PROCEEDINGS OF LOCAL BRANCHES OF THESOCIETY OF AMERICAN BACTERIOLOGISTS

CENTRAL NEW YORK BRANCHSENECA CASTLE, NEW YoRK, NOVEMBER 1, 1941

THERMOPMLiC BACTERIA ISOLATED FROMPAPER PULP AND PAPERBOARD. Elea-nore Heist and J. R. Sanborn, NewYork State Experiment Station,Geneva.Samples of pulp and paperboard from

seven mills were studied in an attempt tolearn the identity, numbers, and eco-nomic significance of the thermophilicbacterial flora of pulp and paperboard.It was found that the obligate and facul-tative thermophilic organisms presentwere predominatingly spore-forming rods.Eight species of these bacteria wereidentified. Only Bacillus subtilis and anunidentified brown chromogenic, gram-negative rod appeared abundantly in thematerial examined. One species of Acti-nomyces was identified as Actinomycescasei Bernstein and Morton. The ma-jority of the cultures belonged to speciescommonly present in soil, dust, and air,and probably resulted from contamina-tion from such sources. As examina-tions for the purpose of determining thenumber of bacteria present in pulp andpaperboard are routinely carried out at37°C., it seems probable that the obligatethermophilic bacteria escape notice be-cause their optimal temperature forgrowth is 55°C. It is apparent from thepresence of these organisms in pulp sus-pensions that mills that maintain hightemperatures (49°C. to 63°C.) through-out their pulp systems provide suitableconditions for the development of ther-mophilic bacteria. Although types of

thermophilic bacteria are capable ofdestroying cellulose, none of the culturesisolated in the present study was shownto possess this ability.

STREPTOCOCCUS SALIVARIUs IN THE Hu-MAN INTESTINE. C. F. Niven, Jr., K.L. Smiley, and J. M. Sherman, Labora-tory of Bacteriology, College of Agri-culture, Cornell University, Ithaca.Streptococcus salivarius, as the pre-

dominating microorganism in most hu-man throats, must be swallowed daily bythe millions. Because of its biologicalcharacteristics, however, it has beendoubtful whether these streptococci cansurvive the gastric acidity and bile, andthus contribute to the intestinal flora.The present report summarizes brieflythe results of studies showing that a pro-portion of these organisms do survive thegastric acidity and bile and do ultimatelyconstitute much of the intestinal flora.A special medium with both selective anddifferential values for the isolation andenumeration of polysaccharide-synthe-sizing strains of Streptococcus salivariuswas employed in making cultures of fecalsamples from 18 persons. This mediumincorporates sucrose and sodium azide ina suitable nutrient agar (sucrose, 5%;Bacto-Tryptone, 1%; yeast extract,0.5%; dipotassium phosphate, 0.3%;agar, 1.5%; sodium azide, 0.02%; pH7.0). On this medium Streptococcus sali-varius forms large mucoid colonies, whichcontrast sharply with the character-

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istically small colonies of other strepto-cocci, which are made more apparent bythe partial-to-complete inhibition ofcoliform or other groups that form largecolonies typically. It was found thatfecal samples from 15 of the 18 personsyielded Streptococcus salivarius in num-bers ranging from 1200 to 129 million perg. of feces. Moreover, examinations ofthe fecal samples from 7 of these personsshowed that Streptococcus salivarius waspresent in excess of 1 million per g. offeces, and from 5 of the 7 comprised over50 per cent of the total number of strep-tococci that grew on the sucrose sodium-azide agar.

STREPTOCOCCUS SALIVARIUS, "STREPTO-COCCUS CARDIOARTRITIDIS" AND CER-TAIN OTHER "INDIFFERENT STREPTO-cocci." K. L. Smiley, C. F. Niven,Jr., and J. M. Sherman, College ofAgriculture, Cornell University,Ithaca.The "typical" Streptococcus salivarius

is marked by its ability to synthesize alarge amount of a soluble levan fromsucrose. No other known streptococcushas this property, though occasionalstrains of Streptococcus bovis produce aninsoluble dextran. In Streptococcus sali-varius, this property is correlated withthe ability to ferment inulin and theinability to produce appreciable green-ing of blood agar; also, about 40 per centof such strains belong to one serologicaltype (type I).

Small's Streptococcus cardioarthritidisfermented raffinose and inulin, failed toferment mannitol, and produced nochange in blood agar; and 31 strains be-longed to one serological type. A cul-ture of Small's organism obtained fromthe American Type Culture Collectionproved to be a typical polysaccharide-synthesizing strain of Streptococcus sali-varius, and also belonged to our sero-logical type I.

Hitchcock studied inulin-fermenting,indifferent streptococci and found that alarge proportion of them belonged to oneserological type (type I), while the re-maining cultures were serologically het-erogeneous ("group X"). Two strainsof Hitchcock's "group X" have beenfound to be Streptococcus salivarius onthe basis of physiological reactions andthe ability to synthesize levan, but not tobelong to our type I. Although it wouldseem probable that Hitchcock's sero-logical type I and our own are the same,we have been unable to obtain a strainof his type I organism.Burkhaug's inulin-fermenting, indif-

ferent streptococcus from rheumaticfever was identified culturally and sero-logically by Hitchcock with his ownindifferent streptococci, and was there-fore probably Streptococcus salivarius.

SANITIZATION oF BEVERAGE GLASSES.William G. Walter, New York StateAgricultural Experiment Station, Ge-neva, New York.A survey was made of the bacterial

flora of beverage glasses in six tavernswhere three different sanitization pro-cedures were employed within a two-month period. The glasses fell into twogroups: recently washed, and dried withtowelling. The inside and outside rimsof each glass were swabbed and the swabwas placed in 4 ml. of phosphate buffersolution. One and 0.1 ml. samples of theresultant suspensions were transferredto a special sucrose-agar medium, de-scribed by Niven, Smiley, and Shermanfor the isolation of Streptococcus sali-varius. (Dick and Hucker have indi-cated that the presence of this organismon the rims of drinking glasses is a pre-sumptive index of oral contamination.)It was found that no typical mucoidcolonies developed on plates seeded fromglasses which had been cleansed by anyof three sanitization procedures. Not

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infrequently, however, macroscopicalidentification of Streptococcus salivariuswas impossible because the dilutionsused often resulted in Petri plates over-

crowded with coliform colonies andyeasts. It was observed that an alkyl-dimethyl - benzyl - ammonium - chloriderinse solution generally resulted in a

more marked decrease in the nuimber oforganisms and the elimination of coli-form types from the rims of glasses thandid a similar treatment with cold wateror the chlorine compounds available,and that glasses dried with towelling hadfewer organisms on the rims than thosewhich were wet from dipping in chlorineand water.

TOXOPLASMA INFECTIONS IN ANIMALS.Peter Olaf8on, N. Y. State VeterinaryCollege, Cornell University, Ithaca.

AN OUTBREAK OF ANTHRAX IN MINK.W. A. Hagan, N. Y. State VeterinaryCollege, Cornell University, Ithaca.

THE USE OF BACTERIA IN TIE CONTROLOF INJURIOUS INSECTS. Edward H.Smith, N. Y. State Agricultural Ex-periment Station, Geneva.

PREPARATION AND PRESERVATION OFFRUIT JUICES. C. S. Peder8on and H.G. Beattie, New York State Agricul-tural Experiment Station, Geneva.The continued expansion of the fruit-

juice industry is dependent on a pro-gressive improvement in packing meth-ods in order to provide products of highquality. The attainment of this goaldemands a good blend of fruit, the reten-tion of flavor during processing, and theinhibition of fermentation and alterationof flavor or color during storage. Be-cause of differences in inherent proper-ties, each fruit requires an individualizedprocedure. Pasteurization at low tem-peratures is essential to the retention of

the best flavor, but such temperaturesmust be high enough to kill the organismsresponsible for spoilage and to inacti-vate enzymes in the fruit juice. Thefruit juices of the Northeastern states,because of their comparatively high acidcontent, can usually be preserved byheating to 165-175°F. Reducing theoxygen tension by the de-aeration ofjuice and by completely filling containersis an aid in the preservation of quality.

PROBLEMS IN CLASSIFYING COLIFORMBACTERIA. M. W. Yale, N. Y. StateAgricultural Experiment Station,Geneva.

THE CONTENT OF FUNGOUS SPOREMS INTHE AIR IN BUFFALO, NEw YORK.Victor L. Cohen, Children's Hospital,Buffalo.Air-borne fungi were studied at

Buffalo, New York, in an attempt tolearn their identity, incidence, and rela-tionship to season and to fluctuations inatmospheric temperature and humidity.Sabouraud's agar in Petri plates (15cm. in diameter) was exposed for 30minutes at biweekly intervals through-out 1940. After incubation at room

temperature for 3 days, the cultures wereidentified and counted. These fungifall into the following genera, which arelisted according to their relative inci-dence and seasonal occurrence: Yeasts,from Feb. to Nov., peak Oct.; Hormo-dendrum, from March to Dec., peak July;Alternaria, from May to Dec., peakAugust; sterile mycelia, from Feb. toDec., peak August; Penicillium, fromMarch to May, and again from Oct. toDec., peaks April and Nov.; Aspergillus,sporadic throughout the year; Phoma,from March to August, and again Sept.to Dec., peaks May and Nov.; Mucor,from Feb. to July, and August to Dec.,peaks April and Nov.; Fusidium, fromJune to Nov., peak August; Phycomyces,

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from May to Nov., peak June; Hyalopus,Jan. and Feb., peak Jan.; Monilia, fromJune to Sept., peak July; Cephalothe-cium, from Jan. to March and Oct. toDec., peaks Feb. and Nov. It was

found that the prevalence of differentfungi was apparently unrelated to fluc-tuations in either atmospheric tempera-ture or humidity.

MICROSCOPE OBJECTICVMS AND A NEwSTEREO-POLARIZER. L. V. Foster, Sci-entific Bureau, Bausch & Lomb OpticalCompany, Rochester.Stereoscopy on the non-objective bin-

ocular microscope has usually beenaccomplished by introducing stops in theilluminating system and the exit pupil ofthe microscope. This method has re-

sulted in restricting the numerical aper-ture of the microscope to undesirablelimits. By the use of Polaroid in placeof opaque stops, stereoscopy without

sacrifice in definition has been attained.A new stereo-polarizer with Polaroid eyecaps has been used to accomplish thestereoscopic effect.

AN ANAEROGENIC VARIANT OF SALMO-NELLA ENTERITIDIS (GAERTNER's BA-CILLUS) RECOVERED FROM DOMESTICAND WILD RATS. R. A. Bruce and H.E. Dascomb, University of Rochester,Rochester.

THEc ROUTES OF INVASION OF THE: CEN-TRAL NERVOUS SYSTEM IN YOUNGRABBITS AND MICE BY HERPES VIRUSINSTILLED INTRANASALLY. C. A.Evans, H. B. Slavin, and G. P. Berry,University of Rochester, Rochester.

NEW TRENDS IN RESEARCH ON PLANTDISEASES. G. L. McNew, N. Y. StateAgricultural Experiment Station, Ge-neva.

EASTERN MISSOURI BRANCH

ST. LOUIS UNIVERSITY MEDICAL SCOOL, OCTOBER 21, 1941

DETECTION OF NEIUROTROPIC VIRUS"ANTIBODY" BY THE AGGLUTINATIONOF ANTIGEN-COATED BACTERIA. E. C.Roberts and L. R. Jones, Department ofBacteriology, St. Louis UniversitySchool of Medicine, St. Louis,Missouri.A method was developed which con-

sists of the coating of heat-killed bac-terial cells with virus antigen and theirsubsequent agglutination by an anti-serum which is specific for the adsorbedantigen. In applying this test for thepresence of "antibody" in the sera ofconvalescent encephalitis patients, thevirus of St. Louis type encephalitis was

adsorbed to Serratia marce8cens, and sus-pensions of such virus-coated cells were

agglutinated by these sera. The sera ofexperimentally infected monkeys (Ma-

cacus rhesus) and hamsters likewise gavepositive agglutination reactions.With the sera of 10 or 12 poliomyelitis

cases positive agglutination reactionshave been obtained when tested with a

suspension of cells coated with polio-myelitis virus. In experimental pol-iomyelitis in the monkey, we haveobserved the initial appearance of thisagglutinating factor in the blood serum

as early as the second day after inocula-tion, and 4 or 5 days prior to the advent.of symptoms.

Further study is required to determinethe possible utility of this reaction as an

aid in establishing an early and exactdiagnosis of poliomyelitis.

THE EFFECT OF PASTEURIZATION ON THE

NuTRITIVE REQuiREMENTS OF MILK

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BACTERIA. J. B. Hershey, HealthDivision Laboratory, St. Louis, Mo.The Standard Methods Committee for

the examination of dairy products, onadopting an enriched standard mediumfor plate counts, has observed a rela-tively greater increase in counts of pas-teurized than of raw milk. The reportsthat heat shock causes certain species tobecome more demanding nutritionallysuggest an explanation that can betested.Raw milks from varied sources were

counted on extract-peptone agar (oldstandard) and tryptone-glucose-extract-milk agar (new standard), heated in testtubes under time and temperature condi-tions approximating those of commercialpasteurization, and recounted on bothmedia. The ratios of counts on the two

media were the same before and aftreheating, with an equal increase in thecounts of laboratory-pasteurized and ofraw milk.

Cultures of various non-sporeformingorganisms isolated from milk, counted inthe same way before and after heating,either gave the same counts on bothmedia or failed entirely to grow on ex-tract-peptone agar. Newly isolated andstock strains of Escherichia coli similarlycounted on both agars and by the dilu-tion method in TGEM broth, had iden-tical counts in all three regardless ofheating.Under the conditions of laboratory

pasteurization, no change in the nutri-tive requirements of milk organismscould be detected by the new standardmedium as compared with the old.

INDIANA BRANCHINDIANA UNIVERSITY, NOVEMBER 14, 1941

STUDIES ON DRY STERILIZATION. K. B.Riddle and P. S. Prickett, Bacteriologi-cal Laboratory, Mead Johnson & Co.,Evansville, Ind.The necessity for dry sterilization in

certain commercial applications whereheat can not be used is pointed out.Comparisons of the results obtained byvarious methods of dry sterilizationshow that the most satisfactory onestudied is based on an insecticidalmethod which uses ethylene oxide. Byadapting this method it is shown thateither pure ethylene oxide or a mixtureof this gas with carbon dioxide can beused to effect dry sterilization of micro-organisms. With this adaptation bothfood and non-food products were shownto be successfully sterilized.

Soil samples of high counts, among thenon-food products, were sterilized whentreated by this method, using pureethylene oxide, in determining its germi-cidal effects under severe conditions.

ISOLATION OF A NEW CLOSTRIDIUM PRO-DUCING A BLUE PIGMENT. L. S. Mc-Clung, Indiana University, Bacteri-ological Laboratories, Bloomington,Indiana.From a sample of mud taken at the

Hadlyme Ferry on the Connecticut River,in southern Connecticut, a Clostridiumwas isolated which produces a blue-greenpigment which is selectively absorbed bythe germ of a grain of corn. The pig-ment, which may be demonstrated intubes of 5 per cent white corn mealmedium, requires several days for forma-tion and is further characterized by thefact that exposure to air causes a changeto the leuco form. Pigmented colonieson carbohydrate yeast water agar losetheir color within 30 minutes after beingremoved from an anaerobic environment.The organism is a slender bacillus, withoval spores, and best pigmentation isobtained at temperatures near 30°C.Preliminary studies, which are being

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continued, indicate that the isolationrepresents a new species. It has notbeen encountered in approximately 150other samples from various locations inthis country nor from samples from thegarden of the Pasteur Institute of Paris,a jungle in India, soil from the ArcticCircle, and beach sands from Cuba.

SEXUAL HORMONES IN THE FUNGI. JohnR. Raper, Botany Department, In-diana University.Sexual hormones in the Fungi were

first demonstrated by Burgeff (1924) inMucor mucedo. Since that time his ob-servations have been confirmed andextended to other heterothallic membersof the Mucorales by Verkaik (1930),Ronsdorf (1931), Kohler (1935), andKrafczyk (1935). Most significant ofthese is the work of Krafczyk whopresented evidence that the sexual re-productive process in Pilobolus crystal-linus is under hormonal control until thetime of gametangial differentiation.More recently sexual hormones have

been demonstrated in the aquatic Phy-comycetes. In Achlya Raper (1939 etseq.) showed that at least four specificsubstances are involved in initiating andcoordinating the several stages of thesexual reaction between c' and 9 plants.It has now been demonstrated that afifth hormone is involved in a mutuallycomplementary manner with the firstof the four originally postulated. Thesecretion and activity of at least onespecific substance has been demonstratedin Sapromyces by Bishop (1940).A substance secreted by A8pergillus

niger and one liberated from dead yeastcells bring about conjugation in Zygosac-charomyces, recently reported by Nick-erson and Thimann (1941).

RIBOFLAVIN ASSAY OF CEREALS BY THEMICROBIOLOGICAL METHOD. K. B.

Riddle, L. J. Paul, H. J. Miller andP. S. Prickett. Mead Johnson.

THE EFFECT OF FLAsH PASTEURIZATIONAND SUBSEQUENT TREATMENT ON THEPHOSPHATASE VALUE OF CREAM. W.H. Brown and P. R. Ellikey, PurdueUniversity, Agricultural ExperimentStation.Repeated trials have shown that when

cream is pasteurized by the vat method,the phosphatase test can be used to de-termine the adequacy of heat treatmenton both sweet and neutralized, sourcream. Comparable results were ob-tained with the laboratory tests ofScharer and Neave's modification of theKay and Graham method.When the flash systems of pasteurizing

cream were used, considerable variationoccurred in the phosphatase reaction ofthe pasteurized creams. In many casesthe phosphatase value immediately afterpasteurization was negative or slightlypositive, but when the cream was heldin storage the phosphatase value in-creased. This increase was encounteredat temperatures as low as 4.4°C. Whenthe cream was stored at higher tempera-tures for the same period, the increase inthe phosphatase value was more pro-nounced. This same phenomenon hasoccurred in cream as a result of prolongedcooling after the completion of thepasteurization process.Recent studies with "vacreated" neu-

tralized, sour cream have shown that theScharer test and the modified Kay andGraham test may not yield comparableresults when NaCl is added to the creamafter pasteurization and the cream storedfor 24 hours at temperatures of 15.6, 21.0,26.8, and 32.1°C. The phosphatasevalues obtained with the Scharer testwere the same for the salted and unsaltedcream. With the modified Kay andGraham test the phosphatase value ofthe salted cream was greater than that

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of the unsalted cream when the sampleswere stored at the above four tempera-tures following pasteurization. Thisdifference was more apparent in the sam-ples stored at the highest temperatures.No differences in phosphatase valueswere obtained with the modified Kay andGraham test on unsalted and saltedsamples of cream when they were storedat a temperature of 4.4°C. or lower for24 hours.

SIGNIFICANT DIFFERENCES IN X-RAYSENSITIVITY AMONG VARIOUS SPECIESAND STRAINS OF YEASTS. Raymond E.Zirkle, Department of Botany andBacteriology, Indiana University.It has been found that, among the

many factors which influence the sensi-tivity of living cells to ionizing radia-tions, small differences in taxonomicposition may be quite significant.Graded doses of 200 kilovolt x-rays wereadministered to samples of yeast cellssuspended in dilute salt solution (Ring-er's), and the percentage of survivors ofeach dose was determined by the platecount method. From the resultingsurvival curves it was found that thedoses necessary to kill 50 per cent of thecells of various yeast types were asfollows:Zygosaccharomyces barkeri Saccardo

3,300 rTorulopsis fermentans Mrak &McClung (Strain A)........... 15,500 r

Torulopsis fermentans Mrak &McClung (Strain B)........... 15,500 r

Torulopsis californicus Mrak &McClung......................27,500 r

Saccharomyces cerevisiae Hansen(Strain A)..................7,600r

Saccharomyces cerevisiae Hansen(StrainB)..................... 14,900 r

It is therefore evident that not only mayspecies of the same genus (Torulopsis)differ widely in radiosensitivity butstrains of the same species (Saccha-

romyces cerevisiae) may exhibit similardifferences. The two strains of S.cerevisiae were of rather widely differentorigin, while the two strains of T. fer-mentans which were equal in radiosensi-tivity were isolated from the samelaboratory stock.

RAPID PERTUSSIS AGGLUTINATION TESTSOF CIRCULATING ANTIBODY IN STOCKAND SWISS MICE TREATED WITH PHASEI PERTUSSIS VACCINE. H. M. Powell,Lilly Research Laboratories, Indian-apolis, Indiana.These experiments utilized a recently

prepared rapid pertussis agglutinatingantigen capable of giving specific posi-tive results with a single droplet ofimmune blood in one minute (Jour. Im-munology, in press). These rapid per-tussis agglutination tests may be donevery quickly with blood from the tip ofthe tail of white mice with no obviousinjury to the mice.Ordinary stock white mice and Swiss

mice have been treated with stand-ardized amounts of Phase I pertussisvaccine. About 50 per cent of the stockmice show agglutinin at two to eightweeks after immunization, with the re-actions becoming stronger in the latertests. About 90 per cent of the Swissmice show agglutinin under the sameconditions, and more of these reactionsare stronger than those exhibited by thestock mice. Several comparative im-munity tests have been done with dif-ferent lots of pertussis vaccine.

THE IMMUNIZING VALUE IN GUINEAPIGS OF FOUR TYPES OF TYPHuSVACCINES. H. A. Dettwiler, Lilly Re-search Laboratories, Indianapo-lis, Ind.A comparative study of vaccines pre-

pared by different methods was made by

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measuring the protection afforded tovaccinated guinea pigs. Vaccines ofsimilar rickettsial concentration wereprepared as follows: (1) yolk sac vaccinemade with the epidemic strain of typhus,(2) yolk sac vaccine prepared with theendemic strain, (3) material preparedfrom rat lungs infected with the endemicstrain of typhus, and (4) from mouselungs infected with the epidemic strain.

Different groups of guinea pigs werevaccinated with each preparation andlater, along with control animals, sub-

jected to a challenging dose of epidemictyphus rickettsiae.The results, as determined by the

temperature curves of the experimentalanimals, indicate that vaccines madewith the endemic strain of typhus protectguinea pigs against an epidemic infectionbut to a lesser degree than vaccinesprepared with the epidemic strain.

OBSERVATIONS ON PuBLIC HEALTH INENGLAND AT WAR. Dr. N. Paul Hud-son, Ohio State University.

MARYLAND BRANCH

BALTIMORE, OCTOBER 9, 1941

MORPHOLOGICAL STUDIES OF FUSIFORMORGANISMS. M. S. Aisenberg, Univer-sity of Maryland, School of Dentistry,Department of Bacteriology.The organisms commonly called fusi-

form bacilli in active Vincent's diseaseare shown, from studies of their morpho-logical appearance, to be spores of ahigher organism. By means of their

septate stems, a life cycle in theirdevelopment is demonstrated. Becauseof their morphological appearance, it isbelieved that these organisms were neverisolated in pure culture.

RAT BITE FEVER. Thomas McP. Brom,Johns Hopkins University, Depart-ment of Medicine.

NORTHERN CALIFORNIA-HAWAIIAN BRANCH

NOVEMBER 4, 1941

CARBON ASSIMILATION OF YEAST.Morris J. Pickett, Department ofBacteriology, Stanford, California.Under aerobic conditions approxi-

mately 31 per cent of the total carbon inthe glucose utilized by resting suspen-sions of Saccharomyces cerevisiae is givenoff as C02, the R.Q. being 1.00. Using amicrocombustion technique, it has beendemonstrated that approximately 30 percent of the original carbon entered thecell as synthesized material, apparently apolysaccharide as suggested by acidhydrolysis followed by reducing sugardeterminations. Twenty-nine per centof the carbon is present as non-volatilematerial in the suspension medium and

8 per cent as volatile material, 98 percent of the added carbon thus beingrecovered. Approximately one-half ofthe carbon in the suspension mediumcan be accounted for as glycerol, alcohol,succinic acid and traces of residualglucose. Tests for 2,3-butylene glycol,acetyl-methyl-carbinol, diacetyl, phos-phate esters, and volatile, pyruvic andlactic acids were negative or showed onlytraces of these compounds.

THE FATE OF PASTEUIRELLA PESTIS INTHE FLEA. J. R. Douglas and C. M.Wheeler, The George Williams HooperFoundation for Medical Research,

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University of California, San Fran-CisCO.The following observations were made

in connection with studies of the relativevector efficiency of two species of flea,Xenopsylla cheopis (Roth.) and Dia-manuts montanus (Bak.).The animal chosen for transmission

studies was the white mouse. It was

found to be very susceptible to smallnumbers of Pasteurella pestis and todevelop a high degree of bacteraemia.The maximal bacteraemia approidmated1 X 107 organisms per cubic millimeter ofblood. In this phase there is one

organism per red blood cell.The capacity of the stomach of D.

montanus and X. cheopi8 as calculatedfrom the number of organisms ingestedwas found to average 0.030 c. mm.

Under conditions of maximal bacterae-mia either of these species will ingest an

average of 3 X 101 P. pestis.Approximately 60 per cent of the

infected D. montanus and 2 per cent ofthe X. cheopis were able to clear them-selves of P. pestis. In the former thistakes place within twenty-four hoursafter feeding and in the latter the processrequires more than forty-eight hours.P. pestis may multiply in the oesopha-

gus, proventriculus, stomach and rectalsac of the two species studied. Eightyper cent of infected D. montanus and

40 per cent of infected X. cheopis haveplugs of plague bacilli in the oesophagus.In infective individuals the oesophagus isinvaded in 95 per cent of D. montanusand 62 per cent of X. cheopis. In thesame individuals the proventriculus isplugged in 95 per cent of D. montanusand 100 per |cent of X. cheopis. Fiveper cent of the D. montanus were able totransmit the infection and later force a

canal through the proventricular plug.In D. montanus the period from infec-

tion to transmission, or in other wordsthe period necessary for the developmentof the oesophageal and proventricularplugs, averaged ten days and the periodfrom transmission to death five days. InX. cheopis the developmental periodaveraged sixteen days and the fleas diedin three days. It is quite obvious that a

species which survives longer in theinfective state will be able to effect moretransmissions than a species whichsurvives a shorter time.The daily fecal cultures of infected

fleas were found to be irregularly positivefor plague. Fifty-six per cent of D.montanu and 25 per cent of X. cheopissamples contained viable P. pesti8.The number of organisms excreted was

found to be very small, the average was

200 in a twenty-four hour period with a

maximum of 400 and a minimum of lessthan ten.

TEXAS BRANCHFIRST MEETING, THE UNIVERSITY OF TExAs, AUSTIN, TEXAS, NOvEMBER 1, 1941

MODE OF SELECTVE AcTION OF E.M.B.AGAR IN DIFFERENTIATION WITHN THE

ENTERIC GRoup. E. S. Wynne, L.J. Rode and A. E. Hayward, Depart-ment of Bacteriology, The Universityof Texas, Austin, Texas.The actual mechanism of the differen-

tiation of lactose-fermenting and non-

lactose-fermenting organisms on eosin-methylene-blue agar is not reported in

the literature. The present study offersan explanation of this mechanism.The color of lactose-fermenting organ-

isms on E.M.B. plates is dependent on

two factors: (1) The reaction of eosinwith methylene-blue to form a meth-ylene-blue eosinate which acts as an

acidic dye, and (2) the production, bythe lactose fermenters, of a sufficientlylow pH so that the acidic dye is taken up

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by individual cells of the colonies. Thenon-lactose-fermenters produce colorlesscolonies because the acidic dye will notstain cells in the alkaline medium pro-

duced by such colonies.The methylene-blue eosinate has been

shown to consist of one molecule of eosinand one of methylene-blue.The results of this investigation are to

be published in the January, 1942 issueof Stain Technology.

THE SALMONELLA TYPING CENTER AT

BAYLOR COLLEGE OF MEDICINE. MacD. Fulton, Baylor University Collegeof Medicine, Dallas, Texas.

NON-COUFOiam BACTERIA IN STOOLSSUBMITTED FOR PARASITOLOGICAL Ex-AMNATION. W. M. Fisher, BaylorUniversity College of Medicine, Dal-las, Texas.

BACTERIOPHAGE V Fonit TYPEs OFTYPHOID BACILLI IN TEXuS. I. Dis-TRIBUTON OF TYPES. E. B. M. Cookand M. M. Sigel, Bureau of Labora-tories, Texas State Department ofHealth, Austin, Texas.Six hundred-and-sixty-four strains of

typhoid bacilli isolated from patients in84 counties in Texas during the past 8years have been tested by the V formbacteriophage typing method of Craigieand Yen. 331 strains have been typed.It is of interest that although some ofthese cultures were 8 years old the Vform still persisted and the organismscould be typed. Types A, E1 and C were

predominant among the strains typed.

OBSERVATIONS ON STREET RABIES INWHITz MCE. J. V. Irons and S. W.Bohls, Bureau of Laboratories, TexasState Department of Health, Austin,Texas; and J. D. English, John Tarle-ton College, Stephenville, Texas.Two strains of white mice, one of which

was a Swiss strain, were comparably sus-

ceptible to intracerebral injections ofstreet rabies virus. The incubationperiods varied considerably but were

possibly slightly decreased with theSwiss strain. Paralytic symptoms were

constantly observed in animals whichsuccumbed; the mortality rate was ap-proximately 90 per cent with each strainof mice.Both the virus content of the Ammon's

horn in dog brains and of the dog's sub-maxillary gland frequently varied ap-

preciably. In the submaxillary glandvirus oftentimes was not detected.

THEE ISOLATION AND CULTURE OF A

CELLULOSE-DECOMPOSING BACTERiUMProM THE RumEN OF CATTLE. R. E.Hungate, Department of Zoology, TheUniversity of Texas, Austin, Texas.An obligate anaerobic, spore-forming,

cellulose-decomposing bacterium hasbeen isolated in pure culture from therumen of cattle. In contrast to thedifficulties usually encountered, the or-

ganism is easy to culture in anaerobicshake tubes containing cellulose, agar,

and an inorganic salt medium includingammonium sulfate. Transfer to a glu-cose agar medium gives growth and disc-shaped colonies. Return to celluloseagain results in cellulose digestion.Clear spots around the colony in whichno bacteria are present indicate theformation of an extracellular enzyme.

In old cultures in which the cells havebecome inactive, presumably due tostaling products, the enzyme continuesto act and dissolves the cellulose, form-ing substances which give a distinct cop-

per reduction. Fermentation productsinclude CO2, H2, ethyl alcohol, formicacid, acetic acid and lactic acid. Thenutritional requirements are met bysupplying biotin in addition to am-

monium salts and cellulose.

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PROCEEDINGS OF I

THE BACTERIA INVOLVED IN THE RIPEN-

ING OF' BRICE CHEESE. E. M. Foster,Department of Botany and Bacteri-ology, the University of Texas, Austin,Texas.

PNEumOCOCCUS TYPE INCIDENCE IN

TExAS DURING 1940-1941. J. T.Kroulik and W. E. Ragsdale, Bureauof Laboratories, Texas State Depart-ment of Health, Austin, Texas.Pneumococcus type incidence in a

group of 1,160 typed cases of clinicalpneumonia which occurred in Texasduring 1940-1941 and received the serv-

ices of the Texas Pneumonia ControlProgram were studied from the stand-point of general type incidence, incidencein varying races, incidence in varying age

limits and incidence in deaths andbacteremias.Types I, VII, III, XIV and XIX were

the most frequently occurring types,being present in 18.4, 9.9, 7.8, 7.4 and6.9 per cent of the cases respectively.Among the 634 cases in whites and the

423 cases in Negroes, Types I and VIIwere the most prevalent in each group.

Types XIX and VI were the most preva-lent in the 103 cases reported in Mexicansbut considering the small number ofcases reported in this group a change inthe order of occurrence might be ex-

pected with the study of additionalcases.

In 576 cases reported in adults and 317cases in children from 2 to 13 years of ageinclusive, Type I was the most prevalentwith Type VII being second in occur-

rence in the adult group and Type VIin that of the children. Type XIV was

the most prevalent in 267 cases in infantsand children under two years of age, withType XIX being second in occurrence.

Bacteremias were reported in 0.93 percent of the total cases typed and themortality rate in the same group was

8.1 per cent.

LOCAL BRANCHES 123

TOXIN PRODUCTION BY STAPHYLOCOCCUISVARIANTS. Sol Haberman and AlvinL. Miller, Bacteriology Laboratory,Baylor University Hospital, Dallas,Texas, and De Lamar Institute ofPublic Health, Columbia University,College of Physicians and Surgeons,New York, New York.One hundred and thirty-three variants

of seven staphylococcus cultures werestudied as to their ability to producedemonstrable toxic filtrates. The toxicmanifestations under observation werethe lethal effects in mice, dermonecrosisin rabbits, and the hemolysis of rabbit,sheep, and human erythrocytes.The most common observation was the

loss of toxic manifestations among thevariants as compared to the parentstrains. Twenty-two variants lost theability to produce a demonstrable lethaltoxin, seventeen lost the dermonecrosin,twenty-four failed to lyse rabbit erythro-cytes, twenty-one failed to lyse sheeperythrocytes, seventeen failed to lysehuman erythrocytes, and twenty-one didnot form any hemolysins. Two parentcultures which did not produce sheep orhuman hemolysins yielded three variantswhich lysed sheep erythrocytes and fourvariants which lysed human erythro-cytes. Also, two parent cultures whichformed no dermonecrosins yielded twen-ty-one variants that produced dermone-crotic filtrates.The independent appearance and dis-

appearance of these manifestations sug-gests that the toxin is composed of amultiplicity of factors.

OBSERVATIONS ON NATURALLY INDUCEDHImAN RELAPSING FEVER. J. L. Ter-rell, Bureau of Laboratories, TexasState Department of Health, Austin,Texas.Relapsing fever was used as a thera-

peutic fever at the Austin State Hospitalon a total of 147 patients between the

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PROCEEDINGS OF LOCAL BRANCHES

years 1934 and 1940. This group com-prized 131 colored and 16 white patients.Over 75 per cent of the group were diag-nosed as paretics.

Infected ticks of the genus Ornitho-doru turicata collected from a cave incentral Texas were used in the inocula-tions and were propagated in a "tick-torium" for this purpose. With one

exception no untoward effects followedthe tick bites.The average incubation period was

6.55 days, varying from 4 to 9 days.Prodromal symptoms were common,

while the onset of the clinical symptomswas abrupt. Chills were rare; nausea

was fairly common; and drenchingsweats were frequently seen during at-tacks. The main complaints were head-aches, pains in the joints and muscles,and lack of appetite. During the apy-rexial periods the symptoms abated. Amacular rash was seen on seven of thewhite patients. No serious complica-tions were observed.

The daily blood picture on ten of thepatients showed no abnormal changeswith the exception of a mild fluctuatingleucocytosis and a marked increase inmonocytes. There was no anemia.The blood smears showed the spiro-

chetes to be most numerous one-and-one-half to two days after the onset of thefirst paroxysm, rapidly decreasing innumbers at the decline of the paroxysm.

The organisms were rare in the relapsesmears, while none were found duringthe apyrexial periods. The organismstaken from onset blood appeared a littlelonger and more slender than the declineorganisms.

ANTIGENIC VARIATION OF BORRELIA RE-CURRENTIS IN THE RELAPSE PHENOM-ENON IN THE WmTE RAT. V. T.Schuhardt, Director of the ClaytonLaboratory for the Study of InfectiousDiseases, Department of Bacteriology,University of Texas, Austin, Texas.

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