APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1978, p. 506-5120099-2240/78/0036-0506$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 36, No. 3

Printed in U.S.A.

Autoradiography and Epifluorescence Microscopy Combinedfor the Determination of Number and Spectrum of Actively

Metabolizing Bacteria in Natural WaterstLUTZ-AREND MEYER-REIL

Institut fur Meereskunde an der Universitat Kiel, Abteilung Marine Mikrobiologie, Kiel, Federal Republicof Germany

Received for publication 30 March 1978

A technique is described for the determination of bacterial numbers and thespectrum of actively metabolizing cells on the same microscopic preparation bya combined autoradiography/epifluorescence microscopy technique. Natural bac-terial populations incubated with [3H]glucose were filtered onto 0.2-,um Nucleporepolycarbonate membranes. The filters were cut into quarters and fixed on thesurface of glass slides, coated with NTB-2 nuclear track emulsion (Kodak), andexposed to the radiation. After processing, the autoradiographs were stained withacridine orange. A combination of overstaining on the slightly alkaline side andgradual destaining on the acid side of neutrality gave the best results. Epifluores-cence microscopy revealed bright-orange fluorescent cells with dark-silver grainsassociated against a greenish-to-grayish background. Based on the standardizationcurves, detection of actually metabolizing cells was optimal when cells wereincubated with 1 to 5 ytCi of [3H]glucose per ml of sample for 4 h and theautoradiographs were exposed to NTB-2 emulsion at 7°C for 3 days. In watersamples taken immediately above sandy sediments at beaches of the Kiel Fjordand the Kiel Bight (Baltic Sea, FRG), between 2.3 and 56.2% (average, 31.3%) ofthe total number of bacteria were actually metabolizing cells. Spearman rankcorrelation analysis revealed significant interrelationships between the number ofactive bacteria and the actual uptake rate of glucose.

Ecological studies of the role of microorga-nisms in aquatic systems are limited by thenumber and validity of methods available. Al-though each of the methods has its own impor-tance, the data obtained with different methodsare difficult to compare and to interpret. Only avery limited number of bacteria actually occur-ring are capable of growth on agar plates. Epi-fluorescence microscopy permits information onthe bacterial standing crop (number, biomass),which does not necessarily reflect bacterial ac-tivity. The same holds true for chemical meth-ods (ATP, DNA, lipopolysaccharide) for deter-mining standing crop. The results obtained fromuptake studies of organic solutes (tracer tech-nique) provide information on the uptake of thetotal bacterial population, regardless of the spec-trum of actually metabolizing cells.

In addition to the above-mentioned tech-niques, autoradiography becomes a useful toolin ecological studies, since it enables investiga-

t Publication no. 209 of the Joint Research Program at KielUniversity (Sonderforschungsbereich 95 der Deutschen For-schungsgemeinschaft).

tors to relate activity to individual cells (2, 14).The problems involved in grain density autora-diography have been pointed out recently (7).Determination of the number of actually metab-olizing cells by spot counting (6, 12) is limitedby the difficulty of relating spots to individualcells. Single silver grains or small groups ofgrains often regarded as "background" seem tobe an additional problem. As an alternative,autoradiographs of either smears or filtered sam-ples were stained with traditional dyes (4, 11,13). The identification of cells and their relationto silver grains becomes extremely difficult insome cases. By staining autoradiographs withfluorescent antibodies (immunofluorescence),much better microscopic preparations can beobtained (5).

Epifluorescence microscopy of samples fil-tered onto Nuclepore polycarbonate membranesand stained with acridine orange permits a reli-able picture of number and spectrum of naturalbacterial populations (16). This paper deals withthe coupling of autoradiography and epifluores-cence microscopy to determine which organismsare metabolizing in an ecological situation.

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AUTORADIOGRAPHY/EPIFLUORESCENCE MICROSCOPY 507

MATERIALS AND METHODS

Sample preparation. Water samples were takenin sterile glass flasks immediately above sandy sedi-ments of beaches of the Kiel Fjord and the Kiel Bight(Table 1) during a joint research program (in situwater temperatures, 18 to 200C; incubation at in situtemperature). Standard curves were run with watersamples taken from the inner part of the Kiel Fjord(in situ water temperature, 5 to 80C; incubation tem-perature, 1000).

Duplicate 7-rnl water samples were incubated in 50-ml bottles (Sovirel) with the addition of 35 pL of D-[2-3H(N)]glucose (5 ACi/ml; specific activity, 10 to 20Ci/mmol; New England Nuclear) and aerated by shak-ing (100 rpm). After 4 h of incubation at a temperaturewithin 1 to 20C of the in situ water temperature, thesamples were fixed with 35 p1 of concentrated Formalin(35%). In pilot experiments this concentration was

found to be sufficient to stop uptake. Portions of thefixed samples were filtered by applying low vacuum

(0.2 kPa/cm2) through 0.2-,um Nuclepore polycarbon-ate membranes (shiny side up; filters prestained in 2g of Irgalan Black per liter in 2% acetic acid; Watson,personal communication) and rinsed 10 times with a

total of 100 ml of filter-sterilized 1% sodium chloridesolution. The filters were then dried and cut intoquarters. Chromic acid-washed glass slides were

dipped into filtered, prewarmed (4500) subbing solu-tion (2.5 g of gelatin, 0.025 g of chrome alum, 50 ml ofdouble-distilled water). After draining off the excessgelatin, the gelatin was wiped off the back side, andthe filter quarters were fixed on the coated slides.Prior to drying, the slides were placed horizontally on

a cold metal tray to solidify the gelatin.Autoradiographic preparations. Kodak NTB-2

nuclear track emulsion (diluted to a concentration of1:3 with filtered double-distilled water, stored at 400)

was heated to 430C in a constant-temperature waterbath for 45 min. The emulsion was gently poured into

a dipping jar (beaker, 20-ml content) and checked fora uniform emulsion free of bubbles (14). Slides were

immersed in the emulsion in a reproducible manner.

The excess emulsion was allowed to drain off, and theback of the slides was carefully wiped clean. The slideswere then placed on a cold metal tray for 20 min to gelthe emulsion, after which the slides were transferredto a dry tray for a further 1 h. The slides were thenmounted on a rack, dried overnight at room tempera-ture in the presence of dried silica gel, placed in blackplastic boxes (containing silica gel), sealed, and ex-

posed at 70C for 3 days. The time at which the boxeswere sealed was regarded as zero time. Each dippingprocedure included a slide with a blank filter throughwhich double-distilled water instead of the sample wasfiltered as a control for the quality of the emulsion.All manipulations involving NTB-2 emulsion wereperformed in total darkness.Processing the autoradiographs. The slides

were developed for 1 min in Kodak D-19 developerdiluted to a final concentration of 1:3 with double-distilled water, fixed for 4 min in 30% sodium thiosul-fate, washed in tap water, and air dried. All solutionswere held at 230C (5).

Staining. Dried slides were soaked for 5 min incitrate buffer (pH 6.6) and stained through the devel-oped and fixed emulsion for 20 min with acridineorange (Merck no. 1333; concentration, 1:2,500 in cit-rate buffer, pH 6.6). The slides were destained byimmersing in citrate buffer of gradually decreasing pHvalues (6.6, 5,4) and rinsed with double-distilled water.The destaining procedure must be controlled visuallywith regard to a uniform destaining.

Microscopic analysis. The dried slides were

viewed with a drop of Cargille immersion oil (type A)by epifluorescence microscopy (Zeiss Universal micro-scope; BG 12, X2; FL 500; BF 50; epifluorescencecondensor III RS; Osram HBO 200). Microphoto-graphs were taken with a Zeiss CS-matic camera, usingAgfapan Professional 100 film (ASA 100; exposure

TABLE 1. Microbiological variables measured in water samples taken above sandy sediments at beaches ofthe Kiel Fjord and the Kiel Bight (4 to 13 July 1977)0Colony- Total no. of Total bio- No. of active Active bac- Actual uptakeforming bacteria/ml x mass bacteria/ml x teria (% of rate of glucose

Station unmts/mi x 105 (direct (mg/ml, x 105 (autora- total no. of (pg/ml per h,

CoUnt() counts) 10-5) diography) bacteria) x 10-3)

A Hindenburgufer 261 41.7 50.2 19.8 47.5 18.9A' Monkeberg 188 57.8 65.4 32.5 56.2 23.5B Falckenstein 81.4 26.5 32.1 11.0 41.5 11.9B' Moltenort 187 64.2 62.4 29.9 46.6 10.3C Strande 433 68.8 74.2 3.1 4.5 2.6C' Laboe 98.2 67.2 70.9 23.8 35.4 6.6D D.-Nienhof 3.4 52.2 58.0 1.2 2.3 1.2D' Stein 3.1 51.4 40.9 6.1 11.9 7.8E Surendorf 5.5 39.4 43.6 16.6 42.1 6.9E' Heidkate 2.9 65.8 60.1 19.2 29.2 4.8F Krusendorf 7.4 56.1 60.8 28.3 50.5 3.8F' Schonberg 1.7 50.0 44.7 3.9 7.8 5.2

a A to F mark stations at the west side; A' to F' are corresponding stations at the east side of the Kiel Fjordand the Kiel Bight, respectively. Stations A/A' are located at the inner part, B/B' at the center part, and C/C'at the outer part of the Kiel Fjord. Stations D/D', E/E', and F/F' are located at the Kiel Bight.

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508 MEYER-REIL

time, 45 s). The microscopic image displays a greenish-to-grayish background with orange or green fluores-cent cells and dark-silver grains, both in similar focalplanes. Only bodies with distinct fluorescence, clearoutline, and recognizable bacterial shape were re-garded as bacterial cells. The question of lysis of cellsduring filtration cannot be answered as yet. Fluores-cent cell debris (obviously cell envelopes) were recog-nized very seldom. However, in some samples with arelatively high number of active cells, silver-grain ag-gregations of bacterial shape and size were observed,which might be a visible expression of cell lysis. Ac-tually metabolizing cells are characterized by silvergrains above or attached to the cells. Even if the cellsare heavily coated with silver grains, the bright-orangefluorescence remains visible. No attempt was made tocount single silver grains. At least 400 cells werecounted, chosen at approximately even intervals be-tween the periphery and the center of the filter. Theanalysis comprises the total number of cells and totalbiomass (8) and the number of active cells and back-ground silver grains on duplicate filters and on thecontrols. For the final analysis, the number of "active"cells in the controls (normally less than 1% of the totalnumber of cells) was subtracted from the number ofactive cells in the samples. A standard error of 11.8%was calculated for counting eight parallel filters. Gen-erally, the total number of cells counted on the auto-radiography/epifluorescence microscopy preparationswas in agreement with the total number of cellscounted on Nuclepore membranes prepared for thenormal enumeration procedure (10). The detectionand differentiation of active cells is limited by theproblems of fluorescence microscopy in general. Atcertain seasons the differentiation between bacterialand algal cells may cause problems, which may besignificant if transfer studies of isotopically labeledsubstrate between trophic levels are desired. Otherlimitations are the differentiation between bacteriaand detritus and the subjectivity in the counting pro-cedure.

RESULTS AND DISCUSSIONFilter preparation. Although different glues

were tried to fix the filter quarters onto the glassslides, only gelatin gave satisfactory results. Thedisadvantages with other glues were either aninherent background fluorescence (glues withsilicone basis) or an indistinct microscopic image(nail polish), obviously due to differences in therefraction index caused by the layer of nail polishbetween the focal plane and the objective. Byusing Ulrich adhesive (2), yellow-orange fluores-cent spots could be observed, which might beaggregations of silicate. Different filter systemswere tried, but only Nuclepore polycarbonatemembranes were acceptable. Cellulose estermembranes with their spongy structure trapbacterial cells in all filter layers. The associationbetween bacteria and silver grains became diffi-cult, because the inherent background fluores-cence interfered mainly with the recognition of

APPL. ENVIRON. MICROBIOL.

smaller cells. Thorough destaining of the filterbackground resulted in destaining of smallercells. It should be mentioned that difficultieswere observed with the distribution of cells onthe filter surface. To our knowledge, the manu-facturing procedure of Nuclepore filters hadbeen changed several times. This resulted inuneven wettable batches of filters rolling upafter fitration. It is therefore recommended tocheck the filters before use according to evenwettability.Staining procedure. The stain can be intro-

duced before applying the emulsion (prestain-ing) or after processing the emulsion (poststain-ing). In prestaining possible loss and/or trans-location of radioactivity are two of the majorproblems that must be considered (14). Cellsprestained with acridine orange lost their fluo-rescence during the subsequent procedures, al-though the background fluorescence was consid-erably reduced. The loss of fluorescence ismainly due to the removal of acridine orangefrom the cells by gelatin, which could be dem-onstrated by coating prestained filters with gel-atin. Staining through the undeveloped and un-fixed emulsion after exposure worked well as faras the fluorescence of the cells and the back-ground was concerned, but one should be awareof possible interactions between the undevel-oped emulsion and the staining solutions. Be-cause of the limitations mentioned above, post-staining is preferable. To obtain optimal results,different buffer systems, acridine orange concen-trations, and destaining techniques were tried.Generally, the application of black Nucleporemembranes (prestained in Irgalan Black; seeMaterials and Methods) is recommended. Afterexposure, photographic processing, and stainingwith acridine orange, untreated filters displayeda cloudy light-green background, whichdarkened only after prolonged illumination, thusleading to a fading of the fluorescence of thecells. However, filters prestained with IrgalanBlack should be thoroughly washed in double-distilled water prior to filtration to avoid un-bound residues of stain on the filter surface.Staining of the autoradiographs with acridineorange dissolved in phosphate buffer (pH 6.6, 5,or 4) was not suitable because of the dark-red-dish filter background, which interfered with thefluorescence of the cells. Acridine orange dis-solved in double-distilled water gave similar un-satisfactory results.A significant improvement was obtained when

autoradiographs were stained with acridine or-ange dissolved in citrate buffer (pH 6.6). Bright-orange fluorescent cells and dark-silver grainsare easily detectable against a greenish back-

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AUTORADIOGRAPHY/EPIFLUORESCENCE MICROSCOPY 509

ground, which darkens to gray with time. Stain-ing at lower pH values (5, 4, or 3) led to a light-greenish background which interfered with thefluorescence of the cells, decreasing with de-creasing pH values. Prior to staining, the slideswere immersed for 5 min in citrate buffer (6.6),which seemed to make the staining of the cellsless variable. Optimal results were obtained bypreparing a 1:2,500 solution of acridine orange incitrate buffer. Lower concentrations (1:5,000;1:10,000) were sufficient to stain the cells butresulted in a more rapid fading. Higher concen-trations of acridine orange (up to 1:1,000) causeda grossly overstained background, whichdarkened only after prolonged illmination, thusleading to a fading of the fluorescence of thecells. If a sufficient concentration of acridineorange is used, the period of staining (between10 and 20 min) does not seem to influence thequality of the microscopic image to any degree.Treatment ofthe specimen with citrate buffer ofdecreasing pH values (6.6, 5, 4) washes out theexcess of stain from the background withoutinfluencing the fluorescence of the cells. Subse-quent rinsing of the slides with double-distilledwater seems to increase the contrast betweenthe fluorescence of the cells and the background.Generally, a combination of overstaining on theslightly alkaline side and gradual destaining onthe acid side of neutrality is recommended.Other fluorescent dyes (such as fluorescein iso-thiocyanate) were not applied to the autoradi-ographic technique. From former experiments,acridine orange was found to be superior tofluorescein isothiocyanate because ofits brighterfluorescence, which is necessary for analyzingautoradiographs.

Standardization. The method requires thestandardization of both substrate uptake (influ-ence of incubation time and substrate concentra-tion) and exposure of the autoradiographs. Stan-dard curves were repeated at least once withsimilar results. Because of the relatively shortincubation periods (see below) and the low de-composition rate of [3H]glucose (radiochemicalspecifications; New England Nuclear), the prob-lem of possible exchange of tritium can be re-garded as not significant.

In activity studies short incubation periodsrequire the addition of relatively high substrateconcentrations, whereas lower substrate concen-trations can be applied in combination with ex-tended incubation periods. However, high sub-strate concentrations might increase the naturalconcentration considerably, possibly leading tothe induction of transport systems. With ex-tended incubation periods, the "natural" envi-ronment enclosed in the incubation bottles will

change rapidly. The effect of different substrateconcentrations and incubation periods on thepercentage of labeled bacteria is demonstratedin Fig. 1. Autoradiographs were exposed for 3days at 7°C. By applying high substrate concen-trations (5 and 10 lCi/ml of sample; correspond-ing to 24 and 48 jig of C per liter, respectively),cells were maximally labeled within 2 to 4 h,whereas at 1 ,uCi/ml (corresponding to 4.8 ytg ofC per liter) 4 to 6 h of incubation was required.However, incubation at high substrate concen-tration causes an increase in the number ofbackground silver grains.The effect of different substrate concentra-

tions and exposure periods on the percentage oflabeled cells is shown in Fig. 2. The water samplewas incubated with 1, 5, and 10 ,uCi/ml for 4 h.Parallel filters were exposed to the radiation fordifferent periods (0 to 3 days). Although highsubstrate concentrations enhance early labelingof the cells, the maximum percentage of labeledcells obtained after 2 to 3 days of exposure issirnilar for the different substrate concentrationsadded. However, the number of background sil-ver grains increases significantly with high sub-strate concentration (10 ,uCi/ml).Most autoradiographs were exposed at 4°C (4,

6, 13, 14) or even at room temperature (2, 5).Brock and Brock (2) mentioned the use of scin-tillator fluid to detect relatively weak amountsof radioactivity after short incubation periods.From the work of Durie and Salmon (3) it be-comes obvious that use of a scintillator andexposure at low temperature (-85°C) greatlyenhance early labeling of the cells. Based on thisinformation, different filter treatments and ex-posure temperatures were tried (Fig. 3). A watersample was incubated with 5 ,uCi/ml for 4 h.Parallel filters were treated after coating with

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FIG. 1. Standardization ofautoradiographic tech-nique. Effect of different ['Hlglucose concentrationsand incubation periods on percentage of labeled bac-teria (above) and on background level (below). Auto-radiographs were exposed for 3 days at 7°C.

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FIG. 2. Standardization ofautoradiographic tech-nique. Effect of different [3H]glucose concentrationsand exposure periods on percentage of labeled bac-teria (above) and on background level (below). Thewater sample was incubated with different [3H]-glucose concentrations for 4 h.

emulsion in the dark with and without scintil-lator (2) and exposed at 7 and -25°C, respec-tively. By applying the scintillator, the fluores-cence of the background remains unchanged. Nostimulatory effect of either temperature or scin-tillator on the percentage of labeled bacteriacould be observed. However, the lower temper-ature significantly reduced the number of back-ground silver grains. Since treatment of the fil-ters with scintillator introduces another step intothe procedure, the exposure of untreated filtersis preferable.From the standardization curves, a 2- to 4-h

incubation period with 1 to 5,uCi of [3H]glucoseper ml of sample added can be recommended,after which the autoradiographs can be exposedfor 3 days at 7°C.Application to ecological studies. During

a joint research program, the method was ap-plied to water samples taken immediately abovesandy sediments at beaches of the Kiel Fjordand the Kiel Bight (Table 1). Depending uponlocation, between 2.3 and 56.2% of the totalnumber of cells were actually metabolizing (cellswith associated silver grains) with regard to [3H]-glucose uptake (absolute values: between 1.2 x105 and 32.5 x 105 cells per ml). The averagepercentage of active cells (31%) agrees with the29% [14C]glucose active cells reported by Hoppe(6). Generally, the number of active cells de-creased from the inner part to the outer part ofthe Kiel Fjord (stations A/A' to D/D'). At thosestations located at the Kiel Bight (stations E/E'and F/F'), the number of active cells generallyincreased (Table 1). This might be dependenton differences in the bacterial populations andtheir substrate preference.

For the purpose of this paper, the relationshipbetween the number of actually metabolizingcells and a few "key" variables will be discussedvery briefly. A detailed report on the interrela-tionships between all of the variables measuredis scheduled to appear separately (L.-A. Meyer-Reil and M. Bolter, manuscript in preparation).The number of colony-forming units varied bya factor of 200, with highest values in the innerpart of the Kiel Fjord. In contrast, total bacterialnumber and biomass showed only small varia-tions (maximally by a factor of 2). Parallel to thevariations in the number of actually metaboliz-ing cells, the actual uptake rate of glucose (de-termined according to a method described byMeyer-Reil [9]) varied by a factor of 20. Again,high values were found in the inner part andlower values were found towards the outer partof the Kiel Fjord.Spearman rank correlation analysis (15) re-

vealed significant correlations between the bac-terial biomass and bacterial numbers and col-ony-forming units (Table 2). These interrela-tionships could be expected from previous inves-tigations (1, 10). For the evaluation of themethod described, the significant correlation be-tween the number of actually metabolizing cellsand the actual uptake rate of glucose should bepointed out. This means that the total amountof glucose taken up by an unknown number ofcells (tracer technique) can be related directly to

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FIG. 3. Standardization ofautoradiographic tech-nique. Effect of different exposure temperatures (7°C,-25'C) and autoradiograph treatments (+, with scin-tillator fluid; -, without scintillator fluid) on per-centage of labeled bacteria (above) and on back-ground level (below). The water sample was incu-bated with 5 ILCi of [3H]glucose per ml for 4 h.

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AUTORADIOGRAPHY/EPIFLUORESCENCE MICROSCOPY 511

the number of actually metabolizing individualcells (autoradiography/epifluorescence micros-copy technique). The absence of a significantcorrelation between standing crop variables (col-ony-forming units, total number of cells andbiomass) and activity variables (actual uptakerate of glucose, number of actually metabolizingcells) in this study is not surprising, since bac-terial standing crop does not necessarily reflectmetabolic activity (10).

Figure 4 presents photomicrographs of theautoradiographs obtained. Unfortunately, thephotomicrographs presented in black and whitedo not reflect the quality and the informationcontent of the colored microscopic images show-ing bright-orange fluorescent cells and dark-sil-ver grains. From the microscopic observations it

TABLE 2. Spearman rank correlation matrix formicrobiological variables measured in water

samples taken above sandy sediments at beaches ofthe Kiel Fjord and the Kiel Bight'

Parameter CFU C B AC AUp

CFU 1.00 0.29 0.55b 0.38 0.36C 1.00 0.90c 0.21 -0.39B 1.00 0.37 -0.25AC 1.00 0.56"AUp 1.00

" CFU, Colony-forming units; C, number of bacteria;B, bacterial biomass; AC, number of active bacteria;AUp, actual uptake rate of glucose.

b Significant at the 0.05 leavel (rank correlation).'Significant at the 0.001 level (rank correlation).

becomes obvious that uptake of [3H]glucose isspread over the total spectrum of the naturalbacterial population. Rods and curved cells, aswell as small cocci (0.2 to 0.4 ,pm in diameter),take part in the uptake of [3H]glucose. Filamen-tous cells, which are typical for samples taken atthose locations in early summer (8), accountedfor a maximum of 20% of the total number ofcells. In comparison with nonfilamentous cells,filamentous cells were heavily coated with silvergrains (Fig. 4). Therefore, the filamentous cellscan be expected to account for much higherpercentages of the total actual uptake rates thanthe nonfilamentous cells. Generally, the numberof background silver grains was significantlyhigher in the samples than in the controls. Thismeans that bacterial activity causes an increasein the number of background silver grains, pos-sibly due to the release of highly labeled sub-stances into the water.The method described enables the determi-

nation of standing crop variables (bacterial num-ber, biomass) and the spectrum of actually me-tabolizing cells on the same autoradiographic/epifluorescence preparation. Since activity canbe related directly to individual cells, themethod is a useful tool for other studies inaddition to ecological studies.

ACKNOWLEDGMENTSThe initial development of the technique described in this

paper was developed in the laboratory of R. Y. Morita, OregonState University, Corvallis. I greatly appreciate the comments

FIG. 4. Combined technique of autoradiography and epifluorescence microscopy. The photomicrographsshow the spectrum of unlabeled and labeled (with associated silver grains) bacterial cells ofa natural watersample incubated with 5 gCi of [3HJglucose per ml for 4 h. In comparison with nonfilamentous cells (upperleft), filamentous cells (lower left, lower right) are heavily coated with silver grains. The control (fixed withFormalin before labeling) displays only unlabeled cells (upper right). Bar = 2 mn.

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of R. Y. Morita on the manuscript and thank W. Schmidt forvaluable technical assistance.

LITERATURE CITED1. Bolter, M., L.-A. Meyer-Reil, and B. Probst. 1977.

Comparative analysis of data measured in the brackishwater of the Kiel Fjord and the Kiel Bight, p. 250-280.In G. Rheinheimer (ed.), Microbial ecology ofa brackishwater environment. Springer Verlag, Berlin.

2. Brock, M. L., and T. D. Brock. 1968. The application ofmicro-autoradiographic techniques to ecological stud-ies. Mitt. Int. Ver. Theor. Angew. Limnol. 15:1-29.

3. Durie, B. G. M., and S. E. Salmon. 1975. High speedscintillation autoradiography. Science 190:1093-1095.

4. Faust, M. A., and D. L. Correll. 1977. Autoradiographicstudy to detect metabolically active phytoplankton andbacteria in the Rhode River estuary. Mar. Biol.41:293-305.

5. Fliermans, C. B., and E. L. Schmidt. 1975. Autoradiog-raphy and immunofluorescence combined for autecol-ogical study of single cell activity with Nitrobacter as amodel system. Appl. Microbiol. 30:676-684.

6. Hoppe, H.-G. 1976. Determination and properties of ac-tively metabolizing heterotrophic bacteria in the sea,investigated by means of micro-autoradiography. Mar.Biol. 36:291-302.

7. Knoechel, R., and J. Kalff. 1976. The applicability ofgrain density autoradiography to the quantitative de-

termination of algal species production: a critique. Lim-nol. Oceanogr. 21:583-590.

8. Meyer-Reil, L.-A. 1977. Bacterial growth rates and bio-mass production, p. 223-236. In G. Rheinheimer (ed.),Microbial ecology of a brackish water environment.Springer-Verlag, Berlin.

9. Meyer-Reil, L.-A. 1978. Uptake of glucose by bacteria inthe sediment. Mar. Biol. 44:293-298.

10. Meyer-Reil, L.-A., R. Dawson, G. Liebezeit, and H.Tiedge. 1978. Fluctuations and interactions of bacterialactivity in sandy sediments and overlying waters. Mar.Biol., in press.

11. Paerl, H. W. 1974. Bacterial uptake of dissolved organicmatter in relation to detrital aggregation in marine andfreshwater systems. Limnol. Oceanogr. 19:966-972.

12. Peroni, C., and 0. Lavarello. 1975. Microbial activitiesas a function of water depth in the Ligurian Sea: anautoradiographic study. Mar. Biol. 30:37-50.

13. Ramsay, A. J. 1974. The use of autoradiography todetermine the proportion of bacteria metabolizing in anaquatic habitat. J. Gen. Microbiol. 80:363-373.

14. Rogers, A. W. 1973. Techniques for autoradiography.Elsevier Scientific Publishing Co., New York.

15. Sachs, L. 1974. Angewandte Statistik, p. 309-311. Sprin-ger Verlag, Berlin.

16. Zimmermann, R., and L.-A. Meyer-Reil. 1974. A newmethod for fluorescence staining of bacterial popula-tions on membrane filters. Kiel. Meeresforsch.30:24-27.

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/jour

nal/a

em o

n 25

Feb

ruar

y 20

22 b

y 18

7.10

2.16

.198

.