Tentative Identification of Methanogenic Bacteria by Fluorescence Microscopy

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1977, p Copyright (C 1977 American Society for Microbiology Vol. 33, No. 3 Printed in U.S.A. Tentative Identification of Methanogenic Bacteria by Fluorescence Microscopy RONALD W. MINK' AND PATRICK R. DUGAN* Department of Microbiology, The Ohio State University, Columbus, Ohio, Received for publication 8 September 1976 Methanogenic bacteria, which are presently identified on the basis of cell morphology and substrate conversion to CH,, can be differentiated from nonmethanogens and identified in pure or mixed culture on the basis of their autofluorescence under ultraviolet illumination. Methane fermentation of complex natural organic substrates is the end of a food chain process involving a wide variety of anaerobic bacteria. That is, the methanogenic bacteria are limited in substrate range to H.,, CO2, formate, methanol, acetate, and possibly carbon monoxide; and they depend upon non-methanogenic anaerobes in mixed culture to produce these substrates via fermentation reactions. Methanogenic bacteria, taxonomically placed in the family Methanobacteriaceae, are identified primarily on the basis of ability to produce CH4 gas from the substrates listed above and on the basis of cell morphology. The pathway of electrons serving as reducing power for conversion of one-carbon compounds to CH4 was partially revealed when an unidentified fluorescent compound was isolated from Methanobacterium strain M.o.H. (1). This substance, which exhibited bluegreen fluorescence in the oxidized state and was nonfluorescent in the reduced form, was given the trivial name of factor42(, due to its strong absorption at 420 nm when oxidized. The autofluorescence exhibited by colonies of methanogenic bacteria has been used as a means of detecting methanogenic bacteria when cultivated on solid growth media. Edwards and McBride (2) used colony fluorescence as presumptive evidence of methanogenic bacteria, because all methanogenic colonies isolated from sewage sludge exhibited the typical blue-green fluorescence and, conversely, all fluorescent colonies were methanogenic, although not all colonies were free of contaminants. They also noted that the bluegreen autofluorescence of the methanogenic bacteria was easily distinguishable from the white-yellow fluorescence observed in many non-methanogenic cultures and thereby sug- I Present address: Department of Dairy Science, University of Illinois, Urbana, Illinois gested the potential for fluorescence microscopic observation of methanogens. This fluorescence has been shown to exist in colonies of all methanogenic bacteria thus far examined, including Methanobacterium, Methanospirillum, and Methanosarcina. The purpose of this report is to extend the observation to microscopic identification of pure or mixed cultures and to illustrate that methanogenic species can be tentatively identified and enumerated within a mixed population on the basis of their autofluorescence. MATERIALS AND METHODS Maintenance and source of cultures. A mixed cellulolytic culture was obtained from a 5-gallon (ca liter) carboy of fermenting sawdust that was actively producing methane. The sawdust mixture was originally inoculated with both rumen fluid obtained from a fistulated sheep and fluid from another carboy containing fermenting sawdust from the natural environment. Enrichment cultures were obtained by culturing the mixed cellulolytic culture in successive transfers into 5 ml of the MS culture medium of Ferry et al. (3) held in test tubes. Methanobacterium ruminantium strain PS, Methanobacterium strain M.o.H., and Methanospirillum hungatii strain JF were obtained from M. P. Bryant, University of Illinois, Urbana. Methanobacterium formicicum strain JF and Methanosarcina barkeri strain JF were obtained from R. S. Wolfe, University of Illinois, Urbana. All cultures were maintained in MS medium either in broth or on agar slants under hydrogen and carbon dioxide (4:1). Microscopy. Phase-contrast and fluorescence photomicrographs were taken with a Zeiss Universal microscope equipped with an epi-illuminant ultraviolet lamp and a x 100 Neofluor objective lens. All photomicrographs were taken with the no. 1 exciter filter and the no. 47 barrier filter in place. The camera mounted on the microscope was a Nikon reflex attachment with an automatic exposure. The film used was Kodak 3200K High Speed Ektachrome, which was specially processed after exposure to increase the relative ASA rating to the film's 713

2 714 MINK AND DUGAN limit. All photomicrographs were made of wet mounts except those of strain M.o.H., which were dry mounts. RESULTS Phase-contrast and fluorescence photomicrographs of M. formicicum, M. barkeri, M. ruminantium, and Methanobacterium strain M.o.H. are presented in Fig. 1 through 8. Although all species initially exhibited cellular autofluorescence of comparable intensity when observed under ultraviolet light, the autofluorescence of Methanobacterium strain M.o.H. and M. hungatii decreased very rapidly. Fluorescence of M. hungatii decreased so rapidly that adequate photographic exposures were not obtained. Figures 9 through 12 show two fields of the enrichment culture under APPL. ENVIRON. MICROBIOL. both phase contrast and ultraviolet light, where autofluorescent cells are evident. Figures 13 through 16 represent two fields of rumen fluid photographed under both phase contrast and ultraviolet light. The latter exhibited cells with both blue-green and whiteyellow autofluorescence. DISCUSSION Due to the difficulty and slowness with which methanogens are grown, identification and/or enumeration is a long and tedious process. Methods used to date require that the organisms grow sufficiently to be visualized as either broth turbidity or agar colonies (2, 4, 5). Thus, a rapid means to tentatively identify and enumerate methanogenic bacteria would be beneficial. All of the methanogenic bacteria observed exhibited autofluorescence, although the rate at which the fluorescence faded varied from species to species. The technique has the added advantage that the anaerobic precau- Figures 1 through 16 are pairs ofphotomicrographs taken under phase contrast and then with ultraviolet epi-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.

3 VOL. 33, 1977 IDENTIFICATION OF METHANOGENIC BACTERIA 715 -UU FIG. 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.

4 716 MINK AND DUGAN APPL. ENVIRON. MICROBIOL. 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 methanogens are unnecessary for microscopic observation, since the pigments fluoresce in their oxidized state. This also indicates, however, that nonviable cells may exhibit autofluorescence. Preliminary studies indicate that fluorescence is still present 24 h after exposure of the cells to oxygen. Although the fluorescence does fade after exposure to ultraviolet light, removing the light for a few minutes and then returning the light generally results in the return of fluorescence. It is possible that there may be non-methanogenic bacteria in these methaneproducing systems that also exhibit bluegreen autofluorescence. Although no such anaerobes have yet been observed, the technique will remain tentative until correlations are established between numbers of fluorescent anaerobes and methanogens enumerated by plate counting or serial dilution techniques. On the basis of this technique, at least one species of Methanobacterium was tentatively identified in the enrichment culture (Fig. 9 through 12). The organism was morphologically similar to the M.o.H. strain or to M. formicicum bacterium, although the possibil-

5 VOL. 33, 1977 IDENTIFICATION OF METHANOGENIC BACTERIA 717 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 the enrichment was another methanogen that has not yet been classified. ACKNOWLEDGMENTS This investigation was supported by a grant from The Ohio State University Graduate School. LITERATURE CITED 1. Cheeseman, P., A. Toms-Wood, and R. S. Wolfe Isolation and properties of a fluorescent compound, factor420, from Methanobacterium strain M.o.H. J. Bacteriol. 112: Edwards, T., and B. C. McBride New method for the isolation and identification of methanogenic bacteria. Appl. Microbiol. 29: Ferry, J. G., P. H. Smith, and R. S. Wolfe Methanospirillum, a new genus of methanogenic bacteria, and characterization ofmethanospirillum hungatii sp.nov. Int. J. Syst. Bacteriol. 24: Siebert, M. L., and W. H. J. Hattingh Estimation of methane producing bacterial numbers by the most probable number (MPN) technique. Water Res. 1: Toerien, D. F., and M. L. Siebert A method for the enumeration and cultivation of anaerobic acid forming bacteria present in digesting sludge. Water Res. 1: