APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1977, p. 713-717Copyright (C 1977 American Society for Microbiology

Vol. 33, No. 3Printed in U.S.A.

Tentative Identification of Methanogenic Bacteria byFluorescence Microscopy

RONALD W. MINK' AND PATRICK R. DUGAN*Department of Microbiology, The Ohio State University, Columbus, Ohio, 43210

Received for publication 8 September 1976

Methanogenic bacteria, which are presently identified on the basis of cellmorphology and substrate conversion to CH,, can be differentiated from non-

methanogens and identified in pure or mixed culture on the basis of theirautofluorescence under ultraviolet illumination.

Methane fermentation ofcomplex natural or-ganic substrates is the end of a food chain proc-ess involving a wide variety of anaerobic bacte-ria. That is, the methanogenic bacteria are lim-ited in substrate range to H.,, CO2, formate,methanol, acetate, and possibly carbon monox-ide; and they depend upon non-methanogenicanaerobes in mixed culture to produce thesesubstrates via fermentation reactions.

Methanogenic bacteria, taxonomically placedin the family Methanobacteriaceae, are identi-fied primarily on the basis of ability to produceCH4 gas from the substrates listed above andon the basis of cell morphology.The pathway of electrons serving as reduc-

ing power for conversion of one-carbon com-pounds to CH4 was partially revealed when anunidentified fluorescent compound was iso-lated from Methanobacterium strain M.o.H.(1). This substance, which exhibited blue-green fluorescence in the oxidized state andwas nonfluorescent in the reduced form, wasgiven the trivial name of factor42(, due to itsstrong absorption at 420 nm when oxidized.The autofluorescence exhibited by colonies ofmethanogenic bacteria has been used as ameans of detecting methanogenic bacteriawhen cultivated on solid growth media. Ed-wards and McBride (2) used colony fluores-cence as presumptive evidence of methano-genic bacteria, because all methanogenic colo-nies isolated from sewage sludge exhibited thetypical blue-green fluorescence and, con-versely, all fluorescent colonies were methan-ogenic, although not all colonies were free ofcontaminants. They also noted that the blue-green autofluorescence of the methanogenicbacteria was easily distinguishable from thewhite-yellow fluorescence observed in manynon-methanogenic cultures and thereby sug-

I Present address: Department of Dairy Science, Univer-sity of Illinois, Urbana, Illinois 61801.

gested the potential for fluorescence micro-scopic observation of methanogens. This fluo-rescence has been shown to exist in colonies ofall methanogenic bacteria thus far examined,including Methanobacterium, Methanospiril-lum, and Methanosarcina.The purpose of this report is to extend the

observation to microscopic identification ofpure or mixed cultures and to illustrate thatmethanogenic species can be tentatively iden-tified and enumerated within a mixed popula-tion on the basis of their autofluorescence.

MATERIALS AND METHODSMaintenance and source of cultures. A mixed

cellulolytic culture was obtained from a 5-gallon (ca.18.9-liter) carboy of fermenting sawdust that wasactively producing methane. The sawdust mixturewas originally inoculated with both rumen fluidobtained from a fistulated sheep and fluid from an-other carboy containing fermenting sawdust fromthe natural environment. Enrichment cultures wereobtained by culturing the mixed cellulolytic culturein successive transfers into 5 ml of the MS culturemedium of Ferry et al. (3) held in test tubes. Meth-anobacterium ruminantium strain PS, Methanobac-terium strain M.o.H., and Methanospirillum hunga-tii strain JF were obtained from M. P. Bryant, Uni-versity of Illinois, Urbana. Methanobacterium formi-cicum strain JF and Methanosarcina barkeri strainJF were obtained from R. S. Wolfe, University ofIllinois, Urbana. All cultures were maintained inMS medium either in broth or on agar slants underhydrogen and carbon dioxide (4:1).

Microscopy. Phase-contrast and fluorescencephotomicrographs were taken with a Zeiss Univer-sal microscope equipped with an epi-illuminant ul-traviolet lamp and a x 100 Neofluor objective lens.All photomicrographs were taken with the no. 1exciter filter and the no. 47 barrier filter in place.The camera mounted on the microscope was a Nikonreflex attachment with an automatic exposure. Thefilm used was Kodak 3200K High Speed Ekta-chrome, which was specially processed after expo-sure to increase the relative ASA rating to the film's

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limit. All photomicrographs were made of wetmounts except those of strain M.o.H., which weredry mounts.

RESULTSPhase-contrast and fluorescence photomi-

crographs of M. formicicum, M. barkeri, M.ruminantium, and Methanobacterium strainM.o.H. are presented in Fig. 1 through 8. Al-though all species initially exhibited cellularautofluorescence of comparable intensitywhen observed under ultraviolet light, the au-tofluorescence of Methanobacterium strainM.o.H. and M. hungatii decreased very rap-idly. Fluorescence ofM. hungatii decreased sorapidly that adequate photographic exposureswere not obtained. Figures 9 through 12 showtwo fields of the enrichment culture underboth phase contrast and ultraviolet light,where autofluorescent cells are evident. Fig-

ures 13 through 16 represent two fields of ru-men fluid photographed under both phase con-trast and ultraviolet light. The latter ex-hibited cells with both blue-green and white-yellow autofluorescence.

DISCUSSIONDue to the difficulty and slowness with

which methanogens are grown, identificationand/or enumeration is a long and tedious proc-ess. Methods used to date require that theorganisms grow sufficiently to be visualized aseither broth turbidity or agar colonies (2, 4, 5).Thus, a rapid means to tentatively identifyand enumerate methanogenic bacteria wouldbe beneficial. All ofthe methanogenic bacteriaobserved exhibited autofluorescence, althoughthe rate at which the fluorescence faded variedfrom species to species. The technique has theadded advantage that the anaerobic precau-

Figures 1 through 16 are pairs ofphotomicrographs taken under phase contrast and then with ultravioletepi-illumination. All bar markers represent 5 um.

FIG. 1. M. formicicum, phase contrast.FIG. 2. Same field as Fig. 1, photographed under ultraviolet.FIG. 3. M. barkerii, phase contrast.FIG. 4. Same field as Fig. 3, ultraviolet.

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-UUFIG. 5. Methanobacterium strain M.oJH., phase contrast.FIG. 6. Same field as Fig. 5, ultraviolet.FIG. 7. M. ruminantium, phase contrast.FIG. 8. Same field as Fig. 7, ultraviolet.

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FIG. 9 and 11. Enrichment cultures, phase contrast.FIG. 10 and 12. Respective fields of Fig. 9 and 11, ultraviolet.

tions essential for cultivation of methanogensare unnecessary for microscopic observation,since the pigments fluoresce in their oxidizedstate. This also indicates, however, that non-viable cells may exhibit autofluorescence. Pre-liminary studies indicate that fluorescence isstill present 24 h after exposure of the cells tooxygen. Although the fluorescence does fadeafter exposure to ultraviolet light, removingthe light for a few minutes and then returningthe light generally results in the return offluorescence. It is possible that there may benon-methanogenic bacteria in these methane-

producing systems that also exhibit blue-green autofluorescence. Although no such an-aerobes have yet been observed, the techniquewill remain tentative until correlations areestablished between numbers of fluorescentanaerobes and methanogens enumerated byplate counting or serial dilution techniques.On the basis of this technique, at least one

species of Methanobacterium was tentativelyidentified in the enrichment culture (Fig. 9through 12). The organism was morphologi-cally similar to the M.o.H. strain or to M.formicicum bacterium, although the possibil-

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FIG. 13 and 15. Rumen fluid, phase contrast.FIG. 14 and 16. Respective fields of Fig. 13 and 15, ultraviolet.

ity exists that the organism observed in theenrichment was another methanogen that hasnot yet been classified.

ACKNOWLEDGMENTSThis investigation was supported by a grant from The

Ohio State University Graduate School.

LITERATURE CITED1. Cheeseman, P., A. Toms-Wood, and R. S. Wolfe. 1972.

Isolation and properties of a fluorescent compound,factor420, from Methanobacterium strain M.o.H. J.Bacteriol. 112:527-531.

2. Edwards, T., and B. C. McBride. 1975. New method forthe isolation and identification of methanogenic bac-teria. Appl. Microbiol. 29:540-545.

3. Ferry, J. G., P. H. Smith, and R. S. Wolfe. 1974.Methanospirillum, a new genus ofmethanogenic bac-teria, and characterization ofMethanospirillum hun-gatii sp.nov. Int. J. Syst. Bacteriol. 24:465-469.

4. Siebert, M. L., and W. H. J. Hattingh. 1967. Estimationof methane producing bacterial numbers by the mostprobable number (MPN) technique. Water Res. 1:13-19.

5. Toerien, D. F., and M. L. Siebert. 1967. A method forthe enumeration and cultivation of anaerobic acidforming bacteria present in digesting sludge. WaterRes. 1:397-404.

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