JOURNAL OF BACTERIOLOGY, Mar. 1971, p. 820-825Copyright © 1971 American Society for Microbiology

Vol. 105. No. 3Printed in U.S.A.

Isolation, Culture, and FermentationCharacteristics of Selenomonas ruminantium var.

bryanti var. n. from the Rumen of SheepR. A. PRINS'

Department of Bacteriology, University of California, Davis, California 95616

Received for publication 23 November 1970

Large forms of Selenomonas sp. were isolated from the sheep rumen on a rumenfluid-glucose-agar medium by using a differential centrifugation technique to purifythe inoculum. The cells from the six isolated strains were curved, gram-negative,strictly anaerobic crescents, and rapidly motile by flagella attached to the concaveside of the cell. One or more of the volatile fatty acids were essential for growth.None of the strains produced indole or reduced nitrate. All strains grew on fructose,glucose, mannose, cellobiose, maltose, sucrose, and salicin. Fermentation end prod-ucts from glucose were mainly lactate, acetate, propionate, and formate. Smallamounts of succinate were formed. The final pH in a glucose medium ranged be-tween 4.3 and 4.5. On the basis of the sugar fermentation characteristics and thecapacity to form hydrogen sulfide from cysteine, it is suggested that one of thestrains is a large form of Selenomonas ruminantium. The other five strains are des-ignated S. ruminantium var. bryanti, var. n.

Although many rumen bacteria have been iso-lated and identified (8), the large species in therumen of sheep (13) have not yet been culturedand virtually nothing is known of their nutritionand physiology.Woodcock and Lapage (18) described Seleno-

mastix ruminantium from the rumen of goats asincluding crescentic and oval forms, both motile.Subsequently the designation Selenomonas ru-minantium (Certes) Wenyon (12) has been ac-cepted for the large crescentic forms in the rumen(14). The oval form has not been named. Bothlarge forms also occur in sheep. Crescentic bac-teria cultured from the bovine and ovine rumenand classified as Selenomonas ruminantium (1, 5)are usually smaller than the large selenomonadsin sheep. They are serologically related to someof the large selenomonads of sheep but not toothers (6). Attempts to obtain pure cultures oflarge selenomonads by picking single cells havebeen unsuccessful (16).

In the present work, pure cultures of largeselenomonads were obtained by classical dilutionmethods after eliminating most of the small cellsby centrifugation.

I Present address: Laboratory of Veterinary Biochemistry,Utrecht, The Netherlands.

820

MATERIALS AND METHODS

Rumen fluid was removed from one of two tistulatedsheep fed a pelleted alfalfa hay ration at maintenancelevel. Equal portions of the daily ration were fed at 2-hrintervals by means of a mechanical feeder. The rumenfluid (5 ml) was placed in a small culture tube, flushedout with CO2, and stoppered. The tube was centrifugedfor 2 min at 150 x g to sediment feed particles and pro-tozoa. The supernatant fluid, drawn off with a pipetteconnected to a mouth tube, was placed in a fresh tubeunder CO2. This was stoppered and centrifuged at500 x g for 5 min. The creamy-white sediment wastaken up in a l-ml syringe and injected through the re-cessed stopper (9) into a tube containing 6.5 ml ofanaerobic sterile wash medium (Table 1). This tubewas centrifuged for 5 min at 500 x g, the supernatantfluid was discarded, and the sediment was taken up andagain diluted into a fresh tube of wash medium. A sus-pension containing 90% large cells was obtained by re-peating the washing procedure 8 to 10 times. Reducingthe centrifugal speed or the centrifugation time slightlyimproved the quality of the suspension, but it was neverpossible to remove all small cells.

During all steps in the preparation of the suspensionof large cells, extreme anaerobic precautions were taken.The sediment was finally suspended in 6.5 ml of freshwash medium, and the number of cells per milliliterwas counted with a Petroff-Hauser counting chamberunder a phase-contrast microscope at a magnificationof l,000x. Based upon the number of large cells in thesuspension, the inoculum was suitably diluted through aseries of culture tubes (12 x 70 mm) containing 3.4 ml

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of rumen fluid-glucose agar (Table 1). The final agar

(Oxoid lonagar no. 2) concentration was 0.53%. Agarshake cultures (not roll tubes) were prepared, and thetubes were incubated in an upright position at 39 C.Growth was followed by examining the tubes daily undera dissecting microscope for colonies.

All medium ingredients were heat-sterilized exceptsodium bicarbonate, glucose, and rumen fluid, whichwere sterilized by filtration. To obtain filtered rumen

fluid, fresh rumen fluid was bubbled with oxygen-freeH2 for about 15 min, and the fluid was dispensed anaer-

obically into 50-ml cellulose acetate centrifuge tubeswhich were capped with tight-fitting caps. The tubeswere centrifuged for 30 min at 20,000 x g in a coldroom. The top half of the supernatant fraction was

carefully removed with a pipette connected to mouthtubing and transferred to an Erlenmeyer flask underH2. The flask was incubated at 45 C with occasionalshaking for about I hr, during which time a precipitateformed. The precipitate was removed by centrifugationunder anaerobic conditions for 30 min at 20,000 x g,

and the supernatant fluid was drawn off and saturatedwith oxygen-free CO2. This liquid was then passedthrough a filter (Gelman GA-8, 0.2 tim), by using suc-tion. The sterile filtration funnel and the suction flaskwere both completely filled with oxygen-free CO2 be-fore the fluid was added, and air was excluded from thefunnel with a gentle stream of CO2. The sterile rumenfluid, dispensed anaerobically in sterile culture tubes (16by 150 mm) fitted with butyl rubber stoppers, was

stored in the refrigerator until used. Stringent anaerobictechniques (9) were used throughout.

The stock agar medium containing one part of solu-tion A, one part of solution B (15), and two parts ofdeionized water, was autoclaved separately in smallanaerobic culture tubes; other ingredients were addedlater by injection through the rubber stopper.

Culture purity of the isolated strains was assured bysubculturing through agar dilution series at least threesuccessive times. At each transfer colonies were pickedfrom the highest dilution showing growth. If all the col-onies and the bacteria were identical in morphology, theculture was considered pure.

Glucose was used as the energy source in all testsother than the tests on the fermentation of variouscarbon sources. The composition of the liquid mediumused to determine fermentation products was the sameas that of the agar medium (Table 1), except that agarwas omitted and glucose was supplied at a final concen-tration of 0.5% (w/v). Tubes containing 10 ml of thisliquid medium were inoculated with one drop of a vig-orously growing culture in the same medium. Fermen-tation products from glucose were determined in thespent liquid cultures.

Acid fermentation products were first analyzed bypartition chromatography on cellulose columns usingmixtures of acetone and n-hexane as eluents (10). Bythis method n-butyrate was not separated from itsbranched isomer or from higher volatile fatty acids.Samples were analyzed also for volatile fatty acids witha flame ionization gas chromatograph equipped with a

column of Chromosorb W-DCMS (80 to 100 mesh)coated with neopentylglycolsuccinate and orthophos-phoric acid in a ratio of 10: 1:0.1. Spent cultures were

also analyzed for neutral fermentation products by thin-

TABLE 1. Composition ofmedia used in the isolationand cultivation of large selenomonads

from the sheep rumen

Agar Wash-Ingredients medium medium

(ml) (ml)

Rumen fluid, FSa ............. 1.00 2.00NaHCOs, 10% (w/v), FS ....... 0.18 0.36Cysteine * HCI, 3% (w/v) ....... 0.035 0.070Na2S *9H2O, 3% (w/v) ......... 0.035Casamino Acids, 2% (w/v) ..... 0.050Yeast extract, 2% (w/v) ........ 0.050Glucose, 10% (w/v), FS ........ 0.030Carbon dioxide atmosphere ..... 100% 100%

Mineral solution A (15) ........ 0.50 1.00Mineral solution B ............ 0.50 1.00Deionized water ............... 1.00 2.00Agar (mg) ................... 18

aFS, sterilized by filtration through a membranefilter (Millipore; porosity, 0.20 Arm); other ingredientswere autoclaved.layer chromatography on Silica Gel G (3). Sum valuesof free carbon dioxide and residual bicarbonate in spentcultures and controls were determined by the syringetechnique (Hungate, personal communication). Onemilliliter of 2.0 N H2SO4 was injected into a tube, andthe syringe was quickly withdrawn. An empty 20-mlglass syringe was then rapidly inserted through thestopper of this tube, and tube and syringe were shakenvigorously several times to equilibrate gas and liquidphases. The glass syringe was lubricated with sterile dis-tilled water. The amount of excess gas was recordedand compared with the control values. Values obtainedwere corrected to standard temperature and pressure.Fermentation gases were separated on a Perkin ElmerVapor Fractometer with a silica gel column and ni-trogen as carrier gas.

Glucose in the spent cultures was determined withthe glucose oxidase method (Biochimica Test Combina-tion; Boehringer, C.F. & Soehne, Germany).

Growth was measured in liquid cultures as opticaldensity at 600 nm on a Spectronic-20 colorimeter(Bausch & Lomb, Inc., Rochester, N.Y.).

Growth at different temperatures was assessed bymeasuring optical density of liquid cultures that wereplaced for 40 hr in different incubators with tempera-tures ranging from 24 to 45 C with 3 C intervals.

The medium used to detect H2S production was theagar medium (Table 1) plus 0.05% (w/v) ferric ammo-nium citrate and 1.5% Trypticase (w/v). This mediumwas inoculated and incubated for 6 days. A black colorindicated that H2S was produced. Nitrate reduction wastested for in the liquid medium with 0.5% (w/v) glucoseand 0.2% (w/v) KNO, added. Indole and Voges-Pros-kauer tests were made in the liquid medium with 1%(w/v) Casitone added, and rumen fluid was decreasedto 10% (v/v).

RESULTSIn successful experiments, the number of colo-

nies of large selenomonads appearing in the

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higher dilutions agreed with the number expectedon the basis of the dilution and the cell count inthe washed inoculum. Direct counts ranged be-tween 1.1 and 3.6 x 107 of large selenomonads/ml and culture counts averaged not less than 80%of the direct counts. Strains A22, E22 and L22were isolated from sheep no. 1, and strains NSIand NS2 were isolated from sheep no. 2. StrainSS was isolated from the rumen of a female black-tailed deer (Odocoileus hemionus columbianus)shot at the Hopland Field Station, Hopland, Calif.

Morphology. The deep colonies in agar werecreamy white, lenticular, and sometimes um-bonate at both sides. They were visible after 1 to2 days of incubation as glassy, round, translucentcolonies with the appearance of tiny droplets ofwater. After 2 to 3 days, colonies changed fromtranslucent to opaque and grew in 5 or 6 days to

a maximum diameter of 6 mm. Growth in agaror gelatin stabs was filiform. Strain L22 differedfrom the other strains by slower growth in agarmedia, colonies usually not appearing before thethird day of incubation.The cells measured 2 to 3 ,um in width and 5 to

10 um in length. In liquid culture, cells wereslightly thicker than in agar, were more uniformin size, and helicoidal filaments were never seen.

Cells were curved, crescentic, gram-negative,rapidly motile with bluntly tapered or roundedends, occurring mostly as singles (Fig. 1). Theorganisms moved by means of flagella, attachedto the concave side of the cells in one or whatseemed sometimes two tufts (dividing organisms).In some electron micrographs they were distrib-uted over the concave side of the cell.

Physiological characteristics. All six strains

L.

E._

*...:...... .........

_r

.d_

4&

V. ;."NI..WE. o.:. : F

.. .-...

4'; .. X Ne

* *b-.:̂ j,,$<*F; .: Av {.: : t ... j

4_

FIG. 1. Electron micrograph of the large selenomonad A 22 isolated from the sheep rumen (x 10,000). Fixed withosmic acid.

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SELENOMONADS FROM SHEEP RUMEN

were strict anaerobes; no growth occurred inmedia in which the resazurin was oxidized. Op-timal growth was obtained at 39 C. No growthoccurred at or below 33 C, at 42 C or at 45 C in40 hr. Growth in a liquid medium fortified withfermentable sugars was heavily turbid with slightsediment formation.As judged from optical density, only slight

growth occurred in the liquid medium with bicar-bonate, cysteine, sodium sulfide, CasaminoAcids, and yeast extract omitted. Bicarbonatestimulated growth when added to such a medium.This effect would be mainly a pH effect. Cysteineand sodium sulfide were inhibitory when added tothe complete medium in concentrations higherthan the 0.03% normally employed. The mediumwas well reduced even without their addition.Yeast extract stimulated growth appreciably buthydrolyzed casein or other organic nitrogensources had no effect on growth when added tothe liquid medium containing all other ingredi-ents (Table 1).

Ovine rumen fluid could be replaced by bovinerumen fluid in the medium, and the cells retainedtheir large size even after repeated transfers withthe bovine medium. Filtered rumen fluid could bereplaced by autoclaved rumen fluid or by thesteam distillate of the same amount of acidifiedrumen fluid. Acidified rumen fluid was steam dis-tilled after removal of cells by using the proce-dure of Fenner and Elliot (4). The steam distil-late was neutralized with 0.1 N NaOH and con-centrated with a rotatory evaporator. The con-centrated solution thus obtained was filter steri-lized and added to the liquid medium withoutrumen fluid to replace the rumen fluid.No growth occurred in the liquid medium con-

taining all other ingredients (Table 1) but norumen fluid or volatile fatty acids. The requiredacids were not identified but a considerable up-take of n-valeric acid from the rumen fluid-glu-cose-broth was evident from a comparison of gaschromatographic analyses of the spent culturesand the inoculated but unincubated medium. Iso-butyrate and iso-valerate concentrations re-mained constant during incubation.None of the strains produced indole or reduced

nitrate. Only strain SS produced H2S from cys-teine, the other strains did not.

All strains fermented fructose, glucose, man-

nose, cellobiose, maltose, sucrose, and salicin.None of the strains grew on arabinose, xylose,fucose, galactose, rhamnose, melibiose, lactose,melezitose, trehalose, glycerol, arabitol, dulcitol,sorbitol, inositol, or pinitol (5-O-methyl-D-inosi-tol). Pectin and cellulose were not hydrolyzed byany of the strains, and starch was only hydro-lyzed by strain SS. Lactate was not fermented,

when it was the only carbon source nor withsmall amounts of glucose. In their sugar fermen-tation characteristics, strains A22, E22, L22,NSI and NS2 were alike but differed from strainSS in some respects (Table 2). It took 40 to 50 hrto reach maximal optical density values on 0.5%concentrations of the sugars that were fermented.Strain L22 grew more slowly on agar, but wasnot noticeably slower in liquid cultures.

Fermentation products. The fermentation prod-ucts of the strains grown in a 0.5% glucose-30%rumen fluid broth are shown in Table 3. Controlvalues from inoculated tubes with the same me-dium minus glucose were subtracted. The patternof products is much the same for the differentstrains. All of the glucose was fermented; nonecould be detected in the spent cultures. Final pHvalues ranged between 4.3 and 4.5.The principal acids formed were lactate, ace-

tate, propionate, and formate, with smallamounts of succinate and butyrate produced inmost cultures. Strain SS did not form butyrate.Insignificant amounts of carbon dioxide (0.01mmole/mmole of glucose fermented) were fixed.Carbon recoveries from glucose corrected forcontrol values and the oxidation-reduction indexvalues, indicating the sum of oxidized productsdivided by the sum of reduced products, are alsogiven in Table 3.The Voges-Proskauer test was negative on all

strains and neutral fermentation products werenot formed.

Products formed by strain L22 on differentenergy sources at the 0.5% level are shown inTable 4. With mannitol and salicin production ofpropionate and acetate was favored at the ex-pense of lactate.To test the strains for iodophilly, cells were

grown on 0.2% glucose for 24 hr in the liquid

TABLE 2. Sugarfermentation characteristics oflargeselenomonadsa

Strain

Substrate A22, E22,L22, NS I, SSand NS2

Raffinose .............. _ +Dextrin ................ _ +Starch ................. _ +Inulin ................. _ +Xylan ................. _ +Mannitol ............. +

a All strains grew on fructose, glucose, mannose, su-crose, maltose, cellobiose, and salicin. None of thestrains grew on arabinose, xylose, galactose, rhamnose,melibiose, lactose, trehalose, melezitose, glycerol, inosi-tol, pinitol, arabitol, dulcitol, sorbitol, fucose, sorbose,or lactate. Pectin and cellulose were not hydrolyzed.

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TABLE 3. Fermentation products ofstrains oflargeselenomonads isolated from the sheep rumena

StrainProducts

A22 E22 4122 NSI SS

Formic acid ...... 0.57 0.62 0.49 0.42 0.50Acetic acid ....... 1.56 1.12 1.20 2.00 1.41Propionic acid .... 0.51 0.35 0.70 0.40 0.75Butyric acid ...... 0.05 0.09 0.05 0.10 0Lactic acid ....... 2.86 4.01 2.60 2.50 3.01Succinic acid ..... 0.11 0.02 0.30 0.21 0.20

Carbon recovery (%) 86.7 98.3 88.8 86.2 92.4Oxidation-reduction

index. 1.11 1.21 0.98 1.05 0.93

a Values expressed as millimoles per 100 ml of rumenfluid-glucose medium.

medium after which they were harvested by cen-trifugation and transferred to fresh mediumwithout an energy source. After incubation in thismedium for another 5 hr to deplete the cells, fer-mentable sugars were added at the 0.1% level,and tests were done after 10 min of incubation.All strains were strongly iodophilic, and the addi-tion of fermentable sugars resulted in a rapiddeposition of reserve polysaccharide. This storagepolysaccharide gave a violet-brown color whenstained with Lugol's solution.Selenomonas ruminantium var. bryanti var. n.

The name Selenomonas ruminantium var.bryanti is proposed for the strains A22, E22, L22,NSI and NS2.

It is a gram-negative, motile, crescentic rodwith rounded ends and is 2 to 3 um wide by 5 to10 gm long. In liquid culture cells are slightlythicker than on agar and more uniform in size.The organism occurs as singles and is motile bymeans of flagella attached to the concave side ofthe cell. The cells contain reserve polysaccharidesthat stain violet-brown with Lugol's iodine solu-tion.

Deep colonies are lenticular, creamy white, andreach a diameter of 6 mm. Growth in agar stabs

is filiform.Growth in liquid medium is heavily turbid with

slight sediment formation.Final pH in glucose medium is 4.3.Good growth occurs at 36 or 39 C but no

growth is obtained at 33, 42, or 45 C after 40 hrof incubation.The organism is a strict anaerobe and will not

grow in media in which resazurin is oxidized.No growth occurs in a medium without rumen

fluid.Indole or H2S are not produced.Nitrate is not reduced.Starch is not hydrolyzed.The Voges-Proskauer test is negative.The organism ferments fructose, glucose, man-

nose, cellobiose, maltose, sucrose, salicin, andmannitol. Arabinose, xylose, fucose, galactose,rhamnose, melibiose, trehalose, raffinose, dex-trin, starch, inulin, xylan, glycerol, arabitol, dul-citol, sorbitol, inositol, pinitol, lactate, pectin,and cellulose are not fermented.

It does not form gas splits in glucose agarshake cultures.

Fermentation products from glucose includelactic, acetic, propionic, formic, butyric and suc-

cinic acids. Carbon dioxide or hydrogen are notproduced.

Gelatin is not liquefied.The source is the reticulo-rumen of sheep.

DISCUSSIONThe cultured organisms were morphologically

similar to the large crescentic cells seen duringmicroscopic examination of rumen contents ofsheep and deer. No diminuition in size nor

change in morphology was observed when thecells were subcultured for over 7 months. This is

in contrast to the results of Purdom (16), whostated that all large curved gram-negative rodswhich grew in pure culture after isolation with a

micromanipulator were smaller than the originalselenomonads. Strains of S. ruminantium cul-

TABLE 4. Fermentation products by a large strain (L22) ofSelenomonas isolatedfrom the sheep rumena

Product Glucose Sucrose Cellobiose Maltose Mannitol Salicin

Formic acid ................................. 0.49 0.65 0.51 0.19 0.30 0.72Acetic acid .................................. 1.20 2.43 1.96 1.89 3.12 2.28Propionic acid ............................... 0.70 0.82 0.69 0.86 1.54 1.05Butyric acid ................................. 0.05 0.05 0.14 0.09 0.23 0.06Lactic acid .................................. 2.60 2.87 1.92 2.17 0.54 0.22Succinic acid ................................. 0.30 0.22 0.40 0.87 0.44 0.30

Carbon recovery (%) ........... ............... 88.8 100.7 82.2 96.3 93.8 100.3Oxidation-reduction index ........ ............. 0.98 0.94 0.94 1.02 0.87

a Values expressed as millimoles per 100 ml of rumen fluid-sugar media.

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tured earlier (1, 5, 7) were all smaller in size thanthe present isolates.Growth was relatively slow. When the inoc-

ulum was not sufficiently washed, the largeforms could not be isolated because they were

rapidly overgrown by small cells, including smallforms that resembled S. ruminantium. Some ofthese small selenomonads were isolated andgrown in the same medium as was used for thelarge isolates. The composition of the mediumhad no effect on size or shape of the small cells,and they did not become large under these cultureconditions.

Strains of S. ruminantium have been shown tobe stimulated or to require one or more of thefollowing acids: isobutyric, n-valeric, isovaleric,and DL-2-methyl-butyric (2). Small forms of lac-tate-fermenting S. ruminantium var. lactilyticafrom the sheep rumen required straight-chainsaturated fatty acids with a C5 to C10 carbon skel-eton, n-valerate being most effective in low con-

centrations (1 1). A small bovine strain also grewwith n-valeric acid as the only volatile acid added(17). Thus, it is of interest that the large formsrequired volatile acid and that n-valerate was

decreased in the medium during growth.The abnormal morphology observed with n-

valerate-deficient media (l1) could explain our

observation of long helicoidal filaments of cells inolder agar cultures, since it may be assumed thatn-valerate was rapidly exhausted at the site of thecolony.Hobson et al. (6) studied the relationship be-

tween rumen selenomonads in vitro and in vivowith the fluorescent-antibody technique. Some ofthe large forms were serologically similar to thesmall form but others were not. The results ob-tained in the present study suggest that there are

at least two types of large selenomonads. Onetype (strain SS) shows the characteristics of S.ruminantium and differs from the describedstrains only by its larger size. Another type(strains A22, E22, L22, NSl, and NS2) differsfrom S. ruminantium by not producing H2S fromcysteine and in fermentation of arabinose, xylose,galactose, lactose, and dulcitol. It differs from S.sputigena by fermenting cellobiose and salicinand by its size. None of the large isolates grew onglycerol or lactate so they cannot be classified as

S. ruminantium var. lactilytica. They are consid-ered to be a hitherto undescribed variety forwhich the name Selenomonas ruminantium var.

bryanti is proposed.

:)M SHEEP RUMEN 825

ACKNOWLEDGMENTS

I am indebted to R. E. Hungate for his guidance and for hishelp in preparing the manuscript. Leave of absence from theUniversity of Utrecht and the award of Public Health Servicepostdoctoral Fellowship I F05 TW 1444 from the Fogarty Inter-national Center are gratefully acknowledged.

LITERATURE CITED

1. Bryant, M. P. 1956. The characteristics of strains of Seleno-monas isolated from bovine rumen contents. J. Bac-teriol. 72:162-167.

2. Bryant, M. P., and 1. M. Robinson. 1962. Some nutritionalcharacteristics of predominant culturable ruminal bac-teria. J. Bacteriol. 84:605-614.

3. Drucker, D. B., and T. H. Melville. 1966. A high resolutionsystem for chromatographic analysis of end products ofbacterial fermentations. J. AppI. Bacteriol. 29:536-539.

4. Fenner, H., and J. M. Elliot. 1963. Quantitative method fordetermining the steam volatile fatty acids in rumen fluidby gas chromatography. J. Animal Sci. 22:624-627.

5. Hobson, P. N., and S. 0. Mann. 1961. The isolation ofglycerol-fermenting and lipolytic bacteria from therumen of the sheep. J. Gen. Microbiol. 25:227-240.

6. Hobson, P. N., S. 0. Mann, and W. Smith. 1962. Serolog-ical tests of a relationship between rumen selenomonadsin vitro and in vivo. J. Gen. Microbiol. 29:265-270.

7. Hobson, P. N., S. 0. Mann, and W. Smith. 1963. Growthfactors for Selenomonas ruminantium. Nature (London)198:213.

8. Hungate, R. E. 1966. The rumen and its microbes. Aca-demic Press Inc., New York.

9. Hungate, R. E. 1969. A roll tube method for cultivation ofstrict anaerobes, p. 117-132. In J. R. Norris and D. W.Ribbons, (ed.) Methods in microbiology, vol. 3B. Aca-demic Press Inc., New York.

10. Hungate, R. E., W. Smith, T. Bauchop, 1. Yu, and J. C.Rabinowitz. 1970. Formate as an intermediate in thebovine rumen fermentation. J. Bacteriol. 102:389-398.

11. Kanegasaki, S., and H. Takahashi. 1967. Function ofgrowth factors for rumen microorganisms. 1. Nutritionalcharacteristics of Selenomonas ruminantium. J. Bac-teriol. 93:456-463.

12. Lessel, E. F., Jr., and R. S. Breed. 1954. SelenomonasBoskamp, 1922-a genus that includes species showingan unusual type of flagellation. Bacteriol. Rev. 18:165-167.

13. Moir, R. J., and M. J. Masson. 1952. An illustratedscheme for the microscopic identification of the rumenmicro-organisms of sheep. J. Pathol. Bacteriol. 64:343-350.

14. Oxford, A. E. 1955. Some observations upon the status ofthe generic name Selenomonas Prowazek. Int. Bull. Bac-teriol. Nomencl. Taxon. 5:131-132.

15. Paynter, M. J. B., and R. E. Hungate. 1968. Characteris-tics of Methanobacterium mobilis, sp. n., isolated fromthe bovine rumen. J. Bacteriol. 95:1943-1951.

16. Purdom, M. R. 1963. Micromanipulation in the examina-tion of rumen bacteria. Nature (London) 198:307-308.

17. Tiwari, A. D., M. P. Bryant, and R. S. Wolfe. 1969.Simple method for isolation of Selenomonas ruminan-tium and some nutritional characteristics of the species.J. Dairy Sci. 52:2054-2056.

18. Woodcock, H. M., and G. Lapage. 1914. On a remarkabletype of protistan parasite. Quart. J. Microscop. Sci. 59:43 1-458.

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