,  but  peristalsis  aids  in  dislodgment  of  micro- 
organisms. 

Immunity  is  the  ability  to  resist  infection  and  intoxica- 
tion.    It  is  always  relative  and  never  absolute. 


f  Natural 
Immunity 

Acquired 


Active. 
Passive. 


Natural  immunity  is  a  natural  inherited  resistance  against 
infection  or  intoxication,  peculiar  to  certain  groups  of  animals, 
but  common  to  all  the  mdividuals  of  these  groups.  It  is 
peculiar  to  the  kind  of  animal,  not  to  the  individual.  Thus 
the  field  mouse  is  susceptible  to  glanders;  the  house  mouse  is 
slightly  immune,  and  the  white  mouse  is  immune. 

Acquired  immunity  is  resistance  to  infection  or  intoxica- 
tion possessed  by  certain  animals  of  a  naturally  susceptible 
kind,  in  consequence  of  circumstances  peculiar  to  them  as 
individuals.  Active  acquired  immunity  arises  from  the  activ- 
ities performed  by  the  organism  itself.  It  depends  on  infec- 
tion or  intoxication,  which  may  have  been  accidental  or 
intentional;  i.  e.,  for  the  purpose  of  producing  immunity. 
Some  accidental  infections,  recovery  from  which  renders  the 
individual  immune,  are  measles,  scarlet  fever,  and  smallpox. 
Other  infections  are  followed  by  an  immunity  of  short  dura- 
tion, as  typhoid  fever  and  pneumonia. 

Immunity  from  intentional  infection  or  intoxication  is  pro- 
duced by — {A)  bringing  about  a  different  disease,  as  in  the 
production  of  vaccinia  to  bring  about  immunity  to  small- 
pox. (B)  Inoculation  with  killed  bacteria,  as  in  the  protec- 
tive inoculation  against  typhoid  fever  or  bubonic  plague. 
(C)  Inoculation  with  bacterial  products,  as  diphtheria  or  tetanus  ■ 
toxin.  (D)  Inoculation  with  attenuated  cultures  of  micro- 
organisms, as  in  Pasteur's  anthrax  vaccine  or  Haffkine's 
cholera  vaccine.  (E)  Inoculation  with  virus  of  increasing  viru- 
lence, as  in  the  protective  inoculations  against  hydrophobia. 


36  ESSENTIALS   OF   BACTERIOLOGY 

(F)  Inoculation  with  sublethal  doses  of  virulent  bacteridy 
beginning  with  small  doses,  and  gradually  increasing  their 
size.  Guinea-pigs  inoculated  in  this  way  have  acquired  a 
marked  degree  of  immunity  to  tuberculosis. 

Passive  acquired  immunity  is  always  artificially  sup- 
plied to  the  animal.  It  follows  when  antibodies  are  supplied 
from  an  immunized  animal  to  one  normally  susceptible. 
Immunization  against  diphtheria  by  the  injection  of  diph- 
theria antitoxin  is  a  good  example. 

Theories  of  Immunity. — Phagocytic  Theory  of  Metchni- 
kojf. — Immunity  is  dependent  on  the  action  of  the  phago- 
cytes and  their  ferments.  The  phagocytes  are  of  two  kinds 
— macrophages,  which  include  endothelia  and  connective-tis- 
sue cells,  and  micro  phages,  the  polymorphonuclear  leukocytes. 
These  phagocytes  liberate  ferments — macrocytase  and  micro- 
cytase  respectively.  Infecting  organisms  and  their  toxins 
are  destroyed  by  the  phagocytes  and  their  ferments.  This 
theory  has  been  replaced  by  the  lateral  or  side-chain  theory  of 
Ehrlich. 

Ehrlich^s  Lateral  Chain  Theory. — This  derives  its  name 
from  th  e  fact  that  it  presents  an  analogy  to  what  happens 
in  the  benzol  ring  of  organic  chemistry  when  its  replaceable 
atoms  of  hydrogen  are  substituted  by  "side  chains"  of  more 
or  less  complex  nature.  The  molecule  of  protoplasm  is  sup- 
posed to  consist  of  a  central  atom  group,  provided  with  a 
large  number  of  side  chains  which  subserve  the  vital  processes 
of  the  molecule  by  combining  with  other  organic  molecules. 
These  side  chains  are  called  receptors,  and  are  of  many  dif- 
ferent kinds,  so  as  to  fit  them  for  combination  with  many 
different  varieties  of  extraneous  groups. 

Three  orders  of  receptors  are  described:  Receptors  of  the  first 
order,  which  concern  themselves  with  the  assimilation  of 
simple  substances  (toxins,  ferments,  and  other  cell  secre- 
tions), utilizing  a  single  haptophore.  Antitoxins,  as  an 
example. 

Receptors  of  the  second  order,  which,  in  addition  to  the 
haptophore  group,  possess  a  second  group,  which  affects  the 


IMMUNITY  37 

coagulation.  Toxins  may  be  regarded  as  receptors  of  the 
second  order  thrust  off  by  the  bacteria. 

Receptors  of  the  third  order,  which  possess  two  haptophore 
groups,  one  of  which  effects  the  union  with  the  food-stuffs, 
whereas  the  other  lays  hold  on  certain  substances  circulating 
in  the  blood  plasma,  the  complements,  which  cause  ferment- 
like actions — cytolysins,  as  an  example. 

The  Formation  of  Antitoxin  According  to  the  Lateral  Chain 
Theory. — The  toxin  molecule  consists  of  two  groups:  (A) 


m 


Fig.  6. — Graphic  representation  of  receptors  of  the  first  order  and  of 
toxin  uniting  with  the  cell-receptor:  a,  Cell-receptor;  b,  toxin  molecule; 
c,  haptophore  of  toxin  molecule;  d,  toxophore  of  to:dn  molecule;  e,  hapto- 
phore of  the  cell-receptor  (Ehrlich). 

The  haptophore  or  combining  group,  by  which  the  toxin 
molecule  can  join  the  receptor  of  the  cell,  and  (B)  the  toxo- 
phore, or  poisoning  group,  by  which  means  it  can  attack  the 
cell  protoplasm  after  having  been  fixed  to  it  by  the  hapto- 
phore group. 

The  effect  of  the  toxin  depends  on  the  number  of  mole- 
cules attached  to  the  cell.    A  great  number  would  bring 


38  ESSENTIALS   OF   BACTERIOLOGY 

about  death  of  the  cell,  while  a  few  would  act  as  an  irri- 
tant. 

Weigerfs  Law. — When  a  cell  is  attacked  by  a  few  mole- 
cules of  toxin,  it  reacts  by  forming  new  side  chains  or  recep- 
tors, and,  in  accordance  with  the  law  of  Weigert,  always  in 
excess.  Repeated  injections  of  toxins  in  increasing  doses 
cause  such  an  overproduction  of  receptors  of  the  first  order 
that  they  are  thrust  from  the  cell  and  float  free  in  the  blood- 


Fig.  7. — Graphic  representation  of  receptors  of  the  second  order  and 
of  some  substances  uniting  with  one  of  them:  c,  Cell-receptor  of  the 
second  order;  d,  toxophore  or  zymophorous  group  of  the  receptor;  e, 
haptophore  of  the  receptor;  /,  food  substance  or  product  of  bacterial 
disintegration  uniting  with  the  haptophore  of  the  cell-receptor  (Ehrhch). 


stream.  Here  they  can  combine  with  toxin  molecules,  just 
as  when  they  are  attached  to  the  cell.  By  thus  combining, 
they  prevent  the  toxin  from  reaching  the  cells. 

Antitoxins  are  specific  in  their  action;  that  is,  each  anti- 
toxin will  neutralize  only  a  certain  toxin.  Thus  diphtheria 
antitoxin  will  not  neutralize  tetanus  toxin  or  snake  venom, 


IMMUNITY  39 

nor  will   tetanus  antitoxin  neutralize   diphtheria  toxin  or 
snake  venom. 

Lock  and  Key  Theory. — This  specific  action  is  explained  by 
supposing  the  molecule  of  toxin  to  have  a  shape  peculiar  to 
itself.  The  molecule  of  diphtheria  toxin  is  of  such  shape 
that  the  haptophore  end  will  fit  only  on  certain  receptors 
of  a  cell;  the  molecule  of  tetanus  toxin  will  fit  on  only  certain 
other  receptors. 


Fig.  8. — Graphic  representation  of  receptors  of  the  third  order,  and 
of  some  substance  uniting  with  one  of  them:  c,  Cell-receptor  of  the  third 
order,  amboceptor;  e,  one  of  the  haptophores  of  the  amboceptor  with 
which  some  food  substance  or  product  of  bacterial  disintegration,  /,  may- 
unite;  g,  the  other  haptophore  of  the  amboceptor  with  which  complement 
may  unite;  k,  complement;  h,  the  haptophore,  and  z,  the  zymotoxic 
group  of  the  complement  (Ehrlich). 


An  antitoxic  serum  is  a  suspension  of  receptors  of  the  first 
order  in  blood-serum.  Antitoxins  for  diphtheria  and  tetanus 
are  the  most  common. 

Precipitins  are  bodies  in  serum  which,  when  added  to  a 
protein  in  solution,  will  cause  a  precipitate  to  form.  The 
precipitins  are  specific  and  act  only  with  similar  proteins. 

When  a  protein  or   food   substance  is  injected  into  an 


40  ESSENTIALS   OF  BACTERIOLOGY 

animal  and  becomes  attached  to  the  cell  receptors  of  the 
second  order  by  means  of  its  haptophore  group,  the  cell  is 
irritated  and  new  receptors  are  formed.  Further  injection 
of  larger  amounts  of  protein  stimulate  the  cell  to  such  an 
excessive  formation  of  these  receptors  that  they  are  thrust 
free  into  the  blood-stream. 

A  precipitin  serum  is  a  suspension  of  receptors  of  the  second 
order  in  blood-serum. 

The  phenomenon  of  precipitation  has  found  forensic 
application  in  the  identification  of  blood-stains. 

Agglutinins  are  bodies  present  in  a  serum  which,  when 
added  to  bacterial  cells,  cause  them  to  clump,  and,  if  motile, 
to  lose  their  motility.  They  are  specific  when  diluted,  and 
of  value  in  diagnosis  in  such  diseases  as  typhoid  and  Malta 
fever. 

Agglutinins  are  formed  in  response  to  the  stimulus  given  the 
cells  of  a  body  by  the  union  of  antigenic  cell-receptors  with 
receptors  of  the  second  order  of  the  cells  of  the  animal  re- 
ceiving the  injection.  Repeated  injections  stimulate  the 
cells  to  the  formation  of  such  excessive  quantities  of  these 
receptors  that  they  are  thrown  from  the  cells  into  the  blood- 
stream. 

Agglutinins  bear  no  relation  to  the  degree  of  immunity, 
and  should  never  be  used  as  an  index  to  immunity. 

Cytolysins  are  bodies  present  in  a  serum  which  will  dis- 
solve or  destroy  cells  (corpuscles,  bacteria,  etc.). 

They  are  formed  in  the  same  manner  as  the  agglutinins, 
except  that  receptors  of  the  third  order  are  involved.  Recep- 
tors of  the  third  order  have  a  double  combining  affinity. 
One  part  attaches  itself  to  the  receptor  of  the  cell  injected, 
and  the  other  combines  with  complement. 

Complement  {alexin  or  cytase)  is  a  thermolabile,  ferment- 
like body  found  in  all  normal  sera. 

Amboceptors y  ^^ substance  sensibilatrice,''  fixateur,  copula ^ 
and  desmon  are  names  given  to  receptors  of  the  third  order. 

Cytolytic  sera  are  of  little  use  in  medicine.  Sera  have  been 
prepared  against  staphylococci,  pneumococci,  streptococci, 


IMMUNITY  41 

and  others.  Wassermann  has  made  a  very  efficacious  anti- 
meningococcus  serum. 

Opsonins. — Opsonins  are  substances  in  the  blood-serum 
which  act  on  bacteria  and  prepare  them  for  phagocytosis. 
Opsonins  can  be  increased  by  whatever  increases  immunity. 
An  increase  is  coincident  with  increased  immunity.  The 
most  common  method  of  bringing  about  an  increase  is  by  the 
injection  of  killed  cultures  of  bacteria. 

Opsonins  normally  present  in  the  serum  are  not  specific. 
Opsonins  resulting  from  reaction  to  infection  or  inoculation 
are  specific. 

The  opsonic  index  is  the  ratio  between  the  number  of 
bacteria  ingested  by  living  leukocytes  when  operating  in  the 
serum  of  a  test  and  in  normal  serum  respectively. 

After  the  injection  of  bacteria  the  opsonic  index  falls  for 
a  short  time.  This  period  is  called  the  negative  phase,  and 
is  followed  by  a  rise  in  the  index — the  positive  phase. 

The  "estimation  of  the  opsonic  index  is  a  very  complicated 
way  of  finding  out  very  little,"  and  has  been  abandoned  by 
the  great  majority  of  workers. 

Antigens. — Any  substance  that  has,  when  injected  into 
the  body,  power  to  produce  an  antitoxin  or  antibacterial 
body  is  called  an  antigen. 

The  toxin  of  diphtheria,  if  injected,  stimulates  the  normal 
cells  to  produce  chemic  substances  (free  receptors)  which 
are  at  liberty  to  attach  themselves  to  the  active  toxin  mole- 
cules and  thus  save  the  body  cells  from  being  acted  upon; 
toxin  is,  therefore,  an  antigen. 

Substances  which  have  the  power  of  destroying  bacteria 
are  called  bactericides;  those  which  dissolve  them  merely  are 
called  hacteriolysins. 

Hemolysis. — ^When  the  hemoglobin  of  the  red  blood-cells 
is  liberated,  hemolysis  is  said  to  occur.  This  is  brought  about 
by  the  injection  of  certain  substances,  or  hemolysins;  these 
are  present  normally  in  some  sera,  and  can  be  developed  in 
others.  Lysins  and  bactericidal  substances  seem  to  have 
two  parts — one  destroyed  by  heat   (thermolabile),    called 


42  ESSENTIALS   OF  BACTERIOLOGY 

complement  (the  completor),  and  one,  more  resistant,  called 
the  amboceptor,  or  combinor,  which  unites  with  complement 
and  with  the  cell.  For  lysis,  therefore,  it  is  necessary  that 
ambocepter  be  united  to  the  bacteria  or  cell,  and  that  com- 
plement be  present  or  added  to  join  with  amboceptor,  com- 
pleting the  circuit.  Complement  may  be  prevented  from  com- 
bining with  amboceptor  by  ^^ deviation  of  the  complement.^'' 
The  amboceptor  may  be  in  excess,  and  the  free  group  absorb 
or  attach  itself  to  all  the  available  complement,  leaving  none 
to  join  the  amboceptor;  or  anti-complements  may  be  present 
to  monopohze  all  this  complement  and  leave  none  free  to  unite 
with  amboceptor.   This  deviation  prevents  lysis. 

Fixation  of  Complement. — By  adding  a  definite  standardized 
complement  to  a  mixture  of  antigen  and  amboceptor  of  a 
similar  kind  the  complement  is  bound  or  fixed,  and  none  is 
left  free.  If  the  amboceptor  is  not  like  the  antigen,  the  com- 
plement will  not  unite  the  two,  will  not  be  bound,  and  is  free 
to  unite  with  any  other  amboceptor  that  may  be  introduced. 
If  this  be  a  hemolytic  amboceptor,  and  red  corpuscles  are 
added  as  an  indicator,  the  cells  will  lose  their  hemoglobin, 
because  hemolysis  will  occur  from  the  completing  of  the  re- 
action. The  complement  will  unite  to  hemolytic  ambocep- 
tor, since  it  is  not  fixed  or  bound  by  the  other  amboceptor, 
and  the  other  amboceptor  is  not  of  the  same  nature  as  the 
antigen.  This  is  the  principle  of  the  Wassermann  serum  re- 
action or  test. 

Anaphylaxis  or  Allergy. — Under  certam  circumsta,nces 
the  second  injection  of  a  proteid  as  antigen  instead  of  render- 
ing immune,  produces  hyper  sensitiveness.  Behring,  in  1892, 
noticed  this  with  injections  of  antitoxin,  and  called  it  ^'hyper- 
susceptibility.'^ 

Richet,  in  1904,  called  a  similar  condition  anaphylaxis,  or 
the  reverse  of  prophylaxis,  and  von  Pirquet  introduced  the 
term  ''allergy,'"  "altered  reactivity,''''  to  express  the  same  thing. 
Guinea-pigs  may  be  rendered  so  sensitive  by  o.ooi  c.c.  of 
horse-serum  that  a  second  dose  within  a  week  or  a  few  days 
produces  fatal  shock. 


METHODS    OF    STUDYING    BACTERIA  43 

Other  proteins,  like  beef-serum,  egg-albumin,  red  blood- 
corpuscles,  have  produced  similar  results,  varying  doses  and 
periods  of  incubation.  Human  beings  may  be  sensitized  by 
single  injections  of  horse-serum. 

Hay-fever,  asthma,  puerperal  convulsions,  and  sympathetic 
ophthalmia  partake  of  the  nature  of  anaphylactic  reactions, 
and  the  peculiar  intolerances  to  certain  articles  of  food  may 
be  better  explained  by  the  same  theory. 

The  sudden  attacks  of  collapse  and  death  which  have 
followed  the  injection  of  even  small  doses  of  antitoxins  made 
from  horse-serum  are  believed  to  come  from  this  condition 
of  hypersensitiveness. 

The  use  of  globulins  instead  of  the  entire  serum  has  lessened 
the  danger  from  anaphylaxis. 


CHAPTER  V 
METHODS  OF  STUDYING  BACTERIA*— MICROSCOPE 

Microscope. — Most  clinical  instruments  now  on  the  mar- 
ket have  all  the  necessary  appliances  for  bacterial  examina- 
tion. Three  objectives  are  advisable — 16  mm.  (^  inch); 
4  mm.  06  inch);  2  mm.  (tV  inch).  It  is  not  so  much 
required  to  have  a  picture  very  large,  as  to  have  it  sharp  and 
clear. 

Oil-immersion  Lens. — ^The  penetration  and  clearness  of 
a  lens  are  very  much  influenced  by  the  absorption  of  the  rays 
of  Hght  emerging  from  the  picture.  In  the  ordinary  dry 
system  many  of  the  light  rays,  being  bent  outward  by  the  air 
which  is  between  the  object  and  the  lens,  do  not  enter  the  lens, 
and  are  lost.  By  interposing  an  agent  which  has  the  same 
refractive  index  as  glass,  cedar-oil  or  clove-oil,  for  example, 
all  the  rays  of  light  from  the  object  enter  directly  into  the  lens. 

The  "homogeneous  system,"  or  oil-immersion  lens,  con- 


44 


ESSENTIALS    OF   BACTERIOLOGY 


sists  of  a  system  of  lenses  which  can  be  dipped  into  a  drop  of 
cedar-oil  placed  upon  the  cover-glass,  and  which  is  then  ready 
for  use. 

Abbe's  Condenser. — The  second  necessary  adjunct  is  a 
combination    of    lenses    placed    underneath    the    stage,    for 
bringing  wide  rays  of  light  directly  under  the  object.     It 
serves  to  intensify  the  colored  pic- 
tures by  absorbing  or  hiding  the 
unstained  structure. 

This  is  very  useful  in  searching 
a  specimen  for  bacteria,  since  it 
clears  the  field  of  everything  that 
is  not  stained.  It  is  called  Abbe's 
condenser  (Fig.  9).  Together  with  it  is  usually  found  an  in- 
strument for  shutting  off  part  of  the  light — a  blender  or  dia- 
phragm (Fig.  10).  When  the  bacteria  have  been  found,  and 
their  relation  to  the  structure  is  to  be  studied,  the  "Abbe"  is 
generally  shut  out  by  the  iris  blender, ,  and  the  structure 
comes  more  plainly  into  view.     A  white  light  (daylight  or  a 


Fig.  9. — Abbe's  condenser. 


Iris  blender. 


Welsbach  burner)  is  best  for  bacterial  study:  use  the  plane 
mirror  with  the  condenser. 

For  all  stained  bacteria  the  oil-immersion  lens  and  Abbe  con- 
denser, without  the  use  of  blender.  For  unstained  specimens^ 
oil-immersion  and  the  narrowed  blender. 

When  examining  with  low-power  objective,  use  a  strong 


METHODS   OF   STUDYING  BACTERIA  45 

ocular.  When  using  high-power  objective  use  weak  ocular. 
A  revolving  nose-piece  will  be  found  very  useful,  since  it  is 
sometimes  necessary  to  change  the  objective  on  the  same 
field,  and  this  insures  a  great  steadiness  of  the  object. 

Great  cleanliness  is  needed  in  all  bacteriologic  methods, 
but  nowhere  more  so  than  in  the  microscopic  examination. 

The  cover-glass  should  be  very  carefully  washed  in  alcohol, 
and  dried  with  a  soft  linen  rag.  To  remove  the  stains  on  the 
cover-glasses  that  have  been  used  they  should  be  soaked  in 
hydrochloric  acid  or  placed  in  a  6  per  cent,  aqueous  solution 
of  potassium  dichromate  with  6  per  cent,  of  strong  sulphuric 
acid,  washed  in  water,  and  kept  in  absolute  alcohol. 

Examination  of  Unstained  Bacteria. — As  the  coloring  of 
bacteria  kills  them  and  changes  their  shape  to  soine  extent,  it 


Fig,  II. — Platinum  needles  for  transferring  bacteria,  made  from  No.  27 
platinum  wire  inserted  in  glass  rods:  a,  Looped  needle;  b,  straight- 
pointed  needle  (McFarland) . 


is  preferable  to  examine  bacteria,  when  possible,  in  their 
natural  state. 

We  obtain  the  bacteria  for  examination  either  from  liquid 
or  solid  media. 

From  Liquids. — With  a  long  platinum  needle  the  end  of 
which  is  bent  into  a  loop  (Fig.  11,  a)  obtain  a  small  drop  from 
the  liquid  containing  the  bacteria,  and  place  it  on  a  cover- 
glass  or  slide,  careful  that  no  bubbles  remain. 

Sterilize  Instruments. — Right  here  w^e  might  say  that 
it  is  best  to  accustom  one's  self  to  pass  all  instruments, 
needles,  etc.,  through  the  flame  before  and  after  each  proce- 
dure; it  insures  safety;  and  once  in  the  habit,  it  will  be  done 
automatically. 

From  Solid  Media. — With  a  straight-pointed  platinum 
needle  (Fig.  11,  Z>)  a  small  speck  of  the  medium  is  taken  and 


46 


ESSENTIALS    OF   BACTERIOLOGY 


rubbed  upon  a  glass  slide  with  a  drop  of  sterilized  water  or 
bouillon,  and  from  this  a  little  is  taken  on  cover-glass,  as 
before. 

The  cover-glass  with  its  drop  is  now  placed  on  the  glass 
slide,  carefully  pressing  out  all  bubbles.  Then  a  drop  of 
cedar-oil  is  laid  on  top  of  the  cover-glass,  and  the  oil-immer- 
sion lens  dipped  gently  down  into  it  as  close  as  possible  to  the 
cover-glass,  the  narrow  blender  shutting  of  the  Abbe  conden- 
ser, for  this  being  an  unstained  specimen,  we  want  but  little 
light.  We  now  apply  the  eye,  and  if  not  in  focus,  use  the 
fine  adjustment  or  the  coarse,  but  always  away  from  the 
object — i.  e.,  toward  us — since  the  distance  between  the  speci- 


Fig.   12. — A  "concave  slide"  with  "hanging  drop"   (McFarland). 


men  and  the  lens  is  very  slight,  it  does  not  require  much 
turning  to  break  the  cover-glass  and  ruin  the  specimen. 
Having  found  the  bacterium,  we  see  whether  it  is  bacillus, 
micrococcus,  or  spirillum,  discover  if  it  is  motile  or  not. 
The  phenomenon  of  agglutination  is  observed  in  this  way. 

Hanging  Drop  (Fig.  12). — When  the  looped  platinum 
needle  is  dipped  into  a  liquid,  a  very  finely  formed  globule  will 
hang  to  it;  this  can  be  brought  into  a  little  cupped  glass  slide 
(an  ordinary  microscopic  glass  slide  with  a  circular  depression 
in  the  center)  in  the  following  manner:  The  drop  is  first 
brought  upon  a  cover-glass;  the  edges  of  the  concavity  on  the 
glass  slide  are  smeared  with  vaselin,  and  the  slide  inverted 
over  the  drop;  the  cover-glass  sticks  to  the  smeared  slide, 


METHODS   OF   STUDYING   BACTERIA  47 

which,  when  turned  over,  holds  the  drop  in  the  depression 
covered  by  the  cover-glass,  thus  forming  an  air-tight  cell; 
here  the  drop  cannot  evaporate.  Both  slide  and  cover-glass 
should  first  be  sterilized  by  heat. 

Search  for  the  bacteria  with  a  weak  lens;  having  found 
them,  place  a  drop  of  cedar-oil  upon  the  cover-glass,  and 
bring  the  oil  immersion  into  place  (here  is  where  a  nose-piece 
comes  in  very  useful),  careful  not  to  press  against  the  cell, 
for  the  cover-glasses  are  very  fragile  in  this  position. 

Search  the  edges  of  the  drop  rather  than  the  middle;  the 
bacteria  will  usually  be  very  thick  in  the  center  and  not  so 
easily  distinguished. 

Spores,  automatic  movements,  fission,  and  cultivation  in 
general  can  be  studied  for  several  days.  This  moist  chamber 
can  be  placed  in  a  brood-oven  or  on  the  ordinary  warming 
stage  attachment  of  the  microscope. 

Hanging  Block. — A  small  slice  of  agar  containing  some 
of  the  growth  seared  to  the  glass  slide  with  a  hot  needle. 

Agglutination  as  observed  in  Widal's  test  is  best  seen  in  the 
hanging  drop. 


CHAPTER  VI 


METHODS  OF  STUDYING  BACTERIA  (Continued),— 
SOLUTIONS  AND  FORMULAS  FOR  STAINING 

Staining  or  coloring  bacteria  is  done  in  order  to  make  them 
prominent  and  to  obtain  permanent  specimens.  It  is  also 
necessary  to  bring  out  the  structure  of  the  bacteria,  and 
serves  in  many  instances  as  a  means  of  diagnosis ;  it  would  be 
well-nigh  impossible  to  discover  them  in  the  tissues  without 
staining. 

Anilin  Colors. — Of  the  numerous  dyes  in  the  market, 
nearly  all  have,  at  one  time  or  other,  been  used  in  staining 


48  ESSENTIALS    OF   BACTERIOLOGY 

bacteria.  But  now  only  a  very  few  find  general  use,  and  with 
methylene-blue  and  fuchsin  nearly  every  object  can  be 
accomplished. 

Basic  and  Acid  Dyes. — Ehrlich  was  the  first  to  divide  the 
anilin  dyes  into  two  groups,  the  basic  colors  to  which  belong — 
Gentian-violet,  or  pyoktanin.  Basic  fuchsin. 

Methyl- violet,  or  dahlia.  Bismarck-brown 

Methylene-blue  {not  methyl  blue).  Thionin. 
Safranin. 
And  the  acid  colors  to  which  eosin  and  acid  fuchsin  belong. 

The  basic  anilme  dyes  stain  the  bacteria  and  the  nuclei  of 
cells;  the  acid  dyes  stain  chiefly  the  tissue,  leaving  the  bac- 
teria almost  untouched.  Carmin  and  hematoxylin  are  also 
useful  as  contrast  stains,  affecting  bacteria  very  slightly.  The 
anilin  dyes  are  soluble  in  alcohol  or  water  or  a  mixture  of  the 
two. 

Staining  Solutions. — A  saturated  solution  of  the  dye  is 
made  with  alcohol.  This  is  called  the  stock  or  concentrated 
solution;  i  part  of  this  solution  to  about  lo  parts  of  distilled 
water  constitutes  the  ordinary  aqueous  solution  in  use  or 
weak  solution. 

It  is  readily  made  by  adding  to  an  ounce  bottle  of  distilled 
water  enough  of  the  strong  solution  until  the  fluid  is  still 
opaque  in  the  body  of  the  bottle,  but  clear  in  the  neck  of  the 
same. 

These  weak  solutions  should  be  renewed  every  three  or  four 
weeks,  otherwise  the  precipitates  formed  will  interfere  with 
the  staining. 

Compound  Solutions. — By  means  of  certain  chemic 
agents  the  intensity  of  the  anilin  dyes  can  be  greatly  increased. 

Intensifiers  or  Mordants. — Agents  that  ''bite''  into  the 
specimen,  carrying  the  stain  with  them,  depositing  it  in  the 
deeper  layers,  are  called  mordants  or  etchers. 

Various  metallic  salts  and  vegetable  acids  are  used  for  such 
purpose. 

The  mother  liquid  of  the  anilin  dyes,  anilin-oil,  a  member 
of  the  aromatic  benzol  group,  has  also  this  property. 


METHODS    OF    STUDYING   BACTERIA  49 

Anilin-oil  Water. — Anilin-oil  is  shaken  up  with  water  and 
then  filtered;  the  aniUn  water  so  obtained  is  mixed  with  the 
dyes,  forming  the  "aniUn- water  gentian- violet "  or  anilin- 
water  fuchsin,  etc. 

Carbolfuchsin. — Carbolic  acid  or  phenol  can  be  used 
instead  of  anilin-oil,  and  forms  one  of  the  main  ingredients  of 
Ziehl's  or  Neelsen's  solution,  used  principally  in  staining 
Bacillus  tuberculosis.  Kuhne  has  a  carbol-methylene-blue 
made  similar  to  the  carbolfuchsin. 

Alkaline  Stains. — Alkalis  have  the  same  object  as  the 
above  agents,  namely,  to  intensify  the  picture.  Potassium 
hydroxid,  ammonium  carbonate,  and  sodium  hydroxid  are 
used. 

Loffler's  alkaline  blue  and  Koch's  weak  alkaline  blue  are 
made  with  potassium. 

Heat. — Warming  or  boiling  the  stains  during  the  process 
of  staining  increases  their  intensity. 

Decolorizing  Agents. — The  object  after  staining  is  usu- 
ally overcolored  in  some  part,  and  then  decolorizing  agents  are 
employed.  Water  is  sufficient  in  many  cases;  alcohol  and 
strong  mineral  acids  combined  are  necessary  in  some. 

lodin  as  Used  in  Gram's  Method. — Belonging  to  this 
group,  but  used  more  in  the  sense  of  a  protective,  is  tincture 
of  iodin.  It  picks  out  certain  bacteria,  which  it  coats;  pre- 
vents them  from  being  decolorized,  but  fades  the  rest  of  the 
picture.  Then,  by  using  one  of  the  acid  or  tissue  dyes,  a  con- 
trast color  or  double  staining  is  obtained.  Many  of  the  more 
important  bacteria  are  not  acted  upon  by  the  iodin,  and  it 
thus  becomes  a  very  useful  means  of  diagnosis. 


FORMULAS  OF  DIFFERENT  STAINING  SOLUTIONS 

I.  Saturated  Alcoholic  Solution 

Place  about  lo  grams  of  the  powdered  dye  in  a  bottle  and 
add  40  grams  of  alcohol.  Shake  well  and  allow  to  settle. 
This  can  be  used  as  the  stock  bottle. 


5©  ESSENTIALS   OF   BACTERIOLOGY 

II.  Weak  Solutions 
Made  by  adding  about  i  part  of  stock  solution  (I)  to  lo 
parts  of  distilled  water.     This  is  the  ordinary  solution  in  use. 

III.  Anilin-oil  Water 

Anilin-oil 5  parts 

Distilled  water 100      "  — M. 

Shake  well  and  filter.     To  be  made  fresh  each  time. 

IV.  Anilin-oil  Water  Dyes 
Saturated  alcoholic  solution  of  the 

dye II  parts 

Anilin-oil  water 100      '* 

Absolute  alcohol 10      "    — M. 

Can  be  kept  ten  days. 

V.  Alkaline  Methylene-blue 

A.  Lqffler^s: 

Saturated  alcoholic  solution  methy- 
lene-blue       30  parts 

Solution  potassium  hydroxid  (i  per 

cent.) I  part 

Water. q.  s.   100  parts — M. 

B.  Koch's: 

Solution  potassium  hydroxid  (10  per 
cent.) 2  parts 

Saturated  alcoholic  solution  methy- 
lene-blue         10     " 

Distilled  water 2000     "    — M. 

VI.  Phenol  Solutions 

A.  Ziehl-N eelsen: 

Fuchsin  (powdered) i  part 

Alcohol 10  parts 

5  per  cent,  solution  phenol 100     "   — M. 

Filter.     The  older  the  solution,  the  better. 


METHODS   OF   STUDYING   BACTERIA  5I 

B.  Kiihne: 

Methylene-blue 1.5  parts 

Alcohol lo.o     '' 

5  per  cent,  solution  phenol loo.o     " 

Add  the  phenol  gradually.     This  solution  loses  strength 
with  age. 

VII.  Gramas  lodin  Solution 

lodin I  part 

Potassium  iodid 2  parts 

Distilled  water 300     "     — M. 

VIII.  Lqffler^s  Mordant  {for  Flagella) 
Aqueous  solution  of  tannin  (20  per 

cent.) 10  parts 

Aqueous  solution  ferric  sulphate  (5 

per  cent.) i  part     ■ 

Aqueous  decoction  of  logwood  (1:8)       4  parts. — M. 
Keep  in  well-corked  bottle. 

IX.  Unna's  Borax  Methyl-blue 

Borax i  part 

Methyl  blue i    " 

Water 100  parts. — M. 

X.  Gabbefs  Acid  Blue  {Rapid  Stain) 
Methylene-blue 2  parts 

20  per  cent,  sulphuric  acid 100     "     — M. 

XI.  Alkaline  Anilin-water  Solutions 
Sodium  hydroxid  (i  per  cent.) ....       i  part 
Anilin-oil  water 100  parts. — M. 

And  add — 

Fuchsin,  or  methyl-violet  powdered       4  parts 
Cork  well.     Filter  before  using. 


52  ESSENTIALS    OF   BACTERIOLOGY 

XII.  Roux^s  Double  Stain 

Dahlia  or  gentian- violet 0.5  part 

Methyl-green 1.5  parts 

Distilled  water 200.0     "  — M. 

Use  as  other  stains,  without  acid. 

XIII.  Neisser^s  Stain  {for  Diphtheria) 
Solution  I 

Methylene-blue i  part 

Alcohol  (96  per  cent.) 20  parts 

Dissolve  and  add — 

Water 950  parts 

Glacial  acetic  acid 50     "    — M. 

Solution  II 

Vesuvin 2  parts 

Water 1000     "    — M. 

Stain  cover-glasses — (i)  Three  seconds  in  solution  I;  (2) 
wash  in  water;  (3)  three  seconds  in  No.  II;  (4)  wash  in  water. 
Body  of  bacillus,  brown;  oval  granules  at  each  end,  blue. 

XIV.  Carholthionin  {Nicolle) 
Saturated  solution  thionin  in  alcohol 

(90  per  cent.) 10  parts 

Aqueous    solution    phenol    (i    per 

cent.) 100     "    — M. 

Stain  sections  one-half  to  one  minute. 

XV.  Capsule  Stain  of  Hiss 
Use    the   following,    heated    until    it 
steams:  Saturated  alcoholic  solu- 
tion of  gentian- violet  or  f uchsin .  .       5  parts 

Distilled  water 95     "    — M. 

Wash  in  20  per  cent,  solution  of  cupric  sulphate  crystals. 


METHODS   OF   STUDYING  BACTERIA  53 

XVI.  Capsule  Stain  of  Welch 
(i)  Pour  glacial  acetic  acid  on  film.  After  a  few  seconds 
replace  with  anilin-water  gentian-violet  without  washing  in 
water.  (2)  Remove  all  acid  by  several  additions  of  stain, 
and  allow  it  to  act  for  three  to  four  minutes.  (3)  Wash  and 
examine  in  salt  solution  0.8-2.0  per  cent. 

XVII.  Romanowsky  Stains 

A  compound  dye  originally  used  for  malarial  parasites,  but 
now  employed  in  some  of  its  modifications  in  staining  blood- 
films,  bacteria  in  tissues,  and  protozoa  generally. 

The  stain  is  difficult  to  prepare,  and  can  be  purchased  of 
supply  houses  to  better  advantage. 

The  chief  modifications  are: 

Leishman^s  stain,  consisting  of  a  i  per  cent,  solution  methyl- 
ene-blue,  to  which  0.5  per  cent,  sodium  carbonate  has  been 
added  and  allowed  to  stand  for  twelve  hours  in  incubator  at 
65°  C,  and  then  ten  days  at  room  temperature,  and  a  solu- 
tion of  eosin  (i:  1000)  in  water.  Equal  parts  of  these  solu- 
tions are  mixed  and  allowed  to  stand  for  six  hours.  After  it 
has  been  washed  and  dried,  the  precipitate  is  dissolved  in 
methyl-alcohol. 

Giemsa  Stain: 

Azur  II. — eosin 3  parts 

Azur  II 8      '' 

Glycerin  (pure) 250      " 

Methyl-alcohol 250      "    — M. 

Azur  is  a  mixture  of  methylene-blue  and  eosin  prepared  in 
a  special  way. 

Jenner^s  Stain. — 1.2  per  cent,  aqueous  solution  of  water- 
soluble  eosin;  i  per  cent,  aqueous  solution  methylene-blue 
(Grubler) ;  equal  parts  of  each.  Mix;  allow  to  stand  twenty- 
four  hours,  wash  the  precipitate,  dry  it,  dissolve  0.5  gm.  in 
100  c.c.  methyl-alcohol. 

/.  H.  Wright's  Stain. — Made  in  much  the  same  way  as 
Leishman's.    The  precipitate  is  not  washed,  but  the  satur- 


54  ESSENTIALS    OF   BACTERIOLOGY 

ated  methyl-alcohol  solution  is  filtered  and  further  diluted 
with  methyl-alcohol.  The  stains  are  used  in  very  dilute 
form.  Where  the  blood-films  or  exudates  are  not  first  fixed 
in  alcohol,  the  concentrated  stain  is  allowed  to  cover  the 
preparation  for  five  to  twenty  seconds  to  fijc;  then  water  is 
poured  on  to  dilute  and  from  five  to  fifteen  minutes  allowed 
for  staining,  the  excess  removed  with  water.  The  stains  can 
be  purchased  in  powder  or  tablet  form,  and  need  only  be 
mixed  with  methyl-alcohol  to  be  ready  for  use. 


CHAPTER  VII 
GENERAL  METHOD  OF  STAINING  SPECIMENS 

Cover-glass  Preparations. — ^The  material  is  evenly 
spread  in  as  thin  a  layer  as  possible  upon  a  cover-glass;  then, 
to  spread  it  still  more  finely,  a  second  cover-glass  is  pressed 
down  upon  the  first  and  the  two  slid  apart.  This  also  secures 
two  specimens.  Before  they  can  be  stained,  they  must  be 
perfectly  dry,  otherwise  deformities  will  arise  in  the  structure. 

Drying  the  Specimen. — The  cover-glass  can  be  set  aside 
to  dry,  or  held  in  the  fingers  over  the  Bunsen  burner  (the 
fingers  preventing  too  great  a  degree  of  heat).  Since  most  of 
the  specimens  contain  a  certain  amount  of  albuminoid  mater- 
ial, it  is  best  in  all  cases  to  "fix" — i.  e.,  to  coagulate  the 
albumin.  This  is  accomplished  by  passing  the  cover-glass 
(after  the  specimen  is  dry)  three  times  through  the  flame  of 
the  burner,  about  three  seconds  being  consumed  in  so  doing, 
the  glass  being  held  in  a  small  forceps,  smeared  side  up. 

The  best  forceps  for  grasping  cover-glasses  is  a  bent  one, 
bent  again  upward,  near  the  ends  (Fig.  13).  It  prevents  the 
flame  or  staining  fluid  from  reaching  the  fingers. 

The  object  is  now  ready  for  staining. 

Staining. — A  few  drops  of  the  staining  solution  are  placed 


GENERAL  METHOD   OF   STAINING  SPECIMENS  55 

upon  the  cover-glass  so  that  the  whole  specimen  is  covered, 
and  the  stain  is  left  on  a  few  minutes,  the  time  depending 
upon  the  variety,  the  strength  of  stain,  and  the  object  de- 
sired. Instead  of  placing  the  dye  upon  the  object,  the  cover- 
glass  can  be  immersed  in  a  small  glass  dish  containing  the 
solution;  or,  if  heat  is  desired  to  intensify  or  hasten  the  proc- 
ess, a  watch-crystal  holding  the  stain  is  placed  over  a  Bun- 
sen  burner  and  in  it  the  cover-glass ;  the  cover-glass  may  be 
held  directly  in  the  flame  with  the  staining  fluid  upon  it, 
which  must  be  constantly  renewed  until  the  process  is  com- 
pleted, or  the  cover-glass  can  be  heated  in  a  test-tube,  con- 
taining stain  solution. 

Removing  Excess  of  Stain. — The  surplus  stain  is  washed 
off  by  dipping  the  glass  in  distilled  water. 


Fig.  13. — Author's  bent  forceps  for  holding  cover-glass  over  flame. 

The  water  is  removed  by  drying  between  filter-paper  or 
simply  allowed  to  run  off  by  standing  the  cover-glass  slant- 
wise against  an  object.  When  the  specimen  is  to  be  examined 
in  water  (which  is  always  best  with  the  first  preparation  of 
the  specimen,  as  the  Canada  balsam  destroys  to  some  extent 
the  natural  appearance  of  the  bacteria) ,  a  small  drop  of  ster- 
ilized water  is  placed  upon  the  glass  slide,  and  the  cover-glass 
dropped  gently  down  upon  it,  so  that  the  cover-glass  remains 
adherent  to  the  slide. 

The  dry  system  or  the  oil  immersion  can  now  be  used. 

When  the  object  has  been  sufiiciently  examined,  it  can  be 
permanently  mounted  by  lifting  the  cover-glass  off  the  slide 
(this  is  facilitated  by  letting  a  little  water  flow  under  it,  one 


56  ESSENTIALS  OF  BACTERIOLOGY 

end  being  slightly  elevated).  The  water  that  still  adheres 
is  dried  off  in  the  air  or  gently  over  the  flame,  and  when  per- 
fectly dry,  the  cover-glass  is  placed  upon  the  drop  of  Canada 
balsam  which  has  been  put  upon  the  glass  sHde. 

In  placing  the  cover-glass  in  the  staining  solutions  one 
must  be  careful  to  remember  which  is  the  spread  side,  by 
holding  it  between  one's  self  and  the  window  and  scraping  the 
sides  carefully  with  the  sharp  point  of  the  forceps,  the  side 
having  the  specimen  on  it  will  show  the  marks  of  the  instru- 
ment. 

Little  glass  dishes,  about  one-half  dozen,  should  be  at  hand 
for  containing  the  various  stains  and  decolorants. 

Tissue  Preparations. — In  order  to  obtain  suitable  speci- 
mens for  staining,  very  thin  sections  of  the  tissue  must  be 
made. 

As  with  histologic  preparations,  the  tissue  must  be  hardened 
before  it  can  be  cut  thin  enough.  Alcohol  is  the  best  agent 
for  this  purpose. 

Pieces  of  the  tissue  one-quarter  inch  in  size  are  covered  with 
alcohol  for  twenty-four  to  forty-eight  hours. 

When  hardened,  it  must  be  fixed  upon  or  in  some  firm 
object.     A  paste  composed  of — 

Gelatin i  part 

Glycerin 4  parts 

Water 2      " 

will  make  it  adhere  firmly  to  a  cork  in  about  two  hours,  or  it 
can  be  embedded  in  a  small  block  of  paraffin  and  covered 
over  with  melted  paraffin.  Celloidin  may  be  used  as  an 
embedding  agent,  and  formalin  is  useful  to  harden  tissue 
quickly. 

Cutting. — ^The  microtome  should  be  able  to  cut  sections 
Tutu  inch  in  thickness;  this  is  the  fineness  usually  required. 

The  sections  are  brought  into  alcohol  as  soon  as  cut,  unless 
they  have  been  embedded  in  paraffin,  when  they  are  first 
washed  in  chloroform  to  dissolve  out  the  paraffin. 


GENERAL   METHOD   OF    STAINING   SPECIMENS  57 

Staining. — All  the  various  solutions  should  be  in  readiness, 
best  placed  in  the  little  dishes  in  the  order  in  which  they  are  to 
be  used,  as  a  short  delay  in  one  of  the  steps  may  spoil  the 
specimen. 

A  very  useful  instrument  for  transferring  the  deHcate 
sections  from  one  solution  to  another  is  a  little  metal  spatula, 
the  blade  being  flexible  (Fig.  14). 

A  still  better  plan,  especially  when  the  tissue  is  ''crum- 
bling," is  to  carry  out  the  whole  procedure  on  the  glass  slide. 

General  Principles. — The  section  is  transferred  from  the 
alcohol  in  which  it  has  been  kept  into  water,  which  removes 
the  excess  of  alcohol,  from  here  into — 

Dish  /,  containing  the  stain,  where  it  remains  five  to  fifteen 
minutes.     Then — 

Dish  II,  containing  5  per  cent,  acetic  acid  (1:20),  where  it 


Spatula  for  lifting  sections. 


remains  one-half  to  one  minute.  The  acid  removes  the 
excess  of  stain. 

Dish  III,  water,  to  rinse  off  the  acid.  The  section  can  now 
be  placed  under  the  microscope,  covered  with  cover-glass  to 
see  if  the  intensity  of  the  stain  is  sufficient  or  too  great.  A 
second  section  is  then  taken,  avoiding  the  errors,  if  any;  and 
having  reached  this  stage,  proceeded  with  as  follows: 

Dish  IV,  alcohol,  two  to  three  seconds,  to  remove  the  water 
in  the  tissue. 

V.  A  few  drops  of  oil  of  cloves,  just  long  enough  to  clear  the 
specimen  to  make  it  transparent  (so  that  an  object  placed 
underneath  will  shine  through). 

VI.  Remove  excess  with  filter-paper. 

VII.  Mount  in  Canada  balsam  (xylol  balsam). 
Staining  Blood  Specimens. — A  drop  of  blood  is  spread  on 


58  ESSENTIALS   OF   BACTERIOLOGY 

a  cover-glass  and  stained  with  the  ordinary  dyes ;  but  in  order 
to  eliminate  the  coloring-matter  of  the  red  corpuscles  and 
bring  the  stained  bacteria  more  prominently  into  view, 
Gunther  recommends  that  the  blood,  after  drying  and  fixing, 
should  be  rinsed  in  a  dilute  solution  of  acetic  acid  (i  to  5  per 
cent.).  The  hemoglobin  is  thereby  extracted,  and  the  cor- 
puscles appear  then  only  as  faint  outlines. 

Instead  of  "fixing"  by  heat,  Canon  employs  alcohol  for  five 
minutes,  especially  in  staining  for  influenza  bacilli  which  have 
been  detected  in  the  blood. 


CHAPTER  VIII 

SPECIAL  METHODS  OF  STAINING  AND  MODIFICATIONS 

Gram's  Method  of  Double  Staining  {For  Cover-glass 
Specimens). — I.  A  hot  solution  of  anilin- water  gentian- violet 
two  to  ten  minutes. 

II.  Directly,  without  washing,  into  Gram's  solution  of 
iodin  potassium  iodid  one  to  three  minutes  (the  cover-glass 
looks  black). 

III.  Wash  in  alcohol  60  per  cent,  until  only  a  light  brown 
shade  remains  (as  if  the  glass  were  smeared  with  dried  blood). 

IV.  Rinse  off  alcohol  with  water. 

V.  Contrast  color  with  either  eosin,  picrocarmin,  or  Bis- 
marck-brown. The  bacteria  will  appear  deep  blue,  all  else 
red  or  brown  on  a  very  faint  brown  background. 

Gram's  Method  for  Tissues  {Modified  by  Gunther) 
I.  Stain  in  anilin- water  gentian- violet  .  .     i  minute 
II.  Dry  between  filter-paper. 

III.  Iodin  potassium  iodid  solution 2  minutes 

IV.  Alcohol i^  minute 

V.  3  per  cent,  solution  hydrochloric  acid 

in  alcohol 10  seconds 

VI.  Alcohol,  oil  of  cloves,  and  Canada  balsam. 


SPECIAL   METHODS    OF   STAINING   AND   MODIFICATIONS     59 

Behavior  of  the  More  Important  Bacteria  to  Gram's  Stain. — 
Positive  means  that  the  bacteria  retain  the  primary  color,  or 
gentian- violet;  negative,  that  they  do  not. 

Positive.  Negative. 

Tubercle  bacillus.  Colon  bacillus. 

Smegma  bacillus.  Typhoid  bacillus. 

Lepra  bacillus.  Cholera  bacillus. 

Anthrax  bacillus.  Influenza  bacillus. 

Tetanus  bacillus.  Friedlander's  bacillus. 

Diphtheria  bacillus.  Plague  bacillus. 

Pneumococcus.  Diplococcus  intracellularis. 

Streptococcus.  Gonococcus. 

Staphylococcus.  Koch- Weeks  bacillus. 

Cocci  of  the  urethra.  Conjunctivitis  bacillus  of  Morax. 

Loffler's  Method  for  Tissues 

Alkaline  methylene-blue 5~30  minutes 

I  per  cent,  acetic  acid few  seconds. 

Absolute  alcohol,  xylol,  Canada  balsam. 
Bacteria  dark  blue,  nuclei  blue,  cell-bodies  light  blue. 

To  Stain  Spores. — Since  spores  have  a  very  firm  capsule, 
which  tends  to  keep  out  all  external  agents,  a  very  intensive 
stain  is  required  to  penetrate  them,  but  once  this  object  is 
attained,  it  is  equally  as  difficult  to  decolorize  them. 

A  cover-glass  prepared  in  the  usual  way,  i.  e.,  drying  and 
passing  the  specimen  through  the  flame  three  times,  is  placed 
in  a  watch-crystal  containing  Ziehl's  carbolfuchsin  solution, 
and  the  same  placed  upon  a  rack  over  a  Bunsen  burner,  where 
it  is  kept  at  boiling-point  for  one  hour,  careful  to  supply  fresh 
solution  at  short  intervals  lest  it  dry  up. 

The  bacilli  are  now  decolorized  in  alcohol  containing  0.5 
per  cent,  hydrochloric  acid.  A  contrast  color,  preferably 
methylene-blue,  is  added  for  a  few  minutes. 


6o  ESSENTIALS   OF   BACTERIOLOGY 

The  spores  will  appear  as  little  red  beads  in  the  blue-stained 
bacteria,  and  loose  spores  lying  about  outside  the  cell-wall. 

Spore  Stain  {Modified). — I.  Carbolfuchsin  on  cover-glass 
and  heated  in  the  flame  to  boiling-point  20  to  30  times. 

II.  25  per  cent,  sulphuric  acid,  two  seconds;  rinsed  in 
water. 

III.  Methylene-blue  contrast. 

Alex.  Klein  recommends  the  following  spore  method:  mix  a 
little  of  the  culture  (potato)  with  three  drops  of  physiologic 
salt  solution,  and  heat  gently  with  an  equal  quantity  of 
carbolfuchsin  for  a  period  of  six  minutes.  Spread  then  on 
cover-glasses,  dry  in  the  air,  and  fix  by  passing  three  times 
through  Bunsen-burner  flame.  Decolorize  in  i  per  cent, 
sulphuric  acid  for  one  to  two  seconds;  contrast  in  weak 
methylene-blue. 

Bowhill's  Orcein  Stain 

Saturated  alcoholic  solution  of  orcein  .  15  c.c. 

20  per  cent,  aqueous  solution  tannin  .  10  c.c. 

Distilled  water 30  c.c. — M. 

Filter. 

Use  orcein  solution  in  watch-glass,  float  cover-glass  in  it, 
and  heat  gently,  not  boil,  for  ten  minutes.  Wash  in  water. 
Dry  and  mount  in  balsam. 

Five  per  cent,  chromium  trioxid  applied  for  fifteen  minutes 
has  been  recommended  in  staining  spores.  This  is  followed 
by  the  carbolfuchsin  stain  as  above. 

Sporogenic  bodies  stain  quite  readily,  and  in  order  to  distin- 
guish them  from  spores  Ernst  uses  alkaline  methylene-blue, 
slightly  warmed.  Then  rinse  in  water.  Contrast  with  cold 
Bismarck-brown.  The  spores  are  colored  bright  blue,  the 
spore  granules  a  dirty  blue,  being  mixed  with  the  brown, 
which  colors  also  the  bacteria. 

Kuhne's  Method.— In  sections  the  alcohol  used  sometimes 
decolorizes  too  much.    To  obviate  this  Kuhne  mixes  the  alco- 


SPECIAL   METHODS    OF    STAINING   AND   MODIFICATIONS     6l 

hol  with  the  stain,  so  that  while  the  section  is  being  anhy- 
drated,  it  is  constantly  supplied  with  fresh  dye. 

Weigert  uses  anilin-oil  to  dehydrate  instead  of  alcohol,  and 
here,  too,  it  can  be  used  mixed  with  the  dye. 

Capsule  Stain  {Buerger). — I.  Spread  culture  by  means  of 
a  drop  of  ascitic  fluid  on  cover-glass. 

II.  Fix  in  Miiller's  fluid,  which  has  been  saturated  with 
5  per  cent,  bichlorid  of  mercury,  and  warm  for  three  seconds. 

III.  Wash  quickly  in  water;  rinse  in  alcohol. 

IV.  Cover  with  tincture  of  iodin  for  one  minute. 
V.  Wash  in  alcohol  and  dry  in  air. 

VI.  Stain  in  anilin-water  gentian-violet  for  two  seconds. 
VII.  Wash  in  2  per  cent,  salt  solution. 

VIII.  Mount  in  salt  solution  ringed  with  vaselin. 

Hiss*  Method  for  Capsule. — Smear  on  cover-glass  the 
organisms  mixed  with  a  drop  of  animal  serum  (beef-blood 
serum  or  ascitic  fluid).  Dry  in  air.  Fix  by  heat.  Stain  for 
few  seconds  in  Hiss'  stain  (p.  52).  Wash  in  20  per  cent,  cop- 
per sulphate  solution.  Dry  and  mount.  Capsule  appears 
as  faint  blue  halo  about  dark-purple  cell. 

Flagella  Stain,  with  Loffler's  Mordant. — I.  A  few  drops 
of  the  mordant  stain  (p.  51)  are  placed  upon  the  spread 
cover-glass  and  heated  until  it  steams. 

II.  Wash  with  water  until  the  cover-glass  looks  almost 
clean,  using  a  small  piece  of  filter-paper  to  rub  off  the  crusts 
which  have  gathered  around  the  edges. 

III.  Anilin-water  fuchsin  (neutral)  held  in  flame  about  one 
and  one-half  minutes. 

IV.  Wash  in  water. 

If  the  stain  is  properly  made,  the  bacteria  are  deeply 
colored  and  the  flagella  seen  as  little  dark  lines  attached  to 
them. 

Unna's  Method  for  Fungi. — Especially  useful  for  epi- 
dermic scales.  Moisten  horny  scale  or  crust  with  acetic  acid; 
macerate  between  two  glass  slides;  dry  in  flame;  wash  out  fat 
with  ether  and  alcohol  (equal  parts) ;  stain  in  horax  methyl-hlue 


62  ESSENTIALS   OF   BACTERIOLOGY 

for  ten  seconds  (over  flame) ;  bleach  with  glycerin  and  ether 
(equal  parts) ;  rinse  in  water,  alcohol,  dry,  and  mount. 


CHAPTER  IX 
CULTIVATION  OF  BACTERU 

Artificial  Cultivation. — The  objects  of  cultivation  are  to 
obtain  germs  in  pure  culture,  free  from  all  foreign  matter, 
isolated,  and  so  developed  as  to  be  readily  used  either  for 
microscopic  examination  or  animal  experimentation. 

To  develop  bacteria  properly  we  supply,  as  nearly  as  possi- 
ble, the  conditions  which  hold  for  the  especial  germ  in  nature. 
With  the  aid  of  solid  nutrient  media  the  bacteria  can  be  easily 
separated,  and  the  methods  have  been  gradually  evolved 
from  those  originally  devised  by  Pasteur  and  Koch. 

Sterilization  of  Culture-media,  etc. — If  we  place  our 
nutrient  material  in  vessels  that  have  not  been  properly  dis- 
infected, we  will  obtain  growths  of  bacteria  without  having 
sown  any. 

If  we  have  thoroughly  cleaned  our  utensils  and  then  not 
taken  care  to  protect  them  from  further  exposure,  the  germs 
we  have  sown  will  be  effaced  or  contaminated  by  multitudes 
of  others  that  are  constantly  about  us.  We,  therefore,  have 
two  necessary  precautions  to  take: 

First,  thoroughly  to  clean  and  sterilize  every  object  that  enters 
into,  or  in  any  way  comes  in  contact  with,  the  culture. 

Second,  to  maintain  this  degree  of  sterility  throughout  the 
phole  course  of  the  growth,  and  prevent,  by  proper  containers, 
the  entrance  of  foreign  germs. 

Disinfectants. — Corrosive  sublimate  (bichlorid  of  mercury), 
which  is  the  most  effective  agent  we  possess,  cannot  be  gener- 
ally used  because  it  renders  the  soil  unproductive,  and,  there- 
fore, must  be  employed  only  in  washing  dishes,  to  destroy  the 


CULTIVATION   OF    BACTERIA 


63 


old  cultures.  Even  after  washing  a  few  drops  of  the  solution 
may  remain  and  prevent  growth,  so  that  one  must  be  careful 
to  have  the  glassware  that  comes  in  contact  with  the  nutrient 
media  free  from  the  sublimate. 


Fig.  15. — Hot-air  sterilizer.  The  gas-jets  are  inclosed  within  the 
space  between  the  outer  and  middle  walls,  C,  and  can  be  seen  at  F.  The 
heat  ascends,  warming  the  air  between  the  two  inner  walls,  which  ascends 
between  the  walls,  K,  K,  then  descends  over  the  contents,/,  and  escapes 
through  perforations  in  the  bottom,  B,  to  supply  the  draft  at  F,  and 
eventually  escapes  again  at  S;  R,  gas  regulator;  T,  thermometer. 


Heat. — Heat  is  the  best  agent  we  possess  for  general  use. 
Dry  heat  and  moist  heat  are  the  two  forms  employed,  but 
these  differ  greatly  in  effectiveness.     Thus  Koch  found  that 


64  ESSENTIALS    OF   BACTERIOLOGY 

while  moist  heat  at  ioo°  C.  killed  the  spores  of  the  anthrax 
bacillus  in  one  hour,  it  required  three  hours  of  dry  heat  at 
140°  C.  to  produce  death. 

For  obtaining  dry  heat — that  is,  a  temperature  of  150°  C. 
(about  300°  F.) — a  sheet-iron  oven  (Fig.  15)  is  used  which 
can  be  heated  by  a  gas-burner.  If  it  have  double  walls  (air 
circulating  between),  the  desired  temperature  is  much  more 
quickly  obtained.  A  small  opening  in  the  top  to  admit  a 
thermometer  is  necessary.  These  chests  are  usually  about 
I  foot  high,  i}i  feet  wide,  and  ^  foot  deep.  In  them  glass- 
ware, cotton,  and  paper  can  be  sterilized.  When  the  cotton 
is  turned  slightly  brown,  it  usually  denotes  sufficient  steriliza- 
tion. All  instruments,  where  practicable,  should  be  drawn 
through  the  flame  of  an  alcohol  lamp  or  Bunsen  burner.  One 
hour  in  the  oven  at  170°  C.  usually  sterihzes  glassware,  while 
the  ordinary  germs  in  Hquids  may  be  killed  by  boiling  for 
five  minutes  if  no  spores  are  present.  The  boiling  of  any 
fluid  at  100°  C.  for  one  and  one-half  hours  nearly  always 
insures  sterilization. 

Moist  Heat. — Steam  at  100°  C.  in  circulation  has  been 
shown  to  be  a  very  effective  application  of  heat. 

The  steam  chest  devised  by  Koch  consisted  of  a  long 
double  boiler  divided  by  a  perforated  shelf  on  which  the 
material  could  rest  while  subjected  to  streaming  steam. 

Arnold's  steam  sterilizer  will  answer  every  purpose  of  the 
Koch  steam-chest.  It  is  cheaper,  also  requiring  less  fuel  to 
keep  it  going.  The  steam  does  not  escape,  but  is  condensed 
in  the  outer  chamber. 

The  autoclave  (Fig.  16),  which  produces  steam  under 
pressure  and  allows  a  temperature  of  120°  C.  to  be  obtained, 
is  a  most  effective  method  of  sterilization,  but  the  higher 
temperatures  are  not  suitable  for  gelatin  or  sugar  solution. 
Gelatin  loses  its  power  of  solidifying  if  the  boiling  is  pro- 
longed. 

Instead  of  sterilizing  for  a  long  time  at  once,  successive 
sterilization  is  practised  with  nutrient  media,  so  that  the 
albumin  will  not  be  too  strongly  coagulated.     Fifteen  minutes 


CULTIVATION   OF    BACTERIA 


6s 


each  day  for  three  days  in  succession  in  the  Arnold  sterilizer, 
or  one  exposure  in  the  autoclave,  five  to  fifteen  minutes,  at 
15  pounds  pressure;  120°  C.  is  sufficient  to  sterilize  most 
culture-media. 

Fractional  Sterilization  of  T3mdall. — Granted  that  so 
many  spores  originally  exist  in  the  object  to  be  sterilized,  it 


^       li 


Fig.  16. — Autoclave.     Horizontal  form. 

is  subjected  to  60°  C.  for  four  hours,  in  which  time  a  part  at 
least  of  those  spores  have  developed  into  bacteria,  and  the 
bacteria  destroyed  by  the  further  application  of  the  heat. 
The  next  day  more  bacteria  will  have  formed,  and  four 
hours'  subjection  to  60°  C.  heat  will  destroy  them,  and  so, 
at  the  end  of  a  week,  using  four  hours'  application  each  day, 
all  the  spores  originally  present  will  have  germinated  and  the 
bacteria  be  destroyed. 
5 


66 


ESSENTIALS   OF   BACTERIOLOGY 


As  modified,  and  in  use  in  most  laboratories,  fifteen  minutes, 
sterilization  in  steam,  at  ioo°  C,  in  the  Arnold  sterilizer  on 
three  successive  days,  has  been  found  sufficient,  while  one 
steriHzation  in  the  autoclave  at  120°  C.  for  fifteen  minutes 
will  serve  in  most  cases,  especially  if  the  medium  is  for  imme- 
diate use,  and  does  not  contain  gelatin  or  sugar. 

Cotton  Plugs  or  Corks. — All  the  glass  vessels  (test-tubes, 
flasks,  etc.)  must  be  closed  with  cotton  plugs,  cotton-wool, 


Fig.  17. — ^Wire  cage.  Fig.  18. — Cotton-plugged  test-tubes. 


or  a  good  quality  of  non-absorbent  cotton),  the  cotton  being 
easily  sterilized  and  preventing  the  entrance  of  germs  from 
the  air. 

Tin-foil  may  be  used  to  cover  the  cotton,  or  caps  made  of 
india-rubber. 

Test-tubes. — New  test-tubes  are  washed  with  hydro- 
chloric acid  and  water  to  neutralize  the  alkalinity  often  pres- 
ent in  fresh  glass,  or  in  chromic  acid  cleaning  mixture  one 
hour.  (Potassium  dichromate,  6;  water,  30;  sulphuric  acid, 
46.)     They  are  then  well  washed  and  rubbed  with  a  brush, 


PREPARATION   OF   NUTRIENT  CULTURE-MEDIA  67 

placed  obliquely  to  drain,  and  when  dry,  corked  with  cotton 
plugs.  Then  put  in  the  hot-air  oven  (little  wire  cages,  Fig. 
17,  being  used  to  contain  them)  for  fifteen  minutes,  after 
which  they  are  ready  to  be  filled  with  the  nutrient  mediiun. 
(The  cotton  should  fit  firmly  in  the  tube  and  extend  a  short 
space  beyond  it.) 

Test-tubes  without  flaring  edges  are  more  desirable,  since 
the  edges  can  easily  be  drawn  out  so  as  to  seal  the  tube. 

Instead  of  test-tubes,  ordinary  3-ounce  panel  medicine 
bottles  can  be  used  for  retaining  the  nutrient  media  and 
cultures. 

According  to  investigations,  the  glass  tubes  become  suffi- 
ciently sterile  in  the  steam-chest  without  the  preliminary 
sterilization  in  the  dry  oven. 

Sterilization  by  Filtration. — Germ  Filters. — Kaolin  or  por- 
celain bougies,  such  as  are  used  in  the  Berkefeld,  Chamber- 
land,  and  Pasteur  filters,  restrain  most  bacteria,  except  those 
now  known  as  ultramicroscopic.  In  the  making  of  toxins 
this  method  is  used,  heat  or  disinfectants  being  undesirable. 
With  the  knowledge  of  smaller  forms  of  life,  the  filter  will 
need  further  improvement. 


CHAPTER  X 
PREPARATION  OF  NUTRIENT  CULTURE-MEDU 

Of  the  many  different  media  recommended  and  used  since 
bacteriology  became  a  science,  we  can  describe  only  the  more 
important  ones  now  in  use.  Each  investigator  changes  them 
according  to  his  taste. 

Potato  as  Medium. — The  knowledge  of  bacteria  and 
germs  or  molds  settling  and  growing  upon  slices  of  potato 


68 


ESSENTIALS   OF   BACTERIOLOGY 


exposed  to  the  air  led  to  the  use  of  solid  media  for  the 
artificial  culture  of  the  same.  It  was  thus  learned  that  each 
germ  tends  to  form  a  separate  colony  and  remain  isolated, 
and  so  pure  cultures  were  first  obtained. 

Esmarch's  Cubes. — ^The  potato  is  first  well  cleaned  and 
peeled.     It  is  then  cut  in  cubes  Yi  inch  in  size. 

These  are  placed,  each  in  a  little  glass  dish  or  tray,  and  then 
in  steam-chest  for  one-half  hour,  after  which  they  are  ready 
for  inoculation  (the  dishes  first  having  been 
sterilized  in  hot-air  oven). 

Test-tube  Potatoes. — Cones  are  cut  out 
of  the  peeled  potato  and  placed  in  test-tubes, 
which  can  then  be  plugged  and  easily  pre- 
served. 

Roux's  test-tube  (Fig.  19),  specially  de- 
signed for  potato  cultures,  consists  of  a  tube 
with  a  small  constricted  portion  at  the  bot- 
tom, in  which  water  may  be  kept  to  keep  the 
potato  moist. 

Manner  of  Inoculating  Potatoes. — With 
a  platinum  rod  or  a  spatula  (sterilized)  the 
material  is  spread  upon  one  of  the  slices, 
keeping  free  of  the  edges.  The  growth  on 
this  first,  or  original,  potato  will  be  quite  lux- 
uriant, and  the  individual  colonies  often  diffi- 
cult to  recognize;  therefore  dilutions  are  made. 
From  the  original  or  first  slice  a  small  portion,  including 
some  of  the  meat  of  the  potato,  is  spread  upon  the  surface  of 
a  second  slice,  which  is  first  dilution.  From  this  likewise  a 
small  bit  is  taken  and  spread  on  a  third  slice,  or  second  dilu- 
tion, and  here  usually  the  colonies  will  be  sparsely  enough 
settled  to  study  them  in  their  individuality. 

This  is  the  principle  carried  on  in  all  the  cultivations.  It 
is  a  physical  analysis. 

Potato  and  Bread  Mash. — These  pastes  are  used  chiefly 
in  the  culture  of  molds  and  yeasts.  Peeled  potatoes  are 
mashed  with  distilled  water  until  thick,  and  then  sterilized 


Fig.  19. — Tube 
for  potato  cul- 
ture. 


PREPARATION   OF    NUTRIENT   CULTURE-MEDIA  (ig 

in  flasks  three-quarters  of  an  hour  for  three  successive 
days. 

Bread  Mash. — Bread  devoid  of  crust,  dried  in  an  oven,  and 
then  pulverized  and  mixed  with  water  until  thick,  and  steril- 
ized as  above. 

Solid  transparent  media  are  prepared  from  materials 
which  are  transparent  and  which  can  readily  be  converted 
into  liquids.    Such  are  the  gelatin  and  agar  culture-media. 

Gelatin. — Gelatin  is  obtained  from  bones  and  tendons, 
and  consists  chiefly  of  chondrin  and  gluten. 

Agar-agar. — This  agent,  which  is  of  vegetable  origin, 
derived  from  sea-plants  gathered  on  the  coasts  of  India  and 
Japan,  has  many  of  the  properties  of  gelatin,  retaining  its 
solidity  at  a  much  higher  temperature;  it  becomes  liquid  at 
90°  C.  and  congeals  again  at  45°  C.  (gelatin  will  liquefy  at 
35°  C),  whereas  38°  C.  is  the  temperature  at  which  most 
pathogenic  germs  grow  best.  Agar  cultures  can  be  kept  in 
incubator  for  days  and  weeks  without  liquefying. 

Agar  is  not  affected  very  much  by  the  peptonizing  action 
of  the  bacteria. 

The  crude  agar  should  first  be  rinsed  in  water,  and  then  in 
5  per  cent,  acetic  acid  and  clear  water  again,  to  rid  it  of  im- 
purities. If  agar  is  boiled  thoroughly  over  a  hot  flame  or  in 
an  autoclave,  it  can  be  filtered  much  more  readily.  The 
main  point  is  to  see  that  all  the  agar  is  dissolved. 

Glycerin-agar. — The  addition  of  4  to  6  per  cent,  of  gly- 
cerin to  nutrient  agar  greatly  enhances  its  value  as  a  culture- 
medium. 

Gelatin-agar. — A  mixture  of  5  per  cent,  gelatin  and  0.75 
per  cent,  agar  combines  in  it  some  of  the  virtues  of  both 
agents. 

Blood-servun. — Blood-serum,  being  rich  in  albumin,  co- 
agulates very  easily  at  70°  C,  and  if  this  temperature  is  not 
exceeded,  a  transparent  solid  substance  is  obtained  upon 
which  the  majority  of  bacteria  develop,  and  some  with 
preference. 


70  ESSENTIALS    OF   BACTERIOLOGY 

PREPAIIATION  OF  NUTRIENT  CULTURE-MEDIA 

(After  the  recommendations  of  the  American  Public  Health  Association) 

Materials. — All  water  used  should  be  distilled. 
Fresh  meat. 

Dried  peptone,  Witte  brand. 

Best  French  gelatin,  as  free  as  possible  from  impurities. 
Best  commercial  agar  in  threads. 
Sugars,  dextrose,  lactose,  and  saccharose,  all  chemically 

pure. 
Glycerin,  double  distilled. 
Azolitmin  in  place  of  litmus. 
All  other  materials  as  nearly  as  possible  chemically  pure. 

Sterilization. — Preferably  in  the  autoclave  and  in  small 
containers,  at  120°  C.,with  15  pounds  pressure  for  fifteen 
minutes.  The  sterilizer  should  be  hot  before  the  medium 
is  put  in. 

Intermittent. — For  gelatin  or  sugar  media  a  high  tempera- 
ture is  not  suitable.  The  media  are  placed  in  streaming 
steam  for  thirty  minutes  on  three  successive  days. 

Reaction. — One-half  per  cent,  solution  phenolphthalein 
(5  grams  to  i  liter  alcohol)  is  needed  as  an  indicator. 
The  reaction  should  be  -f-i  per  cent.,  i.  e.,  1  per  cent, 
alkaline  solution  required  to  make  it  neutral. 

Method  of  Obtaining  Reaction. — To  5  c.c.  of  medium  add 
45  c.c.  water.  Boil  one  minute.  Add  i  c.c.  solution 
phenolphthalein.  If  the  mixture  is  not  tinted  pink,  the 
medium  is  acid  or  neutral  and  requires  the  gradual  addi- 
tion of  I  :  20  normal  sodium  hydroxid  solution  until  a 
faint  pink  color  remains.  The  soda  should  be  added 
while  the  mixture  is  hot  or  boiling.  Calculate  from  the 
amount  of  alkali  used  for  the  5  c.c.  how  much  will  be 
needed  for  the  whole  quantity  of  media  and  add  the 
same,  using  normal  solution  instead  of  i  :  20  normal. 
Example:  If  2  c.c.  -  NaOH  will  neutralize  5  c.c.  media, 

N  N 

2  c.c.  Y  NaOH  will  neutralize  100  c.c,  or  20  c.c.  -  NaOH 
will  neutralize  1000  c.c.  media. 

If  the  medium  is  very  alkaline,  hydrochloric  acid  must  be 
added  to  reduce  to  -|-  i  per  cent. 


NUTRIENT    CULTURE-MEDIA  7 1 

Nitrate  Broth. — One  gram  peptone  to  one  liter  water  and 

add  0.2  gm.  nitrite  free  potassium  nitrate;    place  ten 

c.c.  in  test-tube,  sterilize  in  autoclave. 
Nutrient  Broth. — i.  Cover   i   pound   (500  gm.)   chopped 

meat  with   1000  c.c.   water  and  place  in  refrigerator 

twelve  hours. 

2.  Strain  through  Canton-flannel  or  cheese-cloth  and 
add  water  to  make  1000  c.c. 

3.  Add  I  per  cent,  peptone,  warming  until  dissolved. 

4.  Heat  over  water-bath  thirty  minutes. 

5.  Restore  loss  of  water. 

6.  Titrate  and  adjust  reaction  to   +1   per  cent,  by- 
adding  alkali  or  acid.     (See  above.) 

7.  Boil  two  minutes  over  free  flame. 

8.  Restore  loss  of  evaporation. 

9.  Filter  through  absorbent  cotton  and  Canton-flannel 
and  refilter  until  clear. 

10.  Titrate  and  record  final  reaction.     If  it  varies  0.2 
per  cent,  from  standard,  readjust. 

11.  Tube,  using  10  c.c.  in  each  tube. 

12.  Sterilize. 

The  nutrient  broth  as  above  prepared  is  used  as  a  basis 
for  most  of  the  other  media.  It  is  practically  the  same 
as  was  devised  by  Lofller  in  the  early  days  of  bacteri- 
ology. 

Sugar  Broths. — Prepared  as  the  standard  broth  with  the 
addition  of  i  per  cent,  dextrose,  lactose,  or  other  sugar 
just  before  final  sterilization. 

Nutrient  Gelatin. — Ten  per  cent,  gelatin  is  added  with  the 
peptone  to  the  meat-water  infusion.  Warm  gently  at 
60°  C.  until  dissolved,  then  adjust  reaction.  Heat 
over  steam-bath  for  forty  minutes.  Restore  loss  of 
evaporation,  readjust  reaction,  and  boil  five  minutes. 
Make  up  loss  from  evaporation  and  record  final  reaction. 
Filter,  tube,  and  sterilize  fifteen  minutes  in  autoclave  at 
120°  C.  Place  at  once  in  ice- water  until  solid  and  store 
in  ice-chest. 

Nutrient  Agar. — Boil  10  to  15  gm.  thread  agar  in  500  c.c. 


72  ESSENTIALS    OF   BACTERIOLOGY 

water  for  half-hour  or  digest  in  autoclave  fifteen  minutes. 
Restore  loss  by  evaporation  and  allow  to  cool  to  60  c.c. 
To  meat-water  infusion  (500  parts  meat  to  500  c.c. 
water)  add  2  per  cent,  peptone,  also  500  c.c.  agar  solu- 
tion. Titrate  after  boiling  one  minute,  and  adjust 
reaction  to  +1.  Heat  in  steam-bath  forty  minutes,  and 
proceed  as  with  nutrient  gelatin,  i.  e.,  restore  loss,  read- 
just reaction,  and  filter  and  refilter  until  clear.  The 
filtering  should  be  done  while  the  solution  is  hot.  Pour 
into  tubes  or  plates,  sterilize  in  au- 
j^  toclave,  and  finally  slant  the  tubes 

<^2Si^  so   2-s   to   obtain   a   larger  surface. 

(Most  agar  tubes  are  used  for  stroke 
cultures.) 

The  addition  of  the  white  of  an  egg 
will  often  clear  it  up;  if  this  avails 
not,  refiltering  several  times  and  at- 
tention to  the  few  points  mentioned 
will  produce  a  clear  solution. 
Lactose  Litmus  Agar. — One  per  cent, 
lactose  added  to  nutrient  agar  just 
before  sterilization.  Reaction  neu- 
tral. One  per  cent,  azolitmin 
(Kahlbaum)  boiled  five  minutes  and 

recdve  '°'~Wood-sl-  ^^^^^  ^i^^^^  ^^  the  tube  before  final 

mm.  sterilization    or,   if   media   used   in 

plates,  added  at  the  time  of  plating. 

Preparation  of  Nutrient  Blood- sertmi. — If  the  slaughter 
of  the  animal  can  be  supervised,  it  were  best  to  have  the  site 
of  the  wound  and  the  knife  sterilized,  and  sterile  flasks  (Fig. 
20)  at  hand  to  receive  the  blood  directly  as  it  flows. 

The  blood  is  placed  on  ice  forty-eight  hours,  and  the 
serum  is  drawn  out  with  sterile  pipets  into  test-tubes,  avoid- 
ing shaking  of  the  jar.  These  are  placed  obliquely  in  an 
oven  where  the  temperature  can  be  controlled  and  main- 
tained.    (See  Fig.  21.) 

Coagulation  of  Blood-seriun. — The  tubes  of  blood-serum 


NUTRIENT    CULTURE-MEDIA  73 

having  been  placed  in  the  thermostat,  are  kept  at  a  temper- 
ature of  65°  to  68°  C.  until  coagulation  occurs;  then  removed 
and  sterilized  by  fractional  sterilization. 

Sterilization  of  Blood-serum. — The  tubes  are  placed 
three  to  four  days  in  incubator  at  58°  C,  and  those  tubes 
which  show  any  evidences  of  organic  growth  are  discarded. 

If,  now,  at  the  end  of  a  week,  the  serum  remains  sterile  at 


Thermostat  or  inspissator  for  blood-serum. 


the  ordinary  temperature  of  the  room,  it  can  be  used  for 
experimental  purposes. 

Perfectly  prepared  blood-serum  is  transparent,  of  a  gelatin- 
like consistence,  and  straw  color.  It  will  not  liquefy  by  heat, 
though  bacteria  can  digest  it.  Water  of  condensation 
always   forms,   which  prevents   the   drying   of   the   serum. 

Short  Method, — Blood-serum  may  be  prepared  in  a  shorter 


74 


ESSENTIALS   OF   BACTERIOLOGY 


way  by  coagulating  the  serum  at  a  temperature  short  of  boil- 
ing-point. Sterilization  is  completed  in  three  days  by  expos- 
ing the  tubes  to  a  temperature  of  about  90°  C.  each  day  for 
five  minutes.    Tubes  so  prepared  are  opaque  and  white. 

Preservation   of  Blood-serum   in   Liquid  State. — Kirchner 
advises  the  use  of  chloroform.    To  a  quantity  of  serum  in  a 

well-stoppered  flask  a  small 
amount  of  chloroform  is 
added — enough  to  form  about 
a  2  mm.  layer  on  the  bot- 
tom. If  the  chloroform  is 
not  allowed  to  evaporate, 
the  serum  remains  sterile 
for  a  long  time.  When 
needed  for  use,  test-tubes  are 
filled  and  placed  in  a  water- 
bath  at  50°  C.  until  all  chlo- 
roform has  been  driven  off 
(determined  by  absence  of 
characteristic  odor) ;  the  se- 
rum is  then  solidified  and 
sterilized  as  in  the  ordinary 
way,  or  may  be  used  in  a 
fluid  state. 

Human  Blood-serum. — 
Blood-serum  derived  from 
placenta,  serimi  from  ascitic 


Incubator. 


fluid  and  ovarian   cysts,   is 

prepared  in  a  similar  manner 

to  the  above. 

Blood  coagulum,  suggested  by  the  author,  is  the  blood 

itself  (not  the  serum  only)  coagulated  in  test-tubes.     It  is 

dark  brown  in  color  and  allows  some  colonies  of  bacteria  to 

be  more  visible.     It  requires  less  time  to  prepare,  and  is  not 

so  likely  to  become  contaminated  as  when  the  serum  is  used. 

Loffler's    Blood-serum    Mixture. — To    3    parts    clear 

serum  add  i  per  cent,  glucose,  beef  infusion,  and  prepare  as 

above;  tube. 


NUTRIENT  CULTURE-MEDIA  75 

Hiss'  Medium  for  Plating 

Agar 15  gin. 

Gelatin 15   " 

Meat  extract 5   " 

Sodium  chlorid 5 

Dextrose 10 

Distilled  water 1000  c.c. 

Digest  agar  in  autoclave,  then  add  the  other  ingredients, 
except  dextrose,  which  is  added  to  the  cleared  and  filtered 
product.  No  neutralization  is  necessary.  Tube  in  regular 
way.  For  tube  cultures  this  medium  is  modified  by  using 
agar  5  gm.  and  gelatin  80  gm.  in  place  of  the  quantities  given 
above.  A  careful  titration  is  made  and  the  reaction  adjusted 
to  1.5  per  cent,  acid  by  adding  HCl.  After  filtration,  dex- 
trose is  added,  then  tubed  and  sterilized. 

Hesse's  Medium  for  Tjrphoid 

Agar Sgm. 

Peptone 10  gm. 

Extract  of  beef 5   " 

Sodium  chlorid 8.5   " 

Water 1000  c.c. 

Digest  agar  in  500  c.c.  water,  add  the  other  ingredients 
dissolved  in  water.  Mix  and  filter.  Adjust  reaction  to  i 
per  cent,  acid,  tube,  and  sterilize  in  autoclave. 

Bile  Salt  Agar  (MacConkey's) 

Sodium  taur-ocholate .- 0.5  part 

Peptone 1.5  parts 

Lactose 3.5      *' 

Agar 1.5      " 

Water q.  s.  loo.o      " 

Agar  and  peptone  dissolved  first.  Lactose  and  bile  salt 
added  before  tubing.     Sterilize  on  three  days  intermittently. 


76  ESSENTIALS   OF   BACTERIOLOGY 

(A)  Conradi-Drigalski  Medium 

Fresh  meat 1500  gm. 

Water 2000  c.c. 

Mix  and  allow  to  stand  twelve  hours.  Strain,  boil  one 
hour,  and  add — 

Peptone 20  gm. 

Nutrose 20    " 

NaCl 10    " 

Boil  one  hour,  filter,  then  add — 
Agar 60  gm. 

Boil  one  hour  in  autoclave  or  until  agar  is  dissolved. 
Render  weakly  alkaline  to  litmus,  filter,  and  boil  one-half 
hour. 

(B) 

Litmus  solution  (Kahlbaum) 300  c.c. 

Lactose 30  gm. 

Boil  fifteen  minutes.  Mix  with  solution  A,  and  make 
slightly  alkaline  with  soda  solution.  Then  add  4  c.c.  10  per 
cent,  soda  carbonate  solution  (hot  sterile)  and  20  c.c.  of 
sterile  i :  1000  crystal  violet  solution  (Hochst  B). 

Lactose-bile  {Jackson). — Sterilized  undiluted  ox-gall, 
98  parts;  or  dry  bile,  10  per  cent,  solution;  peptone,  i  part; 
lactose,  I  part.  M.  Filled  into  fermentation  tubes,  40  c.c. 
each,  sterilized  fractional  method. 

Blood-agar. — Human  or  other  blood  is  obtained  direct 
from  the  body  under  strict  aseptic  conditions,  and  a  few 
drops  smeared  over  the  surface  of  agar  in  tubes  or  plates. 
These  are  then  placed  in  the  incubator  for  a  few  days,  and  the 
contaminated  ones  are  rejected.  This  medium  is  used  for 
influenza  bacilli  and  gonococci. 

Eisner's  Medium  (for  Typhoid)  (Potassitmi  lodid — 
Potato-gelatin). — Five  hundred  grams  of  peeled  and 
washed  potatoes  are  mashed  and  pressed  through  a  fine 
cloth.  The  juice  is  allowed  to  settle,  is  filtered,  and  after 
one  hour's  cooking  has  added  to  it  10  per  cent,  gelatin;  then 


NUTRIENT  CULTURE-MEDIA  77 

2jE^  c.c.  tV  normal  sodium  hydroxid  solution,  and  finally  i  per 
cent,  potassium  iodid. 

Endo  Medium  (Fuchsin-Lactose-Agar). — To  looo  c.c. 
agar  add  lactose,  lo  grams;  fuchsin  (saturated  alcoholic 
solution),  2  c.c;  solution  sodium  sulphite  (lo  per  cent.),  25 
c.c;  sterilize  in  steam,  and  make  acid,  o.i  per  cent. 

Peptone  Water  (Modified  Dunham)  {Mother  Solution): 

Dry  peptone  (Witte) 100  parts 

Sodium  chlorid 100 

Potassium  nitrate i  part 

Sodium  carbonate i 

Distilled  water  (95) q.  s.  ad  1000  parts — ^M. 

When  wanted  for  use,  dilute  ten  times  with  water. 

Dunham's  rosalic  acid  solution  consists  of  the  following: 

Peptone  solution  (Dunham) 100  c.c. 

2  per  cent,  solution  rosalic  acid 0.5  gm. 

Alcohol  (80  per  cent.) 100  c.c. — M. 

To  detect  acids  and  alkalis. 

Dieudonne*s  Mediimi: 

A.  Normal  solution  potassium  hydrate,  defibrinated  ox- 
blood,  equal  parts.  Mix,  sterilize  in  autoclave. 

B.  Nutrient  agar  (neutral).  Mix  3  parts  A  wdth  7  parts 
B,  and  pour  into  Petri  dishes;  allow  to  stand  forty-eight 
hours  at  room  temperature  before  using. 

Milk  Culture-medium. — The  milk  used  should  be  fresh 
and  should  be  placed  on  ice  for  eight  to  ten  hours  to  allow  the 
cream  to  rise;  the  skimmed  milk  is  siphoned  off  into  flasks 
or  tubes  and  sterilized  for  three  successive  days.  Litmus  is 
often  added,  or  sterile  i  per  cent,  azolitmin  solution. 

Fresh  Egg  Cultures  (After  Hueppe). — The  eggs  in  the 
shell  are  carefully  cleaned,  washed  with  sublimate,  and  dried 
with  cotton. 

The  inoculation  occurs  through  a  very  fine  opening  made 
in  the  shell  with  a  hot  platinum  needle;  after  inoculation,  the 
opening  is  covered  with  a  piece  of  sterilized  paper  and  collo- 
dion. 


78  ESSENTIALS   OF   BACTERIOLOGY 

Boiled  Eggs.— Eggs  boiled,  shell  removed  over  small  por- 
tion, and  the  coagulated  albumen  stroked  with  the  material. 

Guinea-pig  Bouillon. — The  flesh  of  guinea-pigs,  as  well 
as  that  of  other  experiment  animals,  is  used  instead  of  beef 
in  the  preparation  of  bouillon,  for  the  growth  of  special  germs. 

The  extracts  of  different  organs  have  been  added  to  the 
various  media  for  experimentation. 

Wertheim's  Medium  for  Gonococcus: 

Nutrient  agar 2  parts 

Human    blood-serum     or    hydrocele 

fluid I  part 

Melt  agar  and  cool  to  45°  C;  then  add  serum.  Tube  on 
slant  or  pour  in  Petri  plate.  Glycerin  or  glucose  can  be  added 
to  enrich. 

Solution  Dried  Blood  Albumin  (King) : 

Blood  albumin  (commercial) 15  parts 

Glucose  bouillon 85     '' 

Dissolve,  tube,  inspissate,  and  sterilize  as  for  blood-serum. 


CHAPTER  XI 

INOCULATION  OF  CULTURE-MEDIA 

Glass  Slide  Cultures. — Formerly  the  gelatin  was  poured 
on  little  glass  slides,  such  as  are  used  for  microscopic  purposes, 
and  after  it  had  become  hard,  inoculated  in  separate  spots  as 
with  potatoes. 

'  Test-tube  Cultures.— The  gelatin,  agar,  or  blood-serum 
having  solidified  in  an  oblique  position  is  smeared  on  the 
surface  with  the  material,  and  the  growth  occurs  along  the 
smear,  or  the  medium  is  punctured  with  a  stab  of  the  plati- 
num rod  containing  the  material,  and  the  growth  follows 
the  line  of  thrust.  The  former  is  called  a  stroke  or  smear 
culture,  the  latter  a  stah  or  thrust  culture. 


INOCULATION   OF   CULTURE-MEDIA  '  79 

Streaked  Surface  Plating. — The  surface  of  the  medium, 
hardened  in  a  Petri  dish,  is  scratched  by  a  needle  containing 
the  inoculating  material,  three  or  more  streaks  being  made 
without  obtaining  fresh  material,  so  that  the  growth  along 
the  streak  or  scratches  will  represent  varying  amounts  of 
the  substance  to  be  tested.  In  removing  the  cotton  plugs 
from  the  sterile  tubes  to  carry  out  the  inoculation  the  plugs 
should  remain  between  the  fingers  in  such  a  way  that  the 
part  which  comes  in  contact  with  the  mouth  of  the  tube  will 
not  touch  anything  (Fig.  23). 

It  is  well  to  pass  the  mouth  of  the  tube  and  the  cotton 
plugs  through  a  flame,  scorching  the  latter  before  reinserting; 


Fig.  23. — Manner  of  holding  plugs. 

Sterilizing  Needle. — Sterilize  needles  by  passing  through 
the  flame  before  and  after  each  inoculation;  also  sterilize  the 
glass  part,  as  it  is  liable  to  become  infected. 

After  the  needle  has  been  withdrawn,  the  plugs  are  rein- 
serted and  the  tubes  labeled  with  the  kind  and  date  of  culture. 

Plate  Cultures. — Several  tubes  of  the  culture-medium 
are  made  liquid  by  heating  in  water-bath,  and  then  inocu- 
lated with  the  material  as  follows.  A  looped  platinum  needle 
is  dipped  into  the  material  and  then  shaken  in  the  tube  of 
liquid  media  (gelatin,  agar,  etc.). 

This  first  tube  is  called  original.  From  this  three  drops 
(taken  with  the  looped  platinum  rod,  Fig.  ii,  p.  45)  are 
placed  in  a  second  tube,  the  rod  being  shaken  somewhat  in  the 


8o 


ESSENTIALS   OF   BACTERIOLOGY 


gelatin  or  agar;  this  is  labeled  first  dilution  (a  colored  pencil 
is  useful  for  such  markings).  From  the  first  dilution  three 
drops  are  taken  into  a  third  tube,  which  becomes  the  second 
dilution. 

The  plugs  of  cotton  must  be  replaced  after  each  inocula- 
tion, and  while  being  held  must  be  carefully  protected  from 
contamination. 

Glass  Plating. — The  larger  the  surface  over  which  the 
nutrient  medium  is  spread,  the  more  isolated  will  the  colonies 
be;  window  glass  cut  in  rectangular  plates  6x4  inches  in 
size  was  formerly  used,  but  now  Petri  dishes  consisting  of 
2  circular  glass  or  porcelain  dishes,  one  fitting  over  the  other 
as  a  cover,  are  universally  employed  (Fig.  24).  They  are 
sterilized,  the  softened  and  inoculated  agar  or  gelatin  is 
poured  from  the  test-tube  into  the  dish  with  as  much  speed 


Fig.  24. — Petri  dish  for  making  plate  cultures. 


as  possible,  and  the  lid  replaced,  avoiding  contamination 
from  the  air  and  surroundings.  They  are  labeled  or  marked 
with  pencil,  and  placed  in  the  incubator  or  kept  at  room 
temperature  for  further  development. 

This  method  is  very  useful  for  transportation,  and  does 
away  with  the  cooling  apparatus  and  moist  chamber  for- 
merly employed;  the  saucers  can  be  viewed  under  micro- 
scope similar  to  the  glass  plates,  and  have  entirely  super- 
seded them. 

Esmarch's  Tubes  or  Rolled  Cultures. — This  method, 
especially  used  in  the  culture  of  anaerobic  germs,  consists  in 
spreading  the  inoculated  gelatin  upon  the  inner  w^alls  of  the 
test-tube  in  which  it  is  contained  and  allowing  it  to  congeal. 


INOCULATION   OF   CULTURE-MEDIA 


The  colonies  then  develop  upon  the  sides  of  the  tube  without 
the  aid  of  other  apparatus.  The  method  is  useful  whenever  a 
very  quick  and  easy  way  is  required.  The  rolling  of  the  tube 
is  done  under  ice-water  or  running  water  from  the  faucet. 
The  tube  is  held  a  little  slanting,  so  as  to  avoid  getting  too 
much  gelatin  around  the  cotton  plug. 

The  tubes  can  be  placed  directly  under  the  microscope  for 
further  examination  of  the  colonies. 

Animals  as  Culture-media. — It  is 
almost  impossible  to  separate  certain 
organisms,  such  as  the  tubercle  bacil- 
lus and  pneumococcus,  from  mixed 
cultures  by  ordinary  plate  methods, 
and  the  plan  of  producing  the  disease 
in  animals  by  inoculation,  and  then 
obtaining  the  organism  in  pure  cul- 
ture, has  to  be  employed. 

Pure  Cultures  by  Boiling. — 
Spored  organisms  may  be  separated 
from  others  by  boiling  the  mixture  for 
a  few  minutes,  when  all  the  non-spored 
forms  will  perish,  and  only  the  spores 
remain  to  germinate  subsequently. 

Fermentation  Tube. — For  show- 
ing the  presence  of  gas  or  fermenta- 
tion the  Smith  tube  (Fig.  25)  or  some 
of  its  modifications  must  be  used. 
The  closed  end  and  part  of  the  bulb 
are  filled  with  the  glucose  or  dextrose 

bouillon  and  sterilized  at  low  temperatures  for  three  succes- 
sive days,  then  inoculated  and  placed  in  the  incubator.  Gas 
forms  gradually,  displacing  the  fluid  in  the  closed  end. 


-Smith's  fer- 
mentation tube. 


82 


ESSENTIALS    OF   BACTERIOLOGY 


CHAPTER  XII 

CULTIVATION  OF  ANAEROBIC  BACTERIA 

Special  methods  are  necessary  for  the  culture  of  the  ana- 
erobic variety  of  bacteria  in  order  to  procure  a  space  devoid 
of  oxygen. 

Liborius's  High  Cultures. — The  tube  is  filled  about 
three-quarters  full  with  gelatin,  which  is  then  steamed  in  a 
water-bath  and  allowed  to  cool  to  40°  C,  when  it  is  inoculated 


^ 


Fig.  26. — Liborius's  method. 


Fig.  27. — Hesse's  method  of  making 
anaerobic  cultures  (McFarland). 


by  means  of  a  long  platinum  rod  with  small  loop,  the  move- 
ment being  a  rotary  vertical  one,  and  the  rod  going  to  the 
bottom  of  the  tube. 

The  gelatin  is  next  quickly  solidified  under  ice;  very  little 
air  is  present.    The  anaerobic  germs  will  grow  from  the 


CULTIVATION  OF  ANAEROBIC   BACTERIA 


83 


bottom  upward,  and  any  aerobins  present  will  develop  first 
on  top,  this  method  being  one  of  isolation. 

From  the  anaerobic  germ  grown  in  the  lower  part  a  stab 
culture  is  made  into  another  tube  containing  three-quarters 
gelatin,  the  material  being  obtained  by  breaking  test-tube 
with  the  culture.     (See  Fig.  26.) 

Hesse's  Method. — A  stab-culture  having  been  made  with 


Fig.  28. — Frankel's  method  of  Fig,  29. — Buchner's  method  of 
making  anaerobic  cultures  (McFar-  making  anaerobic  cultures  (Mc- 
land).  Farland). 


anaerobic  germs,  gelatin  in  a  semisolid  condition  is  poured 
into  the  tube  until  it  is  full,  thus  displacing  the  air  (Fig.  27). 
Esmarch's  Method. — Having  inoculated  a  tube,  the  gela- 
tin is  rolled  out  on  the  walls  of  the  tube,  a  "roll  culture," 
and  the  rest  of  the  interior  is  filled  with  gelatin,  the  tube 
bei^g  held  in  ice-water.  The  colonies  develop  upon  the  sides 
of  the  tube  and  can  be  examined  microscopically. 


84 


ESSENTIALS    OF   BACTERIOLOGY 


Gases  like  Hydrogen  to  Replace  the  Oxygen. — Several 
arrangements  for  passing  a  stream  of  hydrogen  through  the 
culture : 

Frankel  puts  in  the  test-tube  a  rubber  cork  containing  two 
glass  tubes,  one  reaching  to  the  bot- 
tom and  connected  with  a  hydrogen 
apparatus,  the  other  very  short, 
both  bent  at  right  angles.  When 
the  hydrogen  has  passed  through 
from  ten  to  thirty  minutes,  the 
short  tube  is  annealed  and  then  the 
one  in  connection  with  the  hy- 
drogen bottle,  and  the  gelatin 
rolled  out  upon  the  walls  of  the 
tube  (Fig.  28). 

Use  of  Aerobic  Bacteria  to 
Remove  the  Oxygen. — Roux  in- 
oculates an  agar  tube  through  a 
needle-thrust,  after  which  semi- 
solid gelatin  is  poured  in  on  top. 
When  the  gelatin  has  solidified,  the 
surface  is  inoculated  wdth  a  small 
quantity  of  Bacillus  subtilis  or 
some  other  aerobic  germ.  The 
subtilis  does  not  allow  the  oxygen 
to  pass  by,  appropriating  it  to 
itself. 

Buchner's  Method. — The  test- 
tube    containing    the    culture     is 
placed  within  a  larger  tube,   the 
lower  part   of   w^hich  contains  an 
alkaline  solution  of  pyrogallic  acid. 
The  tube  is  then  closed  with  a  rub- 
ber stopper  (Fig.  29). 
Botkin's  Method. — Petri  dishes,  uncovered,  are  placed 
on  a  rack  under  a  large  bell-jar,  into  which  hydrogen  gas  is 
conducted.     Alkaline  pyrogallic  acid  is  placed  in  the  upper 


Fig.  30. — Wright's 
method  for  the  cultivation 
of  anaerobes. 


CULTIVATION   OF  ANAEROBIC  BACTERIA 


8s 


and  lower  dishes  to  absorb  what  oxygen  remains.  The  Novy 
jar  (Fig.  31)  is  used  instead  of  a  bell- jar,  and  sealed  after 
the  oxygen  is  displaced  by  hydrogen  gas. 

Wright's  Method.— Applicable  to  both  fluid  and  solid 
media.  After  the  test-tube  is  inoculated  the  plug,  which 
must  be  of  absorbent  cotton,  is  cut  off  flush  with  the  ex- 
tremity of  the  tube  and  pushed  inward  for  a  distance  of  i  cm. 
It  is  then  impregnated  with  i  c.c.  of  a  watery  solution  of 
pyrogallic  acid  and  i  c.c.  of  5  per  cent,  sodium  hydroxid 


Fig.  31. — Novy's  jars  for  anaerobic  cultures. 

solution.     A  tightly  fitting  rubber  stopper  is  inserted,  and 
the  tube  is  then  ready  for  incubation  (Fig.  30). 

Park's  Method. — An  Erlenmeyer  flask  containing  the 
medium  to  be  used  is  boiled  in  a  water-bath  from  ten  to 
fifteen  minutes  to  drive  off  dissolved  oxygen,  quickly  cooled, 
and  inoculated.  Hot  melted  paraffin  is  then  poured  into  the 
flask,  which  forms  a  layer  over  the  medium,  and  on  congeal- 
ing, provides  an  air-tight  seal  which  does  not  adhere  to  the 
glass  so  closely  as  to  prevent  the  escape  of  any  gases  formed 
by  the  bacterial  growth. 


Requirements  for  a  Small  Laboratory 
Incubator,  with  thermostat  and   thermometers. 
Hot-air  oven. 


86  ESSENTIALS   OF   BACTERIOLOGY 

Arnold  steam  sterilizer. 

Autoclave. 

Bunsen  burners. 

Erlenmeyer  or  liter  glass  flasks,  yi  dozen. 

Test-tubes,  loo. 

One  I  GOO  c.c.  measuring  glass. 

One  I  GO  c.c.  measuring  glass. 

One  5  c.c.  pipet. 

One  I  c.c.  pipet. 

One  accurate  buret. 

One-half  dozen  20  c.c.  porcelain  capsules. 

Glass  stirring  rods. 

Normal  soda  solution. 

Hydrochloric  acid. 

Lactose,  dextrose,  glucose,  and  phenolphthalein. 

A  selection  of  dry  stains,  especially  fuchsin,  methylene- 
blue,  and  eosin. 

Gram's  solution. 

Phenol. 

Alcohol,  methyl  alcohol. 

Cover-glasses,  slides. 

Canada  balsam,  cedar-oil,  xylol. 

A  small  microtome  and  embedding  material. 

Cotton- wool  for  plugs. 

Twenty-five  or  more  Petri  dishes. 

Four  platinum  needles  in  glass  handles. 

One-half  dozen  fermentation  tubes. 

One-half  dozen  tubes  for  potato  culture. 

One  Novy  jar. 

One  animal  holder. 

Three  wire  boxes  for  holding  tubes. 

Test-tube  rack. 

The  materials  must  include  what  is  needed  for  making 
culture-media:  agar,  gelatin,  peptone,  beef-extract,  chemic- 
ally pure  salt. 

And  to  this  there  will  be  added  from  time  to  time  such 
other  apparatus  and  material  as  occasion  demands. 


THE  GROWTH  AND  APPEARANCES  OF  COLONIES     87 

CHAPTER  XIII 

THE  GROWTH  AND  APPEARANCES  OF  COLONIES 

Macroscopic. — Depending  greatly  upon  the  temperature, 
which  should  be  about  65°  F.  (20°  C.)  for  gelatin,  and  40°  C. 
for  agar,  the  colonies  ordinarily  develop  so  as  to  be  visible  to 
the  naked  eye  in  two  to  four  days.  Some  require  ten  to  four- 
teen days,  and  others  grow  rapidly,  covering  the  third  dilu- 
tion in  thirty-six  hours.  The  plate  should  be  looked  at  each 
day. 

The  colonies  present  various  appearances  from  that  of  a 


Fig.  32. — Staphylococcus  pyogenes  aureus:  colony  two  days  old,  seen 
upon  an  agar-agar  plate  (X40)  (Heim). 


small  dot,  like  a  fly-speck,  to  that  resembling  a  small  leaf. 
Some  are  elevated,  some  depressed,  and  some,  like  cholera, 
cup-shaped — umbilicated. 

Then  they  are  variously  pigmented.  Some  liquefy  gelatin 
speedily,  others  not  at  all.  The  appearances  of  a  few  are 
so  characteristic  as  to  be  recognized  at  a  glance.  Some 
produce  gas-bubbles. 


88 


ESSENTIALS   OF   BACTERIOLOGY 


Microscopic. — Use  a  low-power  lens,  with  the  Abbe 
nearly  shut  out — that  is  the  narrowest  blender.  The  stage  of 
the  microscope  should  be  of  such  size  as  to  carry  a  Petri 
saucer  easily  upon  it. 

The  second  dilution  or  third  plate  is  usually  made  use  of — 
that  one  containing  the  colonies  sufficiently  isolated. 

These  isolated  ones  should  be  sought  for,  and  their  appear- 
ance well  noticed. 

There  may  be  two  or  three  forms  from  the  same  germ,  the 
difference  due  to  the  greater  or  less  amount  of  oxygen  that 


Fig.  33- — Microscopic  appear- 
ances of  colonies. 


Fig,  34. — Klatsch  preparations. 


they  have  received,  or  the  greater  or  less  amount  of  space 
that  they  have  had  to  develop  in. 

The  microscopic  picture  varies  greatly;  now  it  is  like  the 
gnarled  roots  of  a  tree,  and  now  like  bits  of  frosted  glass; 
some  bacteria  have  quite  characteristic  colonies  (Fig.  32). 

Impression  or  "Klatsch*'  Preparations.— In  order 
more  thoroughly  to  study  a  certain  colony  and  to  make  a 
permanent  specimen  of  the  same,  we  press  a  clean  cover-glass 
upon  the  particular  colony,  and  it  adheres  to  the  glass.  It 
can  then  be  stained  or  examined.  The  Germans  give  the 
name  of  "Klatsch"  to  such  preparations. 

Fishing. — To  obtain  and  examine  the  individual  members 


THE  GROWTH  AND  APPEARANCES  OF  COLONIES 


89 


of  a  particular  colony  the  process  of  fishing,  as  it  is  called,  is 
resorted  to. 

The  colony  having  been  placed  under  the  field  of  the  micro- 


Fig.  35. — Types  of  growth  in  stab-cultures:  A,  Non-liquefying:  i, 
Filiform  (Bacillus  coli);  2,  beaded  (Streptococcus  pyogenes);  3,  echinate 
(Bacterium  acidi  lactici);  4,  villous  (Bacterium  murisepticum) ;  5,  arbor- 
escent (Bacillus  mycoides).  B,  Liquefying:  6,  Crateriform  (Bacillus 
vulgare,  twenty-four  hours);  7,  napiform  (Bacillus  subtilis,  forty-eight 
hours);  8,  infundibuliform  (Bacillus  prodigiosus) ;  9,  saccate  (Micro- 
sporon  Finkleri);  10,  stratiform  (Psorospermum  fluorescens)  (Frost). 


scope,  a  long  platinum  needle,  the  point  slightly  bent,  is 
passed  between  the  lens  and  the  plate  so  as  to  be  visible 
through  the  microscope,  then  turned  downward  until  the 
colony  is  seen  to  be  disturbed,  and  the  needle  is  dipped  into 


90 


ESSENTIALS    OF   BACTERIOLOGY 


the  colony.  This  procedure  must  be  carefully  done,  lest  a 
different  colony  be  disturbed  than  the  one  looked  at,  and  an 
unknown  or  unwanted  germ  obtained. 

After  the  needle  has  entered  the  particular  colony,  it  is 
withdrawn,  and  the  material  thus  obtained  is  further  exam- 
ined by  staining  and  animal  experimentation.     The  bacteria 


L 


/  -/-    \ 


Fig.  36. — ^Types  of  stroke  cultures:  i,  Filiform  (Bacillus  coli);  2, 
echinulate  (Bacterium  acidi  lactici);  3,  beaded  (Streptococcus  pyogenes); 
4,  effuse  (Bacillus  vulgaris);  5,  arborescent  (Bacillus  mycoides)  (Frost). 


are  further  cultivated  by  inoculating  fresh  gelatin  or  agar, 
making  stab-  and  stroke  cultures. 

It  is  necessary  to  transfer  the  bacteria  to  fresh  media  about 
every  six  weeks,  as  the  products  of  growth  and  decay  given 
off  by  the  organisms  destroy  them.  Stroke  and  stab  test- 
tube  cultures  are  more  characteristic  than  plate  cultures,  as 
the  types  in  Figs.  35  and  36  show. 


ANIMAL  INOCULATION  91 


CHAPTER  XIV 

ANIMAL  INOCULATION 

Used:  (i)  For  obtaining  pure  cultures;  (2)  to  determine 
virulence;  (3)  to  regain  virulence  of  an  organism  that  has 
become  exhausted  in  artificial  media;  (4)  to  furnish  a  suit- 
able culture-medium  for  bacteria  that  have  so  far  failed  to 
grow  on  other  media. 

The  smaller  rodents  and  birds  are  the  ones  usually  employed 
for  inoculation,  as  rabbits,  guinea-pigs,  rats,  mice,  pigeons, 
and  chickens.  These  are  preferred,  because  easily  affected 
by  the  various  bacteria,  readily  obtained,  and  not  expensive. 
Monkeys  have  been  used  in  recent  years  in  connection  with 
syphihs  and  meningitis. 

The  white  mouse  is  very  prolific  and  easily  kept,  and  is 
therefore  a  favorite  animal  for  experiment.  It  lives  well 
upon  a  little  moistened  bread.  A  small  box,  perforated  with 
holes,  is  filled  partly  with  sawdust,  and  in  this  ten  to  twelve 
mice  can  be  kept.  When  the  female  becomes  pregnant,  she 
should  be  removed  to  a  glass  jar  until  the  young  have  opened 
their  eyes,  because  the  males,  which  have  not  been  raised 
together,  are  apt  to  attack  each  other. 

Guinea-pigs. — When  guinea-pigs  have  plenty  of  light  and 
air,  they  multiply  rapidly.  Therefore  it  is  best  to  have  them 
in  some  large  stall  or  inclosure.  They  can  be  fed  upon  all 
sorts  of  vegetables  and  grasses,  and  require  but  little  atten- 
tion. 

Methods  of  Inoculation. — /.  Inhalation. — Imitating  the 
natural  infection,  either  by  loading  an  atmosphere  with  the 
germs  in  question  or  by  administering  them  with  a  spray. 

//.  Through  skin  or  mucous  membrane. 

III.  With  the  food. 

Method  of  Cutaneous  Inoculation. — The  ear  of  a  mouse 
is  best  suited  for  this  procedure.  A  small  abrasion  is  made  with 
the  point  of  a  lancet  or  needle,  which  has  been  dipped  in  the 
virus  or  material  to  be  inoculated.    The  animal  is  then  sepa- 


92  ESSENTIALS   OF   BACTERIOLOGY 

rated  from  the  rest  and  placed  in  a  glass  jar,  which  is  partly 
filled  with  sawdust  and  covered  with  a  piece  of  wire  gauze. 

Subcutaneous. — The  root  of  the  tail  of  a  mouse  is  used  for 
this  purpose.  The  hair  around  the  root  of  the  tail  is  clipped 
off,  and  with  a  pair  of  scissors  a  very  small  pocket  is  made  in 
the  subcutaneous  connective  tissue,  not  wounding  the  animal 
any  more  than  is  absolutely  necessary,  avoiding  much  blood. 
The  inoculating  material  is  placed  upon  a  platinum  needle 
and  introduced  into  the  pocket;  solid  bodies,  with  a  forceps. 

To  hold  the  mouse  still  while  the  operation  is  going  on  a 
little  cone  made  of  m^etal  is  used.  The  mouse  just  fits  in 
here.  There  is  a  slit  along  the  top  in  which  the  tail  can  be 
fastened,  and  thus  the  animal  is  secure  and  immobile. 

Variously  designed  animal-holders  are  on  the  market  and 
used  in  laboratories. 

Intravenous  Injections. — Rabbits  are  very  easily  in- 
jected through  the  veins.     Mice  are  too  small. 

The  ear  of  the  rabbit  is  usually  taken.  It  is  first  washed 
with  I  :  2000  bichlorid,  w^hich  not  only  disinfects,  but  also 
makes  the  vessels  appear  more  distinct.  The  base  of  the  ear 
is  compressed  to  sw^ell  the  veins.  Then  a  hypodermic 
syringe,  which  can  be  easily  sterilized,  is  filled  with  the  de- 
sired amount  of  virus,  which  is  slowly  injected  into  any  one 
of  the  more  prominent  veins  present  (Fig.  37). 

Intraperitoneal  Injection. — This  is  used  with  guinea- 
pigs  chiefly.  The  abdominal  wall  is  pinched  up  through  its 
entire  thickness,  and  the  needle  of  the  syringe  thrust  directly 
through,  so  that  it  appears  on  the  other  side,  then  the  fold 
let  go,  the  needle  withdrawn  just  far  enough  so  as  to  be  within 
the  cavity. 

Inoculation  in  the  Eye. — The  anterior  chamber  and  the 
cornea  are  the  two  places  used.  The  rabbit  is  fixed  upon  a 
board,  the  eyefids  held  apart  and  head  held  still  by  an  assist- 
ant. A  few  drops  of  cocain  having  first  been  introduced  in 
the  eye,  a  small  cut  is  made  in  the  cornea.  The  material  is 
passed  through  the  opening  with  a  small  forceps,  and  with  a 
few  strokes  of  a  spoon  it  is  pushed  in  the  anterior  chamber. 


ANIMAL   INOCULATION  93 

For  the  cornea  a  few  scratches  made  in  the  corneal  tissue 
will  suffice ;   the  material  is  then  gently  rubbed  in. 

Inoculation  of  the  Cerebral  Membranes. — The  skin 
and  aponeurosis  cut  through  where  the  skull  is  the  thinnest. 
Then  the  bone  carefully  trephined,  and  the  dura  exposed.  In 
rabies  inoculation,  the  syringe  containing  the  hydrophobic 
virus  pierces  the  dura  and  arachnoid,  and  the  virus  is  dis- 
charged beneath  the  latter. 


Fig.  37. — Method  of  making  an  intravenous  injection  into  a  rabbit. 
Observe  that  the  needle  enters  the  posterior  vein  from  the  hairy  sur- 
face. 


Intratracheal. — The  bacteria  can  be  introduced  directly 
into  the  trachea,  thus  coming  in  contact  with  the  lungs. 

Intraduodenal. — Cholera  germs  are  injected  into  the  in- 
testines after  they  have  been  exposed  by  carefully  opening 
the  abdomen.  This  is  done  in  order  to  avoid  the  action  of 
the  gastric  juice. 

Celloidin  sacs  of  small  size  are  sometimes  used  to  intro- 


94  ESSENTIALS   OP   BACTERIOLOGY 

duce  living  cultures  of  bacteria  into  the  bodies  of  animals 
without  their  coming  into  direct  contact  with  the  tissues. 

Obtaining  Material  from  Infected  Animals. — The  ani- 
mal should  be  skinned,  or  the  hairs  plucked  out,  before  it  is 
washed — at  least  the  portion  where  the  incision  is  to  be  made. 
Then  the  entire  body  is  washed  in  sublimate.  Two  sets  of 
instruments  are  required — one  for  coarser  and  one  for  finer 
work:  the  one  sterilized  in  the  flame;  the  other,  to  prevent 
being  damaged,  heated  in  a  hot-air  oven. 

The  animal,  the  mouse,  for  example,  is  stretched  upon  a 
board,  a  nail  or  pin  through  each  leg,  and  the  head  fixed  with 
a  pin  through  the  nose.  The  skin  is  dissected  away  from  the 
belly  without  exposing  the  intestines.  Then  the  ribs,  being 
laid  bare,  the  sternum  is  lifted  up,  and  the  pericardium  ex- 
posed. A  platinum  needle  dipped  into  the  heart  after  the 
pericardium  has  been  slit  will  give  sufficient  material  for 
starting  a  culture.  If  the  other  organs  are  to  be  examined, 
further  dissection  is  made.  If  the  intestines  are  first  to  be 
looked  at,  they  should  be  laid  bare  first. 

In  this  manner  material  is  obtained  and  the  results  of 
inoculation  noted. 

Frequent  sterilization  of  the  instruments  is  desirable. 

Koch's  Rules  in  Regard  to  Bacterial  Cause  of  Disease. 
— Before  a  microbe  can  be  said  to  be  the  cause  of  a  disease,  it 
must — 

First,  be  found  in  the  tissue  or  secretions  of  the  animal  suf- 
fering from,  or  dead  with,  the  disease. 

Second,  it  must  be  cultivated  outside  of  the  body  on  ar- 
tificial media. 

Third,  a  culture  so  obtained  must  produce  the  disease  in 
question  when  it  is  introduced  into  the  body  of  a  healthy 
animal. 

Fourth,  the  same  germ  must  then  again  be  found  in  the 
animal  so  inoculated. 


BACTERINS    (VACCINES)  9$ 

CHAPTER  XV 
BACTERINS  (VACCINES) 

Bacterins  are  sterilized  suspensions  of  bacteria  in  normal 
saline  solution.  The  term  vaccines  or  bacterial  vaccines  is 
frequently  but  erroneously  used  in  place  of  bacterins,  as  the 
word  vaccine  relates  to  a  cow  or  calf.  Bacterins  are  used  in 
the  treatment  of  locaHzed  infections,  and  especially  those  of 
a  chronic  nature,  and  have  been  employed  extensively  to  es- 
tablish immunity  against  infection.  The  best  example  of 
this  is  the  immunization  of  armies  and  inmates  of  institu- 
tions against  typhoid  fever. 

Preparation. — The  organism  is  grown  on  the  surface  of 
the  most  appropriate  medium,  usually  agar-agar,  until  an 
abundant  growth  is  present.  This  ordinarily  requires 
twenty-four  hours.  The  growth  is  then  washed  from  the 
medium  with  sterile  normal  saline  solution,  and  collected  in 
a  small  sterilized  flask  or  bottle  containing  glass  beads  and 
shaken  to  break  up  clumps.  A  sterilized  glass  bulb,  drawn 
to  a  point  (a  test-tube  drawn  out  answers  as  well),  is  filled 
with  the  resulting  emulsion,  the  end  sealed  in  a  flame,  and  the 
bulb  immersed  in  a  water-bath  at  60°  C.  for  one  hour.  The 
neck  of  the  bulb  is  then  broken,  and  a  few  drops  of  the  emul- 
sion sown  on  culture-media  to  determine  the  presence  or  ab- 
sence of  living  organisms. 

Standardization. — The  number  of  bacteria  in  a  cubic  cen- 
timeter of  the  mixture  is  determined  as  follows:  a  portion 
of  the  emulsion  is  reserved  unheated,  and  at  once  mixed 
with  an  equal  volume  of  blood  by  aspirating  into  a  capillary 
tube  any  quantity,  usually  a  column  2.5  cm.  long,  of  the 
emulsion,  followed  by  an  equal  volume  of  blood.  The  blood 
and  emulsion  are  then  mixed  on  a  glass  slide  and  thin  smears 
are  made.  After  air  drying,  the  films  are  fixed  with  satur- 
ated solution  of  bichlorid  of  mercury  and  stained  with  car- 
bolthionin. 


96  ESSENTIALS    OF   BACTERIOLOGY 

Counting. — Two  crossed  hairs  are  placed  on  the  dia- 
phragm in  the  eye-piece  of  the  microscope  and  the  slide 
examined  under  the  oil-immersion  lens.  The  number  of  cor- 
puscles and  bacteria  in  a  number  of  fields  are  counted  until 
at  least  200  red  corpuscles  have  been  enumerated.  As  the 
number  of  corpuscles  per  cubic  centimeter  is  5,cxdo,ooo,ooo 
by  simple  proportion,  the  number  of  bacteria  per  cubic  centi- 
meter can  be  determined.  For  example,  200  red  corpuscles 
and  150  bacteria  are  counted  in  the  same  fields.     Then — 

200  corpuscles  :  150  bacteria  :  :  5,000,000,000  :  x  x  =  3, 750,000,0000 
(Number  of  corpuscles  is  to  number  of  bacteria  as  the  total  number 
of  corpuscles  in  a  cubic  centimeter  is  to  the  quantity  to  be  determined.) 

Any  number  of  bacteria  per  cubic  centimeter  can  then  be 
obtained  by  simple  dilution  with  sterile  normal  saline  solu- 
tion. When  the  final  dilution  is  made,  0.2  per  cent,  of  tri- 
kresol  is  added  as  a  preservative. 


PART  II 
SPECIAL  BACTERIOLOGY 


CHAPTER  XVI 
SOME  COMMON  BACTERIA  SLIGHTLY  PATHOGENIC 

Bacterium  Prodigiosum  (Ehrenberg). — This  bacillus, 
formerly  called  micrococcus,  is  very  common,  and  was  one  of 
the  first  noticed,  because  of  the  brilliant  red  pigment  it 
forms  on  cooked  vegetables  and  starchy  substances.  "The 
bleeding  host"  miracles  are  said  to  have  been  due  to  it. 

Morphology. — Short  rods,  often  in  filaments,  resembling 
cocci,  ends  slightly  pointed,  i  jli  in  size ;  spores  absent. 

Facultative  anaerobic,  that  is,  it  can  grow  without  air;  but 
the  pigment  requires  oxygen  for  its  development. 

Flagella  and  motion  present  in  young  bouillon  cultures. 
Absent  in  older  and  those  grown  on  potato. 

Stain  easily  with  ordinary  watery  stains,  but  not  with 
Gram. 

Cultural  Features. — Agar  stroke:  Growth  limited  to  stroke; 
filiform,  varying  from  a  light  pink  to  dark  purple  in  color, 
due  to  pigment  (prodigiosin)  formed  by  the  growing  colonies. 
Odor  of  trimethylamin  present.  Media  colored  brow  n  under- 
neath growth. 

On  potato,  growth  of  pigment  appears  best.  At  first  rose 
red,  then  in  a  few  days  dark  purple,  with  a  glistening,  green- 
gold  luster,  resembling  the  dry  fuchsin  dye.  Odor  more 
pronounced. 

Gelatin  Stab. — In  six  hours  liquefaction  begins  on  surface, 
and  spreading  downward;  funnel  shape;  the  liquid  portion 
7  97 


98  ESSENTIALS    OF   BACTERIOLOGY 

containing  small  flakes  of  red  pigment  which  settle  at  the 
bottom.     Milk  coagulated  in  twenty-four  hours.   - 

Agar  Colonies. — Small  red  points  in  thirty-six  hours,  irregu- 
lar in  outline.     Granular  in  structure. 

Gelatin  Colonies. — On  the  surface,  round,  granular,  smooth 
edges  which  soon  liquefy  a,nd  have  depression  in  center.  The 
edges  then  become  irregular. 

Biologic  Features. — The  characteristic  red  pigment  is  in- 
soluble in  water,  slightly  soluble  in  alcohol  and  ether;  alka- 
lies turn  it  orange,  acids,  violet  red.  Light  fades  it.  Gases 
of  methylamin  and  ammonia  are  produced.     Gas  and  acid 

produced     in     sugar     solutions. 
Indol  feeble. 

Temperature,  22°-25°  C.;  higher 
temperatures  interfere  with  pig- 
ment. 

Pathogenic  for  small  animals. 
When  injected  intraperitoneally, 
1-2  c.c.  has  proved  fatal;  causes 
intoxication.  Proteids  of  the  cul- 
tures poisonous. 
Fig.  38.— Colony  of  Bacillus  Cancer  Remedy.  —  Used  in 
mesentericus  vulgatus.  Coley's   treatment    mixed    with 

cultures  of  streptococci. 
Bacillus    Mesentericus   Vulgatus    {Bacillus    Vulgatus; 
Potato  Bacillus  of  Flilgge)  (Fig.  38). 

Origin. — Surface  of  the  soil,  on  potatoes,  and  in  milk. 
Form. — Small  thick  rods  with  rounded  ends,  often  in  pairs. 
Very  motile;  produces  abundant  spores. 
Cultures. — Rapid  growth;  stain  with  Gram. 
Agar  Colonies. — Round,  with  transparent  center  at  first, 
then  becoming  opaque.     The  border  is  ciliated;   little  pro- 
jections evenly  arranged. 

Potato. — A  white  covering  at  first,  which  then  changes  to 
a  rough  brown  skin;  the  skin  can  be  detached  in  long 
threads. 

Temperature. — Spores  at  ordinary  temperature. 


SOME   COMMON   BACTERIA   SLIGHTLY  PATHOGENIC 


99 


Spores. — Are  very  resistant;  are  colored  in  the  manner 
described  in  first  part  of  the  book  for  spores  in  general. 

Bacillus  Megaterium  (de  Bary)  (Fig.  39).— Origw.— 
Found  on  rotten  cabbage  and  garden-soil. 

Form. — Large  rods,  four  times  as  long  as  they  are  broad, 
2.5  ju.  Thick,  rounded  ends.  Chains  with  ten  or  more  mem- 
bers often  formed;  granular  cell  contents. 

Abundant  spore  formation;  very  slow  movement. 

Growth. — Strongly  aerobic;  grows  quickly  and  best  at  a 
temperature  of  20°  C. 


Fig.  39. — Bacillus  megaterium,  with  spores. 

Plate  Colonies. — Small,  round,  yellow  points  in  the  depth  of 
the  gelatin.  Under  microscope,  irregular  masses  like  B, 
subtilis.  '     •  \'  ' 

Stab-culture. — Funnel-shaped  from  above  downwa]*4^ 

Potato. — Thick  growth  with  abundance  of  epor^  4ike -.©, 
subtilis.  '        -' '      ,  >'*^  ' 

Bacillus  Ramosus . — Synonyms. ^Bacillus  ^  ^[tnyeotdes 
(Fliigge);  Wurzel  or  root  bacillus.         ^     ^'        '.-  '■ 

Origin. — In  the  upper  layers  of  gardei^  or  fafni  grounds  and 
in  water.  "^^.'^  \'' 

Form. — Short  rods,  with  rourrdM  ends,  sbout  three  times 
as  long  as  they  are  thick;  often  in-  ibn^ 'threads  and  chains. 


100 


ESSENTIALS    OF   BACTERIOLOGY 


ImmoHle. 
Stain. — Gram. 

Agar  Stroke. — Gray  soft  mass,  gnarled  and  twisted;  feath- 
ery extensions  spreading  over  entire  surface. 

Gelatin  Stab. — Arborescent  and  plumose-parallel  projections 
on  either  side  of  the  stab;  a  thick  skin  on  surface  with  slow 
liquefaction  (Fig.  40). 

Colonies. — Twisted  threads,  like  a  bundle  of  hair;   opaque 
center;  the  threads  or  branches  divide  endlessly,  forming  coils. 
Growth. — -At    ordinary    temperature, 
with  plentiful  supply  of  air. 

Staining. — Spores  stain  readily  with 
the  ordinary  spore  stain. 

Bacterium  Zopfii  (Kurth)  (1883).— 
Origin. — Intestines  of  a  fowl. 

Form. — Short  thick  rods  forming  long 
threads  coiled  up,  which  f.nally  break  up 
into  spores,  which  were  cnce  thought  to 
be  micrococci. 

Properties.' — Very   motile;     does  not 

dissolve   or    liquefy  gelatin.      Produces 

putrefaction  in  albuminous  m.edia,  with 

gas  formation. 

Growth. — In  thirty  hours  abundant  growth ;  aerobic;  grows 

best  at  20°  C. 

Agar  Plates. — Small  white  points  which  fcrm  the  center  of  a 
very,  fine  netting.     With  high  power  this  netting  is  found 
composed  of  bacilli  in  coils,  like  braids  of  hair. 
T  YjXfiQ^epX  impress  or  "Klatsch"  preparations  are  obtained 
■froEi  th'es/i  cejonies. 
''  Suiiping s-r-Ordinsiry  dyes  and  Gram. 

Bapillu^  SuHiH^  (Hay  Bacillus)  (Ehrenberg). — Origin. 
— Hay  kifusionsV 'found  also  in  air,  water,  soil,  feces,  and 
putrefying  Jiciui4s.  jV'S'ry  common,  often  contaminates  cul- 
tures. '''-/  ' '      '\''y/ ' 

Form. — Short, ^thiek  rods;' jdiree  times  as  long  as  broad; 
slight  roundness  of  ends^,,  seldom  found  singly;    usually  in 


Fig.     40.  —  Bacillus 
mycoides  (Frost). 


SOME   COMMON   BACTERIA   SLIGHTLY   PATHOGENIC        lOI 

long  threads.  Flagella  are  found  on  the  ends.  Spores  of 
oval  shape,  strongly  shining,  very  resistant. 

Very  motile;   Gram  stain.  , 

Growth. — Rapid;   strongly  aerobic. 

Plate. — Round,  gray  colonies  with  depressed  white  center. 
Under  microscope  the  center  yellow;  the  periphery  like  a 
wreath,  with  tiny  little  rays  projecting;  very  characteristic. 

Agar  Stroke. — Soft,  round,  smooth  edges;  gray. 

Gelatin  Stab. — Gray  on  surface,  sinks  in  thirty-six  hours, 
shallow  crater,  in  which  small  white  particles  are  floating; 
as  gelatin  softens  a  skin  forms  on  surface. 

Potato. — Thick,  dirty-white  growth,  spreading  over  sur- 
face; dull,  raised  edges,  wavy. 

Properties  like  B.  vulgatus. 

Pathogenic. — Has  been  found  present  in  eyeball  suppura- 
tions, especially  panophthalmitis.  Injected  in  guinea-pigs  it 
causes  toxemia  and  death.  Has  been  found  in  acute  conjunc- 
tivitis, and  may  at  times  produce  it. 

Staining. — Rods,  ordinary  stain;  spores,  spore  stain. 

It  is  easily  obtained  by  covering  finely  cut  hay  with  dis- 
tilled water,  and  boiling  a  quarter  of  an  hour.  Set  aside 
forty-eight  hours.  A  thick  scum  will  show  itself  on  the  sur- 
face, composed  of  the  subtilis  bacilli,  whose  spores  alone  have 
survived  the  heat. 

Was  formerly  considered  a  non- virulent  form  of  B.  anthrax. 

Boas-Oppler  Bacillus. — Also  known  as  the  Bacillus 
geniculatus.  Owing  to  the  faculty  possessed  by  this  organ- 
ism of  growing  in  the  presence  of  amounts  of  lactic  acid  suf- 
ficient to  check  the  development  of  all  other  lactic-acid  form- 
ers, it  usually  predominates  in  stomach-contents  containing 
large  amounts  of  this  substance.  The  parent  type  is  com- 
posed of  short  rods,  but  in  the  presence  of  considerable 
amounts  of  lactic  acid  these  change  to  a  longer  form,  which 
occurs  singly  or  in  long  chains.  It  is  stained  brown  by  Gram's 
iodin  solution.  The  bacillus  affords  confirmatory  evidence 
of  the  presence  of  a  new-growth,  like  cancer  of  the  stomach, 
though  it  may  occur  in  benign  conditions. 


I02  ESSENTIALS   OF   BACTERIOLOGY 

Bacillus  Violaceus  (Schrater). — Origin. — Water. 

Synonym. — B.  ianthinum  (Zopf). 

Form. — ^A  slender  rod  with  rounded  ends,  three  times  as 
long  as  it  is  broad,  often  in  threads. 

Spores. 

Motile,  flagelia. 

Stain. — ^With  Gram  and  ordinary  dyes. 

Cultures. — Agar  stroke,  moist,  ghstening,  raised,  at  first 
yellow,  then  violet,  inky  colored. 

On  Potato. — Violet  black,  moist,  abundant  growth. 

Gelatin  Stab. — Rapidly  Uquefying  funnel-shaped  masses  of 
pigment  along  the  stab. 

Colonies. — ^Hairy  outer  zone  with  liquid  center,  and  small 
masses  of  opaque  blue  pigment  floating  about. 

Biology. — Acid  formed  in  sugar  bouillon.  No  gas.  A 
moderate  amount  of  H2S  and  indol.  Pigment  formed  is  in- 
soluble in  water,  slightly  soluble  in  alcohol. 

Facultative  anaerobe. 

Temperature. — 22°-25°  C. 

Microorganisms  Found  in  Urine. — When  freshly  passed, 
urine  of  a  normal  state  contains  no  bacteria.  By  contact 
with  the  air  and  the  urinary  passages  exposed  to  air,  a  great 
number  of  yeasts,  molds  and  bacteria  soon  accumulate  in  the 
fluid.  Bacteria  also  enter  urine  through  the  blood  and  dur- 
ing its  secretion. 

A  number  of  bacteria  have  the  property  of  converting  urea 
into  carbonate  of  ammonia. 

The  urine  should  be  centrifuged  and  the  deposit  then  exam- 
ined. The  drying  and  fixing  must  proceed  very  slowly,  since 
otherwise  crystals  of  salts  will  be  precipitated  and  mar  the 
specimen. 

B.  coli  are  frequently  present,  especially  in  acid  urine. 

T3^hoid  bacilli  in  25  per  cent,  of  patients  affected  with  ty- 
phoid fever. 

Micrococcus  Ureae  (Pasteur  and  Van  Tiegham). — 
Origin. — Decomposed  urine  and  in  the  air. 

Form. — Cocci,  diplococci,  and  streptococci. 


SOME  COMMON  BACTERIA   SLIGHTLY  PATHOGENIC       IO3 

Properties. — Decomposes  urea  into  ammonium  carbon- 
ate; does  not  liquefy  gelatin. 

Growth. — Grows  rapidly,  needing  oxygen;  can  remain  sta- 
tionary below  0°  C,  growing  again  when  a  higher  temperature 
is  reached. 

Colonies  on  Plate. — On  the  surface  like  a  drop  of  wax. 

Stab-cultures. — Looks  like  a  very  delicate  thread  along  the 
needle-thrust. 

Other  bacteria  are  found  in  urine  in  various  pathologic  proc- 
esses, such  as  tubercle  bacilli,  typhoid  bacilU,  gonococci,  and 
other  pyogenic  organisms. 

Spirilla. — A  number  of  non-pathogenic  spirilla  have  been 
described. 

Spirillum  Rubriim  (Esmarch). — Origin. — Body  of  a 
mouse  dead  with  septicemia. 

Form. — Spirals  of  variable  length,  long  joints,  flagella  on 
each  end;  no  spores. 

Properties. — Does  not  liquefy  gelatin;  very  motile;  pro- 
duces a  wine-red  pigment,  which  develops  only  in  absence  of 
oxygen. 

Growth. — Can  grow  with  oxygen,  but  is  then  colorless; 
grows  very  slowly;  ten  to  twelve  days  before  any  sign;  grows 
best  at  37°  C. 

Gelatin  Roll-cultures. — Small,  round;  first  gray,  then  wine- 
red  colonies. 

Stab-cultures. — A  red-colored  growth  along  the  whole  line; 
it  is  deepest  below,  getting  paler  as  it  approaches  the  surface. 

Sarcina. — Cocci  in  cubes  or  packets  of  colonies.  A  great 
number  have  been  isolated,  many  producing  very  beautiful 
pigments.     The  majority  of  them  found  in  the  air. 

Sarcina  Lutea  (Schroter). — Origin. — Air. 

Form. — Very  large  cocci  in  pairs;  tetrads  and  groups  of 
tetrads. 

Properties. — ^Liquefies  gelatin  slowly;  produces  sulphur- 
yellow  pigment. 

Growth. — Slowly,  at  various  temperatures;  strongly  aerobic. 

Plates. — Small,  round,  yellow  colonies. 


I04  ESSENTIALS   OF  BACTERIOLOGY 

Stab-cultures. — Grows  more  rapidly,  the  growth  being 
nearly  all  on  the  surface,  a  few  separated  colonies  following  the 
needle-thrust  for  a  short  distance.  Agar,  a  very  beautiful 
yellow,  along  the  stroked  surface. 

Sarcina  Aurantica. — Flava,  rosea,  and  alba  are  some  of 
the  other  varieties.     Many  are  obtained  from  beer. 

Sarcina  Ventriculi  (Goodsir)  (Fig.  41). — Origin. — Stom- 
ach of  man  and  animals. 


Fig.  41. — Sarcina    ventriculi    from    stomach-contents    (X530)    (Van 
Valzah  and  Nisbet). 


Form. — Colorless  oval  cocci,  in  groups  of  eight  and  packets 
of  eight. 

Properties. — Does  not  liquefy  gelatin;  shows  tne  reaction 
of  cellulose  to  iodin. 

Growth. — Rapid.  At  end  of  thirty-six  hours,  round,  yellow 
colonies,  from  which  colorless  cocci  and  cubes  are  obtained. 

Habitat. — They  are  found  in  many  diseases  of  the  stomach, 
especially  when  dilatation  exists.  Also  normally;  increased 
when  fermentation  occurs. 


BACILLUS   OF   ANTHRAX  I05 

CHAPTER  XVII 
BACILLUS  OF  ANTHRAX 

Bacillus  Anthracis  (Rayer  and  Davaine). — Rayer  and 

Davaine,  in  1850,  first  described  this  bacillus;  but  Pasteur, 
and  later  Koch,  gave  it  the  importance  it  now  has. 

Synonyms. — Bactericie  du  charbon  (Fr.);  Milzbrand  bacil- 
lus (German) ;  bacillus  of  splenic  fever  or  malignant  pustule. 

Origin. — In  blood  of  anthrax-suffering  animals. 


\* 


^ 


-  ,       »' 


\    ; 


Fig.  42. — Bacillus   anthracis,    stained    to   show    the    spores    ( X  1000) 
(Frankel  and  Pfeiffer). 

Form. — Rods  of  variable  length,  largest  of  pathogenic  or- 
ganisms 4  M  to  10  )u  in  length,  nearly  the  size  of  a  human  blood- 
corpuscle;  broad,  cup-shaped  ends;  in  bouillon  cultures 
long  threads  are  formed,  with  large  oval  spores  (Figs.  42,  43). 

Spores. — Single,  large,  very  resistant.  Dry  heat,  140°  C, 
in  three  hours;  steam  in  five  minutes;  necessary  to  kill.     Do 


io6 


ESSENTIALS   OF   BACTERIOLOGY 


not  occur  in  the  circulating  blood,  but  develop  after  death  or 
in  artificial  media  at  30°  C. 


Fig.  43-— Anthrax  bacilli  in  human  blood  (fuchsin  staining)  (Zeiss  one- 
twelfth  oil-immersion;  No.  4  ocular)   (taken  from  Vierordt). 


Fig.  44.— Bacillus  anthracis,  impression  preparation,  edge  of    colony; 
Zettnow  prep.  (KoUe  and  Wassermann). 

Liquefies  gelatin;  immotile. 

Growth.— Grows  rapidly,  between  12°  C.  and  ^5°  C,  and 


BACILLUS   OF   ANTHRAX 


107 


requires  plenty  of  oxygen,  but  may  be  classed  as  a  facultative 
anaerobe;  grows  well  in  all  media. 

Colonies  develop  in  two  days;  white  shiny  spots,  which 
appear  under  microscope  as  slightly  yellowish,  granular, 
twisted  balls,  Uke  a  ball  of  yarn;  each  separate  string  or  hair,  if 
looked  at  under  high  power,  being  composed  of  bacteria  in 
threads.     (See  Fig.  44.) 


Fig.  45-  Fig.  46. 

Figs,  45,  46. — Stab-cultures  of  anthrax  in 'gelatin. 


Agar  Stroke. — Grayish- white,  slightly  wrinkled  layer  with 
irregular  edges. 

Gelatin  Stab-cultures. — A  white  growth  with  thorn-like 
processes  along  the  needle-track  (like  an  "inverted  fir  tree"). 
Later  on,  gelatin  liquefied,  and  flaky  masses  at  the  bottom. 
(See  Figs.  45,  46.) 


I08  ESSENTIAXS   OF   BACTERIOLOGY 

Potato. — ^^A  dry,  creamy  layer,  and  when  placed  in  incu- 
bator, rich  in  spores. 

Staining. — Readily  take  the  anilin  dyes  with  the  ordinary 
methods.  To  bring  out  the  cup-shaped  concave  extremities, 
a  very  weak  watery  solution  of  methylene-blue  is  best. 
Gram  positive. 

Spores  are  stained  by  the  usual  method.  When  several 
bacilli  are  joined  together,  the  place  of  their  joining  looks  like 
a  spore,  because  of  the  hollowed  ends.  Double  staining  will 
differentiate  the  spores.     (See  Fig.  42.) 

Sections  of  tissue  are  stained  according  to  the  ordinary 
methods,  taking  Gram's  method  very  nicely. 

Pathogenesis. — When  mice  are  inoculated  with  anthrax 
material  through  a  wound  in  the  skin,  they  die  in  twenty- 
four  hours  from  an  active  septicemia,  the  point  of  inocula- 
tion remaining  unchanged. 

On  autopsy  will  be  found: 

Peritoneum. — Covered  with  a  gelatinous  exudate. 

Spleen. — Very  much  swollen,  dark  red,  and  friable. 

Liver. — Parenchymatous  degeneration. 

Blood. — Dark  red.  The  bacilli  are  found  wherever  the 
capillaries  are  spread  out,  in  the  spleen,  liver,  intestinal  villi, 
and  glomeruli  of  kidney,  and  in  the  blood  itself.  Only  when  the 
capillaries  burst  are  they  found  in  the  tubules  of  the  kidney. 

Mode  of  Entrance. — The  bacilli  can  be  inhaled,  and  then  a 
pneumonia  is  caused,  the  pulmonary  cells  containing  the 
bacilli;  when  the  spores  are  inhaled,  a  general  infection  occurs. 

Feeding. — The  cattle  graze  upon  the  meadows,  where  the 
blood  of  anthrax  animals  has  flowed  and  becc«ne  dried;  the 
resistant  spores  contaminate  the  grass  and  so  enter  the  ah- 
mentary  tract;  here  they  then  cause  the  intestinal  form  of  the 
disease,  ulcerating  through  the  villi.  Cattle  are  also  in- 
fected by  wading  in  streams  which  tannery  washings  have 
contaminated. 

Local  Infection. — In  man  usually  only  a  local  action  occurs; 
by  reason  of  his  occupation — woolsorter,  cattle-driver, 
tanner,  etc. — he  handles  the  hides  or  wool  of  animals  that 


BACILLUS   OF   ANTHRAX  lOQ 

have  been  infected,  and  through  a  scratch  or  sHght  wound  he 
becomes  infected,  and  local  gangrene  and  necrosis  set  in,  but 
death  follows  in  the  severer  forms  from  a  general  pyemia; 
there  is  severe  edema  of  the  tissues  in  and  about  the  wound, 
and  pulmonary  edema.  Wounds  about  the  face  and  neck  are 
more  fatal. 

Pneumonia  by  inhalation  and  intestinal  ijifection  also 
occur  in  man. 

Woolsorter's  disease  is  the  pulmonary  form  caused  by  in- 
halation of  spores  from  infected  wool. 

Susceptibility  of  Animals. — Dogs,  birds,  and  cold-blooded 
animals  affected  the  least;  white  mice,  sheep,  and  guinea-pigs 
quickly  and  surely. 

Products  of  Anthrax  Bacilli. — ^A  basic  ptomain  has  not  been 
found,  but  a  toxalbumin  or  proteid,  called  anthraxin,  has  been 
obtained.  A  certain  amount  of  acid  is  produced  by  the  viru- 
lent form,  alkali  by  the  weak. 

Attenuation  and  Immunity. — Cultures  left  several  days  in 
an  incubator  at  a  temperature  between  40°  and  42°  C.  soon 
become  innocuous,  and  when  injected  into  animals  protect 
them  against  the  \'irulent  form. 

The  lymph  obtained  from  lymph-sac  of  a  frog  destroys  the 
virulence  of  anthrax  bacilli  and  spores  tem.porarily. 

Hankin  obtained  an  alexin  from  the  blood  and  spicen  of 
rats,  they  being  naturally  immune.  It  destroyed  the  anthrax 
bacilli  in  vitro,  and  used  by  injection  in  susceptible  anim.als, 
made  them  immune.     It  is  insoluble  in  alcohol  or  water. 

Protective  Inoculation. — Animals  have  been  rendered  im- 
mune in  various  ways — by  inoculation  of  successive  atten- 
uated cultures;  also  with  sterilized  cultures — that  is,  cul- 
tures containing  no  bacilli,  and  with  cultures  of  other  bacteria. 

Immune  Serum. — That  obtained  from  animals  rendered 
immune  by  attenuated  cultures  contains  protective  substances 
which  seem  to  have  some  antitoxic  action. 

Habitat. — In  the  serum  about  the  wound  and  in  the  blood 
anthrax  bacilli  are  readily  found. 

The  bacillus  has  never  been  found  free  in  nature. 


no  ESSENTIALS    OF   BACTERIOLOGY 

CHAPTER  XVIII 
BACILLUS  TUBERCULOSIS  AND  ALLIED  ORGANISMS 

This  very  important  bacillus  was  first  described,  demon- 
strated, and  cultivated  by  Robert  Koch,  who  made  his  in- 
vestigations public  before  the  Physiological  Society  of  Berlin 
on  the  twenty-fourth  of  March,  in  the  year  1882. 

Synonyms. — Mycobacterium  tuberculosis . 


V 


> 


Fig.  47. — Tubercle  bacilli  in  sputum;  carbolfuchsin  and  methylene-blue 
(Zeiss  one-twelfth  oil-immersion). 

Origin. — In  various  tuberculous  products  of  man  and  other 
animals  and  in  the  dust  containing  the  discharges. 

Form. — Very  slender  rods,  slightly  curved,  2  /x  to  4  )Lt  in 
length,  about  one-quarter  the  size  of  a  red  blood- corpuscle's 
diameter,  their  ends  rounded,  usually  solitary,  often,  how- 
ever, lying  in  pairs  in  such  a  manner  as  to  form  an  acute 
angle.     Sometimes  they  are  S-shaped.     In  colored  prepara- 


BACILLUS   TUBERCULOSIS  AND  ALLIED  ORGANISMS      III 

tions  little  oval  spaces  are  seen  in^  the  rod  which  resemble 
spores,  but  have  none  of  the  properties  of  spores.     (See  Figs. 

47,  48.) 

Properties. — Does  not  possess  motility. 

Growth. — Requires  special  media  for  its  growth,  and  a  tem- 
perature varying  but  slightly  from  37.5°  C.  It  grows  slowly, 
developing  first  after  teh  days,  reaching  its  maximum  in 
three  weeks.  It  is  facultative  anaerobic.  On  gelatin  it  does 
not  form  a  growth.     The  media  should  be  slightly  acid; 


Fig.  48. — Giant-cell  containing  bacilli  (from  a  photograph  made  by  Dr. 
Wm.  M.  Gray). 


growth  mostly  on  surface.  Subcultures  grow  more  rapidly 
than  those  direct  from  lesions. 

Colonies  on  Blood-serum. — Koch  first  used  blood-serum  for 
culture,  and  obtained  thereon  very  good  growths.  Stroke 
cultures  or  test-tubes  inoculated  with  small  bits  of  tubercu- 
lar tissue  are  placed  in  a  well- ventilated  and  slightly  humid 
incubator  at  37°  C.  for  ten  to  fourteen  days,  when  small 
glistening  white  points  appear,  which  then  coalesce  to  form  a 
dry,  white,  scale-like  growth.  Under  microscope,  composed 
of  many  fine  lines  CQntaining  the  tubercle  bacillus. 

Glycerin-agar. — By  adding  4  to  6  per  cent,  glycerin  to 


112  ESSENTIALS   OF   BACTERIOLOGY 

ordinary  agar-peptone  medium,  Nocard  and  Roux  obtained  a 
culture-medium  upon  which  tubercle  bacilli  grow  much  better 
than  upon  blood-serum,  especially  after  once  obtained  in 
pure  culture.  Bits  of  tissue  are  placed  on  the  surface,  not 
rubbed  in  until  after  several  weeks;  then  gently  crushed  and 
spread  over  surface ;   this  hastens  growth. 

Stroke  cultures  are  used  as  with  blood-serum.  They 
are  placed  in  incubator  after  inoculation,  and  remain  there 
about  ten  days,  at  a  temperature  of  37°  C.  The  cotton  plugs 
of  the  tubes  are  covered  with  rubber  caps,  the  cotton  first 
having  been  passed  through  the  flame,  and  moistened  with  a 
few  drops  of  subHmate  solution.  The  rubber  cap  prevents 
the  evaporation  of  the  water  of  condensation,  which  always 
forms  and  keeps  the  culture  from  drying  up. 

The  growth  which  occurs  resembles  the  rugae  of  the  stom- 
ach, and  sometimes  looks  like  moistened  crumbs  of  bread. 
The  impression  or  "Klatsch"  preparation  shows  under  the 
microscope  a  thick,  curled-up  center  around  which  threads 
are  wound  in  all  directions.  And  these  fine  lines  show  the 
bacilli  in  profusion. 

Potato. — It  can  be  cultivated  on  slices  of  potato  which  are 
placed  in  air-tight  test-tubes  to  which  glycerin  has  been  added. 

Bouillon. — Bouillon  containing  4  per  cent,  glycerin  is  a 
very  good  medium.     Growth  on  the  surface  only. 

Pure  Cultures  from  Sputum. — Kitasato  recommends  the 
thorough  washing,  changing  the  water  ten  times,  of  the  small 
masses  found  in  the  sputum  of  tuberculous  persons.  When 
such  specimens  are  examined,  they  show  tubercle  bacilli 
alone,  and  when  inoculated  in  agar,  give  rise  to  pure  cultures. 

Animal  Inoculation  for  Diagnosis. — When  the  bacilli  are  so 
few  in  number  in  sputum  or  urine  as  to  make  their  detec- 
tion difficult,  and  also  when  doubt  exists  as  to  the  identity 
of  acid-fast  bacilli  found,  several  guinea-pigs  should  be  in- 
jected in  the  groin  and  smears  and  sections  made  from  the 
enlarged  glands  resulting. 

Varieties. — Branching  and  other  aberrant  forms  are  not 
rare,  and  the  tendency  now  is  to  class  the  organism  with  the 


BACILLUS   TUBERCULOSIS   AND   ALLIED   ORGANISMS       II3 

*' higher  bacteria,"  mycobacteria,  similar  to  actinomyces. 
Other  acid-fast  bacilli  exhibit  similar  types,  and  it  is  possi- 
ble that  the  bacillary  parasitic  form  is  only  one  stage  in  the 
life-history  of  the  organism. 

Little  granules,  arranged  like  streptococci,  which  take  the 
characteristic  stain,  and  look  as  if  the  protoplasm  had  been 
destroyed  that  inclosed  them,  are  frequently  found  in  sputum. 
Some  believe  these  ''splinters"  to  develop  into  regular  bacilli 
in  cultures. 


.:^fi> 


Fig.  49. — Tubercle  bacillus  in  sputum  (Frankel  and  Pfeiffer). 

Bovine  tubercle  bacilli  are  about  one-third  smaller  than  hu- 
man tubercle  bacilli. 

Resistance. — Bacilli  in  sputum,  in  dark,  cool  places  may  live 
several  months.  Dried  sputum  in  sunlight  and  dust  is  infec- 
tive not  more  than  ten  days.  The  bacilli  will  resist  in  the  dry 
state  a  temperature  of  ioo°  C.  one  hour.  In  moisture  death 
occurs  at  60°  C.  in  a  few  minutes. 

Chemic  Properties. — A  waxy  substance  found  in  pure  cul- 
tures, due  to  fatty  acids.     The  fat-free  substance  is  nucleo- 


114 


ESSENTIALS   OF   BACTERIOLOGY 


albumin,  and  the  ash  shows  a  large  amount  of  phosphoric 
acid.     Indol  not  found. 

Staining. — The  tubercle  bacilli  require  special  methods  to 
stain  them,  and  a  great  number  have  been  introduced.  They 
are  stained  with  great  difficulty,  but  once  stained,  they  are 
very  resistant  to  decolorizing  agents,  hence  called  acid-proof 
or  acid-fast.  Upon  these  facts  all 
the  methods  are  founded. 

The  resisting  action  of  the  bacil- 
lus to  acids  is  supposed  to  be  due 
to  a  pecuhar  arrangement  of  the 
albumin  and  cellulose  of  the  cell, 
rather  than  to  any  particular  cap- 
sule around  it.  A  waxy  substance, 
made  up  of  fatty  acid,  has  been 
found  and  supposed  to  account  for 
this  resistance.  Others  believe  this 
substance  to  be  an  alcohol. 

It  will  be  necessary  to  describe 
only  those  methods  principally  in 
use;  and  as  the  examination  of 
sputum  for  bacilH  is  of  so  frequent 
an  occurrence  and  so  necessary,  it 
is  well  to  detail  in  particular  the 
method  of  staining. 

Starting  with   the   sputum,  we 

search  for  little  clumps  or  rolled-up 

masses;    if  these  are  not  present, 

the   most   solid    portions   of    the 

mucus  are   brought  with   forceps 

upon  a  clean  cover-glass;  very  little 

suffices.     With  another  cover-glass 

the  mass  is  pressed  and  spread  out  evenly.    Drawing  one  glass 

over  the  other,  we  obtain  two  specimens,  and  these  are  put 

aside  or  held  high  over  the  flame  until  dry. 

When  the  preparation  is  dry  and  has  been  fixed  by  passing 
through  the  flame  three  times,  carbolfuchsin  is  dropped  on 


Fig.  50. — Bacillus  tuber- 
culosis; glycerin  agar-agar 
culture,  several  months  old 
(Curtis). 


BACILLUS   TUBERCULOSIS   AND   ALLIED   ORGANISMS       II5 

the  cover-glass  and  held  over  the  flame  until  the  stain  boils; 
fresh  stain  is  added,  the  boiling  continued  for  a  minute. 
Then  the  excess  of  stain  is  removed  with  edge  of  filter-paper. 
Decolorize  in  25  per  cent,  nitric  or  2  per  cent,  hydrochloric 
acid.  The  excess  of  acid  is  then  washed  out  with  95  per 
cent,  alcohol  until  no  further  color  is  imparted  to  the  alcohol, 
and  the  smear  is  gray  or  light  pink  in  color.  The  preparation 
is  then  washed  with  water  and  counterstained  with  aqueous 
methylene-blue  for  ten  to  thirty  seconds. 

The  Rapid  Method  {B.  FrdnkeVs  Method,  Modified  by  Gab- 
bet), — The  principle  is  to  combine  with  the  contrast  stain  the 
decolorizing  agent;  but  the  preparations  are  not  permanent; 
the  method,  however,  is  very  useful. 

Two  solutions  are  required:  one  of  Ziehl's  carbolfuchsin; 
the  other,  Gabbet's  acid  irethylene-blue.  (See  Formula  No. 
X,  on  p.  51.) 

The  cover-glass  containing  the  dried  sputum  is  passed 
three  times  through  the  flame,  as  described  in  the  general 
directions.  It  is  then  placed  in  the  carbolfuchsin  solution 
five  minutes  (cold),  or  two  minutes  in  the  hot,  immediately 
transferred  to  the  second  solution,  the  acid  blue,  where  it 
remains  one  minute,  then  washed  in  water.  The  preparation 
is  dried  between  filter-paper  and  mounted.  Examined  with 
oil-immersion. 

Slow  Method. — The  above  method  may  also  be  used  with- 
out heating,  though  in  this  case  a  much  longer  time  is  required 
before  the  bacilli  take  up  the  stain.  The  preparation  is  left 
in  a  small  dish  or  beaker  full  of  carbolfuchsin  for  eight  to 
ten  hours,  and  theri  decolorized  and  counterstained  in  the 
way  described  above.  The  method  is  less  liable  to  produce 
artefacts  than  the  quick  method,  but  is  not  much  used  on 
account  of  the  time  it  takes. 

Examination  in  Urine. — In  urine,  owing  to  the  almost  in- 
e\itable  contamination  with  the  smegma  bacillus,  special 
methods  are  necessary  to  avoid  error.  The  preparation  may 
be  left  in  97  per  cent,  alcohol  for  eight  hours,  when  the 
smegma  bacillus  will  have  become  decolorized,  or  Pappenheini's 


Il6  ESSENTIALS   OF   BACTERIOLOGY 

method  may  be  used:  (i)  Smear  and  fix  as  usual;  (2)  stain 
with  hot  carbolfuchsin  for  two  minutes,  pour  off  the  surplus 
dye  without  washing;  (3)  counterstain  and  decolorize  by 
pouring  five  times  over  the  preparation  the  following  solu- 
tion: A  I  per  cent,  alcoholic  solution  of  corallin  is  saturated 
with  methylene-blue  and  20  parts  of  glycerin  added.  Wash 
in  water,  dry  with  blotting-paper,  then  in  the  air,  and  exam- 
ine. The  tubercle  bacilli  are  stained  red,  smegma  bacilli, 
blue. 

Examination  of  Milk  for  Tubercle  Bacilli. — Place  a  drop 
of  the  sample  on  a  cover-glass  and  mix  it  with  two  drops 
of  a  I  per  cent,  solution  of  sodium  carbonate.  The  cover- 
glass  is  then  gently  warmed  until  evaporation  is  complete. 
The  saponified  fat  is  then  stained,  as  the  ordinary  cover-glass 
preparation.  Only  a  few  times  has  any  one  succeeded  in 
discovering  the  bacillus  in  milk. 

Other  Acid-fast  Bacteria. — The  bacillus  of  leprosy  re- 
sembles the  tubercle  bacillus  in  its  staining  properties,  but 
gives  up  the  carbolfuchsin  more  easily  and  is  usually  de- 
colorized by  the  acid  and  alcohol.  It  is  colored  blue  by  Pap- 
penheim's  method. 

Acid-fast  bacilli  have  also  been  obtained  from  timothy 
grass,  butter,  milk,  manure,  and  the  surfaces  of  animal  bodies, 
but  differ  from  the  tubercle  bacillus  in  cultural  characteristics. 

Water  has  been  found  to  contain  acid-fast  bacilli;  care 
should  be  taken  to  test  the  water  used  previously  to  any  im- 
portant examination  for  tubercle  bacilli. 

Biederfs  Method  of  Collecting  Bacilli. — When  the  bacilli 
are  very  few  in  a  great  quantity  of  fluid,  as  urine,  pus,  abun- 
dant mucus,  etc.,  Biedert  advises  to  mix  15  c.c.  of  the  fluid 
with  75  to  100  c.c.  water  and  a  few  drops  of  potassium  or 
sodium  hydroxid,  then  boiling  until  the  solution  is  quite 
thin.  It  is  placed  in  a  conical  glass  for  two  days,  and  bacilli 
with  other  morphologic  elements  sink  to  the  bottom  of  the 
glass;  when  the  supernatant  liquid  is  decanted,  the  residue 
can  be  easily  examined.  In  this  way  bacilli  were  found  that 
had  eluded  detection  examined  in  the  ordinary  manner. 


BACILLUS  TUBERCULOSIS  AND  ALLIED  ORGANISMS      II 7 

The  centrifugal  machine  is  used  either  in  connection  with 
Biedert's  sediment  method  or  without,  to  obtain  the  soUds 
suspended  in  urine  or  serum. 

Antiformin  Method. — A  mixture  of  chlorin  water  and  so- 
dium hydroxid;  chlorin  is  liberated,  and  this  dissolves  most 
of  the  organisms  in  the  sputum  and  the  mucus,  leaving  un- 
altered the  tubercle  bacillus.  Dilute  thick  sputum  with  dis- 
tilled water,  add  one-quarter  volume  antiformin,  mix  until 
solution  is  effected;  add  alcohol,  equal  volume,  and  allow 
mixture  to  stand  eighteen  hours.  Prepare  cover-slip  prepa- 
rations from  this. 

Staining  Bacillus  Tuberculosis  in  Tissue  {Sections). — The 
general  method  of  Gram  can  be  used,  but  the  better  way  is 
to  use  the  following: 

Warm  carbolfuchsin,  fifteen  to  thirty  minutes. 

5  per  cent,  sulphuric  acid,  one  minute. 

Alcohol,  until  a  light-red  tinge  appears. 

Weak  methylene-blue,  three  to  five  minutes. 

Alcohol,  for  a  few  seconds. 

Oil  of  cloves,  until  cleared. 

Canada  balsam,  to  mount  in. 
Instead   of  carbolfuchsin,  alcoholic  solution  of  fuchsin  or 
anilin-water  fuchsin  can  be  used,  but  the  sections  must  re- 
main in  the  stain  overnight. 

Hardened  Sputum  and  Sectioning. — Sputum  can  be  hard- 
ened by  placing  it  in  98  per  cent,  alcohol.  Thin  sections  can 
be  obtained  by  imbedding  the  hardened  sputum  in  celloidin. 
The  sections  are  then  stained  as  ordinary  tissue  sections. 

To  Preserve  Sputum. — Sputum  can  be  preserved  for  future 
use  by  placing  it  in  alcohol,  w^here  it  can  be  kept  for  months. 
Cover-glass  preparations  can  then  be  made  by  softening  the 
coagula  with  a  small  amount  of  liquor  potassa. 

Pathogenesis. — When  a  guinea-pig  has  injected  into  its 
peritoneal  ca\ity  some  of  the  diluted  sputum  containing  tu- 
bercle bacilli,  it  perishes  in  about  three  weeks,  and  the  follow- 
ing picture  presents  itself  at  the  autopsy:  at  the  point  of 
inoculation  there  is  a  local  tuberculosis — little  tuberculous 


Il8  ESSENTIALS  OF  BACTERIOLOGY 

nodules  containing  the  characteristic  bacilli.  In  the  lungs 
and  the  lymphatics  similar  tubercles  are  found — a  general 
tuberculosis. 

If  the  animal  lingers  a  few  weeks  longer,  the  tubercles 
becomes  necrosed  in  the  center  and  degeneration  occurs,  the 
periphery  still  containing  active  bacilli,  cavities  ha\dng  formed 
in  the  center. 

Since  the  bacilH  die  in  course  of  time,  killed  by  their  own 
products,  their  number  forms  no  correct  guide  of  the  dam- 
age present:  even  their  absence  in  the  sputum  does  not  pre- 
clude the  absence  of  a  tuberculous  process.  //  is  their  pres- 
ence only  that  warrants  a  positive  declaration.  The  number  of 
bacilli  in  a  given  specimen  is  no  indication  of  the  severity  of 
the  disease. 

They  are  found  in  the  blood  only  when  a  vessel  has  come 
in  direct  contact  with  a  tuberculous  process  through  rupture 
or  otherwise.  They  have  been  found  occasionally  in  other 
secretions — milk,  urine,  etc. 

Man  is  infected  as  follows: 

Through  Wounds. — Local  tuberculosis. 

Through  Nutrition. — Milk  and  meat  of  tuberculous  ani- 
mals. Phthisical  patients  swallowing  their  own  sputum  and 
causing  an  intestinal  tuberculosis. 

Inhalation. — This  is  the  most  usual  way,  probably  consti- 
tuting the  cause  in  nine-tenths  of  the  cases  in  adults. 

The  sputum  of  phthisical  patients  expectorated  on  the 
floors  of  dwelling-houses,  in  handkerchiefs,  etc.,  dries,  and 
the  bacilli  set  free  are  placed  in  motion  by  the  wind,  or  rising 
with  the  dust,  are  thus  inhaled  by  those  present.  When  the 
sputum  is  kept  from  drying  by  expectoration  in  vessels  con- 
taining water,  this  great  danger  can  be  avoided. 

Intra-uterine  or  placental  infection  has  been  demonstrated, 
but  is  a  great  rarity.  The  ovum  or  human  semen  is  seldom 
if  ever  infected,  although  tubercular  infection  of  the  testicle 
is  common. 

Nearly  all  the  cases  of  supposed  heredity  can  be  explained 
as  follows :  the  young  children,  p  ossessing  very  httle  resist- 


BACILLUS  TUBERCULOSIS  AND  ALLIED  ORGANISMS      II9 

ance,  are  constantly  exposed  to  the  infection  through  inhal- 
ation, and  to  intestinal  infection  through  milk  and  other  foods. 

Immunity. — No  one  can  be  said  to  be  immune,  though  per- 
sons who  have  been  greatly  weakened  offer  less  resistance 
than  healthy  individuals. 

Bovine  and  Human  Tuberculosis. — Tuberculosis  in 
Animals. — Tuberculosis  is  probably  the  most  widely  dis- 
seminated disease  among  domestic  ,  animals,  and  affects 
cattle,  pigs,  horses,  dogs,  cats,  the  smaller  ruminants,  birds, 
and  even  turtles  and  fish.  The  conclusion  of  Koch,  made 
public  in  his  address  to  the  Tuberculosis  Congress  in  1901, 
that  human  and  bovine  tuberculosis  are  distinct  and  that  in- 
fection of  human  beings  from  cattle  occurs  so  seldom  that  no 
general  regulations  to  restrict  it  are  necessary,  has  found 
few  adherents.  In  1908  Koch  reiterated  his  idea  and  chal- 
lenged his  opponents  to  bring  proofs  to  the  contrary.  Con- 
clusions at  this  writing  seem  to  be  that  go  per  cent,  of  all 
puhnonary  cases  in  adult  man  are  not  due  to  bovine  infection. 
In  children  under  five,  however,  10  per  cent,  of  the  intestinal 
tuberculosis  and  cervical  adenitis  are  due  to  the  bovine 
type  of  infection  through  milk  of  diseased  cows.  It  is  true 
that  certain  differences  exist  between  human  and  bovine 
tubercle  bacilli,  the  latter  appearing  to  be  more  virulent  to 
animals,  and  it  is  a  fact  that  cattle  are  very  slightly  suscepti- 
ble to  the  human  bacillus,  but  it  is  not  likely  that  the  con- 
verse is  so.  Children  are  particularly  liable  to  infection 
through  the  gastro-intestinal  tract,  and  it  has  been  shown 
that  the  uninjured  mucosa  of  the  infant's  intestine  is  per- 
meable to  bacilli,  so  that  the  pulmonary  disease  in  the 
young  may  often  be  the  result  of  tuberculous  bronchial  nodes 
secondary  to  tuberculous  glands  of  the  mesentery. 

Various  observations  on  animals  have  shown  that  the 
bacillus  occurring  in  each  species  has  acquired  certain  special 
characteristics  regarding  growth  and  virulence.  The  bacilli 
causing  tuberculosis  in  the  cold-blooded  animals  have  de- 
parted farthest  from  the  human  type,  those  of  birds  to  a  less 
degree,  and  those  of  cattle  least  of  all. 


I20  ESSENTIALS   OF   BACTERIOLOGY 

Products  of  Tubercle  Bacilli. — The  true  nature  of  the 
tubercle  toxin  is  not  yet  clear.  It  is  not  unlikely  that  several 
toxic  bodies  differing  from  one  another  in  their  properties  are 
produced.  Koch's  tuberculin  (1890),  a  bacterioprotein,  was 
obtained  by  filtering  through  unglazed  porcelain,  concen- 
trated glycerin  bouillon  cultures  of  tubercle  bacilli.  It  was 
speedily  shown  to  be  devoid  of  curative  power,  and  is  now 
used  mainly  for  diagnosing  the  disease  in  cattle.  In  healthy 
animals  little  or  no  reaction  is  produced  by  the  injection  of  30 
to  40  eg.  of  tuberculin,  but  if  tuberculous,  the  temperature 
rises  2°  to  3°  F .  in  eight  to  twelve  hours,  and  remains  elevated 
for  a  like  period  of  time  and  may  in  larger  doses  prove  fatal. 
It  is  dangerous  unless  used  carefully. 

Tuberculocidin. — This  is  an  albuminoid  obtained  from  the 
original  tuberculin  by  precipitation  with  alcohol.  Klebs 
used  it  as  a  treatment  for  tuberculosis. 

Tuberculin  residuum,  an  emulsion  from  the  residuum, 
hence  the  name,  T.  R.,  is  an  extract  made  from  dried  and 
powdered  living  bacilli,  and  was  recommended  by  Koch  in 
place  of  the  original  or  old  tuberculin,  O.  T. 

Koch^s  bacillen  emulsion  {B.  E.)  is  similar  to  tuberculin  R, 
and  is  a  glycerin  emulsion  of  crushed  bacteria,  this  being  the 
entire  substance  instead  of  an  extract.  Theo.  Smith  recom- 
mends virulent  uncrushed  bacteria  killed  by  moderate  heat. 

Denys^  B.  F.  (bouillon  filtrate)  tuberculin  is  a  filtrate  of 
Hquid  cultures  to  which  0.25  per  cent,  phenol  has  been 
added  and  allowed  to  stand  two  weeks.  It  is  prepared  in 
eight  dilutions. 

Opsonic  Treatment. — In  recent  years  the  use  of  tuberculin 
R  has  again  been  brought  forward  by  Wright  and  others  and 
curative  claims  made  for  it.  It  is  used  in  very  small  doses — 
TTrV"o  milligram  at  intervals  of  several  days,  and  the  effect  on 
the  opsonic  index  carefully  watched. 

Use  of  Tuberculin. — In  the  use  of  tuberculin  severe  reactions 
are  to  be  avoided.  The  smallest  dose  possible  is  commenced 
with.  Trudeau  uses  for  afebrile  cases  a  solution  containing 
xiy^TFirniilligram,  Hquid  measure,  Koch's  B.  E.,  or  Denys'  B.  F., 


BACILLUS  TUBERCULOSIS  AND  ALLIED  ORGANISMS      121 

increasing  i  decigram  of  the  solution  every  three  days  until 
I  c.c.  of  the  pure  filtrate  can  be  injected  without  causing  any 
reaction.  A  negative  reaction  sometimes  occurs  in  well- 
advanced  cases,  and  is,  therefore,  not  a  proof  of  the  absence 
of  disease.  The  reaction  is  due  to  the  stimulation  of  irri- 
tating proteins.  Yeast  nucleins  and  other  substances  have 
a  similar  action.  This  treatment  must  extend  over  months. 
Tuberculin  immunity  does  not  last  indefinitely.  Under  this 
careful  treatment,  associated  with  open  air,  proper  food,  and 
general  hygiene,  Trudeau  and  his  followers  have  had  some 
very  good  results. 

Von  Pirquet  Test. — TubercuHn  applied  to  the  abraded 
skin  like  a  vaccination  w^ith  cow-pox  causes  a  local  reaction 
in  tuberculous  infants  and  no  reaction  in  healthy  ones.  It 
is  not  applicable  to  children  over  eighteen  months  of  age. 
The  test  is  so  sensitive  that  it  will  be  positive  in  the  majority 
of  instances,  because  the  majority  of  people  have  at  some 
time  been  affected  with  tuberculosis  or  exposed  sufficiently 
to  have  within  them  sensitive  bodies  that  are  easily  stimu- 
lated. 

Ophthalmic  Tuberculin  Reaction  of  Calmette. — A  modified 
form  of  tuberculin  is  placed  on  the  conjunctiva  of  an  indi- 
vidual suspected  of  having  tuberculosis.  In  a  few  hours  a 
congestion,  more  or  less  severe,  results,  and  lasts  several 
days.  In  healthy  persons  no  reaction  occurs.  The  test  is 
claimed  to  be  harmless,  though  severe  reactions  have  been 
reported  in  tuberculous  patients,  and  even  in  healthy  persons 
a  second  appHcation  to  the  same  eye  may  cause  an  inflam- 
matory reaction. 

Morons  Test. — An  ointment  of  tuberculin  and  lanolin,  equal 
parts,  rubbed  in  the  skin  of  the  arm.  A  crop  of  papules  de- 
velops in  twelve  to  twenty-four  hours  if  test  is  positive. 

Agglutination. — Arloing  and  Courmont  have  described 
an  agglutination  reaction  for  the  tubercle  bacillus  similar  to 
the  Widal  reaction  of  typhoid  fever.  (See  p.  140.)  It  is 
very  unreliable,  however,  and  but  little  importance  is  at- 
tached to  it. 


122 


ESSENTIALS   OF   BACTERIOLOGY 


Antituherculous  Serum. — The  attempts  to  produce  an 
effective  serum  have  so  far  been  unsuccessful.  Marmorek, 
by  growing  the  bacillus  on  a  special  serum  obtained  by  in- 
jecting calves  with  the  leukocytes  of  guinea-pigs,  has  secured 
a  toxin  which  he  used  to  immunize  horses,  and  the  serum  so 
obtained  has  been  tried  with  encouraging  results,  but  its 
value  is  still  doubtful.  Several  other  sera  have  been  intro- 
duced, but  none  of  them  has  shown  any  lasting  virtues. 

Lepra  Bacillus  (Hansen). — Origin. — In  1880  Armauer 
Hansen  declared,  as  the  result  of  many  years'  investigation, 

that  he  found  specific  bacil- 
lus in  all  leprous  processes. 

Form. — Small  slender  rods, 
somewhat  shorter  than  tu- 
bercle bacilli,  otherwise  very 
similar  in  appearance. 
Neither  in  the  form  nor 
staining  reactions  can  B. 
lepra  be  distinguished  from 
B.  tuberculosis. 

In  the  interior  of  the  cell 
two  or  three  oval  spaces  are 
usually  seen,  not  believed  tc 
be  spores. 

They  are  immotile. 
Growth.  —  Bordoni-Uffred- 
uzzi  have  obtained  growths 
upon  blood-serum  to  which  peptone  and  glycerin  had  been 
added,  but  the  accuracy  of  this  observation  was  doubted, 
and  not  until  Clegg,  in  1909,  and  Duval,  in  1910,  in  work  in 
the  Philippine  Islands  devised  special  media  was  it  possible 
to  obtain  readily  initial  and  subcultures. 

The  method  depends  upon  supplying  the  organism  with 
albumin  partially  metabolized.  Clegg  prepared  this  by 
planting  the  lepra  bacilH  on  media  containing  ameba  and 
bacteria;  then,  by  short  sterilization,  destroying  these,  while 
the  resistant  B.  lepra  lived  on.     Duval,  by  adding  trypsin  to 


Fig.  51. — Pure  culture  of  bacil- 
lus of  leprosy,  showing  the  charac- 
teristic morphology  and  arrange- 
ment of  the  bacilli  (Duval). 


BACILLUS  TUBERCULOSIS  AND  ALLIED  ORGANISMS      1 23 

egg-albumin  or  blood-serum,  was  able  to  change  the  protein 
sufficiently  without  requiring  ameba  or  bacterial  digestion. 

The  leprous  nodule  is  cut  in  small  sHces  and  spread  over 
an  albumin  slant  or  Petri  plate,  and  the  surface  covered  with 
I  per  cent,  solution  trypsin  and  placed  in  oven  at  37°  C.  for 
ten  days. 

The  growth  is  moist  and  becomes  yellow  after  several 
generations;  it  is  on  surface. 

Staining. — B.  lepra  resist  the  decolorizing  action  of  acids, 
as  the  tubercle  bacilli,  but  they  are  more  easily  stained,  re- 
quiring but  a  few  minutes  more  with  the  ordinary  watery  solu- 
tions.    They  take  Gram's  stain  readily. 

Pathogenesis. — Arning  inoculated  a  prisoner  with  tissue 
obtained  from  leprous  patients  and  produced  true  leprosy, 
but  this  was  a  susceptible  native  and  the  evidence  is  not 
clear.  Duval,  by  repeated  injections  of  large  amounts  of 
pure  culture,  has  produced  leprosy  in  mice,  guinea-pigs,  and 
monkeys. 

Rabbits  which  have  been  infected  through  the  anterior 
chamber  of  the  eye  show^ed  the  lepra  nodules  (containing  the 
lepra  bacilli)  diffused  through  various  organs. 

In  man  the  skin  and  peripheral  nerves  are  principally 
affected,  but  the  lymphatic  glands,  liver,  and  spleen  can  also 
become  the  seat  of  the  lepra  nodules.  The  lepra  cells  which 
compose  these  nodules  contain  the  bacilli  in  large  numbers. 
By  applying  a  vesicant  to  the  leprous  skin,  the  serum  there- 
by obtained  will  contain  great  numbers  of  bacilli.  This  is  a 
simple  diagnostic  test. 

Method  of  Infection. — ^Not  yet  determined;  the  air,  soil, 
water,  and  food  of  leprous  districts  have  been  carefully  ex- 
amined without  result.  The  nasal  secretion  is  very  infectious. 
Intimate  contact  over  a  long  period  seems  necessary,  but  the 
records  of  leper  asylums  show  that  very  few  cases  ever  de- 
velop among  the  attendants. 

Smegma  Bacillus  of  Alvarey  and  Tavel. — Lustgarten,  in 
1885,  through  a  certain  staining  process,  found  peculiar 
bacilU  in  syphilitic  tissues  which  he  thought  had  a  direct 


124  ESSENTIALS   OF   BACTERIOLOGY 

connection  with  the  disease.  But  this  has  been  disproved 
and  the  cause  of  syphiUs  has  been  found  in  a  protozoon 
which  has  been  called  Spirochaeta  palHda,  which  see  (p.  209). 
The  smegma  bacillus  is  found  in  and  about  the  genital  or- 
gans, is  an  acid-fast  bacillus  which  resembles  the  tubercle 
bacillus  in  form,  but  is  easily  decolorized  with  alcohol,  thus 
differing  from  the  latter.  It  has  no  pathogenic  properties,  but 
is  found  at  times  in  the  throat  and  may  be  mistaken  for  B. 


rT 


/ 


^"^1#'^       "^ 


fe% 


Fig.  52. — Bacillus  of  glanders  from  a  culture  upon  glycerin  agar-agar 
(Xiooo)  (Frankel  and  Pfeiffer). 

tuberculosis.  Differentiated  in  staining,  according  to  Pap- 
penheim's  Method. 

Bacillus  of  Glanders  (Bacillus  Mallei  (Loffler-Schiitz) ; 
Rotz-bacillus) . — Origin. — In  the  "farcy  buds"  or  little 
nodules  of  the  disease,  by  Loffler  and  Schiitz,  in  1882. 

Form. — Small  slender  rods,  about  the  size  of  the  tubercle 
bacillus.  The  ends  rounded.  Never  appearing  in  large  col- 
lections, usually  singly  (Fig.  52).  Granules  like  spores 
appear  in  some  cultures;  branched  forms  are  found. 


BACILLUS   TUBERCULOSIS   AND   ALLIED   ORGANISMS       1 25 

Properties. — They  are  not  motile;  do  not  produce  gas; 
some  pigment  on  potato. 

Growth. — The  growth  occurs  between  25°  and  40°  C. — best 
at  37°  C;  it  is  very  sparse  upon  gelatin,  but  on  glycerin-agar 
or  blood-serum  a  very  abundant  growth  occurs.  Easily  de- 
stroyed by  heat,  but  cultures  sealed  and  protected  from 
light  may  live  several  months. 

Colonies. — On  agar  or  glycerin-agar  there  appear  in  two  to 
three  days  small  white  glistening  drops,  which  under  micro- 
scope seem  as  round  granular  masses  with  an  even  periphery, 
similar  to  young  B.  typhi  colonies. 

Stroke  Cultures. — On  glycerin-agar  and  blood-serum  small 
transparent  drops  of  whitish  or  grayish  color,  which  soon 
coalesce  to  form  a  broad  band  like  B.  coli. 

Potato. — An  amber-colored,  honey-like  growth  which  grad- 
ually turns  red,  then  brown,  and  greenish-brown  around  it. 
Weakly  acid  potatoes  are  a  good  medium  and  give  the  most 
typical  growth. 

Staining. — Gram  negative.  Since  the  bacillus  is  very  easily 
decolorized,  some  special  methods  have  been  recommended. 
Loffler's  and  Klihne's  solutions  for  cover-glass  and  sections. 
(See  Staining.) 

Pathogenesis. — If  horses,  field-mice,  or  guinea-pigs  be  inocu- 
lated subcutaneously  with  but  a  very  small  quantity  of  cul- 
ture, a  local  affection  results,  followed  some  time  after  by 
a  general  disturbance;  ulcers  form  at  the  point  of  inocula- 
tion— little  nodules,  which  then  caseate,  leaving  scars  and 
involving  the  lymphatics;  metastatic  abscesses  then  occur  in 
the  spleen  and  lungs,  and  death  from  exhaustion.  Cattle, 
pigs,  and  rabbits  are  not  easily  affected;  man  is  readily 
attacked.  The  bacilli  gain  entrance  to  the  blood  and  urine. 
Nasal  glanders  occurs  whatever  the  mode  of  inoculation.  In 
the  horse  the  type  is  more  chronic  than  in  the  mule.  A  catar- 
rhal nasal  discharge  occurs,  highly  infectious.  In  the  cutane- 
ous variety,  the  enlarged  lymphatics  or  nodes  which  develop 
are  cdiW^di  farcy-huds. 

Manner  of  Infection. — Glanders,  being  a  highly  contagious 


126  ESSENTIALS   OF   BACTERIOLOGY 

disease,  it  requires  but  a  slight  wound  to  allow  it  to  gain 
entrance. 

In  horses  the  primary  sore  seems  to  be  at  the  nasal  mucous 
membrane.  In  man  it  affects  those  attending  horses  and  is 
usually  on  the  fingers,  and  terminates  fatally  within  three 
weeks.  Chronic  glanders  may  last  several  years  and  end  in 
recovery. 

Mallein. — ^A  substance  called  mallein  has  been  obtained 
from  the  cultures  grown  in  glycerin  bouillon.  It  gives  a  re- 
action when  injected  into  cattle  suffering  from  glanders,  and 
is  said  to  be  useful  in  diagnosing  the  disease.  The  reaction  is 
specific  and  never  fails  to  reveal  the  presence  of  infection. 

The  inoculation  of  a  guinea-pig  intraperitoneal^  with  some 
of  the  suspected  discharge  will  produce  an  orchitis  if  the 
glanders  bacillus  is  present,  which  is  quite  characteristic  and 
helpful  in  the  diagnosis. 


CHAPTER  XIX 
DIPHTHERIA  BACILLUS 

Bacillus  of  Diphtheria  (Klebs-LoflBler) . — Origin. — 
Klebs  found  it  in  diphtheritic  membrane  in  1883;  it  was  iso- 
lated by  Loffler  in  1884. 

Form. — Small,  slightly  curved  rods  about  as  long  as  tu- 
bercle bacilli  and  twice  as  broad;  i  /i  to  6  /x  in  length;  the 
ends  are  at  times  swollen;  spores  have  not  been  found.  Their 
form  is,  however,  very  variable — sometimes  much  longer 
than  usual,  one  end  often  greatly  knobbed.  Normal  bacilli 
are  found  only  in  membrane. 

-  Stained  forms  are  characteristic,  since  the  ends  are  more  eas- 
ily colored  than  the  center,  and  usually  the  bacillus  stains  in 
segments,  so  that  it  seems  to  be  made  up  of  very  short  sec- 
tions or  beaded.    At  first  sight  it  appears  like  a  chain  of  cocci. 


DIPHTHERIA   BACILLUS  1 27 

Small  granules,  the  Babes-Ernst  granules,  are  shown  by 
the  special  staining  of  Neisser. 

Properties. — B.  diphtheria  is  immobile;  does  not  liquefy 
gelatin.  Is  not  very  resistant,  being  destroyed  by  a  tempera- 
ture of  50°  C,  but  may  live  on  blood-serum  for  months. 
Acid  is  produced  in  sugar  media. 

Growth. — Grow  readily  on  all  media,  but  best  on  blood- 
serum  mixtures,  between  temperatures  of  20°  and  40°  C. 
They  are  facultative  anaerobic;  they  grow  quite  rapidly  and 


^i 


Fig.  53. — Diphtheria  bacilli  frwni  ^  cliuic  on  blood-serum,  stained 
by  Loffler's  methylene-blue  solution,  showing  deeply  stained  points 
(X2000)     (Wright  and  Brown). 

profusely.  Egg  cultures  (Hueppe's  method)  give  good 
growths.  Passing  currents  of  air  increase  the  growth;  on 
agar,  growth  is  slow. 

Colonies  on  Agar  Plates. — At  24°  C.  little  round  colonies, 
white  under  low  power,  granular  center ;  irregular  borders. 

S tab-cultures. — Small,  white  drops  along  the  needle-track. 
In  glycerin-agar  a  somewhat  profuse  growth.  Media  should 
be  slightly  acid. 

Potato. — On  alkaline  surface,  a  gra)dsh  layer  in  forty-eight 
hours. 


128 


ESSENTIALS  OF  BACTERIOLOGY 


Blood-serum  {After  Loffler). — See  p.  69. 
In  a  few  hours  (eight  to  sixteen)  on  the  white  opaque  sur- 
face a  sUght  moisture  is  noticeable  which,  if  examined,  is 
composed   of   bacilli.     In    twenty-four   hours    small   round 
colonies  are  found  which  seem  to  ar- 
range themselves  concentrically.     The 
growth  becomes  more  abundant,  and 
the  individual  colonies  larger  and  yel- 
lowish (Fig.    54).     On  blood-coagulum 
(see  p.  72)  the  growth  is  usually  gray 
and  the  margins  of  the  culture  crenated. 
Often  a  diagnosis  can  be  made  in  four 
hours  if  the  serum-tubes  are  kept  in  the 
oven  at  37°  C.      In   milk,  abundant 
growth,  without  curdling. 

Bouillon. — In  bouillon  an  abundant 
growth  takes  place,  and  this  medium  is 
used  to  obtain  the  toxins. 

Staining. — Is  positive  by  Gram's 
method.  Stained  best  with  Loflfler's  al- 
kaline methylene-blue.  Neisser's  double 
stain  (see  p.  52)  shows  granules,  blue 
black,  and  body,  brown. 

Pathogenesis, — By  inoculation,  ani- 
mals, which  naturally  are  not  subject 
to  diphtheria,  have  had  diphtheritic 
processes  develop  at  the  site  of  infec- 
tion; hemorrhagic  edema  then  follows, 
and  death. 

No  agglutinins  are  developed  in  the 
serum. 

In   rabbits   paralyses    develop,   and 
when  the  inoculation  occurs  upon  the 
trachea,  all  the  prominent  symptoms  of    diphtheria   show 
themselves. 

Manner  of  Infection  in  Man. — The  exact  way  is  not  yet 
known.     It  is  supposed  that  the  mucous  membrane,  altered  in 


Fig.  54. — Bacillus 
diphtheriae;  agar-agar 
culture  (photograph 
by  Dr.  Henry  Koplik) . 


DIPHTHERIA   BACILLUS  1 29 

some  manner,  the  diphtheria  bacillus  then  gains  entrance  and 
the  disease  develops.  The  bacilli  may  be  found  in  healthy 
indi\dduals  who  may  act  as  a  source  of  infection  to  sus- 
ceptible individuals  without  themselves  becoming  infected. 
They  are  seldom  found  in  blood  or  other  tissues;  the  symp- 
toms arise  mainly  from  the  absorption  of  the  toxin. 

Prevalence  of  Bacillus  DiphthericB. — Examinations  made  on 
a  large  scale  of  the  throats  of  supposedly  healthy  individuals 
have  shown  that  the  Bacillus  diphtheriae  is  rather  widely  dis- 
tributed. Not  only  does  it  linger  for  many  weeks  in  the 
throats  of  persons  recently  recovered  from  the  disease,  but  it 

Fig-  55. — Bacillus  diphtheriae,  from  a  pure  culture. 

is  found  in  the  caretakers,  nurses,  etc.,  and  there  are  allied 
organisms,  with  more  or  less  pathogenicity,  that  have  been 
found  in  atrophic  rhinitis,  in  conjunctivitis,  and  in  the  throats 
of  unexposed  normal  individuals. 

Pseudodiphtheria. — The  pseudohacillus  of  Hoffman  is  be- 
lieved by  some  investigators  to  be  but  a  weakened  diphtheria 
bacillus  that  has  lost  its  toxic  power,  but  its  true  relation  is 
not  settled.  It  is  morphologically  identical  and  at  times  is 
found  side  by  side  with  the  true  bacillus.  It  grows  well 
on  agar,  shows  no  granules  with  Neisser  stain,  and,  contrary 
to  B.  diphthericB,  does  not  produce  as  much  acid  in  dextrose 
broth. 

9 


130  ESSENTIALS   OF   BACTERIOLOGY 

Methods  of  Diagnosis. — A  small  piece  of  exudate  or  some 
secretion  from  phar^^nx,  tonsil,  or  nares  is  obtained  on  a  ster- 
ile cotton  swab  and  transferred,  as  soon  as  possible,  to  the  sur- 
face of  two  or  more  blood-serum  tubes  (if  these  are  not  avail- 
able, the  swab  should  be  placed  in  a  sterile  test-tube  or  bottle, 
and  sent  to  the  laboratory  at  once).  The  inoculated  tubes 
are  placed  in  the  incubator  at  37°  C,  and  examined  in  twelve 
hours.     If  a  growth  is  visible,  a  slide  is  made  and  stained 


-*4 


HT 


Fig.  56. — Bacillus  diphtheriae,  from  a  culture  upon  blood-serum  (Xiooo) 
(Frankel  and  Pfeiffer). 

with  Loffler's  and  Neisser's  stain,  and  if  bacilli  are  present, 
with  characteristic  granules,  the  diagnosis  of  diphtheria  is 
most  probable.  Negative  results  are  not  to  be  depended  on. 
The  use  of  antiseptics,  gargles  or  the  failure  to  obtain  a  portion 
of  the  exudate  may  give  a  negative  culture  result  in  a  case  of 
diphtheria.  If  the  symptoms  are  suggestive,  it  is  best  to  use 
antitoxin  and  isolate  the  patient,  notwithstanding  a  negative 
report  from  the  laboratory.  If  there  are  no  clinical  signs, 
the  growth  should  be  tested  for  toxicity  by  inoculating  a 


DIPHTHERIA  BACILLUS  I3r 

guinea-pig;  it  should  be  grown  in  alkaline  sugar  bouillon  and 
tested  in  two  days  for  acid.  The  xerosis  and  Hoffman's 
bacilH  are  not  pathogenic  for  guinea-pigs. 

Products. — But  it  is  not  the  mere  presence  of  the  bacillus 
that  gives  rise  to  trouble:  certain  products  which  generate  it 
get  into  the  system  and  produce  the  severe  constitutional 
symptoms. 

Toxins  of  Diphtheria. — Roux  and  Yersin,  in  1888,  discov- 
ered the  toxin  and  showed  that  the  injection  of  the  filtered 
culture  bouillon  (that  is,  freed  of  all  diphtheria  bacilli)  gave 
rise  to  the  same  palsies  as  when  the  bacilli  themselves  were 
introduced. 

The  toxins  may  be  separated  from  three-weeks-old  bouillon 
cultures  by  filtration.  They  are  not  albumins  and  are  very 
complex.  Ehrlich  claims  three  forms:  one  he  calls  toxone; 
the  other,  toxin;  the  toxone  produces  paralytic  symptoms 
and  appears  to  be  less  affected  by  antitoxin;  the  third,  toxoid, 
combines  with  antitoxin.  The  toxins  are  highly  poisonous — 
o.ooi  c.c.  may  be  sufficient  to  kill  a  guinea-pig  in  less  than 
twenty-four  hours.  The  substance  is  unstable,  losing  its  toxic 
power  gradually.  Heating  at  58°  C.  for  two  hours  is  destruc- 
tive, but  drying  renders  it  more  stable.  Direct  sunlight 
destroys  its  power  in  a  few  hours.  Boiling  in  five  minutes. 
If  kept  cold  and  in  the  dark,  it  may  remain  active  two  years. 
Alcohol  and  calcium  chlorid  precipitate  the  toxic  element. 

Antitoxin. — Behring,  in  1890,  found  that  animals  rendered 
immune  had  a  principle  in  their  blood  that  was  antagonistic 
to  the  development  of  the  toxin. 

Immunity. — Brieger  and  Frankel,  by  injecting  10  to  20  c.c. 
of  a  three-weeks-old  culture  of  diphtheria  bacilli  which  had 
been  heated  at  70°  C.  for  one  hour,  produced  an  immunity  in 
guinea-pigs  against  the  virulent  form.  This  active  principle 
is  unknown  chemically,  but  has  been  called  antitoxin. 

The  toxin  generated  by  the  germ  is  supposed  to  be  neutral- 
ized by  the  antitoxin  and  prevented  from  injuring  the  body 
tissues.  The  value  of  antitoxin  in  diphtheria  seems  to  be 
established  beyond  a  doubt,  and  it  is  the  claim  of  eminent 


132  ESSENTIALS   OF   BACTERIOLOGY 

sanitarians  that  the  death-rate  from  this  disease  has  been  re- 
duced from  66  per  100,000  to  19  per  100,000  since  the  use  of 
antitoxin  (Park). 

The  strength  commonly  employed  in  human  beings  is  5000 
imits,  and  as  much  as  120,000  units  may  be  given  without 
detriment  in  severe  cases.  If  this  amount  is  injected  sub- 
cutaneously  and  even  intravenously  into  a  child  suffering 
from  diphtheria  in  the  earlier  stages  (second  to  third  day), 
the  disease  is  often  arrested.  The  membrane  begins  to  dis- 
appear, and  in  two  or  three  days  has  vanished.  The  con- 
stitutional symptoms  are  likewise  greatly  influenced  by  the 
injection.  For  prophylaxis  and  immunizing  well  persons 
1000  to  3000  units  are  employed. 

In  such  conditions  as  asthma  severe  and  fatal  results  have 
followed  the  use  of  the  serum,  and  some  cases  of  peculiar  sen- 
sitiveness to  horse  serum  (see  Anaphylaxis)  have  been  re- 
ported, fatal  results  having  occurred,  but  fortunately  such 
mishaps  are  exceedingly  rare. 

The  antitoxin  has  no  influence  on  the  bacteria  themselves; 
their  virulence  and  length  of  residence  in  the  body  are  not 
lessened. 

Preparation  of  Antitoxic  Serum. — Horses  are  rendered 
immune  by  gradually  increased  doses  of  diphtheria  toxin,  the 
power  of  the  toxin  having  first  been  standardized  by  its  neu- 
tralization with  some  standard  antitoxin  in  powdered  form. 

Preparation  of  Toxin. — ^The  bacillus  is  grown  in  veal  broth 
with  an  alkaline  reaction.  (Acids  prevent  toxin  formation.) 
There  should  be  a  free  supply  of  oxygen,  and,  therefore,  large 
shallow  flasks  are  used.  The  maximum  toxicity  is  developed 
in  seven  to  ten  days.  The  strength  should  be  -s^q-  c.c,  fatal 
for  500-gram  guinea-pig. 

The  toxin  is  at  first  injected  subcutaneously,  then  intraven- 
ously, and  after  several  months'  treatment  a  resistance  is  ob- 
tained that  will  withstand  300  to  500  times  the  original  lethal 
dose.  The  horse  is  then  bled,  and  from  five  to  nine  liters 
withdrawn;  this  is  then  allowed  to  coagulate,  and  under  very 
careful  precautions  the  serum  is  placed  in  sterile  packages, 


THE   COLON-TYPHOID   GROUP  I33 

its  strength  having  first  been  compared  with  a  standard  fur- 
nished by  the  United  States  Government.  Unless  kept  in 
the  dark  and  at  low  temperature,  it  loses  strength  rapidly. 

Antitoxic  Unit. — An  immunity  unit,  according  to  Ehr- 
lich,  is  the  amount  of  antitoxic  serum  which  will  neutral- 
ize 100  times  the  minimum  lethal  dose  of  toxin,  when  serum 
and  toxin  mixed  and  injected  into  a  250-gram  guinea-pig 
does  not  cause  death  in  four  days.  Thus,  if  the  serum  will 
protect  in  doses  of  -jV  c.c,  then  each  cubic  centimeter  has 
50  units'  power,  and  20  c.c.  will  contain  1000  units,  or  will  be 
sufficient  to  neutralize  an  amount  of  toxin  that  would  be 
fatal  for  25,000  kilos  (12,500  pounds)  of  guinea-pigs,  or 
100,000  pigs  weighing  250  grams  each.  The  serum  is  con- 
centrated by  precipitation  and  separation  from  the  blood- 
serum  of  the  pseudo-globulins  containing  the  antitoxic  prin- 
ciple, so  that  10  c.c.  contain  more  units  than  formerly.  The 
doses  given  now  are  much  larger  than  when  first  introduced. 
As  much  as  100,000  units  have  been  employed  in  a  single 
case.  (Sera  containing  1000  units  to  i  c.c.  are  now  being 
marketed.) 

Streptococcus  in  Diphtheria. — Streptococci  have  been 
found  quite  constant  in  diphtheria,  but  they  resemble  the 
Streptococcus  pyogenes,  and  have  no  specific  action. 


CHAPTER  XX 

THE  COLON-TYPHOID  GROUP 

In  this  group  are  placed  a  variety  of  organisms  similar  in 
form  and  growth  and  having  many  biologic  properties  in 
common,  but  differing  in  pathogenesis.  The  more  impor- 
tant members  of  this  group  are:  Bacillus  coli,  B.  typhosus ^ 
B.  enteritidis,  B.  dysenteries.  Another  closely  related  organism 
is  the  B.  suipestifer  (hog  cholera).  The  form  is  usually  a 
plump  rod  with  rounded  ends.     Gram-negative.     No  spores. 


134  ESSENTIALS    OF   BACTERIOLOGY 

Motile,  all  possessing  flagella,  on  gelatin  surface,  a  leaf -shaped, 
thin  colony.  They  all  reduce  nitrate  to  nitrite.  Gelatin  not 
liquefied.  Ferment  sugar  broth;  some  produce  acid  in  milk, 
some  do  not.     Some  form  gas  in  sugar,  some  not. 

Bacillus  Coll  (Escherich). — Synonyms. — Bacterium  colt 
commune;  Colon  Bacillus. — Found  (1886)  in  human  feces,  in- 
testinal canal  of  most  animals,  in  pus  and  water. 

Form. — Short  rods,  with  very  slow  movement,  often  asso- 
ciated in  little  masses,  resembling  the  typhoid  germ,  fiagel- 


Fig.  57. — Bacillus  coll  communis,  from  an  agar-agar  culture  (X  1000) 
(Itzerott  and  Niemann). 

lated,  not  forming  spores  (Fig.  57).     Very  short  round  ends; 
oval  forms  are  found  in  animal  tissues. 

Properties. — Does  not  liquefy  gelatin,  causes  fermentation 
in  saccharine  (dextrose)  solutions  in  the  absence  of  oxygen, 
forming  gas.  Two  parts  hydrogen  to  i  part  carbon  dioxid. 
Produces  acid  fermentation  in  milk;  coagulates;  its  optimum 
temperature  for  growth  is  37°  C;  causes  formation  of  indol  in 
peptone  solutions.  In  bouillon,  forms  cloudiness  with  shmy 
precipitate.     Some  cultures  non-motile. 


THE  COLON-TYPHOID   GROUP  I35 

Growth. — On  potato  a  thick,  moist,  yellow-colored  growth; 
on  agar  a  gray- white  growth;  on  gelatin  a  growth  similar  to 
typhoid.  It  can  also  develop  on  phenol-gelatin,  and  with- 
stands a  temperature  of  45°  C. 

Staining. — Ordinary  stains;    does  not  take  Gram. 

Pathogenesis. — Inoculated  into  rabbits  or  guinea-pigs, 
death  follows  in  from  one  to  three  days,  the  symptoms  being 
those  of  diarrhea  and  coma;  after  death  tumefactions  of 
Peyer's  patches  and  other  parts  of  the  intestine;  perforations 
into  peritoneal  cavity,  the  blood  containing  a  large  number  of 
bacilH. 

The  colon  bacillus  by  many  writers  is  held  responsible  for 
most  of  the  complications  of  typhoid  fever,  such  as  peri- 
tonitis, cholangitis,  etc. 

Epidemics  of  a  cholera  or  dysentery  nature,  called  by  Esche- 
rich  colitis  contagiosa,  and  due  to  infection  of  water  and  food, 
have  been  noted  by  a  number  of  writers.  The  onset  is  very 
sudden  and  prostrating,  though  not  fatal. 

Many  other  forms  of  suppuration  are  associated  with  the 
presence  of  Bacillus  coh. 

It  is  supposed  to  give  rise  to  cystitis,  infecting  the  bladder 
either  through  the  urethra  or  the  blood.  The  urine  is  then 
acid. 

Distribution. — ^The  bacillus  has  been  found  very  constant  in 
acute  peritonitis  and  in  cholera  nostras.  All  normal  persons 
harbor  the  B.  coli  in  the  intestine,  where,  under  ordinary  con- 
ditions, it  produces  no  disturbance.  After  death  it  multiplies 
rapidly,  invading  the  tissues. 

In  Water.— Tht  presence  of  B.  coli  in  surface  waters  is 
natural,  owing  to  contamination  with  the  fecal  discharges  of 
man  and  other  animals.  In  well-water  its  presence  denotes 
sewage  or  surface  contamination,  and  such  a  well  should  be 
condemned  until  free  from  coli.     (See  Water  Analysis.) 

Bacillus  of  Typhoid  or  Enteric  Fever  (Eberth-Gafifky). 
— Origin. — Eberth,  in  the  year  1880,  found  this  bacillus  in 
the  spleen  and  lymphatic  glands  of  persons  dying  of  typhoid, 
and  Gaffky  isolated  and  cultivated  the  organism  four  years 
later. 


136  ESSENTIALS   OF   BACTERIOLOGY 

Form. — Rods  with  rounded  ends  about  three  times  as  long 
as  they  are  broad.  Usually  solitary  in  tissue-sections,  but 
in  old  artificial  cultures  found  in  long  threads.  Flagella  on 
all  sides  (Fig.  58). 

Properties. — Very  motile.  Spores  have  not  been  found; 
they  do  not  liquefy  gelatin. 

Growth. — They  are  facultative  anaerobic;  grow  best  at  37° 
C,  but  can  also  develop  at  ordinary  room  temperature.  They 
develop  chiefly  on  the  surface,  and  very  slowly.     Repeated 


Fig.  58. — Bacillus  typhi,  from  an  agar-agar  culture  six  hours  old, 
showing  the  flagella  stained  by  Loffler's  method  (Xiooo)  (Frankel  and 
Pfeiffer). 

freezing  and  thawing  do  not  affect  the  vitality  of  the  germ, 
and  phenol  in  i  to  2  per  cent,  solution  has  no  effect  on  it. 
A  ten-minute  exposure  to  60°  C.  is  invariably  fatal. 

Colonies  on  Gelatin  Plates. — Two  forms:  the  ones  near  the 
surface  spread  out  like  a  leaf,  transparent,  with  bluish  fluor- 
escence. The  deeper  ones  resemble  whetstone  crystals  of 
uric  acid,  with  the  same  yellowish  tinge  (Fig.  59). 

In  five  days  they  attain  to  3  millimeters  in  diameter. 


THE   COLON-TYPHOID   GROUP 


137 


Bile  Salt  Media. — Rapid  growth  without  gas  formation;  a 
number  of  special  media  suited  for  the  growth  of  typhoid, 
namely,  Jackson's,  Hesse,  Hiss,  Conradi-Drigalski,  etc.  (See 
Water  Analysis  and  formula  for  Media.) 

Stab-cultures. — Mainly  on  the  surface,  a  pearly  layer. 

Agar  Stroke  Cultures. — A  transparent  thick  layer. 

Potato. — The  growth  here  is  quite  characteristic.  At  37° 
C.  in  forty-eight  hours  a  moist,  transparent  film  is  formed 
over  the  whole  surface,  but  so  transparent  that  it  can  hardly 
be  seen  without  close  observation.  If  a  small  portion  of  this 
is  placed  under  a  microscope,  it  will  be  seen  swarming  with 
bacilli  (Fig.  60). 

The  growth  never  becomes 
more  prominent;  the  potato 
must  have  a  neutral  or  acid 
reaction. 

On  Potato  Gelatin. — The  colo- 
nies do  not  have  the  yellow 
color;  they  are  transparent; 
later  on  they  become  dark 
brown  with  green  iridescence. 

Milk.  —  The  bacteria  grow 
very  well  in  milk,  producing  a 
slightly  acid  reaction,  but  no 
coagulation. 

Fermentation  Tube. — In  sugar 
broth,  in  the  fermentation  tube,  acids  are  formed  without  gas. 

Glucose  Gelatin. — In  glucose  gelatin  there  is  no  gas-produc- 
tion. Indol  is  likewise  not  generated  by  the  typhoid  bacillus, 
whereas  it  is  by  the  colon  bacillus. 

Staining. — Colored  with  the  ordinary  anilin  dyes,  when 
they  are  warmed;  since  they  are  easily  decolorized,  acids 
should  be  avoided.     Gram  negative. 

Distribution. — Outside  of  the  body  it  is  rarely  found. 
Typhoid  or  enteric  fever  is  a  general  infection,  but  affecting 
chiefly  the  Peyer's  patches  of  the  intestine.  The  bacilli  are 
found  in  the  intestinal  glands  and  in  the  enlarged  and  deeply 


Fig.  59. — Colonies  of  typhoid 
bacilli  three  daj's  old  (Xioo) 
(Frankel  and  Pfeiffer). 


138  ESSENTIALS   OF   BACTERIOLOGY 

congested  spleen.  Metastatic  abscesses  form  in  various  parts 
of  the  body,  and  here  likemse  the  organisms  abound.  They 
occur  in  the  feces  only  in  small  numbers,  more  commonly  in 
the  urine.  The  urine  may  contain  active  bacilli  for  weeks 
after  recovery  from  the  fever. 

Typhoid  Bacilli  in  Water. — Although  all  evidence  shows 
that  the  water-supply  is  a  frequent  source  of  infection,  very 
few  persons  have  ever  isolated  the  typhoid  bacillus  from  such 


Fig.    60. — Bacillus    typhosus.     Impression    preparation    from    gelatin 
plate.     Fuchsin  (Xiooo)  (Hicks). 

an  infected  source.  The  earlier  reports  show  that  no  account 
was  taken  of  Bacillus  coli,  which  is  usually  present  in  pol- 
luted waters.     (See  Water  Analysis.) 

Persistence  in  PFa/er.— Franckland  kept  baciUi  alive  in 
water,  sterilized  by  heat,  seventy-five  days;  in  filtered  water 
at  19°  C,  five  days;  at  6°  C,  twelve  days.  In  ordinary  water 
they  are  likely  to  be  destroyed  in  a  short  time  by  the  over- 
growth of  other  bacteria.  Under  ordinary  conditions  they 
do  not  multiply,  but  decrease  steadily  in  numbers.     In  soil 


THE   COLON-TYPHOID    GROUP  I39 

they  are  more  persistent.  Sewer-gas  or  air  is  never  a  source 
of  infection. 

Mode  of  Infection. — The  bacilU  in  the  dejecta  of  the  dis- 
eased person  find  their  way  into  drinking-water,  milk,  or 
dirty  clothes,  and  so  into  the  aUmentary  tract  of  a  person 
predisposed  to  the  disease.  Flies  act  as  conveyors  by  infect- 
ing food.  The  bacilli  enter  the  blood  through  the  lymphatics, 
and  so  become  lodged  in  various  organs?  They  are  quite 
resistant,  living  for  some  time  in  the  soil  and  water,  and  are 
more  resistant  than  other  organisms  to  the  action  of  phenol. 
An  epidemic  has  been  traced  to  the  eating  of  oysters  taken 
from  contaminated  water.  Milk-cans  washed  in  polluted 
water  may  be  the  origin  of  an  epidemic.    Ice  is  rarely  a  cause. 

Pathogenesis. — Lower  animals  do  not  have  enteric  fever, 
though  their  death  has  been  caused  by  injection  of  the  bacilli 
into  the  veins  of  the  ear  and  peritoneum  due  to  toxic  substance. 

In  man  the  bacillus  has  been  found  in  the  urine,  blood,  spu- 
tum, milk,  intestinal  discharges,  roseolar  spots,  and  in  various 
organs,  as  spleen,  Uver,  lymphatic  glands,  and  intestinal  \'ilU. 

It  is  found  in  secretions  several  days  after  the  attack  has 
subsided.     It  is  found  in  this  disease  only. 

Typhoid  Carriers. — Some  individuals  retain  a  culture  of 
the  bacilli  in  the  gall-bladder  for  years,  and  manufacture,  or 
at  least  expel,  true  virulent  bacilH  through  the  feces  and  urine 
intermittently.  Such  persons  have  infected  other  individuals 
without  suffering  any  inconvenience  themselves.  Some 
forms  of  chronic  inflammation,  as  cholecystitis  and  appen- 
dicitis, have  been  caused  by  the  typhoid  bacillus,  though 
more  often  the  colon  bacillus  is  found. 

Products. — Brieger  found  a  substance  in  the  cultures, 
which  he  named  typhotoxin,  with  the  formula  C9H17NO2. 
It  has  no  specific  action.  A  toxalbumin  insoluble  in  water  has 
also  been  isolated,  but,  as  experiment  animals  are  immune  to 
the  disease,  no  definite  actions  have  yet  been  determined. 

The  cultures,  when  old,  show  an  acid  reaction. 

Antityphoid  bacterins  (vaccines)  have  been  used  very  ex- 
tensively in  armies  and  institutions  as  a  prophylactic  or  pro- 


I40  ESSENTIALS   OF   BACTERIOLOGY 

tective.  Bacterins  are  made  from  a  weakly  virulent  culture. 
The  results  so  far  obtained  would  indicate  that  this  inocula- 
tion has  some  value,  but  the  evidence  is  far  from  conclusive 
and  the  statistics  on  the  subject  require  more  careful  study 
before  they  can  be  accepted  as  positive  proof.  An  eighteen- 
hour  agar  surface  growth  is  w^ashed  in  sterile  salt  solution 
and  killed  by  heating  at  56°  C.  one  hour.  It  is  then  diluted 
so  as  to  contain  one  biUion  bacilli  to  i  c.c.  Tricresol,  0.25  per 
cent.,  is  added  to  preserve,  and  animals  tested  for  purity  of  the 
vaccine.  A  slight  local  reaction  follows  the  inoculation; 
about  three  injections  of  >^,  i,  and  one  billion  bacilli  at  ten- 
day  intervals,  render  the  subject  immune.  General  symp- 
toms rarely  occur. 

The  Gruher-Widal  Blood- serum  Test. — In  1896  Widal  and 
Griinbaum,  working  separately,  developed  what  is  now  spoken 
of  as  the  ''Widal  serum  test,"  or  ^^ Widal  reaction,'''  or  aggluti- 
nation test.  It  consists  in  testing  a  drop  of  blood  of  a  patient 
suspected  of  having  typhoid  fever,  by  mixing  a  dilution  of 
it  with  a  drop  of  a  fresh  bouillon  culture  of  typhoid  bacilli, 
and  examining  the  mixture  in  a  hanging  drop  under  the 
microscope.  Within  fifteen  minutes  to  an  hour  the  motility 
of  the  bacilli  will  cease,  and  they  will  have  arranged  them- 
selves into  clusters,  as  if  stuck  or  glued  together  (Fig.  61). 
If  this  reaction  occurs  within  an  hour,  and  with  the  proper . 
dilution  of  the  serum,  the  patient  has  or  has  had  typhoid 
fever.  Widal  first  used  the  serum  of  the  blood;  this  has 
been  modified  so  that  a  drop  of  dried  blood  is  sufficient. 

Method  of  Test. — The  method  as  applied  in  city  laboratories 
is  as  follows:  The  physician  is  told  to  clean  the  finger  of  the 
patient  with  water  (no  germicides) ,  and  with  a  needle  draw  a 
drop  of  blood  on  to  a  piece  of  ordinary  note-paper.  This  is  - 
then  sent  to  the  laboratory;  the  paper  with  the  dried  blood 
is  soaked  for  a  few  minutes  in  a  watch-glass  containing  4 
drops  of  clean  water,  thus  obtaining  a  dilution  of  i  15.  One 
drop  of  this  is  then  mixed  with  one  drop  of  a  bouillon  culture 
of  typhoid  bacilli  of  about  twenty-four-hours'  growth,  and 
examined  under  the  microscope  in  the  hanging  drop.     Weaker 


THE    COLON-TYPHOID    GROUP  I4I 

dilutions  of  the  serum  have  been  recommended  (i  :  50),  and 
this  should  be  used  in  cases  of  doubt.  So  far,  about  95  per 
cent,  of  the  cases  examined,  and  which  clinically  were  con- 
sidered typhoid  fever,  have  given  a  positive  reaction.  It  is 
not  often  present  until  the  fifth  day  of  the  fever,  and  dis- 
appears usually  within  a  year,  though  in  some  individuals  it 
has  been  found  ten  years  after  an  attack  of  the  disease. 

The  agglutinating  properties  have  been  found  in  nearly  all 
the  secretions  of  the  body — tears,  urine,  milk,  pleuritic  effu- 
sions serous  fluid  from  blisters,  etc. 


Fig.  61. — The  Widai  agglutination  reaction  (Slater  and  Spitta). 

There  is  no  relation  between  the  reaction  and  the  bacteri- 
cidal power  of  the  serum;  the  agglutination  is  not  a  destruc- 
tion. The  agglutinating  power  is  active,  though  the  blood  be 
dried  and  sealed  up  for  months.  It  seems  to  have  no  direct 
relation  with  the  question  of  immunity,  since  it  occurs  at  the 
height  of  the  disease,  and  intense  agglutinating  serum  may 
be  had  in  severe  cases  and  in  cases  with  relapses.  A  negative 
result  does  not  exclude  typhoid. 

The  test  is  quantitative — i.  e.,  it  depends  upon  the  dilution 
of  the  blood-serum,  since  the  serum  of  healthy  persons  in 
strong  dilution  will  cause  agglutination  and  loss  of  motility. 


142  ESSENTIALS   OF   BACTERIOLOGY 

A  serum  in  a  dilution  of  i  :  loo  causing  complete  clumping 
in  half  an  hour  is  undoubtedly  typhoid. 

The  culture  must  be  kept  in  a  vigorous  condition  by  fre- 
quent subplanting,  and  must  be  tested  occasionally  with 
normal  serum.  Cultures  kept  in  an  incubator  for  a  long  time 
tend  to  agglutinate  spontaneously. 

Macroscopic  Agglutination  Test. — Where  laboratory  facili- 
ties are  not  available,  the  sedimentation  test  is  practical. 
It  consists  in  adding  the  diluted  blood  or  serum  to  be  tested 
to  a  suspension  of  dead  typhoid  bacilli  in  salt  solution.  If 
the  reaction  is  positive,  a  flocculent  precipitate  forms  which 
consists  of  masses  of  agglutinated  bacilli.  A  control  tube  con- 
taining normal  serum  and  the  suspension  should  remain 
opaque  and  show  no  flocculi. 

Differentiation  Between  Colon  and  Tjrphoid. — The 
colon  bacillus  and  the  typhoid  bacillus  resemble  each  other 
so  closely  that  much  attention  has  been  paid  to  methods  of 
differentiation. 

Points  of  Resemblance  Between  Bacillus  Typhi  and  Bacillus 
Coli  Communis. — First, microscopic  appearance;  second,  agar 
and  gelatin  cultures;  third,  sometimes  growth  on  potato  the 
same;  fourth,  staining  peculiarities;  fifth,  resistance  to  phenol. 

Points  of  Difference: 

Colon  Bacillus.  Typhoid  Bacillus. 

Bile  media,  gas.  None. 

Less  motile.  Actively  motile. 

Gelatin    colonies    develop  Develop  slowly. 

more  rapidly. 

Produces  gas  on  dextrose  or  Does  not. 

lactose  media. 

Coagulates  milk.  Does  not. 

Produces  indol.  Does  not. 

Growth  on  potato  visible.  Invisible. 

Changes    neutral    red    to  Does  not  reduce  neutral  red. 

yellow. 

Endo-fuchsin  red.  Not. 


THE   COLON-TYPHOID   GROUP  I43 

Differences  are  also  noted  in  the  growth  on  special  media, 
such  as  those  of  Hiss  and  Eisner.  On  Eisner's  potato-gelatin 
the  colon  bacillus  and  the  typhoid  bacillus  both  grow  read- 
ily. The  medium  of  Hiss  is  of  some  assistance  in  isolating 
the  germ.      (See  p.  75.) 

Hiss  Media. — Show^s  B.  colt,  large  colony,  even  borders. 
B.  typhi,  small  colony,  hairy  and  fringy  threads.     (See  p.  75.) 

Endo-fuchsin-lactose  Agar  (see  p.  77). — Incubation  on 
plates  of  this  media  shows  B.  coli  red;  typhoid  as  clear,  color- 
less drops. 

Malachite  green  added  to  agar  permits  the  growth  of  B, 
typhi,  but  not  B.  coli.  The  dye  must  be  as  nearly  neutral  as 
possible. 

Bile  Salt  Media. — Fresh  bile  or  sodium  taurocholate  added 
to  lactose  glucose  agar  or  broth  permits  the  rapid  growth  of 
both  B.  typhi  and  B.  coli,  fermentation  with  gas  formation 
denoting  B.  coli,  growth  without  fermentation  meaning 
typhoid. 

Drigalski  and  Conradi  Media. — Petri  plates  filled  with 
this  media  are  inoculated  on  the  surface  only.  Placed  in 
incubator  sixteen  to  twenty-four  hours.  Typhoid  colonies 
small,  transparent,  and  blue.  Colon  colonies  red,  coarser, 
and  larger. 

Typhoid  Bacilli  from  Blood. — Conradi,  Busquet,  Coleman 
and  Buxton,  and  others  have  found  the  bacilli  in  the  blood  of 
every  patient  by  the  following  method:  A  mixture  of  ox-bile, 
90  c.c,  glycerin,  10  c.c,  and  peptone,  2  gm.,  is  distributed 
into  20  c.c.  flasks  and  sterilized.  Ten  cubic  centimeters  of 
blood  is  drawn  from  the  elbow  into  a  glass  syringe  and  divided 
among  three  flasks.  These  are  incubated,  and  in  twenty-four 
hours  litmus-lactose- agar  plates  are  inoculated  on  the  surface 
by  a  stroke  from  the  flasks.  A  growth  is  obtained  in  five  or 
six  hours. 

If  the  growth  is  a  bacillus  which  has  not  reddened  the 
medium,  it  is  tested  for  the  Widal  reaction  with  immune  se- 
rum.    The  diagnosis  has  been  made  as  early  as  the  second  day. 

Paracolon  or  paratyphoid  bacilli  are  members  of  the 


144  ESSENTIALS   OF   BACTERIOLOGY 

colon  group  described  by  Widal,  Gwyn,  Schottmliller,  and 
others.  They  are  of  importance,  since  they  produce  fevers 
clinically  resembling  a  mild  form  of  typhoid,  but  which  are 
rarely  fatal.  They  may  be  the  sole  cause  of  the  disease,  and 
also  occur  together  with  the  typhoid  bacillus  in  mixed  arid 
secondary  infections.  Morphologically,  they  resemble  the 
typhoid  bacillus,  but  differ  from  it  culturally  and  give  their 
own  serum  reactions  with  the  blood  of  affected  patients. 
They  ferment  glucose,  but  not  lactose  or  saccharose;  litmus 
milk  at  first  becomes  acid,  but  later  grows  alkaline  and  is  not 


Fig.    62. — Bacillus    botulinus,    with    spores.     Pure    culture    on    sugar- 
gelatin.     Van  Ermengem  prep.  (Kolle  and  Wassermann) . 


coagulated.  On  potato  a  slight  visible  growth  occurs;  in- 
dol  is  usually  not  formed.  Typhoid  sera  do  not  agglutinate 
paracolon  bacilli,  and  vice  versa;  also  different  paracolon 
infections  may  not  agglutinate  each  other. 

Bacillus  Botulinus  (Van  Ermengem). — An  anaerobic  ba- 
cillus cultivated  by  Van  Ermengem  in  1896  from  ham  which 
had  caused  poisoning. 

Form. — A  large  bacillus  with  rounded  or  spindle-shaped 
ends,  and  often  with  oval  terminal  spores,  motile,  with  lateral 
flagella  (Fig.  62). 


THE    COLON-TYPHOID   GROUP  I45 

Staining. — Gram  positive,  easily  stained  with  ordinary 
dyes. 

Growth. — Strictly  anaerobic.  Forms  abundant  gas  in  glu- 
cose, gelatin,  and  liquefies  cultures,  producing  butyric  acid 
odor.     Best  temperature  between  20°  and  30°  C. 

Pathogenesis. — Produces  a  powerful  toxin  in  the  tissues, 
like  the  tetanus  bacillus.  This  toxin  may  be  present  in  the 
affected  meat  without  causing  decomposition,  and  thus  give 
rise  to  poisoning. 

Bacillus  Dysenteriae  (Shiga,  1898). — The  term  dysen- 
tery is  applied  to  an  intestinal  disease  displaying  more  or  less 


Fig.  63. — Bacillus  dysenteriae  from  agar  culture.     Fuchsin  stain.     Zett- 
novv  prep.  (KoUe  and  Wassermann) . 

constancy  in  its  clinical  manifestations,  but  having,  as  is  now 
known,  a  variety  of  causative  agents.  It  is  fairly  certain  that 
one  type  is  the  result  of  infection  with  an  ameba,  while  non- 
amebic  forms  can  probably  be  produced  by  several  bacteria. 
Chief  among  these  is  the  bacillus  first  described  by  Shiga  in 
Japan,  and  since  then  found  by  Kruse  in  Germany,  by  Flex- 
ner,  Strong,  and  Harvie  in  the  Philippine  Islands,  and  by 
Vedder  and  Duval  in  the  United  States.  The  fact  that  it  is 
constantly  present  in  the  feces  in  one  type  of  dysentery,  that 


146  ESSENTIALS   OF   BACTERIOLOGY 

such  cases  give  a  positive  agglutination  reaction,  the  produc- 
tion of  a  curative  serum  by  the  immunization  of  animals 
with  pure  cultures,  and  the  results  on  experiment  animals, 
leave  little  doubt  as  to  the  specificity  of  the  organism. 

Origin. — The  dejecta  of  dysenteric  patients. 

Form. — ^A  plump  bacillus  with  rounded  ends,  resembling 
the  typhoid  and  colon  bacilli  (Fig.  63). 

Properties. — Motility  doubtful,  but  numerous  flagella  have 
been  demonstrated.     Does  not  form  spores. 

Staining. — Stains  readily,  negative  to  Gram;  facultative 
anaerobe. 

Growth. — Best  at  37°  C.  Killed  by  ten  minutes'  exposure 
to  55°  C. 

Gelatin. — ^A  white  line  of  growth  along  puncture;  super- 
ficial growth  slight. 

Bouillon. — Uniform  clouding.  Indol  usually  not  produced; 
milk  not  coagulated. 

Agar. — Resembles  typhoid  bacillus. 

Potato. — Thin  whitish  layer,  turning  light  brown. 

No  gas-formation  in  glucose  or  lactose  media. 

Acid  is  formed. 

Pathogenesis. — Mice  and  guinea-pigs  die  in  one  or  two  days 
after  intraperitoneal  inoculation.  Rabbits  usually  recover, 
though  lesions  analogous  to  those  of  human  dysentery  have 
been  produced.  Dogs  die  in  five  or  six  days,  with  well- 
marked  diarrhea. 

Products. — The  patient's  blood-serum  agglutinates  the  ba- 
cillus in  cases  in  which  it  can  be  cultivated  from  the  stools. 
The  reaction  is  absent  from  other  cases.  Shiga  has  reduced 
the  mortality  from  34.7  to  19  per  cent,  by  means  of  a  serum 
obtained  from  immunized  horses,  but  in  more  extensive  tests 
the  antidysenteric  serum  proved  of  little  value. 

In  man  the  organism  or  some  of  its  varieties  is  associated 
with  dysentery  and  is  found  chiefly  in  the  stools;  abscesses 
are  seldom  found;  the  amebic  dysentery  forms  liver  abscess, 
not  in  other  organs.  Polluted  water  is  responsible  for  its 
spread  in  epidemic  form. 


THE    COLON-TYPHOID    GROUP  I47 

In  the  summer  diarrhea  of  infants  associated  with  mucus, 
B.  dysenterice  has  been  found,  and  is  considered  a  causative 
agent. 

Bacterium  Termo  (Cohn). — This  was  a  name  given  to  a 
form  of  microorganism  found  in  decomposing  albuminous 
material,  and  was  supposed  to  be  one  specific  germ.  Hauser, 
in  1885,  found  three  different  distinct  bacilli  which  he  grouped 
under  the  common  name  of  proteus,  which  have  the  putrefy- 
ing properties  ascribed  to  Bacillus  termo. 

Bacillus  Proteus  Vulgaris  (Hauser,  1885). — Origin. — 
In  putrid  animal  matter,  in  the  feces,  and  in  water. 

Form. — Small  rods,  slightly  curved,  of  varying  lengths, 
often  in  twisted  chains,  having  long  cilia  or  flagella. 

Properties. — Very  motile,  and  very  soon  liquefying  gelatin; 
forms  hydrogen  sulphid  gas;  causes  putrefaction  in  meat. 

Growth. — Growth  very  rapid,  best  at  24°  C;  is  facultative 
aerobic. 

Gelatin  Plates. — Yellowish-brown,  irregular  colonies,  with 
prolongations  in  every  direction,  forming  all  sorts  of  figures; 
an  impression  preparation  shows  these  spider-leg  processes  to 
consist  of  bacilli  in  regular  order. 

Stab-culture. — The  gelatin  soon  liquid,  a  gray  layer  on  the 
surface,  but  the  chief  part  of  the  culture  in  small  crumbs  at 
the  bottom. 

Agar. — Rapid,  moist,  gray  growth. 

Milk. — Acid  coagulation. 

Dextrose  Broth. — Gas-production. 

Pathogenesis. — Rabbits  and  guinea-pigs  injected  subcutane- 
ously  die  quickly;  a  form  of  toxemia,  hemorrhagic  condition 
of  lungs  and  intestines,  present.  When  neurin  is  injected 
previously,  the  animals  do  not  die.  This  ptomain  is  sup- 
posed to  be  generated  by  the  Proteus  vulgaris. 

In  man  these  or  similar  bacteria  have  been  associated  with 
food-poisoning  epidemics,  infantile  diarrhea,  infectious  jaun- 
dice (Weil's  disease). 

Proteus  Mirabilis  (Hauser). — Differs  from  Proteus  vul- 


148  ESSENTIALS   OF   BACTERIOLOGY 

garis  in  that  the  gelatin  is  less  rapidly  liquefied.     Found  also 
in  putrid  material. 

Proteus  Zenker!  (Hauser). — Does  not  liquefy  gelatin: 
otherwise  similar  to  the  other  two. 


CHAPTER  XXI 

CHOLERA  BACTERIA 

Spirillum  Cholerae  (Koch)  (Comma  Bacillus  of  Chol- 
era) . — Synonym,  Vibrio  CholercB. — Origin. — Koch,  as  a  mem- 
ber of   the   German  expedition   sent  to  India  in    1883   to 
,^^  ^^  study    cholera,    found    this    micro- 

'^•i^^^^^^^  organism  in  the  intestinal  contents 

l^^^^j^^  ^^/^^S^  of  cholera  patients,  and  by  further 
Jt»'^&^^''\^^%  A  experiments  identified  it  with  the 
^^     "^^-v^^^'^*-^       disease. 

*^^f\  mS^^^tT-  Fortn. — ^The  spirillum  as  seen  or-, 

'^^^^t'i^^'^^^  '         dinarily  appears  as  a  shorty  arc-like 
^"'"     '  '■"^  body,  about  half  the  size  of  a  tuber- 

Fig.  64.— Comma  bacillus,  cle  bacillus,  but  when  seen  in  large 
pure  culture  (X  600).  groups,  spirals  are  formed,  each  little 
arc  appearing  then  as  but  a  segment, 
a  vibrio.  Each  arc  is  about  three  times  as  long  as  it  is  broad, 
and  possesses  a  flagellum  at  one  end.  Old  agar  cultures  show 
straight  forms;  S-shaped  forms  not  uncommon,  made  of  two 
vibrios  end  to  end  (Fig.  64). 

Properties. — The  spirilla  are  very  motile;  liquefy  gelatin. 
They  are  easily  affected  by  heat  and  dryness.  Spores  have 
not  been  found. 

Growth. — ^At  ordinary  temperatures  on  all  nutrient  media 

that  have  an  alkaline  or  neutral  reaction.     Strongly  aerobic. 

Colonies,  Gelatin. — After  twenty-four  hours,  small  w^hite 

points  which  gradually  come  to  the  surface,  the  gelatin  being 


CHOLERA   BACTERIA 


149 


slowly  liquefied,  a  funnel-shaped  cavity  formed,  holding  the 
colony  in  its  narrow  part,  at  the  bottom,  and  on  the  fifth  day 
all  the  gelatin  is  liquid.  If  the  colonies  of  three  days'  growth 
are  placed  under  microscope,  they  appear  as  if  composed  of 
small  bits  of  frosted  glass  with  sharp,  irregular  points. 

Stab-culture. — After  thirty  hours  a  growth  can  be  distin- 
guished along  the  needle- track,  and  on  the  surface  a  little 
cavity  is  formed,  filled  by  a  bubble  of  air,  and  this  liquefaction 


Fig.    65. — Cholera  colonies   after   thirty  hours   (Xioo)    (Frankel  and 

Pfeiffer). 


proceeds  until,  on  the  sixth  day,  it  has  reached  the  sides  of 
the  tube,  tapering,  funnel-shaped,  to  the  bottom  of  the  tube. 
After  several  weeks  the  spirilla  are  found  in  little  collections 
at  the  bottom  of  the  fluid  gelatin.  In  eight  weeks  the  bacilli 
have  perished. 

Agar. — Stroke  cultures.  A  shiny  white  layer  which  lasts 
many  months. 

Alkaline  Agar. — Plates  at  37°  C.  Flat  discs,  transparent, 
grayish  blue. 


ISO 


ESSENTIALS   OF  BACTERIOLOGY 


Potato. — A  yellow,  honey-like,  transparent  layer  if  the 
potato  is  kept  at  animal  heat. 

Bouillon. — A  wrinkled  scum  is  soon  formed  in  bouillon. 
The  spirilla  live  well  and  grow  in  sterilized  milk  and  sterilized 
water,  remaining  virulent  in  the  latter  for  many  months. 


Fig.  66. — Cholera 
bacillus  (forty-eight 
hours;  5  per  cent, 
gelatin). 


Fig.  67. — Cholera 
bacillus  (sixty  hours; 
5  per  cent,  gelatin). 


Fig.  68.— Cholera 
bacillus  (seventy- 
two  hours;  15  per 
cent,  gelatin). 


Figs.  66-68. — Tube-cultures  (from  United  States  Government  Report 
on  Cholera. — Shakespeare) . 


In  ordinary  water  the  bacteria  present  are  destructive  to  the 
comma  bacilli,  and  they  die  in  a  few  days. 

Dunham^ s  Peptone  Solution. — Useful  for  the  development  of 
nitrites  and  the  indol  reaction.  (See  p.  77.)  Also  for  the 
rapid  development  of  the  cholera  vibrio.  In  four  hours  after 
inoculation  of  peptone  water  pure  cultures  may  be  obtained; 


CHOLERA  BACTERIA  15I 

best  to  make  several  plantings  from  the  peptone  to  agar  after 
six  hours'  growth. 

Dieudonne's  Medium. — (See  p.  77.)  In  this  cholera  vibrio 
grow  abundantly;  other  intestinal  bacteria  very  scantily. 
This  medium  valuable  mostly  for  feces,  less  for  infected  water. 

Staining. — They  are  colored  well  with  watery  anilin  solu- 
tions. The  fiagella  can  be  well  seen  by  staining  according  to 
the  fiagella  stain  or  Giemsa. 

Pathogenesis. — Experiment  animals  are  not  subject  to 
cholera  Asiatica,  but,  by  overcoming  two  obstacles,  Koch 
produced  choleraic  symptoms  in  guinea-pigs.  Nicati  and 
Rietsch  prevented  peristalsis  and  avoided  the  acidity  of  the 
stomach-juices  by  direct  injection  into  the  duodenum,  after 
tying  the  gall-duct.  Koch  alkalinized  the  gastric  juice  with 
5  c.c.  of  5  per  cent,  solution  of  sodium  carbonate,  and  then 
injected  2  grams  of  opium  tincture  for  every  300  grams  of 
weight  into  the  peritoneal  cavity,  paralyzing  peristalsis.  The 
cholera  culture  then  introduced  through  a  stomach-tube,  the 
animals  die  in  forty-eight  hours,  presenting  the  same  symp- 
toms in  the  appearance  of  the  intestines  as  in  man,  the  serous 
effusion  containing  great  numbers  of  spirilla.  Rabbits  in- 
jected into  the  ear  veins  with  cholera  cultures  die  very  quickly 
and  present  intestinal  lesions.  The  vibrio  is  met  with  in  the 
layer  of  flaky  mucus  which  coats  the  surface  of  the  intestine. 

It  may  invade  the  biliary  passages. 

Manner  of  Infection  in  Man. — Usually  through  the  alimen- 
tary tract,  with  the  food  or  drink,  the  intestinal  discharges  of 
cholera  patients  having  found  entrance  into  the  source  of 
drinking-water.  Soiled  clothes  to  fingers,  fingers  to  the 
mouth,  etc.;  torpid  catarrhal  affection  of  the  digestive  tract 
predisposing.  The  spirilla  are  not  foimd  in  the  blood  or  any 
organ  other  than  the  intestines — the  tissue  of  the  small  intes- 
tines. They  are  also  found  in  the  vomit  and  the  intestinal 
contents. 

Toxins. — From  broth  cultures  soluble  toxins  which  have  a 
hemolytic  action  have  been  isolated.  The  toxin  is  easily 
destroyed  by  heat  (thermolabile). 


152  ESSENTIALS   OF   BACTERIOLOGY 

Products — ^^ Cholera  red.^'  Indol  Reaction. — Present  in 
peptone  water  cultures  containing  nitrates.  The  indol  is 
shown  by  the  addition  of  a  few  drops  of  pure  sulphuric  acid, 
the  solution  turning  red — the  so-called  ^'cholera  red.''  Once 
thought  distinctive,  but  other  bacteria  also  give  rise  to  indol, 
and  the  same  reaction. 

Serum  Agglutination  Test. — The  agglutination  test  is  made 
in  the  same  way  as  the  Widal  test  for  typhoid  fever.  Ag- 
glutinins appear  in  the  blood  five  to  ten  days  after  infection. 


Fig.  67. — Comma  bacillus  in  mucus,  from  a  case  of  Asiatic  cholera. 

Cultures  in  serum  dilutions  of  1:1000  up  to  1:10,000  are 
agglutinated. 

Detection  of  Cholera  Organisms  in  Drinking-water. — ^When 
a  few  bacteria  are  supposed  to  be  present  in  fecal  matter  or 
drinking-water,  it  is  best  to  add  a  large  quantity  of  the  ma- 
terial (200  c.c.  of  drinking-water)  to  about  10  c.c.  of  bouillon 
or  peptone-water,  and  place  the  mixture  for  twenty-four  hours 
in  an  incubator,  which  will  cause  rapid  reproduction,  and  then 
the  organisms  can  be  readily  discovered. 


CHOLERA   BACTERIA  1 53 

From  Feces. — The  following  technic  is  recommended: 

1.  Examine  mucous  flakes  in  stained  preparations  and 
hanging  drop. 

2.  Isolate  on  agar  media  at  37°  C. 

(a)  Plant  plates  of  alkalinized  agar  and  Dieudonne's 
medium  with  particles  of  feces. 

(b)  Plant  in  50  c.c.  peptone  solution  i  c.c.  fecal  matter. 
After  six  hours  or  longer  in  the  incubator  at  37°  C. 
take  several  loopsful  from  the  surface  and  plant  on 
several  plates  Dieudonne's  and  ordinary  alkaline  agar. 

(c)  Investigate  agglutination  reaction,  using  drops  from 

isolated  colonies  and  secure  pure  cultures. 

3.  Demonstrate  the  reaction  of  Pfeiffer  and  agglutination 
with  the  pure  colonies. 

Protective  Bacterins. — ^Virulent  cultures  killed  by  heat  have 
shown  protective  power  and  were  used  extensively  during  an 
epidemic  in  Japan. 

Haffkine  has  obtained  a  great  reduction  in  mortality  in 
cholera  regions  by  the  use  of  anticholera  bacterins  as  a  pro- 
tective measure. 

Serum  therapy  has  not  been  successful. 

Carriers. — In  some  recent  examinations  of  persons  exposed 
to  cholera,  carriers  of  typical  cholera  vibrio  have  been  found. 
The  vibrio  may  be  found,  as  in  typhoid  fever,  in  the  gall- 
bladder. 

Pfeifer^s  Reaction  and  Agglutination. — The  serum  of  an 
animal  (a  rabbit)  made  iromune  against  cholera  by  the  in- 
jection of  sterile  or  living  cultures,  intravenously,  three  times 
at  intervals  of  a  week,  has  an  action  against  the  cholera  spiril- 
lum. It  first  precipitates  the  bacteria  out  of  an  emulsion,  leav- 
ing a  clear  liquid  (agglutination),  and  then  dissolves  the  bac- 
teria (bacteriolytic  action),  leaving  only  spheric  granules. 
This  action  is  specific,  i.  e.,  the  cholera  immune  sera  will 
affect  only  cholera  vibrio.  Such  serum  is  not  antitoxic,  it 
is  bacteriolytic.  For  diagnostic  purposes  an  agglutination 
in  dilution  of  i  :  1000  by  a  serum  with  an  activity  of  i  :  4000 
is  suspicious  of  cholera.     The  blood-serum  of  convalescents 


154  ESSENTIALS    OF   BACTERIOLOGY 

and  cholera-vaccinated  individuals  contains  the  same  bac- 
tericidal substances. 

Allied  Varieties. — Many  vibrios  resembling  the  spirillum  of 
cholera  have  been  isolated  from  drinking-waters  and  from 
the  stools  of  persons  suffering  with  diarrhea,  and  some  bac- 
teriologists are  inclined  to  consider  them  as  varieties  of  the 
true  cholera  spirillum,  which  under  certain  conditions  become 
pathogenic.  Among  these  are  Spirillum  berolinense,  S.  dun- 
barii,  S.  danubicum,  S.  of  Wernicke,  S.  bonhoffii,  S.  weibeH,  S. 
schuylkilliensis,  S.  milleri,  S.  aquatilis.  The  last  two  are 
non-pathogenic  for  experiment  animals,  also  the  Finkler- 
Prior  vibrio,  \dbrio  Metchnikovii,  and  tyrogenum,  which  have 
historic  interest  because  of  their  close  identity  with  the 
cholera  organism,  but  with  the  agglutination  tests  and 
Pfeiffer  phenomenon  they  have  been  shown  to  be  dissimilar. 

Conclusions  of  International  Committee  of  Public  Hygiene, 
Adopted  October  p,  igii. — Every  choleriform  vibrio  can  be 
considered  as  truly  choleraic  which  presents  agglutination  in 
the  proportion  of  at  least  i:iooo  by  a  cholera  serum  of 
1:4000  activity,  or  a  positive  Pfeiffer  reaction,  and  every 
choleriform  affection  in  which  is  encountered  such  a  vibrio 
should  be  considered  as  a  case  of  cholera. 

Method  of  Pfeifer. — i.  Secure  immune  serum  by  injecting 
into  peritoneal  cavity  of  a  rabbit  an  entire  agar  culture  which 
has  been  killed  by  heating  for  one  hour  at  56°  C.  Four- 
teen days  after  collect  the  blood-serum. 

2.  Dilute  the  suspected  vibrio  by  adding  one  loopful  of  an 
eighteen-hour-old  agar  culture  to  i  c.c.  meat  water. 

3.  Add  to  the  above  about  i  milligram  of  immune  serum 
and  inject  this  into  peritoneal  cavity  of  a  guinea-pig. 

4.  At  the  same  time  a  second  guinea-pig  is  inoculated  with 
diluted  culture  (2),  but  without  the  serum. 

5.  A  third  guinea-pig  is  inoculated  with  a  similar  dilution 
of  culture  to  which  has  been  added  about  10  milligrams 
normal  rabbit  serum. 

6.  At  the  end  of  twenty  minutes,  and  again  at  the  end  of 
one  hour,  some  of  the  peritoneal  fluid  is  examined  from  each 


BACTERIA  IN  PNEUMONIA  15$ 

pig,  under  strong  magnification,  in  hanging  drop  and  dark 
field  illumination. 

7.  The  reaction  is  positive  if  iii  the  fluid  from  No.  i  pig 
the  vibrios  are  dissolved,  while  in  that  from  No.  2  arid  No.  3 
the  vibrios  are  very  motile  and  active  and  form  well  pre- 
served. 

It  is  necessary  that  the  vibrio  be  of  good  virulence. 

Method  of  Bordet. — As  experiment  animals  are  not  always 
available,  Bordet  has  elaborated  a  test-tube  method.  The 
immune  serum  is  diluted  1:50,  1:100,  1:500,  and  1:1000. 
Into  a  series  of  test-tubes  there  are  poured  5  drops  of  a  guinea- 
pig  serum,  5  drops  of  a  mixture  of  suspected  culture  (one  loop- 
ful  of  an  eighteen-hour-old  agar  culture  to  i  c.c.  salt  solution), 
and  enough  of  immune  serum  and  salt  solution  to  make  the 
necessary  dilution  and  up  to  20  drops.  A  series  of  controls 
is  made  with  normal  serum  and  the  same  amount  of  microbic 
culture  and  guinea-pig  serum. 

After  eighteen  hours  the  cholera  vibrios  will  be  active  in  the 
control,  but  dissolved  and  clumped  up  in  the  tubes  containing 
the  immune  serum. 


CHAPTER  XXII 
BACTERIA  IN  PNEUMONIA 

Klebs  in  1875  called  attention  to  the  presence  of  bacteria  in 
pneumonia,  and  in  1882  Friedlander  developed  a  bacillus  from 
the  lung  tissue  of  a  pneumonic  person  which  he  thought  was 
a  coccus,  and  called  it  pneumococcus. 

In  1886  A.  Frankel  and  Weichselbaum  proved  that  this 
organism  was  not  constant — in  fact,  was  rare. 

A.  Frankel  obtained  in  the  majority  of  cases  of  pneumonia 
an  organism  that  he  had  described  in  1884  under  the  name 
of  sputum-septicemia  micrococcus. 

Weichselbaum  called  this  Diplococcus  pneumonic^,  and  be- 


156 


ESSENTIALS    OF   BACTERIOLOGY 


Fig.  70. — Bacillus  pneumuniic  ui  1-  nediaiKler,  from  the  expectoration  of  a 
pneumonia  patient  (Xiooo)  (Frankel  and  Pfeiffer). 


m 


•*  • 


V 


Fig.  71. — Diplococcus  pneumonias  in  exudate  from  human  lung;  anilin- 
water-fuchsin;  Weichselbaum  prep.  (Kolle  and  Wassermann). 


BACTERIA  IN  PNEUMONIA 


157 


lieved  it  to  be  the  real  cause  of  pneumonia.  It  is  the  generally 
accepted  organism  of  the  disease,  and  can  be  isolated  from 
nearly  all  cases  of  acute  croupous  pneumonia.  It  is  found  in 
about  three-quarters  of  all  cases  of  pneumonia. 

Diplococcus  Pneumoniae  (Frankel  and  Weichselbaum, 
1 886) . — Synonyms. — Streptococcus  Lanceolatus;  Pnemno  coccus; 
Diplococcus  Lanceolatus;  M.  of  Sputum  Septicemia;  Fr ankers 
Fneumococcus. 

Origin. — Found  it  in  the  sputum  of  pneumonic  patients. 
It  has  been  found  in  many  other  serous  inflammations,  and 
also  in  the  mouths  of  healthy  persons. 


Fig. 


72. — Diplococcus    of    pneumonia    in    blood    of    rabbit    (Xiooo) 
(Frankel  and  Pfeiffer). 


Form. — ^Large,  lancet-shaped  cocci.  Usually  found  in 
pairs,  sometimes  in  filaments  of  three  and  four  elements.  In 
the  material  from  the  body  a  capsule  surrounds  each  coccus. 
In  the  artificial  cultures  this  is  not  found  (Figs.  71  and  72). 

Froperties. — Variable  in  form,  approaching  the  bacillary 
type.  Do  not  liquefy  gelatin.  There  are  no  spores.  Non- 
motile. 

Growth. — Best  between  27°  C.  and  41°  C,  seldom  below 
25°  C.  Facultative  anaerobic.  The  culture-media  must  be 
slightly  alkaline;  the  growth  is  slow. 


158  ESSENTIALS   OF  BACTERIOLOGY 

Colonies. — Glucose  or  Glycerin  Agar  Plates. — Growth  slow, 
of  small,  round,  moist  colonies,  separated. 

Stab-cultures. — Along  the  needle-track  small  separate  white 
granules,  one  above  the  other,  like  a  string  of  beads. 

Blood  Bouillon. — Bouillon  containing  one-third  blood-serum 
or  ascitic  fluid  favors  the  growth.  They  grow  better  here  than 
in  the  other  media,  remaining  alive  a  longer  period  of  time. 

Blood-serum  or  Blood-agar. — Growth  more  vigorous.  A 
good  growth  on  blood-serum  or  blood-agar. 


Fig.    73. — Pneumobacillus  in  blood  (Xiooo)  (Frankel  and  Pfeiffer). 

Staining. — Takes  Gram's  method  and  the  other  anilin 
stains  very  readily.  The  capsule  stained  by  Hiss  method 
(p.  61)  or  Welch. 

Resistance. — Cultures  in  sugar  media  must  be  frequently 
transplanted,  as  the  organism  is  destroyed  in  a  few  days  by 
the  acid  generated.  In  albumin  alkaline  media  (blood-serum, 
etc.)  the  cultures  can  be  kept  active  two  weeks  or  more.  In 
sputum  the  pneumococcus  may  survive  several  days.  When 
dried  but  exposed  to  sunlight,  death  occurs  in  a  few  hours. 


BACTERIA  IN  PNEUMONIA  1 59 

Pathogenesis. — Rabbits  and  guinea-pigs,  if  subcutaneously 
injected,  die  in  the  course  of  a  couple  of  days  with  septicemia 
(o.i  ex.  of  a  fresh  bouillon  culture  suffices). 

Autopsy  shows  greatly  enlarged  spleen  and  myriads  of 
micrococci  in  the  blood  and  viscera,  the  lungs  not  especially 
affected.  If  injected  into  the  trachea,  a  pneumonia  occurs. 
In  man  they  are  found  in  90  per  cent,  of  croupous  pneumonia, 
and  usually  only  during  the  existence  of  the  rusty  sputimi, 
i.  €.,  the  first  stage.  Found  in  the  tissue  of  the  inflamed 
lung,  and  in  the  blood  in  nearly  all  cases  of  lobar  pneumonia. 

The  pneumococcus  has  also  been  found  in  pleuritis,  peri- 
tonitis, pericarditis,  meningitis,  and  endocarditis.  It  stands 
in  some  intimate  relation  to  all  infectious  inflammations  of 
the  body.  Their  presence  in  healthy  mouth  secretion  does 
not  speak  against  this,  it  requiring  some  slight  injury  or  low- 
ered resistance  to  allow  this  ever-present  germ  to  produce  a 
pneumonia  from  an  infectious  disease  like  measles  or  in- 
fluenza. 

Toxins  and  antitoxins  have  not  been  separated  or  demon- 
strated. The  poisons  are  probably  endotoxins,  and  closely 
connected  with  the  cell-body.  Agglutination  properties  of 
pneumonia  blood  serum,  if  any,  are  very  weak — i  :  50. 

Immunity  and  Serum  Therapy. — One  attack  produces  no 
immunity;  and  no  immune  serum  has  been  found  of  any  value. 
By  growing  in  an  acid  medium,  the  organism  has  been  ren- 
dered less  virulent. 

Bacillus  Pneumoniae  (Friedlander,  1882). — Synonym. — 
Capsule  Bacillus  of  Pfeifer. — Once  supposed  to  be  a  cause  of 
pneumonia.  It  grows  readily  on  ordinary  media;  is  Gram 
negative;  in  form  and  capsule  formation  it  sometimes  re- 
sembles the  pneumococcus  (Fig.  70). 

Bacillus  of  Rhino  scleroma  (Frisch,  1882). — It  was  found 
in  the  tissue  of  a  rhinoscleroma,  but  resembles  the  Fried- 
lander  bacillus  in  nearly  every  respect,  and  as  the  disease 
rhinoscleroma  is  not  reproduced  by  the  inoculation  of  the 
bacillus  in  animals,  it  can  be  considered  identical.    The 


l6o  ESSENTIALS  OF  BACTERIOLOGY 

growth,  cultures,  and  properties  are  the  same  as  the  pneumo- 
bacillus  of  Friedlander. 

Bacillus  of  Influenza  (Pfeiffer,  1892). — Origin. — One  of 
the  smallest  of  the  known  bacilli,  1.5  ^u  by  0.3  /z,  about  one- 
half  the  size  of  the  bacillus  of  mouse  septicemia,  and  ar- 
ranged in  chain  form.  It  develops  upon  blood-serum  agar. 
It  is  aerobic,  without  movement  (Fig.  74). 

Stain. — It  is  best  stained  with  diluted  carbol-fuchsin,  the 


.1* 


.*  "vV 


*K' 


% 


-.?*. 


Fig.  74. — Bacillus  influenzae,  from  a  gelatin  culture  (Xiooo)  (Itzerott 
and  Niemann). 


contrast-Stain  being  Loffler's  methylene-blue ;  does  not  take 
the  Gram  stain. 

Growth. — Upon  blood-agar  or  glycerin-agar,  over  which  a 
drop  of  blood  has  been  spread,  in  an  incubator  at  37°  C.  at 
the  end  of  twenty-four  hours  a  very  delicate  growth  occurs 
which  resembles  condensed  moisture.  Very  small  colonies, 
never  larger  than  a  pinhead,  feebly  resistant.  Subcultures 
must  be  made  every  few  days. 

Pathogenesis. — It  is  found  in  the  sputum  and  in  the  bron- 
chial and  nasal  secretions  and  blood  of  influenza  patients.    It 


BACTERIA  IN   PNEUMONIA  l6l 

has  been  transmitted  to  monkeys;  other  animals  are  not  suscep- 
tible. It  has  never  been  found  outside  the  body.  Its  resistance 
is  very  feeble ;  in  water,  the  bacilli  die  in  twenty-four  hours, 
but  sputa  containing  the  germs  may  be  ejected  for  days  and 
weeks.  Influenza  bacilli  are  found  accompanying  broncho- 
pneumonia, tuberculosis,  meningitis,  and  other  inflammations. 
The  bacillus  is  found  in  healthy  individuals,  to  a  consider- 
able extent  in  the  nasal  secretions,  and  it  is  probably  spread  in 
the  fine  droplets  of  mucus  expelled  in  sneezing  and  coughing. 

Koch-Weeks  Bacillus  (1883-87)  .—Cause  of  epidemic 
conjunctivitis,  or  ''pink  eye";  found  in  the  secretion. 

Form. — ^Very  minute  bacillus,  resembling  the  influenza 
bacillus;  non-motile.     (See  Fig.  84,  p.  173.) 

Growth. — They  grow  best  on  blood-serum  agar,  but  very 
sparsely  in  minute  transparent  colonies;   non-liquefying. 

Stains. — With  carbolfuchsin,  and  is  often  intracellular. 
Does  not  take  Gram. 

Pathogenesis, — Very  contagious,  found  in  10  per  cent,  to  20 
per  cent,  of  all  cases  of  conjunctivitis.  Not  infectious  for 
lower  animals,  and  not  causing  any  other  form  of  disease. 

Bacillus  of  Pertussis  (Whooping-cough)  (Bordet- 
Gengou,  1906). — It  has  been  shown  that  very  minute 
bacilli  resembling  the  influenza  bacillus  occur  in  the  cilia  of 
the  cells  lining  the  trachea  and  bronchi  of  persons  affected 
with  whooping-cough ;  these  bacilli  interfere  with  the  normal 
movement  of  the  cilia,  and  cause  an  irritation  producing 
symptoms  peculiar  to  the  disease. 

Morphology. — Very  minute  bacilli  with  rounded  ends 
(Fig.  75). 

Cultures. — On  potato-blood-agar,  after  twenty-four  hours, 
slight  growth,  sticky,  grayish;  subcultures  made  on  blood- 
serum  and  veal-agar  grow  readily. 

Staining. — Gram-negative,  stain  lightly  with  ordinary  dyes. 

Pathogenesis. — By  inhalation  inoculation  young  rabbits 
were  made  to  develop  a  spasmodic  cough,  and  the  bacillus 
was  recovered  from  the  trachea  and  from  bronchi  in  pure  cul- 
tures.   In  the  sputum  of  persons  affected  with  whooping- 


l62  ESSENTIALS   OF   BACTERIOLOGY 

cough  the  bacillus  is  found  in  large  numbers.  The  recent 
work  of  Mallory,  Henderson,  and  Horner  (Jour.  Med.  Re- 
search, March,  19 13)  seems  to  establish  this  organism  as  the 
real  cause  of  pertussis. 

Bacterins  made  from  the  culture  have  been  recommended 
to  allay  the  spasmodic  cough. 

Bacillus  Melitensis  (Bruce,  1887). — Synonym. — Micro- 
coccus Melitensis. — Malta  fever,  also  known  as  Mediterra- 
nean fever,  occurs  in  the  region  from  which  it  derives  its 


/•-" 


Fig.  75- — The  Bordet-Gengou  bacillus  of  whooping-cough.  Twenty-four- 
hour-old  culture  upon  solid  media  containing  blood  (Bordet-Gengou). 


name,  but  has  been  observed  in  India,  the  Philippine  Islands, 
and  Porto  Rico.  Bruce  cultivated  an  organism  from  the 
spleen  and  proved  its  specificity. 

Origin. — Is  found  most  abundantly  in  the  spleen. 

Form. — Rounded  or  oval,  very  small,  coccus-like  bacilli, 
0.5  jLt  in  diameter,  singly,  in  pairs,  or  short  chains. 

Properties. — Non-motile,  though  flagella  said  to  be  present; 
grows  slowly,  best  at  body-temperature. 

Gelatin. — Not  liquefied;  growth  very  slow. 


PYOGENIC  coca  163 

Bouillon. — Turbid,  with  sediment. 

Agar. — Pearly  white  growths. 

Potato. — Slight  invisible  growth. 

Stained  by  ordinary  anilin  dyes.     Gram  negative. 

Glucose  broth,  unfermented. 

Milk  made  alkaline. 

The  disease  may  be  produced  in  monkeys  by  even  small 
amounts  of  pure  culture.  In  man  a  chronic,  remittent  febrile 
disease  is  produced,  with  sweating  and  arthritis.  The  mor- 
tality is  2  per  cent.  A  reaction  can  be  obtained  and  is 
diagnostic. 

Agglutination — 1:30  dilution  of  serum  will  give  positive 
result,  but  the  complement-fixation  test  considered  more  cer- 
tain {which  see). 

Flies  an  agency  for  transmission. 

Mode  of  Transmission. — Zammitt  found  that  50  per  cent, 
of  the  goats  of  Malta  gave  the  agglutination  reaction  to  the 
micrococcus,  and  it  was  present  in  the  milk  in  10  per  cent. 
Monkeys  fed  on  the  milk  contracted  the  disease. 

Preventive  measures  instituted  in  1906  have  borne  out  the 
theory  that  the  milk  of  goats  is  the  cause  of  Malta  fever,  and 
since  the  practice  of  importing  goats  from  Malta  has  stopped, 
the  disease  has  disappeared  from  Gibraltar.  In  Malta, 
among  the  troops,  the  fever  has  been  greatly  reduced  by 
eliminating  milk  from  the  dietary. 


CHAPTER  XXm 
PYOGENIC  COCCI 

Nearly  all  micro-organisms  can  produce  suppuration,  but 
in  the  acute  abscesses  occurring  in  the  skin  and  lymphatics  and 
accompanying  all  pus  affections  are  found  groups  of  micro- 
cocci so  regularly  that  they  have  been  designated  as  the  pus- 
forming  or  pyogenic  cocci.    The  two  most  important  mem- 


164 


ESSENTIALS   OF   BACTERIOLOGY 


bers  of  this  group  are  the  Staphylococcus  pyogenes,  and  the 
Streptococcus  pyogenes,  so  named  from  the  mode  of  division, 
the  former  being  found  usually  in  clusters  or  bunches,  the  lat- 
ter in  chains. 

Streptococcus  Pyogenes  (Rosenbach) :  Streptococcus 
Erysipelatis  (Fehleisen). — Origin. — Fehleisen  in  1883  dis- 
covered this  microbe  in  the  lymph- 
atics of  the  skin  in  erysipelas,  and 
he  thought  it  the  cause  of  the  same. 
Under  the  name  Streptococcus 
pyogenes,  Rosenbach  described  an 
identical  coccus  which  has  been 
found  in  nearly  all  suppurative 
conditions. 


Fig.    76. — Streptococcus 
pyogenes;  culture  upon  agar- 
agar  two  days  old  (Frankel 
and  Pfeiffer). 


-Streptococcus  pyogenes 
(Jakob). 


Form. — Small  cocci  singly  and  in  chain-like  groups.  Spores 
have  not  been  found  (Fig.  77). 

Properties. — They  are  immotile;   do  not  Hquefy  gelatin. 

Growth. — They  grow  slowly,  usually  on  the  surface,  and 
best  at  higher  temperatures. 


PYOGENIC  COCCI  165 

Colonies. — In  three  days  a  very  small  grayish  speck,  which 
hardly  ever  becomes  much  larger  than  a  pin-head;  under 
microscope,  looking  yellowish,  finely  granular,  the  edges  well 
defined. 

Stab-cultures. — Along  the  needle-track  little  separated  col- 
onies, like  strings  of  beads,  which  after  a  time  become  one 
solid  white  string. 

Stroke-culture  on  Agar. — ^Little  drops,  never  coalescing, 
having  a  bluish  tint,  very  transparent. 

Potato. — No  apparent  growth. 

Bouillon. — At  37°  C.  clouds  are  formed  in  the  bouillon, 
which  then  sink  to  the  bottom,  and  long  chains  of  cocci  found 
in  this  growth. 

Loffler's  Blood-serum  and  Serum  Bouillon. — Development 
more  abundant  in  serum  media. 

Milk. — Good  growth;  produce  lactic  acid  and  coagulate 
milk. 

Preservation  of  Cultures. — In  ice-chest,  the  cultures  may  be 
kept  alive  several  weeks  at  room  temperature;  they  usually 
die  out  in  ten  days. 

Staining. — Easily  colored  with  the  ordinary  stains.  Gram's 
method  is  also  applicable. 

Pathogenesis. — Inoculated  subcutaneously  in  the  ear  of  a 
rabbit,  an  erysipelatous  conditiori  develops  in  a  few  days, 
rapidly  spreading  from  point  of  infection. 

The  micro-organism  acts  variously,  depending  upon  the 
nature  of  the  lesion  from  which  it  originally  was  obtained. 
Injected  into  the  circulation,  septicemia  results.  The  more 
virulent  the  affection,  the  more  virulent  the  strain. 

In  man,  inoculations  have  been  made  to  produce  an  effect 
upon  carcinomatous  growths,  and  erysipelas  has  always  re- 
sulted. When  it  occurs  upon  the  valves  of  the  heart,  endo- 
carditis results.  Puerperal  fever  is  caused  by  the  microbe 
infecting  the  endometrium,  the  Streptococcus  puerperalis  of 
Frankel  being  the  same  germ. 

In  scarlatina,  variola,  yellow  fever,  cerebrospinal  menin- 
gitis, and  many  similar  diseases,  the  microbe  has  been  an 


1 66  ESSENTIALS   OF   BACTERIOLOGY 

almost  constant  attendant.  It  is  often  associated  with  the 
diphtheria  bacillus  in  true  diphtheria,  and  is  the  cause  of 
many  of  the  diphtheritic  complications.  It  is  associated  with 
the  influenza  bacillus  in  acute  ear  suppurations;  with  pneu- 
monia bacteria;  with  tubercle  bacilli,  and  in  such  instances 
usually  causes  high  fever.  In  osteomyelitis  and  mastoiditis 
it  is  usually  the  sole  cause. 

Streptococci  in  Milk. — ^In  milk  streptococci  are  often  found, 
but  it  is  not  considered  an  absolute  indication  of  udder  in- 
flammation. 

Protective  Sera. — An  antistreptococcic  serum  has  been  used 
as  a  curative  agent  in  puerperal  fever,  scarlatina,  and  other 
diseases  supposed  to  be  due  to  this  germ.  The  antistrepto- 
coccic sera  have  been  given  an  extensive  trial  in  a  variety 
of  suppurative  and  inflammatory  diseases,  but  the  results 
are  still  under  discussion. 

Coley's  Fluid. — A  mixture  of  a  culture  of  pyogenes  and 
prodigiosus  has  been  used  as  an  injection,  with  apparent 
benefit,  in  inoperable  cases  of  sarcoma,  and  is  known  as 
Coley's  fluid. 

Immune  Bodies. — Neither  antitoxic  nor  bactericidal  bodies 
have  been  found  in  the  blood  of  animals  made  resistant  by  the 
injection  of  dead  or  attenuated  cultures. 

Polyvalent  {Vaccines)  Bacterins. — ^As  it  is  possible  that  there 
are  several  varieties  of  streptococci,  and  varying  in  patho- 
genic properties,  bacterins  made  from  several  strains  have 
been  used  as  injections  against  suppurative  processes,  and 
with  some  degree  of  success.  Autogenous  bacterins  are 
more  reliable. 

Distribution. — Streptococci  can  often  be  found  in  air,  dust, 
on  the  skin,  on  all  the  mucous  surfaces,  pharynx,  conjunctiva, 
tonsils. 

Staphylococcus  Pyogenes  Aureus  (Rosenbach). — 
Origin. — Found  commonly  in  pus  (80  per  cent,  of  all  suppura- 
tions), in  air,  water,  and  earth;  also  in  sputum  of  healthy 
persons. 

Form. — Micrococci  in  clusters  like  bimched  grapes,  hence 


PYOGENIC  COCCI  ,  1 67 

the  name  staphylo,  which  means  grape.  They  never  form 
chains.  Spores  have  not  been  found,  though  the  cocci  are 
very  resistant  (Fig.  78). 

Properties. — Immotile;  liquefying  gelatin.  Giving  rise  to 
an  orange-yellow  pigment  in  the  various  cultures. 

Growth. — It  grows  moderately  fast  at  ordinary  tempera- 
ture, and  can  live  without  air,  a  facultative  aerobin  and  an- 
aerobin. 

Colonies  on  Gelatin. — On  second  day  small  dots  on  the  sur- 
face, containing  in  their  center  an  orange-yellow  spot.     The 


Fig.  78. — Staphylococcus  pyogenes  albus  (Jakob). 

gelatin  all  around  the  colony  is  liquefied;  the  size  is  never 
much  greater  than  that  attained  the  second  day. 

Colonies  on  Agar. — The  pigment  remains  for  a  long  time. 

Stab-culture. — At  first,  gray  growth  along  the  track,  which, 
after  three  days,  has  settled  at  the  bottom  of  the  tube  in  a 
yellow,  granular  mass,  the  gelatin  being  all  liquid  (Fig.  79). 

Stroke-culture  on  Agar. — The  pigment  diffused  over  the  sur- 
face where  the  growth  is  in  moist  masses. 

Potato. — ^A  thin  white  layer  which  gradually  becomes  yel- 
low and  gives  out  a  doughy  smell. 


1 68 


ESSENTIALS   OF   BACTERIOLOGY 


Staining. — Very  readily  colored  with  ordinary  stains;  also 
with  Gram's  method. 

Pathogenesis. — When  rabbits  are  injected  with  cultures  of 
this  microbe  into  the  knee-joint  or  pleura,  they  die  in  a  day. 
If  injected  subcutaneously,  only  a  local  action  occurs,  namely, 
abscesses. 

If  directly  into  circulation,  a  general  phlegmonous  condi- 
tion arises,  the  capillaries  become  plugged  with  masses  of 
cocci,  infarcts  occur  in  kidney  and  liver, 
and  metastatic  abscesses  form  in  viscera 
and  joints.  Garre,  by  rubbing  the  culture 
on  his  forearm,  caused  carbuncles  to  ap- 
pear. 

Several  varieties  of  the  pyogenic  staph- 
ylococci are  recognized  according  to  their 
color-producing  properties  and  slight  vari- 
ations of  growth.  Of  these,  the  Staph- 
ylococcus pyogenes  aureus  is  the  most 
virulent,  and  is  considered  the  type  of  the 
group.  They  are  always  present  on  the 
surface  of  the  body,  beneath  the  nails,  in 
the  nose  and  mouth,  in  the  dust  of  streets, 
and  on  the  floors  of  houses,  and  are  found 
in  nearly  all  suppurative  processes,  whether 
on  the  surface  or  internally. 

Staphylococcus  pyogenes  albus  dif- 
fers frofn  the  preceding  only  in  the  absence 
of   pigment  and  in   its   slight  virulence. 
Welch  describes  a  variety  constantly  found  both  on  the  skin 
and  in  its  deeper  layers,  which  he  calls  the  Staphylococcus 
epidermidis  albus. 

Specific  Therapy. — Sera  have  been  found  of  no  special 
value. 

Bacterins  {Vaccines). — Twenty-four-hour-old  agar  surface 
culture  killed  by  heating  at  60°  C.  is  emulsified  with  normal 
saline  solutions  and  injected  for  the  treatment  of  boils,  ab- 
scesses, and  acne.    The  cultures  should  be  autogenous,  i.  e., 


Fig.  79. — Stab- 
culture.  Micro- 
coccus pyogenes 
aureus. 


PYOGENIC   COCCI  169 

derived  from  the  person  affected,  although  stock  vaccines 
have  been  used  with  some  success. 

The  Opsonic  Index. — It  was  proposed  by  Wright  that  the 
opsonic  index  should  be  obtained  before  treatment  with  vac- 
cines, although  most  of  the  treatment  is  now  given  without 
such  control. 

Micrococcus  Pyogenes  Citreus  (Passet). — This  lique- 
fies gelatin  less  rapidly  than  the  pyogenes  aureus,  and  forms 
a  citron-yellow  pigment  instead  of  the  orange-yellow  of  the, 
aureus. 

Micrococcus  Cereus  Albus  (Passet). — Differs  from  the 


^f  .d 


^ 


Fig.  80. — Micrococcus  tetragenus  in  sputum  (tubercle  bacillus  also). 


pyogenes  albus  in  the  form  of  colony.  A  white,  shiny  growth, 
like  drops  of  wax;  hence  the  name,  cereus. 

Micrococcus  Cereus  Flavus  (Passet) . — A  lemon-yellow 
colored  growth  after  a  short  time,  otherwise  not  differing  from 
cereus  albus. 

Micrococcus  Pyogenes  Tenuis  (Rosenbach). — Origin. 
— Found  in  the  pus  of  large  inclosed  abscesses. 

Form. — Cocci,  without  any  especial  arrangement. 

Properties. — ^Not  much  studied. 

Growth. — Cultivated  on  agar,  it  forms  clear,  thin  colonies; 
along  the  needle- track  an  opaque  streak,  looking  as  if  var- 
nished over. 


lyo 


ESSENTIALS   OF  BACTERIOLOGY 


V\ 


.'y  )> 


■V- 


WUHc 


Micrococcus  Tetragenus   (Koch;  Ga&ky) .—Origin.— 

Koch  found  this  microbe  in  the  cavity  of  a  tuberculous  lung. 
Gaffky,  in  1883,  studied  its  pathogenic  actions  and  gave  it 
the  name  it  now  bears. 

Form. — Cocci  which  are  gathered  in  the 
tissues  in  groups  of  four,  forming  a  square 
— a  tetrad.  (See  Fig.  80.)  In  artificial 
culture  sometimes  found  in  pairs.  A  cap- 
sule of  light,  gelatinous  consistence  sur- 
rounds each  tetrad. 

Properties. — ^They  are  immobile;  do  not 
liquefy  gelatin. 

Growth. — ^They  grow  well  on  all  nutrient 
media  at  ordinary  temperature;  are  facul- 
tative aerobic.     They  grow  slowly. 

Colonies  in  gelatin  plates.  In  two  days, 
little  white  spots,  which,  when  on  the 
surface,  form  little  elevations  of  a  porce- 
lain-like appearance;  under  low  power 
they  are  seen  very  finely  granulated. 

Stab-culture. — Small,  round,  separated 
colonies  along  the  needle-track,  and  on 
the  surface  a  button-like  elevation — a 
form  of  "nail  culture."     (See  Fig.  81.) 

Potato. — ^A  thick,  slimy  layer  which  can 
be  loosened  in  long  shreds. 

Staining. — Colored  with  the  ordinary 
anilin  stains.     Gram  positive. 

Pathogenesis. — White  mice  and  guinea- 
pigs   die   in   a   few   days   of    septicemia 
when   injected  with  the  tetragenus   cul- 
tures, and  the  micrococcus  is  then  found 
in  large  numbers  in  the  blood  and  viscera.     Field-mice  are 
immune. 

In  the  cavities  of  tuberculous  lungs,  in  the  sputum  of 
phthisical  and  healthy  patients,  it  is  often  found,  but  what 
action  it  has  upon  man  has  not  yet  been  determined. 


Fig.  81.— Stab 
culture.  Micrococ 
cus  tetragenus. 


PYOGENIC   COCCI 


171 


Morax-Axenfeld   Diplobacillus    of    Conjunctivitis. — 

This  bacillus  is  found  in  the  greater  number  of  cases  of  con- 
junctivitis. 

Form. — ^A  short,  plump  bacillus,  usually  in  pairs  and  chains 
of  pairs.     Non-motile  (Fig.  82). 

Growth. — With  difficulty  in  blood-serum  agar,  it  forrns  small 
pitted  colonies  or  lacunae;   liquefies. 

Staining. — Does  not  take  Gram,  but  stains  readily. 

Non-pathogenic  for  lower  animals. 


Fig.  82. — Morax-Axenfcld  diplobacillus  from  conjunctival  exudate 
during  course  of  subacute  conjunctivitis  (obj.  B.  and  L.,  one-twelfth  oil- 
immersion)  (Boston). 


Bacillus  Pyocyaneus  (Gessard). — Synonyms. — Bacillus 
fluorescens  (Schroter) ;   the  bacillus  of  bluish-green  pus. 

Origin. — Found  in  1882  in  green  pus  in  pyemia.  Has  been 
found  in  water,  in  bandage  material,  in  feces  and  street  dust, 
in  the  mouth  of  healthy  individuals,  and  in  all  suppurating 
conditions,  especially  in  middle-ear  discharge. 

Form. — Small  slender  rods  with  rounded  ends,  easily  mis- 
taken for  cocci.  Often  in  groups  of  four  and  six,  without 
spores. 


172  ESSENTIALS   OF   BACTERIOLOGY 

Properties. — Very  motile;  liquefy  gelatin  rapidly;  a  pecu- 
liar sweetish  odor  and  a  blue  pigment  are  produced  in  the 
cultures. 

Growth. — Develops  readily  at  ordinary  temperature,  grow- 
ing quickly  and  mostly  on  the  surface;  it  is  aerobic.  Agar 
plate:  In  two  or  three  days  a  greenish  iridescence  appears 
over  the  whole  plate. 

A  bright  green  at  first,  causing  fluorescence;  then  later  a 
blue  pigment  in  deeper  portion. 


>><A 


»*l\.«* 


Fig.  83. — Bacillus  pyocyaneus,  from   an   agar-agar   culture    (X  1000) 
(Itzerott  and  Niemann). 

Gelatin  Stab-cultures. — Mainly  in  upper  strata,  the  lique- 
faction funnel  shaped,  the  growth  gradually  settling  at  the 
bottom,  a  rich  green  shimmer  forming  on  the  surface,  and  the 
gelatin  having  a  deep  fluorescence. 

Potato. — The  potato  is  soaked  with  the  pigment,  a  deep 
fold  of  green  occurring  on  the  surface. 

Indol  is  produced. 

In  ear  abscesses  pure  cultures  have  been  found. 

Bacillus  fluorescens,  found  in  water,  is  considered  identical 


PYOGENIC  COCCI  1 73 

with  Bacillus  pyocyaneus  and  other  fluorescent  bacteria 
are  believed  to  be  varieties. 

Crystals  develop  on  agar  cultures  in  a  short  time. 

Staining. — With  ordinary  anilin  dyes.     Gram  negative. 

Pathogenesis. — When  animals  are  injected  with  fresh  cul- 
tures in  the  peritoneal  cavities  or  cellular  tissues,  a  rapidly 
spreading  edema  with  general  suppuration  develops.  The 
bacilli  are  found  in  the  viscera  and  blood. 

If  a  small  quantity  is  injected,  a  local  suppuration  occurs, 


Fig.  84. — Koch-Weeks'  bacillus  from  conjunctival  exudate  at  third  day 
of  epidemic  conjunctivitis  (Boston). 

and  if  the  animal  does  not 'die,  it  then  can  withstand  large 
quantities.     It  is  immune. 

The  Pigment. — Pyocyanin. — When  the  pus,  bandages,  and 
dressings  containing  the  Bacillus  pyocyaneus  are  washed  in 
chloroform,  the  pigment  is  dissolved  and  crystallizes  from  the 
chloroform  in  long  needles.  It  is  soluble  in  acidulated  water, 
which  is  turned  red  thereby,  and  when  neutralized,  the  blue 
color  returns.  It  has  no  pathogenic  action.  It  is  an  aromatic 
compound.  The  bacillus  has  no  especial  action  on  the  wound, 
and  is  found  sometimes  in  perspiration  of  healthy  persons. 


174  ESSENTIALS    OF   BACTERIOLOGY 

CHAPTER  XXIV 
GONOCOCCUS.— MENINGOCOCCUS 

Micrococcus  Gonorrhoeae  (Gonococciis  Neisser). — In 

1879  Neisser  demonstrated  the  presence  of  this  germ  in  the 
secretion  of  specific  urethritis. 

Form. — Cocci,  somewhat  triangular  in  form,  found  nearly 

always  in  pairs,  the  base  of  one  coccus  facing  the  base  of  the 

other  and  giving  the  appearance  of  a  Vienna  roll,  hence  the 

German  name,  Semmel  (roll),  form.     Four  to  twelve  such 

pairs  are  often  found  together.    Immotile 

(Fig.  86).     In  pus  usually  within  the  cells. 

Culture. — No  growth  on  ordinary  media ; 

^^:  on    blood-serum   or    agar    smeared   with 

-^  \0  blood,  cultures  have  been  obtained.     The 

^X%  temperature  must  be  between  33°  and  37° 

*^*'  C,  and  the  growth  occurs  very  slow^ly  and 

sparsely. 

Wertheim's  medium   (q.   v.,  p.   78)  has 
Fig.      85.—      given  the  best  results, 
orrheal  pus.    Ani-  Colonies. — Extremely  delicate,  translu- 

lin    -methyl-violet      cent  spots,  separate,  and  of  a  slimy  con- 
^■^  ^°^'  sistence,  appearing  in  one  to  two  days. 

Resistance. — The   cultures   live   only  a 
few  days  at  room  temperature,  but  in  the  ice-chest  they  last 
longer.     A  temperature  of  45°  C.  destroys  the  gonococci  and 
it  is  but  slightly  resistant  to  the  ordinary  chemic  antiseptics. 
From  the  Blood. — In  septicemic  cases  the  gonococcus  has 
been  isolated  from  the  blood  direct  by  drawing  5  to  10  c.c. 
from  a  vein  and  adding  it  in  equal  parts  of  melted  agar. 
The  mixture  is  poured  into  Petri  dishes  and  developed  in  the 
incubator  at  37°  C. 
Staining. — Colored  easily  with  all  ordinary  anilin  stains. 
Gram  negative  is  one  of  its  main  diagnostic  features. 


GONOCOCCUS. — MENINGOCOCCUS 


175 


The  following  method  is  recommended  by  Neisser: 
The  cover-glasses,  with  some  of  the  urethral  discharge 
smeared  upon  them,  are  covered  with  a  few  drops  of  alcoholic 
solution  of  eosin,  and  heated  for  a  few  minutes  over  the  flame. 
The  excess  of  the  dye  is  removed  with  filter-paper,  then  the 
cover-glass  placed  in  concentrated  methylene-blue  (alcoholic 
solution)  for  fifteen  seconds,  and  rinsed  in  water. 
The  gonococci  are  colored  dark  blue,  the  protoplasm  of  the 


^k 


Fig.  86. — Gonococcus  in  urethral  pus  (X  1000)  (Frankel  and  Pfeiffer). 


cell  pink,  and  the  nucleus  a  light  blue,  the  gonococci  lying 
in  the  protoplasm  next  to  the  nucleus  (Fig.  85). 

Bacterial  Diagnosis. — Other  bacteria  are  similar  to  the  gon- 
ococci in  form;  they  are  distinguished  from  the  gonococcus 
in  that  they  are  positive  with  Gram's  method.  The  points 
on  which  the  diagnosis  is  to  be  made  are  the  characteristic 
biscuit  shape,  the  intracellular  position  of  the  organism,  its 
failure  to  stain  with  Gram  and  very  difficult  to  grow  artificially 
on  common  media. 


176  ESSENTIALS   OF  BACTERIOLOGY 

Pathogenesis. — ^The  attempts  to  infect  the  experiment  ani- 
mals with  gonorrhea  have  so  far  been  without  success.  In 
man,  upon  a  healthy  urethra  a  specific  urethritis  was  pro- 
duced with  even  the  twentieth  generation  of  the  culture. 
Gonorrheal  ophthalmia  contains  the  cocci  in  great  numbers, 
and  endocarditis  and  gonorrheal  rheumatism  are  said  to  be 
caused  by  the  cocci. 

The  micrococci  have  been  found  long  after  the  acute  attack, 
when  only  a  very  slight  oozing  remained,  and  the  same  were 
found  very  virulent. 

The  specific  inflammations  of  the  generative  organs  of  the 
female  are  due  to  this  organism  gaining  entrance  through  the 
vagina.  It  is  found  chiefly  in  the  superficial  layers  of  the 
mucous  membrane. 

Bacterins  {Vaccines). — A  number  of  vaccines  have  been  pre- 
pared in  recent  years  for  the  treatment  of  gonorrhea  and  its 
complications.  The  bacterins  are  made  as  described  under 
Bacterins.  This  method  of  treatment  is  still  on  trial.  The 
best  results  have  been  obtained  in  gonorrheal  rheumatism 
and  epididymitis. 

Toxins. — ^True  toxins  not  found,  but  the  cells  contain  poi- 
sons that  produce  suppuration  and  death  when  injected  into 
the  mice  and  guinea-pigs. 

Allied  Varieties. — A  number  of  diplococci  which  resemble 
the  gonococcus  in  form  are  found  in  the  vaginal  secretions 
and  pus  and  may  at  times  lead  to  a  wrong  diagnosis.  The 
meningococcus  is  very  similar,  but  is  easily  cultivated  and  is 
not  apt  to  be  found  in  the  same  secretions  as  the  gonococcus. 

Micrococcus  citreus,  albicans,  and  subflavus,  described  by 
Bumm,  are  all  Gram  positive  and  grow  readily  on  gelatin  and 
agar. 

The  gonococcus  is  distinguished  from  all  these  similar  micro- 
cocci by  the  tests  enumerated  above. 

These  characteristics,  taken  in  toto,  form  sufficient  features 
for  its  ready  recognition,  and  as  it  is  often  a  serious  question  to 
decide,  not  so  much  because  of  the  patient's  health  as  because 
of  his  character,  we  should  be  very  careful  not  to  pronounce  a 


GONOCOCCUS. — MENINGOCOCCUS  1 77 

verdict  until  we  have  tested  the  micro-organism  as  above. 
When  the  germ  is  found  which  answers  to  the  above  descrip- 
tion, the  process  can  be  called  gonorrhea  without  a  doubt. 

Diplococcus  Intracellularis  Meningitidis  (Weichsel- 
baum). — Synonyms. — Meningococcus;  Micrococcus  Meningi- 
tidis. 

Origin. — Found  by  Weichselbaum  in  epidemic  cerebrospinal 
meningitis  in  1887. 


Fig.  87. — Diplococcus  intracellularis  meningitidis  in  leukocytes. 
Cover-glass  preparation  from  peritoneal  exudate  in  a  guinea-pig  (X2000) 
(Wright  and  Brown). 

Form. — ^A  small  coccus  occurring  in  pairs,  flattened  against 
each  other,  and  contained  within  the  leukocytes,  resembling 
gonococcus.     No  capsule  (Fig.  87). 

Properties. — Ferments  sugars,  with  acid  production. 

Growth. — Best  on  blood-agar,  serum-agar,  and  ascitic  glu- 
cose-agar  at  body  temperature;  good  growth  in  twenty-four 
hours.     Sheep  serum  better  medium. 


1 78  ESSENTIALS   OF   BACTERIOLOGY 

Colonies. — Circular  discs,  whitish,  almost  transparent,  mar- 
gins, smooth. 

Stain. — With  basic  anilin.  Gram  negative.  Jenner's 
blood-stain  and  Neisser  stain  best  for  spinal  fluid  specimens. 
Loffler's  alkaline  methylene-blue  a  good  stain. 

Resistance. — Organisms  very  perishable  one  to  three  days. 
Apparently  destroyed  by  a  self-elaborated  ferment.  Sun- 
light destroys  in  a  few  hours. 

Pathogenesis. — Causes  epidemic  cerebrospinal  fever,  prob- 
ably by  infection  through  the  nasopharynx;  the  organism  is 
found  in  the  spinal  fluid  and  in  other  inflammatory  exudates, 
and  can  be  seen  in  fluid  obtained  by  lumbar  puncture. 

Ordinary  laboratory  animals  immune,  but  Flexner  has 
succeeded  in  inoculating  monkeys. 

Agglutination. — On  the  fourth  day;  in  dilution  of  i  :  50 
agglutination  is  had. 

By  the  use  of  large  quantities  of  meningococci  injected  into 
a  horse  agglutinins,  opsonins,  and  specific  immune  bodies 
(amboceptor)  can  be  produced. 

Protective  Serum. — Flexner  has  been  able  to  obtain  an  anti- 
toxin from  monkey  serum  that  has  therapeutic  properties  in 
man.  Such  an  antiserum,  when  injected  directly  into  the 
spinal  canal,  has  a  curative  action,  destroying  the  cocci. 

Bacterial  Diagnosis. — By  means  of  lumbar  puncture  the 
spinal  fluid  is  obtained  and  allowed  to  settle.  Smears  made 
from  sediment.    Examined  for  bacteria. 

Gram-positive  organisms  are  either  pneumococci,  strepto- 
cocci, or  staphylococci. 

Meningococcus  is  Gram  negative  and  within  the  leukocytes, 
and  can  be  readily  grown  on  blood-serum.  If  such  an  or- 
ganism is  present,  the  disease  is  undoubtedly  cerebrospinal 
meningitis. 

Bacillus  of  Soft  Chancre,  Chancroid  (Ducrey-Unna, 
1889). — A  diplobacillus  which  is  specific  has  been  described 
by  Ducrey  as  obtained  from  the  secretion  and  in  the  depth 
and  margins  of  the  chancroid.  Unna's  bacillus  is  narrower 
and  unbroken  in  the  center  (Fig.  ^%). 


GONOCOCCUS. — MENINGOCOCCUS  1 79 

Cultivation. — Cultivation  has  occurred  on  blood-agar,  the 
blood  being  added  in  the  proportion  of  one  to  two.  Colonies 
sue  small,  round  globules. 

Staining. — With  borax,  methylene-blue,  decolorized  with 
weak  acetic  acid. 

Pathogenesis. — Probably  a  mixed  infection  occurs  in  most 


Fig.  88. — Smear  of  pus  of  chancroid  of  penis  (X  1500)  (Davis)  (photo- 
micrograph by  Mr.  L.  S.  Brown). 


chancroids,  especially  if  buboes  result.  The  bacillus  of 
Ducrey  is  not  found  in  unopened  buboes,  though  often  con- 
taminating the  ulcerated  ones. 

The  disease  has  been  reproduced  by  inoculation  of  the 
human  subject.    Laboratory  animals  are  immune. 


l8o  ESSENTIALS    OF   BACTERIOLOGY 


CHAPTER  XXV 

ANAEROBIC  BACTERIA  (BACILLUS  OF  TETANUS;  BACILLUS 
OF  MALIGNANT  EDEMA,  ETC.) 

Similar  in  form  and  cultural  requirements  are  a  group  of 
bacteria  which  are  found  as  a  result  of  injury  or  the  infection 
of  wounds.  They  vary  greatly  in  the  clinical  symptoms 
produced. 

Bacillus  of  Tetanus  (Nicolaier-Kitasato) . — Origin. — 
Nicolaier  found  this  bacillus  in  the  pus  of  a  wound  in  one 
who  had  died  of  tetanus,  describing  it  in  1884. 

Kitasato  isolated  and  cultivated  this  germ  (1889). 

Form. — A  very  slender  rod. 

When  the  spores  form,  a  small  swelling  occurs  at  the  spore 
end,  giving  the  bacillus  a  drum-stick  shape  (Fig.  89) . 

Properties. — Not  very  motile,  though  distinctly  so;  lique- 
fies gelatin  slowly.  The  cultures  give  rise  to  a  foul-smelling 
gas. 

Growth. — Develops  very  slowly,  best  at  36°  to  38°  C,  and 
only  when  all  oxygen  is  excluded — an  obligatory  anaerobin. 
In  an  atmosphere  of  hydrogen  it  flourishes. 

Colonies  on  gelatin  plates  in  an  atmosphere  of  hydrogen. 
Small  colonies.  After  four  days  a  thick  center  and  radiating, 
wreath-like  periphery,  like  the  colonies  of  Bacillus  sub  tills. 
Pure  cultures  not  easy  to  obtain  (Fig.  90). 

High  Stab-culture. — ^The  gelatin  having  2  per  cent,  glucose 
added  and  filling  the  tube.  Along  the  lower  portion  of  the 
needle-track,  a  thorn-like  growth,  little  needle-like  points 
shooting  out  from  a  straight  line.  The  whole  tube  becomes 
clouded  as  the  gelatin  liquefies,  and  then  the  growth  settles 
at  the  bottom  of  the  tube  (Fig.  91). 

Agar. — On  agar,  in  the  incubator,  the  growth  is  quite 
rapid,  and  at  the  end  of  forty-eight  hours  gas-bubbles  have 
formed  and  the  growth  nearly  reached  the  surface. 


ANAEROBIC   BACTERIA  l8l 

Bouillon. — Adding  glucose  to  the  bouillon  gives  a  medium 
in  which  an  abundant  growth  occurs. 

Stab-agar. — Inverted  iir-tree  appearance. 

Milk. — Acid  reaction  and  slow  coagulation. 

Inoculation  of  animals  with  suspected  material  may  be 
necessary  as  preliminary  step. 

Cultivation  from  Spores. — Kitasato,  by  exposing  a  portion 
of  suspected  material  to  a  temperature  of  80°  C.  for  one  hour, 


/  < 


i 


^-y-'- 

'*^/>^  ^ 


•• 

^ 


/ 


\<i 


Fig.   89. — Bacillus  of    tetanus  with    spores  fX    1000)   (Frankel   and 

Pfeiffer). 

killed  off  all  the  other  bacteria,  but  the  spores  of  tetanus 
escaped  and  these  then  vegetated. 

Staining. — All  the  ordinary  stains,  Gram's  method  also, 
the  spores  being  colored  in  the  usual  way. 

Pathogenesis. — A  small  amount  of  the  pure  culture  injected 
under  the  skin  of  experiment  animals  will  cause,  in  two  to 
three  days,  death  from  true  tetenus,  the  tetanic  condition 
starting  from  the  point  of  infection.  At  the  autopsy  nothing 
characteristic  or  abnormal  is  found,  and  the  bacilli  have  dis- 


l82 


ESSENTIALS   OF   BACTERIOLOGY 


appeared,  except  near  the  point  of  entrance.     This  fact  is 
explained  as  follows: 

Toxins. — Several  toxic  products  have  been  obtained  from 
the  cultures,  and  they  are  produced  in  the  body  and  give  rise 


Fig.  90. — Bacillus  tetani:  cul- 
ture four  days  old  in  glucose-gela- 
tin (Frankel  and  Pfeiffer). 


Fig.  91. — Six  days'  culture  of 
bacillus  of  tetanus  in  gelatin  (deep 
stab)  (Frankel  and  Pfeiffer). 


to  the  morbid  symptoms.     These  have  been  isolated,  and 
when  injected  singly,  cause  some  of  the  tetanic  symptoms. 
Tetanospasmin,  the  most  important  for  man. 


ANAEROBIC   BACTERIA  1 83 

Tetanolysin. — ^The  blood  and  the  urine  contain  the  toxin 
and  are  fatal  to  animals. 

The  virus  enters  the  circulation,  but  does  not  remain  in  the 
tissues.  The  toxin  is  most  virulent.  It  acts  on  the  end- 
plates  of  the  muscles,  and  then  on  the  motor  nerve-cells. 
The  incubation  period  is  from  two  to  fourteen  days  after 
receipt  of  injury.  The  spores  are  very  resistant  to  heat, 
drying,  and  chemicals. 

Burns  and  injuries  from  firearms,  cartridges,  powder,  and 
fireworks,  a  common  cause  of  tetanus. 

Immunity. — Kitasato,  by  inoculation  of  sterilized  cultures, 
has  caused  immunity  to  the  effects  of  virulent  bacilli. 

An  antitoxin  obtained  by  Tizzoni  and  Cattani  from  the 
serum  of  animals  made  immune  by  sterilized  cultures  is  used 
with  curative  effects  in  cases  of  tetanus  in  man.  It  is  a 
globulin,  but  differs  from  the  diphtheria  antitoxin.  By  pre- 
cipitation with  alcohol  and  drying  in  vacuo  the  antitoxin  is 
obtained  in  a  solid  state.  The  aqueous  solution  is  used  for 
injection  subcutaneously  or  subdurally  through  a  trephine 
opening.  Its  injection  into  the  spinal  canal  by  lumbar  punc- 
ture has  also  been  recommended.  Antitoxin  is  more  beneficial 
in  chronic  cases  than  in  acute. 

The  dried  antitoxin  has  been  spread  on  the  wound  with 
some  curative  action. 

The  antitetanic  serum,  to  be  effective,  must  be  given  very 
early  and  in  large  doses.  Its  greatest  use  is  in  preventing 
tetanus  in  wounds  liable  to  be  infected.  From  50  c.c.  to  100 
c.c.  of  a  billion-unit  serum  should  be  given  in  divided  doses; 
only  sera  with  very  high  protective  powers  should  be  used. 

United  States  Government  Unit  for  Tetanus  Antitoxin. — 
*'The  immunity  unit  is  ten  times  the  least  quantity  of  anti- 
tetanic  serum  necessary  to  save  the  life  of  a  350-gram  guinea- 
pig  for  ninety-six  hours  against  the  official  test  dose  of  a 
standard  toxin  furnished  by  the  Hygienic  Laboratory  at 
Washington." 

Habitat. — The  bacillus  is  present  in  garden-earth,  in  man- 
ure, and  it  has  been  isolated  even  from  mortar. 


184  ESSENTIALS   OF   BACTERIOLOGY 

The  earth  of  special  districts  seems  to  contain  the  bacilli  in 
greater  quantities. 

Spores  of  tetanus  may  gain  access  to  animal  sera,  and 
if  not  properly  destroyed,  may  produce  tetanus  during  the 
use  of  these  products.  Previous  testing  for  the  tetanus  bacil- 
lus should  be  made  in  the  manufacture  of  all  animal  vac- 
cines, antitoxins,  etc. 

Bacillus  (Edematis  Maligni  (Koch,  1881) ;  Vibrion 
Septique  (Pasteur,  1875). — Synonym. — Bacillus  (Edematis, 


Fig.  92. — Bacillus  of  malignani  edema,  from  the  body-juice  of  a  guinea- 
pig  inoculated  with  garden-earth  (X  looo)  (Frankel  and  Pfeiffer). 


Origin. — ^In  garden-earth,  found  also  in  severe  wounds  in 
man  when  gangrene  with  edema  had  developed.  Identical 
with  the  bacillus  found  in  Pasteur^ s  septicemia. 

Form. — Rods  somewhat  smaller  than  the  anthrax  bacillus, 
the  ends  rounded  very  sharply.  Long  threads  are  formed. 
Very  large  spores  which  cause  the  rods  to  become  spindle 
shaped.   Resembles  in  form  and  culture  B.  chauvei  (Fig.  92). 


ANAEROBIC  BACTERIA 


i8S 


< 


Properties. — Very  motile ;  liquefies  gelatin ;  gas  is  produced 
in  cultures  but  very  little  in  the  body. 

Growth. — Grows  rapidly,  but  only  when  the  air  is  excluded, 
and  best  in  incubator  at  37°  C. 

Roll  Cultures  {After  Esmarch^s 
Method). — Small,  round  colonies  with 
fluid  contents,  under  low  power,  a  mass 
of  motile  threads  in  the  center,  and  at 
the  edges  a  wreath-like  border. 

High  Stab-culture. — With  glucose 
gelatin,  the  growth  at  first  seen  in  the 
bottom  of  the  tube,  with  a  general 
liquefaction  of  the  gelatin;  gases  de- 
velop and  a  somewhat  unpleasant  odor. 

Agar. — The  gases  develop  more 
strongly  in  this  medium,  and  the  odor 
is  more  prominent. 

Guinea-pig  Bouillon. — In  an  atmos- 
phere of  hydrogen  clouding  of  the  en- 
tire culture-medium  without  any  floc- 
culent  precipitate  until  third  day.  Milk 
coagulated.  Glucose  media  marked  gas 
fermentation. 

Staining. — Are  stained  with  the  ordi- 
nary dyes,  but  Gram's  method  negative. 

Pathogenesis. — When  experiment  ani- 
mals, mice  or  guinea-pigs,  are  injected 
with  a  pure  culture  under  the  skin,  they 
die  in  eight  to  fifteen  hours,  and  the 
following  picture  presents  itself  at  the 
autopsy:  In  guinea-pigs  from  the  point 
of  infection,  spreading  over  a  large  area, 
an  edema  of  the  subcutaneous  tissues 
and  muscles,  which  are  saturated  with 
a  clear  red  serous  exudate,  free  from  odor,  and  containing 
great  quantities  of  bacilli. 

The  spleen  is  enlarged,  especially  in  mice.    The  bacilli  are 


Fig.  93.  —  Bacillus 
of  malignant  edema 
growing  in  glucose- 
gelatin  (Frankel  and 
Pfeiffer). 


l86  ESSENTIALS  OF  BACTERIOLOGY 

not  found  in  the  viscera,  but  are  present  in  great  numbers  on 
the  surface,  i.  ^.,  in  the  serous  coverings  of  the  different  organs; 
though  when  any  length  of  time  has  elapsed  between  the 
death  of  the  animal  and  the  examination,  they  can  be  found 
in  the  inner  portions  of  the  organs,  for  they  grow  well  upon 
the  dead  body.  In  man  they  have  been  found  in  rapidly 
spreading  gangrene  following  wounds. 

Habitat. — They  are  present  in  the  soil,  in  putrefactions  of 
various  kinds,  and  in  dirty  water. 

Immunity. — Is  produced  by  injection  of  the  sterilized  cul- 
tures, and  also  the  filtered  blood-serum  of  animals  dead  with 
the  disease. 

Bacillus  Aerogenes  Capsulatus  (Welch,  1891). — 
Synonym. — Bacillus  Welchii;  B.  of  Phlegmonous  Emphysema 
(Frankel). 

Origin. — The  intestine  of  man  and  animals,  soil,  sewage, 
and  water. 

Form. — A  thick  bacillus,  3  to  6  )Li  in  length,  frequently 
capsulated. 

Properties. — Not  motile,  anaerobic,  forms  spores  chiefly  in 
cultures  on  blood-serum.     Gram  positive. 

Growth. — Best  at  37°  C. 

Gelatin. — Liquefied  slowly  or  not  at  all. 

Bouillon. — Forms  gas. 

Milk. — Coagulated  and  becomes  acid.  Under  anaerobic 
conditions. 

Potato. — Thin,  grayish- white  growth  with  gas-production. 

Forms  gas  in  abundance  in  dextrose,  lactose,  or  saccharose 
media. 

Pathogenesis. — Is  not  usually  pathogenic  for  rabbits  and 
mice,  though  in  guinea-pigs  and  birds  it  produces  "gas  phleg- 
mons." It  is  sometimes  found  in  autopsies  on  human  sub- 
jects, producing  bubbles  or  cavities  in  the  viscera  (Schaum- 
organe),  but  this  is  probably  due  to  postmortem  migration 
of  the  germ  from  the  intestine.  It  has  been  recovered  from 
the  blood  during  life,  however,  and  is  the  most  frequent 
cause  of  emphysematous  gangrene.  In  man,  infection  of 
wounds,  through  dirt,  with  this  bacillus  causes  rapid  emphy- 


ANAEROBIC  BACTERIA  187 

sema  of  the  wound  and  a  thin  offensive  discharge  and  fatal 
outcome.  After  death  the  bacillus  develops  rapidly  and 
through  the  blood-vessels  brings  on  general  emphysema,  with 
large  accumulation  of  hydrogen  gas  in  all  the  organs  and 
subcutaneous  tissue.  Various  foreign  observers  have  de- 
scribed organisms  having  similar  properties,  and  have  given 
them  such  names  as  Bacillus  perfringens,  B.  enteritidis  sporo- 
genes,    Granulobacillus    immobilis,    B.     saccharobutyricus. 


Fig.  94. — Bacillus  aerogenes  capsulatus  (from  photograph  by  Professor 
Simon  Flexner). 

but  they  were  probably  dealing  with  the  Bacillus  aerogenes 
capsulatus. 

Bacillus  Enteritidis  Sporogenes  (Klein,  1895). — Re- 
garded as  identical  with  B.  aerogenes  capsulatus  {q.  v.). 

Bacillus  Chauvei. — Synonyms. — Bacillus  of  Symptomatic 
Anthrax  (BolUnger  and  Feser) ;  Rauschhrand  (German) ;  Char- 
bon  symptomatique  (Arloing,  Cornevin,  and  Thomas). 

Origin. — This  bacillus,  described  in  1879,  has  been  isolated, 
and  by  animal  inoculation  shown  to  be  the  cause  of  the 
"black-leg"  or  "quarter-evil"  disease  of  cattle. 


i88 


ESSENTIALS   OF   BACTERIOLOGY 


Form. — Large  slender  rods,  which  swell  up  at  one  end  or  in 
the  middle  for  the  spore  (Fig.  95). 

Properties. — They  are  motile,  and  liquefy  gelatin  quite 
rapidly. 

A  rancid  odor  is  developed  in  the  cultures. 

Cultures. — The  growth  occurs  slowly,  and  only  in  an  atmos- 
phere of  hydrogen,  being  anaerobic;  grows  best  at  38°  C; 
under  15°  C.  no  growth. 

Glucose-gelatin. — In  a  few  days  little  round  colonies  develop, 


'■^^^  *,, 


Fig.  95- 


-Bacilli  of  symptomatic  anthrax,  with  spores  (  X  1000)  (Frankel 
and  Pfeiffer). 


which,  under  low  power,  show  hairy  processes  around  a  com- 
pact center. 

Stab-cultures  in  Full  Test-tubes. — The  first  growth  in  the 
lower  portion  of  the  tube  not  very  characteristic.  Gases 
develop  after  a  few  days,  and  the  gelatin  becomes  liquid. 

Agar  at  brood  temperature,  in  twenty-four  to  forty-eight 
hours,  an  abundant  growth  with  a  sour  odor  and  abundant 
gas-formation. 


ANAEROBIC   BACTERIA  189 

Staining. — Ordinary  methods.  Gram's  method  is  nega- 
tive, but  the  spores  can  be  colored  by  the  regular  double  stain 
for  spores. 

Sugar  Media. — Gas  production. 

Milk. — Rendered  acid  and  coagulated. 

Variability. — Great  variation  in  cultures. 

Toxin  elaborated  in  fluid  media  fatal  for  rabbits  when 
injected  intravenously. 

Pathogenesis. — If  a  small  amount  of  the  culture  be  injected 
under  the  skin  of  a  guinea-pig,  in  twenty  hours  a  rise  of  tem- 
perature, pain  at  the  site  of  injection,  and  a  few  hours  later 
death,  occur.  At  the  autopsy,  the  tissues  are  found  black- 
ened in  color  and  soaked  with  a  bloody,  serous  fluid;  in  the 
connective  tissue  large  collections  of  gas,  but  only  in  the  neigh- 
borhood of  the  point  of  infection.  The  bacilli  are  found  in 
great  numbers  in  the  serum,  but  only  appear  in  the  viscera 
some  time  after  death,  when  spores  have  developed. 

The  animals  are  usually  infected  through  wounds  on  the 
extremities;  the  stalls  or  meadows  having  been  soiled  by  the 
spore-containing  blood  of  animals  previously  dead  of  the  dis- 
ease. '' Rauschbrand'''  is  the  German  name;  "Charbon  symp- 
tomatique,^'  the  French,  from  the  resemblance  in  its  symp- 
toms to  anthrax. 

Feeding  experiments  and  infection  from  animal  to  animal 
negative. 

Dried  virus  inoculation  practised  by  the  United  States 
Government  as  preventive. 

Immunity. — Rabbits,  dogs,  pigs,  and  fowl  are  immune  by 
nature,  but  if  the  bacilli  are  placed  in  a  20  per  cent,  solution  of 
lactic  acid  and  the  mixture  injected,  the  disease  develops  in 
them.  The  lactic  acid  is  supposed  to  destroy  some  of  the 
natural  resistance  of  the  animal's  cells. 

Immunity  is  produced  by  the  injections  of  these  weakened 
cultures,  and  also  by  some  of  the  products  which  have  been 
obtained  from  the  cultures. 


IQO  ESSENTIALS   OF   BACTERIOLOGY 

CHAPTER  XXVI 
HEMORRHAGIC  SEPTICEMIA  GROUP 

Bacillus  of  Bubonic  Plague  (Yersin  and  Kitasato, 
1894). — Synonym. — Bacillus  Pestis. — Bubonic  plague  or  pest 
is  an  extremely  infectious  disease,  more  or  less  common  in 
China  and  the  East,  and  is  believed  to  have  its  origin  in  man 
from  rats  and  other  rodents.     It  spreads  with  great  rapidity, 


Fig.  96. — Bacillus  pestis  in  smear  from    rat's    liver,  showing    bipolar 
staining  (X  720)  (Wherry). 

especially  among  those  living  under  unsanitary  conditions. 
The  ''Black  Death"  of  the  fourteenth  century  and  the  plague 
epidemics  of  the  seventeenth  century  are  said  to  have  been 
the  same  disease. 

Nearly  at  the  same  time  Yersin  and  Kitasato,  working 
independently,  discovered  in  the  bubonic  swellings  and  blood 


HEMORRHAGIC    SEPTICEMIA    GROUP  IQI 

of  affected  persons  a  distinctive  bacillus  which  has  conformed 
to  all  the  conditions  necessary  to  make  it  the  cause  of  the 
disease. 

Origin. — In  the  tissues  and  all  the  body-fluids  and  secre- 
tions of  affected  individuals. 

Form. — Short,  thick  rods  with  an  indistinct  capsule  and 
rounded  ends.    Growing  in  chains  in  fluid  media  (Fig.  96) . 

Properties. — Immotile.  Stains  readily.  No  spores.  Cul- 
tivated best  in  oxygen,  but  is  facultative  anaerobic.  Stains 
stronger  at  the  ends,  producing  bipolar  appearance.  Gela- 
tin not  liquefied.  Easily  destroyed  by  sunlight  and  drying. 
Very  resistant  to  cold. 

Growth. — Best  at  30°  C;    aerobic. 

Gelatin. — At  22°  C,  in  twenty-four  hours,  w^hite,  point- 
like colonies  on  the  plates,  with  broad  and  flat  surface,  turn- 
ing gray  and  then  brown.    Milk  not  curdled;   slightly  acid. 

Stab. — Snow-white,  spreading  out  on  the  surface  to  the 
edge,  and  fluorescent. 

Bouillon. — Granular  precipitate,  with  clear  fluid  above. 
When  covered  with  oil  and  kept  at  rest,  filaments  hang  down 
from  surface  like  stalactites. 

Agar  and  Blood-serum. — Glass-like  colonies  like  drops  of 
dew  at  first,  then  growing  larger  with  iridescent  edges. 

Potato. — ^At  37°  C.  small  white  mass.     Slow  growth. 

No  gas  formation  in  glucose  media. 

Staining  readily  with  all  basic  dyes.  Gram  negative. 
Capsule  found  in  agar  growths. 

Pathogenesis. — ^After  subcutaneous  injection  in  rats  death 
follows  in  forty  to  sixty  hours,  with  symptoms  of  severe  toxe- 
mia and  convulsions.  The  point  of  infection  shows  a  local 
edema  and  inflammation  of  the  lymphatics.  All  the  organs 
congested  and  surrounded  by  a  bloody  exudate.  The  charac- 
teristic bacilli  in  all  the  tissues  and  secretions.  Nearly  all  the 
domestic  animals  are  susceptible.  Mosquitos  and  pigeons, 
however,  are  immune — flies  are  not;  fleas  are  a  very  impor- 
tant element  in  the  transmission,  and  the  rat-flea  may  com- 
mimicate  the  disease  to  the  rat  from  man  or  from  the  rat  to 


192  ESSENTIALS   OF   BACTERIOLOGY 

man.  Infected  ground  squirrels  are  supposed  to  be  a  factor 
in  spreading  the  disease.  Animals  protected  from  the  flea 
may  live  near  infected  animals  without  danger.  Direct  in- 
fection by  dust  or  other  material  seldom  occurs.  The  sputum 
of  patients  having  the  pneumonic  t>pe  is  highly  infectious. 
Close  personal  contact  with  the  infected  is  a  means  of  trans- 
mission. The  main  point  of  entrance  is  the  skin.  Fifty  per 
cent,  of  wild  rats  immune  and  not  easily  affected. 

Products. — ^A  toxin  has  been  obtained  and  immunity  has 
been  effected;  the  serum  of  immune  animals  has  protective 
properties.  The  serum  likewise  shows  agglutinating  powers, 
and  gives  similar  reactions  to  typhoid  and  cholera  sera. 

Habitat. — Not  found  in  water,  but  most  likely  spreads  from 
the  soil  in  damp  and  darkened  areas.  Rats  become  affected 
first,  and  then,  through  fleas,  affect  man  and  other  animals. 
In  man  three  forms  of  the  disease  are  recognized  according  to 
the  mode  of  infection  and  course  of  the  disease — viz.,  bubonic, 
pulmonic,  septicemic. 

Vaccines. — ^The  vaccines  of  Haffkine  and  Terni  and  Bandi 
have  been  used  extensively,  and  with  some  good  results. 

Antitoxins. — ^The  antitoxins  of  Yersin  and  of  Lustig  have 
been  used,  but  without  much  result.  Closely  identified  with 
Bacillus  pestis  is  the  group  known  as  the  hemorrhagic  sep- 
ticemia bacteria 

Bacteria  of  Hemorrhagic  Septicemia  (Hueppe,  1886). 
— Under  this  heading  Hueppe  has  gathered  a  number  of 
.  bacteria  very  similar  to  the  bacillus  of  chicken  cholera,  differ- 
ing from  it  and  each  other  but  very  little.  They  have  been 
described  by  various  observers  and  found  in  different  diseases. 

The  bacteria  of  this  group  color  themselves  strongly  at  the 
poles,  giving  rise  to  the  dumb-bell  shape  (Fig.  97).  They 
do  not  take  the  Gram  stain;  they  are  without  spores,  and  do 
not  liquefy  gelatin. 

They  have  been  divided  into  three  groups.  Bacillus  avi- 
septicus,  as  it  appears  in  fowls;  Bacillus  bovisepticus,  as  it 
attacks  cattle;  Bacillus  suisepticus,  as  it  attacks  swine. 
The  prominent  members  of   each  group  are:    Bacillus  of 


HEMORRHAGIC   SEPTICEMIA   GROUP  1 93 

chicken  cholera  of  Pasteur,  bacillus  of  swine  plague,  and 
bacillus  of  cattle-plague  or  pleuropneumonia. 

Bacillus  of  Chicken  Cholera  (Perroncito,  Pasteur, 
1878). — Synofiyms. — Micrococcus  cholera  gallinarum;  Microbe 
en  huit;  avicidus  bacillus;  bacillus  of  fowl  septicemia. 

Origin. — In  1879  Perroncito  observed  this  coccus-like  ba- 
cillus in  diseases  of  chickens,  and  Pasteur,  in  1880,  isolated 
and  reproduced  the  disease  with  the  bacillus  in  question. 

Form. — At  first  it  was  thought  to  be  a  micrococcus,  but  it 
has  been  found  to  be  a  short  rod,  about  twice  as  long  as  it  is 


Fig.  97. — Bacillus    of    swine-plague   (from    photograph  by  E.   A.  de 

Schweinitz). 

broad,  the  ends  slightly  rounded.  The  center  is  very  slightly 
influenced  by  the  anilin  colors,  the  poles  easily,  so  that  in 
stained  specimens  the  bacillus  looks  like  a  dumb-bell  or  a 
figure-of-8  (Microbe  en  huit). 

Properties. — Does   not   possess   self-movement;    does   not 
liquefy  gelatin;  no  spores. 

Growth. — Occurs  at  ordinary  temperature,  requiring  oxygen 
for  development.     It  grows  very  slowly. 
13 


194  ESSENTIALS   OF   BACTERIOLOGY 

Gelatin  Plates. — In  the  course  of  three  days  little  round, 
white  colonies,  which  seldom  increase  in  size,  having  a  rough 
border  and  very  finely  granulated. 

Stab-cultures. — A  very  delicate  gray  line  along  the  needle- 
track,  which  does  not  become  much  larger. 

Agar  Stroke  Culture. — ^A  moist,  grayish-colored  skin,  more 
appreciable  at  brood-heat. 

Potato. — At  37°  C,  after  several  days,  a  very  thin,  trans- 
parent growth. 

Sugar  Broth. — Acid  fermentation,  no  gas. 
Indol  is  formed. 

Staining. — Methylene-blue  gives  the  best  picture.     Gram's 
method  is  not  applicable.     As  the  bacillus  is  easily  decol- 
orized, anilin-oil  is  used   for  dehydrat- 
ing tissue  sections,  instead  of  alcohol. 

Pathogenesis. — Feeding  the  fowls  with 
the  bacilli  or  injecting  them  under  the 
skin  will  cause  death  in  from  twelve  to 
twenty-four  hours,  the  symptoms  pre- 

Fig.  98.— Chick-  ceding  death  being  those  of  a  severe 
en  cholera  m  blood  ^ .         . 

(X    1000)    (Frankel       septicemia. 

and  Pfeiffer).  The  bacillus  is  then  found  in  the  blood 

and  viscera  and  the  intestinal  discharges, 
the  intestines  presenting  a  hemorrhagic  inflammation. 

Guinea-pigs  and  sheep  are  immune.  Mice  and  rabbits  are 
affected  in  the  same  manner  as  the  fowls. 

Immunity. — Pasteur,  by  injecting  different-aged  cultures 
into  fowls,  produced  in  them  only  a  local  inflammation,  and 
they  were  then  immune.  But  as  the  strength  of  these  cul- 
tures could  not  be  estimated,  many  fowls  died  and  the  healthy 
ones  were  endangered  from  the  in.testinal  excretions,  which  is 
the  chief  manner  of  infection  naturally,  the  feces  becoming 
mixed  with  the  food. 

Bacillus  of  Erysipelas  of  Swine  (Loffler,  Schiitz).— 
Synonyms. — Schweinerotlauf bacillus  (German);  Rouget  du 
Pore  (French). 

Origin. — Found  in  the  spleen  of  an  erysipelatous  swine  by 
Loffler  in  1885. 


HEMORRHAGIC   SEPTICEMIA   GROUP  I95 

Form. — One  of  the  smallest  forms  of  bacilli  known;  very- 
thin,  seldom  longer  than  i  ix,  looking  at  first  like  little  needle- 
like crystals.     Spores  have  not  been  found. 

Properties. — They  are  motile;  do  not  liquefy  gelatin. 

Growth  at  ordinary  temperature  very  slowly,  and  the  less 
oxygen,  the  better  the  growth. 

Gelatin  Plate. — On  third  day  little  silver-gray  specks,  seen 
best  with  a  dark  background,  coalescing  after  a  while,  pro- 
ducing a  clouding  of  the  entire  plate. 

Stab-cultures. — In  a  few  days  a  very  light,  silvery-like  cloud- 
ing, which  gradually  involves  the  entire  gelatin;  held  up 
against  a  dark  object,  it  comes  plainly  into  view. 

Staining. — All  ordinary  dyes  and  Gram's  method  also. 

Tissue  sections  stained  by  Gram's  method  show  the  bacilli 
in  the  cells,  capillaries,  and  arterioles  in  great  numbers. 

Pathogenesis. — Swine,  mice,  rabbits,  and  pigeons  are  sus- 
ceptible; guinea-pigs  and  chickens,  immune. 

When  swine  are  infected  through  food  or  by  injection,  a 
torpidity  develops  with  diarrhea  and  fever,  and  on  the  belly 
and  breast  red  spots  occur  which  coalesce,  but  do  not  give 
rise  to  any  pain  or  swelling.  The  animal  dies  from  exhaus- 
tion in  twenty-four  to  forty-eight  hours.  In  mice  the  lids  are 
glued  together  with  pus. 

At  the  autopsy  the  liver,  spleen,  and  glands  are  enlarged 
and  congested,  little  hemorrhages  occurring  in  the  intestinal 
mucous  membrane  and  that  of  the  stomach. 

Bacilli  are  found  in  the  blood  arid  in  all  the  viscera. 

One  attack,  if  withstood,  protects  against  succeeding  ones. 

Immunity. — Has  also  been  attained  by  injecting  vaccines 
of  two  separate  strengths. 

Bacillus  Murisepticus  (Koch) ;  Mouse  Septicemia. — 
Origin. — Found  in  the  body  of  a  mouse  which  had  died  from 
injection  of  putrid  blood,  and  described  by  Koch  in  1878. 

Form. — Differs  in  no  particular  from  the  bacillus  of  swine 
erysipelas,  excepting  that  it  is  a  very  little  shorter,  making  it 
the  smallest  known  bacillus.  Spores  have  been  found,  the 
cultures  exactly  similar  to  those  of  swine  erysipelas. 


196 


ESSENTIALS    OE   BACTERIOLOGY 


The  pathologic  actions  are  also  similar.  Field-mice  are 
immune,  whereas  for  house  and  white  mice  the  bacillus  is 
fatal  in  two  to  three  days. 

Micrococcus  of  Mai  de  Pis  (Nocard). — Gangrenous 
mastitis  of  sheep. 

Origin. — In  the  milk  and  serum  of  a  sheep  sick  with  the 
^'mal  de  pis." 


.«-— / 
'^/S'^ 


Fig.  99. — Bacillus   of    mouse  septicemia,  from  the  blood  of  a  mouse 
(  X  1000)  (Frankel  and  Pfeiffer). 


Form. — Very  small  cocci,  seldom  in  chains. 
Properties. — Immotile;  liquefying  gelatin. 
Growth. — Growth  occurs  best  between  20°  and  37°  C,  is 
very  rapid,  and  irrespective  of  oxygen. 

Plates  of  Gelatin. — White  round  colonies,  some  on  the  sur- 


PROTOZOA  197 

face  and  some  in  the  deeper  strata,  with  low  power,  appearing 
brown,  surrounded  by  a  transparent  areola. 

Stab-culture. — Very  profuse  along  the  needle-track,  in  the 
form  of  a  cone  after  two  days,  the  colonies  having  gathered 
at  the  apex. 

Potato. — A  dirty  gray,  not  very  abundant,  layer,  somewhat 
viscid. 

Staining. — With  ordinary  methods;  also  Gram's  method. 

Pathogenesis. — If  a  pure  culture  is  injected  into  the  mam- 
mary gland  of  sheep,  a  "mal  de  pis"  is  produced  which 
causes  the  death  of  the  animal  in  twenty-four  to  forty-eight 
hours.  The  breast  is  found  edematous,  likewise  the  thighs 
and  perineum;  the  mammas  very  much  enlarged,  and  at  the 
nipples  a  blue-violet  coloration.  The  spleen  is  small  and 
black ;  other  animals  are  less  susceptible.  In  rabbits  abscesses 
at  the  point  of  infection,  but  no  general  affection. 


CHAPTER  XXVII 
PROTOZOA 

Protozoa  are  unicellular  animal  organisms,  minute  as  bac- 
teria, and  differing  from  bacteria  in  the  methods  of  repro- 
duction. Their  structure  and  functions  are  more  complex, 
although  the  borderland  is  ill  defined.  A  nucleus  is  usually 
present. 

Divisions. — There  are  four  grand  divisions  of  protozoa: 
(i)  Sarcodina,  containing  5500  species;  (2)  mastigophora, 
containing  500  species;  (3)  infusoria,  containing  700  species; 
(4)  sporozoa,  containing  300  species. 

Sarcodina  are  chiefly  marine  forms,  with  processes  change- 
able in  shape.  Examples:  Ameba,  foramnifera,  entameba, 
parasitic  for  man. 

Mastigophora  have  undulating  flagella  and  are  known 
as  flagellates;  to  this  division  the  trypanosomata  belong. 
Example:  Trypanosoma. 


198  ESSENTIALS    OF   BACTERIOLOGY 

Infusoria  have  fine  ciliary  processes  or  numerous  delicate 
flagella.     Example:  Balantidium. 

Sporozoa  have  no  motile  organs,  and  are  reproduced 
by  spores.  To  this  division  belong  the  coccidia  of  malaria 
and  the  organisms  discovered  by  Mallory  in  scarlatina. 
Examples:  Plasmodium,  coccidium. 

Life-cycle. — The  complete  cycle  of  reproduction  has  been 
observed  in  only  one  of  the  pathogenic  protozoa,  namely,  the 
protozoa  of  malaria. 

Methods  of  Cultivation. — Novy,  Clegg  and  others  have 
obtained  pure  cultures  of  protozoa  by  the  use  of  blood-agar 
and  animal  tissue,  or  by  cultivation  with  bacteria,  on  which 
the  ameba  and  other  protozoa  live. 

Entamoeba  Histolytica  (Shaudinn,  1903). — Amoeba 
Dysenteriae. — Found  in  the  intestinal  ulcers,  feces,  and 
secondary  liver  abscesses  in  certain  cases  of  dysentery. 
Kartulis,  in  1886,  definitely  established  the  cause,  although 
amebae  were  noted  in  feces  by  Lamb!  in  i860.  A  non- 
pathogenic form.  Amoeba  coli,  also  occurs.  The  Amoeba  dys- 
enteriae is  a  unicellular  animal  organism,  measuring  25  to  35  )Lt 
in  diameter,  though  larger  and  smaller  forms  occur.  A  nu- 
cleus and  a  nucleolus  are  present;  the  protoplasm  of  the  cell- 
body  is  vacuolated,  and  often  contains  red  blood-cells  and 
bacteria.  In  fresh,  warm  stools  active  ameboid  motion  may 
be  observed.  The  non-pathogenic  form  is  smaller  and  never 
contains  red  blood-cells. 

Examination  for  Amebce. — From  the  slimy  part  of  the 
fresh  feces  a  loopful  is  taken  and  diluted  with  salt  solution 
and  examined  with  moderate  power  on  a  warm  stage.  '  Look 
for  contracting  vacuole  and  motion. 

Staining  with  hematoxylin  eosin  or  eosin  methylene-blue 
after  the  film  on  a  glass  slide  or  cover-glass  has  been  fixed 
in  hot  alcohol  or  methyl  alcohol. 

Cultures. — On  nutrient  agar  a  loopful  of  feces  is  spread  and 
examined  from  day  to  day,  transplanting  the  young  amoebae 
with  their  accompanying  bacteria. 

Pathogenesis. — Inoculation  experiments  with  monkeys  and 


PROTOZOA  199 

dogs  produce  dysentery  and  liver  abscess.  In  man,  50  per 
cent,  of  human  beings  harbor  non-pathogenic  amebae,  but 
the  pathogenic  variety  is  found  mainly  in  tropical  countries, 
where  it  produces  serious  lesions  and  often  occurs  in  wide- 
spread epidemics. 

Source. — It  is  supposed  to  come  from  poor  water  supplies. 
Amebic  dysentery  differs  from  the  bacillary  form  in  that  no 
severe  toxic  symptoms  are  present  and  the  amebic  disease 
is  more  chronic.  The  Shiga  bacillus,  B.  dysenteriae,  is  found 
in  the  bacillary  form  of  dysentery. 

Life  Cycle  of  the  Malarial  Sporozoa. — According  to  its 
situation,  the  parasite  exhibits  two  distinct  phases  of  exist- 
ence :  in  the  human  blood  it  passes  through  an  asexual  repro- 
ductive cycle,  known  as  schizogony,  while  in  the  body  of  the 
mosquito  it  undergoes  an  entirely  different  series  of  sexually 
reproductive  changes,  called  sporogony. 

I.  The  Asexual  Cycle  in  Man. — An  infected  mosquito  con- 
veys the  parasites  into  the  blood  of  man  as  minute  hyaline 
bodies  which  enter  the  blood-cells.  At  first  they  are  small, 
round,  colorless  bodies,  exhibiting  more  or  less  active  ameboid 
motion  in  the  fresh  blood.  Sometimes,  particularly  in  the 
estivo-autumnal  form,  a  ring  shape  is  assumed.  Their  size 
gradually  increases  and  pigment-granules  appear,  while  in 
stained  specimens  a  nucleus  containing  chromatin  granules 
is  visible.  As  the  parasite  approaches  maturity  the  chroma- 
tin becomes  scattered,  and  finally  the  protoplasm  or  mother- 
cell,  known  as  sporocyte,  divides  into  six  to  twenty  spores, 
daughter-cells  or  merozoites,  each  containing  a  portion  of  the 
chromatin.  The  number  of  spores  formed  and  their  arrange- 
ment before  segmentation  takes  place  differ  in  the  three 
varieties  and  will  be  noted  below.  The  spores  burst  through 
the  envelop  of  the  red  corpuscle  and  become  free  in  the  blood, 
but  speedily  enter  fresh  corpuscles  and  pass  through  the 
same  series  of  changes.  The  febrile  stage  is  synchronous 
with  sporulation  and  liberation  of  the  young  forms. 

Certain  of  the  parasites  do  not,  however,  go  on  to  segmenta- 
tion, but  after  reaching  maturity,  remain  quiescent  and  form 


200 


ESSENTIALS  OF  BACTERIOLOGY 


the  so-called  gametes  or  sexual  types.     In  the  tertian  and 
quartan  varieties  these  are  not  very  different  from  the  mature 


*  '  Human  Phase-  [     Jj\ 


■ftn. 


^        THE  EMDOOCHCTUS  OR  'Nw-^v 

U/il    '  (^04    ASEXUAL.  CyCLX.  ^^^1      \ 


THE 

MOSQCJITO    PHA3E 
EXOQENOLJS 


-Sexual-  cvn  f.. 


Fig.  100. — Schema  showing  the  human  and  mosquito  cycles  of  the 
malarial  parasite:  A,  Normal  red  cell;  B,  C,  D,  E,  red  cells  containing 
amebulas  or  myxopods;  F,  G,  H,  sporocytes;  J',  K',  L',  M',  microgame- 
tocytes  or  male  gametes;  J",  K",  L",  M",  O,  macrogametocytes,  or 
female  gametes;  N',  M',  microgametes;  P,  traveling  vermicule;  Q, 
young  zygote;  R,  S,  zygotomeres;  T,  blastophore;  U,  mature  zygote 
(modified  from  Blanchard's  diagram  illustrating  life-cycle  of  Coccidium 
schubergi)  (Rees,  in  "Practitioner,"    March,  1901). 


organisms,  but  the  estivo-autumnal  gametes  are  crescentic  in 
shape  and  very  characteristic. 


PROTOZOA  20I 

2.  The  Sexual  Cycle  in  the  Mosquito. — The  common  mos- 
quito is  known  as  Culex  and  does  not  harbor  the  malarial 
parasite.  The  anopheles  species,  spotted  wings,  is  the  true 
host;  only  the  females  are  bloodsuckers  and  responsible  for 
the  spread  of  the  disease.  They  take  the  infected  blood 
containing  the  male  element  and  which  represents  the  male 
fertilizing  element  {micro gametes).  These  become  detached, 
and,  entering  a  female  gamete  {macro gamete),  a  true  sexual 
fertilizing  process  takes  place.  In  the  alimentary  canal  of 
the  mosquito  these  fertilized  cells  penetrate  the  stomach- 
walls  and  form  cysts  (oocysts)  filled  with  a  large  number  of 
filiform  spores  (sporozoites),  which  are  extruded  into  the 
body  cavity  of  the  insect,  and  some  of  w^hich  reach  the  salivary 
glands,  whence  they  are  ejected  when  the  mosquito  bites. 
This  cycle  of  development  takes  seven  or  eight  days. 

Three  Forms  of  Malarial  Protozoa. — i.  Plasmodium 
Vivax,  or  The  Tertian  Form. — The  adult  forms  are  large,  not 
very  refractile,  and  their  outline  is  somewhat  indistinct. 
There  is  an  abundance  of  fine  pigment-granules,  and  the 
ameboid  motion  is  vigorous.  Segmenting  forms  divide  into 
fifteen  to  twenty  merozoites ;  the  sexual  forms  or  gametes  are 
large  The  red  cell  containing  the  organism  is  swollen  and 
pale.  Sporulation  and,  therefore,  the  malarial  paroxysm 
occur  every  forty-eight  hours. 

2.  Plasmodium  Malarice,  the  Quartan  Form. — The  organism 
is  smaller,  is  more  refractile,  and  ij:s  outline  is  more  distinct. 
The  pigment  is  coarse  and  situated  at  the  periphery  of  the 
organism,  while  the  protoplasmic  motion  is  sluggish.  Seg- 
mentation forms  only  six  to  twelve  spores,  and  has  the  regular 
'Maisy-head"  appearance;  the  gametes  are  small.  The  red 
cells  become  dark  in  color,  and  the  cycle  requires  seventy- 
two  hours. 

3.  Plasmodium  Falciparum,  or  Malignant  Tertian,  or  Estivo- 
autumnal  Form. — The  adult  forms  are  found  mainly  in  the 
spleen  and  other  viscera,  and  do  not  very  often  occur  in  the 
peripheral  blood;  their  outline  is  sharp,  and  they  are  highly 
refractile.     The  pigment  is  scanty  and  fine;  the  motion  is 


202 


ESSENTIALS   OF   BACTERIOLOGY 


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PROTOZOA  203 

active.  A  variable  number  of  merozoites  is  formed — usually 
six  to  twelve.  The  gametes  are  characteristic,  being  cres- 
centic  in  shape  and  very  resistant  to  quinin.  The  red  cell 
becomes  shriveled  and  yellowish.  The  cycle  usually  takes 
forty-eight  hours,  though  it  is  somewhat  variable. 

Mixed  infections  with  the  different  organisms  or  with  two 
or  more  broods  of  the  same  organism  may  occur,  so  that 
quotidian  and  irregular  paroxysms  may  be  produced. 

Transmission. — Malaria  is  spread  by  means  of  a  mosquito, 
the  anopheles,  in  whose  body  the  protozoon  undergoes  its 
highest  development.  Man  is  the  intermediate  host;  the 
mosquito,  the  true  host. 

Methods  of  Examination  for  Malarial  Organisms. — 
I.  Fresh  preparations  are  made  by  placing  a  small  drop  of 
blood  on  a  slide  and  a  cover-glass  over  it,  so  that  only  a  thin 
film  is  formed.  A  ring  of  vaselin  is  smeared  over  the  edges  of 
the  cover-glass  to  prevent  evaporation.  This  is  the  best 
method  for  studying  flagellation  and  fertilization,  but  is  less 
satisfactory  for  routine  clinical  work  than — 

2.  Stained  Smears. — These  are  made  by  spreading  a  drop 
of  blood  in  a  thin  film  over  one  slide  with  the  edge  of  another, 
drying  in  the  air,  and  staining.  Many  stains  have  been 
devised  for  the  malarial  organism,  but  Jenner's  or  Wright's 
is  sufficient  for  ordinary  use: 

(i)  Jenner's  Stain. — This  is  excellent  for  routine  work,  as 
no  preparatory  fixation  is  required.  The  smears  are  dropped 
into  this  stain  for  one  to  three  minutes,  without  previous 
fixation,  and  at  once  rinsed  in  distilled  water.  The  malarial 
parasites  are  stained  blue,  the  cell-bodies  a  reddish  brown. 

Fig.  loi. — Various  forms  of  malarial  parasites  (Thayer  and  Hewet- 
son):  i-io  inclusive,  tertian  organisms;  11-17  inclusive,  quartan  organ- 
isms;  18-27  inclusive,  estivo-autumnal  organisms. 

I,  Young  hyaline  form;  2,  hyaline  form  with  beginning  pigmenta- 
tion; 3,  pigmented  form;  4,  full-grown  pigmented  form;  5,  6,  7,  8,  seg- 
menting forms;  9,  mature  pigmented  form;   10,  flagellate  form. 

II,  Young  hyaline  form;  12,  13,  pigmented  forms;  14,  fully  devel- 
oped form;    15,  16,  segmenting  forms;   17,  flagellate  form. 

18,  19,  20,  Ring-like  and  cross-like  hyaline  forms;  21,  22,  pigmented 
forms;   23,  24,  segmenting  forms;   25,  26,  27,  crescents. 


204  ESSENTIALS   OF   BACTERIOLOGY 

(2)  WrigMs  Chromatin  Stain. — This  is  the  best  of  the 
chromatin  stains.  For  its  preparation,  which  is  quite  com- 
plicated, see  Wright,  Journal  of  Medical  Research,  vol.  vii, 
1902.  It  can  be  purchased  already  made.  It  is  used  as 
follows : 

1.  The  stain  is  poured  over  the  film  and  allowed  to  remain 
for  one  minute  to  secure  fixation. 

2.  Add  distilled  water  drop  by  drop  until,  a  metallic  scum 
is  formed  on  the  surface.  The  staining  now  takes  place  and 
requires  two  to  three  minutes.     Wash  in  distilled  water  until 


Fig.  102. — Pure  culture  of  trypanosomes  of  mosquitos — Crithidia 
fasciculata.  Multiplication  roset  showing  large  and  small  cells.  Nine- 
day  culture  (Gen.  i  X  1500)  (Novy,  MacNeal,  and  Torrey), 

a  pinkish  tint  appears  in  the  thin  portions  of  the  smear.  The 
body  of  the  malarial  parasite  is  stained  blue,  and  its  chromatin 
a  lilac  to  red  color.     The  red  cells  are  orange-pink. 

If  possible,  examinations  for  malarial  organisms  should 
always  be  made  before  quinin  is  administered. 

Trypanosomata. — Trypanosomes  are  flagellate  protozoa 
found  in  the  blood  of  various  animals,  and  causing  a  number 
of  diseases,  such  as  surra,  dourine,  and  nagana,  affecting 
horses  and  cattle,  especially  in  tropical  countries,  and  causing 


PROTOZOA  205 

the  sleeping  sickness  of  Africa,  which  is  very  fatal  for  human 
beings.  About  60  species  have  been  described,  and  10  dis- 
eases are  believed  to  be  due  to  this  form  of  organism. 

Morphology. — A  fusiform  mass,  containing  at  one  end  a 
flagellum  (Fig.  103). 

In  the  livihg  state  these  protozoa  are  very  motile.  In  the 
stained  specimen  chromatin  granules  are  found  and  two  or 
more  nuclei.  From  the  smaller  nucleus  arises  the  undulatory 
membrane,  which  passes  into  the  flagellum  and  assists  in  the 
wave-like  motion. 


Fig.  103. — Pure  culture  of  trypanosomes  of  mosquitos — Crithidia 
fasciculata.  Part  of  roset  of  elongated  crithidia  with  flagella  directed 
centrally  (Gen.  39  X  1500)  (Novy,  MacNeal,  and  Torrey). 


In  the  body  fluids  division  occurs,  first  of  the  nucleus  and 
then  of  the  protoplasm. 

Cultivation. — Novy  and  MacNeal  have  succeeded  in  culti- 
vating these  protozoa  on  blood-agar,  and  multiplication  goes 
on  rapidly,  so  that  rosettes  are  formed  with  the  flagella  ar- 
ranged around  a  common  center.     (See  Figs.  102,  103,  104.) 

Trypanosoma  Lewisi  (Kent,  1878). — Found  in  rats  by 
Lewis;  not  fatal  to  them,  though  often  equaling  the  red  cor- 
puscles in  number.     It  was  one  of  the  first  of  this  group  to 


2o6  ESSENTIALS   OF   BACTERIOLOGY 

be  described.  The  infection  continues  for  two  months  with- 
out producing  any  illness,  and  the  animal  is  then  immune. 

Injection  of  infected  rat  blood  into  healthy  rat  causes  the 
latter  to  become  infected. 

The  injection  of  serum  from  an  immune  rat  will  prevent 
the  disease  in  normal  rats. 

Cultivated  best  at  20°  C.  and  is  very  resistant  to  cold. 
The  rat  is  probably  infected  by  the  bite  of  a  flea  or  louse. 
(See  Fig.  105.) 


/ 


Fig.  104. — Pure  culture  of  trypanosomes  of  mosquitos — Crithidia 
fasciculata.  Elongated  crithidia  from  same  preparation  as  preceding 
(Novy,  MacNeal,  and  Torrey). 

Trypanosoma  Brucei  (Plimmer  and  Bradford,  1894) 

causes  nagana,  or  tsetse-fly  disease,  a  disease  affecting  horses, 
cattle,  and  dogs  in  certain  regions  of  South  Africa.  The 
trypanosome  of  Bruce  is  less  motile  than  that  of  Lewis.  It 
has  been  cultivated  at  25°  C,  and  is  less  resistant  to  cold. 
All  laboratory  animals  subject  to  infection.  The  rat  dies  in 
ten  days. 

In  the  natural  infection  Bruce  discovered  that  the  tsetse- 
fly  transmitted  the  disease,  but  that  it  did  so  by  first  biting 
some  animal  whose  blood  contained  the  trypanosome.     The 


PROTOZOA 


207 


blood  of  infected  animals  contains  the  organism,  and  can,  if 
injected,  produce  the  disease  without  the  agency  of  the  fly. 
So  far  the  tsetse-fly  alone  is  responsible  for  the  spread  of  the 
infection. 

Sleeping  Sickness. — Trypanosoma  Ugandense  Gam- 
biense  (Button,  1904). — {T.  Castellani,  T.  Hominis,  T. 
Neprevi.) — Sleeping  sickness,  or  human  trypanosomiasis,  is  a 
disease  peculiar  to  some  parts  of  Africa.  It  is  accompanied 
by  periods  of  fever,  anemia,  and,  finally,  a  lethargy  deepening 


Fig.  105. — Trypanosome  from  blood  of  gray  rat;    stained  with  a  2  per 
cent,  aqueous  solution  of  methylene-blue   (Boston). 


into  coma  and  death.  The  disease  may  be  rapid,  and  it 
may  last  with  recurrences  for  many  years.  Trypanosomes 
identical  with  those  found  in  nagana  disease  have  been 
found  in  the  blood  of  infected  persons,  and  described  by 
various  observers,  and  given  different  names. 

Monkeys,  when  inoculated  with  cerebrospinal  fluid  from 
affected  persons,  develop  a  similar  disease,  and  the  parasites 
are  found  in  the  blood.  So  far  the  organism  has  not  been 
cultivated. 


208  ESSENTIALS   OF   BACTERIOLOGY 

A  blood-sucking  fly,  known  as  the  Glossina  palpalis,  is  con- 
sidered the  means  of  infection.  The  fly  is  closely  related  to 
the  Glossina  morsitans,  or  tsetse  fly.  The  sleeping  sickness  in 
man  is  most  likely  the  same  thing  as  the  nagana  of  cattle. 

Methods  of  Examinations. — From  Blood. — A  patient  search 
may  fail  to  detect  the  organisms — a  large  amount  of  blood, 
lo  c.c,  obtained  by  venesection — is  centrifuged  and  the  white 
cells  examined  in  hanging  drop  or  stained  smear. 

Cerebrospinal  fluid  will  at  times  give  results. 

Animal  Inoculation. — The  blood  of  suspected  person  in- 
jected into  monkeys  or  rats  and  the  resulting  infection  stu- 
died by  above  methods. 

Staining. — The  organism  is  best  stained  by  Giemsa  stain 
or  the  Romanowsky  method. 

Trypanosoma  Evansi  (Steel,  1880). — Pathogenic  for  all 
animals. 

Discovered  by  Evans  in  the  blood  of  horses  suffering  from 
surra,  a  disease  prevalent  in  India  and  the  Philippine  Islands. 
The  disease  resembles  nagana. 

T.  equiperdum  and  T.  Rougetii  are  names  given  to  similar 
organisms  found  in  dourine,  a  disease  affecting  horses  in 
southern  France  and  Spain.  Trypanosomes  are  found  in  fish, 
oysters,  birds,  and  frogs,  and  many  varieties  have  been 
described. 

Herpetomonas  (Leishman,  1903)  (Leishman-Donovan 
Bodies). — A  disease  called  variously  kala-azar,  dum-dum 
fever,  tropical  splenomegaly,  is  considered  to  be  due  to  an  or- 
ganism somewhat  related  to  the  trypanosomes. 

Smears  are  stained  after  fixation  by  the  Wright  or  Roman- 
owsky stains.  Cultivation  has  succeeded  on  blood-media 
made  acid  with  citric  acid. 

The  bedbug  is  considered  instrumental  in  transmitting  the 
organism. 

Piroplasma  Bovis  (P.  Bigeminum)  (T.  H.  Smith,  1893). 
— Origin. — In  the  blood  of  animals  suffering  from  Texas 
cattle-fever. 

Form. — A  pear-shaped  protozoon,  found  in  pairs  in  the  red 


THE  MICRO-ORGANISM  OF  SYPHILIS  AND  ALLIED  ORGANISMS    209 

cells  of  the  blood,  the  smaller  ends  of  pear  in  opposition; 
coarse  ameboid  movement. 

Transmission. — An  insect  or  tick  (Boophilus  bovis)  be- 
comes infected,  and  by  its  bite  infects  other  animals. 

Other  similar  sporozoa  have  been  found  in  animal  diseases 
and  in  man  in  Rocky  mountain  fever.  The  P.  hominis  has 
been  described,  but  not  definitely  determined. 

Rabies  or  Hydrophobia. — Negri  Bodies  (Negri,  1903). 
— Origin. — Found  in  the  nervous  system  of  animals  dying  of 
rabies  (hydrophobia). 

Form. — Round  and  oval,  hyaline  bodies,  with  a  sharp  out- 
line and  containing  a  nucleolus.  The  plasma  is  slightly 
granular.     They  are  regarded  as  protozoa. 

Staining. — A  smear  from  brain  tissue  is  made  on  a  cover- 
glass  and  fixed  in  methyl-alcohol  for  five  minutes;  then  stained 
by  Giemsa;  stain  for  half-hour  to  three  hours. 

All  mammals  susceptible;  man  chiefly  from  bite  of  dog. 
Only  a  small  percentage  of  persons  bitten  by  rabid  dog  be- 
come infected — 16  per  cent. 

The  virus  resides  in  the  saliva,  and  also  in  the  central 
nervous  system.  The  Pasteur  preventive  is  an  accepted  fact, 
and  depends  for  its  power  on  a  form  of  active  immunization. 
The  virus  used  is  obtained  from  dried  spinal  cord  of  infected 
rabbits,  gradually  increasing  the  virulence,  older  cords  first 
used  and  then  cords  exposed  to  drying  for  lesser  time. 


CHAPTER  XXVIII 

THE  MICRO-ORGANISM  OF  SYPHILIS  AND  ALLIED 
ORGANISMS 

Spirochaeta    Pallida    (Schaudinn,    1905). — Spironema 
Pallidum;  Treponema  Pallidum. — Found  in  hereditary  syph- 
ilis in  all  organs,  in  chancre,  and  lymphatic  glands,  and  in 
secondary  lesions,  mucous  patches,  in   the  internal  organs, 
14 


2IO 


ESSENTIALS    OF   BACTERIOLOGY 


and  likewise  in  the  tertiary  lesions,  the  very  latest  being  the 
brain,  and  cerebrospinal  fluid  in  cases  of  general  paralysis,  and 
establishing  the  identity  of  this  disease  with  cerebral  syphilis. 
Form. — A  minute,  spiral-shaped  organism,  with  6  to  20 
curves,  ends  tapering.  Actively  motile  in  fresh  specimen 
(Fig.  106),  intracellular,  and  affecting  glandular  epithelium. 
Staining. — The  organism  requires  special  staining,  and  a 
number  of  complicated  methods  have  been  introduced  by 

different  investigators. 
The  Giemsa  stain  is 
said  to  give  the  best 
results.  (See  Staining 
Fluids,  p.  47.) 

The  slide  is  fixed, 
dried  in  air,  hardened 
in  absolute  alcohol 
twenty  -  five  minutes, 
stained  with  dilute 
stain  (i  drop  to  i  c.c. 
^•"^^'^'^^'^  .*'  ^         ^^  water)  for  ten  min- 

■""'"'■'"  utes,  washed  in  water, 

and  mounted. 

In  tissues  the  organ- 
ism can  be  shown  by 
fixing  with  silver  ni- 
trate after  the  manner 
of  Ramon  y  Cajal.  The 
tissue  is — (i)  Hardened  in  formalin  for  twenty-four  hours 
(the  sections  should  be  thin) ;  (2)  washed  in  water  for  one 
hour;  (3)  alcohol,  twenty-four  hours;  (4)  i^  per  cent,  silver 
nitrate  solution  in  incubator  at  37°  C,  three  days;  (5)  washed 
in  water  twenty  minutes;  (6)  placed  in  mixture  of  pyrogallic 
acid,  4  parts;  formalin,  5  parts;  distilled  water,  to  make  100 
parts,  and  kept  in  dark  bottle  for  forty-eight  hours;  (7) 
washed  in  water  and  alcohol  and  then  embedded  in  paraffin 
and  sectioned.  Spirochaetae  black,  tissues,  pale  yellow.  Or 
counterstain  of  f  uchsin  can  be  employed. 


Fig,  106. — Spirochseta  pallida.  Micro- 
photograph  made  by  Dr.  R.  E.  Lavenson 
from  a  specimen  prepared  by  H.  Fox 
(Stengel). 


THE  MICRO-ORGANISM  OF  SYPHILIS  AND  ALLIED  ORGANISMS    211 

The  Iftdia  Ink  Method. — A  drop  of  fluid  from  a  lesion  is 
mixed  with  a  drop  of  India  ink  upon  a  clean  glass  slide  and 
allowed  to  dry.  Examine  with  oil-immersion  lens.  The 
spirilla  appear  dark  in  a  mass  of  carbon  particles.  By  using 
dark  ground  illumination,  the  organism  appears  brightly 
refractive. 

Culture  Methods. — Noguchi,  by  using  a  serum  water  (i 
part  sheep  or  horse  serum,  3  parts  water,  and  adding  a  piece 
of  sterile  rabbit's  kidney  or  testicle),  under  strict  anae- 
robic conditions  at  35°  C.  succeeded  in  cultivating  the  organ- 
ism direct  from  lesions  in  man.  After  several  transfers  the 
organism  will  grow  on  agar  containing  the  bit  of  tissue. 

Inoculation  Experiments. — Pure  cultures  inoculated  into 
rabbits  and  monkeys  produce  lesions  resembling  .the  primary 
sores,  and  the  blood  of  such  animals  gives  a  Wassermann 
reaction.  Cutaneous  inoculation  on  eyebrows  and  genitals 
of  material  from  primary  and  secondary  lesions  produces 
results  in  from  fifteen  to  fifty  days. 

Wassermann  Reaction. — In  1906  Wassermann,  Neisser, 
and  Bruck  described  a  method  of  making  the  diagnosis  of 
syphilis  by  demonstrating  in  the  blood  and  spinal  fluid  of 
a  patient  complement-binding  substances  not  present  in 
normal  blood. 

Technic. — The  following  reagents  are  employed:  (i)  Syphi- 
litic antigen;  (2)  serum  to  be  tested;  (3)  fresh  guinea-pig 
serum;  (4)  washed  sheep  corpuscles  and  antisheep  ambo- 
ceptor. 

The  antigen  is  an  alcoholic  extract  of  liver  from  a  congenital 
syphilitic,  and  is  prepared  by  extracting  the  ground-up  liver 
with  five  volumes  of  absolute  alcohol  for  ten  days  and  then 
filtering. 

Complement  is  normal  guinea-pig  serum. 

Antisheep  amboceptor  is  obtained  by  injecting  into  a  rabbit 
2,  4,  6,  8,  and  12  c.c.  of  washed  sheep  corpuscles  on  the  first, 
tenth,  nineteenth,  twenty-eighth,  and  thirty-seventh  days 
respectively.  Nine  days  after  the  last  injection  the  animal 
is  bled  to  death  from  the  carotid  and  the  blood  collected  in 


212  ESSENTIALS    OF   BACTERIOLOGY 

sterile  test-tubes.  After  clotting  has  taken  place  the  clear 
serum  is  removed.     This  is  the  amboceptor  serum. 

Washed  sheep  corpuscles  are  obtained  by  centrifuging  de- 
fibrinated  sheep  blood,  pipeting  off  the  serum,  replacing  it 
with  normal  salt  solution,  shaking,  and  again  centrifuging. 
This  is  repeated  three  times. 

Patient's  serum  obtained  from  blood  from  the  patient's  arm 
is  heated  thirty  minutes  at  56°  C.  to  destroy  complement. 

Titration  or  Testing  of  Reagents. — Titrate  amboceptor. 
One  c.c.  of  a  5  per  cent,  suspension  of  washed  sheep  cor- 
puscles in  salt  solution  and  o.i  c.c.  of  fresh  guinea-pig 
serum  are  added  to  a  series  of  test-tubes.  The  amboceptor 
serum  is  then  added  so  that  each  tube  receives  more  than  the 
preceding  one.  Salt  solution  is  added  to  make  5  c.c.  and 
the  tubes  incubated  for  two  hours  at  37°  C.  with  occasional 
shaking.  That  tube  in  which  complete  hemolysis  has  taken 
place  in  just  two  hours  contains  \  unit  of  amboceptor. 

Titration  of  Complement. — Into  each  of  a  series  of  tubes 
place  I  c.c.  of  the  corpuscle  suspension  and  \  unit  of  ambo- 
ceptor. Next  add  0.6,  0.7,  0.8,  0.9,  i,  i.i,  1.2  c.c.  of  fresh 
guinea-pig  serum  respectively  and  incubate  for  two  hours, 
shaking  occasionally.  Those  tubes  which  show  complete 
hemolysis  in  just  two  hours  contain  i  unit  of  complement. 

Titration  of  Antigen. — Two-tenths  c.c.  of  serum,  pre- 
viously heated  to  56°  C.  for  a  half-hour,  from  a  known,  un- 
treated case  of  secondary  syphilis,  and  i  unit  of  complement 
are  added  to  each  of  a  series  of  test-tubes.  Antigen  is  now 
added,  so  that  each  tube  contains  more  than  the  preceding 
one,  and  salt  solution  added  and  brought  to  3  c.c.  The 
mixture  is  incubated  for  one  hour  at  37°  C,  at  the  end  of 
which  time  2  units  of  amboceptor  and  i  c.c.  of  corpuscle 
suspension  are  added  and  the  tubes  returned  to  the  incubator. 
After  a  short  period  the  tube  containing  the  smallest  amount 
of  antigen  will  show  complete  hemolysis.  As  the  dose  of 
antigen  is  increased  the  amount  of  hemolysis  is  decreased 
until  a  point  is  reached  at  which  no  hemolysis  takes  place 
even  after  twenty-four  hours.     The  first  tube  in  the  series 


THE  MICRO-ORGANISM  OF  SYPHILIS  AND  ALLIED  ORGANISMS    213 

which  shows  no  hemolysis  after  twenty-four  hours  contains 
i'  unit  of  antigen  provided  tw^ice  that  amount  will  not  prevent 
hemolysis  when  no  serum  is  added. 

Having  found  out  the  exact  amount  of  guinea-pig  serum 
(complement)  necessary  to  unite  with  hemolytic  amboceptor 
(rabbit  serum)  in  order  to  hemolyze  blood-corpuscles,  this 
amount  is  mixed  with  syphilitic  antigen  plus  the  suspected 
syphilitic  serum  amboceptor,  and  incubated  for  one  hour  at 
37°  C.  //  the  amboceptor  is  syphilitic,  it  will  combine  with 
the  antigen  and  guinea-pig  complement.  To  find  out  if  the 
complement  has  been  bound,  the  hemolytic  amboceptor  and 
its  antigen  sheep  corpuscles  are  added  to  the  mixture, 
and  if  no  hemolysis  takes  place,  the  complement  is  fixed  and  the 
patient'' s  serum  contains  the  syphilitic  antibodies  or  amboceptors. 

To  Set  Up  Test. — Nine  tubes  needed  for  Wassermann  reac- 
tion and  control.  Into  each  tube  i  c.c.  diluted  complement 
guinea-pig  serum.  Into  tubes  1,2,  and  9,  0.2  c.c.  of  patient's 
serum.  Into  tubes  3  and  4,  control,  syphilitic  serum  0.2  c.c; 
in  5  and  6,  normal  serum  as  control,  0.2  c.c. ;  antigen  extract, 
I  unit  placed  in  i,  3,  5,  and  7. 

To  each  tube  is  now  added  sufficient  normal  salt  solution 
to  make  3  c.c.  Tubes  gently  shaken  and  placed  in  incubator 
at  37°  C.  one  hour.     At  end  of  the  hour  to  each  tube  is  added 

1  unit  of  suspension  sheep  corpuscles,  and  to  all  but  No.  9 

2  units  of  standard  amboceptor,  in  i  c.c.  saline. 

The  tubes  again  placed  in  incubator  for  one  hour,  readings 
taken,  and  then  placed  in  ice-box  twenty-four  hours,  when 
final  results  noted.    If  Wassermann  positive — 

No.  I.  No  hemolysis. 

No.  2.  Complete  hemolysis. 

No.  3.  No  hemolysis. 

No.  4.  Complete. 

No.  5.  Complete. 

No.  6.  Complete. 

No.  7.  Complete. 

No.  8.  Complete. 

No.  9.  No  hemolysis. 


214 


ESSENTIALS    OF   BACTERIOLOGY 
WASSERMANN  SCHEME 


6 

< 

5 

Patient's  Serum 

Known  Syphilitic 
Serum 

Bi 
M 

in 

< 

"Si 
fi 
o 

S 
o 

1 

< 

en 

1 

o 
U 

M 
M 

1 

+  Result 

I 

2 

3 
4 
5 
6 

7 
8 

9 

I  c.c. 
I  c.c. 
I  c.c. 
I  c.c. 
I  c.c. 
I  c.c. 
I  c.c. 
I  c.c. 
I  c.c. 

0.2  C.C. 
0.2  C.c. 

0.2  C.C. 

• 
0.2  C.C. 

0.2  C.C. 
0.2  C.C. 

I 
I 
I 
I 

s 
'il 

1 

1 

1/2 

I 

1 

u 
1— 1 

No  hemolysis. 

+ 
Complete 

hemolysis. 
No  hemolysis. 

+ 
Complete 

hemolysis. 
Complete 

hemolysis. 
Complete 

hemolysis. 
Complete 

hemolysis. 
Complete 

hemolysis. 
As   a   rule,  no 

hemolysis. 

Noguchi  modification  of  the  Wassermann  reaction 

consists  in  using  human  corpuscles  and  antihuman  ambocep- 
tor, and,  as  antigen,  acetone  insoluble  lipoids. 

Antigen. — Extract  a  finely  ground  ox-heart  with  lo  vol- 
umes of  absolute  alcohol  at  37°  C.  for  several  days;  filter 
and  evaporate  the  extract  (using  an  electric  fan  and  not  heat) 
almost  to  dryness.  Extract  the  residue  with  ether;  decant, 
evaporate  the  ether,  and  redissolve  in  the  smallest  quantity  of 
pure  water-free  ether.  To  this  ethereal  solution  add  5 
volumes  of  water-free  acetone.  A  precipitate  forms  which 
is  the  antigen.  The  precipitate  is  dissolved  in  purest  methyl- 
alcohol  in  the  proportion  of  3  per  cent.  For  use,  i  c.c.  of 
this  alcoholic  solution  is  mixed  with  9  c.c.  of  salt  solution. 

Titration  of  Antigen. — (i)  Hemolytic  Action. — A  tube  con- 


THE  MICRO-ORGANISM  OF  SYPHILIS  AND  ALLIED  ORGANISMS   21$ 

taining  0.4  c.c.  of  the  antigen  emulsion,  o.i  c.c.  of  10  per 
cent,  suspension  of  corpuscles,  and  0.6  c.c.  of  salt  solution 
should  show  no  hemolysis  after  two  hours  at  37°  C. 

(2)  Anticomplementary  Bodies. — A  tube  containing  0.4  c.c. 
of  antigen,  o.i  c.c.  of  a  40  per  cent,  dilution  of  complement, 
2  units  of  amboceptor,  and  0.6  c.c.  of  salt  solution  is  incu- 
bated for  one  hour  and  0.1  c.c.  of  10  per  cent,  corpuscle  sus- 
pension added.  In  two  hours  there  should  be  complete 
hemolysis. 

(3)  Antigenic  Properties. — After  incubating  for  one  hour  a 
tube  containing  0.02  c.c.  antigen,  0.02  c.c.  of  a  known  syphilitic 
serum,  0.1  c.c.  of  a  40  per  cent,  dilution  of  complement,  2 
units  of  amboceptor,  and  0.8  c.c.  of  salt  solution,  0.1  c.c.  of  a 
10  per  cent,  corpuscle  suspension  is  added,  and  the  tube 
returned  to  the  incubater.  At  the  end  of  two  hours  there 
should  be  no  hemolysis. 

(4)  Amboceptor  and  complement  are  titrated  the  same  as 
in  the  Wassermann  reaction,  except  that  a  i  per  cent,  sus- 
pension of  human  corpuscles  and  0.02  c.c.  of  complement 
and  antihuman  amboceptor  are  used. 


NOGUCHI  SCHEME 


1 

0 

< 

After  Ten  Hours 

f 

I 

I          .  . 

oT   • 

u 

I   i 

No  hemolysis. 

Patients    ] 

0 

Positive 

2 
3 

I 

I 

d 

I 

1 

Complete    hemol- 
ysis. 
No  hemolysis. 

control  ■ 

4 

I 

r^ 

0 

2'^ 

3 

Complete    hemol- 

Negative  f 

S 

I 

c3 

S  -2 

I 

5 

3 

a  c^ 

(S3 

:3 

ysis. 
Partial,  later  com- 

control 

6 

I 

"a 

1 

1— 1 

plete. 
Complete    hemol- 

^ 

U 

ysis. 

2l6  ESSENTIALS   OF   BACTERIOLOGY 

Explanation  of  Noguchi  Modified. — Requires  six  tubes: 

In  I  and  2,  one  drop  serum  to  be  tested;  in  3  and  4,  one 
drop  known  syphilitic  serum;  in  5  and  6,  one  drop  normal 
serum. 

To  each  tube  add  i  c.c.  i  per  cent,  suspension  washed 
human  blood-corpuscles,  and  o.i  c.c.  40  per  cent,  fresh 
guinea-pig  serum  (complement). 

Into  1,3,  and  5,  one  drop  antigen  solution. 

Incubate  at  37°  C.  one  hour,  then  add  2  units  antihuman 
amboceptor  to  each  tube.  Incubate  two  hours  and  read 
reaction  every  hour  for  next  ten  hours,  keeping  tubes  at 
room  temperature. 

Tubes  2,  4,  6,  complete  hemolysis. 

Tube  5,  complete  hemolysis. 

Tube  3,  no  hemolysis. 

Tube  I,  no  hemolysis. 

Results  of  Wassermann  Test. — Eighty  per  cent,  of  primary 
cases  give  a  positive  result,  but  a  negative  reaction  in  this 
stage  does  not  mean  much,  as  nearly  20  per  cent,  of  cases  are 
negative. 

95  per  cent,  of  secondary  cases  give  a  positive  reaction. 

85  per  cent,  of  tertian  positive. 

90  per  cent,  congenital  forms  strongly  positive. 

100  per  cent,  general  paresis  positive. 
50  per  cent,  locomotor  ataxia  positive. 
65  per  cent,  latent  tertiary  forms. 

Luetin  Reaction  (Noguchi). — An  emulsion  of  a  pure 
culture  of  the  spirochetes  of  syphilis  heated  to  60°  C.  for 
one  hour,  and  made  sterile,  is  called  luetin. 

When  applied  subcutaneously  by  means  of  a  fine  needle, 
an  erythema  lasting  forty-eight  hours  results  in  normal 
persons,  but  in  persons  affected  with  tertiary,  latent,  and 
congenital  syphilis  after  forty-eight  hours  a  small  induration 
or  papule  appears,  which  at  times  becomes  vesicular  and 
pustular,  increasing  in  redness  and  turning  bluish  red  in 
three  or  four  days.     It  is  an  adjunct  to  other  tests  for  syphilis. 


THE  MICRO-ORGANISM  OF  SYPHILIS  AND  ALLIED  ORGANISMS    2X7 

Yaws. — Spirochetes   similar   and  possibly   identical   with 
those  of  syphilis  have  been  found  in  this  tropical  disease. 
Spirillum  of  Relapsing  Fever  (Obenneier,  1873). — 

Synonym. — Spirochceta  Ohermeieri. 

The  definite  classification  of  this  organism  has  not  been 
made.  Some  regard  it  now  as  a  protozoon,  and  one  of  a 
group  in  which  numerous  other  spirilla  belong. 

Origin. — Found  in  the  blood  of  recurrent  fever  patients, 
described  in  1873. 

Form. — Long,  wavy  threads  (16  to  40  /x  long),  a  true  spiril- 
lum; flagella  are  present  (Fig.  107). 


Fig.  107. — Spirochaeta  Obermeieri  from  human  blood  (Kolle  and  Wasser- 

mann). 

Properties. — Very  motile.     Has  not  been  cultivated. 

Staining. — Ordinary  anilin  stains.  Bismarck-brown  best 
for  tissue  sections. 

Pathogenesis. — Found  in  the  organs  and  blood  of  recurrent 
fever.  Man  and  monkeys  inoculated  with  blood  from  one 
suffering  from  this  disease  become  attacked  with  the  fever, 
and  in  their  blood  the  spirillum  is  again  found.  It  is  found  in 
the  blood  only  in  the  relapses  (during  the  fever).  After  the 
attack  the  spirilla  gather  in  the  spleen  and  gradually  die 


215  ESSENTIALS    OF   BACTERIOLOGY 

there.  It  has  been  found  in  the  brain,  spleen,  liver,  and 
kidneys.     In  the  secretions  it  has  not  been  discovered. 

Agglutinating  substances  have  been  developed.  Immu- 
nity has  been  produced  in  rats,  and  the  serum  has  anti- 
toxic properties. 

Transmission. — The  bedbug  retains  the  spirillum  in  its 
blood  and  is  considered  an  important  factor  in  spreading  the 
disease. 

African  Tick  Fever. — A  spirochaete  similar  to  that  of 
relapsing  fever  has  been  observed  in  ticks,  which  conveyed 
a  disease  to  monkeys  similar  to  the  above  fever. 


CHAPTER  XXIX 
FILXERABLE  ORGANISMS 

Filterable  or  Ultra-microscopic  Organisms. — There  are 
many  widely  distributed  infectious  diseases  that  have  all 
the  characteristics  of  germ  or  bacterial  diseases,  but  so  far 
the  organism  has  not  been  found.  It  has  been  suggested 
that  the  bacteria  are  so  small  that  they  pass  through  the 
ordinary  germ-filters  and  are  beyond  the  powers  of  the 
microscope.  By  aid  of  the  ultra-microscope  twice  the  mag- 
nification of  the  usual  oil-immersion  lens  can  be  obtained, 
and  it  is  hoped  that  the  cause  of  some  of  these  diseases  will 
thereby  be  ascertained.  The  instrument  is  still  imperfect, 
though  even  so  it  has  opened  up  a  new  field  of  research. 

Such  diseases  as  measles,  foot  and  mouth  disease  of  cattle, 
typhus  fever,  small-pox,  scarlatina,  and  infantile  poliomyeli- 
tis (epidemic  infantile  paralysis)  are  assumed  to  be  due  to 
these  bacteria. 

Small-pox  and  Vaccinia. — The  exciting  agent  of  small- 
pox is  still  unknown,  but  numerous  bacteria  and  protozoon- 
like  bodies  have  been  described  and  given  etiologic  signiii- 


FILTERABLE   ORGANISMS  219 

cance  by  various  authors.  There  is  some  evidence  in  favor 
of  Funck's  belief  that  vaccinia  is  caused  by  a  protozoon,  the 
Sporidium  vaccinale.  Animals  inoculated  with  this  organ- 
ism developed  both  vaccinia  and  variola.  It  is  possible  that 
the  organism  causing  small-pox  is  a  filterable  one,  and 
beyond  the  present  methods  of  research. 

Yellow  Fever. — For  some  years  it  was  thought  that  a 
bacillus,  called  Bacillus  icteroides  by  Sanarelli,  was  the  cause 
of  yellow  fever.  The  earlier  work  of  Sternberg  was  disproved 
when  it  was  shown  that  his  bacillus,  Bacillus  X,  was  identical 
with  the  colon  group,  and  Reed  and  Carroll  found  that  San- 
arelli's  germ  was  an  allied  organism. 

It  is  now  known  that  a  special  species  of  mosquito,  Ste- 
gomyia  fasciata,  conveys  the  infection  and  acts  as  a  culture- 
medium  for  some  unknown  microorganism,  possibly  a  proto- 
zoon,  which  must  undergo  certain  changes  to  become  virulent. 

Only  by  the  bite  of  a  mosquito  infected  with  the  blood  of  a 
yellow-fever  patient  or  by  direct  inoculation  of  such  blood  can 
yellow  fever  be  transmitted. 

The  experiments  made  so  far  show  that  the  germ  is  de- 
stroyed by  a  temperature  of  55°  C.  for  ten  minutes.  It  can 
pass  through  a  Berkefeld  filter,  and  is,  therefore,  extremely 
minute,  ultra-microscopic,  but  no  one  has  as  yet  been  able  to 
find  any  distinctive  organism  in  the  blood. 

Measles.  —  Recent  experiments  (Anderson  and  Gold- 
berger)  demonstrated  the  virus  in  the  nasal  and  mouth  secre- 
tions, and  this  secretion,  collected  forty-eight  hours  before 
eruption,  when  inoculated  into  monkeys  reproduced  measles 
in  them.  The  infection  was  not  possible  forty-eight  hours 
after  the  eruption  nor  from  the  desquamation. 

Typhus  fever,  or  Brill's  disease,  has  a  virus  which  is 
non-filterable  and  which  resides  in  the  plasma  of  the  blood. 
Monkeys  can  be  inoculated  with  the  disease.  Transmitted 
by  lice. 

Acute  Poliomyelitis. — The  virus  is  contained  in  brain 
and  spinal  cord  and  also  in  the  mucous  membrane  of  the 
nose,  in  the  salivary  glands,  and  cerebrospinal  fluid;  it  is 


220  ESSENTIALS    OF   BACTERIOLOGY 

very  little  resistant  to  heat.  Monkeys  inoculated  through 
the  nose  or  directly  into  the  brain.  Immunity  is  produced 
and  an  immune  serum  as  preventive  is  obtained.  The 
stable-fly  is  supposed  to  act  as  a  carrier  of  infection. 


CHAPTER  XXX 
YEASTS  AND  MOLDS 

In  works  on  bacteria  these  true  fungi,  yeasts  and  molds,  are 
usually  considered.  They  are  so  closely  related  to  bacteria, 
and  so  often  contaminate  the  culture-media,  and  are  so  similar 
in  many  respects,  that  a  description  is  almost  a  necessity. 

But  there  are  several  thousand  varieties,  and  we  cannot 
attempt  to  describe  even  all  the  more  important  ones.  A 
description  of  a  few  of  the  more  common  kinds  must  suffice. 

Blastomycetes  (budding  fungi)  or  yeasts  increase 
through  budding;  the  spores  are  attached  to  the  mother-cell 
like  a  tuber  on  a  potato  (Fig.  io8). 

Yeasts  are  the  cause  of  alcoholic  fermentation  in  the  sac- 
charoses, and  hence  called  saccharomycetes. 

Saccharomyces  Cerevisiae  (Torula  Cerevisiae). — This 
is  the  ordinary  beer-yeast. 

Form. — Round  and  oval  cells;  a  thin  membrane  inclosing  a 
granular  mass,  in  which  usually  can  be  seen  three  or  four 
irregular-shaped  spores.  When  these  become  full  grown, 
they  pass  through  the  cell-wall  and  form  a  daughter-cell. 
Sometimes  long  chains  are  produced  by  the  attached  daugh- 
ter-cells. 

Growth. — They  can  be  cultivated  as  bacteria  are  in  bouil- 
lon, but  grow  best  in  beer. 

Yeasts  are  very  resistant:  cultures  have  been  obtained 
from  material  twelve  years  old  and  dry  as  a  bone. 

There  are  several  varieties  of  beer-yeasts,  each  one  giving  a 


YEASTS   AND   MOLDS  221 

characteristic  taste  to  the  beer.  Brewers,  by  paying  special 
attention  to  the  nutrient  media,  cultivate  yeasts  which  give 
to  their  beers  individual  flavors. 

Mixed  yeast  gives  rise  to  a  poor  quality  of  beer. 

Saccharomyces  Rosaceus;  S.  Niger;  S.  Albicans. — 
These  yeasts  are  found  in  the  air;  and  instead  of  producing 
alcoholic  fermentation,  they  give  rise  to  a  pigment  in  the 
culture-media.  They  grow  upon  gelatin,  which  they  do  not 
liquefy. 


Fig.  io8. — ^Yeast-cells  i^  X  500;  (,r  raiiKcl  and  Pfeiffer). 

Saccharomyces  Mycoderma. — This  yeast  forms  a  mold- 
like growth,  or  skin,  on  the  surface  of  fermented  liquids,  but 
does  not  cause  any  fermentation  itself.  It  forms  the  common 
"mold"  on  wine,  preserves,  and  "sauer-kraut." 

Oidium. — ^A  form  which  seems  to  be  the  bridge  between 
the  yeast  and  the  molds  is  the  oidium.  Sometimes  it  re- 
sembles the  yeasts,  sometimes  the  molds,  and  often  both 
forms  are  found  in  the  same  culture.  Several  are  patho- 
genic for  man. 


222  ESSENTIALS    OF   BACTERIOLOGY 

Oidium  Lactis. — Origin. — In  sour  milk  and  butter. 

Form. — The  branches  or  hyphae  break  up  into  short,  rod- 
like spores.     No  sporangium,  as  in  molds. 

Growth. — In  milk  it  appears  as  a  white  mold. 

Artificially  cultured  on  gelatin  plates,  or  milk-gelatin  plates, 
it  forms  satin-like,  star-shaped  colonies,  which  slowly  liquefy. 
Under  the  microscope  the  form  of  the  fungus  is  well  seen. 

Agar  Stroke  Culture. — The  little  stars,  very  nicely  seen  at 
first;  then  the  culture  becomes  covered  with  them,  causing  a 
smeared  layer  to  appear  over  the  whole  surface,  with  a  sour 
odor. 

Properties. — The  milk  is  not  changed  in  any  special  way. 
It  is  not  pathogenic  for  man  or  animals.  It  is  found  when 
the  milk  begins  to  sour. 

Oidium  Albicans  (Soor ;  Thrush  Fungus,  Langenbeck, 
1839). — Origin. — Mucous  membrane  of  the  mouth,  especi- 
ally of  infants. 

Form. — Taken  from  the  surface  of  the  culture,  a  form  like 
yeasts;  but  in  the  deeper  layers,  mycelia  with  hyphae  occur. 

Growth. — Not  liquefying;  snow-white  colonies  on  gelatin 
plates. 

Stab-culture. — Radiating  yellow  or  white  processes  spring 
from  the  line  made  by  the  needle,  those  near  the  surface 
having  oval  ends. 

Potatoes. — The  yeast  form  develops  as  thick  white  colonies. 

Bread-mash. — Snow-white  veil  over  the  surface. 

Pathogenesis. — In  man  the  parasitic  thrush,  or  "white 
mouth,"  is  caused  by  this  fungus.  In  the  white  patches  the 
spores  and  filaments  of  this  microbe  can  be  found.  Rabbits 
receiving  an  intravenous  injection  perish  in  twenty-four  to 
forty-eight  hours,  the  viscera  being  filled  with  mycelia. 

Pathogenic  Yeasts. — A  number  of  workers  have  inter- 
ested themselves  in  experiments  with  yeasts  in  their  relation 
to  disease;  and  under  the  name  of  blastomycetes,  Sanfelice  has 
grouped  yeasts  that  produce  tumors  resembling  epithelio- 
mata;  and  he  has  tried  to  prove  that  the  so-called  animal 
parasites  found  in  malignant  growths,  and  variously  known 


YEASTS  AND  MOLDS  223 

as  coccidia  and  sporozoa,  are  yeasts.  These  are,  however, 
protozoa. 

Blastomycetic  Dermatitis  or  Oidiomycosis. — A  skin 
disease  described,  in  1894,  by  Gilchrist,  and  since  then  by 
other  writers,  is  due  to  a  fungus  which  resembles  yeast,  and 
which  has  been  called  a  blastomyces;  but  Ophiils  and  Ricketts 
term  it  an  oidium,  and  the  former  calls  the  parasite  O'idium 
coccidioides. 

On  Lofifler's  blood-serum  and  agar  a  growth  occurs  in  from 
three  to  seven  days,  small  white  colonies  made  up  of  branch- 
ing, mold-like  forms.  On  potato  the  growth  is  more  rapid  and 
shows  the  yeast  forms. 

The  disease  is  slow  in  process, — ten  to  twelve  years, — 
leaving  much  deformity.     When  generalized,  it  is  fatal. 

Form. — The  fungus  increases  by  budding,  but  in  culture- 
media  it  may  resemble  a  mold  or  oidium. 

Pathogenesis. — Small  abscesses  form  in  wart-like  lesions, 
which  extend  over  large  areas  of  the  skin,  becoming  later 
on  systemic  and  invading  lungs  and  kidneys;  abscesses  and 
nodules  form  in  these  organs. 

Hyphomycetes  (True  Molds). — Fliigge  has  made  five 
distinct  divisions  of  molds.  It  will,  however,  serve  our 
purpose  to  classify  those  to  be  described  under  three  head- 
ings: Fenicillium,  Mucor,  and  Aspergillus. 

Penicillium  Glaucum. — Origin. — The  most  widely  dis- 
tributed of  all  molds,  found  wherever  molds  can  exist. 

Molds  frequently  contaminate  the  cultures  by  bacteria  and 
culture-media. 

Form. — From  the  mycelium,  h3^h3e  spring  which  divide 
into  basidia  (branches),  from  which  tiny ,  filaments  arise 
(sterigmata),  arranged  like  a  brush  or  tuft.  On  each  sterigma 
a  little  bead  or  conidium  forms,  which  is  the  spore.  In  this 
particular  fungus  the  spores  in  mass  appear  green. 

Growth. — It  develops  only  at  ordinary  temperatures,  form- 
ing thick,  grayish-green  molds  on  bread-mash.  At  first  these 
appear  white,  but  as  soon  as  the  spores  form,  the  green  pre- 
dominates.    Gelatin  is  liquefied  by  it. 


224  ESSENTIALS    OF   BACTERIOLOGY 

Mucor  Mucedo. — Next  to  the  Penicillium  glaucum,  this 
is  the  most  common  mold.  Found  in  horse-dung,  in  nuts  and 
apples,  in  bread  and  potatoes,  as  a  white  mold. 

Form. — The  mycelium  sends  out  several  branches,  on  one 
of  which  a  pointed  stem  is  formed  which  enlarges  to  form  a 
globular  head,  a  spore-bulb,  or  sporangium.  The  spore-bulb 
is  partitioned  off  into  cells  in  which  large  oval  spores  lie. 
When  the  spores  are  ripe,  a  cap  forms  around  the  bulb,  the 


Fig.  109. — Penicillium  glaucum  (  X  500)  (Frankel  and  Pfeiffer). 

walls  break  down,  and  the  wind  scatters  the  spores,  leaving 
the  cap  or  ''columella^'  behind.  The  rounded  sporangium  is 
usually  black. 

Growth. — ^Takes  place  at  higher  temperatures  on  acid  media. 
It  is  not  pathogenic. 

Achorion  Schonleinii.  Trichophyton  Tonsurans. 
Microsporon  Furfur. — These  three  forms  are  similar  to 
each  other  in  nearly  every  particular,  and  resemble  in  some 
respects  the  Oidium  lactis,  in  other  ways,  the  mucors.     The 


YEASTS  AND  MOLDS 


225 


first  one,  Achorion  Schdnleinii,  was  discovered  by  Schonlein 
in  1839,  in  favus,  and  is  now  known  as  the  direct  cause  of  this 
skin  disease. 

Origin. — Found  in  the  scaly  crusts  of  favus  (Fig.   no). 

Form. — Similar  to  Oidium  lactis. 

Growth. — Is  very  sparse.  Agar,  at  body  temperature, 
two  types — waxy,  yellowish  mass,  and  downy,  white-plush- 
like covering. 

In  milk  it  is  destroyed. 

Pathogenesis. — Causes  favus  in  man,  also  in  animals. 

Trichophyton  Tonsurans  ("Ring-worm"). — Found,  in 
1854,  by  Bazin,  in  tinea. 


Fig,   no. — Achorion  Schonleinii  (after  Kaposi). 


Form. — Similar  to  the  achorion  or  favus  fungus. 

Growth. — Somewhat  more  rapid  than  the  favus,  and  the 
gelatin  quickly  liquefied.  Old  cultures  are  of  an  orange- 
yellow  color.     Colonies  have  a  star-shaped  form. 

On  agar  and  potato  the  organism  can  be  cultivated  by 
first  treating  the  infected  hairs  and  scales  with  potassium 
hydroxid  (dilute  solution) ;  this  liberates  the  spores  and  dis- 
solves some  of  the  bacteria  which  usually  contaminates  the 
culture.     Some  of  the  colonies  are  crateriform. 

Pathogenesis. — Herpes  tonsurans  and  the  various  tineae  are 
produced  by  this  fungus. 
IS 


226 


ESSENTIALS   OF  BACTERIOLOGY 


Microsporon  Furfur. — Found  in  tinea  or  pityriasis  versi- 
color, almost  identical  with  the  above;  forms  dry  yellow 
spots,  usually  on  the  chest,  in  persons  suffering  from  wasting 
diseases. 

Aspergillus  Glaucus. — The  aspergillus  is  a  common 
mold  contaminating  bacterial  cultures. 

Origin. — In  saccharine  fruits. 

Form. — The  hypha  has  formed  upon  its  further  end  a  bulb, 
from  which  pear-shaped  sterigmata  arise  and  bear  upon  their 
ends  the  conidia  or  spores. 


Fig.  III. — Aspergillus  fumigatus  (X  soo)  (Frankel  and  Pfeiffer). 


Growth. — Best  upon  fruit-juices.  Non-pathogenic.  The 
mold  is  green.  Aspergillus  flavus  has  the  tufts  and  spores  of 
a  yellow  color. 

Aspergillus  Fumigatus. — Is  pathogenic  for  rabbits  when 
injected  into  them.  At  the  autopsy  their  viscera  are  found 
filled  with  the  mold. 

Examination  of  Yeasts  and  Molds. — Yeasts  and  molds 
are  best  examined  in  the  unstained  condition.  A  small  por- 
tion of  the  colony  rubbed  up  with  a  mixture  of  alcohol  and  a 


YEASTS    AND    MOLDS 


227 


few  drops  of  liquor  ammonia;  of  this,  a  little  is  brought  upon 
the  glass  slide,  covered  with  a  drop  of  glycerin,  and  the  cover- 
glass  pressed  upon  it.  If  the  preparation  is  to  be  saved,  the 
cover-glass  is  secured  by  ringing  around  the  edges  with 
varnish  or  cement.  Yeasts  take  methylene-blue  stain  very 
well. 

Cladothrices  and  Streptothrices. — The  streptothrix  and 
cladothrix  groups  are  classed  with  the  higher  bacteria,  but 
their  exact  status  is  still  undetermined.     They  may  be  con- 


Fig.  112. — Cladothrix  dichomata  from  well-water  (one-twelfth  oil-im- 
mersion.    Fuchsin  stain)  (author's  specimen). 


side  red  as  representing  transition  forms  from  the  bacteria  to 
the  lower  fungi. 

Crenothrix  Ktihniana  (Rabenhorst) . — Long  filaments 
joined  at  one  end;  little  rod-like  bodies  form  in  the  filaments, 
and  these  break  up  into  spores. 

Zooglea  are  also  formed  by  means  of  spores,  and  these  can 
become  so  thick  as  to  plug  up  pipes  and  carriers  of  water. 
They  are  not  injurious  to  health. 

Cladothrix  Dichotoma  (Cohn). — ^Very  common  in  dirty 
waters.  The  filaments  branch  out  at  acute  angles,  otherwise 
resembling  the  crenothrix;    accumulations  of  ocher-colored. 


228  ESSENTIALS    OF   BACTERIOLOGY 

slime,  consisting  of  filaments  of  this  organism,  are  found  in 
springs  and  streams.     (See  Fig.  112.) 

Leptothrix  Buccalis. — In  the  mouth,  long  filaments  or 
threads  resembling  bacteria  are  commonly  found.  At  one 
end  are  seen  numerous  cocci-like  bodies,  which  some  regard 
as  spores.  A  variety  of  this,  or  a  nearly  aUied  organism,  is 
the  most  frequent  cause  of  noma  or  gangrenous  stomatitis. 

With  iodin  the  leptothrix  is  colored  yellow.  At  one  time 
it  was  considered  the  cause  of  "  tartar  "  on  the  teeth,  and  often 
it  fills  the  crypts  of  the  tonsils,  forming  there  small  masses 
which  are  difficult  to  remove.  Miller  distinguishes  three 
varieties — ^Leptothrix  buccalis  innominata,  maxima,  and 
gigantea. 

Beggiatoa  Alba  (Vancher). — The  miost  common  of  this 
species.  The  distinction  between  this  and  the  preceding 
species  lies  in  the  presence  of  sulphur  granules  contained  in  the 
structure,  and  hence  they  are  often  found  where  sulphur  or 
sulphids  exist;  but  where  the  remains  of  organic  life  are  de- 
composing they  can  also  be  found. 

Several  large  spirilla  and  vibrios  live  in  bog  and  rain-water, 
but  our  space  does  not  suffice  to  describe  them.  For  the 
Bacteriologic  Examination  of  Water  see  p.  325. 

Streptothrix  or  Cladothrix  Actinomyces  (Ray-fungus). 
— Actinomycosis  is  a  disease  caused  in  man  and  cattle  by 
an  organism  which  is  commonly  found  in  grain,  particu- 
larly barley.  It  is  probable  that  several  varieties  of  the 
parasite  can  produce  the  characteristic  lesions.  It  has  been 
discovered  in  all  countries  and  in  various  organs  of  the  body, 
although  its  place  of  election  is  about  the  lower  jaw,  where  it 
tends  to  form  hard,  ulcerating  abscesses,  affecting  other 
organs  secondarily. 

Form. — In  the  granular  masses  of  an  abscess  cylindric  fila- 
ments are  matted  together,  and  radiating  outward  from  this 
zone  are  club-shaped  branches,  as  the  petals  of  an  aster. 
(See  Fig.  113.)  In  the  center  of  the  granule  are  numerous 
cocci-like  bodies,  and  some  of  the  ovoid  or  club-shaped 
hyphae  lie  detached  from  the  clusters.     Through  cultivation 


YEASTS   AND   MOLDS  229 

it  is  found  that  the  ovules  give  rise  to  filaments,  and 
they  then  form  the  ovules  again. 

Cultivation. — At  2^'^°  C.  on  glycerin-agar  in  a  period  of  one 
to  two  weeks  pointed  scales  about  the  size  of  a  millet-seed, 
center  dry  and  prominent,  margins  hyaline,  composed  only  of 
filaments,  short  and  long,  massed  together,  but  no  clubbed 
forms. 

The   clubs  have   been   considered   as   spore   organs;    by 


Fig.  113. — Actinomyces  granule  crushed  beneath  a  cover-glass,  show- 
ing radial  striations  in  the  hyaline  masses.  Preparation  not  stained; 
low  magnifying  power  (Wright  and  Brown). 

others,  they  are  thought  to  be  encapsulated  or  thickened 
filaments. 

Pathogenesis, — When  a  portion  of  the  growth  obtained  in 
eggs  is  injected  into  the  abdominal  cavity  of  a  rabbit, 
actinomycotic  processes  develop  upon  the  peritoneum. 

It  usually  gains  access  to  the  living  body  through  a  wound 
in  the  gum  or  some  caries  of  the  teeth.  A  new  growth  is 
formed,  ulceration  being  first  set  up. 

The  new  tissue,  composed  of  round-cells,  then  undergoes 


230  ESSENTIALS    OF   BACTERIOLOGY 

softening,  purulent  collections  form,  and  the  normal  structure 

is  destroyed. 

The  usual  seat  is  in  the  maxillary  bones,  but  the  fungus  has 

been  found  in  the  lungs,  tonsils,  intestines,  and  various  other 

organs  in  man  and  cattle. 

Examination. — Well  seen  in  the  unstained  condition.   From 

the  pus  or  scraping  a  small 

1^       portion      is      taken      and 

^       squeezed    upon    the    glass 

slide;   if  calcareous  matter 

is  present,  a  drop  of  nitric 

acid  will  dissolve  this. 

Glycerin  will  preserve  the 

r>%ij       preparation. 

'h^^i  Staining.  —  Cover -glass 

:       specimens  stained  best 

by  Gram's  method.    Tissue 

sections  should  be  stained 

,  ,      i       as  follows: 

J  Ziehl's  carbol-fuchsin,  ten 

;       minutes.     Rinse  in  water. 

Concentrated      alcoholic 

"' '  solution  of  picric  acid,  five 

minutes.     Rinse  in  water. 

J,  ^ ,  Alcohol,     50    per    cent., 

1  *        %  \       fifteen    minutes.      Alcohol 

'       m  *    '®^^'      \       absolute,  clove-oil,  balsam. 

\^^   --^  J  The  rays  stained  red,  the 

17-       ""     c.      .  .u  •    iiT  T*^  tissue  yellow. 

Fig.  114, — Streptothrix  Madurae  o*        4.   .n.   •        -kit    a 

in  a  section  of  diseased  tissue  (Vin-  btreptothnx      Madurae 

cent).  (Vincent). — Origin. — 

Found  in  the  disease  known 
as  Madura  foot,  or  mycetoma,  an  ulceration  affecting  the 
feet,  especially  of  individuals  living  in  the  tropics.  Two 
varieties,  the  pale  and  the  black,  have  been  described. 

Form. — Branched  filaments   resembling   the   actinomyces 
streptothrix.     In  the  mycelia  spores  are  seen  (Fig.  114). 


1  j  * 

-A  '.  .' 

\ 

'      B  -^  W 

^<, "    ^ 

&ij^^'-t' 

YEASTS  AND  MOLDS  231 

Cultivation. — In  liquid  media  containing  vegetable  infu- 
sions growth  occurs  best.  Temperature  of  37°  C.  most 
suited.     The  colonies  near  the  surface  become  colored  red. 

Agar. — Glazed  colonies,  at  first  colorless,  then  rose-colored, 
about  the  size  of  a  pea,  with  the  central  part  umbilicated  and 
pale.     Gradually  the  rose  color  fades. 

Acid  Potato. — ^A  slow  and  meager  growth. 

Pathogenesis. — Only  local  reaction  has  been  caused  by 
inoculation  in  animals.  In  man  the  disease  usually  follows  a 
slight  injury  and  attacks  the  leg  or  foot,  slowly  forming  a 
nodular  growth,  which  in  the  course  of  months  or  a  year 
begins  to  soften  and  ulcerate,  and  with  the  seropus  are  dis- 
charged numerous  little  granules,  some  black,  some  pink, 
containing  mycelia.  The  limb  becomes  much  deformed,  the 
tissue  vascularized,  and  the  degenerated  area  filled  with  the 
streptothrix  filaments. 

Staining. — ^The  organism  itself  stained  with  ordinary 
stains.     Gram's  method  for  the  tissue. 

Nocardia  (Streptothrix)  Farcinica.(Nocard) ;  Bovine 
Farcin  du  Boeuf . — Origin. — A  disease  affecting  cattle,  and 
giving  rise  to  tubercle-like  lesions  in  the  lungs,  liver,  and 
spleen.     Common  in  France. 

Form. — Small  interwoven  mass  of  threads  arranged  in 
tufts  found  in  the  centers  of  the  tubercles. 

Culture. — ^At  body-temperature  in  various  media. 

Bouillon. — Colorless  masses,  irregular  in  size  and  shape. 

Agar  and  Gelatin. — Small,  rounded,  opaque  colonies, 
thicker  at  the  periphery. 

Potato. — Rapid  growth  of  pale-yellow,  dry  scales,  consist- 
ing of  many  spores. 

Pathogenesis. — Pure  cultures  introduced  into  the  perito- 
neum of  guinea-pigs  give  rise  in  nine  to  twenty  days  to 
tubercle-like  lesions.  Subcutaneous  injections  cause  abscesses 
with  secondary  involvement  of  the  lymphatics,  ending  in 
recovery.     Dogs,  horses,  and  rabbits  are  immune. 

Staining. — Wright's  double  stain  for  tissues;  also  Gram's. 

Plant  Diseases  due  to  Bacteria. — There  are  a  great 


232  ESSENTIALS    OE   BACTERIOLOGY 

variety  of  blights,  rots,  and  new-growths,  such  as  galls  attack- 
ing plants,  which  are  seemingly  due  to  bacteria.  About  30 
varieties  have  so  far  been  more  or  less  accurately  described, 
but  only  a  few  of  the  organisms  have  been  definitely  asso- 
ciated with  the  disease.  The  pear  blight  is  due  to  Bacillus 
amylovorus.  Crown  gall,  which  affects  a  great  many  plants 
and  trees,  is  supposed  to  be  due  to  Bacterium  tumefaciens; 
the  black  rot  of  cabbage  to  a  pseudomonas.  There  is  much 
left  to  be  done  to  place  this  part  of  bacteriology  on  a  par 
with  that  devoted  to  animals  and  man. 


CHAPTER  XXXI 
EXAMINATION  OF  AIR,  SOIL,  AND  WATER 

Air. — Many  germs  are  constantly  found  in  the  atmosphere 
about  us.  Bacteria  unaided  do  not  rise  into  the  air  and  fly 
about;  they  usually  become  mixed  with  small  particles  of  dirt 
or  dust  and  are  moved  with  the  wind.  The  more  dust,  the 
more  bacteria,  and,  therefore,  the  air  in  summer  contains  a 
greater  number  than  the  air  in  winter,  and  all  the  other  dif- 
ferences can  be  attributed  to  the  greater  or  less  quantity  of 
dust  and  velocity  of  the  wind. 

By  the  use  of  balloons,  living  bacteria  have  been  found  at 
an  altitude  of  4000  meters. 

Methods  of  Examination. — ^The  simplest  method  is  to 
expose  a  Petri  dish  with  gelatin  or  agar  in  a  dust-laden  atmos- 
phere or  in  the  place  to  be  examined.  In  the  course  of  twenty- 
four  to  forty-eight  hours  colonies  will  form  wherever  a 
germ  has  fallen.  But  this  method  will  not  give  any  accurate 
results  in  regard  to  the  number  of  bacteria  in  a  given  space; 
for  such  purposes  somewhat  more  complicated  methods  are 
used,  so  that  a  definite  amount  of  air  can  come  in  contact 
with  the  nutrient  medium  at  a  certain  regulated  rate  of  speed. 


EXAMINATION    01   AIR,    SOIL,    AND   WATER 


233 


This  form  of  analysis,  however,  has  not  yielded  any  very 
practical  results,  and  is  not  much  resorted  to. 

Hesse's  Method. — Hesse's  method  requires  an  apparatus 
called  an  aero  scope,  which,  by  means  of  siphoning  bottles 
{aspirator),  sucks  air  through 
a  cylinder  lined  with  gelatin,  tlrPh 

and  by  regulating  the  rate  of  ^J/- 

flow  an  approximate  idea  of  ^i3l 

the  number  of  bacteria  per 
liter  of  air  can  be  obtained. 
A  less  complicated  method  is 
known  as  Petri's  method. 


Fig.    115. — Petri's    sand-filter    for 
air-examination  (McFarland). 


Fig.  116. — Sedgwick's  expanded 
tube  for  air  -  examination  (Mc- 
Farland). 


Sand  is  sterilized  by  heating  to  redness,  and  while  still 
warm  placed  in  test-tubes,  which  are  then  plugged. 


234  ESSENTIALS    OF   BACTERIOLOGY 

The  tube  and  its  contents,  the  ends  having  first  been 
plugged  with  cotton,  are  sterilized  in  a  hot-air  oven  at 
150°  c. 

One  end  of  the  tube  is  then  fitted  with  a  rubber  cork 
through  which  passes  a  glass  tube,  which  is  connected  with 
an  aspirator  (a  hand-pump  with  a  known  capacity). 

If  100  liters  of  air  pass  through  the  tube  in  fifteen  min- 
utes, the  germs  should  all  be  arrested  in  the  first  sand-filter. 
And  when  the  filters  are  removed,  each  filter  for  itself,  and 
thoroughly  mixed  with  gelatin,  there  should  be  no  colonies 
developed  from  the  second  filter,  i.  e.,  the  one  nearest  the 
aspirator. 

Sedgwick-Tucker  Method. — A  special  form  of  tube  is 
used,  called  an  aerobioscope.  It  consists  of  a  neck  2.5  cm.  in 
length,  an  expanded  portion  15  cm.  long,  and  a  long  narrow 
tube  of  15  cm.  After  sterilization  the  tube  is  partly  filled 
with  granulated  sugar,  which  is  the  filtering  material.  By 
means  of  a  vacuum  gage  and  an  air-pump,  or  ordinary  aspirat- 
ing bottles,  the  volume  of  air  passing  through  the  apparatus 
can  be  determined.  After  the  air  has  been  passed  through, 
the  sugar  is  gently  shaken  from  the  narrow  tube  into  the 
expanded  portion,  and  20  c.c.  of  liquefied  gelatin  is  poured  in. 
The  sugar  dissolves,  and  the  mixture  is  then  rolled  on  the 
inner  side  of  the  glass  as  an  Esmarch  tube.  This  part  of  the 
apparatus  is  divided  into  squares  to  make  the  counting  of 
colonies  easy.    The  aerobioscope  is  very  highly  recommended. 

Varieties  Found  in  Air. — The  only  pathogenic  bacteria 
found  with  any  constancy  are  the  Staphylococcus  aureus  and 
citreus;  but  any  bacterium  can,  through  accident,  be  lifted 
into  the  atmosphere,  and  under  certain  conditions  may  be 
always  present — the  Bacillus  tuberculosis,  for  example,  in 
rooms  where  consumptives  are  living. 

Typhoid  fever,  influenza,  pneumonia,  and  diphtheria  may 
be  conveyed  through  the  air  by  the  cough  and  expectora- 
tion of  affected  persons. 

N on- pathogenic. — The  micrococci  predominate.  Sarcinae, 
yeasts,  and  molds  constantly  contaminate  cultures. 


EXAMINATION   OF   AIR,    SOIL,   AND   WATER  23$ 

In  the  ordinary  habitations  the  average  number  of  germs 
to  the  liter  of  air  does  not  exceed  five. 

Around  water-closets,  where  one  would  imagine  a  great 
number  to  exist,  but  few  will  be  found,  owing  to  the  undis- 
turbed condition  of  the  air. 

Sewer  air  seldom,  if  ever,  contains  bacteria,  and  neither 
typhoid  fever,  malaria,  nor  diphtheria  has  ever  been  traced  to 
the  escape  of  so-called  sewer-gas. 

Examination  of  Water. — The  bacteriologic  examination 
of  w^ater  is  today  of  as  much  importance  as  the  chemical 
analysis,  and  must  go  hand  in  hand  with  it. 

A  water  containing  thousands  of  germs  to  the  cubic  centi- 
meter is  far  less  dangerous  than  one  containing  but  two 
germs,  if  one  of  these  two  be  a  typhoid  bacillus.  It  is  not  the 
number  that  proves  dangerous,  it  is  the  kind. 

If  a  natural  water  contains  more  than  500  germs  to  the 
cubic  centimeter,  it  were  well  to  examine  its  source,  and 
consider  it  with  suspicion. 

As  a  means  of  diagnosis  the  examination  is  of  but  little  use. 
An  epidemic  of  typhoid  fever  occurs,  the  water  is  suspected, 
an  examination  is  undertaken;  but  the  period  of  incubation 
and  the  days  passed  before  the  water  is  analyzed  have  given 
the  typhoid  germs,  if  any  had  been  present,  ample  time  to 
disappear,  since  in  water  that  contains  other  bacteria  they  live 
a  few  days  only.  Again,  the  water  tested  one  day  may  be 
entirely  free  and  the  next  day  contain  a  great  number,  and 
before  the  typhoid  germ  can  be  proved  to  be  present  in  that 
particular  water  the  epidemic  may  be  past.  Human  sewage 
contamination  is  determined  by  finding  the  colon  bacillus,  and 
if  this  is  found  in  the  course  of  an  epidemic  of  typhoid  the 
water  containing  it  may  well  be  suspected  as  being  the 
cause. 

Purity  of  Waters. — The  purest  water  we  have  is  the 
natural  spring-water — water  that  has  slowly  filtered  its  way 
through  various  layers  of  gravel  and  sand  and  comes  finally 
clear  and  sparkling  from  the  ground.  It  is  free  from  bac- 
teria, but  let  such  a  water  stand  walled  up  in  cisterns  or 


236  ESSENTIALS    OF   BACTERIOLOGY 

wells,  or  run  through  the  wood,  gathering  the  w^ashings  from 
pastures  and  farm  lands,  it  becomes,  as  surface  water,  open 
to  all  sorts  of  impurities,  and  the  bacterial  nature  of  it 
changes  every  moment. 

Artesian  or  Driven  Well. — The  driven  well  will  secure  to 
a  certain  extent  a  pure  water.  It  is  the  only  form  of  well  or 
cistern  that  will  insure  this,  since  the  water  does  not  become 
stagnant  in  it;  but  it  m^ay  connect  with  an  outhouse — the  soil 
being  very  loose — and  thus  bacteria  and  refuse  water  find 
their  way  into  the  wtII.  The  casing  may  not  be  water-tight 
and  surface  water  can  be  sucked  in. 

Filtered  Water. — Dangerous  as  surface  water  is,  the 
greater  quantity  used  is  such,  the  inhabitants  of  larger  towns 
and  cities  using  chiefly  the  rivers  and  other  large  waters  which 
course  near  them  for  drinking  purposes.  A  purification  or 
filtration  can,  to  a  certain  extent,  render  these  waters 
harmless. 

Filtration  is  carried  on  on  a  large  scale  in  the  water-works 
of  cities  and  towns,  and  bacteriologic  examJnation  is  here  of 
great  service  to  determine  if  a  water  which  has  been  filtered 
and  may  have  a  very  clear  appearance,  and  give  no  harmful 
chemical  reaction,  is  entirely  free,  or  nearly  so,  from  germs; 
in  other  words,  if  the  filter  is  a  germ-filter  or  not;  daily  tests 
are  necessary  in  order  to  insure  safety,  and  if  it  is  performing 
this  function  regularly,  a  good  filter  plant  should  show  99.8 
per  cent,  efficiency,  removing  nearly  all  the  bacteria. 

Filter  Materials. — When  waters  are  muddy  or  when  rapid 
filtration  is  wanted,  mechanical  filters  are  employed.  The 
water  is  first  treated  with  coagulants,  like  alum,  w^hich  forms 
a  flocculent  precipitate  and  carries  down  with  the  suspended 
matter  much  of  the  bacterial  content.  This  is  then  filtered 
through  sand  and  gravel.  Sedimentation  and  filtering 
slowly  through  gravel  and  sand  is  known  as  the  slow  process; 
the  other  as  the  rapid,  filtration. 

Charcoal  sponge  and  asbestos,  the  materials  formerly  in 
use,  are  objectionable  because  germs  readily  develop  on  them 
and  clog  them,  so  that  they  require  frequent  renewal.     In 


EXAMINATION   OF   AIR,    SOIL,    AND   WATER  237 

very  large  filters,  sand  and  gravel  give  the  best  results;  the 
number  of  bacteria  in  a  cubic  centimeter  is  reduced  to  forty  or 
fifty  and  kept  at  that  number.  This  is  a  very  pure  water  for 
a  city  water,  though,  as  we  stated  before,  not  a  safe  one,  for 
among  those  forty  germs  very  dangerous  ones  may  be  found. 
It  is  then  necessary  for  the  users  to  refilter  the  water,  before 
drinking  it,  through  a  material  which  will  not  allow  any 
germs  to  pass,  or,  in  the  presence  of  an  epidemic,  to  boil  all 
water  used  for  drinking  purposes. 


Fig.  117.— Flask  fitted  with  porcelain  bougie  for  filtering  large  quantities 

of  fluid. 


Pasteur-Chamberland  Filter. — This  very  perfect  filter 
consists  of  a  piece  of  polished  porcelain  in  the  form  of  a 
cylinder  closed  at  one  end  and  pointed  at  the  other.  It  is 
placed  in  another  cylinder  of  glass  or  rubber,  and  the  pointed 
portion  connected  with  a  bottle  containing  the  water,  or 
directly  with  the  faucet  of  the  water-pipe.  The  water  courses 
through  the  porcelain  very  slowly  and  comes  out  nearly  free 
from  germs;  pipe-clay,  bisque,  infusorial  earth,  and  kaolin  are 
also  good  filters.  The  only  disadvantage  is  the  long  time  it 
takes  for  the  water  to  pass  through.     Pressure  in  the  form 


238  ESSENTIALS    OF   BACTERIOLOGY 

of  an  aspirator  or  air-pump  is  used  to  accelerate  the  pas- 
sage. 

These  porcelain  cylinders  can  easily  be  sterilized  and  the 
pores  washed  out. 

All  the  cylinders  or  bougies  are  not  germ  proof,  so  that  they 
must  be  tested,  and  most  of  them  must  be  cleaned  every 
fourth  day.  In  recent  years  a  number  of  organisms  have 
been  suspected  of  being  so  minute  as  to  pass  through  a 
Berkefeld  or  Pasteur  filter.  At  least  the  poison  or  virus  is 
filterable,  and,  therefore,  we  cannot  regard  these  as  abso- 
lutely safe. 

Boiling  as  a  Means  of  Purifying. — The  only  safe  measure 
in  times  of  epidemics  and  with  waters  of  unknown  composi- 
tion is  boiling,  not  only  of  the  drinking  water,  but  all  water 
used  for  domestic  purposes;  and  this  should  especially  be 
done  in  times  of  typhoid  and  cholera  epidemics. 

Varieties  Found  in  Water. — The  usual  kinds  found  are 
non-pathogenic,  but,  as  is  well  known,  typhoid,  cholera,  and 
dysentery  are  principally  spread  through  drinking-water, 
and  many  other  germs  may  find  their  way  into  the  water. 
Some  of  the  common  varieties  give  rise  to  fluorescence  or 
produce  pigment. 

Eisenberg  gives  100  different  varieties  as  ordinarily  found. 
Other  intestinal  diseases  besides  those  mentioned  above  are 
supposed  to  be  water  borne.  Diarrheas  in  epidemic  form 
may  come  from  suddenly  changing  a  public  supply,  and 
the  presence  of  the  Bacillus  coli  communis  means  sewage 
contamination  or  fecal  contamination;  such  contamination 
may  come  from  the  droppings  of  birds  or  other  animals  and 
need  not  necessarily  imply  human  sewage,  but  10  colon 
bacilli  in  i  c.c.  water  is  a  serious  pollution.  Ice  supplies 
require  the  same  supervision  as  water  supplies,  for  many 
bacteria,  like  the  typhoid  bacillus,  retain  their  vitality  for 
weeks  after  freezing. 

Method  of  Examination. — (After  that  suggested  by  the 
American  Public  Health  Association,  igi2  report.) — Since 
the  germs  rapidly  multiply  in  stagnant  water,  an  examina- 


EXAMINATION   OF  AIR,    SOIL,   AND   WATER  239 

tion  must  not  be  delayed  longer  than  possible  after  the 
water  has  been  collected.  Every  precaution  must  be  taken 
in  the  way  of  cleanliness  to  prevent  contamination;  sterilized 
flasks  with  glass  stoppers,  pipets,  and  plugs  must  be  at  hand, 
glassware  sterilized  in  autoclave  at  120°  C.  for  fifteen  minutes, 
or  dry  heat  at  160°  C.  for  one  hour,  and  the  gelatin  tubes  or 
agar  dishes  be  inoculated  on  the  spot.  If  this  cannot  be  done, 
the  sample  should  be  packed  in  ice  until  it  arrives  at  the 
laboratory.  If  it  is  necessary  to  send  the  sample  by  rail, 
the  bottle  containing  the  sample  should  be  wrapped  in  steril- 
ized cloth,  or  the  neck  covered  with  tinfoil  and  the  bottles 
placed  in  tin  boxes  (about  4  ounces — 100  c.c. — is  sufficient 
for  bacterial  analysis),  and  then  packed  in  cotton  or  paper 
to  prevent  breakage  and  surrounded  by  plenty  of  ice  until 
it  reaches  its  destination.  As  soon  as  it  arrives  at  the  lab- 
oratory the  sample  is  placed  in  a  sterilized  glass  flask,  and 
the  flask  then  closed  with  a  sterile  cotton  plug.  A  sterilized 
pipet  is  then  dipped  into  the  flask,  and  i  c.c.  of  the  water 
withdrawn  and  added  to  a  Petri  dish.  To  a  second  dish,  a 
dilution  of  i  c.c.  of  the  sample  with  sterile  distilled  water  is 
added,  and  other  dilutions  made  if  desired.  To  each  plate  10 
c.c.  of  standard  agar  at  a  temperature  of  40°  C.  is  added. 
Mix  the  water  and  media  thoroughly  by  tipping  the  dish 
back  and  forth,  and  place  in  incubator  at  37°  C.  for  twenty- 
four  hours.  The  incubator  should  be  in  a  dark,  well-venti- 
lated, and  moist  place.  Then  count  all  the  colonies  present 
on  each  plate,  which  will  give  the  number  per  cubic  centi- 
meter. 

Water  that  is  very  rich  in  germs  requires  dilution  with 
sterilized  water  fifty  to  one  hundred  times.  Fewer  colonies 
will  be  found  on  agar  than  on  gelatin,  even  at  the  same  tem- 
perature. 

Special  Media  and  Preparation. — In  the  preparation  of 
media  for  water  analysis,  sodium  chlorid  must  not  be  used. 
The  reaction  of  most  culture-media  should  be  -j- 1  per  cent, 
to  phenolphthalein. 

Sugar  broths  should  be  neutral,  and  must  be  sterilized  care- 


240  ESSENTIALS    OF   BACTERIOLOGY 

fully  in  steam  and  not  overheated,  so  as  to  prevent  inversion 
of  the  sugar. 
Examination  for  Bacillus  Coli  and  Sewage  Bacteria. 

— Instead  of  examining  for  typhoid  bacilli,  sewage  contamina- 
tion is  best  indicated  by  the  presence  of  the  colon  group  of 
organisms,  although  their  abundance  rather  than  mere  pres- 
ence is  to  be  considered.  There  are  many  closely  related 
bacteria  which  give  reactions  similar  to  the  Bacillus  coli,  but 
they  are  chiefly  of  fecal  origin,  and  for  practical  purposes 
they  can  be  included  in  the  colon  group. 

General  Characteristics  of  Colon  Group. — i.  Fermentation  of 
dextrose  and  lactose  with  gas-production.  2.  Short  bacillus, 
non-liquefying,  Gram  negative. 

The  committee  of  the  Public  Health  Association  recom- 
mends the  following  procedure: 

Two  Methods. — Method  a. — ^Applicable  for  sewage  waters. 
Preparation  of  an  agar  plate  with  a  known  volume  of  w^ater, 
using  lactose  litmus-agar  and  incubating  at  40°  C.  Bacillus 
coli  will  show  its  presence  by  red  colonies  (acid  fermentation 
of  the  sugar) ;  further  testing  is  then  needed  to  fully  identify. 
Not  all  red  colonies  Bacillus  coli. 

Method  h. — Cultivation,  at  40°  C,  of  a  measured  quantity 
of  water  in  a  fermentation  tube  containing  a  sugar  broth. 
If  gas  appears,  a  portion  of  the  liquid  is  plated  as  in  method  a. 

Additional  Details. — If  in  twenty-four  hours  no  red  colonies 
appear  in  the  agar-lactose  litmus  Petri  dishes.  Bacillus  coli 
is  considered  absent,  providing  the  sample  was  a  polluted 
one,  so  that  the  bacilli,  if  present,  would  be  in  a  concentrated 
form.  Only  i  or  2  c.c.  of  water  can  be  used,  because  the 
ordinary  water-bacteria  spread  rapidly  and  contaminate  the 
other  bacteria. 

If  acid-forming  colonies  are  found,  five  or  six  are  fished  for 
subcultures  on  slanted  agar,  in  fermentation  tubes,  milk, 
gelatin,  peptone  solution,  and  nitrate  broth. 

If  the  water  is  not  strongly  contaminated,  an  imderground 
water,  for  instance,  or  a  mountain  stream,  the  better  way  is 
to  inoculate  two  or  three  lactose  or  dextrose  bouillon  fermen- 


EXAMINATION   OF   AIR,    SOIL,   AND   WATER  24I 

taticn  tubes  and  place  in  an  incubator  at  40°  C.  Note  the 
presence  of  gas,  if  any,  at  the  end  of  twelve,  twenty-four, 
thirty-six,  and  forty-eight  hours.  //  no  gas  forms,  sewage 
bacteria  are  absent. 

If  gas  forms,  plate  at  once  a  portion  of  the  sediment  as 
above  on  lactose  litmus-agar.  Test  the  other  fermentation 
tubes  for  acidity,  and  the  nature  of  the  gas,  whether  any,  and 
how  much  is  absorbed  by  a  2  per  cent,  solution  of  sodium 
hydroxid.  Bacillus  colt  should  produce  between  jo  and  yo 
per  cent,  of  gas,  of  which  about  one-third  is  CO2  and  is  ab- 
sorbed by  the  alkali;  the  remainder  is  hydrogen.  The  other 
broth  culture  can  be  tested  for  the  presence  or  absence  of 
unfermented  sugar  by  Fehling's  solution. 

Diagnostic  Points  of  Colon  Bacillus. — Microscopic. — 
Non-spore-bearing  motile  bacillus. 

Gelatin. — Non-liquefactive. 

Dextrose  Broth. — Fifty  per  cent,  gas;  one- third  absorbed, 
CO2;  two-thirds,  hydrogen. 

Milk  (litmus)  coagulated  in  forty-eight  hours  and  rendered 
acid;  litmus  colored  red. 

Peptone  Solution. — Production  of  Indol. — (A  peptone  solu- 
tion tube  is  inoculated  with  the  culture  and  kept  together  with 
a  control  four  days  at  37°  C.  Then  2  drops  of  concentrated 
sulphuric  acid  and  i  centimeter  of  a  0.0 1  per  cent,  solution  of 
sodium  nitrate  are  added.  The  appearance  of  a  pink  color 
at  the  end  of  thrity  minutes  denotes  the  presence  of  indol.) 

Presumptive  Test. — If  a  water  from  a  well  or  spring  pro- 
duces gas  in  the  sugar  broth  and  forms  acid  colonies  on  litmus- 
lactose  agar,  the  presumption  is  strong  that  there  is  sewage 
contamination.  If  gas-production  continues  in  a  series  of 
samples  carefully  collected  for  several  days  or  weeks,  there 
can  be  no  doubt  of  a  contamination,  and  especially  if  the  well 
or  spring  is  protected  from  surface  water.  Algae  w^hich  grow 
in  service  pipes,  reservoirs,  and  deep  wells  may  give  rise  to 
non-acid  gas  fermentation,  but  all  well-water  that,  without 
further  testing, .  forms  acid  colonies  on  litmus-agar  lactose 
plates  and  ferments  sugar  broth,  is  open  to  suspicion,  and  if 
16 


242  ESSENTIALS   OF  BACTERIOLOGY 

there  is  evidence  of  the  presence  of  typhoid  fever  or  diarrheal 
diseases,  the  water  should  be  boiled  and  subjected  to  careful 
analysis  daily.  There  may  be  serious  contamination  and 
the  chemical  tests  show  no  appreciable  increase  in  the  chlorids. 

Bile  Media. — In  recent  years  bile  salts  or  fresh  bile  mixed 
with  lactose  have  been  extensively  used,  as  the  bile  inhibits 
the  action  of  many  bacteria  and  allows  the  colon  and  ty- 
phoid group  to  develop  readily. 

The  Jackson  bile  media  (see  formula  for  media.  Chap.  X) 
is  placed  in  fermentation  tubes  of  40  c.c.  capacity,  and  in- 
oculated with  varying  proportions  of  the  water  to  be  tested. 
Incubated  at  37°  C.,  and  presence  of  gas  looked  for  in  twelve 
hours,  twenty-four  hours,  and  forty-eight  hours,  and  the 
quantity  and  time  noted. 

In  sewage  and  contaminated  waters  the  lactose-bile  gives 
better  results  than  any  other  medimji. 

The  Presumptive  Test  (Modified). — Plant  yq-,  i,  and  10 
c.c.  of  water  into  liver  broth  tubes.  Transplant  from  these 
into  lactose  bile  in  six  and  twelve  hours.  By  using  implan- 
tations of  both  lactose  bile  and  liver  broth,  and  then  trans- 
planting the  liver-broth  cultures  into  other  lactose  bile,  we 
have  in  the  original  bile  the  vigorous  Bacillus  coli.  The  liver- 
broth  dilutions  give  all  the  gas  formers,  strong  and  weak, 
and  the  difference  between  the  original  and  the  transplanted 
gives  an  idea  of  the  attenuated  Bacillus  coli  present.  Thus 
all  the  gas  formers  are  cultured. 

Bacillus  Typhosus. — By  the  use  of  bile  media  and  other 
special  media  as  enrichment  and  then  transplanting  on 
Hesse  Agar,  Conradi-Drigalski,  or  Endo  media,  the  Bacillus 
typhosus  are  increased  in  number  and  the  possibilities  of 
diagnosing  them  made  much  easier.  The  Widal  test  is 
used  to  differentiate  Bacillus  typhosus  from  Bacillus  coli. 

Quantitative  Tests. — The  number  of  acid  colonies  in  i  c.c. 
and  in  5  c.c.  of  water  is  taken  as  a  measure  of  pollution,  to- 
gether with  the  total  number  of  colonies  of  all  bacteria  present. 
Thus  in  i  c.c.  on  the  gelatin  plate  at  20°  C.  there  may  be 


EXAMINATION   OF   AIR,    SOIL,   AND   WATER  243 

fifty  colonies;  on  the  agar  plate  at  37°  C.  ten  colonies,  five  of 
which  were  acid-formers,  or  presumably  Bacillus  coli. 

To  count  the  colonies  which  develop  upon  the  plates,  a 
special  apparatus  has  been  designed,  known  as — 

WolfhiigeVs  Counter. — A  glass  plate  divided  into  squares, 
each  a  centimeter  large,  and  some  of  these  subdivided.  This 
plate  is  placed  above  the  dish  with  the  colonies,  and  the  num- 
ber in  several  quadrants  taken,  a  lens  being  used  to  see  the 
smaller  ones. 

It  is  best  to  count  all  the  colonies  on  the  plate  or  dish. 

Bacterial  Treatment  of  Sewage. — Where  sewage  is  to  be 
rendered  innocuous  before  being  allowed  to  flow  into  streams, 
the  process  of  nature  has  been  imitated  by  the  construction 
of  septic  tanks  in  which  the  sewage  remains  excluded  from 
the  air  and  subject  to  the  action  of  the  anaerobic  bacteria 
present  in  the  sewage.  The  organic  nitrogen  is  reduced,  and 
compounds  of  hydrogen  and  sulphur  are  formed.  The 
effluent  is  then  filtered  through  coke-beds,  w^here  the  aerobic 
bacteria  assist  in  further  purification  and  over  sand  filters,  or 
exposed  to  the  air  on  contact  beds.  No  method  of  sewage 
purification  is  very  practical  or  safe.  Pure  water  should  not 
depend  oh  the  efficiency  of  sewage  filtration,  but  should  be 
obtained  from  a  reasonably  pure  source. 

Sewage  is  also  treated  by  sedimentation  with  alum  and 
filtration  of  the  effluent  over  larger  beds,  or  allowed  to  per- 
colate through  the  soil,  which  is  thereby  enriched  and  utilized 
for  agriculture.  It  is  also  dried  and  sold  in  a  compressed 
form  for  fertilizer. 

The  Examination  of  the  Soil. — The  upper  layers  of  the 
soil  contain  a  great  many  bacteria,  but  because  of  the  diffi- 
culty in  analyzing  the  same,  the  results  are  neither  accurate 
nor  constant.  The  principal  trouble  lies  in  the  mixing  of  the 
earth  with  the  nutrient  medium ;  little  particles  of  ground  will 
cling  to  the  walls  of  the  tube,  or  be  embedded  in  the  gelatin, 
and  may  contain  within  them  myriads  of  bacteria.  As  with 
water,  the  soil  must  be  examined  immediately  or  very  soon 
after  it  is  collected,  the  bacteria  rapidly  multiplying  in  it. 


244  ESSENTIALS   OF   BACTERIOLOGY 

When  the  deeper  layers  are  to  be  examined,  some  precau- 
tions must  be  taken  to  avoid  contamination  with  the  other 
portions  of  the  soil.  One  method,  very  laborious  and  not 
often  practical,  is  to  dig  a  hole  near  the  spot  to  be  examined 
and  take  the  earth  from  the  sides  of  this  excavation. 

Frankel's  Borer. — Frankel  has  devised  a  small  apparatus 
in  the  form  of  a  borer,  which  contains  near  its  lower  end  a 
small  cavity,  which  can  be  closed  up  by  turning  the  handle, 
or  opened  by  turning  in  the  opposite  direction. 

It  is  introduced  with  the  cavity  closed,  and  when  it  is  at 
the  desired  depth,  the  handle  is  turned,  the  earth  enters  the 
cavity,  the  handle  again  turned,  incloses  it  completely,  and 
the  borer  is  then  withdrawn. 

The  earth  can  then  be  mixed  with  the  culture-medium  in  a 
tube,  and  this  gelatin  then  rolled  on  the  walls  of  the  tube  after 
the  manner  of  Esmarch,  or  it  can  be  poured  upon  a  plate, 
and  the  colonies  developed  therein. 

Another  method  is  to  wash  the  earth  with  sterilized  water, 
and  the  water  then  mixed  with  the  culture-medium,  as  many 
of  the  germs  are  taken  up  by  the  water. 

The  roll-cultures  of  Esmarch  give  the  best  results,  many  of 
the  varieties  usually  found  being  anaerobic. 

Animals  inoculated  with  the  soil  around  Berlin  are  said  to 
die  almost  always  of  malignant  edema,  and  the  soil  of  other 
towns  produces  tetanus.  Many  of  the  germs  found  are  nitro- 
gen formers  and  play  a  great  role  in  the  economy  of  the  soil. 

Bacteria  and  Soil  Fertility. — Nitrifying  organisms  are 
found  in  the  superficial  layers  of  the  earth.  Organic  matters 
found  in  sewage  and  in  the  fecal  evacuations  of  animals  form 
the  basis  for  their  activity,  whereby  nitrates,  ammonias,  and 
nitric  acid  result.  The  nitrogen  necessary  for  the  growing 
plant  is  thus  produced.  The  nitromonas  of  Winogradsky 
belongs  to  this  group.  The  soil  tends  to  destroy  ordinary 
disease-bacteria  in  a  short  time,  but  spores  may  remain  dor- 
mant for  a  number  of  years,  as,  for  instance,  the  spores  of 
anthrax. 

As   bacteria   are   instrumental   in   transforming    organic 


EXAMINATION    OP   AIR,    SOIL,   AND    WATER  245 

matter,  their  influence  in  making  the  soil  more  useful  for 
agricultural  purposes  has  been  the  subject  of  much  research. 
The  richer  the  soil,  the  greater  the  number  of  bacteria. 
Most  bacteria  are  found  under  the  surface  between  i  and 
2  inches. 

The  rod-shaped  organisms  predominate. 

From  an  agricultural  standpoint  the  most  important 
bacteria  are  those  capable  of  liberating  nitrogen  and  break- 
ing up  protein  substance. 

Carbohydrates  are  added  to  soil  by  manure,  by  the  growth 
of  grasses  and  crops,  and  these  are  decomposed  by  bacteria 
and  methane  and  hydrogen  produced. 

Ammonia  Production, — Most  soil  bacteria  can  produce 
ammonia;  a  few,  the  so-called  urea  bacteria,  are  capable  of 
rapid  transformation — nitrification. 

Ammonia,  oxidized  into  nitrites  or  nitrates,  is  possible 
through  the  agency  of  a  group  of  micro-organisms  given 
especial  prominence  by  Winogradski.  Moisture  conditions 
and  the  presence  of  lime  and  mineral  carbonates  influence  the 
nitrifying  organisms. 

The  character  of  the  growing  crop  affects  the  accumulation 
of  nitrates;  legumes  assimilate  nitrogen  more  rapidly  than 
non-legumes. 

Denitrification. — The  reduction  of  nitrates  to  nitrites  and 
ammonia  is  accomplished  by  a  number  of  bacteria.  Nitrate 
reduction  is  of  little  importance  in  the  field,  but  under  exces- 
sive manuring  it  may  become  so.  Bacteria  play  the  impor- 
tant part  of  making  available  to  vegetation  the  nitrogen  of 
the  air. 

Azofication. — Certain  bacteria  can  fix  atmospheric  nitrogen 
and  make  it  serve,  but  the  energy  necessary  must  be  fur- 
nished by  carbohydrates. 

The  enrichment  of  the  soil  by  the  growth  of  legumes  has 
been  shown  to  be  due  to  the  bacteria  contained  in  the  nodules 
or  tubercles  of  the  plant,  these  bacteria  having  the  power  to 
fix  nitrogen  and  deriving  their  energy  from  the  plant  juices, 


246  ESSENTIALS   OF  BACTERIOLOGY 

the  plants  in  turn  utilizing  the  nitrogen  compounds  created 
by  the  bacteria. 

Soil  Inoculation. — Artificial  help  to  soils  deficient  in  nitro- 
gen-fixing organisms  has  been  the  subject  of  much  experiment. 

Nitragin. — Pure  cultures  of  legume  bacteria  under  the 
above  name  have  been  tried.  Dried  cultures  under  the  name 
of  nitro-bacterine  have  likewise  been  marketed,  but  neither 
of  these  methods  has  proved  valuable;  the  matter  is  still  in 
the  experimental  stage. 


CHAPTER  XXXII 
BACTERIA  IN  MILK  AND  FOOD 

The  Bacteria  of  Milk. — Milk  as  secreted  is  sterile,  but 
at  every  step  in  its  passage  from  the  cow  to  the  consumer  it 
is  liable  to  contamination.  Even  the  lower  portion  of  th6 
teat  is  a  source  of  infection,  owing  to  the  presence  of  stag- 
nated milk  from  the  former  milking,  and,  as  milk  ready  for 
consumption  usually  contains  thousands  to  millions  of  bac- 
teria to  the  cubic  centimeter,  sterilization  or  pasteurization 
and  supervision  of  the  dairies  should  always  be  enforced  for 
milk  used  for  infant  feeding. 

A  standard  milk  should  he  free  from  pus  and  should  not 
contain  more  than  10,000  bacteria  to  thz  cubic  centimeter. 

Leukocytes  are  normally  found  in  milk,  and  only  when 
their  number  exceeds  one  million  and  pyogenic  organisms  are 
also  present  can  pus  be  said  to  exist.  Pasteurization  of  un- 
clean milk  sometimes  renders  it  more  dangerous  as  a  food 
than  untreated  milk,  because,  by  preventing  the  action  of 
lactic-acid  formers,  other  bacteria  are  permitted  to  develop 
and  produce  pathogenic  toxins. 

Pure  Milk. — A  pure  milk  is  one  that  is  obtained  from  a 
healthy  cow,  well  groomed,  in  a  clean  room,  by  a  healthy, 
clean  person,  in  clean  cans  or  bottles,  and  transported  to  the 


BACTERIA   IN   MILK   AND   FOOD  247 

consumer  in  as  short  time  as  possible  without  further  hand- 
ling,  keeping  the  container  in  the  mean  time  at  a  low  tern- 
perature  and  protected  from  the  air.  Such  treatment  is  safer 
than  any  form  of  sterilization. 

Classification  of  Milk. — {Abstract  of  resolutions  adopted 
by  the  Commission  on  Milk  Standards  at  Richmond,  Va., 
May  2-j,  191 3.) : 

Milk  shall  be  divided  into  three  grades,  which  shall  be  the 
same  for  both  large  and  small  cities  and  towns. 

Grade  A. — Raw  milk. — Milk  of  this  class  shall  come  from 
cows  free  from  disease  as  determined  by  tuberculin  tests  and 
physical  examinations  by  a  qualified  veterinarian,  and  shall 
be  produced  and  handled  by  employees  free  from  disease 
as  determined  by  medical  inspection  of  a  qualified  physician, 
under  sanitary  conditions  such  that  the  bacteria  count  shall 
not  exceed  100,000  per  cubic  centimeter  at  the  time  of  de- 
livery to  the  consumer.  It  is  recommended  that  dairies 
from  which  this  supply  is  obtained  shall  score  at. least  80  on 
the  United  States  Bureau  of  Animal  Industry  score  card. 

Pasteurized  Milk. — Milk  of  this  class  shall  come  from  cows 
frefe  from  disease  as  determined  by  physical  examinations 
by  a  qualified  veterinarian  and  shall  be  produced  and  handled 
imder  sanitary  conditions  such  that  the  bacteria  count  at  no 
time  exceeds  200,000  per  cubic  centimeter.  All  milk  of  this 
class  shall  be  pasteurized  under  official  supervision,  and  the 
bacteria  count  shall  not  exceed  10,000  per  cubic  centimeter 
at  the  time  of  delivery  to  the  consumer.  It  is  recommended 
that  dairies  from  which  this  supply  is  obtained  should  score 
65  on  the  United  States  Bureau  of  Animal  Industry  score 
card. 

The  above  represents  only  the  minimum  standards  under 
which  milk  may  be  classified  in  grade  A. 

Grade  B. — Milk  of  this  class  shall  come  from  cows  free 
from  disease,  as  determined  by  physical  examinations,  of 
which  one  each  year  shall  be  by  a  qualified  veterinarian,  and 
shall  be  produced  and  handled  under  sanitary  conditions 
such  that  the  bacteria  count  at  no  time  exceeds  1,000,000  per 


248  ESSENTIALS   OF   BACTERIOLOGY 

cubic  centimeter.  All  milk  of  this  class  shall  be  pasteurized 
under  official  supervision,  and  the  bacteria  count  shall  not 
exceed  50,000  per  cubic  centimeter  when  delivered  to  the 
consumer. 

It  is  recommended  that  dairies  producing  grade  B  milk 
should  be  scored  and  that  the  health  departments  or  the 
controlling  departments,  whatever  they  may  be,  strive  to 
bring  these  scores  up  as  rapidly  as  possible. 

Grade  C. — Milk  of  this  class  shall  come  from  cow^s  free 
from  disease  as  determined  by  physical  examinations  and 
shall  include  all  milk  that  is  produced  under  conditions  such 
that  the  bacteria  count  is  in  excess  of  1,000,000  per  cubic 
centimeter. 

All  milk  of  this  class  shall  be  pasteurized,  or  heated  to  a 
higher  temperature,  and  shall  contain  less  than  50,000  bac- 
teria per  cubic  centimeter  when  delivered  to  the  customer. 
It  is  recommended  that  this  milk  be  used  for  cooking  or  manu- 
facturing purposes  only. 

Whenever  any  large  city  or  community  finds  it  necessary, 
on  account  of  the  length  of  haul  or  other  peculiar  conditions, 
to  allow  the  sale  of  grade  C  milk,  its  sale  shall  be  surrounded 
by  safeguards  such  as  to  insure  the  restriction  of  its  use  to 
cooking  and  manufacturing  purposes. 

Classification  of  Cream.— Cream  should  be  classified 
in  the  same  grades  as  milk,  in  accordance  with  the  require- 
ments for  the  grades  of  milk,  excepting  the  bacterial  standards, 
which  in  20  per  cent,  cream  shall  not  exceed  five  times  the 
bacterial  standard  allowed  in  the  grade  of  milk. 

Ice  Cream. — Made  and  handled  under  sanitary  conditions 
it  contains  mostly  Bacillus  lactis  acidi  type,  not  dangerous; 
but  if  made  from  milk  and  cream  containing  putrefactive 
bacteria,  freezing  wall  not  prevent  further  growlh  and  bac- 
terial poisons  may  be  developed,  causing  sickness  and  death. 
An  examination  of  specimens  collected  gave  as  the  lowest 
count  50,000  bacteria  per  cubic  centimeter,  and  the  highest 
150,000,000  per  cubic  centimeter. 


BACTERIA   IN   MILK   AND   FOOD  249 

SOME  BACTERIA  FOUND  IN  MILK 

Fermentation  of  Milk. — Lactic  Acid  Lactose. — Fermenta- 
tion of  miik  is  due  to  the  conversion  of  milk-sugar  into  lactic 
acid.  This  can  be  accomplished  by  a  number  of  different 
bacteria,  such  as  Bacillus  coli,  streptococci  and  staphylo- 
cocci, which  are  apt  to  be  present  about  the  dairy.  The 
lactic-acid  bacteria  are  commonly  present  in  sour  milk,  and 
are  chiefly  concerned  with  fermentation.  There  are  several 
varieties,  but  principally  three  groups. 

The  first  group,  like  the  Streptococcus  pyogenes,  is  called 
the  Bacterium  lactis  acidi  group.  Milk  is  curdled  within 
twenty-four  hours  without  gas-formation.  The  milk  has  a 
mild  acid  taste  and  agreeable  odor.  The  curd  is  even,  a 
true  lactic  fermentation. 

The  second  group  resembles  the  Bacillus  coli — Bacillus 
lactis  aerogenes.  Indol  and  hydrogen  sulphid  often  formed. 
Milk  curdles,  but  the  curd  shrinks.  Not  easUy  emulsified. 
This  fermentation  undesirable. 

The  third  group,  true  lactic  bacteria — Bacterium  bulgari- 
cum;  exclusively  lactic  acid;  curd  easily  broken. 

Bacterium  Acidi  Lactici  (Hiippe) . — Belongs  to  the  same 
group  as  the  Bacillus  coli  conununis  (see  page  134). 

Synonyms. — Bacillus  acidi  lactici;  B.  lactis  aerogenes 
(Escherich), 

Origin. — ^In  sour  milk. 

Form. — Short  thick  rods,  nearly  as  broad  as  they  are  long, 
usually  in  pairs,  resembling  B.  coli. 

Properties. — Immotile.  Does  not  liquefy  gelatin.  Breaks 
up  the  sugar  of  milk  into  lactic  acid  and  carbonic  acid  gas, 
the  casein  being  thereby  precipitated.  The  fermentation  of 
milk  produced  by  this  group  is  offensive;  taste  undesirable. 
Curd  is  firm. 

Stain. — Does  not  take  Gram. 

Growth. — Rapid  and  abundant;  is  facultative  anaerobic. 
Grows  at  10°  C.  Grows  in  all  media  and  in  absence  of  car- 
bohydrates. 

Stab-culture. — A  thick  dry  crust  with  cracks  in  it  forms  on 
the  surface  after  a  couple  of  weeks. 


250  ESSENTIALS   OF  BACTERIOLOGY 

Attenuation. — If  grown  through  successive  generations, 
it  loses  power  to  produce  fermentation. 

Streptococcus  Acidi  Lactici  (Grotenfeld)  (1889). — 
Widely  distributed  in  nature. 

Synonyms. — Bacterium  lactis  acidi;  Bact.  Giintheri. 

Origin. — In  sour  milk. 

Appearance. — Very  short  cells,  often  as  large  as  oval  cocci, 
in  pairs  or  small  chains,  outer  ends  pointed. 

Properties. — Immotile.  Stain  with  Gram.  Growth  best 
at  3o°-35°  C. 

Growth. — Facultative  anaerobic.  Delicate,  opaque,  re- 
sembling dewdrops.  Bouillon  containing  glucose  grows 
cloudy.  Gelatin  not  liquefied.  Milk  coagulated.  Strong 
acid  reaction.  Curd  is  soft  and  easily  mixed  within  twenty- 
four  hours.     Gas  is  not  formed. 

In  lactose-agar  stab  no  surface  growth,  but  all  along  the 
line. 

Potato. — Scant  growth. 

Origin. — Almost  always  in  sour  milk,  and  the  chief  cause  of 
lactic  acid  formation.  Found  at  times  in  combination  with 
B.  acidi  lactici  and  other  bacteria.  Sauer-kraut  fermentation 
is  due  to  streptococcus  of  lactic  acid  and  yeasts,  the  latter 
producing  gas. 

Bacterium  Bulgaricum. 

Synonym. — Bacterium  caucasicus  {v.  Freudenreich) . 

Origin. — Present  in  milk.  Thought  to  be  a  product  cf 
eastern  countries,  but  now  recognized  as  universal.  Arises 
from  aHmentary  tract. 

Properties. — Produces  large  amount  of  acid  at  higher  tem- 
perature;   non-motile. 

Form. — Slender  rods,  2  ju  to  4  /x  long,  tending  to  form 
threads. 

Staining. — Gram  positive. 

Growth. — Best  growth  at  40°  C.  Very  meager  colonies, 
hardly  visible.  Curdling  homogeneous,  changed  later  into 
soluble  products.  Gelatin  not  liquefied.  Used  to  produce 
artificial  buttermilks. 


BACTERIA   IN   MILK   AND   FOOD  251 

Potato. — Growth  wrinkled  and  many-folded,  gray  changing 
to  brown,  extending  over  the  entire  surface  as  a  thick  cover- 
ing or  skin. 

Agar  Stroke. — Abundant,  grayish,  fatty,  later  on  wrinkled 
skin. 

Gelatin  Stab. — On  surface,  grayish,  fatty  exudate  covered 
with  skin  which  slowly  sinks  as  the  media  liquefy.  Gelatin 
liquefied.  No  gas  in  sugar  bouillon;  acid  is  formed;  no 
indol.  Has  been  found  in  ropy  or  gelatinous  bread  and  is 
considered  the  cause. 

Bacillus  Butyricus  (Hiippe). — This  bacillus  causes  bu- 
tyric-acid  fermentation.      Supposed   to 
be  identical  with  Bacillus  mesentericus. 

Bacillus  Amylobacter  (Van  Tieg- 
ham) . — Synonyms. — Clostridium  butyri- 
cum  (Frasmowsky);  Vibrion  butyrique  of 
Pasteur;  Bacterium  saccharobutyricus 
(Klecki)  (Fig.  ii8). — Origin. — Found  in 
putrefying  plant-infusions,  in  fossils  and 
conifera  of  the  coal  period,  in  cheese, 
water,  earth. 

Form. — ^Large,   thick   rods,   with 

rounded  ends,  often  found    in   chains,      t^-        „       t>    .., 
.   ,  ,       .'  ^  ,    ^,         Fig.  118.  — Bacillus 

A  large  glancmg  spore  at  one  end,  the  amylobacter. 

bacillus  becoming  spindle  shaped  in  or- 
der to  allow  the  spore  to  grow;  hence  the  name,  Clostridium. 

Properties. — Very  motile;  gases  arise  with  butyric  smell. 
In  solutions  of  sugars,  lactates,  and  cellulose-containing 
plants  and  vegetables  it  gives  rise  to  decompositions  in 
which  butyric  acid  is  often  formed.     Casein  is  also  dissolved.' 

A  watery  solution  of  iodin  will  give  the  starch  reaction 
and  color  blue  some  portions  of  the  bacillus;  therefore 
it  has  been  called  amylobacter. 

Growth  in  Glucose  Agar. — Rapid  at  37°.  Small  indefinite 
colonies  with  gas-bubbles.     No  growth  in  gelatin. 

Bacillus  Cyanogenes  {Bacterium  Syncyanum)  (Hiippe). 
— Origin. — Found  in  blue  milk. 


252  ESSENTIALS    OF   BACTERIOLOGY 

Form. — Small  narrow  rods  about  three  times  longer  than 
they  are  broad;  usually  found  in  pairs.  The  ends  are 
rounded. 

Properties. — They  are  very  motile;  do  not  liquefy  gelatin. 
A  bluish-gray  pigment  is  formed  outside  of  the  cell, 
around  the  medium.  The  less  alkaline  the  medium,  the 
deeper  the  color.  It  does  not  act  upon  the  milk  otherwise 
than  to  color  it  blue. 

Growth. — Grows  rapidly,  obligate  aerobe. 

Colonies  on  Plate. — Depressed  center,  surrounded  by  ring 
of  porcelain-like  bluish  growth.  Dark-brown  appearance 
under  microscope. 

Stab-culture. — Grows  mainly  on  surface ;  a  nail-like  growth. 
The  surrounding  gelatin  becomes  colored  brown. 

Potato. — The  surface  covered  with  a  dirty  blue  scum. 

Attenuation. — ^After  prolonged  artificial  cultivation  loses 
the  power  to  produce  pigment. 

Staining. — By  ordinary  methods.     Gram  positive. 

Red  milk  and  yellow  milk  are  due  to  other  chromogenic 
organisms,  as,  for  instance,  B.  erythrogenes. 

Examination  of  Milk. — American  Standard. — Some  bac- 
teria are  found  in  all  milk  as  ordinarily  handled.  Strepto- 
cocci and  colon  group,  when  present,  always  regarded  with 
suspicion.  A  high-cell  leukocyte  count,  when  accompanied 
by  chain  bacteria,  is  an  indication  of  udder  disease.  There 
should  be  several  samples  taken  one  week  apart  and  an 
average  made.  Bacteria  present  may  be  counted  in  one  of 
three  ways. 

Stewart-Slack  Method. — Centrifuge  i  to  2  c.c.  of  milk; 
smear  sediment  on  slide,  and  stain  with  Jenner  or  Wright 
stain  and  count  bacteria  in  field. 

Prescott-Breed  Method. — In  a  special  capillary  tube  y^ 
c.c.  of  milk  is  sucked  up  and  spread  over  a  square  centimeter 
on  a  microscopic  slide,  dried  and  fixed  with  methyl-alcohol. 
Flood  with  xylol  to  dissolve  fat,  stain  with  methylene-blue 
or  Jenner,  and  decolorize  slightly  with  alcohol.  Focus  15 
mm.  of  the  specimen  and  count  bacteria  and  cells  present. 


BACTERIA   IN   MILK   AND   FOOD  253 

Multiply  by  5000.  This  equals  the  number  in  y-J-Q-  c.c. 
Count  several  fields  and  average  the  result. 

The  Plate  Method. — Microscopic  examination,  while  not 
to  be  relied  upon  wholly,  gives  valuable  and  quick  informa- 
tion as  to  the  general  character  of  bacteria,  their  apparent 
number,  the  presence  or  absence  of  barn-dirt  and  chain 
bacteria.  The  microscopic  count  differs  greatly  from  plate 
count,  because  dead  cells  as  well  as  living  are  shown. 

Certified  milk  should  have  less  than  10,000  bacteria  to  the 
cubic  centimeter.  According  to  the  average  taken  from  a 
count  of  four  specimens,  a  rating  is  given  to  the  milk,  and 
this  rating  is  to  be  interpreted  only  as  other  conditions  are 
considered,  such  as  cleanliness  of  the  cattle  and  stalls,  and 
chemic  composition  and  method  of  handling  the  product. 

Temperature. — Milk  kept  at  10°  F.  or  lower  will  not  allow 
ordinary  bacteria  to  develop  to  any  considerable  extent; 
kept  at  a  higher  temperature,  bacteria  develop  rapidly. 

Separating  or  centrifuging  permits  the  bacteria  to  be  con- 
centrated, and  top-milk  and  cream  contain  more  bacteria  per 
cubic  centimeter  than  whole  milk. 

Time,  an  element. 

Milk  freshly  drawn,  under  proper  precautions,  may  con- 
tain but  few  bacteria,  but  in  forty-eight  to  seventy-two  hours 
on  ice  bacteria  will  increase  enormously.  Market  milk  as 
ordinarily  found  in  cities  may  contain  millions  of  bacteria 
per  cubic  centimeter. 

Pasteurization. — Milk  heated  to  60°  C.  for  twenty  minutes  is 
called  pasteurized.  This  increases  the  keeping  quality  and 
tends  to  destroy  the  vegetative  forms  of  pathogenic  bacteria. 

To  kill  lactic  acid,  the  instantaneous  method,  higher  tem- 
perature, a  few  seconds  only  for  pathogenic  organisms  is 
required.  Pasteurization  is  beneficial  only  when  there  are 
supervision  and  inspection  of  original  supply. 

Milk  as  Source  of  Contagion. — Harmless  Varieties. — 
Sour  milk  contains  the  Bacterium  lactis  acidi  and  is  not 
dangerous,  and  is  even  considered  beneficial,  as,  for  instance, 
buttermilk. 


254  ESSENTIALS    OF   BACTERIOLOGY 

Neutral  Forms. — Many  species  of  air  and  chromogenic 
varieties  found  in  milk  have  no  pathogenic  properties, 
neither  do  they  affect  the  composition  of  the  milk. 

Injurious  Organisms. — Human  diseases,  like  typhoid, 
diphtheria,  and  scarlet  fever,  may  be  conveyed  through 
milk,  the  infection  coming  from  some  one  concerned  in 
handling  the  particular  supply.  The  milk  acts  as  a  favorable 
medium  for  the  pathogenic  organisms  that  accidentally  find 
their  way  into  it.  Animals  wading  in  infected  water  have 
infected  the  milk.  Utensils  washed  in  polluted  water  have 
been  found  to  be  the  cause  in  some  epidemics  of  typhoid. 
Carriers,  persons  who  harbor  the  diphtheria  and  typhoid 
bacteria,  but  who  are  not  affected  with  illness,  may  likewise 
start  epidemics  of  a  kind,  especially  if  working  about  dairies. 
Bacteria  may  enter  milk  from  the  animal,  as  Bacillus  tuber- 
culosis from  diseased  udder.  Infantile  diarrheas  from  the 
putrefactive  Bacillus  coli  group,  streptococcic  sore  throat 
from  udder  disease,  are  other  forms  of  disease  originating  in 
milk. 

Butter  and  Cheese. — Butter  is  milk-fat  separated  by 
creaming  and  churning,  and  as  such  partakes  somewhat  of 
the  bacterial  nature  of  the  milk  from  w^hich  it  is  derived. 
The  flavor  of  butter  is  due  to  the  character  of  the  acid  bac- 
teria used  in  souring  the  milk.  By  eliminating  the  gas-form- 
ing bacteria  and  by  keeping  his  starting  cultures  pure  the 
butter-maker  can  control  and  develop  flavors  as  easily  as 
the  wine-maker.  Pure  cultures  of  lactic  acid  are  supplied 
to  butter-makers  and  used  in  creameries  to  inoculate  sweet 
cream  and  milk.  Bacteria  coming  from  unclean  utensils, 
polluted  water,  or  dirty  milk  undoubtedly  affect  the  flavor 
and  often  produce  a  poor  quality  of  butter.  Disease  bacteria 
are  not  often  conveyed  through  butter,  although  it  is  claimed 
that  Bacillus  tuberculosis  has  been  found  in  salted  butter. 

Cheese. — The  fat  and  casein  salts  and  sugar-of-milk  sepa- 
rated by  curdling  from  the  bulk  of  soluble  portion  of  milk 
constitutes  cheese.  The  curdling  is  accomplished  by  acid 
bacteria  normally  in  milk,  so-called  acid  curd  cheeses,  or  by 


BACTERIA   IN   MILK   AND   FOOD  2$$ 

the  use  of  rennet  to  form  a  curd,  rennet  curd  cheese,  to  which 
all  the  important  varieties  belong.  Milk  for  cheese  should 
be  free  from  Bacillus  coli  or  other  deleterious  bacteria.  The 
milk  for  cheese  cannot  be  pasteurized  as  for  butter. 

Testing  Milk  for  Bacillus  Coli. — ^A  sample  of  milk  is  incu- 
bated at  35°  C.  for  a  few  hours,  noting  the  curd,  whether 
firm  or  soft  and  gassy. 

Wisconsin  Test. — Milk  curdled  by  rennet;  curd  cut  and 
drained  and  jars  kept  at  30°  C.  to  40°  C.  The  curd  should 
have  clean  acid  odor  and  taste. 

After  the  curd  has  been  formed,  the  cheese  is  allowed  to 
ripen,  and  this  is  due  to  acid-forming  bacteria,  which  permit 
the  pepsin  in  the  rennet  to  act.  Various  molds,  notably 
Penicillium  and  Oidium  lactis,  are  used  to  give  certain 
foreign  cheeses  their  characteristic  flavor. 

Condensed  milk  has  few  bacteria  in  it.  The  sugar  and 
condensation  heat  tend  to  prevent  further  growth  of  micro- 
organisms. 

Concentrated  unsweetened  milk  is  a  form  of  pasteurized 
milk  which  is  reduced  in  volume  one-fourth.  It  is  not  always 
sterile,  and  bacteria  may  develop  in  it  if  exposed  to  warmth 
and  air. 

Buttermilk  and  similar  fermented  drinks  depend  on 
Bacillus  lactis  acidi  and  added  yeasts.  Bacillus  bulgaricus 
gives  more  acid  and  allows  partial  sterilization. 

Foods  as  a  Source  of  Infection. — Foods  eaten  after  little 
or  no  cooking,  such  as  fruits,  salads,  and  the  like,  and  also, 
oysters,  are  possible  sources  of  bacterial  diseases,  and  the 
so-called  ptomain  poisoning  observed  after  the  consumption 
of  ice-cream,  sausage,  canned  meats,  etc.,  is  the  result  of  the 
action  of  bacteria  or  their  products. 

Oysters  and  fish  from  sewage-polluted  waters  have  pro- 
duced typhoid.  Vegetables  grown  in  manured  ground  or 
sprinkled  with  polluted  water  may  be  a  possible  source  of 
disease.  The  practice  of  exposing  meats  and  other  food  to 
street  dust  and  flies  is  no  doubt  responsible  for  some  disease. 

Alcohol  and  Vinegar  Fermentations. — On  grapes  are  to 


256  ESSENTIALS    OF   BACTERIOLOGY 

be  found  all  forms  of  air  bacteria  as  well  as  molds  and  yeasts, 
some  beneficial,  some  harmful.  The  acid  of  grape- juice  de- 
stroys many  of  the  harmful  forms,  but  some  persist  and 
must  be  dealt  with  by  the  wine-maker. 

The  various  yeasts  produce  alcohol  from  the  sugar  of  the 
grape.  Vinegar  bacteria  likewise  form  a  small  amount  of 
acetic  acid.  The  wine-maker's  success  lies  in  obtaining  a 
clean,  unbruised  grape,  aiding  the  work  of  the  wine  yeasts,  and 
preventing  the  injurious  forms  from  working.  The  grapes  are 
crushed  and  the  juice  allowed  to  settle.  Pure  cultures  of 
tested  yeast  are  used  as  starters  of  fermentation. 

Fermentation  is  regulated  by  burning  sulphur,  which  in- 
hibits the  growth  of  molds  and  harmful  bacteria.  After 
fermentation  is  completed  the  wine  is  cleared  and  freed  from 
all  organisms  and  kept  as  nearly  as  possible  in  a  sterile  con- 
dition. 

Beer. — The  fermentation  is  produced  by  yeasts  and  with 
a  mixture  of  grains.  Barley  or  other  grains  which  have  been 
allowed  to  germinate  produce  malt.  The  malt  contains  the 
enzjrmes  which  change  starch  into  sugar.  Then,  by  boiling, 
the  enzyme  is  destroyed  and  fermentation  by  yeasts  is  per- 
mitted.    The  yeasts  in  modern  breweries  are  pure  cultures. 

Wild  yeasts  or  lactic-acid  bacteria  may  contaminate  beer. 

Alcohol,  brandy,  and  whisky  are  likewise  the  product  of 
yeast  fermentation,  some  sugary  substance  furnishing  the 
material. 


ORGANS   AND   CAVITIES   OF   THE   HUMAN   BODY  257 


CHAPTER  XXXIII 

BACTERIOLCGIC  EXAMINATION  OF  THE  ORGANS  AND 
CAVITIES  OF  THE  HUMAN  BODY 

The  body,  on  account  of  its  constant  contact  with  the  sur- 
rounding air,  is  necessarily  exposed  to  infection,  and  we  would 
be  likely  to  find  on  the  skin  and  in  the  oral,  anal,  and  nasal 
cavities  the  varieties  of  microorganisms  commonly  around  us. 
Through  the  water  and  food  the  body  is  also  contaminated, 
but  some  organisms  by  predilection  inhabit  the  mouth,  intes- 
tine, and  other  cavities,  and  form  there  a  flora  distinctly  their 
own. 

The  Skin. — The  majority  of  microorganisms  met  with  on 
the  skin  are  non-pathogenic,  although  underneath  the  nails 
and  in  the  hair  pus-forming  microorganisms  often  occur, 
producing  sometimes  serious  abscesses  on  other  parts  of  the 
body. 

In  the  sweat-glands  and  the  sebaceous  glands  various 
organisms  have  been  found.  The  Staphylococcus  pyogenes 
seems  to  be  present  constantly. 

In  foul-smelling  perspiration  of  the  feet  Rosenbach  found 
microorganisms  pathogenic  for  rabbits. 

Micrococcus  cereus  albus  and  flavus,  Diplococcus  liquefa- 
ciens  albus  and  flavus.  Staphylococcus  pyogenes  aureus,  and 
Streptococcus  pyogenes  are  found  underneath  the  nails. 

In  eczema,  Diplococcus  albicans  tardus,  Diplococcus 
citreus  liquefaciens,  Diplococcus  flavus  liquefaciens,  and 
Ascobacillus  citreus. 

In  colored  sweat.  Micrococcus  haematoides,  Bacillus 
pyocyaneus. 

A  diplococcus  is  found  in  acute  pemphigus. 

The  lepra  bacillus,  the  tubercle  bacillus  in  lupus,  and  the 
t3rphoid  bacillus  in  the  eruption  of  typhoid  fever  are  a  few  of 
the  specific  germs  found  on  the  skin  during  the  disease  stage. 
17 


258  ESSENTIALS    OF   BACTERIOLOGY 

Infection  results  through  some  damage  of  the  superficial 
layers.  The  injury  may  be  very  slight — an  expanded  hair- 
foJlicle  may  suffice  to  permit  entrance  of  suppurative  organ- 
isms. 

The  Conjunctiva. — The  micrococcus  of  trachoma,  the 
Kocli-Weeks  bacillus,  considered  to  be  the  specific  cause  of 
acute  catarrhal  conjunctivitis,  or  "pink  eye,"  and  the  Bacillus 
xerosis,  are  special  germs  found  on  the  conjunctiva;  the  other 
varieties  of  air-  and  water-organisms,  and  those  usually 
present  on  the  skin,  are  also  found.  Loffler's  bacillus  and 
the  pneumococcus  have  been  found  in  some  forms  of  con- 
junctivitis.    The  Koch-Weeks  bacillus  is  the  most  contagious. 

A  special  diplobacillus,  known  as  the  bacillus  of  Morax- 
Axenfeld,  produces  a  stubborn  form  of  conjunctivitis. 

The  gonococcus  is  found  in  ophthalmia  of  the  new-born. 

The  Mouth. — The  mouth  is  a  favorite  seat  for  the  devel- 
opment of  bacteria.  The  alkaline  saliva,  the  particles  of 
food  left  in  the  teeth,  the  decayed  teetji  themselves,  all  fur- 
nish suitable  soil  for  their  growth. 

Quite  a  number  of  germs  have  been  isolated  and  their 
properties  partly  studied.  Many  have  some  connection  with 
the  production  of  caries  of  the  teeth,  as  Miller  has  well  shown 
in  his  careful  studies.  The  Leptothrix  buccalis,  found  in 
nearly  all  mouths,  is  a  long  chain  or  filamentous  bacillus  which 
stains  blue  with  iodin.  It  was  formerly  considered  the  cause 
of  tartar  on  the  teeth. 

The  Spirillum  sputigenum,  Spirocha?ta  dentium,  Micro- 
coccus gingivae  pyogenes,  Bacillus  dentalis  viridans,  Bacillus 
pulpae  pyogenes,  micrococcus  of  sputum  septicemia,  and 
Micrococcus  salivarus  septicus  are  a  few  of  the  organisms 
cultivated  by  Miller  and  Biondi  from  the  mouth  and  sup- 
posed to  be  separate  varieties.  Besides  these,  the  pneumo- 
bacteria,  diphtheria  bacillus,  and  tubercle  bacillus  are  often 
met  with,  the  first  two  in  the  mouths  of  healthy  persons. 
The  expired  air  in  quiet  respiration  is  free  from  bacteria,  but 
in  coughing,  sneezing,  etc.,  large  numbers  of  organisms  are 
violently    ejected    and    the    atmosphere    about    tubercular 


ORGANS   AND   CAVITIES   OF   THE   HUMAN   BODY  259 

patients  is  often  saturated  with  tubercle  bacilli.  The  bac- 
teria may  enter  the  system  from  the  pharynx  to  the  tonsils 
and  cervical  glands  by  means  of  the  lymphatics. 

Ear. — In  the  middle  ear  of  new-born  infants  no  pathogenic 
organisms  have  been  found,  but  quite  a  number  of  non- 
pathogenic ones.  In  affections  of  the  ear  the  pnemnobacillus 
and  the  Staphylococcus  pyogenes  are  most  frequent. 

When  the  streptococcus  is  present  in  acute  suppurations, 
there  is  great  danger  of  mastoiditis.  In  chronic  otitis  the 
gas-forming  bacteria,  as  well  as  Bacillus  pyocyaneus,  is  often 
found. 

Nasal  Cavity. — The  nasal  secretion,  containing  as  it  does 
dead  cells  and  being  alkaline  in  reaction,  forms  a  good  soil  for 
the  growth  of  germs. 

Diplococcus  coryzae.  Micrococcus  nasalis.  Bacillus  fcetidus 
ozaenae,  Bacillus  striatus  albus  et  flavus,  Bacillus  capsulatus 
mucosus,  and  Vibrio  nasalis  are  some  of  the  organisms  de- 
scribed by  various  observers. 

Stomach  and  Intestine. — The  secretion  of  the  stomach 
is  in  its  normal  state  not  a  favorable  soil  for  the  development 
of  bacteria,  yet  some  germs  resist  the  action  of  the  gastric 
juice  and  flourish  in  it.  When  the  acids  of  the  stomach  are 
diminished  in  quantity  or  absent  altogether,  the  conditions 
for  the  growth  of  bacteria  are  more  favorable.  The  alimen- 
tary canal  of  the  new^-born  infant  is  sterile,  but  in  a  few 
hours  after  birth  microorganisms  begin  to  appear. 

Some  gastric  bacteria  normally  present  are  Sarcina  ventric- 
uli,  Bacterium  lactis  aerogenes.  Bacillus  subtilis,  Bacillus 
amylobacter,  Bacillus  megaterium. 

The  intestinal  organisms  are  more  numerous,  and  the 
mucous  lining  of  the  intestines  and  the  secretions  there  present 
are  favorable  to  germ-growth. 

Bacillus  geniculatus  Boas  considers  a  sign  of  carcinoma  of 
the  stomach,  and  is  always  present,  he  claims,  when  the  con- 
tents contain  lactic  acid. 

Some  investigators  consider  digestion  dependent  on  micro- 
bic  activity,  but  experiments  with  animals  have  shown  that 


26o  ESSENTIALS    OF   BACTERIOLOGY 

life  and  digestion  can  proceed  in  a  perfectly  sterile  condition. 
Food  and  air  sterilized  will  not  develop  bacteria  in  the  feces. 

In  the  feces  of  the  young  a  great  many  bacteria  have  been 
found  that  are  supposed  to  stand  in  close  relation  with  the 
intestinal  disorders  common  to  nurslings.  The  majority  of 
bacteria  usually  present  in  the  intestines  are  non-pathogenic. 
The  following  varieties  may  be  met  with  in  the  feces:  Micro- 
coccus aerogenes,  Bacillus  subtilis,  Bacillus  butyricus,  Bacil- 
lus putrificus  coli,  Bacillus  lactis  aerogenes,  Bacillus  coli 
commune,  Bacillus  subtiliformis,  and  the  bacteria  of  cholera, 
dysentery,  and  typhoid,  besides  many  yeast-cells. 

Genito-urinary  Passages. — In  vaginal  secretion  Bumm 
has  been  able  to  find  a  number  of  organisms,  some  of  which 
closely  resemble  the  gonococcus;  thus,  there  is  the  Diplococ- 
cus  subflavus.  Micrococcus  lacteus  faviformis,  Diplococcus 
albicans  amplus,  and  the  vaginal  bacillus. 

In  the  urethra  of  healthy  persons  bacteria  are  sometimes 
found,  usually  having  entered  from  the  air. 

In  the  normal  secretions  around  the  prepuce  a  bacillus 
called  the  smegma  bacillus  has  been  discovered.  The  spiro- 
chaete  of  syphilis  can  be  obtained  from  lesions  about  the 
genitalia. 

From  urethral  pus  a  number  of  diplococci  other  than  the 
gonococci  have  been  isolated. 

From  the  urine  itself  a  great  number  of  bacteria  have  been 
obtained,  but  mostly  derived  from  the  air,  finding  in  the 
urine  a  suitable  soil.  The  colon  and  typhoid  bacilli  gain 
entrance  into  the  bladder,  possibly  by  way  of  the  urethra, 
and  produce  cystitis.  In  a  larger  number  of  typhoid  fever 
patients  the  bacilli  are  found  in  the  urine. 

Microorganisms  of  the  Blood. — Many  of  the  bacteria 
described  in  this  book  are  found  in  the  blood  of  the  animal 
infected;  anthrax  bacilli  are  always  found  in  the  blood. 

When  animals  are  subcutaneously  injected  with  pneumo- 
cocci  they  are  found  in  large  quantities  in  the  blood.  The 
diseases  of  a  hemorrhagic  nature  affecting  fowls  and  swine 
usually  show  the  presence  of  bacteria  in  the  vascular  system. 


GERMICIDES,   ANTISEPTICS,  AND  ANTISEPSIS  261 

Bacteria  may  be  recovered  from  the  blood  in  all  forms  of 
septic  infection,  such  as  general  sepsis,  malignant  endocar- 
ditis, puerperal  sepsis,  and  typhoid  fever.  Tubercle  bacilli 
are  rarely  if  ever  obtained  from  the  blood. 

Staining  Blood  Specimens. — A  drop  of  blood  is  spread  on 
a  cover-glass  and  stained  with  the  ordinary  dyes;  but  in 
order  to  eliminate  the  coloring-matter  of  the  red  corpuscles 
and  bring  the  stained  bacteria  more  prominently  into  view, 
Gunther  recommends  that  the  blood,  after  drying  and  fixing, 
should  be  rinsed  in  a  dilute  solution  of  acetic  acid  (i  to  5  per 
cent.).  The  hemoglobin  is  thereby  extracted,  and  the  cor- 
puscles appear  then  only  as  faint  outlines. 

Instead  of  "fixing"  by  heat,  Canon  employs  alcohol  for 
five  minutes,  especially  in  staining  for  influenza  bacilli,  which 
have  been  detected  in  the  blood. 

Blood  Cultures. — ^As  large  a  quantity  of  blood  as  pos- 
sible— never  less  than  10  c.c. — is  taken  from  a  superficial 
vein,  the  median  basilic,  for  example,  by  means  of  a  sterile 
antitoxin  syringe,  a  small  incision  being  made  through  the 
skin  over  the  vein  in  order  to  avoid  skin  infection.  The 
blood  so  obtained  is  immediately  transferred  to  culture-tubes, 
where  the  organisms  are  allowed  to  develop,  and  are  then 
studied  in-  the  customary  manner. 


CHAPTER  XXXIV 
GERMICIDES,  ANTISEPTICS,  AND  ANTISEPSIS 

Sunlight,  pure  air,  and  ordinary  soap  and  water  are  effec- 
tive disinfectants.  Too  often  the  burning  of  chemicals  and 
the  dipping  of  hands  into  antiseptic  solutions  partake  of  the 
nature  of  religious  sacrifice,  and  the  more  nauseous  the  odor, 
the  more  effective  is  the  incense  supposed  to  be.  Much  of 
the  perfunctory  fumigation  by  the  boards  of  health  after  the 


262  ESSENTIALS    OF   BACTERIOLOGY 

minor  contagious  diseases,  instead  of  teaching  the  people  a 
lesson,  create  a  false  impression  of  security,  and  permit  them 
to  neglect  the  commoner  means  of  ordinary  cleanliness 
because  of  this  assumed  virtue  of  fumigation.  The  whole 
subject  of  fumigation  and  quarantine  regulation  needs  more 
careful  investigation  and  study. 
.  A  germicide  is  an  agent  capable  of  destroying  bacterial  life. 

An  antiseptic  solution  or  substance  is  one  that  can  inhibit 
or  prevent  the  growth  of  bacteria  without  necessarily  de- 
stroying them. 

A  disinfectant  must  be  germicidal. 

A  deodorant  may  have  no  germicidal  or  antiseptic  properties. 

Preservative's  are  substances  which  prevent  fermentation, 
but  they  are  not  always  germicides. 

In  considering  the  value  of  a  germicide,  the  strength  in 
which  it  acts  is  the  main  consideration.  Some  very  weak 
chemicals  will  inhibit  and  destroy  the  growth  of  bacteria  if 
used  in  sufficiently  concentrated  solutions.  Some  bacteria 
will  die  in  an  acid  medium;  others  are  destroyed  by  too  much 
alkali.  Some  bacteria  are  very  readily  destroyed  in  pure 
cultures,  but  are  resistant  to  a  considerable  degree  in  the 
body  tissues.  Again,  a  germicide  may  be  ideal  in  laboratory 
experiments,  but  wholly  impractical  at  the  clinic. 

A  I  :  300,000  solution  of  mercuric  chlorid  (corrosive  sub- 
limate) will  prevent  the  development  of  anthrax  spores,  but 
a  I  :  1000  solution  is  needed  to  destroy  them. 

Germicides  are  tested  by  action  in  various  dilutions  or  in 
gaseous  form  on  threads  impregnated  with  virulent  and  spore- 
forming  organisms.  The  length  of  time  is  noted  that  it  takes 
to  destroy  anthrax  bacilli  or  pyogenic  organisms. 

The  infected  material  is  subjected  to  the  solution  and  then 
inoculated  on  media  and  compared  with  control,  or  tested  for 
virulence  on  animals.  Spore-forming  organisms  are  very 
resistant  to  the  most  potent  agents. 

Heat  is  perhaps  the  best  general  germicide.  For  all  articles 
that  can  be  subjected  to  boiling  or  the  direct  flame  there  is 
no  safer  agent. 


GERMICIDES,  ANTISEPTICS,   AND  ANTISEPSIS  263 

Superheated  steam ,  or  steam  under  pressure,  is  now  in 
general  use  in  sterilizing  surgical  dressings  and  instruments, 
and  requires  less  time  than  ordinary  steam. 

The  salts  of  metals  of  high  atomic  weights  come  next  in  order. 
Bichlorid  of  mercury  and  cyanid  of  mercury  are  the  most 
powerful  of  chemical  germicides,  but  in  the  human  body  they 
can  be  used  in  dilute  solutions  only,  and  in  contact  with 
highly  albuminous  solutions,  insoluble  and  inert  albuminates 
are  liable  to  form,  lessening  the  germicidal  value.  A  i  :  200 
solution  combined  with  an  acid  like  citric  will  destroy  the 
spores  of  anthrax  in  one  hour,  but  much  weaker  solutions  will 
destroy  the  anthrax  bacilli  in  the  blood,  and  for  all  practical 
purposes  a  i  :  2000  solution  is  suflScient,  destrojdng  bacterial 
life  in  a  few  minutes. 

One  per  cent,  solution  soda  lye,  NaOH,  kills  most  bacteria 
in  a  few  minutes,  and,  therefore,  hot  soapsuds  is  quite  effec- 
tive as  a  germicide. 

Phenol  in  5  per  cent,  solution  will  destroy  most  of  the  bac- 
teria in  less  than  five  minutes.  Tricresol,  a  combination  of 
cresols,  has  three  times  the  disinfecting  power  of  phenol. 

Formaldehyd,  in  gaseous. form  or  in  a  liquid  spray,  is  a  very 
efficient  germicide,  and  from  the  fact  that  it  is  not  destruc- 
tive to  fabrics  or  paper  has^come  into  general  use  as  a  dis- 
infectant. In  combination  with  potassium  permanganate 
or  in  suitable  generators  it  is  employed  in  houses  after  infec- 
tious diseases.  It  has  no  effect  on  insects,  and  where  it  is 
necessary  to  destroy  these,  other  agents,  known  as  insecti- 
cides, must  be  used  in  connection  with  the  gas.  The  gas 
should  be  in  a  moist  state — from  6  to  16  ounces  for  an  ordi- 
nary room  are  needed;  the  room  should  be  made  as  air-tight  as 
possible,  and  the  gas  evolved  as  speedily  as  possible. 

In  the  permanganate  method  8  ounces  (by  weight)  of 
potassium  permanganate  crystals  are  placed  in  a  large  tin 
vessel  ten  times  the  capacity  of  the  disinfectant  used.  One 
pint  of  formaldehyd  solution  is  quickly  poured  over  the 
crystals.     Formaldehyd  gas  is  thereby  generated  at  once. 


264  ESSENTIALS    OF   BACTERIOLOGY 

This  will  produce  enough  gas  for  disinfection  of  1000  cubic 
feet. 

Solid  formaldehyd  in  the  form  of  candles  is  useful  for  small 
rooms,  and  some  health  boards  employ  it  exclusively. 

Sulphur  dioxid,  or  sulphurous  acid  gas,  is  a  germicide  and 
insecticide,  and  is  much  used  in  disinfecting  ships  after  yellow 
fever  and  malaria.  It  is  obtained  by  burning  sulphur  in  a  pan 
over  water,  and  about  3  pounds  to  1000  cubic  feet  are 
necessary. 

Copper  sulphate,  i  part  to  1,000,000  of  water,  is  effective 
in  destroying  algce,  and  is  useful  in  large  reservoirs  as  a  tem- 
porary disinfectant. 

Alcohol,  iodin,  chlorin,  potassium  permanganate,  hydrogen 
dioxid,  the  salts  of  silver,  lead,  and  zinc,  salicylic  acid,  boric  acid, 
anilin  dyes  {methyl-violet  and  methylene-blue) ,  naphthalin,  and 
creosols  are  a  few  of  the  substances  in  use  as  antiseptics  and 
germicides  in  surgery.  Their  power  varies  with  the  strength 
of  the  solution  and  all  have  limitations. 

In  surgical  operations  more  dependence  is  placed  today 
on  securing  and  maintaining  a  germ-free  or  aseptic  condition 
than  on  the  attempt  to  destroy  germ  life  by  chemicals.  The 
irritation  of  antiseptics  in  some  instances  prevents  the  natural 
body  defenses  (phagocytes)  from  acting,  and  in  abdominal 
operations,  where  no  pus  has  been  encountered,  the  blood- 
serum  is  sufficient  or  normal  salt  solution  is  alone  used. 

Sterilization  of  Hands,  etc, — It  has  been  shown  by  elaborate 
experiments  that  the  skin,  the  hair,  and  clothing  harbor  many 
bacteria,  some  of  a  pathogenic  nature.  The  surgeon  who  is 
anxious  to  secure  good  results  should  carefully  attend  to  his 
toilet;  the  use  of  operating  gowns,  rubber  gloves,  operating 
shoes,  face  guards  is  now  universal.  The  toilet  of  the  hands 
of  the  surgeon  is  as  important  as  that  of  the  field  of  operation, 
but  with  the  use  of  rubber  gloves  the  painstaking  directions  as 
to  the  employment  of  a  half-dozen  or  more  cleansing  agents 
and  germicides  are  no  longer  followed. 

Soap  is  an  efficient  germicide,  the  lye  being  in  most  cases 
powerful  enough  to  prevent  the  growth  of  germs. 


GERMICIDES,   ANTISEPTICS,  AND  ANTISEPSIS  265 

Filtration. — In  the  laboratory,  and  on  a  larger  scale  in  the 
management  of  water-works,  filtration  is  a  method  of  steril- 
ization, acting  as  it  does  by  mechanically  separating  bacteria 
from  a  solution. 

General  Measures  for  Disinfection. — For  discharges — 
urine,  feces,  sputum,  vomitus — solution  of  phenol,  5  per  cent., 
also  fresh  milk  of  lime,  i  part  lime  to  4  parts  water.  Lime 
is  of  value  only  when  sufficient  alkali  present.  Blankets,  woolen 
clothing,  soiled  handkerchiefs,  linen,  boiling  in  steam,  for- 
maldehyd  gas,  or  hot-air  exposure. 

Articles  of  little  value  should  be  burned.  Books  can  be  sub- 
jected to  formaldehyd  vapor  or  immersed  in  gasolene. 

The  hands  and  body  w^ashed  in  strong  soapsuds  and  then 
in  I  :  1000  mercuric  chlorid  solution. 

Tincture  of  iodin,  for  the  skin  and  hairy  parts,  painted 
over  the  field  of  operation,  has  come  into  vogue  as  a  very 
efficient  antiseptic. 

Woodwork  and  floors  should  be  washed  with  soapsuds 
and  I  :  1000  solution  of  mercuric  chlorid,  the  room  itself 
subjected  to  formaldehyd  vapor. 

Testing  the  Value  of  Disinfectants. — Rideal-Walker 
Standard. — For  comparing  one  disinfectant  with  another, 
they  are  compared  with  phenol  solutions  of  known  strength 
in  their  action  on  a  culture  of  some  microorganism  (the 
Bacillus  typhosus  is  now  used  in  most  laboratories).  A 
Standard  temperature  of  20°  C.  has  been  adopted  by  the 
workers  of  the  United  States  Hygienic  Laboratory,  and  some 
changes  have  been  made  by  them  in  the  Rideal-Walker 
method,  so  that  it  is  referred  to  as  the  "Hygienic  Labor- 
atory Phenol  Coefficient.^' 

The  medium  is  made  of  beef-extract,  according  to  the 
American  Health  Association  standard,  and  must  have  a 
reaction  of  +1.5  in  test-tubes  containing  10  c.c.  each  of  the 
medium. 

The  organism  is  a  twenty-four-hour-old  filtered  broth 
culture  of  the  Bacillus  typhosus.  Temperature  of  cultures 
and  dilutions  must  be  brought  up  to  20°  C. 


266 


ESSENTIALS    OF   BACTERIOLOGY 


One- tenth  of  a  cubic  centimeter  of  the  culture  is 
added  to  5  c.c.  of  the  disinfectant  dilution.  The  phenol 
control  is  made  of  different  dilutions,  from  5  to  10  strengths 
being  employed.  The  disinfectant  to  be  tested  is  likewise 
diluted,  depending  on  the  solubility,  etc.  An  accurately 
graduated  pipet  distributes  yV  ^.c.  of  the  culture  to  each 
one  of  the  dilutions,  both  of  the  phenol  control  and  the  test, 
and  the  tubes  are  then  shaken  gently  three  times.  At 
intervals  of  two  and  one-half  minutes  a  platinum  loopful 
(the  loop  4  mm.  in  diameter)  is  transferred  from  each  tube 
and  planted  in  the  tube  of  broth  medium.  The  inoculated 
tubes  are  then  placed  in  an  incubator  at  37°  C.  for  forty- 
eight  hours,  and  at  the  end  of  this  time  results  are  recorded. 

The  coefficient  is  determined  and  recorded  as  in  the  ex- 
ample here  given. 

Example. 

Name  of  disinfectant  to  be  tested,  A. 
Temperature,  20°  C. 
Culture  used.  Bacillus  typhosus,  o.i 
fectant. 


c.c.  to  5  c.c.  disin- 


DlLUTION 


Phenol: 

I  :  80 .  . 

I  :  90.  . 

I  :  100. 

I  :  no. 
Disinfectant 

I  :350- 

I  :375- 

I  :  400 . 

I  :  500. 

I  :  650. 


Time  Exposed  in  Minutes 


+  — 

+      i      + 

+      I      + 


iy2 


15 


The  weakest  disinfectant  dilution  that  kills  within  two  and 
one-half  minutes  (1-375)  is  divided  by  the  weakest  phenol 


GERMICIDES,   ANTISEPTICS,   AND   ANTISEPSIS  267 

dilution  (i:8o),  thus  -^  =  4.69,  and  the  same  is  done  for 
the  strength  that  kills  in  fifteen  minutes,  namely: 

^-  5.9X 

no 

The  average  of  these,  ^^^^-^^^  =  5.30,  is  called  the  co- 
efficient. In  other  words,  disinfectant  A  has  a  value  of 
5.30  times  that  of  phenol.  A  disinfectant  with  a  phenol 
coefl5cient  less  than  i  is  of  very  low  germicidal  value. 


268 


CHIEF   CHARACTERISTICS 


CHIEF  CHARACTERISTICS 
PART  I.— 


This  classification  into  non-pathogenic  and  pathogenic  is  not  strictly  correct,  as 

special 

Name. 

Genus. 

Biology. 

Product 

ACETI. 

Bacillus. 

Short  motile  rods  in 
zooglea;  aerobic. 

Ferment. 

ACIDI  LACTICI. 

Bacillus. 

Short,  immotile  rods; 
aerobic. 

ACIDI  LACTICI. 

Streptococcus. 

Short,  immotile,  oval 
cocci. 

.... 

ACTINOBACTER, 

Bacillus. 

Immotile   rods   with 
capsule;    facul.  an- 
aerob. 

Aerogenes. 

Bacillus. 

Identical     with      B. 
acid  lactici. 

Aerophilus. 

Bacillus. 

Slender  rods  in  threads ; 
immotile;     oval 
spores;    aerobic. 

Agilis. 

Micrococcus. 

Mobile    diplococci 
with  fine  flagella. 

Red  pigment. 

Alba, 

Beggiatoa. 

Cocci  and  spirals  with 
sulphur. 

Alba. 

Sarcina. 

Small  cocci  in  packets 

White  pigment. 

Albicans  amplus. 

Micrococcus. 

Large  cocci  and  dip- 
lococci. 

.... 

Albicans  tardissi- 

Micrococcus. 

Diplococci  colored  by- 

MUS. 

Gram. 

Albicans  tardus. 

Micrococcus. 

Diplococci  not  motile. 

.... 

Allii. 

Bacillus. 

Very  small  rods. 

Alkaloid  pigment. 

Amyliferum. 

Spirillum. 

Rigid     spirilla    with 
spores;     turns  blue 
with  iodin. 

Amylobacter. 

Bacillus. 

See  Butyriaim,  with 

which  it  is  identical. 

Aquatilis. 

Micrococcus. 

Very  small  cocci  in  ir- 
regular groups. 

Arachnoidea. 

Beggiatoa. 

Very  thick  filaments 
containing  sulphur; 
motile. 

Arborescens. 

Bacillus. 

Thin  rods,  with  round- 
ed ends  in  threads, 
andsingly  ;immotile. 

Yellow  pigment. 

Attenuatum. 

Spirillum. 

Threads  with  narrow- 
ed ends. 
Small   cocci   in   pairs 

AURANTIACA. 

Sarcina. 

Orange-yellow    pig- 

and tetrads;  strongly 

ment. 

aerobic. 

AURANTIACUS. 

-Bacillus. 

Motile,    short    thick 

'  Orange-yellow  pig- 

rods, often  in  long 

ment. 

threads. 

i 

OF    THE   PRINCIPAL   BACTERIA 


269 


OF  THE  PRINCIPAL  BACTERIA. 
NON-PATHOGENIC  BACTERIA. 

many  of  the  non-pathogenic  varieties   have  disease-producing  properties  under 
conditions. 


Culture  Characters. 


Not  liquefy;  membran- 
ous growth. 

Not  liquefy;  small  white 
points,  porcelain-like; 
slow. 

Growth  faster  than  above 
appearance  same. 


Liquefy  rapidly;  small 
yellow-gray  colonies. 

Slowly  liquefying,  form- 
ing a  cone  with  rose- 
red  color. 


Slow    growth    in    small 

white  colonies. 
Slowly  liquefy;  gray  col- 
onies;     growth  fairly 

rapid. 
Small  white  points,  not 

Hauefying;    very  slow 

growth. 
Grows  slowly  on  surface, 

the   boundary   raised; 

twice  as  large  as  above. 
Bright-green  pellicle  on 

agar. 


Light-yellow      colonies; 
serrated  edges. 


Colonies,  radiating  fr;ara 
an  oval  center  like 
roots;  later  on  colored 
yellow;  slowly  liquefy. 


Rapidly  liquefy;  little 
orange-yellow  colonies, 
not  growing  in  high 
temperature. 

Slowly  growing;  nail  cul- 
tures; shining  and 
orange-yellow;  not  li- 
quefy. 


Actions. 


Produces  acetic-acid 
fermentation. 

Lactic-acid  fermenta- 
tion;    precipitates 
casein. 

Alcohol  is  formed  af- 
ter the  lactic-acid 
fermentation. 

Causes  fermentation 
with  gas  and  alcohol. 


Is  colored  by  Gram' 
method. 


Decomposes  albumin. 


Habitat. 


Air. 

Air;    sour  milk 

Sour  milk. 

Air. 

Old  cultures. 

Drinking-water. 

Sulphur  springs. 
Air  and  water. 
Vaginal  secretion. 

Urethral  pus. 

Skin  in  eczema. 


Green     slime     of 

onions. 
Water. 


Old  distilled  water. 
Sulphur  water. 


London  Water- 
works. 


Stagnant  water. 
Air  and  water. 


Water. 


Discoverer. 


Kiitzing. 
Pasteur. 

Grotenfeldt. 

Duclaux. 

Miller. 
Liborius. 

Ali-Cohen. 

Vauch. 

Zimmerman. 

Bumm. 

Bumm. 

Unna, 
Tommasoli. 

Griffiths. 

Van  Tiegham. 

Bolton. 
Agardh. 

Frankland. 

Warming. 
Koch. 

Frankland. 


27© 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 


Name. 

Genus. 

Biology.            ,            Product. 

AURANTIACUS. 

Micrococcus. 

Oval  cocci  in  pairs  and   Orange-yellow  Dig- 

singly;    immotile. 

ment  in  water,  al- 
cohol, and  ether; 
insoluble. 

Aurea. 

Sarcina. 

Cocci  in  packets. 

1  Golden-colored      pig- 

'    ment;       soluble    in 

alcohol. 

Aureus. 

Bacillus. 

;  Straight  motile  rods  :  Golden-yellow       pig- 

lying  parallel. 

ment. 

Balticus. 

Bacillus. 

Short  rod. 

Phosphorescence. 

BlENSTOCKII. 

Bacillus. 

See  Putrificiis,  colt. 

BiFIDUS. 

Bacillus. 

Slender  diplococcus, 
pointed  ends,  non- 
motile,  anaerobic. 

BiLLROTHII. 

Micrococcus 

Groups  of  cocci  sur- 

(ascococcus). 

rounded  with  cap- 
sule; zooglea,  aero- 
bic. 

Brunneus. 

Bacillus. 

Motile  rods. 

Brown  pigment. 

Butyric-acid  fer- 

Bacillus. 

Large,  slender  motile 

Diastase. 

mentation. 

rods  in  pairs ;spores; 
facul.  anaerobin. 

BuTYRicuM    (amy- 

Clostridium. 

Thick  motile  rods  en- 

Amyloid  substance. 

lobacter). 

1  a  r  g  i  n  g    for    the 
spores;  obligate 
.    aerobic. 

C^RULEUS. 

Bacillus. 

Rods  in  long  chains. 

Blue  pigment,  not  sol- 
uble in  water,  alco- 
hol, or  acid. 

Candicans 

Micrococcus. 

Masses  of  cocci. 

(candidus). 

Carotarum. 

Bacillus. 

Threads  of  rods  that 
bend  in  various  di- 
rections; oval  spores 


Catenula. 

Bacillus. 

Motile      rods     with 
spores. 

.... 

Caucasicus. 

Bacillus. 

Motile     rods,      with 
spores  in  each  end. 


Cerasinus  siccus. 

Micrococcus. 

Very     small      cocci, 
singly  and  in  pairs; 
aerobic. 

Cherry-red  pigment. 

Cereus  albus. 

Micrococcus. 

Cocci  in  short  chains 
and  bunches.colored 
by  Gram. 

Cereus  flavus. 

Micrococcus. 

Staphylococcus    and 
streptococcus,    and 
in  zooglea,  colored 
by  Gram. 


Chlorinus. 

Bacillus. 

Large    rods,    motile. 

Green    pigment,    sol- 

green-colored,   due 

uble      in      alcohol. 

to  chlorophyll; 

aerobic. 

OF   THE   PRINCIPAL   BACTERIA 
Bacteeia. — (Continued.) 


271 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Round  orange-yellow  col- 

Water. 

Cohn. 

onies,  mostly  on  sur- 

face; slow  growth;  not 

liquefying. 

Liquefy;     bright  golden 


Exudate  of  pneu- 

Mace. 

layer  on  potato. 

monia. 

Slow-growing,     chrome- 

Water  and  skin  of 

Adametz  and 

yellow,    whetstone   in 

eczema. 

Unha. 

shape;    not  liquefy. 

Do  not  liquefy;    require 

Baltic  Sea. 

Fischer. 

glucose  for  growth. 

Oval  colonies  after  three 

Feces    of    infants 

Tissier. 

days  on  glucose  agar. 

breast-fed. 

Creamy  layer  on  surface 

Putrid  broth. 

Cohn. 

of  gelatin. 

Maize. 

Schroter. 

Liquefy  rapidly;  gray  veil 

Casein      precipitates 

Air. 

Hueppe. 

on  surface  of  potato. 

and    changed     into 
butyric    acid;    am- 
monia set  free. 

N3t  cultivated. 

Forms  butyric  acid  in 

Air,    earth,    and 

Prazmowski 

presence    of     lactic 

water. 

and  Van 

acid. 

Tiegham. 

Liquefy;  a  deep-blue  lay- 


Water. 

Smith. 

er  on  potato. 

Not  liquefy;  nail-shaped 

Air  around  old  cul- 

Fliigge. 

in  test-tube. 

tures. 

Rapidly  liquefy  on  sur- 

Cooked carrots 

A.  Koch. 

face,  a  network  center 

and  beets. 

on  potato;  round,  light 

gray;  grow  rapidly. 

Causes     albumin     to- 

Old  cheese. 

Duclaux. 

ferment. 

Ferments  milk,   pro- 

Kefir;   grain. 

Kern. 

ducing     the      kefir 

drink. 

On  potato;  rapidly  form- 

Water. 

List. 

ing   cherry-red    scum, 

not  developed  on  gela- 

tin. 
Not  liquefy;  small,  wax- 

Pus. 

Passet. 

like  drops;   thick  gray 

layer     on     potato; 

growth  rapid. 

Not  liquefy;  dark-yellow 

Pus. 

Passet. 

colonies;   wax-like  ap- 

pearance. 

Liquefy;   greenish-yellow 

Water. 

Engelman. 

colonies. 

272 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Chlorinus. 

Micrococcus. 

Cocci  in  zooglea. 

Green  pigment,  sol- 
uble in  alcohol  and 
water. 

CiNNABAREUS. 

Micrococcus. 

Large  oval  cocci  in 

Brown-red     pigment; 

pairs ;    aerobic. 

foul  odor. 

ClTREUS. 

Bacillus  (asco- 

Straight    and     bent 

Citron  -  yellow       pig- 

coccus). 

rods     in     bundles; 
motile. 

ment. 

ClTREUS. 

Micrococcus. 

Large  round  cocci  in 

Cream-colored  pig- 

chains of  eight  and 

ment. 

more. 

ClTREUS  CONGLOM- 

Micrococcus. 

Diplococci   and    tet- 

ERATUS. 

rads;    aerobic. 

Claviformis. 

Bacillus 

Small    rods;    spores; 

(tyrothrix). 

true  anaerobin. 

Cloac.e. 

See  Proteus. 

CONCENTRICUM. 

Spirillum. 

Thick  motile  spirals 
with      flagella; 
aerobic. 


CORONATUS. 

Micrococcus. 

Cocci  singlyandstrep- 
tococci;    aerobic. 

CORYZ^. 

Micrococcus. 

Large  diplococci  with 
rounded    ends,    the 
contact  surfaces  fiat. 

Crepesculum. 

Micrococcus. 

Round  and  oval  cocci, 
singly  and    in    zo- 
oglea. 

Cyaneus. 

Micrococcus. 

Oval  cells. 

Blue  pigment. 

Cyanogenus  (blue 

Bacillus. 

Motile  rods  in  chains; 

Alkali  and  a  pigment 

milk). 

spores;   aerobic. 

deepened  by  acids. 

DiCHOTOMA. 

Cladothrix. 

Various  forms — rods, 
spirals,  and  cocci,  in 
long  threads. 

DiFFLUENS. 

Micrococcus. 

Oval  cocci;   aerobic. 

Fluorescent  pigment, 
soluble  in  water. 

DiSTORTUS. 

Bacillus 

Motile  rods;    spores; 

Alkali. 

(tyrothrix) . 

aerobic. 

Dysodes. 

Bacillus. 

Long  and  short  rods; 

An    odor    resembling 

spores. 

peppermint  and 
turpentine. 

Endoparagogicum. 

Spirillum. 

Dry    motile    spirals, 
joined    in    peculiar 
shapes. 

Erythrosporus. 

Bacillus. 

Motile      rods      and 

Greenish-yellow  pig- 

threads;    spores, 

ment. 

slender. 

FiGURANS 

Bacillus. 

Large    motile    rods; 

(mycoides). 

spores ;long  threads; 
aerobic. 

FiLIFORMIS. 

Bacillus 

Short     motile     rods; 

(tyrothrix). 

spores  in  one  end. 

or   THE   PRINCIPAL  BACTERIA 


273 


Bacteria. — {Continued.) 


Culture  Characters. 


Actions. 


Yellow-green  layer  on 
gelatin. 

Not  liquefy;  slow  growth; 
bright-red  points. 

Slow  growth;  after  two 
weeks  small  yellow 
points  which  take  va- 
rious shapes  on  potato; 
citron-yellow  layer; 
growth  more  rapid. 

Dirty,  cream-colored  col- 
onies, which  are  raised 
and  moist. 

Lemon-yellow  colonies. 


Not  liquefying;  concen- 
trically disposed  colon- 
ies; very  slow  growth; 
not  growing  on  potato. 

A  halo  formed  around 
the  colonies. 

White,  raised  glassy  col- 
onies, at  first  like  pneu- 
mococci,  later  culture 
flattened;  not  lique- 
fying. 


Bluish-green  colonies. 
Not    liquefying;      small 

white  colonies. 
Cultivated  in  infusion  of 

plants. 

Do  not  liquefy;  small 
granular,  yellow  col- 
onies; green  fluores- 
cence. 


Does  not  liquefy;  green 
fluorescence;  white  col- 
onies. 

Liquefying;  root-like  pro- 
cesses extending  in  the 
gelatin;  feather  form  in 
test-tube. 


Ferments  milk,  giving 
rise  to  alcohol. 


No  pathogenic  action. 


Changes  milk  to  deep- 
blue  color. 


Milk  made  viscid  and 
casein  precipitated. 


Causes  casein  to  be 
precipitated  from 
milk. 


Habitat. 


Boiled  eggs. 

Air  and  water. 
Skin  in  eczema. 


Water. 


Dust  and  blennor- 
rhagic  pus. 

Fermenting  albu- 
min. 

Putrefying  blood. 


Air. 

Acute  coryzal 
cretion. 


Putrefying     infu- 
sions. 

Cooked  potatoes. 
Air   of  certain 

countries. 
Water. 


Air. 


Air. 

Bread  and  yeast. 


Trunk  of  worm- 
eaten  tree. 

Air  and  putrefying 
substances. 

Garden-earth. 


Discoverer. 


Cohn. 


Flugge. 


Unna  and 
Tommasoli. 


List. 

Bumm. 
Duclaux. 

Kitasato. 

Flugge. 
Hajek. 

Cohn. 

Cohn. 
Fuchs. 

Cohn. 
Schroter. 

Duclaux. 
Zopf. 

Sorokin. 

Cohn. 

FlUgge. 

Duclaux. 


18 


274 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 


Name. 

GEIfUS. 

FiSCHERI. 

Bacillus. 

FiTZIANUS. 

Bacillus. 

Flava. 

Sarcina. 

Flavus. 

Bacillus. 

Flavus  desidens. 

Streptococcus. 

Flavus    liquefa- 

CIENS. 

Flavus      tardi- 

gradus. 
Fluorescens   f<e- 

TIpUS. 


Fluorescens 
liquefaciens. 

Fluorescens  niva- 
lis. 


Micrococcus. 
Micrococcus. 
Micrococcus. 


Bacillus. 
Bacillus. 


Fluorescens    pu-    Bacillus. 

TRIDUS. 


Fcetidum. 


Fcetidus. 
Fuscescens. 

FULVUS. 

FUSCUS  LIMBATUS. 


FUSIFORME. 

GlTNICULATUS. 

GiGANTEUS     URE- 
THRA. 

Grass.     See  Tim- 

othy. 
Graveolens. 


H/EMATODES. 


Cladothrix. 
Clostridium. 


Micrococcus. 
Sarcina. 
Micrococcus. 
Bacillus. 


Bacillus. 

Bacillus 

(tyrothrix). 
Micrococcus. 


Bacillus. 


Micrococcus. 


Biology. 


Product. 


Short  rods  in  threads; 
spores  as  large  as 
the  rods. 

Small  cocci  in  pack- 
ets. 

Small  rods;  immotile. 


Phosphorescence. 

Pigment. 
Pigment. 


Cocci  and  diplococci    Yellow-brown   pig- 
in  chains;   aerobic,    j     ment. 

Cocci  and  diplococci     Pigment, 
in  zooglea. 

Cocci  in  short  chains,     Chrome-yellow      pig- 


and  diplococci. 
Small  diplococci. 


Short  motile  rods; 

very  thin. 
Short  rods;    motile. 


Motile  rods;     short, 
with  rounded  ends. 


Threads  twisted  in 
spirals;  very  irreg- 
ular. 

Rods  of  varying 
length;  very  motile; 
a  large  spore  in  one 
end ;    anaerobic. 

See  Crepesculum,  with 

Round  cocci. 

Short  rods;  very  mo- 
tile; facultatively 
anaerobic. 

Spindle-shaped,  with 
pointed  ends. 

Rods  variable  length; 
spores. 

Streptococci  in  thick 
knots. 


Small  rods,  nearly  as 
broad  as  they  are 
long. 


ment. 
Blue-green  pigment: 
acids  turn  red. 


Green     fluorescent 

pigment. 
Blue-green  pigment. 


Green     fluorescent 
pigment. 


Strong      gas-produc- 
tion; very  foul  odor. 


which  it  is  identical. 
Brown  pigment. 


A  bitter  substance. 


Foul  gas. 


Cocci  in  little  zooglea.    Red  pigment. 


or   THE   PRINCIPAL   BACTERIA 
Bacteria. — {Continued.) 


275 


Culture  Characters. 

Actions, 

Habitat. 

Discoverer. 

Not  liquefying;  requires 

Beyerinck. 

peptone  for  growth. 

Transparent  on  surface; 

Produces  ethylic  alco- 

Unboiled hay-in- 

Zopf. 

dark  center  in  t.ie  deei) ; 

hol  in  meat  extract. 

fusion. 

not  liquefying. 

Liquefying. 

Vomited  matter. 

de  Bary. 

Liquefying;  yellow  viscid 

Drinking-water. 

Mace. 

colonies;   foul  odor. 

Yellow     porcelain-white 

Air  and  old   cul- 

Flugge. 

colonies. 

tures;   water. 

Liquefying  rapidly;  yel- 

Air and  old   cul- 

Flugge. 

low  colonies. 

tures;   water. 

Softens  gelatin;     yellow 

Air. 

Flugge. 

beads,  isolated. 

Little  button-like  colon- 

Post-nasal space. 

Klamann. 

ies  that  later  on  sink 

in,  surrounded  by  vio- 

let-green color;    lique- 

fying; growth  rapid. 

Liquefying;  white,  sunk- 

.... 

Water  and  air; 

Flugge. 

en,  iridescent  colonies. 

conjunctival  sac. 

Quickly  liquefying; 

Colors  the  glacial  wa- 

In snow  and  ice  of 

Schmolck. 

growth  rapid;      small 

ters  green. 

Norway. 

white  points;  later  on, 

surrounded    by    blue- 

green  fluorescence. 

Not  liquefying;  transpar- 

All putrefactions. 

Flugge. 

ent  at  first,  then  green 

fluorescence  and  urin- 

ary odor. 


Lacrimal  canal. 

Cohn. 

Liquefying;  growth  rapid; 

Old  cheese  and  se- 

Liborius. 

small  colonies  that  soon 

rum  of  mice  in- 

become filled  up  with 

oculated       with 

fluid    and    assume    a 

garden-earth. 

spherical  form. 

Conic  rusty-red  colonies. 

Excrement  of  horse. 

Cohn. 

Small     brown     colonies, 

In  foul  eggs. 

Scheiben- 

along  needle-track  little 

zuber. 

branches;  not  liquefy. 

Spongy   layer  on 

Warming. 

sea-water. 

Air  and  milk. 

Duclaux. 

No  growth  on  gelatin;  on 

Normal  urine  and 

Lustgarten. 

agar,  thin  drops;  nearly 

urethra. 

transparent ;  very  slow 

growth;  in  bouillon,  a 

flaky  precipitate. 
Liquefying;    irregular 

Skin  between  toes. 

Bordoni- 

grayish,    later    green- 

Uffreduzzi. 

ish,  colonies,  with  very 

foul  odor. 

Grows  best  on  white  of 

Sweat  of  man. 

Zopf. 

egg  at  37°  C;  red  layer. 

276 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 

/ 


Name. 

Genus. 

Biology. 

Product. 

Hansenii. 

Bacillus. 

Medium  large  rods. 

Yellow  pigment;  in- 
soluble. 

Hay.     See  SubtiUs. 

Hoffman's.       See 

Pseudodiphtheri 

a. 

Hyacinthi. 

Bacillus. 

Short  rods  in  dumb- 
bell shapes. 

.... 

Hyalina. 

Sarcina. 

Round  cocci  in  groups 
of  4  to  34. 

Ianthinus. 

Bacillus. 

See  Bacillus  violaceus. 

Indicus. 

Bacillus. 

Short,  motile  rods;  no 

Scarlet     pigment    al- 

spores;     anaerobin 
facul. 
Very  regular  packets 

tered  by  heat. 

Intestinalis. 

Sarcina. 

of  cocci,  eight  in  each. 

Jequirity. 

Bacillus. 

Medium-sized    rods; 

Ferment   called 

spores. 

abrin. 

KUHNIANA. 

Crenothrix. 

Long  threads,  break- 
ing  up    into   cocci. 
Theyareensheathed. 

Lacteus  favifor- 

Micrococcus. 

Diplococci;  not  decol- 

MIS. 

orized  by  Gram. 

Lactis  erythrog- 

Bacillus. 

Short  immotile  rods; 

Yellow   pigment   and 

enes. 

round  ends. 

red  pigment. 

Leptomitiformis. 

Beggiatoa 

Filaments      medium 
size. 

Leucomel^num. 

Spirillum. 

Two  or  three  spirals; 
dark  granular  con- 
tents;   clear  spaces 
between. 

LiNEOLA. 

Bacillus. 

Short  motile  rods  in 
zooglea,  with  flagel- 
la. 

Short    motile     rods; 

LlODERMOS. 

Bacillus. 

rounded  ends. 

LiTORALIS. 

Merismopedia. 

Cocci    in    groups    of 
fours,   containing 
sulphur. 

LiTOREUS. 

Bacillus. 

Oval  rods,  never  in 
chains  or  zooglea. 

LiVIDUS. 

Bacillus. 

Medium-sized    rods; 

Deep    blue-black 

motile. 

pigment. 

LUTEA. 

Sarcina. 

Cocci  singly  and  in 

Pigment      citron-yel- 

fours. 

low. 

LUTEUS. 

Bacillus. 

Short  immotile  rods. 

Pigment;     soluble  in 

with      large    .  oval 

water;    acids  inten- 

spores. 

sify. 

LUTEUS. 

Micrococcus. 

Oval  cocci. 

Pigment,  not  acted 
upon  by  acid  or  al- 
kali. 

OF   THE   PRINCIPAL   BACTERIA 
Bacteria. — (Continued.) 


277 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

On     potato,     a     yellow 
growth  which  changes 
with  age. 

Yellow     skin     of 
nutrient       infu- 
sions. 

Rasmussen. 

•••• 

Slime  of  diseased 
hyacinth-bulbs. 

Wakker.    ' 

Marshes. 

Kutzing. 

Liquefying;  oval  colon- 
ies;   scarlet-colored. 

Intestine  of  mon- 
key. 

Koch. 

.... 

Intestine  of  fowls. 

Zopf. 

Colonies    brick  -  colored 
from  oxid  of  iron. 

Ferment  causes  oph- 
thalmia. 

Infusion  of  jequir- 

ity  bean. 
Drinking-water  of 

wells. 

Sattler. 
Rabenhorst. 

Not  liquefying;  white  col- 
onies; grow  well  on  po- 

.... 

Mucus  of  vagina 
and  uterus. 

Bumm. 

tato. 
Small,  round  yellow  dots, 
later    on    cup-shaped, 
with   rose-colored  pe- 
riphery; liquefying. 

In  red   milk  and 
feces. 

Sulphur  waters. 

Grotenfeldt. 
Tr^visan. 

Water  over  rot- 
ting plants. 

Perty. 

Slimy  layer  on  potatoes. 

Stagnant  water. 

Mullet. 

Liquefying;   transparent, 
then  thick  layer  on  po- 
tato;  like  gum. 

Air  and  potatoes. 
Sea-water. 

Fliigge. 

Oersted  and 
Rabenhorst. 

Sea-water. 

Warming. 

Ink-spot  at  first,  slowly 
liquefying;  blue- violet 
colored  later  on;   slow 

Berlin  Water- 
works. 

Flugge  and 
Proskauer. 

growth. 

Not  liquefying;  little  ele- 
vations; citron-yellow 
center;    yellow    layer 
on  potato. 

Not  liquefying;     irregu- 
lar in  form;     golden- 
yellow  colored. 

Do    not    liquefy;    small 
citron-yellow  colonies 
on  potato. 

.... 

Air. 

Air. 
Air. 

Schroter. 

Fliigge. 
SchrSter. 

278 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 


Name. 


LUTEUS. 


Maidis. 


Marsh. 

Megaterium. 

Melanosporus. 


Merismopedi- 

OIDES. 


M  esentericus  fus 
cus  (potato). 


Mesentericus 

VULGATUS  (potato) 


Mesenteroides. 
Miller's. 

MiNUTA. 

Mir.\rii.is. 

Multipediculosus 
Mycoides.      See 
Nasalis. 

Navicula. 

NiTRIFICANS. 

Nivea. 
nodosus  parvus. 


Genus. 


Micrococcus. 

Bacillus. 

Spirillum. 

Bacillus. 

Bacillus. 

Bacillus. 

Bacillus. 
Bacillus. 

Leukonostoc. 

Bacillus. 
Sarcina. 
Beggiatoa. 

Bacillus. 

Rasmosus. 

Micrococcus. 

Bacillus. 
Micrococcus. 
Beggiatoa. 
Bacillus. 


Biology. 


Diplococci  very  mo- 
tile. 


Rods  with  pointed 
ends,  very  motile; 
seldom  in  threads; 
oval  spores. 

See  Plicatile. 

Large    motile    rods; 

spores;    aerobic. 
Rods;   aerobic. 


Threads  of  rods  which 
are  formed  from 
cocci-like  spores; 
zooglea  in  packets. 

Small  motile  rods 
with  spores. 

Thick  motile  rods  in 
threads;   spores. 


Masses  of  cartilagin- 
ous zooglea,  com- 
posed of  rods  and 
cocci;  arthrospores. 

Delicate  rods,  slightly 

curved;  immotile. 
Cube-shaped  packets 

Very    wide    threads, 
rounded   ends   and 
curled;      sulphur 
granules. 

Long,  slender  rods. 


Diplococci,  motile; 
also  streptococci. 

Spindle-shaped  rods. 

Small  cocci. 

Very  thin  filaments. 

Rods  formed  at  an- 
gles;  immotile. 


Product. 


Yellow  pigment,  turn- 
ing brown-red. 


Black  pigment,  not 
acted  upon  by  acids 
or  alkalis. 


Diastase. 


Amyloid  material. 
Forms  saltpeter. 


OF   THE   PRINCIPAL   BACTERIA 
Bacteria. — (Continued.) 


279 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Round,  light-yellow  col- 

Water. 

Adametz. 

onies,   growing   larger 

in  a  few  days;  on  pota- 

to   a    slimy    covering 

with      moldy      odor; 

slowly  liquefying. 

Gray  points  in  deep,  veil- 

In solutions  of  sugar 

In  maize  and  in 

Paltauf  and 

like  on  surface;  lique- 

an aldehyd   is  pro- 

pellegra;   feces. 

Heider. 

fying;    on    potato,    a 

duced. 

wrinkled    skin     of 

brownish  color. 

Yellow  irregular  masses; 

Cooked  cabbage. 

De  Bary. 

thick  layer  on  potato. 

First  gray,   then  black. 

Air  and  potatoes. 

Eidam. 

pellicle. 

.... 

Stagnant  water. 

Zopf. 

Liquefying;    white    colo- 

Potato. 

Flugge. 

nies,  ray-like  periphery 

brown  layer  on  potato. 

Yellow  colonies,  dark  cen- 

Coagulates milk  and 

Air  and  old  pota- 

Flugge. 

ter,  ciliary  processes  at 

forms  diastase  out  of 

toes. 

periphery;  brown  layer 

starch. 

on  potato,  penetrating 

the  substance. 

Converts  molasses  in- 

Beet-root juice. 

Cienkowski, 

to  a  gelatinous  mass. 

Liquefies;     not  growing 

Caries  of  teeth. 

Miller. 

on  the  surface. 

Grows  slowly;   reacts  to 

Sour  milk. 

De  Bary. 

iodin,  turning  blue. 

Sea-water. 

Cohn. 

Insect-shaped  colonies. 

Potatoes. 

■ 
Flugge. 

Grayish    points,    raised. 

Nasal  space  and 

Hack. 

opaque;  rapid  growth; 

secretion. 

not  liquefying. 


Potatoes. 

Reinke  and 
Berthold. 


Soil. 

VanTiegham 

White  flakes. 


Sulphur  waters. 

Rabenhorst. 

Slow  growth  at  37°  C; 

Urethral  secretion. 

Lustgarten. 

in  agar  a  white  line. 

which    in    the    center 

becomes  poroiis. 

28o 


CHIEF  CHARACTERISTICS 


Non-Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Oblongus. 

Micrococcus. 

Motile   cocci,    singly 
and    in    filaments; 
aerobic. 

.... 

OCHROLEUCUS, 

Micrococcus. 

Cocci    in    pairs    and 
packets;    spores. 

Yellow  pigment. 

Paludosa. 

Sarcina. 

Spheric,  transparent, 
colorless  cocci. 

Pasteurianus. 

Bacillus. 

Differs  from  Bacillus 
aceti    in    that    the 
cells     contain     an 
amyloid  matter. 

Pflugeri. 

Bacillus. 

Short  rods  in  threads. 

Phosphorescence. 

Phosphorescens 

Bacillus. 

Motile;  round,  short 

Phosphorescence. 

GELIDUS. 

rods;    aerobic. 

Phosphorescens 

Bacillus. 

Large  motile  rods. 

Phosphorescence. 

INDICUS. 

Phosphorescens, 

Bacillus. 

Motile  rods. 

Phosphorescence. 

North  Sea. 

Photometricus. 

Bacillus. 

Motile,     red-colored 

Sulphur  and  red  pig- 

rods. 

ment  caused  by 
light. 

Plicatile. 

Spirillum. 

Long     motile,     thin 
spirals;  round  ends. 

POLYMYXA. 

Clostridium. 

Motile  rods  in  threads 

Amyloid       colored 

with  spores. 

blue  by  iodin. 

PRODIGIOSUS. 

Bacillus. 

Short     motile    rods; 

Red      pigment,      sol- 

aerobic. 

uble  in  alcohol 
trimethylamin. 

Proteus  mirabilis. 

Bacillus. 

Very    motile,    short 
rods;    aerobic. 

Proteus  vulgaris. 

Bacillus. 

Rods    sometimes 
curved  as  spirillum. 

.... 

Proteus  Zenkeri. 

Bacillus. 

Motile  rods. 


PSEUDO-DIPHTHE- 

Bacillus. 

Small  rods,  similar  to 

RLE(Hoflfnjan). 

the    true    bacillus; 
immotile. 

PUTRIFICUS  COLI. 

Bacillus. 

Slender  motile  rods; 
long  threads;  spores. 

Pyogenes  tenuis. 

Micrococcus. 

Radiatus. 

Bacillus. 

Motile     rods     with 
rounded  ends;    an- 
aerobic oval  spores. 

Strong-smelling  gas. 

or   THE   PRINCIPAL  BACTERIA 


281 


BACTEmA.^iContinued.) 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Grows  best  in  cultures  to 

Causes   gluconic  fer- 

Beer. 

Boutroux. 

which  glucose  and  am- 

mentation. 

monium  tartrate  have 

been  added. 

Liquefying;  slow  growth; 

Urine. 

Prove. 

thin  yellow  membrane; 

sulphurous  odor. 

.... 

Water  from  sugars 

Schroter. 

factory. 


Heavy  beers. 

Hansen. 

Not  liquefying;  requires 

Putrid  meat  and 

Ludwig. 

glucose;  grows  well  on 

fish. 

potato. 

Not  liquefying;      grows 

.... 

Salt  fish. 

Forster. 

best  with  glucose  and 

salt. 

Liquefying;    grows  best 

Tropical  seas. 

Fischer. 

at  30°  C. 

Liquefying;  colonies  look 

Water    around 

Fischer. 

as    if    punched    out; 

Kiel. 

grows  best  at  15°  C. 

Movements  depend  upon 

.... 

Engelman. 

light. 

Stagnant  water. 

Ehrenberg. 

Thick  skin  on  potato. 

Causes  fermentation  in 
dextrin  solutions. 

Prazmowski. 

Little  red  colonies;  lique- 

Bread  and   pota- 

Ehrenberg. 

fying    rapidly;     espe- 

toes. 

cially    abundant      on 
potatoes. 
Liquefying  slowly; 

Putrefaction. 

Hauser. 

opaque  center,  irregu- 

lar processes. 

Liquefying  quickly. 

Putrefaction. 

Hauser. 

Not    liquefying;      thick 

Putrefaction. 

Hau.ser. 

white  layer  on  potato. 

Grows  at  ordinary  tem- 

Not virulent. 

In  diphtheric 

Wellenhof. 

perature, rapidly  form- 

membrane   and 

(Hoffman.) 

ing  on  surface  a  brown- 

normal pharynx. 

ish  growth;    pin-head 

colonies  raised   above 

surface;  not  liquefying. 

Decomposes  albumin. 

Human  feces. 

Bienstock. 

On  agar,  a  glassy  growth. 

Closed  abscesses. 

Rosenbach. 

Liquefying;  growth  rap- 

Not pathogenic. 

In  serum  of  white 

Luderitz. 

id  ;  colonies  like  molds, 

mice  inoculated 

from  center  radiating 

with  earth. 

in   all   directions   and 

through    the    gelatin; 

the   air   must   be  ex- 

cluded. 

282 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Radiatus. 

Streptococcus. 

Small  cocci  in  chains. 

Ramosus  liquefa- 

Bacillus. 

Motile  rods. 

CIENS. 

Reitenbachii. 

Merismopedia. 

Cocci  in  packets  or 
plates;  colorless  cell- 
w  a  1 1       containing 
chlorophyll. 

RosACEtrs. 

Micrococcus. 

Large  cocci  in  pairs 
and  tetrads. 

Red  pigment. 

Rosea. 

Sarcina. 

Spheric  cocci  in  cubi- 
cal packets. 

.... 

Rosea  perseina. 

Beggiatoa. 

Long  rods  with  cocci- 

Pigment    called    bac- 

shaped     bodies     in 

teriopurpurin. 

them,      containing 

sulphur  and  a  red 

pigment. 

ROSEUM. 

Spirillum. 

Very    short    curved 

Pigment  soluble  in  al- 

rods;     motile   and 

cohol. 

spores. 

Ruber. 

Bacillus. 

Motile  rods  in  groups. 

Brick-red  pigment. 

Rub  RUM. 

Spirillum. 

Motile;  short  spirilla; 
aerobic. 

Pale- rose  pigment. 

RUFUM. 

Spirillum. 

Long  motile  spirals. 

Red-rose  pigment. 

RUGULA. 

Spirillum 

Motile  rods,  in  long 

(vibrio) . 

spirals,   singly  and 
in  chains,  with  flag- 
ellaand  spores;  an- 
aerobic. 

Saprogenes. 

Bacillus. 

Large  rods,  terminal 
spores ;  facultatively 
anaerobic. 

Scaber. 

Bacillus 

Short  motile  rods  in 

Tyrosin     and     leucin 

(tyrothrix) . 

chains;      spores; 
aerobic. 

are  formed. 

Scheurlen's. 

Bacillus. 

Short    motile    rods; 
spores. 

Septicus. 

Bacillus. 

Non-motile    rods    in 
threads  and  spores; 
anaerobic. 

.... 

Serpens. 

Spirillum. 

Long,  lively  threads, 
with  three  windings. 

SiMILIS. 

Bacillus. 

Immotile  rods;  trans- 
parent spores. 

Spinosus. 

Bacillus. 

Large    motile    rods; 
spores;  true  anaero- 
bin. 

SUBFLAVUS. 

Micrococcus. 

Diplococci  colored  by 
Gram's  fluid. 

SUBTILIFORMIS. 

Bacillus. 

Immotile      rods      in 

threads;  transparent 

• 

spores. 

OF   THE   PRINCIPAL  BACTERIA 


283 


Bacteria. — (Continued.) 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Liquefying;  white  colon- 

Air. 

Flugge. 

ies  with  greenish  tinge; 

funnel-shaped  in  test- 

tube. 

Liquefying;     concentric 

Air. 

Flugge. 

colonies;  funnel-shaped 

in  test-tube. 

Caspary. 

Not  liquefying;  small  red 

Air. 

Flugge. 

knobs,  with  fecal  odor. 

Marshes. 

Schroter. 

.... 

Marshes. 

Zopf. 

Not   liquefying;      thick 

Blennorrhagic  pus. 

Mace. 

violet  colonies;     deep 

red  on  potato. 

Boiled  ri&e. 

Frank. 

Not  liquefying;      grows 

Dead  mice. 

Esmarch. 

slowly;   pale-rose  col- 

onies. 

Stagnant  water. 

Perty. 

Liquefying  rapidly ;  round 

Causes    cellulose    to 

Vegetable      infu- 

MiiUer. 

yellow  dots  with  zone; 

ferment. 

sions  and  tartar 

fecal  odor. 

of  teeth. 

Grows  slowly;  foul  odor. 

.... 

Putrefaction. 

Rosenbach. 
Duclaux. 

Growth  best  at  39°  C; 

In  carcinomatous 

Scheurlen. 

slowly    liquefying    on 

and      normal 

potato;         a     yellow 

mamma. 

wrinkled  skin,  under- 

neath whicha  red  color. 

Putrid  blood. 

Klein. 

.... 

Stagnant  water. 

Miiller. 

Grows  rapidly. 

Human  feces. 

Bienstock. 

Liquefying;  spiny  periph- 

Albuminous    decom- 

Garden-earth. 

Luderitz. 

ery;   foul  odor  due  to 

position. 

methylmercaptan. 

Liquefying;  yellow  dots. 

.... 

Vaginal  secretion 
and  lochial  dis- 
cbarges. 

Bumm. 

Grows  best  at  37°  C. 


Human  feces. 

Bienstock. 

284 


CHIEF  CHARACTERISTICS 


Non-Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

SuBTiLis   (hay  ba- 
cillus). 

Bacillus. 

Large    motile    rods, 
three  times  longer 
than      broad,       in 
threads,  with  flag- 
ella      and     spores; 
aerobic. 

Syncyaneus. 

Bacillus. 

Same  as  Cyanogenus. 

Synxanthus   (yel- 
low milk). 

Bacillus. 

Short,     thin     motile 
rods. 

Yellow  pigment,  solu- 
ble in  water;  simi- 
lar to  anilin  colors. 

Tenue. 
Tenuis. 

Spirillum. 

Bacillus 
(tyrothrix). 

Large  motile  spirals 

with  flagella. 
Motile  rods  in  long 

chains;   spores. 

.... 

Termo. 

Bacillus. 

Short    motile,   cocci- 
like  rods  in  zooglea. 

TUMESCENS. 

Bacillus. 

Shortrodswith  spores. 

TuRcrous. 
Ulna. 

Undula. 

URE.E. 

Bacillus 
(tyrothrix). 

Bacillus. 

Spirillum. 
Bacillus. 

Short  immotile  rods 

in      long      chains; 

spores;  aerobic. 
Very    large    rods    in 

chains  and   singly; 

not     very     motile; 

large  spores. 
Long  motile  spirals, 

with  flagella. 
Short  rods;     spores; 

aerobic. 

Carbonate  of  ammo- 
nium. 

Ferment,       propyla- 
min. 

Urin^. 
Urocephalus. 

Sarcina. 

Bacillus 
(tyrothrix). 

Small  cocci  in  fami- 
lies. 

Cylindric  motile  rods 
with  spores;    anae- 
robic. 

Cubic  packets   of   8 
to  64  cocci. 

Rods  motile,  often  in 
bundles  of  four. 

.... 

Ventricula. 
Ventriculi. 

Sarcina. 
Bacillus. 

.... 

Versicolor. 

Micrococcus. 

Small  cocci. 

.... 

ViOLACEUS.      ■ 

Bacillus. 

Motile    rods,    round 
end;   spores. 

Violet  pigment,  sol- 
uble in  alcohol. 

Violaceus. 

Bacillus. 

Immotile  rods,  form- 
ing large  spores. 

Violet  pigment,  like 
anilin. 

ViRENS. 

Bacillus. 

Straight  rods;-spores; 
immotile;         green 
tinged. 

Supposed  to  contain 
chlorophyll. 

OF  THE   PRINCIPAL  BACTERIA 
Bacteria. — (Continued.) 


285 


Culture  Characters. 


Liquefying;  gray  center, 
wreath  -  like  border; 
thick  layer  on  potato. 


In  boiled  milk  a  yellow 
pigment  is  formed. 


Liquefying;  opaque  cen- 
ter, yellow  layer  next, 
and  the  periphery 
lobed ;  funnel-shaped 
in  test-tube. 


On  boiled  carrots  a  wrin- 
kled, gelatinous  disk. 

A  pellicle  formed  on  sur- 
face of  milk;  a  heavy 
precipitate  beneath. 

On  boiled  egg  little  zoog- 
lea. 


Resembling  a  globule  of 
fat;  grows  well  in  mu- 
cous urine. 


Not  liquefying. 

Round  colonies  with  dark 
center;  slow  growth; 
not  liquefying. 

Not  liquefying;  irides- 
cent yellow  surface. 

Not  liquefying;  center 
deep  violet;  color  re- 
mains on  agar  a  long 
time. 

Liquefying;  transparent 
colonies,  surrounded 
by  violet  zone. 


Actions. 


Precipitates  casein; 
forms  a  pellicle  on 
milk. 


Splits  urea  into  ara- 
monii  carbonas. 


Peptonizes  albumin. 


Habitat. 


Soil  and  dust,  hay, 
etc. 


Boiled  milk  and 
potatoes. 

Stagnant  water. 

Fermenting  cheese 
and  milk. 

Connected  with 
putrefaction  of 
plants. 


Boiled  carrots. 

Fermenting    milk 
and  cheese. 

Putrefying  water 
and  boiled  eggs. 


Discoverer. 


Vegetable 

sions. 
Stale  urine. 


infu- 


Bladder. 
Fermenting  milk 


Contents  of  stom- 
ach. 

Stomach  of  dogs 
fed  on  meat. 


Air. 
Water. 


Boiled  potato  and 
water. 

Stagnant  water. 


Ehrenberg. 


Ehrenberg. 

Ehrenberg. 
Duclaux. 

Dujardin. 


Zopf. 
Duclaux. 

Cohn. 

Mailer. 
Miquel. 

Welcker. 
Duclaux. 

Goodsir. 
Raczynssky. 

Flugge. 
Zopf. 

Schrotcr. 
VanTiegham 


286 


CHIEF   CHARACTERISTICS 


Non-Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

ViRESCENS. 

Bacillus. 

Short      motile     rods 

Deep-green    pigment. 

with    flagella    very 

turning           yellow- 

broad. 

brown. 

ViRGULA. 

Bacillus 

Slender         im  motile 

(tyrothrix) . 

rods;  spores  aerobic. 

ViRIDIS. 

Bacillus. 

Little  immotile  rods; 
oval  spore,  which  is 
tinged  green. 

Viscosus. 

Bacillus. 

Motile  rods,  rounded 
ends,      usually     in 
pairs. 

Green  pigment. 

Viscosus. 

Micrococcus. 

Streptococci  of  glob- 

Gummy       substance 

ular  cells. 

called    viscosa,    and 
ferment. 

VlTICULOSUS. 

Micrococcus. 

Oval  cocci    in  large 
groups. 

VOLUTANS. 

Spirillum. 

Long    spirals      with 
flagella. 

.... 

ZOPFII. 

Bacillus. 

Long     motile     rods, 
breaking     up     into 
spores  like  cocci. 

PART  II.— 


Name. 


Aerogenes  Capsu- 

LATUS. 


Alkaligenes. 
Alvei. 


Amylovorus. 


Anthracis    Symp- 
tomatici. 


Anthracis. 


Articulorum 
(diphtheriticus). 


Genus. 


Bacillus. 

Bacillus. 
Bacillus. 

Micrococcus. 
Bacillus. 

Bacillus. 


Micrococcus. 


Biology. 


Usuallyfound  in  pairs, 
resembling  diplo- 
cocci,  capsulated; 
obligate    anaerobe. 

Rods  like  colon  and 
typhoid,  motile. 

Rods  with  large 
spores. 


Oval  cells,  never  in 
chains. 

Large  slender  rods 
with  swellings  at 
spore ;    anaerobic. 

Straight  rods,  slightly 
concave  ends;  im- 
motile ;  aerobic; 
spores. 


Oval  cocci  in  long 
chains,  identical 
with  pyogenes. 


Product. 


Gas    with    character- 
istic odor. 


Produces     alkali     in 
mannite  and  milk. 


Forms  butyric  acid. 
Rancid  odor. 


Toxalbumin. 


OF   THE   PRINCIPAL   BACTERIA 
Bacteria. — {Concluded.) 


287 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Deep  round  colonies,  the 

Green  sputum. 

Frick. 

vicinity  colored  green; 

grows  on  surface;  slow 

growth;  not  liquefying. 

Milk. 

Duclaux. 

.... 

Water. 

VanTiegham. 

Rapid  growth,  liquefying ; 

Water  and  earth. 

Frankland. 

small     hair-like     pro- 

cesses  from    colonies; 

later  on,  viscid  and  in 

threads,     with     green 

fluorescence. 

Mucoid  fermentation 

Beer  and  wine. 

Pasteur. 

in  wine  and  beer. 

Not  liquefying;     a  fine 

Air. 

Fliigge. 

network  in  the  colony; 

mucoidlayer  on  potato. 

Marshes. 

Ehrenberg. 

Not  liquefying;      forms 

Intestinal      c  0  n- 

Kurth. 

thick  coils  like  braided 

tents  of  fowls. 

hair. 

PATHOGENIC  BACTERIA. 


Culture  Characters 


Acid  reaction  in  litmus 
milk;  coagulates  casein 
with  cavity-formation 
due  to  gas. 

Colonies  like  typhoid. 

Liquefying;  growths  ra- 
diating from  center 
downward;  on  potato 
a  dry  yellow  layer. 


Liquefy  gelatin;  grow 
only  in  atmosphere  of 
hydrogen. 

Liquefying;  granular  col- 
onies with  irregular 
border;  on  potato  a 
dry,  creamy  layer;  in 
test-tube  a  thorny, 
prickly  track. 

Grows  well  on  gelatin; 
pale-gray  colonies;  not 
liquefying;  slow 
growth  on  potato. 


Actions. 


Causes  fermentation; 
can  produce  gas 
from  proteid  alone. 

Feces  and  water. 

Produces  a  disease  in 
bees  called  "foul 
brood." 

"Fire-blight"  in  pear 

trees. 
Causes  quarter  evil  in 

animals. 

Causes  splenic  fever  in 
animals;  malignant 
pustule  in  man. 


Fatal  in  mice  and  rab- 
bits. 


Habitat. 


Discoverer. 


Intestinal  con-  |  Welch. 

tents;  earth; 

water;  raw 
foods. 


Larvae  of  bees. 


Blood  and  tissues. 


Petruschky. 


Cheshire  and 
Cheyne. 


Burrill. 
Bollinger- 


Found  in  tissues  Rayer  and 
and  excreta  of  i  Davaine. 
diseased  animals. 


Mucous  membrane 
of  diphtheria. 


Loffler  and 
Cohn. 


288 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name. 


AVISEPTICUS. 
BOIVtBYCIS. 


See 


Bordet-Gengou, 
botulinus, 


BoviSEPTicus.   See 
Bubonic     Plague 

(Pestis). 


BUCCALIS. 


Catarrh  ALis. 


Cattle    Plague 

(Texas  fever). 
Cavicida. 


Chauv^i.  See 

Cholera  asiatice, 


CHOLERiE       GALLI- 

NARUM    (chicken 
cholera) . 

Cholera  nostras 
(Finckler). 


COLI  COMMUNIS. 


Crassus     sputig- 
enus. 


Decalvans. 


Genus. 


Biology. 


Hemorrhagic  Se 
Micrococcus. 

See  Whooping- 
Bacillus. 


Hemorrhagic  Se 
Bacillus. 


Leptothrix. 


Micrococcus. 


Bacillus. 


Symptomatic 
Spirillum. 


Bacillus. 


Spirillum. 


Bacillus. 


Bacillus. 


Micrococcus. 


pticemia. 
Oval  cocci  in  chains 

and  zooglea;  motile. 
cough. 
Large  rounded  ends; 

motile;   flagellated; 

anaerobic. 
pticemia. 
Short  thick  rods  with 

indistinct  capsule. 


Long  threads  in  thick 
bundles,  containing 
masses  of  cocci  and 
spirals. 

Diplococci  at  times 
resembling  gono- 
coccus. 

See  Hemorrhagic  Sep 

Little  rods  twice  as 
long  as  broad. 

Anthrax  (Rauschbra 
Motile,  spiral-shaped 
rods,  often  in  chains; 
very  short  fiagella 
on  ends,  and  strictly 
aerobic;  spores  have 
not  been  found. 


Immotile,  cocci-like 
rods ;  without  spores ; 
strictly  aerobic. 

Motile.comma-shaped 
rods;  strictly  aero- 
bic. 


Short  motile  rods, 
slightly  curved, 
without  spores;  fac- 
ultatively anaerobic. 


Short,  thick  rods  with 
rounded  ends. 


Spheric  cells  in  great 
numbers. 


Product. 


Butyric  acid;     and 
powerful  toxin. 


ticemia  and  Swine 

Propionic      acid 
through  decomposi- 
tion of  sugar, 
nd^. 

Ptomain-like  mus- 
carin  and  toxalbu- 
min,  soluble  in 
water. 


Toxalbumin. 


OF   THE   PRINCIPAL   BACTERIA 

Bacteria. — {Continued.) 


289 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Causes  "fiacherie"  in 

Intestines  of  silk- 

B6champ. 

silkworms. 

worms. 

Gelatin  colonies  appear 

Sausage     and     meat 

Intestine  of  pig. 

Van  Ermen- 

as    small    semi-trans- 

poisoning. 

gem. 

parent  spheres. 

Does  not  liquefy  gelatin; 

Causes  bubonic  plague 

Tissues.         body 

Yersin  and 

white,   point-like   col- 

fluids, and  secre- 

Kitasato. 

onies  turning  gray  and 

tions  of  plague 

then  brown. 

'    patients. 

Causes  dental  caries. 

Teeth  slime. 

Robin. 

At  37°  on  agar,  round. 

Not  pathogenic. 

Mucous  secretions 

R.  Pfeiffer. 

gray  colonies,  serrated 

healthy  persons. 

edges. 

Plague. 

Not  liquefying;  irregular 

Kills  guinea-pigs. 

Human  feces. 

Brieger. 

scale-like  colonies, mak- 

ing the  gelatin  viscid. 

Liquefying  slowly,  small 

Causes  cholera  Asia- 

Feces    of   cholera 

Koch. 

depressed  scars  giving 

tica  in  man  and  a 

patients. 

a  frosted  appearance. 

similar    trouble    in 

or  like  ground   glass; 

animals. 

on  potato, aithin  brown 

layer;  in  test-tube,  a 

funnel-shaped      lique- 

faction, with  a  bubble 

of  air  in  the  top,  the 

funnel   taking   six   or 

seven    days   to   form 

well. 

Not  liquefying;  small  iso- 

Causes chicken  chol- 

Blood and  feces  of 

Pasteur. 

lated  white  disks;    in 

era  in  fowls;      not 

diseased  fowls. 

test-tube,   a   granular 

acting  on  man. 

track:    very  faint. 

Liquefying  rapidly;  col- 

Harmless in  man;  fatal 

Feces    of   cholera 

Finckler  and 

onies        yellow-brown 

to  guinea-pigs. 

nostras  and  ca- 

Prior. 

thick  masses;  in  test- 

ries  of  teeth. 

tube,  funnel  formed  in 

twenty-four        hours. 

dissolving   all    gelatin 

in  two  days;    profuse 

gray  mass  on  potato. 

Not  liquefying;  dark  cen- 

Fatal to   guinea-pigs 

Feces  of  nursing 

Escherich. 

ter,     undulated      per- 

and rabbits;   causes 

infants;    water; 

iphery;    green-colored 

diarrhea     in     man; 

choleraic  stools. 

layer  on  potato;  milky 

ferments  sugar. 

layer    on    surface    of 

test-tube. 

Not     liquefN-ing;,    oval. 

Mice  and  rabbits  die 

Sputum. 

Kreibohm. 

grayish,  slimy  colonies; 

in  forty-eight  hours 

nail-shaped  growth  in 

with  gastro-enteritis. 

test-tube. 

.... 

Causes  alopecia  area- 
ta. 

In  roots  of  hair. 

Thin. 

19 


290 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name, 

Genus. 

Biology. 

Product. 

Dentalis       viri- 
dans. 

Bacillus. 

Slightly  curved  rods, 
round  ends. 

Gray  pigment. 

Diarrhea    of  In- 

Bacillus. 

Motile,  medium-sized 

Toxalbumin. 

fants. 

rods;  spores;  aero- 
bic. 

Diarrhea  of 

Meat-poisoning 
(Enterilidis    sporo- 

genes). 
Diphtheria. 

Diphtheria        of 

Calves      (Vitu- 

lorum). 
Diphtheria        in 

Pigeons  (Colum- 

barum). 
Diplobacillus  of 

Conjunctivitis. 

Bacillus. 

Bacillus. 

Bacillus. 
Bacillus. 
Bacillus. 

Rods  in  groups  of  two 
and   singly;    round 
ends;   spores. 

Immotile,       middle- 
sized  rods,  rounded 
ends;       facultative 
anaerobic. 

Long  rods  in  threads. 

Short  rods  in  groups. 

Non-motile;    usually 
occurs  in  pairs. 

Toxalbumin. 

Duck  Cholera. 

Bacillus. 

Similar     to    chicken 
cholera        bacillus; 
immotile. 

.... 

Dysenteric. 

Dysentery     (epi- 
demic). 

Bacillus. 
Bacillus. 

Resembles     typhoid 

bacillus. 
Short     motile    rods; 

very  thin. 

First     slightly 
then  alkaline. 

acid, 

Enteritidis. 

Bacillus. 

Resembles     typhoid 
bacillus. 

.... 

Enteritidis  sporo- 
genes.     See  Diar 

Erysipelas         of 
SwiNE(Rothlauf; 
rouget  du  pore). 

rhea  of  Meat  Poi 
Bacillus. 

soning. 

Small,  slender  motile 
rods;    facultatively 
anaerobic. 

Two  vaccines, 
give  immunity 

which 

FCETIDUS  OZiENA. 

Bacillus. 

Short  rods,  very  mo- 
tile;    in  pairs  and 
chains. 

Foul  gas. 

Frog  Plague. 
Gangrene. 

Bacillus. 
Micrococcus. 

See  Swine  Plague. 
Oval  cocci  in  zooglea. 

Gigantea. 

Leptothrix. 

Long  rods,  cocci  and 
short  rods  in  one; 
thread  also  spiral. 

OF   THE   PRINCIPAL   BACTERIA 


291 


Bacteria. — (Continued.) 


Culture  Characters, 

Actions. 

Habitat. 

Discoverer. 

Not  liquefyini:;     round, 

Septic  processes  and 

In  caries  of  teeth. 

Miller. 

sharply   outlined    col- 

death in  mice  and 

onies,  with  bluish  gray 

pigs. 

opalescence. 

Not   liquefying;      green 

Causes  green  diarrhea 

Feces    of    infants 

Lesage. 

colonies  with  foul  odor. 

in  animals  when  in- 

suffering     from 

~ 

travenously  injected, 
and  is  the  cause  of 
green  diarrhea  in  in- 
fants. 

green  diarrhea. 

.... 

Causes  death  in  ani- 

Blood and  juices 

Klein. 

mals,  with  symptoms 

of  choleraic  diar- 

of septicemia. 

rhea. 

Not  liquefying;  little  yel- 

Gives rise  to  diphthe- 

Diphtheric    exu- 

Loffler 

lowish  colonies;  a  mem- 

ria in  man  and  ani- 

date. 

(Klebs). 

branous  layer  on  po- 

mals. 

tato. 

When    inoculated    in 

Diphtheric  mem- 

Loffler. 

mice  causes  death. 

brane  of  calf. 

Whitish  patches. 

Necrosis    in    pigeons 

Diphtheric   mem- 

Loffler. 

and  other  animals. 

brane  in  pigeons. 

Addition  of  blood-serum 

Found    in    subacute 

Conjunctival    se- 

Morax. 

to    media    necessary; 

conjunctivitis. 

cretion. 

liquefying. 

Small  round  yellow  col- 

Fatal for  ducks,  but 

Blood  of  diseased 

Cornil      and 

onies   like   wax-drops; 

not  for  chickens  or 

ducks. 

Toupet. 

not  liquefying. 

pigeons;  less  active 
than   chicken   chol- 
era; causes  diarrhea 
and  exhaustion. 

Resembles  typhoid  bacil- 

Produces one  variety 

In          dysenteric 

Shiga. 

lus  in  many  respects. 

of  dysentery. 

stools. 

Not  liquefying;  concen- 

The cause  of  epidemic 

In  feces  and  mes- 

Chantemesse 

trically  arranged  colon- 

dysentery  in    man; 

enteric  glands. 

and  Widal. 

ies;  dry  yellow  mem- 

enteritis in  guinea- 

brane  on  potato. 

pigs. 

Resembles   typhoid    ex- 

Produces enteritis  in 

Intestinal        con- 

Gartner. 

cept  that  it  ferments 

man  and  animals. 

tents;   its   toxin 

dextrose. 

in  meat  of  dis- 
eased animals. 

Very  delicate  silver-gray 

Causes  erysipelas  in 

Blood  and  organs 

Loflfler. 

clouds  on  the  gelatin, 

swine  and  other  ani- 

of diseased  ani- 

like bone-cells;  not  liq- 

mals;    the  German 

mals. 

uefying;  in  test-tube  a 

"Rotlauf,"    French 

very  faint  clouding. 

"rouget  du  pore." 

Small    greenish   colonies 

Mice  are  killed  by  in- 

Secretion of  per- 

Hajek. 

which     soon     become 

jection;  rabbits  af- 

sons      suflFering 

liquefied  and  indistin- 

fected with  progres- 

from ozena. 

guishable;  a  foul  odor 

sive  gangrene. 

produced. 

Grayish    colonies     with 
foul  odor. 


Gangrenous  tissue. 

Eberth. 

Causes  caries  of  teeth. 

Diseased  teeth  of 

Miller. 

animals. 

292 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name, 

Genus. 

Biology. 

Product. 

GiNGIV/E       P  Y  0  G- 

Bacillus. 

Short  thick  rods  with 

ENES. 

rounded  ends. 

Glanders  (Rotz, 

Bacillus. 

Slender,         immotile 

Mallei). 

rods;  usually  singly; 
spores;  facultative- 
ly anaerobic. 

'G0NORRHCE.E 

Micrococcus. 

Diplococci      kidney- 

(Gonococcus). 

shaped;  motile;  do 
not  color  with  Gram. 

Grouse  Disease. 

Bacillus. 

Small  rods  and  oval 
cocci  in  chains;  im- 
motile. 

.... 

H^MATOCOCCUS 

Diplococcus. 

Cocci       seldom       in 

BOVIS. 

chains;  surrounded 
by  a  pale  zone 

HEMOPHILIA  NEO- 

Micrococcus. 

NATORUM. 

Hemorrhagic 

Bacillus. 

Short  rods,  twice  as 

.... 

Septicemia  (In- 

lon<? as  broad;    im- 

fectious   Pleuro- 

motile. 

pneumonia,  Wild 

Plague,    German 

Swine       Plague, 

Cattle       Plague, 

Steer         Plague, 

Rabbit    Septice- 

mia). 

Hog   Cholera 

Bacillus. 

Very  motile  oval  rods, 

Peptonizes  milk  with. 

(Swedish     swine 

similar    to    hemor- 

out coagulation. 

plague). 

rhagic  septicemia. 

Icteroides. 

Bacterium. 

Once  supposed  to  be 
the  cause  of  yellow 
fever.  Identicalwth 
Sanarelli. 

Influenza. 

Bacillus. 

Very  minute  rods  or 
in  clumps. 

Insectorum. 

Micrococcus. 

Oval  cells  in  chains 
and  zooglea;  strep- 
tococci. 

Intracellularis 

Diplococcus. 

Resembles    gonococ- 

Meningitidis. 

cus  in  morphology 
and  arrangement  in 
interior    of    leuko- 
cytes. 

Koch-Weeks. 

Bacillus. 

Resembles    influenza 
bacillus. 

Lactis  aerogenes. 

Bacillus. 

Short,  thick  immotile 
rods. 

OF   THE   PRIJJCIPAL   BACTERIA 


293 


Bacteria. — {Contin  ued . ) 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Growth  rapid;  liquefying; 

Fatal   to   mice,   with 

Suppurating  pulp 

Miller. 

round  colonies,  visible 

septic  processes. 

of  tooth. 

to  naked  eye  in  twenty- 

four  hours. 

Light  yellow,  like  honey. 

Glanders  is  caused  by 

In  epithelium  and 

Lofiier. 

colonies,  turning  red- 

the  bacillus  in  man 

ulcerated  glands. 

brown,  in  a  few  days. 

and  animals. 

Grow  on  blood-serum. 

Gonorrhea  in  man. 

Gonorrheal     pus; 
in  pus-cells  and 
epithelium. 

Neisser. 

Not   liquefying;      small 

Fatal    for   mice   and 

In  blood  and  or- 

Klein. 

scales  which  turn  gray 

guinea-pigs. 

gans  of  diseased 

in  a  few  days,  the  edges 

grouse. 

serrated. 

Best  at  38°  C;  not  lique- 

Fatal for  rabbits  and 

Blood  and  organs 

Babes. 

fying;       small    white 

rats;    hyperemia  of 

of   animals   dis- 

points; sparse  growth 

lungs    and     spleen; 

eased  with  hemo- 

on potato;  transparent. 

blood  -  exudate    in 
peritoneal  cavity. 

globinuria. 

.... 

Supposed   to   be   the 

Found  in  this  dis- 

Klebs. 

cause  of  the  disease. 

ease. 

White  isolated   pinhead 

A  disease  having  dif- 

Blood and  serum 

Huppe. 

points,  not  growing  on 

ferent  names  in  dif- 

of diseased  ani- 

potato; best  at  37°  C. 

ferent  animals.char- 

mals. 

not  liquefying. 

acterized  by  edema, 
hemorrhage,        and 
septicemia. 

Very  good  growth  on  gel- 

In   experiment,    ani- 

Not spread  through 

Salmon  and 

atin  and  potatoes;     a 

mal's  death  in  four 

tissue, butin  cap- 

Selander. 

yellow-brown  color. 

to  eight  days;    bac- 

illaries   of     dis- 

teria in  little  emboli 

eased  swine. 

in  capillaries. 

Grow  best  on  blood-agar; 

Produces  epidemic  in- 

Secretions of  res- 

Pfeiffer. 

colonies    very    small. 

fluenza. 

piratory  tract. 

Kitasato, 

almost  transparent. 

Canon. 

A  contagious  disease 

Stomach   of 

Burrill. 

in  the  chinch-bug. 

chinch-bug. 

Transparent       colonies, 

Causes  epidemic  cere- 

In    cerebrospinal 

Weichsel- 

forming  thin  layer  on 

brospinal     meningi- 

fluid  and   nasal 

baum. 

Loffler's    blood-serum 

tis. 

secretions. 

and  glycerin  agar. 

Rarely  grows,  except  on 

Most  common  cause  of 

Conjunctival    se- 

Koch and 

serum  agar. 

acute        contagious 
conjunctivitis. 

cretion. 

Weeks. 

Small,          porcelain-like 

Fatal   to  guinea-pigs 

Feces  of   nursing 

Escherich. 

disks   with    depressed 

and  rabbits;   coagu- 

infants   and    of 

center;  funnel-shaped 

lates  milk;    decom- 

cholerine. 

In  test-tube  with  gas. 

poses    sugary    solu- 
tions. 

294 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Lepr^. 

Bacillus. 

1  Slender,         immotile 
rods    with    pointed 
ends. 

LiQUEFACIENS  CON- 
JUNCTIVA. 

Micrococcus. 

Single  cocci;     never 
in  threads. 

Lupus. 

Bacillus. 

Same  as  Tuberculosis. 

Malignant  Edema 
(Gangrenous  Sep- 
ticemia,     Vibrio 
Septique). 

Bacillus. 

Large,   slender  rods, 
rounded  ends,  often 
in  threads;    motile, 
with,    flagella    and 
spores;         strongly 
anaerobic. 

Soluble  vaccine. 

Mammitis  of  Cows. 

Micrococcus. 

Oval  cocci  in  chains; 
streptococci;  facul- 
tatively anaerobic. 

Mammitis  of 
Sheep. 

Micrococcus. 

Streptococci    and   in 
fours. 

Melitensis 
(Malta  Fever). 

Metchnikovi 

Micrococcus. 

Spirillum 
(vibrio). 

S/x  in  diameter;  occurs 
singly  or  in  chains 
of  two  or  more;  said 
to  be  flagellated. 

Motile    spirals    with 
flagella;   aerobic. 

An  alkaline  vaccine 
which  will  cause  im- 
munity. 

Neapolitanus. 

Bacillus. 

Small  immotile  rods, 
with  rounded  ends; 
no  spores;    faculta- 
tively anaerobic. 

Produces  acids  in  gel- 
atin cultures. 

NOMiE. 

Bacillus. 

Small     rods,      with 
rounded  ends,  grow- 
ing   often    in    long 
threads. 

Ole^. 

Oxytocus    perni- 

CIOSUS. 

Bacillus. 
Bacillus. 

Motile  aerobic;   does 
not  liquefy  gelatin. 

Short  rods  with  round 
ends. 

Alkali  in  milk. 

Paratyphoid. 

Bacillus. 

Resembles    typhoid 
bacillus. 

Indol  sometimes  pro- 
duced. 

Perfringens.    See 
Pestis.    See  Bubon 

Aerogenes  capsu 
ic  Plague. 

latus. 

OF   THE  PRINCIPAL  BACTERIA 
Bacteria. — (Continued.) 


295 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

On    blood-serum    round 

Causes  leprosy  in  man 

Leprous  tissue. 

Hansen. 

white  plaques  with  ir- 

and animals. 

regular  borders. 

Liquefying;  growth  rap- 

On cornea  of  rabbits 

Normal     human 

Gombert. 

id;  colonies  on  surface. 

.causes  slight  cloud- 

conjunctiva. 

with    little    radiating 

ing. 

branches  from  a  dark 

center;  those  in  deep, 

berry-shaped. 

Liquefying;  thick  center, 

Animals   quickly  die 

Garden-earth. 

Pasteur. 

radiating  periphery;  in 

with  extensive  gan- 

high  culture   in   test- 

grene  and  edema. 

tube,  gas-bubbles  arise. 

with  foul  odor. 

'. 

Not  liquefying;    brown, 

Causes        contagious 

Mammary  gland. 

Nocard  and 

round  granular  colon- 

marrrritis in  cows: 

Mollereau. 

ies;   grows   slowly;   in 

coagulates  milk. 

test-tube,    heavy    de- 

posit along  the  needle's 

track. 

Liquefying;  round    cen- 

Causes contagious  gan- 

Found in  the  milk 

Nocard 

ters  with  zone  of  lique- 

grenous    mammitis 

of           diseased 

faction;     cone-shaped 

in  sheep. 

sheep. 

in  test-tube. 

Small,     round,     slightly 

Causes  Malta  fever. 

Best        obtained 

Bruce. 

raised  disks;     do  not 

from  spleen. 

liquefy. 

Grows  quickly;  colonies. 

Causes  vibrion  septi- 

Feces of  fowls. 

Gamaleia. 

some  like  cholera  As- 

cemia  in  guinea-pigs 

iatica,  others  like  chol- 

and pigeons. 

era  nostras;  liquefying. 

Not  liquefying;  thin  pearl 

Causes  death  in  some 

Cholera  epidemic 

Emmerich. 

like   scales   in  several 

animals;       not    the 

of  Naples,  1884. 

layers;     wrinkled  and 

cause  of  cholera. 

mucous  layers  on  po- 

tato. 

Granular  spheric  colonies 

No  action  on  mice  or 

In  necrotic  tissue 

Schimmel- 

in  the  deep,  fiat  on  the 

rabbits. 

of  noma. 

busch. 

surface;  not  liquefying; 

growth  rapid;    best  at 
35°  C. 

Whitish  growth  on  cul- 

Causes  olive-gall   on 

Savastano. 

ture-media. 

olive  plant. 

Small    yellow    granular 

Intravenous  injection 

Sour  milk. 

Wyssokow- 

colonies;     nail-culture 

causes  death  in  mice 

itsch. 

in  test-tube. 

and  rabbits;     turns 
milk  acid. 

Ferments  glucose,  but  not 

Causes         continued 

Intestinal      con- 

Widal, Gwyn, 

lactose  or  saccharose; 

fevers. 

tents. 

Schott- 

does     not     coagulate 

muUer. 

milk. 

296 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Pneumonia  (Pneu- 

Bacillus. 

Short,  immotile  rods, 

mococcus  of  Fried- 

singly  or  in  diplococ- 

lander). 

ci,  surrounded  with 
capsule;   no  spores; 
not     colored     with 
Gram;  facultatively 
anaerobic. 

Pneumonia  (Pneu- 

Bacillus. 

Short,  oval  rods,  of- 

.... 

mococcus  of  Fran- 

ten  in  chains;  immo- 

kel;   Micrococcus 

tile  ;  no  spores ;  in  the 

of  Pasteur). 

tissue     surrounded 
with    capsule,    col- 
ored    with    Gram; 
facultatively  anae- 
robic. 

Pneumonicis 

Bacillus. 

Short,    thick    motile 

.... 

AGILIS. 

rods  in  pairs. 

Proteus  septicus. 

Bacillus. 

Slightly  curved  rods, 
swelled  in  portions, 
sometimes   in   long 
threads;    motile. 

Foul  gas. 

PsiTTACi       (perni- 

Micrococcus. 

Streptococci         and 

ciosus). 

zooglea. 

Pyocyaneus. 

Bacillus. 

Thin,  motile  rods;  fa- 

Pyocyanin, a  non- 

, 

cultatively  anaero- 
bic. 

poisonous  pigment. 

Pyocyaneus  /3. 

Bacillus. 

Forms  a  brown-yel- 
low pigment ;  other- 
wise identical  with 
above. 

.... 

Pyogenes  (Strepto- 

Micrococcus. 

Streptococci          and 

coccus  erysipela- 

zooglea. 

tis— Fehleisen). 

Pyogenes  albus. 

Micrococcus. 

Staphylococci       and 
streptococci;  facul- 
tatively anaerobic. 

.... 

Pyogenes  aureus 

Micrococcus. 

Staphylococci       and 

Ptomain,  toxalbumin, 

(micrococcus  of 

zooglea;       faculta- 

and pigment. 

osteomyelitis, 

tively  anaerobic. 

Becker). 

Pyogenes  citreus. 

Micrococcus. 

Same  as  Pyogenes  au- 
reus. 

Pyogenes  fcetidus. 

Bacillus. 

Short  motile  rods  in 
pairs. 

Pyogenes  tenuis. 

Micrococcus. 

Cocci  without  defin- 
ite arrangement. 

Relapsing  Fever 

Spirillum. 

Long,  wavy  spirals; 

(Obei-meier). 

motile. 

OF  THE  PRINCIPAL  BACTERIA 

Bacteria. — {Continued.) 


297 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Does  not  liquefy;  grows 

An  accompaniment  of 

Pneumonic      and 

Friedlander. 

quickly;  a  button-like 

pneumonia,    not    a 

other     sputum, 

colony;  in  test-tube,  as 

cause;    animals  not 

and  lung  tissue. 

if  a  nail  driven  in  the 

affected. 

gelatin  with  head  on 

surface. 

Does  not  liquefy;   grows 

Causes  pneumonia  in 

Sputum    of    lung 

A.  Frankel. 

slowly;  small,  well-de- 

man, septicemia  in 

affections  and  se- 

fined masses;    in  test- 

animals;  also  serous 

rous    inflamma- 

tube,   little    separate 

inflammations       in 

tions. 

globules,    one    above 

man,     as     pleurisy. 

the  other. 

peritonitis,  etc. 

Liquefying;  dark  granu- 

Pneumonia in  rabbits. 

From          rabbits' 

Schon. 

lar  colonies;  thick  sed- 

pneumonia. 

iment  in  test-tube. 

Growth    rapid;   liquefy- 

Fatal for  mice  in  one 

From  a  child  dy- 

Babes. 

ing  ;  colonies  have  foul 

to  three  days. 

ing  of  intestinal 

odor,  are  small,  thick 

gangrene. 

branches,  but  soon  all 

liquid. 

Causes  disease  in  gray 

In  blood  of  par- 

WolfT. 

parrots. 

rot's  disease. 

Liquefying;     large,    flat 

Fatal  for  animals;  col- 

Pus. 

Gessard. 

colonies  with  greenish 

ors      the   dressings 

fluorescence;      on  po- 

green. 

tato,          yellow-green 

skin;    deeply  coloring 

the  pulp. 

.... 

.... 

Ernst. 

Not  liquefying;     round 

Suppuration  and  sep- 

Pus. 

Rosenbach. 

punctiform     colonies; 

ticemia  in  animals. 

slow-growing. 

Liquefying;  white  opaque 

Suppuration  and  ab- 

Pus. 

Rosenbach. 

colonies. 

scess. 

Liquefying;  small  colon- 

Causes abscesses  and 

Pus. 

Rosenbach. 

ies     with     a     yellow- 

suppuration  in  man 

orange  pigment  in  cen- 

and animals. 

ter;    yeast-like  smell; 

a  moist  layer  on  potato. 

Colonies,     citron-yellow 

.Suppuration. 

Pus. 

Passet. 

color. 

Not  liquefying;    mucous 

Fatal  to  animals. 

Pus. 

Passet. 

layer  on  potato;    very 

thick;    in  test-tube,  a 

slicht  layer  on  surface, 

and  small  points  along 

the  track. 

On  surface,  transparent; 

Pus  of  abscesses. 

Rosenbach. 

thin    growth;      grows 

slowly. 

Cannot  be  cultivated. 

Causes  fever  in  man 

Blood  of  man  dur- 

Obermeier. 

and  animals,  and  is 

ing  an  attack  of 

the  cause  of  relaps- 

the disease. 

ing  fever. 

298 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Rhinoscleroma. 

Bacillus. 

See  Pneumococcus  of 

Friedlander,    with 

Sahvarius    pyog- 
enes. 

Micrococcus. 

Very  small  round  coc- 
ci and  staphylococ- 
ci. 

Salivarius  septi- 
cus. 

Salivarius   septi- 
cus. 

Bacillus. 
Micrococcus. 

Short,  immotile  rods, 
encapsulated         in 
pairs,       sometimes 
long  chain;  aerobic. 

Cocci   singly   and   in 
zooglea;    aerobic. 

: 

Saprogenes     No. 
II. 

Bacillus. 

■ 

Short  rods;    faculta- 
tively anaerobic. 

Foul  gas. 

Saprogenes     No. 
III. 

Bacillus. 

Very  short  rods;  fac- 
ultatively    anaero- 
bic. 

Foul  gas. 

Saprogenes  fceti- 

DUS. 

Bacillus. 

I  mmotile    rods; 
spores. 

Foul  gas. 

Senile  Gangrene. 

Septicemia  after 
Anthrax. 

Bacilli 
Micrococcus. 

Thin  rods;  immotile; 

singly  and  in  pairs; 

ends          somewhat 

thickened;  aerobic; 

spores. 
Motile  streptococci. 

Septicemia         of 
Mice. 

Bacillus. 

Smallest          bacillus 
known;    immotile. 

Septicemia         of 
Rabbits    (Cuni- 
culicida). 

Septicus  acumina- 

TUS. 

Bacillus. 
Bacillus. 

See  Hemorrhagic  Septi 

Thin,    lancet-shaped 
rods;    very  slender. 

cemia. 

Septicus      agrig- 

ENUS. 

Bacillus. 

Very  short  rods. 

.... 

Septicus  liquefa- 

CIENS. 

Micrococcus. 

Streptococci  and  dip- 
lococci. 


Septicus  ulceris. 

Bacillus. 

1 
1 

Oval  rods;   motile. 

Gas;  no  odor.      • 

OF  THE  PRINCIPAL   BACTERIA 
Bacteria. — {Contin  tied.) 


299 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

which  it  is  identical. 

Frischl. 

Slowly  liquefying;  small 

Locad  abscess  in  ani- 

Saliva. 

Biondi. 

white  opalescent  col- 

mals. 

onies. 

Not  liquefying;  gray  cir- 

Fatal to  animals. 

Saliva  of  healthy 

Biondi. 

cular  colonies;    trans- 

persons. 

parent  zone;    in  test- 

tube,   separated. 

Not  liquefying;     round 

Fatal  to  animals. 

Saliva     of     puer- 

Biondi. 

colonies;        separated 

peral  women. 

dots  in  test-tube. 

Grows  quickly;  on  agar. 

Produces  septicemia 

Sweat  of  feet. 

Rosenbach. 

hyaline    drops    which 

in  rabbits. 

quickly  coalesce,  and 

form  a  mucoid  layer 

with  a  foul  odor,  that 

of  perspiring  feet. 

Forms  a  fluid  gray  band 

Suppuration  in  rabbit. 

Putrid  marrow  of 

Rosenbach, 

on  agar;    odor  of  pu- 

bone. 

trefaction. 

Not    Hquelving;       thin. 

Rabbits    killed    with 

Mesenteric  glands 

SchotteUus. 

transparent  layer;  pu- 

large doses. 

of    swine     with 

trid  odor. 

erysipelas  and  of 
healthy  swine. 

Round    yellow  colonies; 

Causes    gangrene    in 

In  gangrenous  tis- 

Tricomi. 

liquefying  in  thirty-six 

mice,  similar  to  se- 

sue and  blood  of 

hours;   best  growth  at 

nile      gangrene      of 

senile  gangrene. 

37°  C. 

man. 

In  bouillon  virulence  de- 

Septicemia in  rabbits. 

Blood    of   animal 

Charrin. 

stroyed. 

but  not  in  chickens 

dead    from    an- 

or guinea-pigs. 

thrax. 

Not    liquefying;      small 

Septicemia  in  house- 

Putrefying  liquids. 

Koch. 

flocculent    masses    in 

mice,  but  not  field- 

the  deep;    grows  very 

mice. 

slowly;      in  the  test- 

tube  producing  a  faint 

cloud. 

At  37°  C.  on  blood-serum 

Pathogenic  for  rabbits 

Navel    stump    of 

Babes. 

small          transparent 

and  guinea-pigs; fev- 

child    dead     of 

plates;   later  on.  turn- 

er;   and     bacilli    in 

septicemia. 

ing  yellow. 

blood  and  organs. 

Not  liquefying;     brown 

Septicemia    in    mice 

Earth  of  recently 

Nicolaier. 

center,    a    ring,    then 

and  rabbits. 

plowed  fields. 

yellow  zone. 

Liquefying;  a  thin  gran- 

Pathogenic  for  mice 

Blood  and  organs 

Babes. 

ular  streak,  the  surface 

and  rabbits,  produc- 

of child  dying  of 

sunken  in;  later,  cone- 

ing   edema,  in    the 

septicemia. 

like,  the  walls  covered 

serum  of  which  the 

with   leaf-shaped   col- 

cocci abound. 

onies. 

Liquefying;    yellow  col- 

An ulcer  in  inoculated 

In  blood  of  child 

Babes. 

onies,  taken  up  with 

animals,  followed  by 

with  gangrenous 

gas  later  on. 

paralysis  and  death. 

ulcer. 

300 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

SePTICUS  VESIC/E. 

Bacillus. 

Rods  always  single; 
very  motile;     oval 
spores. 

Smegma. 

Bacillus. 

Slender  curved  rods, 

identical  with  what 

•  -  »• 

was  known  as  syph- 

ilis bacillus  of  Lust- 

garten. 

Soft  Chancre. 

Bacillus. 

Minute     oval    rods, 
chiefly  in  groups  or 
chains. 

Sputigenxjm. 

Spirillum. 

Curved,          comma- 
shaped  rods;  motile. 

Subflavus. 

Micrococcus. 

Diplococci  like  gono- 
cocci;      colored   by 
Gram. 

Swine  Plague 

Bacillus. 

Motile,     oval     rods. 

Causes  casein  precipi- 

(American   and 

similar  to   that   of 

tate  in  milk  and  acid 

French) . 

hog  cholera. 

formation. 

Sycosiferus  fceti- 

Bacillus. 

Short,  straight  immo- 

On    potatoes    a    foul 

DUS. 

tile   rods,   often   in 
threads. 

odor. 

Syphilis    (Spiro- 

Spirocheta. 

Small  delicate  spirals. 

cheta  pallida). 

dif^cult  to  stain. 

Tetanus. 

Bacillus. 

Large,  slender  motile 

Ptomains,        tetanin. 

rods,  with  spores  in 

tetanotoxin,      spas- 

one end,  drumstick 

motoxin;  alsoatox- 

shape,      often      in 

albumin. 

threads;  true  anae- 

robic. 

Tetragenus. 

Micrococcus. 

Large  round  cells,  uni- 
ted in  groups,  usual- 

ly of  four,  and  sur- 
rounded by  a  cap- 
sule;         immotile; 
aerobic. 

Timothy  Grass. 

Bacillus. 

Extremely  acid-fast; 
resembles    tubercle 
bacillus:  in  cultures 
may  show  club  for- 
mation and  branches 

TOXICATUS. 

Micrococcus. 

Cocci  singly  and  in 
pairs. 

OF  THE  PRINCIPAL  BACTERIA 
Bacteria. — (Continued.) 


301 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Not  liquefying;  small  pin- 

Pathogenic   for   mice 

In   urine  of  cys- 

Clado. 

head  colonies,  growing 

and  rabbits,  produc- 

titis. 

slowly;  never  larger;  a 

ing  death. 

brown  center,   yellow 

periphery. 

Not  cultivated. 

Normal  preputial 

Alvarez  and 

secretions. 

Tavel. 

Has  not  been  cultivated. 

Produces  soft  chancre. 

In  the  sore. 

Ducrey. 

Not  cultivated. 

Causes  death  in  ani- 

In caries  of  teeth 

Lewis. 

mals. 

and  saliva. 

Growth  slow;  liquefying; 

No  result  on  mucous 

Normal  secretion 

Bumm. 

on   tenth   day   yellow 

membrane;  injected 

of    vagina    and 

points    with    thready 

under   skin,  abscess 

urethra. 

boundary; on  potato, a 

results. 

brown,     thread  -  like 

growth  aftertwo  weeks. 

Not  liquefying;    growth 

Found    in    American 

Found    in    capil- 

Billings, 

similar     to      typhoid 

and    French    swine 

laries    in    little 

Rietsch,  and 

germ ;       on    potatoes 

plague,       in       frog 

emboli;          not 

Eberth. 

good  growth. 

plague,    and    Texas 

spread  in  organs 

fever-     animals  af- 
fected locally. 

of  diseased  ani- 

mals. 

Slow  growth;  not  liquefy- 

On human  skin  causes 

From    sycosis    of 

Tommasoli. 

ing;  after  four  days,  lit- 

eruption,   vesicular 

the  beard. 

tle  white  points,  which 

around    hairs,    then 

do  not  change  for  sev- 

it becomes  pustular; 

eral   weeks,   then  the 

similar  to  sycosis. 

superficial     ones     are 

mucus-like;    nail 

growth;    on  potatoes. 

^. 

rapid  growth. 

On     blood-serum     thin 

Supposed    to    cause 

In  tissue  and  se- 

Schaudinn. 

growth. 

syphilis. 

cretions  of  syph- 
ilitics. 

Liquefy   gelatin   slowly; 

Produces   tetanus   in 

Earth  and  manure. 

Nicolaier  and 

colonies  have  radiated 

man  and  animals. 

Kitasato. 

appearance;    a  thorny 

growth  along  the  track 

in  test-tube. 

•« 

Not  liquefying;  little  por- 

Fatal to  guinea-pigs 

Found  in  cavern- 

Gaffky. 

celain-like  disks;  thick 

and  white  mice. 

ous      phthisical 

slimy  layer  on  potato. 

lungs. 

Colonies  visible  in  thirty- 

May  produce  tuber- 

Infusions of  tim- 

Moeller. 

six     hours,     scale-like 

cles. 

othy  grass. 

and  grayish  white. 

Supposed   to  be  the 

Found  in  the  Rhus 

BurriU. 

cause  of  Rhus  (pois- 

toxicodendron. 

on  ivy)  poisoning. 

302 


CHIEF   CHARACTERISTICS 


Pathogenic 


Name. 

Genus. 

Biology. 

Product. 

Tuberculosis. 

Bacillus. 

Slender  rods,  usually 
in  pairs;  not  motile; 
spores  not  definitely 
determined ;    facul- 
tatively anaerobic. 

Kochin  or  paratolin, 
a  glycerin  extract  of 
the  pure  culture  (tu- 
berculin). 

Typhoid. 

Bacillus. 

Slender  motile  rods, 
sometimes      in 
threads ;       flagella, 
but       no      spores; 
facultatively  anae- 
robic. 

Typhotoxin  and 
toxalbumin. 

Typhoid  of  Swine 

(swine  plague). 
Tyrogenum. 

Spirillum 
(vibrio) . 

See  Swine  Plague. 

Spiral-shaped     rods; 
aerobic. 

.... 

Whooping-cough. 

Bacillus. 

Short  oval. 

.... 

Welchii. 
Xerosis. 

See  ASrogenes 
Bacillus. 

capsulatus. 

Similar  to  diphtheria 
bacillus. 

OF    THE   PRINCIPAL   BACTERIA 


305 


Bacteria. — (Concluded.) 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Grows  best  on  blood-ser- 

Causes   tuberculosis, 

In  all  organs  and 

Koch. 

um  and  glycerin  agar 

local  and  general,  in 

secretions  of  tu- 

at 37°  C,  forming  little 

man  and  lower  ani- 

bercular persons. 

white  crumbs  on  the 

mals. 

surface;  under  micro- 

scope a  hairy,  matted 

coil  is  seen;     growths 

on  potatoes  when  air- 

tight   have    been   ob- 

tained. 

Not    liquefying;       little 

Gives  rise  to  enteric 

Found  in  dejecta 

Eberth. 

whetstone-shaped  yel- 

or typhoid  fever  in 

and  spleen  and 

low  colonies  inthe  deep, 

man. 

urine  of  typhoid 

and   leaf-shaped  ones 

patients. 

on  the  surface;  on  po- 

tato, a  very  transpar- 

ent, moist  layer. 

Liquefy  rapidly;    small 

Several  animals  have 

From  old  cheese. 

Deneke. 

round   colonies;  dark 

died   from  inocula- 

funnel-shaped liquefac- 

tions. 

tion  in  test-tube. 

Blood     agar.        White 

Produces    spasmodic 

Found  in  whoop- 

Bordet- 

growth  on  surface. 

cough  in  animals. 

ing  cough. 

Gengou. 

Differs  from   diphtheria 

Found   in   patho- 

Kuschbert 

bacillus  in  not  produc- 

logic conditions 

and 

ing  acid  in  bouillon. 

of    conjunctiva, 
sometimes       in 
normal  eye. 

Neisser. 

INDEX 


Abbe's  condenser,  44 
Achorion  schonleinii,  224 
Acid,  boric,  264 

curd  cheese,  254 

dyes,  48 

salicylic,  264 
Acid-fast  bacteria,  116 
Actinomyces,  cladothrix,  228 

strep tothrix,  228 
Actinomycosis,  228 
Aerobes,  28 

facultative,  28 

obligate,  28 
Aerobioscope,  234 
African  tick  fever,  218 
Agar,  bile  salt,  MacConkey's,  75 

blood-,  76 

fuchsin-lactose,  77 

glycerin-,  69 

lactose  litmus,  72 

nutrient,  71 
Agar-agar,  69 

Age,  influence  of,  on  bacteria,  30 
Agglutination  test  for  tubercle  ba- 
cilli, 121 
in  cholera,  152 
in  typhoid  fever,  140 
Pfeiffer's  cholera,  153,  154 
Agglutinins,  40 
Aggressins,  32 
Air,  bacteria  in,  232 

Hesse's  method  of  collecting, 

233 
Petri    method    of   collecting, 

232,  233 
Sedgwick-Tucker  method   of 

collecting,  234 
varieties,  234 
examination  of,  232 
sewer,  bacteria  in,  235 


Albumin,  blood,  solution  dried,  78 
Alcohol,  264 

fermentation,  255 
Alcoholic  solution,  saturated,  49 
Alexin,  40 
Algae,  fission,  21 

Alkaline  anilin- water  solutions,  51 
methylene-blue,  50 
stains,  49 
Alkaloids,  cadaveric,  34 
Allergy,  42 
Altered  reactivity,  42 
Alum,  236 
Amboceptors,  40,  42 
Amebae,  examination  for,  199 
Ammonia  production,  245 
Amoeba  coli,  198 
dysenteriae,  198 
Anaerobes,  28 
facultative,  28 
obligate,  28 
true,  28 
Anaerobic  bacteria,  180 
cultivation,  82 

Botkin's  method,  84 
Buchner's  method,  84 
Esmarch's  method,  83 
Frankel's  method,  84 
Hesse's  method,  83 
Liborius's  method,  82 
Park's  method,  85 
Roux's  method,  84 
Wright's  method,  85 
Anaphylaxis,  42 
Anilin  dyes,  47,  264 
acid,  48 
basic,  48 
Anilin-oil,  48 
water,  49,  50 
dyes,  so 


30s 


3o6 


INDEX 


Animal  inoculation,  91 

celloidin  sacs,  93 

cerebral  membranes,  93 

cutaneous,  91 

eye,  92 

guinea-pigs,  91 

intraduodenal,  93 

intraperitoneal,  92 

intratracheal,  93 

intravenous,  92 

methods,  91 

obtaining  material  after,  94 

subcutaneous,  92 
Animals  as  culture-media,  81 

tuberculosis  in,  119 
Anthrax   105 

symptomatic,  187 
Anthraxin,  109 
Antiformin   method   of   collecting 

tubercle  bacilli,  117 
Antigens,  41 
Antisepsis,  261,  262 
Antiseptics,  261,  262 
Antistreptococcic  bacterins,  166 
serum,  166 
vaccines,  166 
Antitoxic  serum,  39 

unit,  133 
Antitoxin,    formation   of,  accord- 
ing  to   lateral-chain    theory, 

of  diphtheria,  131 

specific  action,  38 

tetanus,  183 
unit  for,  183 
Antituberculous  serum,  122 
Antitj^phoid  bacterins,  139 

vaccines,  139 
Arnold's  steam  sterilizer,  64 
Artesian  well  water,  236 
Arthrosporous  bacteria,  26 
Artificial  cultivation,  62 
Asbestos,  236 
Asexual  cycle  in  man,  199 
Aspergillus  flavus,  226 

fumigatus,  226 

glaucus,  226 
Asporogenic  bacteria,  26 
Autoclave,  64,  65 
Avicidus  bacillus,  193 
Azofication,  245 


Bacillary  dysentery,  199 
Bacillus  acidi  lactici,  249 

aerogenes  capsulatus,  186 

amylobacter,  251 

amylovorus,  232 

anthracis,  105 

avicidus,  193 

avisepticus,  192 

Boas-Oppler,  loi 

botulinus,  144 

bovisepticus,  192 

butyricus,  251 

capsule,  of  Pfeiffer,  159 

chauvei,  187 

coli,  134 

diagnostic  points  of,  241 

in    milk,  Wisconsin    test    for, 

255 

in  urine,  102 

in  water,  examination  for,  240 
colon,  134 

bacillus  typhosus  and,  differ- 
entiation, 142 
comma,  of  cholera,  148 
cyanogenes,  251 
diphtheria,  126 

products  of,  131 
dysenteriae,  145,  199 

products,  147 
enteritidis,  135 

sporogenes,  187 
fluorescens,  171 
hay,  TOO 
icteroides,  219 
Klebs-LofiEler  126, 
Koch-Weeks,  161 
lactis  aerogenes,  249 
lepra,  122 
mallei,  124 
megaterium,  99 
melitensis,  162 
mesentericus  vulgatus,  98 
Milzbrand,  105 
murisepticus,  195 
mycoides,  99 
oedematis,  184 

maligni,  184 
of  anthrax,  105 
of  bluish-green  pus,  171 
of  bubonic  plague,  190 
of  chancroid,  178 


INDEX 


307 


Bacillus  of  chicken  cholera,  193 
of  cholera,  148 
of  diphtheria,  126 

products,  131 
of  dysentery,  145 

products,  146 
of  enteric  fever,  135 
of  erysipelas  of  swine,  194 
of  fowl  septicemia,  193 
of  glanders,  1 24 
of  influenza,  160 
of  lepra,  122 

of  malignant  pustule,  105 
of  pertussis,  161 
of     phlegmonous    emphysema, 

186 
of  rhinoscleroma,  159 
of  soft  chancre,  178 
of  splenic  fever,  105 
of  symptomatic  anthrax,  187 
of  tetanus,  180 
of  typhoid  fever,  135 
paracolon,  143 
paratyphoid,  143 
perfringens,  187 
pestis,  190 
pneumoniae,  159 
potato,  of  Fliigge,  98 
proteus  vulgaris,  147 
pyocyaneus,  171 
ramosus,  99 
root,  99 
rotz-,  124 

saccharobutyricus,  187 
Shiga,  199 
smegma,  123 
subtilis,  100 
suipestifer,  133 
suisepticus,  192 
tubercle,  no 

products  of,  1 20 
tuberculosis,  no 

products  of,  1 20 
typhosus,  135 

colon  bacillus  and,  differentia- 
tion, 142 

from  blood,  143 

in  water,  138 

diagnostic  points,  242 

products,  139 
violaceus,  102 


Bacillus  vulgatus,  98 

Welchii,  186 

Wurzel,  99 

X,  219 
Bacteremia,  33 
Bacteria,  acid-fast,  116 

aerobic,  28 

anaerobic,  28,  180 
cultivation  of,  82 
Botkin's  method,  84 
Buchner's  method,  84 
Esmarch's  method,  83 
Frankel's  method,  84 
Hesse's  method,  83 
Liborius's  method,  .82 
Park's  method,  85 
Roux's  method,  84 
Wright's  method,  85 

and  soil  fertility,  244 

arthrosporous,  26 

asporogenic,  26 

avenues  of  entrance,  32 

biologic  activities,  26,  28 

Brownian  movements,  23 

causing  disease,  29 

chemic  activities,  26 
composition,  22 
products,  29 

chief  characteristics,  268-303 

cholera,  148 

classification,  22 

colonies  of,  growth  and  appear- 
ances, 87 

colon-typhoid  group,  133 

cultivation,  62 

defenses  to  invasion,  34 

development  of,  21 

diseases  from,  29 

distribution,  26 

effects  of,  general,  33 
local,  ss 

endosporous,  24 

facultative,  27 

fermentation  by,  29 

filterable,  218 

fluorescence  of,  30 

gas-forming,  30 

general  effects,  $^ 

growth  of,  27 

how  cause  disease,  30 

in  air,  232 


3o8 


INDEX 


Bacteria  in  air,  Hesse's  method  of 
collecting,  233 

Petri    method    of    collecting, 
232,  233 

Sedgwick-Tucker   method   of 
collecting,  234 

varieties,  234 
in  artesian  well  water.  236 
in  blood,  260 
in  butter,  254 
in  buttermilk,  255 
in  cheese,  254 
in  condensed  milk,  255 
in  conjunctiva,  258 
in  cream,  248 
in  ear,  259 
in  feces,  260 
in  filtered  water,  236 
in  food,  246 

in  geni to-urinary  passages,  260 
in  ice-cream,  248 
in  intestine,  259 
in  milk,  246,  249 
in  mouth,  258 
in  nasal  cavity,  259 
in  pneumonia,  155 
in  sewer  air,  235 
in  skin,  257 
in  soil,  232,  243 
in  stomach,  259 
in  urethra,  260 
in  urine,  102,  260 
in  vagina,  260 

in  water,  method  of  examination, 
2_38_ 

varieties,  238 
in  well  water,  236 
infective,  33 
influence  of  age  on,  30 

of  electricity  on,  28 

of  heat  on,  28 

of  light  on,  28 

of  moisture  on,  28 

of  oxygen  on  life  and  growth 
of,  27 

of  Rontgen  rays  on,  28 

of   temperature   on   life   and 
growth,  27 
local  effects,  33 
locomotion,  23 
methods  of  studying,  43 


Bacteria,  nitrification  by,  29 

non-pathogenic,  29 
table  of,  268-287 

odors  from,  30 

of  hemorrhagic  septicemia,  192 

origin,  26 

oxidation  by,  29 

parasitic,  27 

pathogenic,  29,  97 
table  of,  287-303 

phosphorescence  by,  30 

pigmentation  by,  30 

plant  diseases  due  to,  231 

pyogenic,  33 

quantity  of,   infection  depend- 
ing on,  32 

reduction  by,  29 

reproduction,  24 

sewage,  examination  for,  240 

specific,  ss 
nature,  27 

spore  contents,  25 
formations,  24 

staining  of,  47 

structure  of,  21,  22 

suppurative,  33 

tables  of,  268-303 

types,  21 

ultra-microscopic,  218 

unstained,  examination  of,  45 

vibratory  movements,  23 
Bacterial  vaccines,  95 

treatment  of  sewage,  243 
Bactericides,  41 
Bactericie  du  charbon,  105 
Bacterins,  95 

anticholera,  153 

antistreptococcic,  166 

antityphoid,  139 

counting  of,  96 

preparation  of,  95 

standardization  of,  95 
Bacteriologic   examination  of  or- 
gans and  cavities,  257 
Bacteriolysins,  41 
Bacterium  acidi  lactici,  249 

bulgaricum,  250 

caucasicus,  250 

coH  commune,  134 

guntheri,  250 

lactis  acidi,  250 


INDEX 


309 


Bacterium  prodigiosum,  97 
as  cancer  remedy,  98 
saccharobutyricus,  251 
syncyanum,  251 
termo,  147 
tumefaciens,  232 
Zopfii,  100 

Basic  dyes,  48 

Beer  fermentation,  256 

Beggiatoa  alba,  228 

Bichlorid  of  mercury,  62 

Biedert's    method    of    collecting 
tubercle  bacilli,  116 

Bile,  lactose-,  76 
salt  agar,  MacConkey's,  75 

Biologic  activities,  26,  28 

Black-leg,  187 

Blastomyces,  223 

Blastomycetes,  220,  222 

Blastomycetic  dermatitis,  223 

Blender,  44 

Blood  albumin,  solution  dried,  78 
bacillus  typhosus  from,  143 
bacteria  in,  260 
coagulum,  74 
cultures,  261 
specimens,  staining,  57,  261 

Blood-agar,  76 

Blood-serum,  69 
coagulation  of,  72 
human,  preparation  of,  74 
mixture,  Loflfler's  74 
nutrient,  preparation  of,  72 
preservation,  in  liquid  state,  74 
sterilization  of,  73 
test,  Gruber-Widal,  140 

Bluish-green  pus,  bacillus  of,  171 

Boas-Oppler  bacillus,  loi 

Boiled  eggs,  78 

Boiling  as  means  of  purifying  wa- 
ter, 238 

Bordet's  cholera  test,  155 

Borer,  Frankel's,  244 

Boric  acid,  264 

Botkin's  method  of  cultivating  an- 
aerobic bacteria,  84 

Bouillon  filtrate,  Denys',  120 
guinea-pig,  78 

Bovine  farcin  du  boeuf,  231 
tuberculosis,  human   tuberculo- 
sis and,  relation,  119 


Bowhill's  orcein  stain,  60 

Bread  mash,  69 

Brill's  disease,  219 

Broth,  nitrate,  70,  71 
nutrient,  71 
sugar,  71 

Brownian  movements,  23 

Bubonic  plague,  190 

Buchner's  method  of  cultivating 
anaerobic  bacteria,  84 

Budding  fungi,  220 

Buerger's  method  of  staining  cap- 
sule, 61 

Butter,  bacteria  in,  254 

Buttermilk,  bacteria  in,  255 


Cadaveric  alkaloids,  34 

Calmette's  ophthalmic  tuberculin 
reaction,  121 

Cancer  remedy,  bacterium  prodigi- 
osum as,  98 

Capsule  bacillus  of  Pfeiffer,  159 
of  spore,  25 
stain,  61 

Buerger's  method,  61 
Hiss'  method,  52,  61 
of  Welch,  53 

Carbolfuchsin,  49 

Carbolthionin  stain,  52 

Carriers,  cholera,  153 
typhoid,  139 

Catarrhal  conjunctivitis,  258 

Cattle-fever,  Texas,  208 

Cell-contents,  22 

Celloidin  sacs  for  animal  inocula- 
tion, 93 

Cell-wall,  22 

Cerebral   membranes,   animal   in- 
oculation by,  93 

Cerebrospinal     meningitis,      epi- 
demic, 177 

Chancre,  soft,  178 

Chancroid,  178 

Charbon  symptomatique,  187,  189 

Charcoal  sponge,  236 

Cheese,  bacteria  in,  254 

Chemic  activities,  26 
composition,  22 
products  of  bacteria,  29 

Chicken  cholera,  193 


3IO 


INDEX 


Chlorin,  264 
Cholera,  148 

bacteria,  148 

carriers,  153 

chicken,  193 

comma  bacillus  of,  148 

hog, 133 

red,  152 
Chromatin  stain,  Wright's,  for  ma- 
larial organisms,  204 
Chromium    trioxid    for    staining 

spores,  60 
Cladothrices,  227 
Cladothrix  actinomyces,  228 

dichotoma,  227 
Classification,  22 
Clostridium  butyricum,  251 
Coagulating  ferments,  30 
Coagulation  of  blood-serum,  72 
Coagulum,  blood,  74 
Cocci,  pyogenic,  163 
Coley's  fluid,  166 
Colitis  contagiosa,  135 
Colon  bacillus,  134 

bacillus  typhosus  and,  differ- 
entiation, 142 
Colon-typhoid  group,  133 
Colonies,  growth  and  appearances, 

.    ^7      . 

impression  of,  88 

macroscopic  appearance,  87 

microscopic  appearance,  88 
Combinor,  42 

Comma  bacillus  of  cholera,  148 
Complement,  40,  42 

deviation  of,  42 

fixation  of,  42 
Completor,  42 

Concentrated  staining  solutions,  48 
Condensed  milk,  bacteria  in,  255 
Condenser,  Abbe's,  44 
Conjunctiva,  bacteria  in,  258 
Conjunctivitis,  catarrhal,  258 

diplobacillus  of,  171 

epidemic,  161 
Conradi-Drigalski  medium,  76 
Copper  sulphate,  264 
Copula,  40 

Corrosive  sublimate,  262 
Cotton  plugs  or  corks,  66 
Cover-glass  preparations,  54 


Cream,  classification  of,  248 
Crenothrix  kiihniana,  227 
Cresols,  264 
Crown  gall,  232 
Cultivation,  62 
artificial,  62 

of  anaerobic  bacteria,  82 
Botkin's  method,  84 
Buchner's  method,  84 
Esmarch's  method,  83 
Frankel's  method,  84 
Hesse's  method,  83 
Liborius's  method,  82 
Park's  method,  85 
Roux's  method,  84 
Wright's  method,  85 
Culture-media,  inoculation  of,  78 
nutrient,  preparation  of,  67,  70 
reaction,  70 
solid  transparent,  69 
sterilization  of,  62,  70 
Cultures,  blood,  261 
fresh  egg,  77 
glass  slide,  78 
plate,  79 

pure,  by  boiUng,  81 
rolled,  80 
stab,  78 
stroke,  78 
test-tube,  78 
thrust,  78 
Cutaneous  inoculation  of  animals, 

91 
Cutting  sections,  56 
Cytase,  40 
Cytolysins,  40 
Cytolytic  serum,  40 


Decolorizing  agents,  49 

Denitrification,  245 

Denys'  B.  F.  tuberculin,  120 

Deodorants,  262 

Dermatitis,  blastomycetic,  223 

Dermo-tubercuHn  reaction  of  von 

Pirquet,  121 
Desmon,  40 
Development,  21 
Deviation  of  complement,  42 
Diastatic  ferments,  29 


INDEX 


3" 


Dieudonne's  medium,  77 

for  spirillum  cholerae,  151 
Diphtheria,  126 

antitoxin  of,  131 

methods  of  diagnosis,  130 

Neisser's  stain  for,  5  2 

streptococcus  in,  133 

toxins  of,  131 
Diplobacillus     of     conjunctivitis, 

171 
Diplococcus    intracellularis    men- 
ingitidis, 177 

lanceolatus,  157 

pneumoniae,  155,  157 
Discharges,  disinfection  of,  265 
Disinfectants,  62,  262 

testing  value  of,  265 
Disinfection,  general  measures  for, 
265 

of  discharges,  265 
Distribution,  26 
Dourine,  204 
Dry  heat,  64 
Drying  specimens,  54 
Dum-dum  fever,  208 
Dunham's  modified  peptone  wa- 
ter, 77 

peptone   solution    for   spirillum 
cholerae,  150 

rosalic  acid  solution,  77 
Dyes,  acid,  48 

aniUn,  47,  264 

anilin-oil  water,  50 

basic,  48 
Dysentery,  145,  198 

bacillary,  199 


Ear,  bacteria  in,  259 

Edema,  malignant,  184 

Eggs,  boiled,  78 
fresh,  cultures,  77 

Ehrlich's   lateral-chain   theory   of 
immunity,  36 

Electricity,  influence  of,   on  bac- 
teria, 28 

Eisner's  typhoid  medium,  76 

Emphysema,  phlegmonous,  186 

Emphysematous  gangrene,  186 

Endo  medium,  77 

Endogenous  infection,  31 


Endosporous  bacteria,  24 

Entamoeba  histolytica,  198 

Enteric  fever,  135 

Epidemic  cerebrospinal    meningi- 
tis, 177 
conjunctivitis,  161 

Erysipelas,  164 
of  swine,  194 

Esmarch's  cubes,  68 
method  of  cultivating  anaerobic 

bacteria,  83 
tubes,  80 

Estivo-autumnal    form   of    mala- 
rial protozoa,  201 

Exogenous  infection,  31 

Extracellular  toxins,  31 

Eye,  inoculation    of    animals    by, 
92 


Facultative  aerobes,  28 

anaerobes,  28 

bacteria,  27 
Farcin  du  boeuf,  bovine,  231 
Farcy-buds,  125 
Fat-splitting  ferments,  30 
Favus,  225 
Feces,  bacteria  in,  260 

cholera  bacteria  in,  153 
Fermentation,  29 

alcohol,  255 

beer,  256 

tube,  81 

vinegar,  255 
Ferments,  29 

coagulating,  30 

diastatic,  29 

fat-splitting,  30 

hydrolytic,  30 

inverting,  29 

proteolytic,  29 
Fertility,  soil,  bacteria  and,  244 
Filter  materials,  236 

Pasteur-Chamberland,  237 

Petri's  sand,  233 
Filterable  organisms,  218 
Filtered  water,  236 
Filters,  67 
Filtration,  265 

sterilization  by,  67 
Finkler-Prior  vibrio,  154 


312 


INDEX 


Fishing,  88 
Fission  algae,  21 

fungi,  21 
Fixateur,  40 

Fixation  of  complement,  42 
Flagella,  23 

Loffler's  mordant  for,  51 

stain,  with  Loffler's  mordant,  61 
Flagellates,  197 

Flask  to  receive  blood-serum,  72 
Fliigge's  potato  bacillus,  98 
Fluorescence,  30 
Food,  bacteria  in,  246 
Foods  as  source  of  infection,  255 
Foot,  Madura,  230 
Formaldehyd,  263 

solid,  264 
Fowl  septicemia,  193 
Fractional    steriHzation    of    Tyn- 

dall,  65 
Frankel's  borer,  244 

method  of  cultivating  anaerobic 
bacteria,  84 
of  staining  tubercle  bacilli,  115 

pneumococcus,  157 
Fresh  egg  cultures,  77 
Fuchsin-lactose-agar,  77 
Fungi,  budding,  220 

fission,  21 

ray-,  228 

staining,  Unna's  method,  61 

thrush,  222 


Gabbet's  acid  blue,  51 

modification  of  Frankel's  method 
of  staining  tubercle  bacilH,  115 
Gametes,  200 

Gangrene,  emphysematous,  186 
Gangrenous  mastitis  of  sheep,  196 
Gas  phlegmons,  186 
Gas-formati©n,  30 
Gelatin,  69 

nutrient,  71 

potato,  76 
Gelatinous  membrane,  22 
Genito-urinary  passages,  bacteria 

in,  260 
Germicides,  261,  262 
Germination,  25 
Giemsa's  stain,  53 


Glanders,  124 

Glass  plating,  80 
slide  cultures,  78 

Glossina  morsitans,  208 
palpalis,  208 

Glycerin-agar,  69 

Gonococcus,  174 
allied  varieties,  176 
Neisser,  174 
Neisser's  stain  for,  175 
Wertheim's  medium  for,  78 

Gram's  iodin  solution,  51 

method  of  double  staining,  58 
of  tissue  staining,  58 

Granulobacillus  immobihs,  187 

Growth,  27 

Gruber-Widal    blood-serum    test, 
140 

Guinea-pig  bouillon,  78 

Guinea-pigs,  91 

Giinther's   stain   for  blood   speci- 
mens, 261 


Hands,  sterilization  of,  264 
Hanging  block,  47 

drop,  46 
Haptophore  group,  37 
Hay  bacillus,  100 
Heat,  49 

as  disinfectant,  63 

as  germicide,  262 

dry,  64 

influence  of,  on  bacteria,  28 

moist,  64 
Hemolysins,  41 
Hemolysis,  41 
Hemorrhagic  septicemia,  190 

bacteria  of,  192 
Herpes  tonsurans,  225 
Herpetomonas,  208 
Hesse's  medium  for  t3rphoid,  75 

method    of    collecting    bacteria 
from  air,  233 
of  cultivating  anaerobic  bac- 
teria, 83 
Hiss'  capsule  stain,  52,  61 

medium  for  plating,  75 
Hoffman,  pseudobacillus  of,  129 
Hog  cholera,  133 
Homogeneous  system,  43 


INDEX 


313 


Host,  susceptibility  of,  33 
Hot-air  sterilizer,  63 
Hueppe's  fresh  egg  cultures,  77 
Hydrogen  dioxid,  264 
Hydrolytic  ferments,  30 
Hydrophobia,  209 
Hygienic  laboratory  phenol  coeffi- 
cient, 265 
Hypersensitiveness,  42 
Hypersusceptibility,  42 
Hyphomycetes,  223 


Ice  cream,  248 
Immunity,  34,  35 
acquired,  35 
active,  35 
passive,  36 
Ehrlich's    lateral-chain    theory, 

36 
from  intentional  infection  or  in- 
toxication, 35 
lock  and  key  theory,  39 
Metchnikoff 's  phagocytic  theory 

of,  36 
natural,  35 
theories  of,  36 
unit,  133 
Impression  of  colonies,  88 
Incubator,  74 
Index,  opsonic,  41 

negative  phase,  41 
positive  phase,  41 
India  ink  method  of  identifying 

spirochseta  pallida,  211 
Indol  reaction  in  cholera,  152 
Infantile  paralysis,  epidemic,    218, 

219 
Infection,  30,  31 
avenues  of,  1,2 
cardinal  conditions  for,  32 
depending  on  quantity  of  bac- 
teria, 32 
endogenous,  31 
exogenous,  31 
foods  as  source  of,  255 
mixed,  33 
pathogenesis,  31 
sources  of,  31 
susceptibility  to,  33 
virulence  of,  32 


Infective  bacteria,  33 
Influenza,  160 
Infusoria,  197,  198 
Inoculating  potatoes,  manner  of, 
■     68 

Inoculation,  animal,  91.     See  also 
Animal  inoculation. 
of  culture-media,  78 
Insecticides,  263 
Insoluble  toxins,  31 
Inspissator  for  blood-serum,  73 
Instruments,  sterilization  of,  45 
Intensifiers,  48 
Intestine,  bacteria  in,  259 
Intracellular  toxins,  31 
Intraduodenal  animal  inoculation, 

93 

Intraperitoneal  injections  of  ani- 
mals, 92 

Intratracheal  inoculation   of  ani- 
mals, 93 

Intravenous  injections  of  animals, 
92 

Inverting  ferments,  29 

lodin,  264,  265 

as  used  in  Gram's  method,  49 
solution,  Gram's,  51 

Iris  blender,  44 


Jackson  bile  media,  242 
Jenner's  stain,  53 

for  malarial  organisms,  203 


Kala-azar,  208 

King's  solution  dried  blood  albu- 
min, 78 

Klatsch  preparations,  88 

Klebs-Loffler  bacillus,  126 

Klein's  method  of  staining  spores, 
60 

Koch's  alkaline  methylene-blue,  50 
bacillen  emulsion,  120 
rules  in  regard  to  bacterial  cause 
of  disease,  94 

Koch- Weeks  bacillus,  161 

Kiihne's  method  of  staining  spores,. 
60 


stam,  51 


314 


INDEX 


Laboratory,    small  requirements 

of,  85 
Lactose  litmus  agar,  72 
Lactose-bile,  76 
Lateral-chain  theory  of  immunity, 

Ehrlich's  36 
Law,  Weigert's,  38 
Leishman-Donovan  bodies,  208 
Leishman's  stain,  53 
Lens,  oil-immersion,  43 
Lepra  bacillus,  122 
Leprosy,  122 
Leptothrix  buccalis,  228 

gigantea,  228 

innominata,  228 

maxima,  228 
Liborius's  method  of   cultivating 

anaerobic  bacteria,  82 
Life   cycle  of  malarial   sporozoa, 
199 

of  protozoa,  198 
Light,  influence  of,  on  bacteria,  28 
Lime,  265 

Litmus  agar,  lactose,  72 
Lock  and  key  theory  of  immunity, 

39 
Locomotion,  23 

Loflfler's  alkaline  methylene-blue, 
50 

blood-serum  mixture,  74 

mordant  for  flagella,  51,  61 

stain  for  tissues,  59 
Luetin  reaction,  216 
Lye,  soda,  263 
Lysis,  42 


MacConkey's  bile  salt  agar,  75 
Macrocytase,  36 
Macrogamete,  201 
Macrophages,  36 
Madura  foot,  230 
Mai  de  pis,  196 

Malarial  organisms,     methods     of 
examination  for,  203 
protozoa,  estivo-autumnal  form, 
201 
forms  of,  201 
quartan  form,  201 
tertian  form,  201 
sporozoa,  life  cycle  of,  199 


Malignant  edema,  184 

pustule,  105 

tertian    form   of   malarial   pro- 
tozoa, 201 
Mallein,  126 
Malta  fever,  162 
Mash,  bread,  69 

potato,  68 
Mastigophora,  197 
Mastitis,  gangrenous,  of  sheep,  196 
Measles,  219 
Media,    culture-,    inoculation    of, 

nutrient,  preparation  of,  67 
preparation  of,  70 
reaction  of,  70 
solid  transparent,  69 
sterilization  of,  70 
Mediterranean  fever,  162 
Membrane,  gelatinous,  22 
Meningitis,    epidemic    cerebro- 
spinal, 177 
Meningococcus,  174,  177 
Mercuric  chlorid,  262 
Mercury,  bichlorid  of,  62 
Merozoites,  199 
Metals,  salts  of,  263,  264 
MetchnikofT's  theory  of  immunity, 

36 
Methylene-blue,  alkaline,  50 
Microbe  en  huit,  193 
Micrococcus  albicans,  176 

cereus  albus,  169 
fiavus,  169 

cholera  gallinarum,  193 

citreus,  176 

gonorrhoeae,  174 

melitensis,  162 

meningitidis,  177 

of  mal  de  pis,  196 

of  sputum  septicemia,  157 

pyogenes  citreus,  169 
tenuis,  169 

subflavus,  176 

tetragenus,  170 

ureae,  102 
Microcytase,  36 
Microgametes,  201 
Microphages,  36 
Microscope,  43 
Microsporon  furfur,  224,  226 


INDEX 


315 


Milk  as  source  of  contagion,  253 

bacillus  coli  in,  Wisconsin  test 
for,  255 

bacteria  in,  246,  249 

classification  of,  247 

condensed,  bacteria  in,  255 

culture-medium,  77 

examination  of,  252 
for  tubercle  bacilli,  116 

pasteurized,  247,  253 

pure,  246 

raw,  247 

red,  252 

yellow,  252 
Milzbrand  bacillus,  105 
Mixed  infection,  ;^^ 
Moist  heat,  64 

Moisture,    influence   of,    on    bac- 
teria, 28 
Molds,  220,  221 

examination  of,  226 

true,  223 
Morax-Axenfeld    diplobacillus    of 

conjunctivitis,  171 
Mordant,  Loffler's,  for  flagellaj  51 

for  flagella,  61 
Mordants,  48 

Moro's  tuberculin  test,  121 
Mouse  septicemia,  195 
Mouth,  bacteria  in,  258 

white,  222 
Mucor  mucedo,  224 
Mycetoma,  230 
Mycobacterium  tuberculosis,  no 


Nagana,  204,  206 
Naphthahn,  264 
Nasal  cavity,  bacteria  in,  259 
NeedlQs,  platinum,  45 

sterilization  of,  79 
Negri  bodies,  209 
Neisser's  gonococcus,  174 

stain  for  diphtheria,  52 
for  gonococcus,  175 
NicoUe's  stain,  52 
Nitiagin,  246 
Nitrate  broth,  70,  71    - 
Nitrification,  245 

by  bacteria,  29 
Nitro-bacterine,  246 


Nocardia  farcinica,  231 
Noguchi's  luetin  reaction,  216 
Novy's  jars,  85 
Nutrient  agar,  71 

blood-serum,  preparation  of,  72 

broth,  71 

culture-media,  preparation,   67, 
70 

gelatin,  71 


Obligate  aerobes,  28 

anaerobes,  28 
Odors  from  bacteria,  30 
Oidiomycosis,  223 
Oidium,  221,  223 

albicans,  222 

coccidioides,  223 

lactis,  222 
Oil-immersion  lens,  43 
Oocysts,  201 
Ophthalmic  tuberculin  reaction  of 

Calmette,  121 
Opsonic  index,  41 

negative  phase,  41 
positive  phase,  41 
Opsonins,  41 

Orcein  stain,  Bowhill's,  60 
Origin,  26 

Oxidation  by  bacteria,  29 
Oxygen,  influence  of,  on  life  and 

growth  of  bacteria,  27 


Pappenheim's  method  for  staining 
tubercle  bacilli  in  urine,  115 

Paracolon  bacillus,  143 

Paralysis,  epidemic  infantile,  218, 
219 

Parasites,  27 

Parasitic  bacteria,  27 
thrush,  222 

Paratyphoid  bacilU,  143 

Park's  method  of  cultivating  an- 
aerobic bacteria,  85 

Pasteur-Chamberland  filter,  237 

Pasteurized  milk,  247,  253 

Pathogenic  bacteria,  29,  97 
table  of,  287-303 
yeasts,  222 

Pear  blight,  232 


3i6 


INDEX 


Penicillium  glaucum,  223 
Peptone  water,  modified  Dunham, 

77 
Pertussis,  161 
Pest,  190 
Petri  dish,  80 

method    of    collecting    bacteria 
from  air,  232,  233 

sand-filter,  233 
Pfeiffer's  capsule  bacillus,  159 

cholera  reaction  and  agglutina- 
tion, 153,  154 
Phagocytes,  36 
Phagocytic   theory  of  immunity, 

Metchnikoff's,  36 
Phenol,  263 

coefficient,     Hygienic     Labora- 
tory, 265 

solutions,  50 
Phlegnjonous  emphysema,  186 
Phlegmons,  gas,  186 
Phosphorescence,  30 
Pigmentation,  30 
Pink  eye,  161,  258 
Piroplasma  bigeminum,  208 

bovis,  208 

hominis,  209 
Pityriasis  versicolor,  226 
Plague,  bubonic,  190 
Plant  diseases  due  to  bacteria,  231 
Plants,  splitting,  21 
Plasmodium  falciparum,  201 

malariae,  201 

vivax,  201 
Plate  cultures,  79 

method  of  examining  milk  for 
bacteria,  253 
Plating,  glass,  80 

streaked  surface,  79 
Platinum  needles,  45 
Pneumococcus,  157 

Frankel's,  157 
Pneumonia,  155 

bacteria  in,  155 
Poliomyelitis,  infantile,  218,  219 
Potassium  iodid  medium,  76 

permanganate,  264 
Potato  as  culture-media,  67 

as  medium,  67 

bacillus  of  Fliigge,  98 

mash,  68 


Potatoes,  manner  of  inoculating, 
68 

test-tube.  68 
Potato-gelatin,  76 
Precipitin  serum,  40 
Precipitins,  39 

Prescott-Breed  method  of  exam- 
ining milk  for  bacteria,  252 
Preservatives,  262 
Protein  contents  of  bacterial  cell, 

29 
Proteolytic  ferments,  29 
Proteus  mirabilis,  147 

Zenkeri,  148 
Protozoa,  197 

life-cycle  of,  198 

malarial,  estivo-autumnal  form, 
201 
forms  of,  201 
quartan  form,  201 
tertian  form.  201 
Pseudobacillus  of  Hoffman,  129 
Pseudodiphtheria,  129 
Ptomains,  29,  34 
Puerperal  fever,  165 
Pure  cultures  by  boiling,  81 
Pus,  bluish-green,  bacillus  of,  171 
Pustule,  malignant,  105 
Putrefaction,  30 
Pyemia,  33 
Pyocyanin,  173 
Pyogenic  bacteria,  33 

cocci,  163 


Quartan  form  of  malarial  pro- 
tozoa, 201 
Quarter-evil,  187 


Rabies,  209 

RauschlDrand,  187,  189 

Ray-fungus,  228 

Reaction.     See  Test. 
of  culture-media,  70 

Reactivity,  altered,  42 

Receptors,  36 
free,  41 

of  first  order,  36 
of  second  order,  36 
of  third  order,  37 


INDEX 


317 


Red  milk,  252 

Reduction  by  bacteria,  29 

Relapsing  fever,  217 

Removing  excess  of  stain,  55 

Rennet  curd  cheese,  255 

Reproduction,  24 

Rhinoscleroma,  159 

Rideal-Walker  standard  for  dis- 
infectants, 265 

Ring- worm,  225 

Rolled  cultures,  80 

Romanowsky's  stain,  53 

Rontgen  rays,  influence  of,  on  bac- 
teria, 28 

Root  bacillus,  99 

Rosalie  acid  solution,  Dunham's, 

77 
Rotz-bacillus,  124 
Rouget  du  pore,  194 
Roux's  double  stain,  52 

method  of  cultivating  anaerobic 
bacteria,  84 

test-tube,  68 


Saccharomyces  albicans,  221 
cerevisiae,  220 
mycoderma,  221 
niger,  221 
rosaceus,  221 

Saccharomycetes,  220 

Salicylic  acid,  264 

Salts  of  metals,  263,  264 

Sand-filter,  Petri's,  233 

Saprophytes,  27 

Sarcina,  103 
aurantica,  104 
lutea,  103 
ventriculi,  104 

Sarcodina,  197 

Schizogony,  199 

Schizomycetes,  21 

Schizophyceae,  21 

Schizophyta,  21 

Schweinerotlaufbacillus,  194 

Sedgwick's  expanded  tube  for  air- 
examination,  233 

Sedgwick-Tucker  method  of  col- 
lecting bacteria  from  air,  233 

Sedimentation    test    in    typhoid 
fever,  142 


Septicemia,  33 

fowl,  193 

hemorrhagic,  190 
bacteria  of,  192 

mouse,  195 

sputum,  micrococcus  of,  147 
Serum,  antistreptococcic,  166 ' 

antitoxic,  39 

antituberculous,  122 

blood-,  69 

coagulation  of,  72 
human,  preparation  of,  74 
nutrient,  preparation  of,  72 
preservation     of,     in     liquid 

state,  74 
sterilization  of,  73 

cytolytic,  40 

precipitin,  40 

test,  Wassermann,  42 
Sewage  bacteria,  examination  for, 
240 

bacterial  treatment  of,  243 
Sewer  air,  bacteria  in,  235 
Sexual  cycle  in  mosquito,  201 
Sheep,  gangrenous  mastitis  of,  196 
Shiga  bacillus,  199 
Side-chain    theory    of    immunity, 

EhrUch's,  36 
Skin,  bacteria  on,  257 
Sleeping  sickness,  205,  207 
Small-pox,  218 
Smear  culture,  78 
Smegma  bacillus,  123 
Smith's  fermentation  tube,  81 
Soap,  264 
Soda  lye,  263 
Soil  bacteria  in,  232,  243 

examination  of,  232,  243 

fertility,  bacteria  and,  244 
Soluble  toxins,  31 
Soor,  222 

Spatula  for  lifting  sections,  57 
Specimens,  drying,  54 
Spirillum,  103 

aquatilis,  154 

berolinense,  154 

bonhoffii,  154 

cholerae,  148 

allied  varieties,  154 
products,  152 

danubicum,  154 


3i8 


INDEX 


Spirillum  dunbarii,  154 

milleri,  154 

obermeieri,  217 

of  relapsing  fever,  217 

of  Wernicke,  154 

rubrum,  103 

schuylkilliensis,  154 

weibeli,  154 
Spirochaeta  pallida,  209 
Spironema  pallidum,  209 
Splenic  fever,  105 
Splenomegaly,  tropical,  208 
Splitting  plants,  21 
Sporangium,  224 
Spore  contents,  25 

formations,  24 
requisites  for,  25 
Spores,  resistance  of,  26 

staining,  59 

Klein's  method,  60 
Kiihne's  method,  60 
Weigert's  method,  61 
Sporidium  vaccinale,  219 
Sporocyte,  199 
Sporogenic  bodies,  25 
Sporogony,  199 
Sporozoa,  197,  198 

malarial,  life  cycle  of,  199 
Sporozoites,  201 
Sputum    septicemia,    micrococcus 

of,  157 
Stab  culture,  78 
Stain,  alkaline,  49 

Bowhill's  orcein,  60 

capsule,  61 

Buerger's  method,  61 
Hiss'  method,  61 

carbolthionin,  52 

flagella,  with  Loflfler's  mordant, 
61 

Frankel's,  for    tubercle    bacilli, 

Giemsa's,  53 

Giinther's,  for  blood  specimens, 

261 
Hiss'  capsule,  52,  61 
Jenner's,  53 

for  malarial  organisms,  203 
Koch's,  50 
Kiihne's,  51 
Leishman's,  53 


Stain,  Loflfler's,  50 
for  tissues,  59 
Neisser's,  for  diphtheria,  52 

for  gonococcus,  175 
NicoUe's,  52 
removing  excess  of,  55 
Romanowsky's,  53 
Roux's  double,  52 
Welch's  capsule,  53 
Wright's,  53 

for  malarial  organisms,  204 
Ziehl-Neelsen,  50 
Staining,  47 

blood  specimens,  57,  261 
fungi,  Unna's  method,  61 
general  method,  54 
Gram's  double  method,  58 

for  tissues,  58 
malarial  organisms,  203 
of  tissue  sections,  54,  56,  57 
solutions,  48 
compound,  48 
concentrated,  48 
formulas  of,  49 
stock,  48 
special  methods,  58 
spores,  59 

Klein's  method,  60 
Kuhn's  method,  60 
Weigert's  method,  61 
tubercle  bacilli  in  sputum,  117 
Staphylococcus,  23 
epidermidis  albus,  168 
pyogenes,  164 
albus,  168 
aureus,  166 
Steam,  superheated,  263 
Stegomyia  fasciata,  219 
Sterilization  by  filtration,  67 
fractional,  of  Tyndall,  65 
of  blood-serum,  73 
of  culture-media,  62,  70 
of  hands,  264 
of  instruments,  45 
of  needles,  79 
Sterilizer,  Arnold's  steam,  64 

hot-air,  63 
Stewart-Slack  method  of  examin- 
ing milk  for  bacteria,  252 
Stock  staining  solutions,  48 
Stomach,  bacteria  in,  259 


INDEX 


319 


Streaked  surface  plating,  79 
Streptococcus,  23 

acidi  lactici,  250 

erysipelatis,  164 

in  diphtheria,  133' 

lanceolatus,  157 

puerperaHs,  165 

pyogenes,  164 
Streptothrices,  227 
Streptothrix  actinomyces,  228 

farcinica,  231 

madurae,  230 
Stroke  culture,  78 
Structure,  21,  22 

Subcutaneous  inoculation  of  ani- 
mals, 92 
Substance  sensibilatrice,  40 
Sugar  broths,  71 
Sulphur  dioxid,  264 
Sulphurous  acid  gas,  264 
Suppurative  bacteria,  ^$ 
Surra,  204 
Susceptibihty,  S3 

acquired,  ss 

inherited,  S3 

natural,  37, 
Swine,  erysipelas  of,  194 
Symptomatic  anthrax,  187 
Syphilis,  209 

luetin  reaction  in,  216 

Wassermann  reaction  in,  211 
results,  216 
Noguchi  modification,  214 


Temperature,    influence    of,    on 

life  and  growth  of  bacteria,  27 
Tertian  form  of  malarial  protozoa, 

201 
Test,  agglutination,  for  tubercle  ba- 
cilli, 121 
in  cholera,  152 
in  typhoid  fever,  140 
Pfeiffer's  cholera,  153,  154 
Bordet's  cholera,  155 
Gruber-Widal  blood-serum,  140 
luetin,  216 

Pfeiffer's  cholera,  153,  154 
sedimentation,  in  typhoid  fever, 
142 


Test,  tuberculin,  Calmette's  oph- 
thalmic, 121 
Moro's,  121 
von  Pirquet's,  121 

Wassermann,  in  syphilis,  211 
Noguchi's  modification,  214 
results,  216 

Widal,  in  typhoid  fever,  140 

Wisconsin,   for  bacillus  coli   in 
milk,  255 
Test-tube,  66 

cultures,  78 

potatoes,  68 

Roux's,  68 
Tetanolysin,  183 
Tetanospasmin,  182 
Tetanus,  180 

antitoxin,  183 
unit  for,  183 
Texas  cattle-fever,  208 
Thermostat  for  blood-serum,  73 
Thrush  fungus,  222 

parasitic,  222 
Thrust-culture,  78 
Tick  fever,  African,  218 
Tinea,  225,  226 
Tissue  preparations,  56 
Torula  cerevisiae,  220 
Toxalbumins,  30,  34 
Toxemia,  33 
Toxins,  30,  31,  34 

of  diphtheria,  131 

extracellular,  31 

insoluble,  31 

intracellular,  31 

nature  of,  34 

soluble,  31 
Toxoid  of  diphtheria,  131 
Toxone  of  diphtheria,  131 
Toxophore  group,  37 
Treponema  pallidum,  209 
Trichophyton  tonsurans,  224,  225 
Tricresol,  263 

Tropical  splenomegaly,  208 
Trypanosoma,  204 

brucei,  206 

castellani,  207 

equiperdum,  208 

Evansi,  208 

hominis,  207 

lewisi,  205 


320 


INDEX 


Trypanosoma  neprevi,  207 

Rougetii,  208 

ugandense  gambiense,  207 
Trypanosomes,  204 
Trypanosomiasis,  human,  207 
Tsetse  fly,  208 
Tsetse-fly  disease,  206 
Tubercle  bacillus,  no 
products  of,  120 
Tuberculin  B.  E.,  120 

Denys'  B.  F.,  120 

Koch's  B.  E.,  120 

R,  120 

reaction,   ophthalmic,    of    Cal- 
mette,  121 

residuum,  120 

test,  Moro's,  121 
von  Pirquet's,  121 

use  of,  120 
Tuberculocidin,  120 
Tuberculosis,  no 

bovine,  human  tuberculosis  and, 
relation,  119 

in  animals,  119 
Tubes,  Esmarch's,  80 
Tyndall's   fractional   sterilization, 

65 
Types,  22 

Typhoid  carriers,  139 
fever,  135 

sedimentation  test  in,  142 
Widal  reaction  in,  140 
medium,  Eisner's,  76 
for  typhoid,  75 
Typho  toxin,  139 
Typhus  fever,  219 


Ultra-microscopic  organisms 

218 
Unna's  borax  methyl-blue,  51 

method  for  staining  fungi,  61 
Urethra,  bacteria  in,  260 
Urine,  bacillus  coli  in,  102 

bacteria  in,  102,  260 

examination  of,  for  tubercle  be 
cilli,  115 


Vaccines,  95 

antistreptococcic,  166 


Vaccines,  antityphoid,  139 

bacterial,  95 
Vaccinia,  218 
Vagina,  bacteria  in,  260 
Variola,  218 

Vibratory  movements,  23 
Vibrio  cholerae,  148 

Finkler-Prior,  154 

Metchnikovii,  154 

tyrogenum,  154 
Vibrion  butyrique  of  Pasteur,  251 

septique,  184 
Vinegar  fermentation,  255 
Virulence  of  infection,  32 
von     Pirquet's     dermo-tuberculin 

test,  121 


Wassermann  reaction  in  syphilis, 
211 
Noguchi  modification,  214 
results,  216 
Water,  artesian  well,  236 

bacillus    coli     in,     examination 
for,  240 
typhosus  in,  diagnostic  points 
of,  242 
bacteria  in,  method  of  examina- 
tion, 238 
varieties,  238 
boiUng  of,  as  means  of  purifying, 

238 
cholera  bacillus  in,  152 
examination  of ,  232,  235,  238 
filtered,  236 
purity  of,  235 
well,  236 
Weak  solutions,  50 
Weigert's  law,  38 

method  of  staining  spores,  61 
Welch's  capsule  stain,  53 
Well  water,  236 
Wernicke's  spirillum,  154 
Wertheim's  medium  for  gonococ- 

cus,  78 
White  mouth,  222 
Whooping-cough,  161 
Widal  reaction  in  typhoid  fever, 

140 
Wire  cage,  66 


INDEX 


321 


Wisconsin  test  for  bacillus  coli  in 

milk,  255 
Wolfhugel's  counter,  243 
Woolsorter's  disease,  109 
Wright's  chromatin  stain  for  ma- 
larial organisms,  204 

method  of  cultivating  anaerobic 
bacteria,  85 

stain,  53 
Wurzel  bacillus,  99 


Yaws,  217 

Yeasts,  220 

examination  of,  226 
pathogenic,  222 

Yellow  fever,  219 
milk,  25 


Ziehl-Neelsen  stain,  50 
Zooglea,  23 


21 


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Jefferson  Medical  College,  Philadelphia.  Octavo  of  1309  pages, 
with  356  text-cuts  and  ^^  plates.     Cloth,  $6.50  net. 

Eighth  Edition  published  November,  1916 

The  demand  for  eight  editions  of  this  work  in  such  a  short  period  indicates 
the  practical  character  of  the  book.  In  this  edition  the  articles  on  Frambesia, 
Oriental  Sore,  and  other  tropical  diseases  have  been  entirely  rewritten.  The  new 
subjects  include  Occupational  Dermatoses,  Paraflfinoma,  Purpura  Annularis,  Telan- 
giectodes, Xanthoma  Elasticum,  and  Ulerythema  Ophryogenes.  George  T.  Elliot, 
M.  D.,  Professor  of  Dermatology,  Cornell  University,  says:  "It  is  a  book  that  I 
recommend  to  my  class  at  Cornell,  because  for  conservative  judgment,  for  accurate 
observation,  and  for  a  thorough  appreciation  of  the  essential  position  of  dermatol- 
ogy, I  think  it  holds  first  place." 

Our  books  are  revised  frequently,  so  that  the  editions 
you  find  here  may  not  be  the  latest.  Write  us 
about     any    books    in    which     you     are     interested 


SAUXDEJ^S'    BOOKS   ON 


Schamber^'s  Diseases  of   the 
Skin  and  Eruptive  Fevers 

Diseases  of  the   Skin   and    Eruptive   Fevers.     By  Jay 

F.  ScHAMBERG,  M.  D.,  Profcssor  of  Dermatology  and  the  In- 
fectious Eruptive  Diseases,  Philadelphia  Polyclinic.  Octavo  of 
585  pages,  illustrated.     Cloth,  $3.25  net. 

THIRD  EDITION— published  September.  1915 

Dr.  Schamberg  takes  up  all  diseases  of  the  skin,  giving  special 
emphasis  to  those  diseases  met  most  frequently  in  general  practice. 
The  work  is  particularly  full  on  actinotherapy,  rontgenotherapy,  and 
radium,  these  modern  measures  being  discussed  in  a  separate  chap- 
ter as  well  as  under  the  various  diseases.  The  exanthemata  are 
considered  in  a  special  chapter,  diagnosis  and  treatment  being  given 
unusual  space.  In  addition,  there  are  described  the  usual  and  the 
accidental  eruptions  occurring  in  such  diseases  as  typhoid,  epidemic 
cerebrospinal  meningitis,  influenza,  malaria,  tonsilHtis,  etc.  This 
is  an  important  feature.  All  the  new  vaccines  and  serums  are  con- 
sidered— their  use  both  in  diagnosis  and  treatment.  The  many 
comparative  tables  of  symptoms  and  the  wealth  of  reHable  pre- 
scriptions make  "  Schamberg  "  a  most  practical  work  for  the  gen- 
eral practitioner  as  well  as  for  the  specialist. 

Johns  Hopkins  Hospital  Bulletin 

"The  descriptions  of  the  eruptions  are  so  clear  and  concise  that  the  appearance  of  a 
disease  can  readily  be  imagined.  The  arrangement  of  diagnosis  of  many  of  the  diseases  is 
excellent,  the  points  considered  being  placed  opposite  one  another  in  parallel  rows." 

Asher's  Chemistry  (b  Toxicology  for  Nurses 

Chemistry  and  Toxicology  for  Nurses.  By  Philip  Asher, 
Ph.  G.,  M.  D.,  Dean  and  Professor  of  Chemistry,  New  Orleans  Col- 
lege of  Pharmacy.     l2mo  of  190  pages.  Cloth,  ;?i.25  net. 

Dr.  Asher's  one  aim  in  writing  this  book  was  to  emphasize  throughout  the  applica- 
tion of  chemical  and  toxicologic  knowledge  in  the  practice  of  nursing.  This  he  has 
succeeded  in  doing.  The  nurse,  both  in  training-school  and  in  graduate  practice, 
will  find  it  extremely  helpful  because  the  subject  is  made  so  clear.        October,  1914 


GENirO-URINARY  DISEASES. 


Norris*  Gonorrhea  in  Women 

Gonorrhea  in  Women.  By  Charles  C.  Norris,  M.  D., 
Instructor  in  Gynecology,  University  of  Pennsylvania.  With  an 
Introduction  by  John  G.  Clark,  M.  D.,  Professor  of  Gynecology, 
University  of  Pennsylvania.  Large  octavo  of  520  pages,  illus- 
trated.    Cloth,  $6.50  net. 

A  CLASSIC 

Dr.  Norris  here  presents  a  work  that  is  destined  to  take  high  place  among 
publications  on  this  subject.  He  has  clone  his  work  thoroughly.  He  has 
searched  the  important  literature  very  carefully,  over  2300  references  being 
utilized.  This,  coupled  with  Dr.  Norris'  long  experience,  gives  his  work  the 
stamp  of  authority.  The  chapter  on  serum  and  vaccine  therapy  and  organo- 
therapy is  particularly  valuable  because  it  expresses  the  newest  advances. 
Every  phase  of  the  subject  is  considered :  History,  bacteriology,  pathology 
sociology,  prophylaxis,  treatment  (operative  and  vci&^xzwLdX)^  gonorrhea  during 
pregnancy,  parturition  and  the  puerperiuviy  diffuse  gonorrheal  peritonitis,  and 
all  other  phases.  Further,  Dr.  Norris  considers  the  rare  varieties  of  gonorrhea 
occurring  in  men,  women,  and  children.  Published  May,  1913 


Coolid^e  on  Nose  and  Throat 

Manual  of  Diseases  of  the  Nose  and  Throat.  By  Algernon 
CooLiDGE,  M.  D.,  Professor  of  laryngology.  Harvard  Medical 
School.     Octavo  of  360  pages,  illustrated.     Cloth,  $1.50  net. 

READY  REFERENCE 


This  new  book  furnishes  the  student  and  practitioner  a  guide  and  ready 
reference  to  the  important  details  of  examination,  diagnosis,  and  treatment. 
P^stablished  facts  are  emphasized  and  unproved  statements  avoided.  Anat- 
omy and  physiology  of  the  different  regions  are  reviewed. 

PubUshed  September,  191S 


SAUNDERS'  BOOKS  ON 


Braasch's  Pyelography 

Pyelography  (Pyelo-Ureterography).  By  William  F. 
Braasch,  M.  D.,  Mayo  Clinic,  Rochester,  Minn.  Octavo  of 
323  pages,  with  296  pyelograms.     Cloth,  $5.00  net. 

296  PYELOGRAMS 

This  new  work  is  the  first  comprehensive  collection  of  pyelograms  ever 
issued  in  book  form.  The  300  pyelograms  included  were  selected  from 
several  thousand  made  at  the  Mayo  Clinic  during  the  past  five  years.  You  get 
the  outlines  of  normal  pelves,  those  of  pathologic  conditions,  and  those  of  con- 
genitally  abnormal  pelves.  In  addition  to  the  pyelograms,  you  get  a  des- 
criptive text,  intrepreting  the  outlines,  pointing  out  their  great  value  in  diagnosis. 
You  get  the  history  of  pyelography  and  the  exact  technic — selection  of 
medium  to  be  injected,  preparation  of  solution,  method  of  injection,  sources 
of  error,  etc.  The  work  is  a  most  complete  one,  beautifully  gotten  up,  and 
contains  much  matter  of  great  diagnostic  value.  Published  March,  1915 


Og'den  on  the  Urine  TWrd  Edition 

Clinical  Examination  of  Urine  and  Urinary  Diag- 
nosis. A  Clinical  Guide  (or  the  Use  of  Practitioners  and 
Students  of  Medicine  and  Surgery.  By  J.  Bergen  Ogden, 
M.  D.,  Medical  Chemist  to  the  Metropolitan  Life  In- 
surance Company,  New  York.  Octavo,  418  pages,  54  text- 
illustrations,  and  a  number  of  colored  plates.     Cloth,  $3.00 

net.  Published  October,  1909 

"We  consider  this  manual  to  have  been  well  compiled  ;  and  the  author's  own 
experience,  so  clearly  stated,  renders  the  volume  a  useful  one  both  for  study 
and  reference." — The  Lancet,  London. 

Vecki's  Sexual  Impotence  Fifth  Edition 

Sexual  Impotence.  By  Victor  G.  Vecki,  M.  D. 
i2mo  volume  of  400  pages.     Cloth,  ;g2.25  net. 

"A  scientific  treatise  upon  an  important  and  much  neglected  subject.  .  .  .  The 
treatment  of  impotence  in  greneral  and  of  sexual  neurasthenia  is  discriminating 
and  judicious."— /^A«j  Hopkins  Hospital  Bulletin.     Published  December,  1915 


DISEASES   OF   THE   EYE. 


DeSchweinitzV 
Diseases  of  the  Eye 

The  New  (8th)  Edition 

Diseases  of  the  Eye:  A  Handbook  of  Ophthalmic 
Practice.  By  G.  E.  deSchweinitz,  M.  D.,  Professor  of 
Ophthalmology  in  the  University  of  Pennsylvania,  Philadelphia, 
etc.  Handsome  octavo  of  754  pages,  386  text-illustrations, 
and  7  chromo-lithographic  plates.       Cloth,  $6.00  net. 

Published  June,  1916 

WITH   386  TEXT-ILLUSTRATIONS  AND  7  COLORED  PLATES 

Dr.  deSchweinitz's  book  has  long  been  recognized  as  a  standard  authority 
upon  eye  diseases,  the  reputation  of  its  author  for  accuracy  of  statement 
placing  it  far  in  the  front  of  works  on  this  subject.  For  this  edition  Dr. 
deSchweinitz  has  subjected  his  book  to  a  most  thorough  revision.  Many 
new  subjects  have  been  added,  a  number  in  the  former  edition  have  been 
rewritten,  and  throughout  the  book  reference  has  been  made  to  vaccine  and 
serum  therapy,  to  the  relation  of  tuberculosis  to  ocular  disease,  and  to  the 
value  of  tuberculin  as  a  diagnostic  and  therapeutic  agent. 

The  text  is  fully  illustrated  with  black  and  white  cuts  and  colored  plates, 
and  in  every  way  the  book  maintains  its  reputation  as  an  authority. 

Johns  Hopkins  Hospital  Bulletin 

"  No  single  chapter  can  be  selected  as  the  best.  They  are  all  the  product  of  a  finished 
authorship  and  the  work  of  an  exceptional  ophthalmologist.  The  work  is  certainly  one  of 
the  best  on  ophthalmology  extant,  and  probably  the  best  by  an  American  author." 

deSchweinitz    and    Holloway    on    Pulsating 

£X0phthaIm0S  published  August,  1908 

Pulsating  Exophthalmos.  An  analysis  of  sixty-nine  cases  not  pre- 
viously analyzed.  By  George  E.  deSchweinitz,  M.  D.,  and  Thomas 
B.  Holloway,  M.  D.     Octavo  of  125  pages.     Cloth,  $2.00  net. 

"The  book  deals  very  thoroughly  with  the  whole  subject,  and  in  it  the  most  com 
plete  account  of  the  disease  will  be  found."—  British  Medical  Journal. 

Jackson's  Essentials  of  Eye         Fourth  Revised  Edition 

Essentials  of  Refraction  and  of  Diseases  of  the  Eye.  By 
Edward  Jackson,  A.  M.,  M.  D.,  Emeritus  Professor  of  Diseases  of  the 
Eye,  Philadelphia  Polyclinic.  Post-octavo  of  261  pages,  82  illustra- 
tions.    Cloth,  $1.25  net.     In  Saunders^  Question-Compend  Series. 

"The  entire  ground  is  covered,  and  the  points  that  most  need  careful  elucidation  are 
made  clear  and  ea.sy."— Johns  Hopkins  Hospital  Bulletin.  Published  AprU,  1906 


SAUNDERS'    BOOKS   OX 


GET  A*^^-«I^#v*  THE     NEW 

THE  BEST  /\II16riCan  STANDARD 

Illustrated  Dictionary 

The  New  (9th)  Edition 


The  American  Illustrated  Medical  Dictionary.    A  new 

and  complete  dictionary  of  the  terms  used  in  Medicine,  Surgery, 
Dentistry,  Pharmacy,  Chemistry.  Veterinary  Science,  Nursing, 
and  all  kindred  branches;  with  over  loo  new  and  elaborate 
tables  and  many  handsome  illustrations.  By  W.  A.  Newman 
Borland,  M.D.     Large  octavo  of  1 179  pages.     Flexible  leather, 

I5.OO  net;    with  thumb  index,  ^5.50  net.       Published  September,  1917 
OVER  2000  NEW  WORDS  IN  THIS  EDITION 

For  this  edition  the  book  has  been  subjected  to  a  thorough  revision  and 
entirely  reset,  adding  thousands  of  important  new  terms.  This  work  is  more 
than  a  medical  dictionary — it  is  a  Medical  Encyclopedia. 

Howard  A.  Kelly,  M.D., 

Professor  of  Gynecologic  Surgery,  Johns  Hopkins  University,  Baltimore 

"Dr.  Dorland's  Dictionary  is  admirable.     It  is  so  well  gotten  up  and  of  such  conve- 
nient size.     No  errors  have  been  found  in  my  use  of  it." 


Pilcher's  Practical  Cystoscopy 

Practical  Cystoscopy.  By  Paul  M.  Pilcher,  M.D.,  Con- 
sulting Surgeon  to  the  Eastern  Long  Island  Hospital.  Octavo  of 
504  pages,  with  299  illustrations,  29  in  colors.    Cloth,  $6.00  net. 

SECOND  EDITION— published  November,  1915 

To  be  properly  equipped,  you  must  have  at  your  instant  command  the 
information  this  book  gives  you.  It  explains  away  all  difficulty,  telling  you 
7v/iy  you  do  not  see  something  when  something  is  there  to  see,  and  telling  you 
/io7a  to  see  it.  All  theory  has  been  uncompromisingly  eliminated,  devoting 
every  line  to  practical,  needed  every-day  facts,  telling  you  how  and  when  to 
use   the  cystoscope  and  catheter — telling  you  in  a  way  to  make  you  hio7C'. 

Bransford  Lewis*  M.  D.,  S^.  Louis  University 

"  I  am  very  much  pleased  with  Dr.  Pilcher's  '  Practical  Cystoscopy.'  I  think  it  is  the 
best  in  the  English  language  now." 


DISEASES   OF   THE   EYE. 


Haab  and  DeSchweinitz's 
External  Diseases  qf  the  Eye 

(Published  February,  1909) 

Atlas  and  Epitome  of  External  Diseases  of  the  Eye. 

By  Dr  O.  Haab,  of  Zurich.  Edited,  with  additions,  by  G.  E. 
deSchweinitz,  M.  D.,  Professor  of  Ophthalmology,  University  of 
Pennsylvania.  loi  colored  illustrations  and  244  pages  of  text. 
Cloth,  ;^3.oo  net.  Third  Edition.  Saumiefs'  Atlases. 


Stokes*  The  Third  Great  Plague 

The  Third  Great  Plague:   A  Discussion  of  Syphilis  for 
Every=day  People.     By  John  H.  Stokes,  A.  B.,  M.  D.,  Head 

of  Section  on  Dermatology  and  Syphilology,  Division  of  Med- 
icine, The  Mayo  Clinic.     i2mo  of  204  pages.     Cloth,  $1.50 

net.  Published  October,  1917 

American  Pocket  Dictionary      New  (loth)  Edition 

The  American  Pocket  Medical  Dictioxary.  Edited  by  W.  A. 
Newman  Borland,  M.  D.  Containing  the  definition  of  the  principal 
words  used  in  medicine  and  kindred  sciences.  707  pages.  Flexible 
leather,  with  gold  edges,  $1.25  net;  with  thumb  index,  $1.50  net. 

"  I  am  struck  at  once  with  admiration  at  the  compact  size  and  attractive  exterior. 
I  can  recommend  it  to  our  students  without  reserve." — James  W.  Holland,  M.  D.. 
Emeritus  Professor  of  Medical  Chemistry  and  Toxicology  at  the  Jeferson  Medical  College, 
Philadelphia.  Published  September,  1917 

Jackson  on  the  Eye  Prepanngf-New  (3d)  Edition 

A  Manual  of  the  Diagnosis  and  Treatment  of  Diseases  of 
THE  Eye.  By  Edward  Jackson,  A.  M.,  M.  D.,  Professor  of  Ophthal- 
mology, University  of  Colorado.  i2mo  of  615  pages,  with  184  illus- 
trations. 


SAUNOEKS'    BOOKS    ON 


Barnhill  and   Wales' 
Modern     Otology 

A  Text-Book  of  Modern  Otology.  By  John  F.  Barn- 
hill,  M.  D.,  Professor  of  Otology,  Laryngology,  and  Rhinology, 
and  Earnest  de  W.  Wales,  M.  D.,  Clinical  Professor  of 
Otology,  Laryngology,  and  Rhinology,  Indiana  University  School 
of  Medicine,  Indianapolis.  Octavo  of  598  pages,  with  314  original 
illustrations.     Cloth,  $5.50  net. 

SECOND  EDITION 

This  work  represents  the  resuUs  of  personal  experience  as  practitioners  and 
teachers,  influenced  by  the  instruction  given  by  such  authorities  as  Sheppard, 
Dundas  Grant,  Percy  Jakins,  Jansen,  and  Alt,  Much  space  is  devoted  to 
prophylaxis,  diagnosis,  and  treatment,  botli  medical  and  surgical.  There  is  a 
special  chapter  on  the  bacteriology  of  ear  affections — a  feature  not  to  Ijc  found 
in  any  other  work  on  otology.  Great  pains  have  been  taken  with  the  illus- 
trations.    A  large  number  represent  the  best  work  of  Mr.  H.  F.  Aitken. 

Frank  AUport.  M.  D., 

Professor  of  Otology,  Northwestern  University,  Chicago. 

"I  regard  it  as  one  of  the  best  books  in  the  English  language  on  this  subject.  The 
pictures  are  especially  good,  particularly  as  they  are  practically  all  original  and  not  the  old 
reproduced  pictures  so  frequently  seen."  Published  January,  1911 


Davis*  Accessory  Sinuses 

Development  and  Anatomy  of  the  Nasal  Accessory 
Sinuses  in  Man.  By  Warren  B.  Davis,  M.  D.,  Corinna 
Borden  Keen  Research  Fellow  of  the  Jefferson  Medical  College, 
Philadelphia.  Octavo  of  172  pages,  with  57  original  illustra- 
tions. Published  March,  1914  Cloth,  $3.50  net. 
ORIGINAL  DISSECTIONS 

This  book  is  based  on  the  study  of  two  hundred  and  ninety  lateral  nasal 
walls,  presenting  the  anatomy  and  physiology  of  the  nasal  accessory  sinuses 
from  the  sixtieth  day  of  fetal  life  to  advanced  maturity.  It  was  necessary  for 
Dr.  Davis  to  develop  a  ne7v  technic  by  which  the  accessory  sinus  areas  could 
be  removed  en  fuasse  at  the  time  of  postmortem  examinations,  and  still  per- 
mit of  reconstruction  of  the  face  without  marked  disfigurement. 


GEXITO  URINARY  AND  NOSE,  THROAT,  Etc.  9 

Greene  and  Brooks' 
Genito-Urinary  Diseases 

A  Text-Book  of  Genito-Urinary  Diseases.  By  Robert 
H.  Greene,  M.D.,  Professor  of  Genito-Urinary  Surgery  at 
Fordham  University;  and  Harlow  Brooks,  M.  D.,  Assistant  Pro- 
fessor of  Clinical  Medicine,  Universityand  Belleviie  Hospital  Medi- 
cal School.     Octavo  of  666  pages,  illustrated.     Cloth,  $5.50  net. 

FOURTH  EDITION— published  May.  1917 

This  new  work  covers  completely  the  subject  of  genito-urinary  diseases, 
presenting  both  the  medical  and  surgical  sides.  Kidney  diseases  are  very  elabo- 
rately detailed. 

New  York  Medical  Journal 

"  As  .a  whole  the  book  is  one  of  the  most  satisfactory  and  useful  works  on  genito- 
urinary   diseases    now  extant,  and  will  undoubtedly  be  popular  among  practitioners  and 


students." 


Gleason  on  Nose,  Tliroat, 
and  Ear 

A  Manual  of  Diseases  of  the  Nose,  Throat,  and  Ear.     By 

E.  Baldwin  Gleason,  M.  D.,  LL.  D.,  Professor  of  Otology, 
Medico-Chirurgical  College,  Graduate  School  of  Medicine,  Uni- 
versity of  Pennsylvania,  Philadelphia.  i2mo  of  590  pages,  pro- 
fusely illustrated.      Cloth,  $2.75  net.  Published  October,  1914 

THIRD  EDITION 

Methods  of  treatment  have  been  simplified  as  much  as  possible,  so  that  in 
most  instances  only  those  methods,  drugs,  and  operations  have  been  advised 
which  have  proved  essential.     A  feature  consists  of  the  collection  of  formulas. 

American  Journal  of  the  Medical  Sciences 

"  For  the  practitioner  who  wishes  a  reliable  guide  in  laryns^ology  and  otology  there  ar 
few  books  which  can  be  more  heartily  commended." 

Wilcox  on  Genito-Urinary  and  Venereal  Dis- 
eases Second  Edition,  published  January.  1909 

Essentials  of  Genito-Urtnary  and  Venereal  Diseases.  By 
St.arling  S.  Wilcox,  M.  D.,  Lecturer  on  Genito-Urinary  Diseases  and 
Syphilology,  Starling-Ohio  Medical  College,  Columbus,  Ohio.  i2mo  of 
321  pages,  illustrated.  Cloth,  $1.25  net.  In  Saunders'  Quest ion-Com- 
pends. 


SAUNDERS'    BOOKS   ON 


Head's  Modern  Dentistry 

Modern  Dentistry.  By  Joseph  Head,  M.  D.,  D.D.S.,  Den- 
tist to  the  Jefferson  Hospital,  Philadelphia.  Octavo  of  374 
pages,  with  309  illustrations.     Cloth,  $5.00  net. 

Published  December,  1917 

Dr.  Head's  book  is  a  complete  and  up-to-date  text-book  on  dentistry. 
It  gives  you  the  principles  upon  which  successful  work  must  be  based — 
the  technic  in  full,  and  the  results  of  original  experiments,  with  formulae, 
instruments,  and  methods.  It  brings  out  clearly  the  various  factors  which 
influence  the  diagnosis,  and  carefully  details  the  methods  of  treatment. 
The  subject  of  vaccines  is  gone  into  very  fully,  giving  you  preparation  and 
use  of  autogenous,  stock,  and  special  vaccines.  Particularly  useful  are 
the  sections  on  mouth  hygiene,  local  anesthesia  by  novocain,  electrolysis, 
tooth  discoloration,  care  of  children's  teeth  and  gums,  orthodontia  for 
the  general  practitioner  of  dentistr}^,  cement,  .v-ray  study,  and  the  use  of 
emetin.  Over  three  hundred  original. illustrations  show  the  student  just 
how  the  procedures  are  to  be  carried  out. 


Kyle's  Nose  and  Throat 

Diseases  of  the  Nose  and  Throat.  By  D.  Braden  Kyle, 
M.D.,  formerly  Professor  of  Laryngology  in  the  Jefferson  Medical 
College,  Philadelphia;  Consulting  Laryngologist,  Rhinologist, 
and  Otologist,  St.  Agnes'  Hospital.  Octavo,  856  pages;  with 
272   illustrations  and  27  lithographic  plates   in  colors.      Cloth, 

;^  4. 50  net.  Published  November,  1914 

FIFTH  EDITION 

This  work  has  now  reached  its  fifth  edition.  With  the  practical  purpose 
of  the  book  in  mind,  extended  consideration  has  been  given  to  treatment,  each 
disease  being  considered  in  full,  and  definite  courses  being  laid  down  to 
meet  special  conditions  and  symjitoms. 

Pennsylvania  Medical  Journal 

"  Dr.  Kyle's  crisp,  terse  diction  has  enabled  the  inclusion  of  all  needful  nose  and  throat 
knowledge  in  this  book.  The  practical  man,  be  he  special  or  general,  will  not  search  in 
vain  for  anything  he  needs." 


CHEMISTRY  AMD    DENTISrii 


Holland's 
Chemistry  and  Toxicology 

A  Text-Book  of    Medical  Chemistry   and   Toxicology, 

By  James  W.  Holland,  M.  D.,  Emeritus  Professor  of  Medical 
Chemistry  and  Toxicology  ,  Jefferson  Medical  College,  Phila- 
delphia.    Octavo,  678  pages,  illustrated.     Cloth,  ^3.00  net. 

FOURTH  EDITION— published  April.  1915 

Dr.  Holland's  work  is  an  entirely  new  one,  and  is  based  on  his  thirty-five 
years'  practical  experience  in  teaching  chemistry  and  medicine.  Recognizing 
that  to  understand  physiologic  chemistry  students  must  first  be  informed  upon 
points  not  referred  to  in  most  medical  text-books,  the  author  has  included  in  his 
work  the  latest  views  of  equilibrium  of  equations,  mass-action,  cryoscopy,  os- 
motic pressure,  etc.     Much  space  is  given  to  toxicology. 

American  Medicine 

"  Its  statements  are  clear  and  terse  ;  its  illustrations  well  chosen;  its  development  lojfi- 
cal,  systematic,  and  comparatively  easy  to  follow.  .  .  .  We  heartily  commend  the  work." 


Ivy's  Applied  Anatomy  and  Oral  Surgery 

Applied  Anatomy  and  Oral  Surgery  for  Dental  Students. 
By  Robert  H.  Ivy,  M.  D.,  D.  D.  S.,  Assistant  Oral  Surgeon  to  the 
Philadelphia  General  Hospital.  1 2mo  of  290  pages,  illustrated.  Cloth, 
$1.75  net.  Second  Edition  published  July,  1917 

This  work  is  just  what  dental  students  have  long  wanted — a  concise,  practical  work 
on  applied  anatomy  and  oral  surgery,  written  with  their  needs  solely  in  mind.  No 
one  could  be  better  fitted  for  this  task  than  Dr.  Ivy,  who  is  a  graduate  in  both  den- 
tistry and  medicine.  The  text  is  well  illustrated  with  pictures  that  you  will  find  ex- 
tremely helpful. 

"I  am  delighted  with  this  compact  little  treatise.  It  seems  to  me  just  to  fill  the 
bill."— H.  P.  KuHN,  M.  D.,  Western  Dental  College,  Kansas  City. 

Oertel  on  Bri^ht's  Disease 

The  Anatomic  Histological  Processes  of  Bright's  Disease.  By 
HoRST  Oertel,  M.  D.,  Director  of  the  Russell  Sage  Institute  of  Path- 
ology, New  York,  Octavo  of  227  pages,  with  44  illustrations  and  6 
colored  plates.     Cloth,  jJIS-OO  net. 

These  lectures  deal  with  the  anatomic  histological  processes  of  Bright's  disease,  and 
in  a  somewhat  different  way  from  the  usual  manner.  Everywhere  relations  are  em- 
phasized and  an  endeavor  made  to  reconstruct  the  whole  as  a  unit  of  interwoven 
processes.  Published  December,  1910 

"  Dr.  Oertel  gives  a  clear  and  intelligent  idea  of  nephritis  as  a  continuous  process. 
"  We  can  strongly  recommend  this  book  as  thoughtful,  scientific,  and  suggestive." — 
The  Lancet,  London. 


SAU.XDEKS'    BOOK'S    ON 


Goepp's  Dental  State  Boards 


E>ental    State  Board   Questions  and  Answers.     By  R. 

Max  Goepp,  M.  ]).,  Professor  of  Clinical  Medicine  at  the  Phila- 
delphia Polyclinic.      Octavo  of  428  pages.     Cloth,  ;^3. 00  net. 

SECOND  EDITION 

This  new  work  is  along  the  same  practical  lines  as  Dr.  Goepp's  successful 
work  on  Medical  State  Boards.  The  questions  included  have  been  gathered 
from  reliable  sources,  and  embrace  all  those  likely  to  be  asked  in  any  State 
Board  examination  in  any  State.  They  have  been  arranged  and  classified  in 
a  way  that  makes  for  a  rapid  resume  of  every  branch  of  dental  ])ractice.  and 
the  answers  are  couched  in  language  unusually  explicit — concise,  definite, 
accurate.  Published  February,  1916 


McConnell's  Pathology  and  Bacteriology  Dent&l 

General  Pathology  and  Bacteriology"  for  Dental 
Students.  By  Guthrie  McConnell,  M.  D.,  Assistant 
Surgeon,   Medical  Reserve  Corps,   U.  S.  N.     121110  of  314 

pages,  illustrated.  Second  Edition  published  January,  1918 

This  work  was  written  expressly  for  dentists  and  denial  students,  em- 
phasizing throughout  the  application  of  pathology  and  bacteriology  in 
dental  study  and  practice.  There  are  chapters  on  disorders  of  metab- 
olism and  circulation  ;  retrogressive  processes,  cell  division,  inflam- 
mation and  regeneration,  granulomas,  progressive  processes,  tumors, 
special  mouth  pathology,  sterilization  and  disinfection,  bacteriologic 
methods,  specific  micro-organisms,  infection  and  immunity,  and  labora- 
tory technic. 


FA'E,  EAR,  NOSE,  AXD  THROAT.  13 

Bass  and  Johns'  Alveolodental  Pyorrhea 

Alvkolodental  Pyorrhea.  By  Charles  C.  Bass,  M.D.,  Professor 
of  Experimental  Medicine,  and  Foster  M.  Johns,  M.  D.,  Instructor  in 
the  Laboratories  of  Clinical  Medicine,  Tulane  Medical  College.  Octavo 
of  168  pages,  illustrated.     Cloth,  $2.50  net.  Published  June,  1915 

This  work  discusses  alveolodental  pyorrhea  from  the  viewpoint  of  infeqtion  by  the 
Endamoeba  buccalis  in  a  simple,  concise  way,  in  the  light  of  recent  information. 

Gleason's  Nose  and  Throat      Fourth  Edition.  Revised 

Essentials  of  Diseases  of  the  Nose  and  Throat.  By  E.  B. 
Gleason,  S.B.,  M.D.,  Clinical  Professor  of  Otology,  Medico-Chirurgical 
College,  Graduate  School  of  Medicine,  University  of  Pennsylvania, 
Post-octavo,  241  pages,  112  illustrations.  Cloth,  $1.25  net.  In 
Saunders^  Question-Compend  Series.  Published  October,  1914 

"The  careful  description  which  is  given  of  the  various  procedures  would  be  sufficient 
to  enable  most  people  of  average  intelligence  and  of  slight  anatomical  knowledge  to 
make  a  very  good  attempt  at  laryngoscopy."— JAe  Lancet,  London. 

Grant  on  the  Face,  Mouth,  and  Jaws 

A  Text-Book  of  the  Surgical  Principles  and  Surgical  Dis- 
eases OF  the  Face,  Mouth,  and  Jaws.  For  Dental  Students.  By 
H.  Horace  Grant,  A.  M.,  M.  D.,  Professor  of  Surgery  and  of  Clinical 
Surgery,  Hospital  College  of  Medicine,  Louisville.  Octavo  of  231  pages, 
with  68  illustrations.      Cloth,  $2.50  net.  Published  September,  1911 

Preiswerk  and  Warren's  Dentistry 

Atlas  and  Epitome  of  Dentistry.  By  Prof.  G.  Preiswerk,  of 
Basil.  Edited,  with  additions,  by  George  W.  Warren,  D.  D.  S.,  Pro- 
fessor of  Operative  Dentistry,  Pennsylvania  College  of  Dental  Surgery, 
Philadelphia.  With  44  lithographic  plates,  152  text-cuts,  and  343  pages 
of  text.     Clotli,  $3.50  net.      Saunders'  Hand-Atlases.  August,  1906 

Grunwald  and  Grayson  on  the  Larynx 

Atlas  and  Epitome  of  Diseases  of  the  Larynx.  By  Dr.  L. 
GRtJNWALD,  of  Munich.  Edited,  with  additions,  by  Charles  P. 
Grayson,  M.  D.,  University  of  Pennsylvania.  With  107  colored  figures 
on  44  plates,  25  text-cuts,  and  103  pages  of  text.  Cloth,  1^2.50  net.  In 
Saunders*  Hand-Atlas  Series.  Published  1898 

Mracek  and  Stelwagon's  Atlas  of  Skin    1'^';^;^ 

Atlas  and  Epitome  of  Diseases  of  the  Skin.  By  Prof.  Dr. 
Franz  Mracek,  of  Vienna.  Edited,  with  additions,  by  Henry  W. 
Stelwagon,  M.  D.,  Jefferson  Medical  College.  With  77  colored 
plates,  50  half-tone  illustrations,  and  280  pages  of  text.  Cloth,  $4.00 
net.     In  Saunders'  Hand-Atlas  Series.  Published  July,  1903 


14  SAUNDEXS'-  BOOKS  ON 

Theobald's 
Prevalent  Diseases  of  the  Eye 


Prevalent  Diseases  of  the  Eye.  By  Samuel  Theobald, 
M.  D.,  Clinical  Professor  of  Ophthalmology  and  Otology,  Johns 
Hopkins  University.  Octavo  of  550  pages,  with  219  text-illustra- 
tions and   10  plates.       Cloth,  $4.50  net.  Published  July,  1906 

Chas.  A.  Oliver,  M.  D.. 

Cli7iical  Professor  of  Ophthalmology,  Woman's  Medical  College,  Fhila. 
"  I  feel   I  can  conscientiously  recommend   it,  not   only  to  the  general  physician  and 
medical  student,  but  also  to  the  experienced  ophthalmologist." 


Wells*  Chemical  Patholo^ 

Chemical  Pathology.  By  H.  Gideon  Wells,  Ph.D., 
M.  D.,  Professor  of  Pathology  in  the  University  of  Chicago. 
Octavo  of  616  pages.      Cloth,  $3.25  net.  Second  Edition 

Published  March,  1914 
Wm.  H.  Welch,  M.D.,y<?/««^  Hopkms  University. 

"  The  work  fills  a  real  need  in  the  English  literature  of  a  very  important  subject,  and  I 
shall  be  glad  to  recommend  it  to  my  students." 


Stelwa|»on's  Essentials  of  Skin        seventh  Edition 

Essentials  of  Diseases  of  the  Skin.  By  Henry  W.  Stelwagox, 
M.  D.,  Ph.  D.,  Professor  of  Dermatology  in  the  Jefferson  Medical 
College,  Philadelphia.  Post-octavo  of  292  pages,  with  72  text-illustra- 
tions and  8  plates.  Cloth,  $1.25  net.  In  Saunders'  Question-Compend 
Series.  Published  August,  1909 

"  In  line  with  our  present  knowledge  of  diseases  of  the  skin.  .  .  .  Continues  to  main- 
tain the  high  standard  of  excellence  for  which  these  question  cornpends  have  beea 
noted." — The  Medical  News. 


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DATE  DUE  SLIP 

UOTVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL  LIBRARY 

THIS  BOOK  IS  DUE  ON  THE  LAST  DATE 
STAMPED  BELOW 


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Saunders'  Compends 


ESSENTIALS  OF  GYNECOLCXiY.    8th  ed.    With  57  illustrations. 
By  Edwin  B.  Cragin,  M.  D.    Revised  by  F.  S.  Mathews,  M.  D. 

.-.>^^~^'^'=^^-'*^    OF   THE    SKIN.     7th  edition. 

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