PROCEEDINGS OF LOCAL BRANCHES OF THE SOCIETYOF AMERICAN BACTERIOLOGISTSEASTERN PENNSYLVANIA CHAPTER

ONE HUNDRED AND SIXTY-FIRST MEETING, PHILADELPHIA COUNTY MEDICAL SOCIETYBUILDING, OCTOBER 27, 1942, PHILADELPHIA, PA.

STUDIES ON THE ALLERGIC AND ANTIGENICACTIVITY OF SONIC FILTRATES OF BRU-CELLA ABORTUS. I. Live, School of Veter-inary Medicine, University of Pennsyl-vania, Philadelphia, Pa.Sonic filtrates of Brucella abortus were

prepared by exposing dense suspensions ofthe organisms for two hours to vibrations ofaudible frequency at 9,000 cycles persecond. The magnetostriction oscillatorof Chambers and Flosdorf was used. Rab-bits and guinea pigs were sensitized by aninjection of about 900,000,000live organisms.An intravenous injection of the organismsinto rabbits resulted in a more uniform skinsensitivity to the intracutaneous injectionof sonic filtrate than an intraperitonealinjection. Guinea pigs were sensitized byan intraperitoneal injection of the organ-isms. The animals usually showed satis-factory allergic skin sensitivity to the sonicfiltrate when tested one month after in-jection. In some instances an intracutane-ous injection of as little as 0.0015 mg ofprotein in the filtrate produced skin reac-tions in sensitized animals. The allergicskin reactions were usually at their height48 to 72 hours after injection of sonic filtrate,and persisted in some cases for as long as10 to 14 days. The infected animals de-veloped agglutinins, precipitins, and op-sonins as determined by serological tests.Single intracutaneous injections of as highas 0.05 mg of protein in the sonic filtrate didnot sensitize non-infected rabbits to subse-quent skin tests. However, they developedagglutinins as a result of the injection offiltrate, and the agglutinative titers as wellas the length of time for which they per-sisted were directly proportional to thequantity of protein injected.The stability of sonic filtrates of B.

abortus was studied by comparing the al-lergic activity of lyophilized material withthat of non-lyophilized filtrate. Tests atfrequent intervals over a period of two

years did not show any change in the ac-tivity of the untreated filtrate. Of threepreservatives used, 0.5% phenol, 0.25%formalin, and 1:10,000 dilution of merthio-late, none was found to impair the allergicactivity of sonic filtrate in the course ofcomparisons over a period of one year.

THE ANTIGENIC STRUCTURE OF HEMOPHILUSPERTUSSIS. E. W. Flosdorf, A. Bondi,Jr., Harriet M. Felton, and A. C. Mc-Guinness, Department of Bacteriologyand Pediatrics, School of Medicine, Uni-versity of Pennsylvania, Philadelphia,Pa.Hemophilus pertussis is of a single sero-

logic type. Only organisms freshly isolatedfrom active cases of whooping-cough, knownas strains in phase I, are of clinical sig-nificance.Two toxins occur in H. pertussis in all

phases; one toxin is thermolabile and theother is thermostable. These toxins aloneare apparently not adequate for establish-ment of immunity by their use as toxoidalimmunizing agents unsupported by reagentsto produce antibacterial immunity as well.Vaccines of killed whole organisms are be-lieved to be successful in production ofactive prophylaxis as the result of the anti-bacterial antibodies which are elicited.Because of the lack of thermolabile toxinpresent in such vaccines now currentlyused, one would not expect much fromtoxin as a reagent in a test for suscepti-bility to whooping-cough. It might dis-tinguish those who have had the disease,but it would not distinguish those whohave been vaccinated from the susceptibleindividuals. In clinical trials, the skintest using agglutinogen is being confirmedas of value for determination of suscepti-bility.

CLINICAL RESULTS OF THE USE OF AG-GLUTINOGEN FROM PHASE I HEMOPHILUS

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PERTUSSIS AS A SKIN TEST FOR SUS-CEPTIBILITY TO WHOOPING-COUGH. Har-riet M. Felton and E. W. Flosdorf, De-partments of Pediatrics and Bacteriology,School of Medicine, University of Penn-sylvania, Philadelphia, Pa.Results were given of a clinical study with

a total of 776 cases, using the purifiedagglutinogen as a skin test for suscepti-bility to whooping-cough. The test seems

to classify immune and susceptible indi-viduals according to the histories of diseaseand vaccination in a satisfactory manner.

This material in single skin test doses pro-

duces a marked increase in the agglutinatingtiter of individuals with existing immunityor partial immunity to pertussis. Repeateddoses produce a reversal of the test and a

detectable titer in individuals who had no

initial immunity. A small institutionalepidemic was studied and the results indi-cate that susceptibility can be accuratelypredicted by the use of this skin test.

THE ANTIGENIC STRUCTURE OF HEMO-PHILUS PARAPERTUSSIS AND ITS PROBABLE

CLINICAL SiGNIFICANCE. A. Bondi, Jr.,Harriet M. Felton, and E. W. Flosdorf,Departments of Bacteriology and Pedi-atrics, School of Medicine, Universityof Pennsylvania, Philadelphia, Pa.By means of agglutinative absorptive

tests it has been shown that cross agglutina-tion between H. parapertussis and H.pertussis in Phase I is due to a common

minor antigen. The wide incidence ofparapertussis agglutinins among the generalpopulation as shown by Miller has beenconfirmed. These agglutinins are specific,in large measure, presumably as a result ofspecific infection by H. parapertussis as

shown by means of agglutinin absorption.This suggests some prevalent disease inchildren and adults specifically related toH. parapertussis which clinically and diag-nostically is not recognized as such. Al-though intensive immunization increasesthe agglutinative titer against the heterol-ogous species, it is not known whethersuch agglutinins are effective in cross pro-tection against either disease.

ONE HUNDRED AND SIXTY-SECOND MEETING, PHILADELPHIA COUNTY MEDICAL SOCIETYBUILDING, NOVEMBER 24, 1942, PHILADELPHIA, PA.

TEE USE OF THE SYRIAN HAMSTER AS A

LABORATORY ANIMAL. Harry E. Morton,Department of Bacteriology, School ofMedicine, University of Pennsylvania,Philadelphia, Pa.The syrian hamsters has been found to be a

satisfactory laboratory animal for the studyof numerous viruses, as follows: encephalitis(Linnette, 1941; Broun, LeGier, Mezera,and Muether, 1941), equine encephalomye-litis (Shahan and Creech, 1942), influenza(Taylor, 1940; Taylor and Parodi, 1942, andWheeler and Nungester, 1942), lymphocyticchoriomeningitis (Smadel and Wall, 1942),mare abortion (Anderson and Goodpasture,1942), and poliomyelitis (Plotz, Reagan,and Hamilton, 1942). They have also beenfound useful in the study of dental caries(Arnold, 1942), leptospirosis (Morton, 1942),various vitamin deficiencies (Routh andHouchin, 1942), and are susceptible to thehuman, bovine, avian, and vole strains oftubercle bacilli (Griffity, 1941) and toleishmania infections (Adler and Tcherno-

meretz, 1941). We have found the animalshardy, free from the usual laboratory in-fectious diseases, and capable of bearingyoung when only 67 days old. The gesta-tion period is 21 days with 6 or 7 youngcomprising an average litter.

THE EFFECT OF PROMIN ON EXPERIMENTALTtUBERCUrLOSIS IN THE RABBIT. Max B.

Lurie, The Henry Phipps Institute, Uni-versity of Pennsylvania, and JosephStokes, Jr., The Children's Hospital,Philadelphia, Pa.Twelve rabbits were fed twice daily with

particulate chow which had been thoroughlymixed with a solution of promin in watercontaining Karo syrup. The amount ofpromin consumed by each rabbit variedbetween 350 to 750 mg per kilo per day.These rabbits, together with an equalnumber of control animals, were infectedintracutaneously with 0.2 mg of a virulentstrain of bovine tubercle bacilli (Ravenel).The control animals were given the same

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diet, in exactly the same manner, in thesame quantities including the syrup, exceptthat no promin was added. The progressof the disease at the site of inoculation andin the draining lymph nodes was carefullymeasured at weekly intervals. During thefirst four weeks following the infection no

significant difference was observed in theprogress of the tuberculosis at these sitesbetween the promin-treated and controlanimals. On the thirty-second day afterinoculation the local lesion in both groups

had ulcerated. The pus from each lesionwas evacuated and finely ground crystals ofpromin were sprinkled, at weekly intervals,into the wounds of the experimental ani-mals while glucose, the constituent ofpromin outside the diphenol-sulfone groupof the drug, was likewise sprinkled into thelesion of the control animals after theevacuation of its pus.

It was found that all of the local lesionsof all of the experimental animals withoutexception had healed completely by the15th week after infection while not a singlelocal lesion in the control animals was

healed at the end of 140 days, when allsurviving animals of both groups were

sacrificed. The extent of the disease in the

lymph nodes draining the site of inoculationwas about five times greater in the controlanimals than in the rabbits treated locallywith promin; apparently because of thedrainage of large amounts of the drug fromthe primary lesion to the local nodes. Asimilar though less pronounced retardationof the tuberculous process in the lungs,kidneys, pleura, bones, and joints and otherinternal organs was clearly discernible in thepromin-treated group. The concentrationof promin in the blood of these animalsvaried between 2 to 6 mg per cent. Thereis evidence that the glucose administered tothe control animals was not responsible forthe failure of the local tuberculosis to heal.The efficacy of promin in the local treatmentof tuberculosis in rabbits is evident fromthese experiments.

CHARACTERISTICS AND THERAPEUTIC USESOF SUCCINYLSULFATHIAZOLE. W. F. Ver-way, Sharp and Dohme Laboratories,Glenolden, Pa.

LABORATORY STUDIES IN SPOTTED FEVERIMMUNE SERUM. Florence Fitzpatrick,Sharp and Dohme Laboratories, Glen-olden, Pa.

NEW JERSEY BRANCH (THEOBALD SMITH SOCIETY)NEW BRUNSWICK, N. J., NOVEMBER 5, 1942

CULTIVATION OF HEMOPHILUS INFLUENZAE

IN YOLK-SAC OF DEVELOPING CHICKEMBRYO. E. Alture-Werber, Division ofMicrobiology, The Squibb Institute forMedical Research, New Brunswick, N. J.Six- and seven-day-old chick embryos

proved to be very susceptible hosts for H.influenza. Given these organisms by theyolk sac method in a highly diluted culture6 to 60 organisms were sufficient to kill allembryos. Virulence tests suggested thateven a single organism would infect andkill the embryo. The bacilli multiply andspread rapidly in the egg and kill theembryo in 2 to 3 days. Yolk and amnioticfluid are teeming with bacilli. The sus-

ceptibility to H. influenzae appears todiminish with the age of the embryo.Microscopic preparations of the head of theembryos showed that H. influenzae invadesthe blood vessels and causes severe hemor-

rhages. No meningitis was observed inthese sections.

CHEMOTHERAPEUTIC STUDIES WITH HEMO-PHILUS INFLUENZAE IN MICE AND CHICKEMBRYOS. E. Alture-Werber, Division ofMicrobiology, The Squibb Institute forMedical Research, New Brunswick, N. J.Mice were infected intraperitoneally with

10.000 to 100.000 organisms suspended inmucin. Of mice fed on sulfonamides mixedin Sherman diet 48 hours before infection,74% were protected by 1% sulfathiozole and91 by 0.1% sulfadiazine. Ten mice fed on

1% promin survived. The untreated in-fected controls had a survival rate of 13%.Of mice treated intraperitoneally immedi-ately after infection with the sodium salt ofthe compounds, 42% of those with sulfa-thiozole (600 mg./kg. daily) and 80% ofthose with sulfadiazine (500 mg./kg. daily)

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survived. The untreated controls belongingto this group showed a survival rate of 10%.

Six- and seven-day-old chick embryosinfected with 6 to 60 H. influenza bacilliby the yolk sac method and treated 30 to45 hours later with various amounts of thesodium salts of the drugs by the same routegave the following results: most of theembryos survived for the 8-day period ofobservation when treated with 1 mg. to3 mg. sulfathiozole. One mg. to 3 mg. ofsulfadiazine protected most of the 6- and7-day embryos. Smaller doses of the drugdelayed the death of the embryos. Prominhad no influence on the course of infection.Untreated infected chick embryos have a

mortality rate of 100%; their yolk andamniotic fluid are teeming with bacilli,while in treated eggs, amniotic fluid andembryo, depending on the protective powerand dosage of drug, are little or not at allaffected by the infection.

FUNGI TOLERANT TO EXTREME ACIDITY ANDHIGH CONCENTRATIONS OF COPPER SUL-FATE. R. L. Starkey and S. A. Waksman,Agricultural Experiment Station, RutgersUniversity, New Brunswick, N. J.Two fungi were isolated from acid solu-

tions (pH 0.2 to 0.7) containing 4 per centcopper sulfate. One form was found to beclosely related to Cephalosporium and wasidentified as Acontium velatum Morgan; theother was a dark green fungus belonging tothe Dematiaceae; accurate identificationwas impossible since it did not produce anytrue spores.Both fungi were tolerant to extremely

high concentrations of hydrogen ions andcopper sulfate. They grew quite well in a

synthetic medium with pH 0.3, 0.4, 0.5 and1.0 and made limited development even atpH 0.1. The green fungus likewise grew atpH 0. The most acid medium which sup-

ported growth had an acidity of approxi-mately 2.5 normal H2SO4. Both of the fungiwere able to grow in media saturated withcopper sulfate, although they grew better

in media to which no copper salt was added.They developed quite well in saturatedsolutions at pH 2.0 to 0.3 and produced somegrowth at a reaction level of approximatelypH 0.These fungi appeared to be as tolerant to

acidity as any organism known so far; itis believed that these two organisms showthe greatest tolerance to the combinedeffects of high concentrations of copper

sulfate and of extreme acidity yet recorded.

TUMOR FORMATION BY ATTENUATED CROWN-GALL BACTERIA IN THE PRESENCE OFGROWTH-PROMOTING SUBSTANCES. Ar-min C. Braun and Thomas Laskaris.An attenuated culture of Phytomonas

tumefaciens formed large tumors on tomatoplants when supplemented with growth-promoting substances. The growth sub-stance reaction was not specific. The threesynthetic growth substances used as well as

the plant growth hormones served as stimu-lating agents. Fragments of these arti-ficially induced tumors when implanted intohealthy tomato plants developed intotypical large tumors in 4 to 5 weeks. Thetumor tissues have now successfully beencarried through 5 successive passages intomato plants involving a period of sixmonths. One clone of tumor tissue was

isolated that was entirely free of crown

gall bacteria. Fragments of this bacteria-free tissue grew rapidly into large neoplasticgrowths similar to the bacteria-containingtumors. The host cells when acted upon bythe growth substances alone were foundincapable of inducing the formation oftumors. It would appear, therefore, thatthe attenuated culture is capable of alteringthe host cells to tumor cells. The attenu-ated culture alone, however, is unable tofurther stimulate these altered cells to any

appreciable extent. When supplementedwith growth substances these altered cellsare capable, like those stimulated by thevirulent culture, of uncontrolled growth inthe host.

CONNECTICUT VALLEY BRANCHYALE SCHOOL OF MEDICINE, NEW HAVEN, NOVEMBER 28, 1942

CULTIVATION OF A BACTERIA-FREE STRAINOF TRICHOMONAS FETUS. W. N. Plast-ridge, Department of Animal Diseases,

Storrs Agricultural Experiment Station,Storrs, Connecticut.A bacteria-free strain of Trichomonas fetus

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was isolated postmortem from the uterineexudate of a cow affected with pyometra.The strain has been maintained for a periodof 8 months in a medium composed of beefinfusion, 1.0 per cent peptone, 1.0 per centglucose, 0.07 per cent agar and 2 per centsterile inactivated ox-serum, and adjustedto an initial pH of 7.2 All of the ingredientsmentioned were necessary for growth. Thesize of the inoculum required for growth inthe freshly prepared medium may be assmall as 1 motile trichomonad per 10 ml ofmedium. Counts as high as 12,000,000motile trichomonads per ml were obtained.Cells grown in the clear medium providesatisfactory suspensions for serologicaltests.The presence of a utilizable carbohydrate

was found to be essential for growth. Gooddevelopment, with acid production was ob-tained in a basal medium to which wereadded Andrade's indicator and 1.0 per centof one of the following monosaccharides:galactose, glucose, levulose and mannose.Moderate growth took place in a mediumprepared with arabinose, and slight growthin the one containing xylose. No develop-ment occurred in media prepared withrhamnose, maltose, lactose, sucrose, raffi-nose, dextrin, glycogen, inulin, starch, glyc-erol, dulcitol, mannitol and sorbitol.The optimum initial pH of the medium

was found to be between 7.0 and 7.2; theoptimum concentration of serum was be-tween 2.0 and 20.0 per cent, and of glucose1.0 per cent.Attempts to grow the culture in the

presence of staphylococci and coliform or-

ganisms failed. The inclusion of differentconcentrations of crystal violet and sodiumazide in the medium did not permit thesuccessful cultivation of T. fetus in thepresence of the organisms mentioned.

A STUDY OF THE SPREAD OF H. INFLUENZAE,TYPE B. Roswell D. Johnson and MildredD. Fousek, Department of Pediatrics,Yale School of Medicine.In 1931 Pittman described a "smooth"

variant of Hemophilus influenzae whichshowed capsular formation and was the formto be found in clinical invasions. This or-

ganism she designated type "b." By 1939Alexander developed a rabbit antiserumagainst this organism. From 1922-1939,

22 cases of influenza meningitis were treatedhere with 100% mortality, and, from 1939-1942, 20 cases were treated with this serumand sulfonamides with but 3 deaths, two ofthese in infants under 6 months of age. Be-cause of an apparent increase in the mor-bidity rate due to H. influenzae type "b,"cultures of the throat and nasopharynx infamilies of children harboring the organismwere made to find the carriers.Ten families were studied, comprising a

total of 46 individuals, of whom 14 were 8years of age or less. Of this younger group,7 (50%) were positive, while in the groupover 8 years of age (including parents), therewere 32 individuals with but one motherpositive.

Six families studied as controls, with a

total of 24 members (6 of whom were under6 years of age) were all negative.A convalescent hospital was studied,

where 1 child had a febrile illness. Culturesof his nose and throat revealed the type "b"influenza bacillus. The rest of the wardwas cultured, and positive cultures obtainedin 0 of 5 infants, 3 of 4 toddlers, 4 of 4 olderchildren (to ten years), and 1 of 6 nurses, atotal of 8 out of 19 individuals. Repeatedone week later, one additional child pre-

viously negative was positive, and anothernurse, a newcomer, was positive.

Seventeen older children in an adjoiningward were all negative, although they were

cared for by the same nurses.The organism apparently attacks children

more frequently than it does adults, butclose contact with the latter, as in the caseof mothers or nurses, may induce a carrierstate in them. Local treatment of thecarriers may be considered, but is not beingdone at present.

NON-HEMOLYTIC VARIANTS OF HEMOLYTICSTREPTOCOCCI. Paul L. Boisvert andMildred D. Fousek, Department of Pedi-atrics, Yale School of Medicine.Recently non-hemolytic variants of hemo-

lytic streptococci were recovered from threechildren with scarlet fever. In two in-stances only non-hemolytic colonies werepresent in cultures from the nose and throat.Cultures from one child showed both hemo-lytic and non-hemolytic colonies.These strains were members of Lance-

field's human pathogenic Group A, and their

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fermentation and biochemical reactionswere those of human pathogens. Theybelonged in Griffith's Type 14, a type whichis prevalent in New Haven at this time.The organisms were non-hemolytic on

original cultivation on rabbit's blood agarplates incubated aerobically. Incubationof blood agar plates in an atmosphere ofCO2 produced slightly hemolytic colonies.The strains were hemolytic in blood brothafter several transfers, and subcultures onblood agar plates produced either hemolyticand non-hemolytic colonies or only hemo-lytic colonies.These non-hemolytic variants are of some

importance to the clinical bacteriologistsince such colonies might be overlooked.Although the colonies are not hemolyticthey appear otherwise to be typical mattcolonies of hemolytic streptococci. Wecan attribute no special qualities to thesevariants.

SALMONELLA TYPING IN A PuBnc HEALTHLABORATORY. Earle K. Borman, KennethM. Wheeler, D. Evelyn West, and FriendLee Mickle, Assistant Director, ResearchMicrobiologist, Chief Microbiologist andDirector Bureau of Laboratories, Con-necticut State Department of Health,Hartford, Conn.The value of Salmonella typing in a public

health laboratory is discussed and themethods used in Connecticut are presented.A public health laboratory should render atleast a minimum typing service by whichhuman types may be differentiated fromanimal types and the main somatic groups(B, C, D and E) may be recognized. Themethods involved are no more complicatedthan those for pneumococcus typing. Addi-tions to or departure from this basic planmay be desirable in a given locality butshould be made only after a sufficiently longexperience to obtain a general idea of thetypes commonly encountered. The assist-ance of a National Salmonella Center shouldbe utilized in any case.In Connecticut, Salmonella typing, orig-

inally the minimum stated above, wasstarted in 1939. The results of this serviceextending over 45 months for a total of 358cultures have shown that: 43.3 per cent of allnew Salmonella isolations were strains of

animal origin, belonging to 23 serotypes;56.7 per cent were of human origin, repre-sented by 2 serotypes, S. typhi and S. schott-muelleri. The most common serotypeswere as follows: typhi, 40.8 per cent; 8chott-muelleri, 15.9 per cent; typhimurium, 14.8per cent. Only 1.1 per cent of the serotypesfell into somatic groups other than B, C, Dand E. Organisms isolated for the first timefrom man were S. california, S. pullorum,S. Worthington, S. kentucky and two newtypes, S. hartford and S. simsbury. Con-cerning the animal strains, the carrier statein man was rarely prolonged either in con-valescents or in healthy individuals and thechronic carrier state is by no means so fre-quently found as with types of humanorigin.Typing of a Salmonella culture by means

of antigenic analysis is the only procedureby which the laboratory can furnish an au-thentic report and so save time and effort inthe ensuing epidemiological investigation bystressing the relative importance in eachcase of (1) looking for the human carrier or(2) establishing a chain of events leadingfrom the infected or carrier animal to man.THE BIOCHEMICAL AND SEROLOGICAL RE-LATIONSHIPS OF THE ORGANISMS OF THEGENUS PROTEUS. Robert Rustigian andC. A. Stuart, Biological Laboratory,Brown University, Providence, R. I.Biochemical and serological studies were

madewith several hundred Proteu8, proteus-like and other cultures to determine thecardinal characteristics of the genusProteus.

Culturally P. vulgaris members of thegenus could not be distinguished from P.mirabilis since all motile strains readilyswarmed on nutrient agar and eosin methyl-ene-blue agar. Two strains each of P.vulgaris and P. mirabilis were isolated whichwere non-motile and formed compactcolonies. Swarming of P. vulgaris or P.mirabilis was never observed on SS agar, butwell isolated colonies generally developedblack centers in from 24 to 48 hours. Exceptfor one fecal strain all P. morganii isolateswere motile and 18 of 76 strains exhibitedswarming under suitable conditions.The outstanding physiological criteria for

the genus Proteus appeared to be urea de-composition and slight gas formation

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(bubble to ten per cent) in fermentablecarbohydrates even after several days incu-bation. P. vulgaris, 55 strains, P. mira-bilis, 143 strains, P. morganii, 76 strains,and one presumably new type tentativelycalled "P. entericus", 37 strains, (Cope andKilander, 1942*) decomposed urea andproduced small gas volumes. Strains ofP. ammonia and P. americanus which alsodecomposed urea could not be distinguishedbiochemically or serologically from P. mira-bilis. P. icthyosmius and P. hydrophilusfailed to decompose urea and after severaldays incubation produced large volumes ofgas (50 per cent or more) in fermentablecarbohydrates and it is recommended thatthese types be classed as aberrant coliforms.Both P. vulgaris and P. mirabilis produced

acid and gas in glucose, sucrose and somestrains in salicin. Lactose and mannitolwere not attacked. Biochemically P. vul-garis differed from P. mirabilis in that itfermented sucrose and maltose in 24 to 48hours and formed indole. A majority of P.vulgaris strains were Voges-Proskauernegative and freshly isolated strains gener-ally failed to grow on citrate agar. On theother hand, a majority of P. mirabilis cul-tures gave a positive Voges-Proskauer reac-tion and utilized citrate. Atypical bio-chemical reactions were encountered,particularly with old laboratory cultures ofP. vulgaris and P. mirabilis. P. morganiifermented glucose, and occasionally sucrosebut failed to ferment lactose, maltose,mannitol and salicin. Indole was formed,

but negative reactions were obtained forH2S production, gelatin liquefaction, Voges-Proskauer test and citrate utilization."P. entericus" formed acid, occasionallyacid and a bubble of gas in glucose andmannitol and some strains acid in salicin,most strains produced acid in sucrose slowlywhile lactose and maltose were not fer-mented.P. vulgaris, P. mirabilis, P. morganii and

"P. entericus" exhibited serological hetero-geneity in that several antigenic types wereestablished within each species. The inci-dence of agglutination was relatively highwith strains of each species, indicating afrequent distribution of common antigens.No simple serological division could be madebetween vulgaris and mirabilis since com-mon H and 0 antigens were not infre-quently encountered. In addition therewere H antigens common to mirabilis,"entericus" and morganii, and H and 0antigens common to vulgaris and morganii.Other than the serological distinction of theX strains sero-typing in the genus Proteusdoes not appear to be of any taxonomic orpractical value.

It is suggested that urea decompositionand slight gas formation be considered astwo of the cardinal characteristics of Pro-teus. It is recommended that the term"morganii" be employed solely for theorganisms described as P. morganii (Mor-gan's Bacillus No. I) and that its usage todesignate various "morganii types" be dis-continued.

THE TEXAS BRANCHTEE UNIVERSITY OF TEXAS, AUSTIN, OCTOBER 31, 1942

THE EFFECT OF HEAT STERILIZATION ON THEGROWTH-FACTOR ACTIVITY OF PYRI-DOXINE FOR STREPTOCOCCUS LACTiS R.Esmond E. Snell, Department of Chemis-try, University of Texas, Austin, Texas.Pyridoxine has been recognized for some

time as an essential growth-factor for cer-tain species of lactic acid bacteria. In in-vestigating the growth of Streptococcus lactic

*We feel it imperative to include thisreference since these authors were the firstto describe this organism completely. Am.J. Pub. Health, 1942, 32, 352-354.

R on a pyridoxine-free medium, it was foundthat crystalline pyridoxine (vitamin B.) wasalmost inactive in permitting growth whenheat sterilization was avoided. Auto-claving media which contained pyridoxineincreased its growth-promoting activity forthis organism from 10 to 100 times dependingon the time and temperature of heating.The same effect was achieved in varyingdegrees by autoclaving pyridoxine at neu-trality with individual amino acids.Cystine was the most effective in this re-spect, while glycine was somewhat less effec-tive. The effect is not a specific one given

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only by amino acids, since activation is alsoeffected by heating pyridoxine with variousother compounds.

THE RELATION OF STRUCTURE TO ANTI-SEPTIC ACTION OF CERTAIN CHLORINATED4-PHENYLPHENOLS. C. M. S. Savoy, J. L.Abernethy, and 0. B. Williams, Depart-ments of Chemistry and Bacteriology,The University of Texas, Austin, Texas.The phenol coefficients of five chlorinated

4-phenylphenols (4-hydroxybiphenyls) havebeen determined using the standard F. D. A.method of determination. The organismsused were Staphylococcus aureus and Eber-thella typhosa. From these results it be-comes evident that two chlorines ortho tothe hydroxyl group are more effective thanone. Preliminary tests show that againstE. typhosa a remarkable increase in activityexists when the chlorine atom is substitutedin the most remote position to the hydroxylgroup, namely the 4'-position. However,introduction of a second chlorine in the 3-position decreases the activity, and a thirdchlorine in the 5-position further decreasesthe activity.

OBSERVATIONS ON SALMONELLA TYPHI-MURIUM. Oleta Beck and W. B. Coffee,State Health Laboratory, Austin, Texas.An epizootic has existed among the guinea

pig stock of the State Health Laboratorycaused by Salmonella typhimurium, and thisinfection still persists enzootically amongthe stock. No pathogenic organism otherthan this Salmonella species was encoun-tered, and the possibility of a virus involve-ment was investigated with negativefindings. The causative organism iscapable of being disseminated by roaches ofthe genus Periplaneta americana but not ofthe genus Blatella germanica. The arthro-pods appear to harbor the organism in theintestine as well as mechanically on theappendages.

TRIATOMA SANGUISUGA (LE CONTE) ANDTRIATOMA AMBIGUA (NEIVA) AS NATURALCARRIERS OF TRYPANOSOMA CRUZI INTEXAS. D. J. Davis, National Instituteof Health, Washington, D. C., T.McGregor and T. DeShazo, State Dep't.of Health Laboratories, Austin, Texas.

VIRUs-NEUTRALIZING ANTIBODY CONTENTOF HUMAN SERA FOLLOWING EQUINEENCEPHALOMYELITIS VACCINATION. J. V.Irons, S. W. Bohls, and Dorothy Albert,Bureau of Laboratories, Texas State De-partment of Health, Austin, Texas.Fifteen laboratory workers received

"equine encephalomyelitis" vaccine pre-pared for humans, in two one ml. doses eacha week apart. Sera from eleven laboratoryworkers were examined in neutralizationtests both before and after vaccination.Negative findings were obtained before vac-cination. Neutralizing antibodies protec-tive against 100 to 1000 mouse-braininfective units were obtained followingWestern type vaccination with personsworking intimately with this virus. Feebleor even negative responses were obtainedwith controls who were not working withthis virus.

SOME PHYSIOLOGICAL FACTORS IN SPOREPRODUCTION. A. E. Hayward, Depart-ment of Botany and Bacteriology, TheUniversity of Texas, Austin, Texas.This report presents results of an investi-

gation of the effect of nutrilites and pentoseand hexose sugars on Bacillus subtilis(Kahn) grown in a vitamin-free casein hy-drolysate medium. Thiamin, nicotinicacid, pantothenic acid, riboflavine, pyri-doxine, and biotin did not serve to increasethe percentage sporulation. Inositol wasfound to have a slight stimulatory effect.A correlation between the percentage

sporulation and configuration of the carbo-hydrate molecule is indicated. Furtherinvestigation of this phase of the problem isnow in progress.

ORNITHODOROS TALAJE (GUERIN-MENE-VILLE), A POSSIBLE VECTOR OF RELAPSINGFEVER IN TExAS. T. McGregor, R. B.Eads, and D. C. Thurman, Texas StateDepartment of Health, Bureau of Lab-oratories, Austin, Texas.Ornithodorus talaje has been found natu-

rally infected with spirochetes in an endemicrelapsing fever area in Southwest Texas.Ornithodorus turicata is apparently theprincipal vector of relapsing fever in Texas.After small groups of 0. talaje were fed on

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white rats, a characteristic relapsing spiro-chetosis occurred. 0. turicata was not foundin the immediate locality where infected 0.talaje were collected. In another localityspecimens of 0. talaje were not infected,although these were collected from the samerat nest in which infected 0. turicata werefound.

STUDIES OF SULFONAMIDE RESISTANCE OFTHE GoNococcuS. C. E. Lankford,Virginia Scott, and Willard R. Cooke,Dept. of Obstetrics & Gynecology, TheUniversity of Texas Medical Branch,Galveston, Texas.Strains of gonococcus from 100 patients

have been tested for sulfonamide sensitivityby plating on Proteose-starch-agar con-taining sulfonamides. The range of sulfa-thiazole tolerance is wide, varying from0.0005 to 5 mgm. per cent, and correspondsin general to the clinical response to sulfa-thiazole therapy.

Sulfonamide-resistant variants of sensi-tive strains may be selected by platingheavy suspensions on inhibitory concentra-tions of sulfonamide-agar. The colonieswhich develop are 5-10 or 50-100 times asresistant as the parent strain, and on platingthese, occasional variants obtained are 1000times as resistant as the original, therebyindicating near-logarithmic, step-wiseacquisition of tolerance. Although greatdifferences in strain capacity to produceresistant variants were observed, the num-ber of variants produced by a single strainin the presence of different sulfonamidesdoes not vary significantly, a fact support-ing the theory that acquisition of sulfon-amide-resistance is a sudden, spontaneous"mutation", occurring continuously, butbecoming evident only when conditions aresuitable for selective propagation of theresistant variant cells.Further observations: 1), a sulfonamide-

fast variant selected in the presence of onesulfonamide is proportionately resistant toothers; 2), the bacteriostatic efficiency ratio-sulfathiazole: sulfapyridine : sulfanil-amide-for parent strains and variantstested is usually 100:10:1 or 50:5:1; 3),simultaneous production of variants of in-creased and decreased tolerance may occur.

THE RH ANTIGEN IN SPONTANEOUS ABOR-TIONS AND ERYTHROBLASTOSIS FOETALIS.Sol Haberman, William Buchanan Blood,Plasma, and Serum Center, Baylor Uni-versity Hospital, Dallas, Texas.One clinical case of erythroblastosis and

eight cases of spontaneous abortion weretested for the presence of the Rh factor inthe parents and the foeti. In the eightcases of spontaneous abortion, syphilis,sepsis, eclampsia, and injury were ruled outas etiological factors. The erythroblastosiscase showed the Rh antigen in both thefather and the child, while the mother wasnegative for the antigen and had a weakRh agglutinin titer. In one case of abor-tion, the foetus was aborted at six months ofgestation. The father and the foetus hadthe Rh antigen in their erythrocytes, whilethe mother was negative for the antigen andhad a weak Rh agglutinin in her serum. Intwo of the cases, the Rh antigen was presentin the father and the foetus, while themother was negative in each instance. Noagglutinins were found in the mother'sserum or the foetal serum. Two other casesshowed positive Rh antigens in the fatherand none in the mother. The foeti of thesecases were not available for tests. Threecases of abortion failed to demonstrate theproper incidence of the Rh antigen in theparents or foeti to incriminate this antigenin the cases. The proper distribution of theRh antigen in the parents and the foeti inthree cases of abortion, and the proper dis-tribution in the two cases in which the foetiwere not available, lend some evidence tothe theory that the Rh factor may be con-cerned in repeated spontaneous abortions.

IDENTIFICATION OF PROTEUS AMMONIAE ANDRELATED SPECIES. Preston E. Harrison,Dept. Bacteriology, Hygiene and Preven-tive Medicine, Baylor University Collegeof Medicine, Dallas, Texas.Of 17 cultures of Proteus ammonia and

41 cultures of Proteu8 morganii studied, mostP. ammoniae strains were isolated fromurine while most of the P. morganii cultureswere obtained from feces.On S-S agar P. ammoniae produces a small

colony with a black center whereas P.morganii produces a colorless colony.

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P. ammonia produces a spreading growthon moist agar while P. morganji does not.

Lactose, sucrose, maltose and mannitolare not fermented by either species. AllP. ammoniae strains ferment glucose, galac-tose, trehalose, xylose, and are able toutilize citrate, liquefy gelatin, produce H2S,peptonize milk and rapidly decompose urea,but do not attack levulose and mannose, andfail to produce indole. P. morganii culturesferment glucose, galactose, levulose andmannose, and produce indole, but do notattack trehalose and xylose, and fail toutilize citrate, liquefy gelatin, produce H2Sor peptonize milk. Urea is decomposedslowly.

SALMONELLA AND SALMONELLA-LIKE TYPESFROM TExAS. MacDonald Fulton, BaylorCollege of Medicine Salmonella Center,Dallas.

A collection of 93 strains including 31 fromTexas was typed using Kauffmann's sera.

The most frequent type was Salmonellatyphimurium. The Texas types also in-cluded typhi, panama, newport, london,reading, kunzendorf, enteritidis, san diego,cholerae suis, onderstepoort, shangani, and2 strains related to tel aviv. Many strainssubmitted for study appear to be "morgan"bacilli, paracolon bacilli, proteus, andsimilar types, some of which have antigenscorresponding to those of Salmonella.These other groups must be more specifi-cally defined in order to decide the limits ofthe genus Salmonella.

JAUNDICE FOLLOWING YELLOW FEVER VAC-CINATION. A. Packchanian, Departmentof Public Health and Preventive Medi-cine, The University of Texas MedicalSchool, Galveston, Texas.

JOINT MEETING, NEW YORK CITY BRANCH AND THE THEOBALD SMITHSOCIETY (NEW JERSEY BRANCH)

COLLEGE OF THE CITY OF NEW YORK, DECEMBER 29, 1942

(Most of the papers listed below were prepared for the Annual Meeting of the Society atColumbus, and abstracts of such papers were published in the January issue of the

Journal on the pages listed after each title.)

THE GROWTH OF THERMOPHILIC ORGANISMSIN PASTEURIZING EQUIPMENT. M. L.Isaacs and A. Gordon, Yeshiva College,New York, N. Y. (J. Bact., 45, 51.)

NORMAL BACTERIAL FLORA OF CUTTING OILEMULSIONS. Nicholas D. Duffett, SydneyH. Gold and Clarence L. Weirich, The C. B.Dodge Co., Westport, Conn. (J. Bact.,45, 37.)

THE EVALUATION OF ANTISEPTICS AND

OTHER ANTI-INFECTIOUS AGENTS. WardJ. MacNeal and Nancy C. Farnsworth,New York Post-Graduate Medical Schooland Hospital, New York, N. Y. (J.Bact., 45, 41.)

THE MOUSE-PROTECTIVE TEST AS A UNI-FORM METHOD OF ASSAY FOR ANTI-BACTERIAL SERA. Paul A. Little, LederleLaboratories, Pearl River, N. Y. (J.Bact., 45, 70.)

FORMATION OF TYROTHRICIN IN SUBMERGEDCULTURES OF BACILLUS BREVIS. J. L.

Stokes and C. R. Woodward, Jr., Merckand Co., Rahway, N. J. (J. Bact., 45, 29.)

CULTIVATION OF ACTINOMYCETES, UNDER

SUBMERGED CONDITIONS WITH SPECIALREFERENCE TO THE FORMATION OF STREP-

TOTHRICIN. H. B. Woodruff and J. W.Foster, Merck and Co., Rahway, N. J.(J. Bact., 45, 30.)

ANTI-BIOTIN ACTIVITY OF METHIONINE.C. Virginia Fisher and Gustav J. Martin,Warner Institute for Therapeutic Re-

search, New York, N. Y. (J. Bact.,45, 33.)

CORRELATIONS BETWEEN OXIDATION-RE-DUCTION POTENTIALS AND THE DEVELOP-MENT OF SULPHATE-REDUCING BACTERIA.R. L. Starkey and Kent M. Wight, NewJersey Agricultural Experiment Station,New Brunswick, N. J. (J. Bact., 45, 39.)

THE NATURE OF THE CHANGES IN OPTIMALTEMPERATURE OF LUMINESCENCE CAUSEDBY ENZYME-SULFANILAMIDE AND ENZYME-

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URETHANE EQUILIBRIA. Frank H. John-son and Henry Eyring, Princeton, N. J.(J. Bact., 45, 24.)

ELECTRON MICROSCOPE STUDIES OF LUMI-NOUS BACTERIAL CELLS AND CYTOLYSATES.Nina Zworykin, Frank H. Johnson andGeorge Warren, Princeton University andR. C. A. Research Laboratory, Princeton,

N.J.ELECTRON MICROSCOPE STUDIES OF BAC-TERIOPHAGE. S. E. Luria, M. Delbrickand T. F. Anderson, College of Physiciansand Surgeons, Columbia University,

New York, N. Y.THERAPEUTIC AND PROPHYLACTIC USE OF

BACTERIOPHAGES. Ward J. MacNeal,Post-Graduate Medical School and Hos-

pital, New York, N. Y. (J. Bact., 45, 76.)

DEMONSTRATION OF SEROLOGICAL TYPESWITHIN THE NON-HEMOLYTIC PASTEU-RELLA. Paul A. Little and B. M. Lyon,Lederle Laboratories, Pearl River, N. Y.

THE SEROLOGICAL DIAGNOSIS OF RELAPSING

FEVER. George J. Stein, New YorkUniversity College Of Medicine, New

York, N. Y. (J. Bact., 45, 58.)CULTIVATION OF THE VIRUS OF MOUSEENCEPHALOMYELITIS (THEILER'S VIRUS)

IN THE CHICK EMBRYO. Wolcott B.Dunham and Sue Parker, New York Post-Graduate Medical School and Hospital,

New York, N. Y. (J. Bact., 45, 80.)THE OCCURRENCE OF TUMOR-HEMORRHAGE

AGENTS AMONG BACTERIA. Paul A. Zahland S. H. Hutner, The Haskins Labora-tory, New York, N. Y. (J. Bact., 45, 81.)

MODE OF ACTION OF ANTI-BACTERIAL SUBSTANCES-A SYMPOSIUM

NATURE AND MODE OF ACTION OF ANTI-BIOTIC SUBSTANCES. Selman A. Waks-man, New Jersey Agricultural Experiment

Station, New Brunswick, N. J. (J.

Bact., 45, 64.)THE NATURE OF GRAMICIDIN AND TYRO-CIDINE AND OF THEIR ACTION ON BAC-TERIA. Rollin D. Hotchkiss, Hospital ofthe Rockefeller Institute for Medical

Research, New York, N. Y. (J. Bact.,45, 64.)

THE NATURE AND ACTION OF PENICILLIN.Gladys L. Hobby, Karl Meyer, EleanorChaffee and Martin H. Dawson, College ofPhysicians and Surgeons, Columbia Uni-versity, and Presbyterian Hospital, NewYork, N. Y. (J. Bact., 45, 65.)

THE MICROBIOLOGY OF PENICILLIN FORMA-TION. Jackson W. Foster, Merck andCompany, Rahway, N. J. (J. Bact.,45, 65.)

MODE OF ACTION OF THE SULFONAMIDEDRUGS IN VITRO. George S. Mirick,Hospital of the Rockefeller Institute for

Medical Research, New York, N. Y.

(J. Bact., 45, 66.)THE MECHANISM OF SULFONAMIDE POTEN-

TIATION THROUGH IONIZATION AND OXI-DATION. Franz C. Schmelkes, Wallaceand Tiernan Products, Inc., Belleville,

N. J. (Paper to be read by Dr. Orville

Wyss.) (J. Bact., 45, 67.)

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