APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1976, p. 623-632Copyright © 1976 American Society for Microbiology

Vol. 32, No. 4Printed in U.S.A.

Morphological and Physiological Characteristics ofGemmigerformicilis Isolated from Chicken CecaJ. P. SALANITRO,* P. A. MUIRHEAD, AND J. R. GOODMAN

Shell Development Company, Biological Sciences Research Center, Modesto, California 95352,* andVeterans Administration Hospital, San Francisco, California 94121

Received for publication 26 January 1976

Morphological and physiological studies were made on chicken cecal isolatesof the strictly anaerobic bacterial species Gemmiger formicilis. Structural fea-tures (phase-contrast and electron microscopy) of these microorganisms indicatethey (i) are highly pleomorphic, (ii) possess a trilaminar cell wall like gram-negative bacteria, (iii) exhibit an unusual growth process characterized by polarswelling (resembling budding bacteria), and (iv) grow into elongated cells whenexposed to a subinhibitory concentration of penicillin. The morphological datapresented suggest that this species has a rod-shaped structure. These bacteriaferment a variety of sugars to produce formic, butyric, and lactic acids. Thereappear to be two groups of Gemmiger, one producing primarily lactate and theother producing formate as major fermentation metabolites. Growth of sixstrains in a basal medium, consisting of Trypticase, minerals, carbohydrate,Na2CO:, buffer, and cysteine as reducing agent, was stimulated by rumen fluidand yeast extract. Volatile fatty acids partially replaced the requirement forrumen fluid with some strains. Single deletions of vitamins (from a definedvitamin mixture) indicated that pantothenate, riboflavin, and thiamine werehighly stimulatory to growth of the organism in a medium containing rumenfluid and Trypticase as source ofvitamins. Other vitamin requirements were notstudied.

In previous studies we reported on a gram-negative, strictly anaerobic, pleomorphic, bud-ding-like organism isolated from chicken cecalcontents (16, 17). These bacteria were recoveredin 75% of the birds studied, were present inhigh numbers (0.10 x 10'° to 1.34 x 1010 per g[wet wt]), and constituted from 2 to 18% (meanof 6%) of the total isolates. Microscope analysisof frozen tissue sections from several regions ofthe intestinal tract of many broiler chickens (1to 6 weeks of age) indicates that these orga-nisms are primarily found in the luminal con-tents of the cecum and are not associated withthe mucosal surface. In addition, these bacteriahave been isolated from chicken ceca by work-ers in England (2) and from human feces (12,15). Gossling and Moore (11) have recentlynamed and described this organism as an an-aerobic budding bacterium (Gemmiger formi-cilis). The present investigation was under-taken to obtain definitive morphological (elec-tron microscope observations) and physiologicaldata that would establish similarities withknown anaerobic species.

MATERIALS AND METHODSBacterial strains. Representative chicken cecal

strains were isolated from four 5-week-old commer-

cial broilers (16) and were designated as strains P9,Q32, R15, R48d, S17, and S41. Cultures were main-tained in serum-stoppered vials (5-ml capacity,Wheaton Scientific) containing 3 ml of prereducedrumen fluid medium (Table 1) with raffinose asenergy source.

Light and electron microscopy. Cultures wereexamined in wet mounts by phase-contrast micros-copy using a microscope (Zeiss RA) and photographsmade with a 35-mm camera (Nikon F). For electronmicroscopy, strains were grown in culture media (3to 5 ml), centrifuged (4°C, 5,000 rpm, 10 min), andresuspended in 2 ml of fixative of similar osmolarityas the growth medium. Primary fixative consisted of0.5% glutaraldehyde in 0.05 M cacodylate buffer, pH6.0 (ca. 160 mosM/kg). Fixation was carried out for10 min in screw-cap tubes at ambient temperature.Samples were centrifuged, washed twice in cacodyl-ate buffer (0.10 M, pH 7.3), and postfixed by suspen-sion in 1 ml of 1% OsO4-O. 10 M cacodylate buffer, pH7.3, for 10 min at room temperature. Cells werewashed again to remove fixative, embedded in agar(2%), dehydrated through a series of increasing con-centrations of ethanol solutions followed by propyl-ene oxide, and embedded in Epon-Araldite mixture.Thin sections were cut with an ultramicrotome(LKB), mounted on copper grids, and finally stainedwith uranyl acetate and lead citrate. Sections wereexamined in an electron microscope (RCA-EMU-3F)operating at 50 kV.Growth media and nutrition experiments.

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624 SALANITRO, MUIRHEAD, AND GOODMAN

TABLE 1. Composition of basic growth mediuma

Component % in mediumbRumen fluid, clarifiedc (vol/vol) ......20Carbon source (sugar) ............... (20 mM)Trypticase (BBL) ....................0.40Yeast extract (Difco) .................0.40Minerals 1 and 2c (vol/vol; 7.5% each) 15Resazurin ....................... 0.0001Na2CO3........................ 0.16Cysteine-hydrochloride ............... 0.05

a Final pH, 6.8 to 7.0, 10% CO2-90% N2 gas phase.Medium was equilibrated and dispensed into rub-ber-stoppered tubes (13 by 100 mm) (Bellco Glass)under CO2/N2 gas mixture.

bFinal percentage as weight to volume or as indi-cated.rSee reference 4 for preparation.

Strictly anaerobic techniques were employed in allculture procedures (3, 4). The rumen fluid mediumused for growing cultures for electron microscopy isas given in Table 1, except glucose (20 mM) was theenergy source. In experiments designed to deter-mine the effects of penicillin on growth, penicillin G(sodium salt, Sigma Chemical Co.) was added tomedia. The six strains studied were also tested todetermine factors required for growth. The inoculaused in these experiments were obtained by cul-turing strains at 37°C in growth medium (Table 1)with raffinose to late-log growth phase (ca. 64 h).Cultures were centrifuged anaerobically under CO2gas phase (4°C, 5,000 x g, 10 min), washed once byresuspending the pellet in 5 ml of anaerobic dilutionsolution (4), centrifuged again, and finally resus-pended in anaerobic dilution solution to an opticaldensity (OD) of 0.20 to 0.23 units. The adjustedcultures were used as the inocula (0.05 ml) for 4 mlof the various nutritional media, which are given inthe Results section. Growth was monitored at 24-hintervals by measuring turbidity in OD units with aBausch & Lomb Spectronic 20 colorimeter set at 600nm. In most experiments, maximum turbidity wasrecorded at 120 or 160 h of incubation at 37°C.Media for nutritional experiments were prepared

by equilibrating components in oxygen-free gas for20 min, adjusting the pH to 6.8 to 7.0, and dispens-ing 4 ml/tube into rubber-stoppered Bellco culturetubes (13 by 100 mm) under the same gas phaseusing an automatic pipette fitted with gassingneedles. Tubed media were placed in presses andautoclave-sterilized for 15 min at 15 lb/in2 steampressure.

Analysis of fermentation products by gas chro-matography. Fermentation acids (formic, acetic,butyric, and lactic) produced from carbohydrateswere analyzed according to previously describedchromatographic methods (18). Gas samples for hy-drogen analysis were taken from cultures grown(rumen fluid-raffinose medium) in serum-stopperedvials. Hydrogen was determined by gas chromatog-raphy on a Porapak Q column, 60 to 80 mesh (Ap-plied Science), using pure hydrogen as a standard(6).

RESULTSMorphological observations. The organisms

were gram negative and typically exhibited"bowling pin"- and "dumbbell"-shaped mor-phology under light microscopy (Fig. 1 and 2).The cell dimensions determined with wetmounts under phase-contrast microscopy var-ied from 0.5 ,um wide to 1 to 1.5 ,um long. Moststrains appeared as singles, pairs, or shortchains (Fig. 2). One strain, P9 (Fig. 1), charac-teristically exhibited extensive chain formationand clumping at most stages of growth. Cul-tures of this strain grew as a ropy-type sedi-ment in liquid media. No noticeable differencein morphology of P9 and R15 was observedwhen these strains were grown under a widevariety of culture conditions of gas phase, car-bon source, or nutrient growth factor limita-tion.

Electron micrographs of thin sections aregiven in Fig. 3 through 9. In initial attempts atelectron microscopy, we considered it importantto preserve the morphological features of thecells by fixing the bacteria under anaerobicconditions and in media of the same osmolarityas the growth media. This preliminary worksuggested, however, that no obvious distortionof the cells was evident either when cells werefixed under anaerobic or aerobic conditions orwhen the osmolarity of the fixing medium wasnot adjusted exactly to that of the incubationmedia.

Figure 3 shows that these organisms possessa trilaminar type of cell wall and cell mem-brane structure characteristic ofgram-negativebacteria. The typical dumbbell-shaped cell hasone end larger than the other with a constric-tion in between. The size of these cells variedfrom 0.5 to 0.8 ,um at the larger end to 0.3 to 0.6,tm at the smaller end and 0.2 to 0.4 ,am at theconstriction, whereas the length of the dumb-bell varied from 0.90 to 1.5 ,/m.

Figure 4 shows two cells in a chain undergo-ing what appears to be a "constrictive" type ofdivision.Examination of many cultures andphases of growth by light and electron micros-copy suggests to us, however, that in this orga-nism, cell division takes place when dumbbell-shaped cells elongate and divide into two bowl-ing pin-like cells (Fig. 5). Although we havebeen unable to demonstrate septa in cells, crosswall formation presumably occurs in these bac-teria in a manner similar to that observed forother gram-negative bacteria (5, 10).One method of differentiating cocci from

short rods has been to observe the morphologi-cal effects of subinhibitory concentrations ofpenicillin. Exposure of rod-shaped bacteria to

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FIG. 1 AND 2. Phase-contrast photomicrographs of log- and late-log-phase cultures of chicken isolates ofGemmiger (x1,680). Fig. 1, strain P9; Fig. 2, R15.

3 cw/Jcm

0O5FIG. 3. Electron microscopy of a thin section of strain R15 (stationary-phase culture) with dumbbell-

shaped morphology. Note the trilaminar cell envelope structure with cell wall (cw) and cytoplasmic membrane(cm).

4

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0.5pFIG. 4. Thin section of log-phase culture ofP9 in chain formation. Note points of cell wall invagination

(arrows) where cell division has initiated.625

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626 SALANITRO, MUIRHEAD, AND GOODMAN

small amounts of penicillin (1 to 10 UIml) pref-erentially inhibits septum formation over cellwall synthesis and allows cells to grow intofilaments (5, 7). Cocci, in contrast, when ex-posed to these low penicillin concentrations,

5

develop enlarged and multiseptate cells (7, 14).We have therefore utilized this technique todetermine if these unusual bacterial forms arerods or cocci. The effect of penicillin on growthis shown in Fig. 6 and 7. Cultures of strain P9

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FIG. 5. Thin section ofP9 prepared from an early-log-phase culture showing typical bowling pin-shapedcells.

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*v@'' s*s'.';tk~~~~~~IIff X- 4 2;'' '_tO =;E0.5ps

FIG. 6 AND 7. Exposure ofP9 cells to 1 U ofpenicillin per ml during log-phase growth. Bulges (arrows) incells indicate where cell wall growth occurs, while septum formation is presumably inhibited.

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CHICKEN STRAINS OF GEMMIGER 627

growing in the presence of 1 U of penicillin perml developed into elongated cells, some meas-uring at least 5 Am long. Little or no inhibitionof growth was observed at 1 U/ml of medium,but 10 U/ml almost completely inhibitedstrains P9 and R15.

In our initial studies on these bacteria, weconsidered the possibility that the cell envelopestructure was less rigid and perhaps highlyplastic, thereby undergoing changes in cellshape and size that might account for its pleo-morphism. Log-phase cultures of P9 and R15were placed (2 h, 37°C) into hypertonic rumenfluid-glucose medium containing 0.68 M glyc-erol, 0.40 M NaCl, or 0.60 M sucrose. Differ-ences in cell shape or plasmolysis were notobserved when cells were incubated in glycerol-containing media (Fig. 8); however, translucent

granules appeared to be more obvious in elec-tron micrographs of these cells. Cells incubatedin NaCl or sucrose-containing media developedsome plasmolysis; the cytoplasmic membranepartially pulled away from the rigid cell wall,maintaining several points of contact with themurein layer and outer wall. NaCl also causedintracellular vesicles to form (Fig. 9). Fromthese studies it was concluded that high ionic orhypertonic osmotic media have no gross effectson the shape and structure of these organismsand are similar to those reported for Esche-richia coli (1).

Preliminary work on the isolation and identi-fication of the electron-translucent granulesseen in thin sections of these strains (Fig. 4-8)has indicated that the particles may consist of astarch- or glycogen-like substance. The mate-

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..,, _

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0.5,tFIG. 8. Thin section of a log-phase culture of P9 resuspended in glycerol (0.68 M). Notice the large

numbers of electron-translucent grains (glycogen granules?) in several cells.

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628 SALANITRO, MUIRHEAD, AND GOODMAN

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FIG. 9. Electron micrograph of log-phase cultures ofP9 resuspended in NaCl (0.40 M). Many cells haveundergone partial plasmolysis (arrow) while maintaining several areas of contact between the cell wall andcell membrane.

rial extracted and partially purified accordingto the method of Cheng et al. (9) gave a strongreddish-brown color with iodine, a positive an-throne reaction, and comprised about 9% (R15)and as much as 20% (P9) of the cellular dryweight. These glycogen granules are also simi-lar morphologically to the cytoplasmic inclu-sions observed in electron micrographs of therumen bacteria (9).

Nutrient factors affecting growth. Resultson growth, fermentation, and the formation ofacid products from several carbohydrate energysources are given in Table 2. Considerable vari-ation in fermentation among strains was ob-

tained with most sugars tested; however, allstrains fermented (terminal pH, 6 or less) glu-cose, raffinose, and salicin. Good correlationwas observed between the amount of growth(turbidity in OD units) on a sugar and theterminal pH of the media after 7 days of incuba-tion. Substrates not fermented by any strainincluded amygdalin, arabinose, erythritol, es-culin, glycogen, inositol, mannitol, mannose,melezitose, rhamnose, ribose, starch, and lac-tate. Pyruvate was fermented (pH 5.8) by onlyone strain, R48d, with the production of largeamounts of formic and acetic acids. Esculin washydrolyzed by most strains.

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CHICKEN STRAINS OF GEMMIGER 629

TABLE 2. Effect of carbon source on growth and fermentation products

Growtha Terminal pHb Acids Reac-Carbon source _ - - tive

P9 Q32 S41 R15 R48d S17 P9 Q32 S41 R15 R48d S17 P9 Q32 S41 R15 R48d S17 scored

Cellobiose 60 60 65 65 10 10 5.1 5.1 5.0 5.2 6.7 6.7 bl abL abL FbL _e fab +(4/6)Galactose 35 40 30 20 50 40 5.6 5.6 5.6 6.2 5.1 5.3 fabL abL fbL fabl fab Fabl +(5/6)Glucose 35 40 30 30 80 50 5.7 5.9 5.8 5.8 5.4 5.5 fabL fbL bL fabL Fab Fabl +(6/6)Lactose 10 5 5 40 10 70 6.7 6.8 6.8 5.4 6.6 5.4 fal fl fal FaB - FaBl -(4/6)Maltose 40 35 40 55 10 50 5.2 5.3 5.3 5.5 6.7 5.2 BL fbL fbL FaBl fb Fabl + (5/6)Mannose 40 25 25 50 0 0 6.0 6.4 6.0 5.3 6.9 6.9 fBl fabl fbl fab - - + (3/6)Melibiose 35 30 30 10 50 45 5.6 6.0 6.0 6.7 5.3 5.2 fbL bl bl - FB fBL +(5/6)Raffinose 75 80 75 85 90 75 5.0 4.8 4.9 5.1 5.1 5.2 fbL abL fbL FaBl FaBl FaBL +(6/6)Salicin 70 70 70 85 95 50 5.5 5.5 5.3 5.2 5.1 5.9 FabL fabL FabL FaB FaB FaL +(6/6)Sucrose 80 80 80 80 0 0 4.9 4.9 4.8 5.2 6.8 6.8 bL bL bL fabl - - +(4/6)Trehalose 75 75 80 85 0 0 5.0 4.9 4.9 5.3 6.9 6.9 bL bL fbL Fb - - +(4/6)Xylose 20 25 15 15 0 0 5.9 6.0 5.9 5.8 6.8 6.8 fAb fab fAb Fab - - +(4/6)

a Measured in OD units x 100 after 160 h of incubation in rumen fluid medium (Table 1) with different sugars.bMeasured after 160 h of incubation.c Acids produced from growth on sugars: F,f (formic); A,a (acetic); B,b (butyric); L,l (lactic). Uppercase letters refer to

amounts of 10 ,Lmol/ml of medium or greater, and lowercase letters refer to amounts less than 10 j±mol/ml.d Symbols: -, Little or no fermentation, terminal pH greater than 6.0; +, acid reaction, terminal pH 6.0 or less. Figures

in parentheses refer to the number of strains positive or negative/number of strains tested.e No products formed.

The fermentation pattern presented in Table2 agrees qualitatively with those of Gosslingand Moore (11), except that raffinose and sali-cin are fermented by chicken strains in a ru-men fluid basal medium, whereas these sugarsare not readily utilized by human fecal strainsin the PY basal medium used by the VirginiaPolytechnic Institute Anaerobe Lab. Formate,butyrate, and lactate were the major acids(with small amounts of acetate) produced by allsix isolates in varying amounts depending onth substrate and extent of growth (Table 2).Differences in the distribution of fermentationproducts were also noted (Table 3). With glu-cose and raffinose, strains P9, Q32, and S41formed lactic acid as the major fermentationmetabolite, with smaller amounts of formic andbutyric acids. In contrast, R15, R48d, and S17grown on the same substrates produced largeramounts of formic and butyric acids withsmaller amounts of lactic acid.Preliminary studies (16) on factors stimula-

tory or essential for growth of these organismssuggested that CO2 and components in rumenfluid and yeast extract may be required. Datain Table 4 on the effect of different gas phasesindicate that CO2 enhances the growth of thesix chicken cecal isolates. Three strains (P9,Q32, S41) appear to have an absolute growthrequirement for CO2, since little or no growthwas observed with nitrogen as the culture gasphase. The presence of CO2 in the gas phasereduced the lag time for initiation of growth by24 to 48 h in all strains.

It has been mentioned previously (11, 16)that growth of Gemmiger strains was stimu-lated by rumen fluid and yeast extract. Table 5

represents data showing the effects of rumenfluid and yeast extract on growth. A basal me-dium containing minerals, raffinose, Trypti-case, reducing agent, and CO2/N2 gas phase didnot support good growth of the six strainstested, even when rumen fluid was also added.Little or no growth was observed in the basalmedium alone. However, when yeast extractwas substituted for rumen fluid, four of sixstrains grew well. With P9, Q32, and S41, thesedata also suggested that there were effects ongrowth by rumen fluid and yeast extract.Growth of P9 and S41 (which were not stimu-lated by yeast extract) was improved when avolatile fatty acid mixture was added in place ofrumen fluid. Straight-chain, but not branched-chain, volatile acids could partially substitutefor rumen fluid. The data presented in Table 5also indicate that there may be other factors inrumen fluid that stimulate growth.The stimulatory effect of yeast extract was

subsequently investigated to determinewhether certain vitamins could substitute foryeast extract. The results of single-vitamindeletion experiments indicated that growth ofthese bacteria was more or less stimulated bythiamine, riboflavin, and pantothenate. Also,growth was as good in the rumen fluid-Trypti-case medium containing these vitamins aswhen a complete mixture was added (Table 6).Possibly because of the presence of vitamins inrumen fluid and Trypticase, no other vitaminrequirements were detected.

Other experiments (using the basal mediumof Table 5) concerning the nutritional require-ments of chicken strains of Gemmiger showthat (i) they could not grow without a readily

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630 SALANITRO, MUIRHEAD, AND GOODMAN

TABLE 3. Distribution offermentation acids"

% of acidb Total acid formedStrain Substrate --- -- (,uomol/ml)c

Formic Acetic Butyric Lactic

P9 Glucose 5 6 14 75 20.7Q32 13 0 6 81 23.8S41 0 0 13 87 19.7R15 21 9 17 53 23.5R48d 48 14 38 0 25.8S17 54 4 29 13 28.1

P9 Raffinose 5 0 15 80 42.3Q32 0 4 13 83 36.6S41 6 0 14 80 46.9R15 57 8 20 15 52.1R48d 58 7 26 9 59.3S17 33 19 27 21 51.0

P9 Salicin 32 6 19 43 36.3Q32 7 19 18 56 29.4S41 30 9 13 48 37.2R15 69 5 26 0 39.5R48d 66 6 28 0 45.1S17 48 14 0 38 35.6

"Results are the mean of at least two to three replicates per strain on each substrate in medium ofTable1.

b Expressed as (,umol/ml of acid)/(,umol/ml of total acid) x 100.c Expressed as ,umol/ml of culture fluid.

TABLE 4. Effect ofgas phase on growth"

Culture turbidityb (OD x 100)

Strain N2 CO2/N2 CO2 H2/C02

(100Y (10/90) (100) 10/90

P9 0 75 60 65Q32 15 85 75 80S41 10 80 75 75R15 50 100 100 110R48d 45 90 85 95S17 55 100 75 70

"Strains were cultured in rumen fluid-raffinosemedium (Table 1), which was equilibrated andtubed with the appropriate gas phase. Na2CO3buffer was absent from media equilibrated with N2but present in media containing C02/N2 (0.16%, wt/vol), CO2 (0.40%, wt/vol), or H2/CO2 (0.40%, wt/vol).

b Average of four replicates measured after 160 hof incubation at 37°C.

r Percentage of composition.

fermentable carbohydrate, (ii) addition of Tryp-ticase (BBL, 0.4%) to the medium did not en-

hance growth, (iii) neither Trypticase (0.4%,wt/vol), Casamino Acids (Difco, 9.2%, wt/vol),an amino acid mixture (50 mM each), NaNO3(0.01%, wt/vol), nor NaNO2 (0.01%, wt/vol)could serve as source of nitrogen for growth inplace of NH4+, and (iv) Casamino Acids andTween 80 (0.01%) were inhibitory to growth.Gossling and Moore (11), however, observed

TABLE 5. Effect of rumen fluid factors on growth

Culture turbidity, maximumMedium addition' OD x 100 at 120 h in strain:

P9 Q32 S41 R15 R48d S17

Rumen fluid (R) + 65 80 75 85 85 105yeast extract (Y)

R 15 10 10 20 20 15Y 0 50 15 80 75 65Y + hemin 10 60 15 80 65 75Y + VFA 50 60 40 70 65 95Y + S-VFA 50 55 40 '70 65 75Y + B-VFA 10 35 10 75 65 75

" Basal medium to which components were added con-sisted of minerals 1 and 2 (7.5% each, vol/vol), raffinose (20mM), Trypticase (0.40%, wt/vol), Na2CO3 (0.16%, wt/vol),cysteine-hydrochloride (0.05%, wt/vol). All componentswere equilibrated and tubed under 10% CO2/90% N2 gasphase. Other medium additions were as follows: rumenfluid (20%, vol/vol); yeast extract (0.40%, wt/vol); hemin(0.0002%, wt/vol); VFA, volatile fatty acids (29.5mM acetic,8.1 mM propionic, 4.4 mM butyric, and 1.1 mM isobutyricacids, and 0.9 mM each of valeric, isovaleric, and 2-methyl-butyric acids); S-VFA, straight-chain VFA (acetic, butyric,propionic, and valeric acids, concentrations as given above);B-VFA, branched-chain VFA (isobutyric, isovaleric, and 2-methylbutyric acids, concentrations as given above).

that the amount of growth with human strainsof Gemmiger was enhanced by Tween 80.

DISCUSSIONOur electron microscope observations of

Gemmiger at different phases of growth sug-

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CHICKEN STRAINS OF GEMMIGER 631

TABLE 6. Effects of vitamins on growth

Culture turbidity, maximum OD

Medium additiona x 100 at 120 h in strain:

P9 Q32 S41 R15 R48d S17

R (rumen fluid) 10 10 5 20 10 15R + yeast extract 70 80 75 80 80 100R + vitamin mixture 65 70 60 60 75 85R + pantothenate (P) 15 20 25 30 20 40R + riboflavin (Rf) 20 15 50 30 20 45R + P + Rf 30 55 55 60 40 85R + P + Rf + thiamine 65 70 65 60 75 110

a See Table 5, footnote a; for basal medium and levels ofmedium additions. Vitamin mixture consisted of the follow-ing (final concentration, gg/ml of medium): thiamine-hy-drochloride, calcium-pantothenate, nicotinamide, ribo-flavin, and pyridoxal-hydrochloride, each 2; p-aminobenzoicacid, 1; biotin, folic acid, and DL-thioctic acid, each 0.05; andvitamin B,2, 0.02. Vitamins were combined, filter-steri-lized, and added to media before autoclaving. Vitaminsadded singly or in combination were incorporated at thesame concentrations as in the vitamin mixture.

gest to us that the process of growth and celldivision may occur as diagramed in Fig. 10. Anewly divided cell (type 1) initiates cell wallgrowth with enlargement ofthe terminal swell-ing. This asymmetric growth proceeds until thetwo cell poles have approximately the samediameter, and the cells appear as dumbbellshapes (types 2 and 3), followed by invaginationof the cell wall (types 4 and 5). This cell divides,forming daughter cells with ends of unequaldiameter (types 1 and 7). Exposure of cell types4 and 5 to penicillin gives rise to cells (type 6)that elongate with bulges of cell wall growthforming near sites of division. The validity ofthis proposed scheme for growth in Gemmigerrequires further examination, perhaps by uti-lizing the technique of high-resolution autora-diography employed by de Chastellier et al. (8)in investigations of cell wall growth sites inBacillus megaterium. Slide culture techniquesand photomicrography might also be used todetermine the mode of cell wall growth andbudding in this organism.We consider Gemmiger as a form that has an

uneven mode of polar cell growth (swelling)and reproduction. Morphologically, it resemblesother gram-negative, nonsporeforming speciesofbacteria such as Rhodopseudomonas and Ni-trobacter (13), or the aquatic budding bacteriadescribed by Whittenbury and Nicoll (19). Allthese forms initiate multiplication by a processcharacterized by a swelling of the cell pole.Rhodopseudomonas is an anaerobic phototroph,and Nitrobacter is an ammonia- or nitrate-oxi-dizing aerobe, but Gemmiger is a nonphotosyn-thetic chemorganotrophic anaerobe and there-fore could not be related to these genera.

2

5

FIG. 10. Schematic diagram of the proposed se-quence ofgrowth and cell division in chicken strainsof Gemmiger. See text for explanation.

The effects of penicillin on cell wall growth ofGemmiger suggest that this organism has arod-shaped morphology. If these bacteria areindeed highly pleomorphic rods rather thancocci, as our data indicate, then they cannot beincluded in the groups of anaerobic cocci (Pep-tococcaceae or Veillonellaceae). On the bases ofrod shape, energy metabolism, lack of flagella-tion, and anaerobiosis, Gemmiger is similar tobacteria in the family Bacteroidaceae. Thisfamily includes the strictly anaerobic, pleo-morphic, gram-negative, nonmotile, nonspore-forming, and fermentative rods. Of the generadescribed under this family, it is not similar toBacteroides, fermenting several carbohydrateswith the production of butyric acid. Their gua-nine plus cytosine content of 59% (11) is higherthan the three recognized genera under theBacteroidaceae (Bacteroides, Fusobacterium,and Leptotrichia). The fermentation of carbo-hydrate by chicken isolates of Gemmiger indi-cates that two groups may exist: one (strainsP9, Q32, and S41) producing lactic acid as themajor metabolite, with smaller amounts offormic and butyric acids, and the other (strainsR15, R48d, and S17) producing formic as themajor acid with lesser amounts of butyric andlactic acids.

Results of nutritional experiments indicatethat both rumen fluid and yeast extract arerequired for maximal growth (Table 5). Addi-tion of rumen fluid to the basal medium al-lowed for minimal growth of all strains,whereas addition of yeast extract supported ad-equate growth of some strains. Maximalgrowth was also obtained when a vitamin mix-ture or a combination of pantothenate, ribo-flavin, and thiamine was substituted for yeastextract in the rumen fluid-Trypticase basal me-dium.

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632 SALANITRO, MUIRHEAD, AND GOODMi

LITERATURE CITED

1. Alemohammad, M. M., and C. J. Knowles. 1974. Os-motically induced volume and turbidity changes ofEscherichia coli due to salts, sucrose, glycerol, withparticular reference to the rapid permeation of glyc-erol into the cell. J. Gen. Microbiol. 82:125-142.

2. Barnes, E. M., G. C. Mead, D. A. Barnum, and G. C.Harry. 1972. The intestinal flora ofthe chicken in theperiod 2 to 6 weeks ofage with particular reference tothe anaerobic bacteria. Br. Poul. Sci. 13:311-326.

3. Bryant, M. P. 1972. Commentary on the Hungate tech-nique for culture of anaerobic bacteria. Am. J. Clin.Nutr. 25:1324-1328.

4. Bryant, M. P., and L. A. Burkey. 1953. Cultural meth-ods and some characteristics of some of the morenumerous groups of bacteria in the bovine rumen. J.Dairy Sci. 26:205-217.

5. Burdett, I. D. J., and R. G. E. Murray. 1974. Septumformation in Escherichia coli: characterization of sep-tal structure and the effects of antibiotics on celldivision. J. Bacteriol. 119:303-324.

6. Carle, G. C. 1970. Gas chromatographic determinationof hydrogen, nitrogen, oxygen, methane, krypton,and carbon dioxide at room temperature. J. Chroma-togr. Sci. 8:550-551.

7. Catlin, B. W. 1975. Cellular elongation under the influ-ence of antibacterial agents: way to differentiate coc-

cobacilli from cocci. J. Clin. Microbiol. 1:102-105.8. de Chastellier, C., R. Hellio, and A. Ryter. 1975. Study

of cell wall growth in Bacillus megaterium by high-resolution autoradiography. J. Bacteriol. 123:1184-1196.

9. Cheng, K. J., R. Hironaka, D. W. A. Roberts, and J. W.Costerton. 1973. Cytoplasmic glycogen inclusions in

APPL. ENVIRON. MICROBIOL.

cells of anaerobic gram-negative rumen bacteria.Can. J. Microbiol. 19:1501-1506.

10. Gilleland, H. E., Jr., and R. G. E. Murray. 1975. Dem-onstration of cell division by septation in a variety ofgram-negative rods. J. Bacteriol. 121:721-725.

11. Gossling, J., and W. E. C. Moore. 1975. Gemmigerformicilis, n. gen., n. sp., an anaerobic budding bac-terium from intestines. Int. J. Syst. Bacteriol.25:202-207.

12. Gossling, J., and J. M. Slack. 1974. Predominant gram-positive bacteria in human feces: numbers, variety,and persistence. Infect. Immun. 9:719-729.

13. Hirsch, P. 1974. Budding bacteria. Annu. Rev. Micro-biol. 28:391-444.

14. Lorian, V. 1975. Some effects of subinhibitory concen-trations of penicillin on the structure and division ofstaphylococci. Antimicrob. Agents Chemother. 7:864-870.

15. Moore, W. E. C., and L. V. Holdeman. 1975. Humanfecal flora: the normal flora of 20 Japanese-Hawaii-ans. Appl. Microbiol. 27:961-979.

16. Salanitro, J. P., I. G. Blake, and P. A. Muirhead. 1974.Studies on the cecal microflora of commercial broilerchickens. Appl. Microbiol. 28:439-447.

17. Salanitro, J. P., I. G. Fairchilds, and Y. D. Zgornicki.1974. Isolation, culture characteristics and identifica-tion of anaerobic bacteria from the chicken cecum.Appl. Microbiol. 27:678-687.

18. Salanitro, J. P., and P. A. Muirhead. 1975. Quantita-tive method for the gas chromatographic analysis ofshort-chain monocarboxylic and dicarboxylic acids infermentation media. Appl. Microbiol. 29:374-381.

19. Whittenbury, R., and J. M. Nicoll. 1971. A new, mush-room-shaped budding bacterium. J. Gen. Microbiol.66:123-126.

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