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Optimization of media for enumeration and isolation of aerobic heterotrophic bacteria from the deep terrestrial subsurface
ELSEVIER
Journal of Microbiological Methods 21 (1995) 293-303
Optimization of media for enumeration and isolation of aerobic heterotrophic bacteria from the deep terrestrial subsurface T.O. Pacific Northwest Laboratory,
Stevens*
P.O. Box 999. m/s P7-54, Richland, WA 99352, USA
Received 7 February 1994; revised and accepted 20 June 1994
Abstract The objective of this study was to develop an agar plate-count medium to maximize the number of colony-forming units (CFU) recovered from terrestrial subsurface sediment samples. A Plackett-Burman experimental design was used to test the effects of 13 possible medium variables, including various ingredients and culture conditions. These experiments used sediment samples from different surface and subsurface areas to test the resulting formulations. Microorganisms in different sediments did not respond uniformly to any variable. Variables that always had a positive effect, when significant, included addition of vitamins, addition of activated charcoal (added as a scavenger of toxic oxygen forms), use of pour plates rather than spread plates, and elevated, rather than in situ, incubation temperature. Lowered water potential always led to a negative response, where significant. Varying concentrations of most common medium components, including dilution of carbon sources, had few significant effects over the ranges tested. A medium formulation for enhanced recovery of aerobic heterotrophic bacteria from subsurface sediments was developed, based on these results. Keywords:
Aquifer;
Bacteria;
Enumeration;
Medium; Subsurface; Vadose
1. Introduction The distribution and abundance of microorganisms in the terrestrial subsurface is a fundamental scientific issue that is being addressed as part of the US Department of Energy’s (DOE) Subsurface Science Program [e.g., l-51. The *Corresponding
author.
[email protected]. Elsevier Science B.V. SSDI 0167-7012(94)00056-5
Tel.:
+ 1 (509) 373-0891. Fax:
+ 1 (509) 376-9650. Internet:
to_
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standard plate-count assay plays an important role in these and other such studies; however, most agar media have been developed for other purposes. It is an axiom of microbiology that all culture media are selective for subsets of the total bacterial community; therefore, development of a variety of culture media is needed to maximize the recovery of diverse microorganisms from terrestrial subsurface samples. This study describes development of an agar plate-count medium for isolation and enumeration of aerobic heterotrophic bacteria from the deep terrestrial subsurface. Many well-characterized heterotrophic microorganisms can regulate their intracellular environment to allow for growth under various nutrient regimes. However, the range of conditions that allow for growth of subsurface microorganisms is not well known. Terrestrial subsurface ecosystems appear to contain the most oligotrophic microbial communities known [6], and it is likely that specialized conditions may be required to recover these organisms from core samples and culture them under laboratory conditions. In studies conducted in eastern coastal plain sediments, numbers of organisms recovered on 1% PTYG [7] agar plates were remarkably similar to numbers observed by direct microscopic counts [1,4]. Microaerophilic bacteria were common in some of these sediments [8]. In more recent borehole explorations at western sites, however, the proportion of microorganisms growing on agar plates to those observed by direct counts was in the range observed for surface organisms (l-10% or less) [2,5]. For many geological formations, few if any microorganisms could be recovered on agar plates, even when microbial respiration could be detected in the same bacteria were samples (J.K. Fredrickson et al., this issue). Microaerophilic detected in several of these low-recovery cores, and isolates from microaerophile enrichments often grew as normal aerobic heterotrophs in subsequent culture transfers. This suggested that specialized conditions might be required for recovery of some subsurface organisms from their in situ starved state to laboratory growth conditions. The objective of this study was to develop a heterotrophic plate-count medium that would maximize the number of CFU detected in subsurface samples. Because of the difficulty and expense of obtaining subsurface core samples, these experiments mostly used unsaturated paleosol and lacustrine sediment samples excavated from a recently exposed eroded sedimentary cliff traversing the same geological formation as nearby subsurface boreholes. The resulting medium formulation was tested on subsequent cores recovered from the deep subsurface. While a universal medium is not likely to be attainable, this optimized plate-count agar (OPCA) is an efficacious general medium for initial isolation and enumeration of aerobic heterotrophic bacteria from subsurface sediments.
2. Materials and methods 2.1. Soil and sediment
samples
Samples of unsaturated
paleosols and lacustrine sediments were obtained from
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the Ringold Formation in S.E. Washington. These sediments, ranging from 2 to 14 million years old, were excavated from the Savage Island Landslide (SIL) in S.E. Washington at latitude N 46”, 34’, longitude W 119”, 17’ [9]. The sediments were recently exposed by a landslide due to undermining of a steep bluff by irrigation water flow. Little or no water is believed to have flowed through this formation during the last 10,000 years, so recent bacterial transport is unlikely. These sediments should be good analogs for subsurface sediments, and bacterial habitats within them should be similar to those encountered in deeply buried formations. Individual samples are described in Table 1. The exposed face of the bluff was excavated back approximately 0.5 m, then samples were removed from the resulting fresh face with sterile tools and placed in sterile plastic bags. Samples were transported to the laboratory in less than one hour and stored at 4°C until use. One sample (YB 1335) was obtained from a depth of 103.5 m from a subsurface borehole and was classified as a paleosol. A well water sample (INEL W,) was used from a well located at the Idaho National Engineering Laboratory [3], at latitude N 43”, 30’, longitude W 112*, 48’, which was transported to the lab and used within 24 h. Another sample (INEL C) consisted of sand from a sand filter [lo] that had entrained microorganisms from this same well. Surface soil samples were loamy sands sampled near Richland, WA, and Idaho Falls, ID. 2.2. Mineral salts medium A mineral salts medium was used as the base for all experimental media in this study, and contained ingredients which were not varied. The mineral salts solution consisted of (per liter): KH,PO,, 0.25 g; K,HPO,, 0.35 g; CaCl,‘2H,O, 0.015 g; MgCl, * 6H,O, 0.02 g; FeSO, - 7H,O, 0.007 g; Na,SO,, 0.005 g. The first two ingredients and the remaining ingredients were prepared as two separate 100 X stock solutions. Table 1 Description of samples used in the medium optimization study and relative bacterial concentrations; numbers of bacteria are reported as both the mean number detected in all medium formulations and the maximum plate-count detected Sample
Description
Source
Bacteria, log cfu .
g-’ soil
SIL 1 SIL 2 SIL 3 SIL 6 SIL 7 YB 1335 Hanf S INEL S INEL W INEL C
Savage Island Landslide Savage Island Landslide Savage Island Landslide Savage Island Landslide Savage Island Landslide subsurface borehole Richard, WA surface Idaho Falls, ID surface Idaho Falls, ID water well Idaho Falls, ID water well
a For the groundwater
lacustrine sediment paleosol paleosol banded lacustrine sediment sandlpaleosol paleosol103.5 m deep loamy sand loamy sand groundwater sample sand filter sample
sample, values are cfu ’ III-‘.
mean
max.
3.90 2.94 2.31 4.12 2.90 6.50 6.93 6.46 3.88 5.06
4.59 3.52 3.30 4.68 3.48 7.10 7.68 6.91 4.65” 5.69
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2.3. Identification of significant variables
The first phase of the study consisted of a 16-variable Plackett-Burman experiment [ 11,121 conducted to identify culture-condition variables with a significant effect on microbial recovery. In this design, a ‘high’ and a ‘low’ value are chosen for each variable. Within the experimental matrix, the effect of alternating between these variables is tested eight times. Experimental variation is tested by including a set of dummy variables, for which no actual alteration is made in the formula. This allows screening of a large number of variables for significant effects without the enormous expense of performing a full factorial matrix. The principal disadvantage to this method is that two-factor effects are confounded and cannot be measured in this experiment. Variables tested in this study are listed in Table 2 with their ‘high’ and ‘low’ settings and a brief rationale for their selection. Several of these variables are standard ingredients in microbiological media. Previous investigations have indicated that lower concentrations of nutrients may aid in recovery of starved environmental isolates [e.g., 7,131. They were varied individually here to determine whether lower concentrations would be effective. The ‘complex nutrients’ solution contained, per ml: 0.05 g proteose peptone, 0.05 g tryptone, and 0.1 g yeast extract. The fatty acid solution contained equimolar concentrations (Table 2) of pyruvic acid, acetic acid, and lactic acid, and was included to test whether subsurface microorganisms might be adapted to substrates typical of a ‘lower’ trophic level (i.e. fermentation products) [14]. The vitamin solution [15] contained, per 100 ml: biotin, 0.002 g; folicin, 0.002 g; B6 (HCl), 0.01 g; riboflavin, 0.01 g; thiamine (HCl), 0.01 g; pantothenic acid, 0.005 g; nicotinamide, 0.005 g; B12, 0.01 g; PABA, 0.005 g; lipoic acid, 0.006 g.
Table 2 Medium components
used as variables in the medium optimization study
Component varied
‘High’ setting
Complex concentration Glucose concentration Fatty acid concentration Vitamin solution Ammonium chloride Glutamate Activated charcoal PH Water potential Temperature Pour plate vs. spread plate Trace minerals Dummy variable Dummy variable Dummv variable
2g.l.’ lg.l_I 1mM lmlg.llg.l_’ lg.1 ’ lg.1 ’
NA. not available
6.5 a =0.96 26°C spread 1 ml.I-’ NA NA NA
‘Low’ setting
Brief rationale
0.2g.I-’
effect of complex nutrients a typical sugar substrate a different trophic level of substrate cofactors for biosynthesis inorganic nitrogen source organic nitrogen source relieve O2 toxicity in agar plates optima may vary possible adaptation to desiccation in situ vs. typical laboratory different diffusional environments cofactors for various enzymes
0.1 g.1-’ 100 /.LM not added o.lg.I-’ 0.lg.l I not added 1.5 a =l.O 3&c pour not added NA NA NA
estimates estimates estimates
variance variance variance
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This solution was filter sterilized and frozen in l-ml aliquots until use, when 1 ml was added, after autoclaving, per liter of medium. Activated charcoal was selected as the most convenient protective additive against oxygen toxicity for microaerophilic bacteria [16]. Aliquots (1 g) were autoclaved separately three times for 30 min with 24 h at room temperature between. If added before autoclaving, the charcoal appeared to protect contaminant bacteria from sterilization. One pre-sterilized ahquot was added to media after autoclaving, and with vigorous stirring. The effect of varying osmotic water potential was tested because bacteria in the vadose zone might be expected to experience dehydration. Shock from sudden rehydration might result in loss of viability. For the ‘high’ settings of a, = 0.97, water potential was adjusted by adding 160 ml of glycerol per liter of medium. For the ‘low’ settings, no osmolyte addition was made, resulting in a, = - 1.0. A standard solution of trace minerals was used [17] containing, per 100 ml: MnCl, - 4H,O, 0.5 g; H,BO,, 0.05 g; ZnCl,, 0.05 g; CoCl, - 6H,O, 0.05 g; NiSO, - 6H,O, 0.05 g; CuCl, .2H,O, 0.03 g; NaMoO, - 2H,O, 0.01 g. When used, 1 ml of this solution was added per liter of medium. Three dummy variables were added to the matrix in order to determine experimental variability. No amendment was made to the medium for these variables. To remove experimental bias, each of the 15 variables was assigned a letter identifier using a random-number generator before constructing the experimental matrix (Table 3). A second random number was assigned to each medium recipe to determine the medium ID number and the order of preparation. All media were prepared a second time, and the experiment repeated after re-randomizing the experimental matrix. Table 3 Randomized Plackett-Burman design for medium optimization experiments. for variable, - indicates low setting) Variables
Randomized identity
Randomized medium ID
Variable settings ABCDEFGHI
1
Dummy Complex NH,CI Temperature Plate type H,Opoten Glutamate PH Vitamins Fatty acids Trace element Dummy Charcoal Glucose
B C J M L H G K A E 0 I N F
2 3 4 5 6 7 8 9 10 11 12 13 15 16
(+ indicates high setting
-
-
-++++-+-++--+
-++++-+-++--+--++++-+-++--+- + -++--+---++++ +---++++-+-++ ++++-+-++--+--__-__---------+-+-++--+---+++ ++--+---++++-•+++-+-++--+---++ -++--+---++++-+ +-++--+---++++-+---++++-+-+++--+---++++-+-+ +++-+-++--+---+
JKLMNO
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For each sediment sample, 10 g was added to 100 ml 0.1% sodium pyrophosphate, pH 7, and shaken vigorously for 20 min. Two further decimal dilutions were made in this buffer. Three replicate subsamples from each dilution were plated by the indicated method for each of the 16 medium combinations. Plates were incubated at the indicated temperatures and colonies counted after 2, 6, and 14 days. During preliminary experiments, plates were incubated for more than 45 days. No additional colonies appeared after about 10 days, so the 14-day counts were used for subsequent analyses. 2.4. Statistical melhoak The Plackett-Burman design for 15 variables was obtained from reference [12]. In this method, the effect of each variable was determined by computing the mean plate-count number (not log-transformed) of all media for which the variable was set ‘high’ and subtracting the mean plate-count number of all media for which the variable was set ‘low.’ Standard error was determined by computing the square root of the mean sum of squares of the effects of the dummy variables. A Student’s t-test was evaluated by dividing the effect of each variable by the standard error and comparing this value to a two-sided t table. Effects with values of p = 0.1 or less were assumed to be significant.
3. Results Results of the Placket-Burman experiment are shown in Table 4. There was considerable variation among the different samples tested. Variation of any one ingredient did not result in dramatic response of plate-count numbers for all samples tested, and most variations resulted in no significant change. Several ingredients, however, did have consistently positive effects for all samples in which their effects were significant. Addition of vitamins and activated charcoal had a significant positive effect in four and five of the ten samples, respectively. Their effects were never negative, except that sample SIL3 had an insignificant negative response to vitamins. Activated charcoal had the largest effects, ranging from 180% to 3370% increase in plate-count numbers. Elevated incubation temperature (coded as the ‘low’ variable value in Tables 2 and 4) had a positive effect for all subsurface samples, and never had a significant negative effect. Several variables had consistent patterns of effects for vadose-zone samples, but the opposite effect for the saturated sand filter sample. Using a pour-plate technique, rather than a spread-plate technique, had a significant positive effect for four of eight vadose zone samples, but a negative effect for the sand filter sample. Addition of trace elements, high glucose concentration, and elevated pH had positive effects for two or three vadose-zone samples but negative effects for the sand filter sample. Reducing the matric water potential of the medium had significant negative effects for five of ten samples and never had a significant positive effect.
0.01
227
5
147
140
220
144
102
161
226
SIL 2
SIL 3
SIL 6
SIl.7
YB 1335
Hanf S
INELS
INELW
1NELC
115
107
124
57
108
102
134
59
121
0.60
1.00
0.60
0.50
0.80
1.00
0.60
0.80
0.50
1.00
p
139
111
104
161
90
92
185
809
87
87
effect
0.20
1.00
1.w
0.60
0.80
agO
0.50
0.20
0.50
0.50
p
Complex
-
101
530
122
47
140
211
64
1646
101
62
effect
p
1.00
0.50
0.80
0.50
0.50
0.05
0.50
0.20
1.00
0.02
Dummy
-
121 0.50
66 0.80
100 1.00
210 0.50
84 0.60
134 0.50
202 0.50
11259 0.20
139 0.01
76 0.10
effect
p
Fatty acids ~
Glucose
44
175
83
129
95
217
125
177
154
2%
effect
~
0.05
0.80
0.80
0.80
1.w
0.05
0.80
0.80
0.05
0.01
p
169
179
1.03
53
120
1.51
122
83
68
87
effect
0.10
0.80
1.00
0.50
0.60
0.20
0.80
1.00
0.05
0.50
p
Glutamate -
Water
25
46
29
17
29
78
128
132
50
36
effect
-
potent p
69
0.02
0.01
0.60
0.05 101
10
57
280
71
0.50 0.20
63
73
105
113
0.80
0.80
0.01
0.01
effect
p
73
260
0.20
48
126
225
0.50
0.60
0.20
0.50
0.50
0.02
0.50
54
0.50 0.20
209
1.00
p 0.01
1523
120
48
effect
0.20
0.50
0.50
0.20
0.50
0.80
0.80
0.50
-
Ammonium
Dummy ___
pH
411
103
180
72
57
35
75
155
39
15
effect
-
0.01
1.00
0.20
0.80
0.20
1.00
0.60
0.80
0.01
0.01
p
164
25
102
57
87
3.5
18
14
73
15
effect
0.10
0.50
1.00
0.50
a.80
0.01
0.05
0.20
0.10
0.01
p
Plate type ~
122
27
41
210
72
59
71
67
79
101
effect
0.50
0.50
181
464
171
182 0.10
704 0.50
219
0.10 0.50
224
0.60
116
3365
0.20 0.80
298
1.00
p
0.05
0.50
0.50
0.50
0.01
0.02
0.20
1.00
0.01
0.01
54
78
70
117
139
110
260
8
222
190
effect
0.05
1.00
0.50
1.00
0.50
0.80
0.20
0.20
0.01
0.01
p
elements -
effect
_
p
Trace
Charcoal ~
twe
Tempera-
‘Effect’ is reported as mean plate-count numbers at ‘high’ settings divided by those at ‘low’ settings and multiplied by 100. Effects less than 100 are negative. while those greater than 100 are positive. p is reported as the probability that ‘high’ and ‘low’ means are not significantly different. Effects with values of p = 0.1 or less were assumed to be significant.
0.05
0.80
1.W
0.80
0.01
0.20
0.50
0.20
0.01
100
effect
effect
p
Dummy
-
Vitamins
-
229
SIL 1
Sample
Table 4 Results of medium optimization experiment for different samples
300
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Varying ammonium, glutamate, and fatty acid concentrations did not result in any trend and did not give consistent significant results. No variation of any variable had significant effects for surface samples or the groundwater sample.
4. Discussion This experiment studied the effect of varying concentrations of standard heterotrophic medium components, as well as addition of several special-purpose ingredients, on plate-count numbers of organisms from terrestrial surface and subsurface samples. The results indicate several adjustments that can be made to increase viable counts of subsurface bacteria, at least for the vadose zone, but that would probably have little effect for surface bacteria. High variability in two of the three dummy variables indicates that there may be two-factor effects that were neglected by this study. Alternatively, this variability could be due to differences in distribution of microorganisms within the different samples. Subsurface microorganisms tend to be distributed in clumped patterns, and estimating their numbers by plate-count methods is inherently more variable than for surface soils with similar numbers [21]. For instance, sample SIL 3 appears to be much more variable than other samples examined here. All significance levels, however, were calculated using standard variances derived from the dummy variables, so consistent responses to particular variables likely represent a real effect. Addition of activated charcoal had the greatest positive effect on subsurface plate-count numbers. Charcoal has been widely used as a protectant for microaerophilic bacteria [16]. It is assumed to trap superoxide and oxygen radical species that result from oxidation of complex medium components. Many samples from subsurface cores contain microaerophilic bacteria [8], although organisms isolated from microaerophilic bands often grow well on standard media after transfer from the primary enrichment (J.K. Fredrickson, et al., unpublished data). The above results suggest that microorganisms in a starved or dormant state in the subsurface may not maintain protective systems against toxic oxygen species, but do have protective mechanisms that are activated after recovery. The use of pour-plate techniques instead of spread-plate techniques [18] also resulted in greater numbers. The main difference between these techniques is probably restricted diffusional environments around nascent colonies submerged in the agar matrix of the pour plates. Pour plates may also provide protection against oxygen toxicity. Addition of a vitamin solution stimulated growth in most samples, which indicates that pools of these often complex cofactors may be depleted in subsurface microorganisms, which may or may not be capable of synthesizing these compounds. The consistent negative effect of reduced water activity indicates that, at least in the environments tested here, subsurface bacteria are not adapted to dessication, or high-solute niches which might arise by desiccation. Desiccation of a
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sediment system is not uniform across all microenvironments; rather, water is restricted to spaces with smaller pore sizes. This probably affects microorganisms more by reducing their habitat volume and restricting transport and nutrient diffusion than by actually desiccating their immediate environment [5]. Elevated temperatures resulted in greater numbers of microorganisms than the lower temperature that more closely simulated the in situ environment of the samples. This suggests that faster metabolic rates allowed by the higher temperature offer a greater advantage, during growth on agar medium, than any adaptation to low temperature the organisms might have experienced. Although a number of studies have shown that bacteria from low-nutrient environments are recovered better on dilute media than concentrated laboratory media [e.g., 7,13,18], the results of this study do not support that hypothesis. Dilution of complex nutrients or glucose had little effect on CFU. This may be because the ‘high’ levels of nutrients in this study were still relatively low compared to many standard formulations, including those cited above. Also, most previous studies of this nature have focused on aquatic or saturated environments, as opposed to the mainly vadose-zone environments studied here. Some oligotrophic bacteria grow best on medium containing only agar and its impurities as carbon sources, although they can generally be isolated on media containing a few hundred mg - 1-l carbon 1191, as used in these experiments. This study did not determine whether the same populations were counted under the ‘high’ and ‘low’ settings, only the gross numbers of CFU. Clearly, the list of variables tested was not exhaustive of all possible medium components. However, the components of other media commonly used for enumeration of subsurface bacteria [7,13] were included in the test matrix. It is likely that future investigations will identify additional refinements in recovery of microorganisms that were not tested here. Similarly, the samples used were not representative of all possible subsurface environments. and yet it should be noted that they were diverse enough that not all microbial populations responded to the variables in the same way. Still, several factors were identified which had consistently positive effects on bacterial recovery. Based on these results, the following OPCA medium formulation is suggested for recovery of subsurface aerobic heterotrophic bacteria. Add to 1 liter deionized water: KH,PO,, 0.25 g; K,HPO,, 0.35 g; CaCl, .2H,O, 0.015 g; MgCl, .6H,O, 0.02 g; FeSO, -7H,O, 0.007 g; Na,SO,, 0.005 g; NH4C1, 0.5 g; glucose, 1 g; peptone, 0.05 g; tryptone, 0.05 g; yeast extract, 0.1 g. Add 1 ml of the trace element solution described above, adjust pH to 7.5, and autoclave. After autoclaving, stir in 1 g finely ground pre-sterilized activated charcoal and 1 ml of the vitamin solution described above. Dispense fresh medium by standard pourplate techniques and incubate cultures at 30°C. A variation of this medium was later used to enumerate microorganisms in 32 cores retrieved from 172- to 223-m-deep saturated lacustrine sediments and paleosols from the deep subsurface (J.K. Fredrickson et al., unpublished data). The variations were that, for logistic reasons, spread plates were used instead of pour plates, and incubation temperatures were 17°C instead of 30°C. Despite
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these alterations, when compared with 1% PTYG [7] and B-4 [20], the modified OPCA formulation recovered bacteria from more samples than any other. Culturable aerobic heterotrophs were very rare in these samples, mostly less than 100 CFU . g-t, yet the formula above recovered bacteria in 13 of 26 samples, as compared to 10 of 29 for 1% PTYG and 8 of 28 for B-4. OPCA medium yielded more CFU than the other media in 10 of 24 direct comparisons, as compared to 3 of 24 for 1% PTYG and 1 of 24 for B-4.. Insufficient data were collected to determine whether recoverable numbers were significantly different for any of these experiments. This study has identified several factors that can have a positive effect on recovery of aerobic heterotrophic bacterial CFU from subsurface sediment samples when conducting a plate-count assay. Such an assay is commonly one of a suite of bacteriological tests used to characterize a subsurface environment. Because components of the medium suggested above were selected for their positive effect on plate-count numbers for a variety of samples, and performed well for both saturated and unsaturated sediment samples, OPCA should be an efficacious general medium for enumeration of subsurface microorganisms.
Acknowledgements
We thank Heath Watts and Jennifer Walker for technical assistance on this project, as well as Dr. Jay Grimes and Dr. Frank Wobber, whose assistance made this study possible. This research was supported by the Subsurface Science Program, Office of Health and Environmental Research, US Department of Energy (DOE). Pacific Northwest Laboratory is operated for the DOE by Battelle Memorial Institute under Contract DE-AC06-76RL0 1830.
References and morphological characteristics of aerobic, Balkwill, D.L. (1989) Numbers, diversity, chemoheterotrophic bacteria in deep subsurface sediments from a site in South Carolina. Geomicrobiol. J. 7, 33-52. [2] Brockman, F.J., Kieft, T.L., Fredrickson, J.K.. Bjornstad, B.N.. Li, SW., Spangenburg, W. and Long, P.E. (1992) Microbiology of vadose zone paleosols in south-central Washington state. Microb. Ecol. 23, 279-301. [3] Colwell. F.S., Stormberg, G.J., Phelps, T.J., Birnbaum, S.A., McKinley, .I., Rawson, S.A., Veverka, C., Goodwin, S.. Long, P.E., Russell, B.F., Garland, T., Thompson, D., Skinner, P. and Grover. S. (1992) Innovative techniques for collection of saturated and unsaturated subsurface basalts and sediments for microbiological characterization. J. Microbial. Methods 15, 279-292. [l]
]4] Fredrickson, J.K.. Garland, T.R., Hicks. R.J., Thomas, J.M., Li, SW., and McFadden, K.M. (1989) Lithotrophic and heterotrophic bacteria in deep subsurface sediments and their relation to sediment properties. Geomicrobiol. J. 7, 53-66. [S] Kieft, T.L., Amy, P.S., Brockman, F.J., Fredrickson, J.K., Bjornstad, B.N. and Rosacker, L.L. (1993) Microbial abundance and activities in relation to water potential in the vadose zones of arid and semiarid sites. Microb. Ecol. 26, 59-78.
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F.H. and Lovley, D.R. (1990) Rates of microbial metabolism in deep coastal plain aquifers. Appl. Environ. Microbial. 56, 1865-1874. [7] Balkwill, D.L and Ghiorse, W.C. (1985) Characterization of subsurface bacteria associated with two shallow aquifers in Oklahoma. Appl. Environ. Microbial. 50, 580-588. [8] Benoit, R.E. and T.J. Phelps. (1991) Microaerophilic bacteria from subsurface sediments. In Proceedings of the First International Symposium on Microbiology of the Deep Subsurface (C.B. Fliermans and T.C. Hazen, eds.), pp. 4-87 to 4-96, January 14-19, 1990, Orlando, FL. WSRC Information Services, Aiken, SC. [9] Hays, W.H. and Schuster, R.L. (1987) Maps showing ground-failure hazards in the Columbia River valley between Richland and Priest Rapids Dam, South-central Washington. Map I-1699, US Geological Survey, Denver, Colorado. [lo] Stevens, T.O., McKinley, J.P. and Fredrickson, J.K. (1993) Bacteria associated with deep, alkaline, anaerobic groundwaters in southeast Washington. Microb. Ecol. 25, 35-50. [ll] Plackett, R.L. and Burman, J.P. (1946) The design of optimum multifactorial experiments. Biometrika 33, 305-325. [12] Stowe, R.H. and Mayer, R.P. (1966) Efficient screening of process variables. Industr. Eng. Chem. 58, 36-40. [13] Reasoner, D.J. and Geldreich, E.E. (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbial. 49, l-7. [14] McMahon, P.B. and Chapelle, F.H. (1991) Microbial production of organic acids in aquitard sediments and its role in aquifer geochemistry. Nature 349, 233-235. [15] Wolin, E.A., Wolin, M.J. and Wolfe, R.S. (1963) Formation of methane by bacterial extracts. J. Biol. Chem. 238, 2882-2886. [16] Krieg, N.R. and Hoffman, P.S. (1986) Microaerophily and oxygen toxicity. Annu. Rev. Microbial. 40, 107-130. [17] Zehnder, A.J.B. and Wuhrman, K. (1977) Physiology of a Methanobacterium strain AZ. Arch. Microbial. 111, 199-205. [18] Holt, J.G. and Krieg, N.R. (1994) Enrichment and isolation. In Methods for General and Molecular Bacteriology (Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R., (eds.), American Society for Microbiology, Washington, DC. [19] Fry, J.C. (1990) Oligotrophs. In Microbiology of Extreme Environments (Edwards, C., ed.), pp. 93-116, McGraw-Hill, New York. [20] Boquet, E., Boronat, A. and Ramos-Cormenzana, A. (1973) Production of calcite (calcium carbonate) crystals by soil bacteria is a general phenomenon. Nature 246, 527-528. 1211 Stevens, T.O. and Molbert, B.S. Variability and density dependence of bacteria in terrestrial subsurface samples: implications for enumeration. J. Microbial. Methods 21, 283-292.
Journal of Microbiological Methods 21 (1995) 293-303
Optimization of media for enumeration and isolation of aerobic heterotrophic bacteria from the deep terrestrial subsurface T.O. Pacific Northwest Laboratory,
Stevens*
P.O. Box 999. m/s P7-54, Richland, WA 99352, USA
Received 7 February 1994; revised and accepted 20 June 1994
Abstract The objective of this study was to develop an agar plate-count medium to maximize the number of colony-forming units (CFU) recovered from terrestrial subsurface sediment samples. A Plackett-Burman experimental design was used to test the effects of 13 possible medium variables, including various ingredients and culture conditions. These experiments used sediment samples from different surface and subsurface areas to test the resulting formulations. Microorganisms in different sediments did not respond uniformly to any variable. Variables that always had a positive effect, when significant, included addition of vitamins, addition of activated charcoal (added as a scavenger of toxic oxygen forms), use of pour plates rather than spread plates, and elevated, rather than in situ, incubation temperature. Lowered water potential always led to a negative response, where significant. Varying concentrations of most common medium components, including dilution of carbon sources, had few significant effects over the ranges tested. A medium formulation for enhanced recovery of aerobic heterotrophic bacteria from subsurface sediments was developed, based on these results. Keywords:
Aquifer;
Bacteria;
Enumeration;
Medium; Subsurface; Vadose
1. Introduction The distribution and abundance of microorganisms in the terrestrial subsurface is a fundamental scientific issue that is being addressed as part of the US Department of Energy’s (DOE) Subsurface Science Program [e.g., l-51. The *Corresponding
author.
[email protected]. Elsevier Science B.V. SSDI 0167-7012(94)00056-5
Tel.:
+ 1 (509) 373-0891. Fax:
+ 1 (509) 376-9650. Internet:
to_
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standard plate-count assay plays an important role in these and other such studies; however, most agar media have been developed for other purposes. It is an axiom of microbiology that all culture media are selective for subsets of the total bacterial community; therefore, development of a variety of culture media is needed to maximize the recovery of diverse microorganisms from terrestrial subsurface samples. This study describes development of an agar plate-count medium for isolation and enumeration of aerobic heterotrophic bacteria from the deep terrestrial subsurface. Many well-characterized heterotrophic microorganisms can regulate their intracellular environment to allow for growth under various nutrient regimes. However, the range of conditions that allow for growth of subsurface microorganisms is not well known. Terrestrial subsurface ecosystems appear to contain the most oligotrophic microbial communities known [6], and it is likely that specialized conditions may be required to recover these organisms from core samples and culture them under laboratory conditions. In studies conducted in eastern coastal plain sediments, numbers of organisms recovered on 1% PTYG [7] agar plates were remarkably similar to numbers observed by direct microscopic counts [1,4]. Microaerophilic bacteria were common in some of these sediments [8]. In more recent borehole explorations at western sites, however, the proportion of microorganisms growing on agar plates to those observed by direct counts was in the range observed for surface organisms (l-10% or less) [2,5]. For many geological formations, few if any microorganisms could be recovered on agar plates, even when microbial respiration could be detected in the same bacteria were samples (J.K. Fredrickson et al., this issue). Microaerophilic detected in several of these low-recovery cores, and isolates from microaerophile enrichments often grew as normal aerobic heterotrophs in subsequent culture transfers. This suggested that specialized conditions might be required for recovery of some subsurface organisms from their in situ starved state to laboratory growth conditions. The objective of this study was to develop a heterotrophic plate-count medium that would maximize the number of CFU detected in subsurface samples. Because of the difficulty and expense of obtaining subsurface core samples, these experiments mostly used unsaturated paleosol and lacustrine sediment samples excavated from a recently exposed eroded sedimentary cliff traversing the same geological formation as nearby subsurface boreholes. The resulting medium formulation was tested on subsequent cores recovered from the deep subsurface. While a universal medium is not likely to be attainable, this optimized plate-count agar (OPCA) is an efficacious general medium for initial isolation and enumeration of aerobic heterotrophic bacteria from subsurface sediments.
2. Materials and methods 2.1. Soil and sediment
samples
Samples of unsaturated
paleosols and lacustrine sediments were obtained from
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the Ringold Formation in S.E. Washington. These sediments, ranging from 2 to 14 million years old, were excavated from the Savage Island Landslide (SIL) in S.E. Washington at latitude N 46”, 34’, longitude W 119”, 17’ [9]. The sediments were recently exposed by a landslide due to undermining of a steep bluff by irrigation water flow. Little or no water is believed to have flowed through this formation during the last 10,000 years, so recent bacterial transport is unlikely. These sediments should be good analogs for subsurface sediments, and bacterial habitats within them should be similar to those encountered in deeply buried formations. Individual samples are described in Table 1. The exposed face of the bluff was excavated back approximately 0.5 m, then samples were removed from the resulting fresh face with sterile tools and placed in sterile plastic bags. Samples were transported to the laboratory in less than one hour and stored at 4°C until use. One sample (YB 1335) was obtained from a depth of 103.5 m from a subsurface borehole and was classified as a paleosol. A well water sample (INEL W,) was used from a well located at the Idaho National Engineering Laboratory [3], at latitude N 43”, 30’, longitude W 112*, 48’, which was transported to the lab and used within 24 h. Another sample (INEL C) consisted of sand from a sand filter [lo] that had entrained microorganisms from this same well. Surface soil samples were loamy sands sampled near Richland, WA, and Idaho Falls, ID. 2.2. Mineral salts medium A mineral salts medium was used as the base for all experimental media in this study, and contained ingredients which were not varied. The mineral salts solution consisted of (per liter): KH,PO,, 0.25 g; K,HPO,, 0.35 g; CaCl,‘2H,O, 0.015 g; MgCl, * 6H,O, 0.02 g; FeSO, - 7H,O, 0.007 g; Na,SO,, 0.005 g. The first two ingredients and the remaining ingredients were prepared as two separate 100 X stock solutions. Table 1 Description of samples used in the medium optimization study and relative bacterial concentrations; numbers of bacteria are reported as both the mean number detected in all medium formulations and the maximum plate-count detected Sample
Description
Source
Bacteria, log cfu .
g-’ soil
SIL 1 SIL 2 SIL 3 SIL 6 SIL 7 YB 1335 Hanf S INEL S INEL W INEL C
Savage Island Landslide Savage Island Landslide Savage Island Landslide Savage Island Landslide Savage Island Landslide subsurface borehole Richard, WA surface Idaho Falls, ID surface Idaho Falls, ID water well Idaho Falls, ID water well
a For the groundwater
lacustrine sediment paleosol paleosol banded lacustrine sediment sandlpaleosol paleosol103.5 m deep loamy sand loamy sand groundwater sample sand filter sample
sample, values are cfu ’ III-‘.
mean
max.
3.90 2.94 2.31 4.12 2.90 6.50 6.93 6.46 3.88 5.06
4.59 3.52 3.30 4.68 3.48 7.10 7.68 6.91 4.65” 5.69
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2.3. Identification of significant variables
The first phase of the study consisted of a 16-variable Plackett-Burman experiment [ 11,121 conducted to identify culture-condition variables with a significant effect on microbial recovery. In this design, a ‘high’ and a ‘low’ value are chosen for each variable. Within the experimental matrix, the effect of alternating between these variables is tested eight times. Experimental variation is tested by including a set of dummy variables, for which no actual alteration is made in the formula. This allows screening of a large number of variables for significant effects without the enormous expense of performing a full factorial matrix. The principal disadvantage to this method is that two-factor effects are confounded and cannot be measured in this experiment. Variables tested in this study are listed in Table 2 with their ‘high’ and ‘low’ settings and a brief rationale for their selection. Several of these variables are standard ingredients in microbiological media. Previous investigations have indicated that lower concentrations of nutrients may aid in recovery of starved environmental isolates [e.g., 7,131. They were varied individually here to determine whether lower concentrations would be effective. The ‘complex nutrients’ solution contained, per ml: 0.05 g proteose peptone, 0.05 g tryptone, and 0.1 g yeast extract. The fatty acid solution contained equimolar concentrations (Table 2) of pyruvic acid, acetic acid, and lactic acid, and was included to test whether subsurface microorganisms might be adapted to substrates typical of a ‘lower’ trophic level (i.e. fermentation products) [14]. The vitamin solution [15] contained, per 100 ml: biotin, 0.002 g; folicin, 0.002 g; B6 (HCl), 0.01 g; riboflavin, 0.01 g; thiamine (HCl), 0.01 g; pantothenic acid, 0.005 g; nicotinamide, 0.005 g; B12, 0.01 g; PABA, 0.005 g; lipoic acid, 0.006 g.
Table 2 Medium components
used as variables in the medium optimization study
Component varied
‘High’ setting
Complex concentration Glucose concentration Fatty acid concentration Vitamin solution Ammonium chloride Glutamate Activated charcoal PH Water potential Temperature Pour plate vs. spread plate Trace minerals Dummy variable Dummy variable Dummv variable
2g.l.’ lg.l_I 1mM lmlg.llg.l_’ lg.1 ’ lg.1 ’
NA. not available
6.5 a =0.96 26°C spread 1 ml.I-’ NA NA NA
‘Low’ setting
Brief rationale
0.2g.I-’
effect of complex nutrients a typical sugar substrate a different trophic level of substrate cofactors for biosynthesis inorganic nitrogen source organic nitrogen source relieve O2 toxicity in agar plates optima may vary possible adaptation to desiccation in situ vs. typical laboratory different diffusional environments cofactors for various enzymes
0.1 g.1-’ 100 /.LM not added o.lg.I-’ 0.lg.l I not added 1.5 a =l.O 3&c pour not added NA NA NA
estimates estimates estimates
variance variance variance
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297
This solution was filter sterilized and frozen in l-ml aliquots until use, when 1 ml was added, after autoclaving, per liter of medium. Activated charcoal was selected as the most convenient protective additive against oxygen toxicity for microaerophilic bacteria [16]. Aliquots (1 g) were autoclaved separately three times for 30 min with 24 h at room temperature between. If added before autoclaving, the charcoal appeared to protect contaminant bacteria from sterilization. One pre-sterilized ahquot was added to media after autoclaving, and with vigorous stirring. The effect of varying osmotic water potential was tested because bacteria in the vadose zone might be expected to experience dehydration. Shock from sudden rehydration might result in loss of viability. For the ‘high’ settings of a, = 0.97, water potential was adjusted by adding 160 ml of glycerol per liter of medium. For the ‘low’ settings, no osmolyte addition was made, resulting in a, = - 1.0. A standard solution of trace minerals was used [17] containing, per 100 ml: MnCl, - 4H,O, 0.5 g; H,BO,, 0.05 g; ZnCl,, 0.05 g; CoCl, - 6H,O, 0.05 g; NiSO, - 6H,O, 0.05 g; CuCl, .2H,O, 0.03 g; NaMoO, - 2H,O, 0.01 g. When used, 1 ml of this solution was added per liter of medium. Three dummy variables were added to the matrix in order to determine experimental variability. No amendment was made to the medium for these variables. To remove experimental bias, each of the 15 variables was assigned a letter identifier using a random-number generator before constructing the experimental matrix (Table 3). A second random number was assigned to each medium recipe to determine the medium ID number and the order of preparation. All media were prepared a second time, and the experiment repeated after re-randomizing the experimental matrix. Table 3 Randomized Plackett-Burman design for medium optimization experiments. for variable, - indicates low setting) Variables
Randomized identity
Randomized medium ID
Variable settings ABCDEFGHI
1
Dummy Complex NH,CI Temperature Plate type H,Opoten Glutamate PH Vitamins Fatty acids Trace element Dummy Charcoal Glucose
B C J M L H G K A E 0 I N F
2 3 4 5 6 7 8 9 10 11 12 13 15 16
(+ indicates high setting
-
-
-++++-+-++--+
-++++-+-++--+--++++-+-++--+- + -++--+---++++ +---++++-+-++ ++++-+-++--+--__-__---------+-+-++--+---+++ ++--+---++++-•+++-+-++--+---++ -++--+---++++-+ +-++--+---++++-+---++++-+-+++--+---++++-+-+ +++-+-++--+---+
JKLMNO
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For each sediment sample, 10 g was added to 100 ml 0.1% sodium pyrophosphate, pH 7, and shaken vigorously for 20 min. Two further decimal dilutions were made in this buffer. Three replicate subsamples from each dilution were plated by the indicated method for each of the 16 medium combinations. Plates were incubated at the indicated temperatures and colonies counted after 2, 6, and 14 days. During preliminary experiments, plates were incubated for more than 45 days. No additional colonies appeared after about 10 days, so the 14-day counts were used for subsequent analyses. 2.4. Statistical melhoak The Plackett-Burman design for 15 variables was obtained from reference [12]. In this method, the effect of each variable was determined by computing the mean plate-count number (not log-transformed) of all media for which the variable was set ‘high’ and subtracting the mean plate-count number of all media for which the variable was set ‘low.’ Standard error was determined by computing the square root of the mean sum of squares of the effects of the dummy variables. A Student’s t-test was evaluated by dividing the effect of each variable by the standard error and comparing this value to a two-sided t table. Effects with values of p = 0.1 or less were assumed to be significant.
3. Results Results of the Placket-Burman experiment are shown in Table 4. There was considerable variation among the different samples tested. Variation of any one ingredient did not result in dramatic response of plate-count numbers for all samples tested, and most variations resulted in no significant change. Several ingredients, however, did have consistently positive effects for all samples in which their effects were significant. Addition of vitamins and activated charcoal had a significant positive effect in four and five of the ten samples, respectively. Their effects were never negative, except that sample SIL3 had an insignificant negative response to vitamins. Activated charcoal had the largest effects, ranging from 180% to 3370% increase in plate-count numbers. Elevated incubation temperature (coded as the ‘low’ variable value in Tables 2 and 4) had a positive effect for all subsurface samples, and never had a significant negative effect. Several variables had consistent patterns of effects for vadose-zone samples, but the opposite effect for the saturated sand filter sample. Using a pour-plate technique, rather than a spread-plate technique, had a significant positive effect for four of eight vadose zone samples, but a negative effect for the sand filter sample. Addition of trace elements, high glucose concentration, and elevated pH had positive effects for two or three vadose-zone samples but negative effects for the sand filter sample. Reducing the matric water potential of the medium had significant negative effects for five of ten samples and never had a significant positive effect.
0.01
227
5
147
140
220
144
102
161
226
SIL 2
SIL 3
SIL 6
SIl.7
YB 1335
Hanf S
INELS
INELW
1NELC
115
107
124
57
108
102
134
59
121
0.60
1.00
0.60
0.50
0.80
1.00
0.60
0.80
0.50
1.00
p
139
111
104
161
90
92
185
809
87
87
effect
0.20
1.00
1.w
0.60
0.80
agO
0.50
0.20
0.50
0.50
p
Complex
-
101
530
122
47
140
211
64
1646
101
62
effect
p
1.00
0.50
0.80
0.50
0.50
0.05
0.50
0.20
1.00
0.02
Dummy
-
121 0.50
66 0.80
100 1.00
210 0.50
84 0.60
134 0.50
202 0.50
11259 0.20
139 0.01
76 0.10
effect
p
Fatty acids ~
Glucose
44
175
83
129
95
217
125
177
154
2%
effect
~
0.05
0.80
0.80
0.80
1.w
0.05
0.80
0.80
0.05
0.01
p
169
179
1.03
53
120
1.51
122
83
68
87
effect
0.10
0.80
1.00
0.50
0.60
0.20
0.80
1.00
0.05
0.50
p
Glutamate -
Water
25
46
29
17
29
78
128
132
50
36
effect
-
potent p
69
0.02
0.01
0.60
0.05 101
10
57
280
71
0.50 0.20
63
73
105
113
0.80
0.80
0.01
0.01
effect
p
73
260
0.20
48
126
225
0.50
0.60
0.20
0.50
0.50
0.02
0.50
54
0.50 0.20
209
1.00
p 0.01
1523
120
48
effect
0.20
0.50
0.50
0.20
0.50
0.80
0.80
0.50
-
Ammonium
Dummy ___
pH
411
103
180
72
57
35
75
155
39
15
effect
-
0.01
1.00
0.20
0.80
0.20
1.00
0.60
0.80
0.01
0.01
p
164
25
102
57
87
3.5
18
14
73
15
effect
0.10
0.50
1.00
0.50
a.80
0.01
0.05
0.20
0.10
0.01
p
Plate type ~
122
27
41
210
72
59
71
67
79
101
effect
0.50
0.50
181
464
171
182 0.10
704 0.50
219
0.10 0.50
224
0.60
116
3365
0.20 0.80
298
1.00
p
0.05
0.50
0.50
0.50
0.01
0.02
0.20
1.00
0.01
0.01
54
78
70
117
139
110
260
8
222
190
effect
0.05
1.00
0.50
1.00
0.50
0.80
0.20
0.20
0.01
0.01
p
elements -
effect
_
p
Trace
Charcoal ~
twe
Tempera-
‘Effect’ is reported as mean plate-count numbers at ‘high’ settings divided by those at ‘low’ settings and multiplied by 100. Effects less than 100 are negative. while those greater than 100 are positive. p is reported as the probability that ‘high’ and ‘low’ means are not significantly different. Effects with values of p = 0.1 or less were assumed to be significant.
0.05
0.80
1.W
0.80
0.01
0.20
0.50
0.20
0.01
100
effect
effect
p
Dummy
-
Vitamins
-
229
SIL 1
Sample
Table 4 Results of medium optimization experiment for different samples
300
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Varying ammonium, glutamate, and fatty acid concentrations did not result in any trend and did not give consistent significant results. No variation of any variable had significant effects for surface samples or the groundwater sample.
4. Discussion This experiment studied the effect of varying concentrations of standard heterotrophic medium components, as well as addition of several special-purpose ingredients, on plate-count numbers of organisms from terrestrial surface and subsurface samples. The results indicate several adjustments that can be made to increase viable counts of subsurface bacteria, at least for the vadose zone, but that would probably have little effect for surface bacteria. High variability in two of the three dummy variables indicates that there may be two-factor effects that were neglected by this study. Alternatively, this variability could be due to differences in distribution of microorganisms within the different samples. Subsurface microorganisms tend to be distributed in clumped patterns, and estimating their numbers by plate-count methods is inherently more variable than for surface soils with similar numbers [21]. For instance, sample SIL 3 appears to be much more variable than other samples examined here. All significance levels, however, were calculated using standard variances derived from the dummy variables, so consistent responses to particular variables likely represent a real effect. Addition of activated charcoal had the greatest positive effect on subsurface plate-count numbers. Charcoal has been widely used as a protectant for microaerophilic bacteria [16]. It is assumed to trap superoxide and oxygen radical species that result from oxidation of complex medium components. Many samples from subsurface cores contain microaerophilic bacteria [8], although organisms isolated from microaerophilic bands often grow well on standard media after transfer from the primary enrichment (J.K. Fredrickson, et al., unpublished data). The above results suggest that microorganisms in a starved or dormant state in the subsurface may not maintain protective systems against toxic oxygen species, but do have protective mechanisms that are activated after recovery. The use of pour-plate techniques instead of spread-plate techniques [18] also resulted in greater numbers. The main difference between these techniques is probably restricted diffusional environments around nascent colonies submerged in the agar matrix of the pour plates. Pour plates may also provide protection against oxygen toxicity. Addition of a vitamin solution stimulated growth in most samples, which indicates that pools of these often complex cofactors may be depleted in subsurface microorganisms, which may or may not be capable of synthesizing these compounds. The consistent negative effect of reduced water activity indicates that, at least in the environments tested here, subsurface bacteria are not adapted to dessication, or high-solute niches which might arise by desiccation. Desiccation of a
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sediment system is not uniform across all microenvironments; rather, water is restricted to spaces with smaller pore sizes. This probably affects microorganisms more by reducing their habitat volume and restricting transport and nutrient diffusion than by actually desiccating their immediate environment [5]. Elevated temperatures resulted in greater numbers of microorganisms than the lower temperature that more closely simulated the in situ environment of the samples. This suggests that faster metabolic rates allowed by the higher temperature offer a greater advantage, during growth on agar medium, than any adaptation to low temperature the organisms might have experienced. Although a number of studies have shown that bacteria from low-nutrient environments are recovered better on dilute media than concentrated laboratory media [e.g., 7,13,18], the results of this study do not support that hypothesis. Dilution of complex nutrients or glucose had little effect on CFU. This may be because the ‘high’ levels of nutrients in this study were still relatively low compared to many standard formulations, including those cited above. Also, most previous studies of this nature have focused on aquatic or saturated environments, as opposed to the mainly vadose-zone environments studied here. Some oligotrophic bacteria grow best on medium containing only agar and its impurities as carbon sources, although they can generally be isolated on media containing a few hundred mg - 1-l carbon 1191, as used in these experiments. This study did not determine whether the same populations were counted under the ‘high’ and ‘low’ settings, only the gross numbers of CFU. Clearly, the list of variables tested was not exhaustive of all possible medium components. However, the components of other media commonly used for enumeration of subsurface bacteria [7,13] were included in the test matrix. It is likely that future investigations will identify additional refinements in recovery of microorganisms that were not tested here. Similarly, the samples used were not representative of all possible subsurface environments. and yet it should be noted that they were diverse enough that not all microbial populations responded to the variables in the same way. Still, several factors were identified which had consistently positive effects on bacterial recovery. Based on these results, the following OPCA medium formulation is suggested for recovery of subsurface aerobic heterotrophic bacteria. Add to 1 liter deionized water: KH,PO,, 0.25 g; K,HPO,, 0.35 g; CaCl, .2H,O, 0.015 g; MgCl, .6H,O, 0.02 g; FeSO, -7H,O, 0.007 g; Na,SO,, 0.005 g; NH4C1, 0.5 g; glucose, 1 g; peptone, 0.05 g; tryptone, 0.05 g; yeast extract, 0.1 g. Add 1 ml of the trace element solution described above, adjust pH to 7.5, and autoclave. After autoclaving, stir in 1 g finely ground pre-sterilized activated charcoal and 1 ml of the vitamin solution described above. Dispense fresh medium by standard pourplate techniques and incubate cultures at 30°C. A variation of this medium was later used to enumerate microorganisms in 32 cores retrieved from 172- to 223-m-deep saturated lacustrine sediments and paleosols from the deep subsurface (J.K. Fredrickson et al., unpublished data). The variations were that, for logistic reasons, spread plates were used instead of pour plates, and incubation temperatures were 17°C instead of 30°C. Despite
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these alterations, when compared with 1% PTYG [7] and B-4 [20], the modified OPCA formulation recovered bacteria from more samples than any other. Culturable aerobic heterotrophs were very rare in these samples, mostly less than 100 CFU . g-t, yet the formula above recovered bacteria in 13 of 26 samples, as compared to 10 of 29 for 1% PTYG and 8 of 28 for B-4. OPCA medium yielded more CFU than the other media in 10 of 24 direct comparisons, as compared to 3 of 24 for 1% PTYG and 1 of 24 for B-4.. Insufficient data were collected to determine whether recoverable numbers were significantly different for any of these experiments. This study has identified several factors that can have a positive effect on recovery of aerobic heterotrophic bacterial CFU from subsurface sediment samples when conducting a plate-count assay. Such an assay is commonly one of a suite of bacteriological tests used to characterize a subsurface environment. Because components of the medium suggested above were selected for their positive effect on plate-count numbers for a variety of samples, and performed well for both saturated and unsaturated sediment samples, OPCA should be an efficacious general medium for enumeration of subsurface microorganisms.
Acknowledgements
We thank Heath Watts and Jennifer Walker for technical assistance on this project, as well as Dr. Jay Grimes and Dr. Frank Wobber, whose assistance made this study possible. This research was supported by the Subsurface Science Program, Office of Health and Environmental Research, US Department of Energy (DOE). Pacific Northwest Laboratory is operated for the DOE by Battelle Memorial Institute under Contract DE-AC06-76RL0 1830.
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