SCIENTIFIC PROCEEDINGS

FORTY-SIXTH GENERAL MEETING OF THE SOCIETY

OF AMERICAN BACTERIOLOGISTS
DETROIT, MAY 21 To 24, 1946

ABSTRACTS'
Division of General Bacteriology
G 1-6. Test Methods and Miscellaneous. G 36. Protein Metabolism and Im-
G 7-12. Physiology. munity.
G 13-23. Nutrition and Metabolism. G 37. Physical Methods and Bacteri-
G 24-30. Taxonomy. ology.
G 31-35. Induced Resistance. G 38-45. Morphology and Cytology.
Division of Agricultural and Industrial Bacteriology
A 1-8. Sanitary Bacteriology. A 27-32. Miscellaneous Studies.
A 9-17. Antibiotics. A 33-41. Disinfection and Chemotherapy.
A 18-26. Industrial Processes.
Division of Medical Bacteriology, Immunology, and Comparative Pathology
M 1-5. Antibiotics: streptomycin. M 29-36. Immunization Problems.
M 6-15. Miscellaneous Studies. M 37-44. Comparative Pathology.
M 16-28. Viruses. M 45-51. Miscellaneous Studies.
Round Tables and Symposia
RT 1-6. Problems of Teaching. RT 26-31. Taxonomy.
RT 7-14. Michigan History. RT 32-33. Food Processing.
RT 15-21. Influenza. RT 34-38. Air Disinfection.
RT 22-25. Streptomycin. RT 39-46. Immunizing Fractions.

Gl. The Development of Quick Microtechniques for the Identification of Cultures.

R. H. WEAVER, W. M. ARNOLD, JR., AND JOHN HANNAN, University of
Kentucky, Department of Bacteriology, Lexington, Ky.
The use of small tubes, large inocula, and preheating of media makes it possible
to determine the biochemical characteristics of bacteria quickly. An indole
test, based on these principles, has yielded positive results, in some cases, after
six minutes' incubation. It gives reliable results with members of the Entero-
bacteriaceae and with some other organisms within approximately two hours.
Acid and gas production from glucose has been obtained in less than one hour,
although it usually requires between one and two hours. The disaccharides are
fermented somewhat more slowly. Other tests are under study. It is hoped that
a system of techniques can be developed by the use of which it will be possible
1 This number of the Journal has been edited by the Chairman of the Program Com-
mittee. Authors of abstracts have not seen proof due to restrictions of time imposed by
the printing schedule.
565
566 FORTY-SIH GENERAL MEETING
tentatively to identify bacteria which multiply quickly within a few hours after
their isolation.
0s. Applicability of Modifications of the Thin Filter Paper Disc Method to the
Assay of Enzymes. MARION B. SHERWOOD, The Welcome Research
Laboratories, Department of Bacteriology, Tuckahoe 7, N. Y.
That the uniform absorptive capacity of filter paper discs renders them useful
for assay work in fields other than antibiotic testing has not been generally
recognized. Under conditions similar to those developed for penicillin assay the
logarithmic dose-response relationship between amylases from different sources
and various kinds of starches is readily demonstrable. The assay of starch-
splitting enzymes including purified preparations of a and ,B amylase will be
discussed with reference to the establishment of the optimum conditions for
testing. Preliminary work upon proteinases and other types of enzymes for
which concentration-response curves have been established will also be presented.
Gs. Methods of Study of Antiphage Agents Produced by Microorganisms. DoRs
JONES AND ALBERT SCHATZ, Rutgers University, Department of Micro-
biology, New Brunswick, N. J.
For the purpose of testing large numbers of cultures of microorganisms for
their ability to produce agents active against viruses methods similar to those
used in the study of antibacterial substances were used, namely, the phage agar
plate, phage agar streak, and the agar diffusion or cup test methods.
The fundamental principle in all these methods involves the growth of the
antagonist upon, or the diffusion of the active agent into, phage-seeded agar,
followed by flooding the surface with host-seeded agar. Antiphage action is
indicated by a reduced number of plaques or by a zone of bacterial growth
surrounding either the antagonist or the cup containing the active substance.
The rest of the plate shows complete lysis of the host cells by the uninhibited
phage.
The methods used for the isolation and study of antiphage agents inherently
possess greater variability and difficulty than the analogous procedures used in
the study of antibiotics. This is due to the increased complexity of the biological
system by the addition of bacteriophage. It remains to be determined to what
extent these techniques, or certain modifications thereof, can be utilized for the
isolation and study of agents active against plant and animal viruses.
04. Aeration in the Cultivation of Brucella suis. PMLIPP GERDT, 1sT LT.,
INF., AND LYNN L. GEE, 1ST Dr., AC., Camp Detrick, Frederick, Md.
The aeration requirements of Brucella suis in broth culture have been studied
with a view to maximum production of cells in a minimum growth period. Air
was supplied to the substrate either through the turbulence of shaken flasks con-
taming relatively small amounts of medium or through the dispersal of air by
various types of spargers into deep quantities of medium. A tryptose broth
medium, fortified with glucose and thiamine, was employed.
 SOCIETY OF AMERICAN BACTERIOLOGISTS 56f7
Experience gained during the investigation indicated that aeration was as
critical a limiting factor in maimum growth of the organism as nutrients, inocula,
or incubation temperature. With the extent of growth of the organism as the
criterion, optimum conditions were established for shaken flasks and sparger
aeration systems. Employment of a properly constructed reciprocal shaker
provided a high, yet constant and accurately reproducible rate of aeration for
the growing cultures, thus making the apparatus particularly valuable for the
study of other variables where aeration must be constant. Employment of
sparger aeration systems provided an effective method for laboratory production
of comparatively large amounts of cells, if efficient types of spargers, adequate
rates of aeration, and suitable antifoam agents were used. With either of these
systems, the normal growth cycle of the organism was reduced from 50-70 hr
to less than 24 hr, while cell yields were increased from less than 10 billion
bacteria per ml to more than 50 billion bacteria per ml.
Gs. A Critical Evaluation of the Nitrogen Assimilation Tests as Commonly Used
in the Classification of the Yeasts. LYNFERD J. WICKERHAM, Northern
Regional Research Laboratory, U. S. Department of Agriculture,
Peoria, Ill.
The ability of certain closely related groups of yeasts to utilize nitrate nitrogen
for growth has proved to be a valuable characteristic in classification. For the
past 12 years the ability to assimilate ammonium sulfate, urea, asparagine, and
peptone has also been widely adopted in taxonomic studies. Most nonsporo-
genous yeasts are reported to utilize all of these four nitrogen sources, prominent
exceptions being the members of the genus Kloeckera and five species of Torulop-
sis. Monographs on the Candida genus by the different workers have shown
marked variations in urea utilization within the same species. All used the
auxanographic plate technique with only minor variations in the medium. The
medium consisted of glucose, potassium phosphate, magnesium sulfate, agar,
and the nitrogen compound under test.
By additions to this medium of pure vitamins, trace elements, and the salts
calcium chloride and sodium chloride, all yeasts studied were able to utilize
ammonium sulfate, urea, asparagine, and peptone. Included were four of the
five species of Torulopsis previously reported incapable of utilizing ammonium
sulfate, urea, and asparagine; three species of Kloeckera; and all species of the
Candida genus previously reported negative for urea. To obtain growth with
all strains in urea, it was necessary to use a concentration of approximately 0.05
per cent or less of this substance.
It is thus apparent that most, if not all, of the previouisly reported inabilities of
certain yeasts to utilize ammonium sulfate, urea, and asparagine are in error
owing to the poor nutritional quality of the medium used in these studies.
(6. The Viability of Heat-activatable Spores in Distilled Water or Glucose Solution
as Influenced by Prestorage or Poststorage Heating. HAROLD R. CuRRAni
AND FRED R. EVANS, Bureau of Dairy Industry, U. S. Department of
* Agriculture, Washington 25, D. C.
568 FORTY-SIXTH GENERAL MEETIG
Changes in the apparent viability of heat-activatable spores were observed in
two separate fluid mediums: in one the cells were provided a source of energy
but no nitrogen, the other providing neither nitrogen nor a source of energy.
Exhaustively washed spores of a heat-activatable strain of Bacillus 8ubtilis
(LB) were suspended in glucose solution (0.1 or 1.0 per cent, Baker's cp) or in
distilled water. Plate counts on glucose tryptone agar were made before and
after heating at 95 C for 15 minutes and again after storage at 37 C for varied
periods with and without poststorage heating at 95 C for 15 minutes. In dis-
tilled water without prestorage heating, there was no significant change in the
number of spores that produced colonies over a period of one to two months.
With prestorage heating, most of the spores germinated within a month, but
poststorage heating of this suspension materially increased the count. In
glucose solution without prestorage heating, the count decreased progressively
with the length of the storage period; with prestorage heating, the vast majority
of spores became unable to form colonies after two days of storage, the count
declining further with the time of storage. Poststorage heating of the latter
sample materially increased the count. Other brands of glucose and an espe-
cially purified preparation yielded similar results. One per cent was only
slightly less effective than one-tenth per cent glucose in reducing the count.
A provisional interpretation of results is made based in part upon the microscopi-
cal examination of stained and unstained spore suspensions at different stages
of storage.
07. Production of Vitamin B2 by Mycobacterium smegmatis. R. L. MAYER AND
MARCELFLE RODBART, Ciba Pharmaceutical Products, Inc., Summit,
N. J.
Mycobacterium smegmatis produces, when grown in Long's medium, varying
amounts of vitamin B2 (up to 135 pg/ml, or 36 mg/g dry bacteria), as identified
by biological and physicochemical methods. The different environmental fac-
tors influencing its formation have been investigated. A study of nitrogen
sources showed that the greatest amounts of vitamin B2 are formed if only in-
organic nitrogen is present; the presence of organic nitrogen (for example,
asparagine) results in increased growth, but decreased vitamin B2 production.
The influence of the various carbon sources is more complex. Citrate appears
to effect chiefly the rate of growth, while glycerine chiefly effects the amount of
vitamin produced. Glucose can replace glycerine as the carbon source, but only
if asparagine also is present. Greater yields of vitamin B2 are obtained when
glycerine is replaced by certain sugars, such as arabinose or levulose. Citrate
as a growth factor can be replaced by various acids. The amount of magnesium
and potassium in the mediuim proportionately influences the growth and vitamnm
production, whereas sodium and chlorine have no effect. The influence of iron
was slight. The optimum pH was found to be about 6.0.
08. Influence of Iron Concentration and Attenuation on the Metabolism of Cos-
tridium acetobutylicum. AUSTIN M. HANSON AND NELSON E. RODGERS,
Western Condensing Company, Research Laboratories, Appleton, Wis.
 SOCIETY OF AMERICAN BACTERIOLOGISTS 569
A study of the fermentation of whey by a derived strain of Clostridium aceto-
butylicum revealed a relation between iron concentration and distribution of
products and pronounced differences in the metabolism of "normal" and "at-
tenuated" strains. The basal medium consisted of rennet whey containing about
4 X 10- M iron supplemented with 2 X 10-5 M ZnSO4, 2.5 X 10-5 M MnSO4,
10 parts per billion p-aminobenzoic acid, 0.15 per cent CaCO3, 0.15 per cent
Ca3(PO4)2, and iron as indicated. The inoculum for the normal culture was
taken from the fifth transfer in the basal medium supplemented with 2 X 10-i
M iron. The attenuated strain was derived from the normal culture by 50
transfers at- 24-hour intervals in the same medium. The fermentations were
analyzed after 38 hours' incubation at 37 C. When the normal culture was
tested in the presence of 0.75, 2.0, and 8.0 X 10-5 M iron, lactate; formate, and
ethanol production per unit of sugar fermented diminished as the iron concen-
tration was increased; whereas hydrogen, carbon dioxide, acetone, acetoin,
butyrate, and acetate production increased with iron concentration. Butanol
production was not affected by iron concentration. Carbon recoveries and
oxidation-reduction balances were good. The attenuated strain grown in the
presence of 2 X 10- M iron fermented sugar very incompletely, and lactate was
the principal product.
These findings parallel the observations of high lactate production by Clos-
tridium perfringens (Pappenheimer and Shaskan, 1944) and Aerobacter indolo-
genes (Waring and Werkman, 1944) when grown in iron-deficient media.
G9. Recovery of Biotin from Cultures of Acetone, Butyl Alcohol Bacteria. ROSARIO
REYES-TEODORO AND MILO N. MICKELSON, University of Michigan,
Department of Bacteriology, Ann Arbor, Mich.
An attempt was made to determine the fate of biotin added to a synthetic
medium in which were cultured acetone, butyl alcohol bacteria, for which this
vitamin is an essential growth factor. Assays of the bacterial cells and the cell-
free medium for biotin were carried out using the titrimetric procedure of Shull,
Hutchings, and Peterson. Four methods were employed to liberate biotin from
the cells: (1) autolysis, (2) hydrolysis with hydrochloric acid, (3) digestion with
papain, and (4) digestion with a mixture of papain and takadiastase.
From the data obtained the following findings seemed evident: (1) that the
maximum biotin recovery amounted only to 75 to 80 per cent of the quantity
added to the medium; (2) that about 15 to 20 per cent of the biotin recovered
was present in the medium and the remainder was extracted from the cells; (3)
that about 20 to 25 per cent of the biotin could not be recovered at all; and (4)
that acid hydrolysis and digestion with both papain and takadiastase simultane-
ously offer the best methods of extracting biotin from the bacterial cells.
G10. Relation of Strain Variation and Culture History to the Synthesis of Ribo-
flavin by Clostridium acetobutylicum in Whey. NELSON E. RODGERS,
RICHARD H. HENIKA, AND AUSTIN M. HANSON, Western Condensing
Company, Research Laboratories, Appleton, Wis.
570 FORTY-SIXTH GENERAL I

The production of practically significant quantities of riboflavin by fermenta-

tion of whey with Clostridium acetobutylicum is dependent largely on selection of
suitable strains, maintenance of culture stability, and adjustment of the iron
concentration in the medium to a level optimum for growth but not inhibitory
to riboflavin synthesis. Because of its exceptionally low iron content (about
0.2 ppm) a suitably supplemented whey is an excellent medium for the synthesis
of riboflavin. However, since iron contamination is difficult to control in large-
scale operations, it is desirable to select strains not only for superior riboflavin
production, but also for a high threshold of inhibition of riboflavin synthesis by
iron.
Tests on 13 strains revealed iron optima ranging from 0.5 to 1.6 ppm and
riboflavin yields of 8 to 23 ,ug per ml. Modification of these strains by special
culture treatment resulted in iron optima of 1.0 to 1.6 ppm and riboflavin yields
of 18 to 78 ug per ml. Although in most cases increased iron tolerance was
associated with increased riboflavin synthesis, two cultures showed improved
synthesis unaccompanied by a change in iron optimum, suggesting that the two
characters are not genetically linked.
The capacity to synthesize riboflavin is an extremely unstable character and
is attenuated rapidly by serial transfer of aged cultures. In the development of
inocula for large-scale fermentations, attenuation can be prevented by adjust-
ment of the transfer schedule to fit certain physiological. phases of the culture
cycle.
Gll. The Amino Acid Composition of Microorganisms. J. L. SToxEs AND
MARION GUNNEss, Merck and Co., Inc., Microbiological Research and
Development Department, Rahway, N. J.
The quantities of ten amino acids, namely, histidine, arginine, lysine, leucine,
isoleucine, valine, methionine, threonine, phenylalanine, and tryptophane, were
determined microbiologically in the acid or alkaline hydrolyzates of the dried
cells of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptomymcs
grieus, Saccharomyces cereviae, Rhodotorula rubra, Rhizopus nigrican , Asper-
gillu niger, and PeniciUium notatum grown under a variety of cultural conditions.
The amino acid composition of an organism is, qualitatively and quantitatively,
a stable and characteristic property of the cell under fixed conditions of growth.
Although striking quantitative differences occur between microorganis, the
results, in general, emphasize the similarities rather than the differencee in their
amino acid composition. Certainly no fundamental differences im that some
amino acids are present in one.organism but not another were encountered. The
microbial proteins do not appear to differ materially from plant and animal pro-
teins represented by wheat and beef liver. Fungi contain 10 to 50 per cent les,
per unit of protein, of most of the amino acids than the other microbial groups.
Mold mycelium prior to sporulation compared to that after sporulation.contain
considerably larger quantities of most of the amino acids largely because of its
60 per cent greater protein content. The mycelium and its spores, in general,
have comparable amino acid contents. The quantities.of individual mino
 SOCIETY OF AMERICAN BACTERIOLOGISTS 571
acids in microorganisms may vary with the growth medium, aeration, and age
of the cells.
112. The Amino Acid Composition of Crystalline Botulinus (Type A) Toxin.
HENRY J. BUEHLER, D. H. BORNOR, E. J. SCHANTZ, AND CARL LAMANNA,
Camp Detrick, Frederick, Md.
A method for isolation and crystallization of pure botulinus toxin, type A,
has been reported. In preliminary studies of this material the presence of 14
ino acids has been determined. The toxin appears to be a complete protein
containing the 10 essential amino acids necessary in animal nutrition. There
is no suggestion of an unusual amino acid composition that might be used to
explain the extreme toxicity (32 billion mouse LD5O per g of dry weight). As
with diphtheria toxin the aromatic amino acids appear to be present in
abundance. A dicarboxy amino acid, glutamic acid, is present in greatest
amount.
Positive qualitative chemical tests have been obtained for arginine, lysine,
histidine, threonine, and tryptophane. Negative tests have been obtained for
glycine, alanine, proline, and hydroxyproline. Positive tests have been obtained
for free sulfhydryl groups in the whole protein.
Microbiological assay was finally chosen as the most practical and economic
technique for quantitative study of amino acid content of the toxin. These
assays have confirmed chemical tests for presence of arginine, lysine, histidine,
and threonine. The following amino acids have been shown to be present:
aspartic acid, valine (4.0 per cent), serine (3.0 per cent), threonine (7.3 per cent),
arginine (3.2 per cent), lysine (7.9 per cent), histidine (1.0 per cent), glutamic
acid (14.9 per cent), leucine (8.7 per cent), isoleucine (11.0 per cent), cystine
(0.48 per cent), methionine (0.83 per cent), phenylalanine (1.08 per cent),
tyrosine (9.5 per cent). The quantitative data listed are not to be considered
final.

G1a. Nutritional Studies with Clostridium botulinum, Toxin Types A and B.

W. G. ROESSLER, ENS., USNR, AND C. R. BREWER, Camp Detrick,
Frederick, Md.
The nutritional requirements of Clostridium botulinum, toxin types A and B,
for growth and toxin production were studied. Experiments were designed to
test the effects, interrelationships, and requirements of amino acids, vitamins,
nucleic acid components, and salts. Washed spores were used as the inoculum,
and cultures were grown in Brewer or Fildes jars incubated at 35 C. The
Coleman photoelectric colorimeter was used to evaluate the growth obtained.
All toxin tests were performed on mice. Following the development of a gelatin
hydrolyzate medium, amino acid studies were made in chemically defined media.
The following amino acids are essential for the growth of both types: leucine,
isoleucine, valine, arginine, phenylalanine, tryptophane, tyrosine, and methionine.
In addition, threonine is essential for the growth of type A and histidine for type
B. Nucleic acid or its components are necessary for good growth and toxin
572 FORTY-SIXTH GENERAL MEETING
production of type A. With type B, nucleic acid is less important. Biotin is
the only essential vitamin for both types; however, better growth occurs if
riboflavin, para-aminobenzoic acid, and niacinamide are included. Type A
utilized desthiobiotin in place of biotin but type B did not. Calcium panto-
thenate, pyridoxine, thiamine, inositol, and folic acid are not required for growth
by either type. Toxin titers in excess of 105 mouse MLD/ml with type A and
5 X 103 mouse MLD/ml with type B were obtained.
014. The Nutrition of Coccidioides immitis in Submerged Culture. W. G. RoEss-
LER, ENS., USNR; E. J. HERBST, ENS., USNR; W. G. MCCULLOUGH,
1sT T., AUS; R. C. MILLS, ENS., USNR; AND C. R. BREWER; Camp
Detrick, Frederick, Md.
Coccidioides immitis, a virulent fungous pathogen, has previously been grown
as a surface mat on solid or liquid culture media. In order to obtain greater
yields, to eliminate the hazard of working with dry spores, and to permit more
accurate evaluation of results, culture techniques and media suitable for sub-
merged growth were developed. Dilute spore suspensions were used as inocula.
Cultures were grown in pyrex flasks. Aeration was provided by continuous
mechanical agitation which also aided in the disintegration of sporulated mycelia
to form free spores. Results were evaluated by poured plates. The nutritional
requirements of C. immitis are relatively simple, as has been reported by other
workers. Yields consisting mainly of spores, occurring singly and in short chains,
in excess of 2 X 108 viable fragments per ml were obtained in chemically defined
media of the following composition: 0.08 M ammonium acetate, 0.11 M glu-
cose, 0.008 M MgSO4, 0.015 M K2HPO4, 0.015 M KH2PO4, and 0.00003 M ZnSO4.
Magnesium and zinc were the only metallic ions found to be required for growth.
No vitamn s were required. In submerged cultures the following sequence of
development was observed: spore germination, growth of nonseptate mycelia,
septation, rounding of septa to form spores, maturation of spores, and finally
disintegration of spore chains with liberation of free spores. Maximum turbidi-
ties-were attained after five days' incubation; ten dayswere required for maximum
plate counts. The spores produced were fully virulent for guinea pigs and closely
resembled C. immitis grown on surface cultures in morphology.
015. The Nutritional Requirements of Bacillus anthracis. C. R. BREWER; W. G.
MCCULLOUGH, 1ST LT., AUS; R. C. MILLS, ENS., USNR; W. G.
ROESSLER, ENS., USNR; E. J. HERBST, ENS., USNR; AND A. F. HOWE,
2ND LT., AUS.; Camp Detrick, Frederick, Md.
Because of scanty and confficting- information in the literature, an investiga-
tion was undertaken to determine the nutritional requirements of Bacillus
anthracis. The object was to produce free mature virulent spores in media of
known chemical composition with yields equal to those obtained in the usual
natural media. The accepted microbiological nutrition techniques were used to
elimiate extraneous factors as chemical impurities, carry-over of growth factors
in the inocula, and uncontrolled physical environment. Cultures were grown m
 SOCIETY OF AMERICAN BACTERIOLOGISTS 573
pyrex bottles aerated by continuous mechanical shaking. Results were evalu-
ated by plate counts. Yields in excess of 109 virulent spores per ml in a medium
composed of amino acids (synthetic where available), glucose, thiamine, gluta-
mine, uracil, adenine, guanine, bicarbonate, phosphate, and salts of potassium,
calcium, iron, magnesium, and manganese. No B complex vitamins other than
thiamine were required. Uracil, adenine, and guanine in combination, but not
singly, stimulated growth considerably. Yields were increased greatly by adjust-
ing the concentrations of the inorganic constituents of the media to optimal levels.
The results of the fundamental nutritional studies were applied to the develop-
ment of simplified natural media (tryptic digest of casein, autolyzed yeast, glu-
cose, and salts) which have produced yields in excess of 2.25 X 109 spores per
ml. These media have been valuable in immunological studies by others.
016. Plating Media Requirements for Certain Strains of Bacterium tularense.
F. B. ENGLEY, JR., 2ND Lr., SNC, AND T. L. SNYDER, 1ST Dr., SNC, Camp
Detrick, Frederick, Md.
An extensive survey was made of plating media requirements for Bacterium
tularense strain Schu. The basal medium consisted of bacto peptone, sodium
chloride, cysteine, and glucose. This medium was altered in various ways, and
the results were evaluated on the bases of rate of colonial growth and the vari-
ability among counts of replicate plates.
Bacto peptone could be replaced by proteose peptone, proteose peptone no. 2,
and proteose peptone no. 3 or neopeptone, but not by bacto protone, bacto tryp-
tose, bacto tryptone, Difco beef extract, bacto gelatin, Difco casamino acids,
Wilson peptone USP, Baker peptone USP, or Sheffield pepticase. In the presence
of thiamine, apparently optimum growth was obtained with bacto tryptone, but
this was not true with the remaining peptones. The oxidation-reduction poten-
tial of the medium was adequately adjusted with 0.1 per cent cysteine. Freshly
prepared peptone agar appeared to give more uniform results than plates stored
for several days. The hydrogen-ion concentration had little effect between pH
6.5 and 7.4. The optimum sodium chloride concentration fell between 0.5 and
1 per cent. Glucose was not necessary for growth. Human erythrocytes had
no certain effect other than to prevent inhibition by high concentrations of glu-
cose. Statistical analysis of results failed to demonstrate any differences between
counts on fresh peptone agar and on blood, cysteine, glucose agar.
Of 28 strains tested with the optimum medium for strain Schu, 14 appeared to
give satisfactory results and 14 grew poorly with small inocula.
0l7. The Reduction of Trimethylamine Oxide by Representatives of the Genus
Pseudomonas. E. R. HITCHNER, University of Maine, Department of
Bacteriology and Biochemistry, Orono, Maine.
A study has been made of the ability of representatives of the genus Pseudo-
monas to reduce trimethylamine oxide to trimethylamine. Ninety-eight strains
of polar-flagellated, gram-negative bacteria, representing 15 species including
P. hydrophila and P. icthyosmius and several strains whose specific designation
 574 FOGET-SIXTH GENERAL MEETNG
was not determined, were tested according to the procedure described by Wood
and Baird. The ability to ferment glucose, lactose, sucrose, glycerol, and to
produce acetylmethylcarbinol from glucose was included in the study.
The aerogenic species, P. hydrophilk and P. icthyo8mius and 2 strains listed
as P. putrefaciens, formed trimethylamine, whereas all other cultures, including 2.
strains listed as P. putrefacieiw, which were nonaerogenic, failed to produce this
compound. Those strains which reduced trimethylamine oxide likewise pro-.
duced acetylmethylcarbinol from glucose.
These results indicate that the metabolism of the aerogenic species differs
materially from the nonaerogenic strains and further indicates that the mecha-
nism of the utilization of carbohydrates (true fermentative ability) by aerogenic
istrans of polar-flagellated organisms so differ from the nonaerogenic (oxidative
utilization of carbohydrates) as to warrant their inclusion in the genus Aeromonas
of the family Pseudomonadaceae (Kluyver and van Niel) as suggested by Stanier.
Gl8. The OxWidtion of Glycerol by Escherichia freundii. M. N. MICKELsoN AND
F. E. SHIDEMAN, University of Michigan, Departments of Bacteriology
and Pharmacology, Ann Arbor, Mich.
Relatively little information has been published on the mechanism by which
glycerol is metabolized by bacteria. The present work is concerned with the role
of phosphate in the oxidation of glycerol by Escherichia freundii. Addition of
inorganic phosphate resulted in increased 02 uptake and glycerol consumption.
The respiratory quotient in the absence of added phosphate is les than, but
approaches, the theoretical for complete oxidation of glycerol. In the absence
of added phosphate glycerol oxidation is 68 to 83 per cent complete, in the pres-
ence of phosphate 10 to 40 per cent complete. Addition of inorganic phosphate
produces a marked decrease in the R.Q., and the low R.Q. is associated with in-
creased glycerol utilization. 0.1 M phosphate is the optimum concentration.
There is an uptake of inorganc phosphate when glycerol is oxidized which
reaches a maximum at 40 minutes, during which time 02 uptake is linear. When
02 uptake decreases, inorganic phosphate is released into the medium until at
end of 100 minutes most of the inorganic phosphate esterified has reverted to its
original form. The inorganic phosphate esterified was nearly all found in the
fraction resistant to hydrolysis in normal acid at 100 C for 180 minutes.
Adenosine triphosphate appears to be about 2.5 times as efficient as inorganio
phosphate. 0.075 M NaF caus inhibition of 02 uptake in absence of inorganic
phosphate but not in its presence. 0.001 M cyanide and 0.0005 M iodoaoetate
inhibit glycerol oxidation in presence and absence of phosphate.
G19. A Simplified Medium for the Microbiological Assay for Pantothenic Acid.
HELEN M. KETCHuM AND RAYMOND L. CONKLIN, Miles Laboratories,
Inc., Depaxtment of Medical Research, Elkart, Ind.
A medium for the microbiological assay for pantothenic acid in material of
high potency vitamin content is described. L. arabinosus is used as the test
organism. It appears unnecessary to employ complicated media which can
 SOCIETY OF AMERICAN BACTERIOLGISTS 575
contribute to the possibility of greater errors. By increasing the sugar and so-
dium acetate, as suggested by Stokes and Martin, the original formula of Penning-
ton and Snell and Strong and his associates was found to be very satisfactory with
the following modifications: peptone 1 per cent, glucose 4 per cent, sodium
acetate 3.6 per cent, casein 0.4 per cent, hydrolyzed casein 0.4 per cent, cystine
100 mg, riboflavin 200 mg per L, and inorganic salts. A comparison has been
made of the curves obtained with this medium in contrast to curves obtained with
generally accepted media.
20. Significance in Nutritional Research of Correct Identification of Lactobacilus
casei, L. delbrueckii, and L. bulgaric. MORRISON ROGOsA, Bureau of
Dairy Industry, U. S. Department of Agriculture, Washington, D. C.
The nutritional requirements of lactobacilli designated Lactobacillus delbrueckii
LD5 and L. bulgaric 05 have been described in the literature. Taxonomic
study of these strains by the author proved definitely their identity as L. casei.
As in the case of L. casei, cells are small and colonies smooth. They grow in
defined media; at 10 C; in 4 per cent NaCl; and produce approximately 1.40
to 1.47 per cent acid in milk held at 30 C for two weeks. The acid is predomi-
nantly dextrorotatory. They ferment lactose, maltose, sucrose, mannitol,
trehalose, salicin, sorbitol, and glycerol.
Authentic strains of L. delbrueckii and L. bulgaricus do not form smooth colo-
nies; do not grow at 10 C; do not grow in 4 per cent NaCl; do not produce
dextro acid; do not ferment mannitol, trehalose, salicin, sorbitol, glycerol. L.
delbrueckii does not ferment lactose or curdle milk. It forms rough colonies;
ferments maltose and sucrose; produces mainly levo lactic acid from glucose.
L. bulgarics forms rough colonies; ferments lactose but not sucrose and maltose.
In milk it produces an average of 2.7 per cent lactic acid. The acid is optically
inactive.
Unilike L. casei, neither L. delbrueckii nor L. bulgariu grows in defined media
contaiig all known B vitamins and amino acids. These species require an
unknown factor or factors. Thus, authentic strains of L. delbrueckii and L.
bulgaricus should prove useful as tools in research for unknown growth factors.
Gas. Unidentified Trace Element Requirements of Photosynthetic Purple Bacteria.
S. H. HUTNER, Haskins Laboratories, New York 17, N. Y.
Following identification of the vitamin requirements of purple bacteria (kindly
supplied by Professor van Niel), the inorganic requirements of Rhodospirillum
rubrum and Rhodopseudomonas capsulatus (requiring respectively biotin and
thiamine) were studied. The other organic constituents of the medium were
redistilled ammonium acetate and propionic acid, and 0.025 per cent Na-
citrate * 2H20.
Virtually any assortment of elements such as Fe, Mn, Zn, Cu, Mo, etc., sup-
ported heavy growth if the total concentration exceeded approximately 0.2 mg
per cent. Ash was ineffective. Many substances of biological origin (aspara-
gine, lactate, sugars, etc.) effectively replaced these mixtures of heavy elements.
576 6FORTY-SIXTH GENERAL MEETING
The interchangeability of these elements, and the excessive quantities required,
were traced to the fact that these elements were contaminated with unidentified
essential elements, and that the true requirement for Fe and other recognized
essential trace elements was probably less than 0.001 mg per cent. The platinum
elements, and, significantly, elements susceptible to loss by volatilization on
ashing, e.g., mercury, osmium, rhenium, and ruthenium, proved exceedingly
favorable when supplied in nontoxic concentrations (< 0.001-0.1lug per cent).
The partial interchangeability of these elements suggested that their activity in
turn depended on additional mutual impurities.
The extreme toxicity, in precipitate-free media, of elements proving favorable
in lower concentrations, was a fertile source of misleading results: many elements
formed precipitates in dilute media, and thereby removed toxic elements by pre-
cipitation or adsorption, and hence would seem favorable or even essential.
G22. Fixation of Heavy Carbon Acetaldehyde by Active Juices. NOEL H. GROSS
AND C. H. WER mAN, Iowa State College, Department of Bacteriology,
Ames, Iowa.
There is no general agreement as to the mechanism of the biological formation
of acetylmethylcarbinol. Evidence has been presented to support acetaldehyde
as an intermediate in its formation from glucose. Other evidence has been pre-
sented that acetylmethylcarbinol is formed from pyruvic acid, acetic acid, and
citric acid.
Experimental data have been obtained that contribute to the clarification of
the role of acetaldehyde as an intermediate in the formation of acetylmeythl-
carbinol. Juices prepared from Aerobacter did not utilize synthetic acetaldehyde
in the formation of acetylmethylcarbinol. Heavy carbon acetaldehyde, with
C'3 in both positions, when added to a pyruvic acid fermentation, yielded acetyl-
methylcarbinol with only the normal percentage of heavy carbon.
The yeast juice enzyme preparation, on the other hand, can utilize synthetic
acetaldehyde in the formation of acetylmethylcarbinol. When heavy carbon
enriched acetaldehyde was added to the yeast juice fermentation of pyruvate,
acetylmethylcarbinol was formed containing increased amounts of heavy carbon.
Pig heart juice appears to be quite different from either the bacterial juice
or the yeast juice. This preparation formed large amounts of acetylmethyl-
carbinol from acetaldehyde alone. The addition of pyruvate did not increase
the carbinol production.
The enriched heavy carbon acetylmethylcarbinol formed by the yeast juice
was degraded and heavy carbon was found in all four carbons.
Gas. Mechanism of Pyridoxal Phosphate Function in Bacterial Transamination.
W. W. UMBREIT, D. J. O'KANE, AND I. C. GUNSALUS, Cornell Uni-
versity, College of Agriculture, Laboratory of Bacteriology, Ithaca,
N.Y.
The i#terconversion of pyridoxal and pyridoxamine by heating with amino
and keto acids, respectively, led Snell to suggest that these substances function
 SOCIETY OF AMERICAN BACTERIOLOGISTS 577
in transamination, the compounds themselves undergoing transamination in the
process. This view was further strengthened by a demonstration of the conver-
sion of pyridoxamine into pyridoxal phosphate by incubation with a keto acid
and Streptococcus faecalis cells.
The present study has shown that the glutamic-aspartic apotransaminase pre-
pared from Streptococcus faecalis in a cell-free state can be activated by pyridox-
amine phosphate, in addition to pyridoxal phosphate. The pyridoxamine phos-
phate had been prepared free from pyridoxal phosphate as indicated by the
tyrosine decarboxylase apoenzyme, which responds only to pyridoxal phosphate,
or to pyridoxal in the presence of ATP, but not to pyridoxamine phosphate or
pyridoxamine. The glutamic-aspartic apotransaminase is activated by pyri-
doxal or pyridoxamine under proper conditions, but may be prepared in a state
wlhich responds only to the respective phosphates.
These data constitute biological evidence in support of the proposed mechanism
of the function of pyridoxal phosphate as coenzyme of transamination. Thus
pyridoxamine phosphate and pyridoxamine have been shown to be the equiva-
lents of pyridoxal phosphate and pyridoxal, respectively, for transamination,
though not for amino acid decarboxylation.
Ga4. Factors Influencing the Anaerobic Production of Gas by Bacillus subtilis.
NATHAN R. SMITH AND MARIE E. WENZEL, Plant Industry Station,
U. S. Department of Agriculture, Beltsville, Md.
The anaerobic production of gas by some strains of Bacillus subtilis has been
used recently to divide the species into two sections: one, the so-called Marburg
strain of B. subtilis; the other, the Ford strain of B. subtilis or B. licheniformis.
Using recommended media containing glucose or nitrate, or both together, a
survey was made of some 40 strains of B. subtilis, which were found to correspond
to Ford's broad interpretation of the species. The results showed that certain
strains would not produce gas anaerobically in any of the media tried. Others
would usually produce gas when both glucose and nitrate were present. Gas
from glucose or nitrate alone occurred less often and irregularly. The addition
of metallic iron to certain media containing glucose made conditions more favor-
able for gas formation; in other media it was without effect. The same held true
for yeast extract and for phosphate. Growth was sometimes greatly reduced,
sometimes increased, by anaerobic conditions and bore no relation to the gas
produced. Two strains from Ford formed gas from nitrate in only 50 per cent
of the 12 trials on standard media; but with glucose also added, they nearly
always formed it. Different batches of the same medium failed to give identical
results. From a large number of observations, it is concluded that the anaerobic
production of gas by B. subtilis is a variable character dependent upon a number
of factors, some of which cannot be controlled.
G25. Rapid Identification of Certain Clostridia by Pklte Cultures on Medium Con-
taining Egg Yolk. L. S. MCCLUNG AND RUTH TOABE, Indiana Univer-
sity, Bacteriological Laboratories, Bloomington, Ind.
Addition of sterile egg yolk suspension to 4 per cent proteose peptone no. 2
 578 FORTY-SXT GENERAL MEETING
agar (with mineral salts and glucose) provides a medium for rapid identification
of certain clostridia (Nagler or LV reaction). Colonies of Clostridium perfringen8
(C. welchii) and the C. sordeUi-C. bifermentans group are surrounded by a wide zone
of precipitation. These species may be separated by the rapidity of sporulation
and the inability to ferment lactose by the latter cultures. Colonies of C. oede-
matiens (novyi) type A are surrounded by a smaller, more intense precipitation area
and a metallic luster extending from the edge of the colony outward but not to
the edge of the precipitation zone. This luster is marked with radial striations.
Type B colonies show the precipitate zone but no luster. The colonies of C. para-
botulinum, types A and B, and those of type B C. botulinum are covered with a
luster which extends in a regular circular zone beyond the colony edge and to the
edge of the area of precipitation. Colonies of types C, D, and E of C. botulinum
have the typical wide precipitate zone, but only a narrow luster band following
the contour of the colony edge. C. sporogenes colonies are covered with luster
and show precipitate under the colony, but neither extends beyond the colony
edge. C. tertium, C. tetani, C. histolyticum, C. septicum, C. capitovalis, C. chauvoei,
ad C. cochlearium show no reaction on this medim.
G26. Relationships of Proshigella dispar to Other Proshigella and Shigella
Species. PHiLIP L. CARPENTER, Rhode Island State College, Depart-
ment of Bacteriology, Kingston, R. I.
More than 150 strains of Proshigella dispar and other Proshigella species
isolated in this country and Europe have been studied. These orgamsms are so
diverse physiologically that serological grouping is most feasible. Within a
single serotype, biochemical properties vary widely. Antigenic analysis in-
dicates a multiplicity of antigenic components, many of which are common to
several types. The overlapping of physiological and serological properties re-em-
phasizes the spectrum nature of characteristics within this genus of the En-
Serobacteriaceae.
027. The Classification of Paracolon Bacilli Isolated from Man. MACDONALD
FULTON AND MARTHA L. CHILTON. University of Texas School of
Medicine, Department of Pediatrics, Galveston, Texas.
Paracolon strains were isolated from feces or rectal swab specimens from 239
adults and 31 children, and, in addition, 22 miscellaneous sources. Some of the-
subjects were healthy, others had acute or chronic diarrhea. The IMvIc reac-
tions and other customary bacteriologic tests were studied, together with fer-
mentation tests in adonitol, aesculin, melezitose, and melibiose. Preliminary
serologic studies indicated the possibility of recognizing many paracolon types
promptly by the slide macroscopic agglutination test. Improved methods of
demarking the group from Salmonella and Proteus were investigated.
GQ8. A Study of Adonitol-fermenting Paracolon Bacilli. MARTHA L. CHLTON
AN MACDONALD FLTON. University of Texas School of Medicine,
Department of Pediatrics, Galveston, Texas.
 SOCISTY OF AMERICAN BACTERIOLOGISTS 579
A collection of 50 paracolon strains which fermented adonitol was examined
in detail. There was no bacteriologic or antigenic similarity to Rettger's bacillus.
In Imvic reactions 28 strains were ++-- (Escherichia coli type), 10 were
- - + + (Aerobacter aerogenes type), and 12 were intermediate in reactions.
None of the strains hydrolyzed gelatin or produced sulfide. The limited dis-
tribution among bacteria of the ability to ferment this carbohydrate makes it a
characteristic which appears to be of value in recognizing paracolon bacilli.

G29. A Study of the Genus Microbacterium. R. N. DOETSCH AND O. N. ALLEN,

University of Maryland, Department of Bacteriology, College Park, Md.
Paper withdrawn before presentation.

GSO. Studies on Myxobacteria. ERLING J. ORDAL, University of Washington,

Department of Bacteriology, Seattle, Wash.
Studies have been made on fruiting body production, on the surface charac-
teristics of vegetative cells, and on the virulence of a number of strains of Chondro-
coccus columnaris, a myxobacterium pathogenic to fish. It is concluded that the
fruiting bodies occurring in dilute aqueous solutions may be considered as normal
types. Fruiting bodies may be demonstrated by placing autoclaved fingerling
fish in large flasks containing sterile tap water and inoculating with Chondro-
coccus columnaris. Fruiting body formation is normally evident after a few
days. The fruiting bodies are usually columnar, sometimes highly branched
structures extending a distance of 0.5 to 3 millimeters above the surface of the
fish. They may be packed closely together covering the entire surface of the fish.
Atypical fruiting bodies have been observed with old laboratory cultures, and
with freshly isolated cultures of low virulence. Evidence of a relationship
between virulence and surface characteristics of vegetative cells has been ob-
tained.

G31. Bacterial Variation, Population Dynamics, and Selective Environments.

WERNER BRAUN, University of California, College of Agriculture, De-
partment of Veterinary Science, Berkeley, Calif.
Continued studies on dissociation in Brucella abortus have confirmed previous
reports regarding the role of inherent and certain environmental factors governing
population dynamics in controlling bacterial variation. This information per-
mits better understanding of the apparent instability of bacterial mutants, the
apparent ability of bacteria to adapt themselves to altered environmental condi-
tions, and the occurrence of apparently orderly successive changes. It also sup-
plies experimental evidence for certain general problems of evolution dealing with
the relationship of size of the breeding population, mutation rates, and genera-
tion time to the accumulation of mutations within isolated populations.
Use of selective environments can alter the selection value of arising variants
and prevent their establishment; thus, addition of as little as 2 per cent of
normal serum of rabbits, cows, or hogs to broth cultures will usually prevent
580 FORTY-SIXTH GENERAL MEETING
dissociation from the S type. This is due to the presence in normal serum of
factors suppressive to R and Br variants of B. abortus. If dissociation does
occur, the variants represent types ordinarily not observed; presumably these
types have little chance to establish themselves when in competition with R and
Br. The suppression of dissociation is not due to the general bactericidal action
of sera, since sera heated at 56 C for one hour will still suppress dissociation.
Specifically selective environments, which will permit the establishment of one
chosen variant only, may be produced through the addition of antiserum which
contains antibodies for many variants but has been absorbed by the one desired
variant.
G0S. Induced Resistance of Staphylococcus aureus to Antibiotics. JOHN W. KLIMEK,
CHESTER J. CAVALLITO, AND JOHN HAYS BAILEY, Winthrop Chemical
Company, Inc., Division of Research, Rensselaer, N. Y.
Resistance of Staphylococcus aureus to various antibiotics in vitro has been
studied by subculturing the organisms in the presence of progressively increasing
amounts of antibiotic substance. The rate and degree of resistance acquired
was found to vary with each antibiotic. Slight resistance, even after many
transfers, was developed by cultures exposed to gliotoxin and the active principle
of A Ilium sativum. No appreciable resistance was developed in the presence of
aspergillic acid. Under similar conditions marked increase im resistance was
developed to penicillin, streptomycin, pyocyanin, and the active principle of
Asarum reflexum. In the case of streptomycin, development of resistance oc-
curred most rapidly.
Increase in resistance to any one of the antibiotics in no way affected its
sensitivity to any of the others, regardless of their nature or composition. Re-
versal to sensitive state was found to be rapid, regardless of time required to
develop high or low degrees of resistance.
0ss. The Development of Penicillin Resistance by Meningococcus in Vivo. C.
PHILLIP MILLER AND MARTORIE BOHNHOFF, The University of Chicago,
Department of Medicine, Chicago, Ill.
In a preliminary communication, the authors reported the development of
penicillin resistance in vitro by repeated subcultivation of meningococci on solid
media containing increasing concentrations of penicillin. Continuation of those
studies has raised the level of tolerance previously described; e.g., one strain of
meningococcus has acquired the ability to grow on media containing 65 units of
penicillin per ml. Penicillinase has not been demonstrated in any of the strains
made penicillin-resistant in vitro.
Penicillin resistance was developed in vivo by the following method: mice were
infected with varying numbers of meningococci suspended in mucin and treated
with subcurative doses of penicillin. Cultures of heart blood made at autopsy
on penicillin-free media from mice dying of the infection or killed at con-
venient intervals in its course were used to infect the next group of mice.
 SOCIETY OF AMERICAN BACTERIOLOGISTS 581
At the beginning of the series, 20-40 units of penicillin sufficed to cure mice
infected with approximately 106 meningococci. After 25 passages through peni-
cillin-treated mice this strain has developed sufficient resistance to produce
infections which regularly proceed to fatal termination with positive blood cul-
tures, in spite of treatment with 1,400 units of penicillin. Penicillinase has not
been demonstrated in this strain.

G34. Development of Streptomycin Resistance of the Shigellae. MORTON KLEIN

AND LEONARD J. KIMMELMAN, University of Pennsylvania, Department
of Bacteriology, Philadelphia, Pa.
Eleven strains of Shigella paradysenteriae and one strain of Shigella dysenteriae
have been tested for their susceptibility to streptomycin. In extract broth,
pH 7.2, all of the strains were inhibited in the range of 2 to 7 units of streptomycin
per ml. Ten strains were tested and each was found to become rapidly strepto-
mycin-resistant. Following a series of 7 broth subcultures of 0.1 ml inocula in
increasing concentrations of streptomycin, 7 of the strains grew in 1,000 units per
ml of streptomycin, the highest concentration tested. The 3 remaining strains
became resistant at a slower rate.
By testing 400 one-tenth-ml samples from the original susceptible culture of
S. dysenteriae (0.1 ml of an 18-hour broth culture inhibited by 5 units of strep-
tomycin) it was possible to isolate in approximately 1 per cent of the 0.1-ml
samples, bacteria which grew directly in 1,000 units of streptomycin. Continuous
selection of streptomycin-susceptible colonies revealed that the susceptible cul-
tures consistently gave rise to a small percentage of naturally resistant variants.
Unless one tested a relatively large portion of a culture, the few highly resistant
forms in a "susceptible" culture would readily be missed.
The streptomycin-resistant strains showed no change in their susceptibility to
penicillin or sulfonamides. A combination of streptomycin and sulfadiazine or
streptomycin and penicillin was more effective than either agent alone.

G3a. The Susceptibility of Penicillinase-producing Bacteria to Penicillin. AME-

DEO BONDI, JR., AND CATHERINE COLLINS DIETZ, Temple University
School of Medicine, Department of Bacteriology and Immunology,
Philadelphia, Pa.
Penicillinase-producing bacteria vary considerably in their sensitivity to peni-
cillin. In spite of their potential ability to produce large amounts of this enzyme
which destroys penicillin, some of these organisms are quite sensitive to this anti-
biotic agent. A number of factors including inoculum size and media ingredients
influence the sensitivity of these organisms. Of great importance is the rate of
growth of the organism under study. The slower-growing organisms are more
sensitive due to the slow rate at which the enzyme is produced. Growth of such
organisms is inhibited before sufficient quantity of the enzyme accumulates to
destroy the penicillin present.
582 FORTY-SnITH GENERAL MEETING
There is some evidence that if a suitable agent were available which would
poison this enzyme or inhibit its production the susceptibility of certain of these
organisms to penicillin could be increased.

GS6. Immunologic Aspects of Protein Metabolism. PAUL R. CANNON, University

of Chicago, Department of Pathology, Chicago, Ill.
Evidence will be presented indicating the importance of protein nutrition in
the retention of resistance to bacterial infection, and for the production of anti-
bodies. The basic role of essential amino acids in the fabrication of tissue pro-
tein and antibody globulin will be particularly emphasized, and experiments will
be described illustrating ways in which the problem of the imnologic relation-
ships of diet to resistance may be studied.

G87. Some Applications of Physical Methods to Problems of Bacteriology. RALPH

W. G. WYCKOFF, National Institute of Health, Bethesda 14, Md.
During the last years a group of physical techniques has been developing for
the detection of particles of submicroscopic and macromolecular dimensions, and
for the determination of their essential physical properties and the physico-
chemical characteristics of their suspensions. These tools of a molecular bio-
physics, as illustrated by ultracentrifugation, electrophoresis, and electron mi-
croscopy, have thus far been chiefly used in preparing and characterizing purified
viruses, but they are equally applicable to many problems of bacteriology. The
most obvious of these deal with the fine structure of bacterial cells, with the gross
morphology of the smallest microorganisms, with the nature and properties of
the products of bacterial metabolism, and with the intimate mechanism of the
immune reaction. This paper is a discussion of the kinds of bacteriological prob-
lems to which such a biophysical approach has already been made, of the types
of results that have been attained, of some of the potentialities and limitations
of existing physical tools, and of some indications of the way this type of research
may be expected to develop in the immediate future. Especial attention will be
given to illustrating, through lantern slides, certain recent results of the electron
microscopic study of bacteria and bacterial products.

G88. Electron Microscopy of Bacterium tularense. HENRY T. EIGELSBACH,

ENS., H(S), USNR, LESLIE A. CHAMBERS, AND LEwIs L. CoRmELL,
CAPTr., MC, AUS, Camp Detrick, Frederick, Md.
The morphology of Bacterium tularense as determined with the electron
microscope is in agreement with a recent systematic study of the morphology
of this organism by means of vital staining techniques and dark-field
examination.
In general, B. tularense possesses multiple morphological units including large
and small coccoid and bacillary, oval, minute, filamented, bean-shaped, dumb-
 SOCIETY OF AMERICAN BACTERIOLOGISTS 583
bell, bizarre, and so-called "involution" forms. The suggestion of the existence
of minute morphological units of less than 300, in diameter was confirmed. The
typical cell possesses little opacity to the electron beam and presents a semi-
transparent, nebulous appearance. Critical examination for the presence of a
cell wall revealed an extremely delicate structure of very low electronic density,
which possibly accounts for the low survival rate when subjected to sonic vibra-
tion or the lyophilization process.

GC9. The Structure of Spirochaeta novyi as Revealed by the Electron Microscope.

RUTH LOFGREN AND MALCOLM H. SOULE, University of Michigan, De-
partment of Bacteriology, Ann Arbor, Mich.
Our early observations on the spirochetes of relapsing fever with the electron
microscope revealed marked differences in structure depending upon the methods
of preparing the specimens. Since pure cultures of these organisms are not avail-
able, it is necessary to resort to the use of infected blood as a source of material.
The removal of the blood constituents by washing and repeated centrifugation
was unsatisfactory and necessitated the development of a simpler procedure,
namely, electrodialysis and suspension in distilled water. Organisms prepared
in this manner possessed a uniform outline, pointed tips less dense than the cell
contents, and granular or mottled protoplasm. A few of the forms had a long,
rather heavy terminal filament extending from one end.
Following electrodialysis, suspensions of Spirochaeta novyi were allowed to stand
48 hr in distilled water. Examination of these cells revealed no apparent struc-
tural changes. The addition of distilled water with subsequent centrifugation
and resuspension was repeated four times, with examination of the spirochetes
after each washing. There was a progressive destruction of the cells; fragmenta-
tion of the periplast resulted in the production of numerous flagellalike fibers
along the bodies of most of the organisms; and areas of pronounced swelling and
granulation of the protoplasm were common. Large, dense granules, similar to
those detected in frozen preparations of this organism, were frequently observed.
It appears possible that the flagella attached to spirochetes observed by other
investigators may be formed by the mechanical shredding of the cell wall as a
result of the treatment of the specimens.

G40. Electron Microscope Studies of Organisms of the Pleuropneumonia Group.

WILLIAM E. SMITH, JAMES HILLIER, AND STUART MUDD, Rockefeller
Institute, New York, N. Y.; RCA Laboratories, Radio Corporation of
America, Princeton, N. J.; and University of Pennsylvania, School of
Medicine, Philadelphia, Pa.
The diversity of morphological forms exhibited by organisms of the pleuro-
pneumonia group has led to the conception that these organisms are essentially
different from the bacteria. Spherical bodies are prominent in all strains, and
branched structures and filamentous forms have also been described, as well as
5845ORTY-SIXTH GENERAL MEETING
smnall rod-shaped bodies. The electron microscope was employed to gain further
knowledge of these forms, and micrographs were made of a strain of organisms of
the pleuropneumonia group isolated from the human cervix. This strain, L 50,
had been accepted as a member of the group by all investigators who examined it.
The electron micrographs show spherical forms and short filaments, and, more
important, many small rod-shaped forms are present which have a well-defined
bacillary structure with cell wall, cytoplasm, and various intracellular details.
The findings indicate that the strain studied is essentially a highly pleomorphic
bacillus. This is in accord with the view of Dienes concerning the nature of
organisms of the pleurbpneumonia group.

G41. A Comparison of Electron Micrographs with Photomicrographs of Young

Bacterial Cultures Stained to Demonstrate Desoxyribosenucleic Acid.
RUTH A. C. FOSTER AND LELAND L. ANTEs, University of Texas, De-
partment of Bacteriology and Bureau of Engineering Research, Austin,
Texas.
Young cultures of Escherichia, Salmonella, Proteus, Serratia, and Corynebac-
terium species, grown on solid medium and photographed with the electron
microscope, show a spiral structure, opaque to electrons. This spiral is most
convincingly demonstrated during the early logarithmic phase of bacterial
growth when the cells are of maximum size. When these photographs are com-
pared with light microscope photographs of cells of comparable age, stained with
Wright's stain following osmic acid fixation and acid hydrolysis, the dark bodies
variously called nucleoids, chromatinic bodies, or dumbbell-shaped bodies appear
within the cells in positions which suggest that they are analogous to the spiral
structure observed with the electron microscope.

G42. Gram-positive Characteristics of the Neisseria. JAMEs W. BARTHOLOMEW,

University of Southern California, Department of Bacteriology, Los
Angeles 7, Calif.
Henry and Stacy (1943) and Bartholomew and Umbreit (1944) showed that
gram-positive cells could be made to stain either gram-negative or gram-positive
by removing or replating magnesium ribonucleate onto the cell. True gram-
negative celLs could not be rendered gram-positive in this manner. This was the
principal approach for this investigation, although resistance to digestive agents
and dye sensitivjty were included.
Suspensions of Neisseria catarrhalis, Escherichia coli, and Serratia marcescens
were prepared, and 0.1 per cent formaldehyde and 1 per cent magnesium ribo-
nucleate were added. Slides were prepared at intervals and gram-stained.
Suspensions of N. catarrhalis, Staphylococcus aureus, Bacillus subtilis, S. mar-
cescens, and E. coli were digested with 1 per cent solutions of trypsin, pepsin,
and potassium hydroxide. Dye sensitivity was determined by presence or ab-
sence of growth on nutrient agar containing .001 per cent gentian violet.
 SOCIETY OF AMERICAN BACTERIOLOGISTS 585
N. catarrhalis accepted the magnesium ribonucleate and stained gram-positive
after fifteen minutes' exposure. E. coli and S. marcescens remained gram-nega-
tive. N. catarrhalis was more resistant to the digestive agents than E. coli or
S. marcescens, but was less resistant than B. subtilis or S. aureus. N. catarrhalis,
B. subtilis, and S. aureus did not grow on the gentian violet agar; E. coli and
S. marcescens did. Hence, N. catarrhalisisstructurallyandphysiologicallyrelated
to gram-positive bacteria.
The implications of these results on the division of bacteria into gram-positive
and gram-negative groups will be presented later.

G43. Reproductive Processes in Proteus Cultures. L. DIENES, Massachusetts

General Hospital, Department of Pathology and Bacteriology, Boston,
Mass.
Proteus starts to grow in the first few hours in short bacillary forms without
tendency to spread. At a certain moment the bacteria grow into filaments, are
provided abundantly with flagella, and begin to swarm on the surface of the agar.
In nearly all strains a few bacilli develop large round bodies attached to their
sides. The swarming filaments of such strains transferred to tap water show
bacterioptysis (the contents of the filaments are extruded in the form of droplets
attached to the filament). Some of these enlarge within a few minutes to a large
round body. This is not a purely physical phenomenon because the round bodies
have a resistant membrane and germinate in transplants. Like the round bodies
in other species, they usually produce a tiny "L"-type colony; rarely they frac-
tionate and reproduce bacteria of the usual morphology. When the spreading
filaments of two appropriate strains meet on the surface of the agar, in the area
of juncture the majority of the long filaments are transformed quickly into large
bodies. These increase in size until they fractionate and reproduce typical
bacterial forms. "L"-type colonies, in contrast to the large bodies produced in
tap water, are rarely formed. These phenomena are produced only when spread-
ing filaments meet spreading filaments; meeting colonies of other strains growing
as small bacillary forms does not produce this effect. Such observations suggest
a sexual process but a union either between bacterial filaments or between large
bodies was not observed.

G44. The Size of Living Bacteria, Measured with the Phase Microscope. OSCAR W.
RICHARDS, American Optical Co., Scientific Instrument Division, Re-
search Division, Buffalo 11, N. Y.
Bacteria living in a suitable culture medium are revealed clearly without stain-
ing with the Spencer phase microscope, either brighter or darker than their
environment. The diffraction patterns which obscure unstained bacteria ob-
served with bright-field microscopy are avoided. The phase microscope uses
optical path differences introduced into its lenses to make visible regions of
different refractive index in the specimen. By the use of an intense light source
686 8FORTY4IXTH GENERAL MEETING
the photographic exposures can be made short enough to avoid blurring from
brownian movement. Measurements of Bacillus megatherium will illustrate
the method.

G45. Nuclear Staining of Escherichia coli. JAN LovF, PALMER, Carnegie Insti-
tution of Washington, Department of Genetics, Cold Spring Harbor,
N.Y.
The Robinow technique for staining bacterial nuclei was modified to yield
reproducible results with Escherichia coli (strain B). The length of each step
in the procedure (fixation in 0s04 vapor, hydrolysis in HCI, neutralization in
buffer, staining in Giemsa solution) must be carefully chosen, for best results,
with organisms from both liquid and solid media.
Changes in nuclear morphology during the growth of a culture were studied.
Cells from fully grown cultures appear lightly but uniformly stained; nuclear
differentiation begins to appear soon after transfer to a fresh medium while cell
elongation without division takes place. In actively growing cells, the nuclei
seem to divide before the bacterial cell itself does, so that the common longer
cell forms show at least two nuclei, while the small, recently divided individuals
contain one nucleus only.
The nuclear structures of abnormal cells produced by various methods have
also been examined.

Al. Is Rinse Water at 170 F or Higher Essential to Produce "Sterile" Eating and
Drinking UTtensils? MURRAY P. HoRwoOD, Massachusetts Institute of
Technology, Department of Civil and Sanitary Engineering, Cambridge
39, Mass.
The Massachusetts Institute of Technology Graduate House dining service
has been under technical supervision since 1943. Eating and drinking utensils
are soaked and thoroughly desoiled in clean soapy water at 110-120 F, and
passed through a mechanical dishwashing machine for washing and rinsing. The
temperature of the water varies between 160-180 F and usually is at 170 F.
The total time in the dishwasher is 35 seconds. All utensils are allowed to dry
in the air. Bacteriological results have been consistently satisfactory and ap-
proximated sterility. In the course of the work it became obvious that the
excellent bacteriological results were due to the effective preliminary desoiling
of all utensils. Since the temperature of the wash and rinse water could be regu-
lated, a comparative study, using rinse water at 145-150 F and rinse water at
160-180 F, was undertaken. All other factors were kept constant. Five test
runs over weekly intervals clearly demonstrated that "sterile" utensils can be
obtained with rinse water at 145-150 F, as with rinse water at 160-180 F, if the
preliminary desoiling is complete and effective. The emphasis in restaurant
sanitation should therefore be on adequate preliminary desoiling of all utensils
rather than on the use of high temperature rinse waters.