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A fluorochrome-staining technique for enumeration of bacteria in saline, organically enriched, alkaline lakes.

A procedure is described for epifluorescence direct-counting of planktonic bacteria in an alkaline, hypersaline, organically enriched lake. To preclude precipitation and staining of dissolved organic material (DOM), the procedure involved isotonic
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  L~mnol. ceanogr., 32(4), 1987, 993-995  O 1987, by the American Society of Limnology and Oceanography, Inc.  A fluorochrome-staining technique for counting bacteria insaline, organically enriched, alkaline lakes Abstract-A procedure is described for epiflu-orescence direct-counting of planktonic bacteriain an alkaline, hypersaline, organically enrichedlake. To preclude precipitation and staining ofdissolved organic material (DOM), the procedureinvolved isotonic dilution of unfixed samples atin situ pH and filtration before fluorochromestaining. Fluorochrome-DOM complexes re-maining on the filter were removed by sequentialrinses with isotonic 0.1 M citrate (pH 6.6). Withthe modified procedure, the dye acridine orange(AO) yielded better specimen-background con-trast and higher overall cell counts than did ethid-ium bromide, Hoechst dye No. 33258, or 4,6- diamidino-2-phenylindole (DAPI). Saline lakes harbor remarkably largenumbers of bacteria (Hamilton-Galat andGalat 1983; Kilham 198 1; Post 1981). Manysaline lakes are meromictic and are inter-esting for microbiological study becausedensity gradients in the water column influ-ence the flux of nutrients essential for mi-crobial growth (Cloern et al. 1983). How-ever, there is little information on thebacterial populations due, in part, to ex-treme chemical conditions which interferewith direct counting techniques.I here report a preparative staining pro-cedure developed for counting bacteria inBig Soda Lake, Nevada. This lake is deepand meromictic with three major, chemi-cally distinct zones: saline and aerobic, sa-line and mildly reducing, and hypersalineand strongly reducing (Cloern et al. 1983;Oremland et al. 1982). The preparative pro-cedure reduces autofluorescence and pre-cipitation problems that occur during rou-tine AO-direct counting of bacteria (Hobbieet al. 1977) in Big Soda Lake samples. Theprecipitation is due to high pH (9.7) andDOM content (up to 60 mg liter-' DOC)(Kharaka et al. 1984). I have also evaluatedfour different fluorochromes for use with themodified preparative procedure and BigSoda Lake samples. The fluorochromestested were: A0 (Curtin Matheson Sci. No.AX0305), DAPI, ethidium bromide (EB),and Hoechst dye No. 33258 (HD) (Sigma(hemCo. N~. 1388, E8 7 5 1, B288 3, re-spectively)' I thank R. Oremland for providing logis-tical support at Big Soda Lake, C. Culbert-Son for help in sampling, and L. George forwith the preparations for bacte-ria1 enumeration and ~~oto~~c~osco~hemanuscript was reviewed by R. Smith.Water samples were collected in July1984, October 1984, February 1985, andMay 1985 with a 7-liter Niskin collectingbottle at a station near the middle of thelake (Zehr et al. 1987). Water samples fromvarious depths between 1 and 60 m weretransferred to sterile 125-ml amber poly-ethylene bottles and stored on ice. Sampleswere processed within 48 h of collection.Organic fixatives such as formaldehyde andglutaraldehyde were not used, since addi-tion of aldehyde fixatives to Big Soda Lakewater results in formation of precipitates.Precipitation was most severe in samplesfrom the monimolimnion (permanently an-oxic, 88% salinity water below the 35-mdepth chemocline).Bacteria were counted with a Leitz Dialux20 microscope, fitted for epifluorescence asdescribed by Harvey et al. (1984). Use ofan ultraflat field, high-resolution 63 x [1.40numerical aperture (NA)] P1 Apo objectivein combination with a before-ocular mag-nification changer improved recognition ofvery small AO-stained bacteria (<0.3 pmin cell length) relative to the 100 x (1.32NA) Fluotar iris objective used in the studycited above. Optical filter combinations,staining times, and final fluorochrome con-centrations are summarized in Table 1.Recognition of individual stained bacte-ria was not possible with A0 and standardtechniques of preparation. There was strongautofluorescence apparently due to thebinding of free A0 to DOM adsorbed ortrapped on the polycarbonate Nuclepore fil-ter. The problem of autofluorescence wasmuch less severe with DAPI and EB, but  994 Notes 2 MINUTE INCUBATION OF tered onto black Nuclepore filters (0.2-pm WITH NeCl SOLUTIONALDEHYDE-FIXED SAMPLE pore size, 25-mm diam, Nuclepore Corp. 0.4' or 1.5"M, pH 9.7)(0.01% Flnal Concenlretlon) 4 No. 110656) before application of fluoro-chrome-staining solution to minimize in- FILTRATION ONTO BLACK.STAINED NUCLEPORE FILTER  teractions of fluorochrome with DOM. Atthe end of the staining interval, the stainingsolutions were drawn through the filter, fol-lowed by sequential rinsing of the filter with0.1 M, pH 6.6 citrate buffer to remove re- t maining extracellular organic-fluorochromecomplexes. Citrate rinses were most critical I during preparation of monimolimnionsamples. All dilution, staining, and buffer CITRATE (O.IM, pH 6.6, 26%or solutions were made up in Milli-Q-purified FILTRATION THROUGH SAMPLE 88.1 S."1.THROUGHSTAINED SAMPLE 1 I water (1 8 Mohm resistivity) made isotonicwith respect to samples by addition of NaCland filter-sterilized with 0.2-pm (pore size)filter disks. Separate sets of solutions were FILTER AND COVER SLIP used for mixolimnion (22% salinity water Fig. I. Protocol for acridine-orange preparations above the 35-m-depth chemocline) and for epifluorescence direct counting of bacterial abun- monimolimnion samples, because of the dance. The standard technique (Hobbie et al. 1977) is fourfold salinity difference between these depicted on the right and the modified procedure forBig Soda Lake samples on the left. Solutions used for two major zones. Figure 1 shows a summary samples from 0-34-m depth are indicated by an as- protocol for this modified preparative tech- terisk and from 35-60-m depth by double asterisks. nique.With the modified procedure, A0yieldedonly when these two fluorochromes were higher bacterial counts on a 15-m-depthapplied directly to the bacteria on the filter. water sample than did the other threefluo-Use of HD resulted in poor bacteria-back-rochromes (Table 1). Differences in appar-ground contrast and the image faded rapidly ent effectiveness between A0 and DAPI,under incident ultraviolet light. However,HD, and EB for detecting bacteria in Bigthe image might be improved by modifi-Soda Lake appear to be due to differencescation of the optics or use of another HD. in contrast. The better contrast afforded byTo obtain reasonable contrast betweenAO, with the modified preparative proce-fluorochrome-stained bacteria and thedure, allowed easier recognition of verybackground, it was necessary to modify the small bacteria (<0.3 pm long). Analysis ofpreparative procedure. Each unfixed sampleunstained sample preparations suggests thatwas diluted with a solution isotonic to Bigautofluorescing particles did not contributeSoda Lake water to reduce formation of or-significantly to apparent bacteria counts, asganic precipitates during subsequent prep-has been observed in two other aquatic hab-arative steps. Diluted samples were then fil- itats (Paul 1982). Table 1. Final concentrations, staining times, optical filter combinations, and relative bacterial enumerationdata for the fluorochromes used to process Big Soda Lake water samples. Stalnlng Additional excl-Concn tlme Ploemopak tation filter Comparative counts*Ruorochrome bM) (mln) designation (nm) (x 106iSD) Acridine orange 270 5 H2t 480 8.16k0.82DAPI290 20A$ - 3.5 1 0.42Ethidium bromide 250 5 H2 455 3.29k0.56Hoechst dye No. 33258 20 30A - 1.41+0.32 * Numbers of bactena In water sample from 15-m depth of Blg Soda Lake as determined w~theach fluorochrome.  t Contalns BP 390-490-nm excltatlon filter, RKP 510 mlrror, LP 515-nm banier filter (Leltz).  f Contalns BP 340-380-nm excltatlon filter, RKP 400 mirror, LP 430-nm bamer filter (Le~tz).   Notes 995 Table 2. Seasonal changes in relative abundance ofbacteria <0.3 pm long (t total bacterial abundance x 100) in the water column of Big Soda Lake. Depth (m) Jul Oct Feb May a SD/x1568.4 33.327.613.635.7 0.653031.4 26.524.3 25.4*26.7 0.124018.8 31.329.0 28.927.0 0.21 *Value IS for 32-m depth. The accuracy of bacteria counts with EB,HD, or DAPI depended, in part, on thenumber of very small bacteria, which variedseasonally and with depth (Table 2). At the15-m depth, relative abundance of bacteria<0.3 pm long varied from 68.4% in July to13.6% in May. In general, very small bac-teria were more predominant at 15 m thanat 30 and 40 m. Hence, EB and DAPI ap-pear to be better suited for bacterial countsin the lower 40 m of Big Soda Lake. Prob-lems with enumerating small bacteria inDOM-enriched water by fluorochrome-staining epifluorescence are not unique tohypersaline lakes. Bergstrom et al. (1986)reported that, due to weak fluorescence ofthe bacteria, the fluorochromes acroflavinand bisbezimide did not work very well forcounting very small bacteria in humic-richlake water.The modified AO-epifluorescence tech-nique worked well for the three chemicallydistinct zones of Big Soda Lake. Althoughthe chemistry and overall bacterial mor-phology changed significantly with depth,contrast between background and stainedbacteria was always good enough for accu-rate bacterial counts. Bacteria in the aerobicmixolimnion (epilimnion) in the upper 20m were generally more difficult to count,since that population was dominated bysmall coccoidal rods10.4 pm in diameter.In contrast, populations below the 35-mchemocline consisted mainly of large rodsand filamentous forms that were easy tocount. However, extreme chemical condi-tions of the monimolimnion necessitatedcare during sample preparation. In general,there were few abiotic particles in Big SodaLake, and they did not interfere with bac-terial enumeration.In summary, A0 appears to be a usefulfluorochrome for counting morphologicallydiverse bacterial populations in a variety ofaquatic habitats, although the standardpreparative technique must be modified foruse in some aquatic environments with ex-treme chemical conditions-particularlythose habitats with high DOM. The above-described procedure also worked well atMono Lake, California (Harvey, which is also hypersaline, alkaline,and organically enriched. R. W. Harvey Water Resources DivisionU.S. Geological Survey 345 Middlefield Road Menlo Park, California 94025  References BERGSTROM, AND K. SALONEN. 986. ., A. HEINANEN,Comparison of acridine orange, acriflavine, andbisbenzimide stains for enumeration of bacteriain clear and humic waters. Appl. Environ. Micro-biol. 51: 664-667.CLOERN, . E., B. E. COLE, ND R. S. OREMLAND.983. Seasonal changes in the chemistry and biology of a meromictic lake (Big Soda Lake), Nevada, U.S.A. Hydrobiologia 105: 195-206. HAMILTON-GALAT, ., AND D. L. GALAT. 1983. Sea-sonal variation of nutrients, organic carbon, ATP,and microbial standing crops in a vertical profileof Pyramid Lake, Nevada. Hydrobiologia 105: 27-43.HARVEY, . W., R. L. SMITH, AND L. GEORGE. 1984.Effect of organic contamination upon microbialdistributions and heterotrophic uptake in a CapeCod, Mass., aquifer. Appl. Environ. Microbiol. 48: 1197-1202.HOBBIE, . E., R. J. DALEY, AND S. JASPER. 1977. Useof Nuclepore filters for counting bacteria by flu-orescence microscopy. Appl. Environ. Microbiol. 33: 1225-1228.KHARAKA, Y. K., S. W. ROBINSON,. M. LAW, AND W. W. CAROTHERS.984. Hydrochemistry of BigSoda Lake, Nevada: An alkaline meromictic des-ert lake. Geochim. Cosmochim. Acta 48: 823-835.KILHAM,P. 1981. Pelagic bacteria: Extreme abun-dances in African saline lakes. Natunvissenschaf-ten 67: 280-38 1.PAUL, J. H. 1982. Use of Hoechst dyes 33258 and33342 for enumeratiog of attached and planktonicbacteria. Appl. Environ. Microbiol. 43: 939-944.OREMLAND,. S., L. MARSH, ND D. J. DESMARAIS.1982. Methanogenesis in Big Soda Lake, Nevada:An alkaline, moderately hypersaline desert lake.Appl. Environ. Microbiol. 43: 462-468.POST, F. J. 198 1.6. Microbiology of the Great SaltLake north arm. Hydrobiologia 81: 59-69.ZEHR, J. P., AND OTHERS.1987. Big Soda Lake (Ne-vada). 1. Pelagic bacterial heterotrophy and bio-mass. Limnol. Oceanogr. 32: 781-793. Submitted: 20 November 1986Accepted: 23 March 1987
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