Comparison of media and diluents for enumeration of aerobic bacteria from Bermuda grass golf course putting greens1

Comparison of media and diluents for enumeration of aerobic bacteria from Bermuda grass golf course putting greens1

Journal of Microbiological Methods 34 (1999) 193–202 Journal of Microbiological Methods Comparison of media and diluents for enumeration of aerobic ...

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Journal of Microbiological Methods 34 (1999) 193–202

Journal of Microbiological Methods

Comparison of media and diluents for enumeration of aerobic bacteria from bermuda grass golf course putting greens q M.L. Elliott*, E.A. Des Jardin Fort Lauderdale Research and Education Center, University of Florida, 3205 College Avenue, Fort Lauderdale, FL 33314, USA Accepted 2 September 1998

Abstract To increase our knowledge of soil and rhizosphere bacteria associated with bermuda grass grown on golf courses, a standardized procedure has been developed. Seven aerobic bacterial groups were selected for enumeration. For each group selected, appropriate media were compared to determine which one was best for enumeration of that group. Six diluents were evaluated across all media for the seven bacterial groups. The best diluent was sodium pyrophosphate with glycerol. The following media were best for enumeration: solidified 10% tryptic soy broth for total culturable aerobic bacteria and heat tolerant bacteria; reduced arginine soluble salts medium for actinomycetes; S1 medium for fluorescent pseudomonads; XMSM for Stenotrophomonas maltophilia; Bacto azide blood agar base for gram-positive bacteria; crystal violet agar for gram-negative bacteria.  1999 Elsevier Science B.V. All rights reserved. Keywords: Bacteria; Bermuda grass; Cynodon spp.; Enumeration; Plate counting

1. Introduction Golf course putting greens are rarely composed of a native soil. The US Golf Association specifications for putting greens essentially exclude the use of native soil (USGA Green Section Staff, 1993). Instead, a 25–30-cm thick layer of root-zone mix is placed either directly on a gravel layer or on a coarse sand layer that has been placed on the gravel layer. A standard root-zone mix is composed of sand, preferably quartz sand, mixed with sphagnum peat; ratios vary with 80–95% sand mixed with 5–20% peat (v / v). Additional organic matter will be added *Corresponding author: Tel.: 1 1-954-475-8990; fax: 1 1-954475-4125; e-mail: [email protected] q Florida Agricultural Experiment Station Journal Series no. R06427.

to the putting green as the turfgrass root system dies and regenerates throughout the years. Unlike traditional agricultural soils, neither microbial enumeration nor diversity in putting greens have been extensively examined. Only three studies have examined these populations. One study was conducted in Arizona, USA, on an 80% sand / 20% sphagnum peat (v / v) putting green planted to bentgrass (Mancino et al., 1993). The second study was conducted in Ontario, Canada, on a native soil putting green planted to bentgrass (Liu et al., 1995). The third study was conducted in Florida, USA, on an 80% sand / 20% sphagnum peat (v / v) putting green planted with a hybrid bermuda grass (Elliott and Des Jardin, 1998). All three studies examined the effect of natural organic materials on microbial populations in the putting greens. Numerous products are available to

0167-7012 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 98 )00088-8

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golf course turfgrass managers that are either live microbial products or products that claim to increase microbial populations in the thatch, soil, or on the root systems. However, there is usually no data available to support such claims. While all three studies used microbial enumeration to follow populations over time, the different media and methods used for enumeration purposes prevent meaningful comparisons among the studies. There are numerous methods for examining bacterial numbers and diversity (reviews: Pankhurst et al., 1996; Skipper et al., 1996; Hurst et al., 1997), and no single method will provide all the information desired by the investigators. The methods can essentially be broken into two categories—culturable methods and non-culturable methods. The primary advantage of the culturable methods is the ability to obtain pure cultures that can be stored for future analysis or screening, including taxonomic, genetic or functional identification. The disadvantage of the culturable methods is the bias that is instantly introduced into the analysis— i.e., one only obtains those organisms that can be cultured on a particular medium under defined conditions. Therefore, if one selects to use culturable methods, it would be beneficial to determine the diluents and media that would be most efficient at enumerating and isolating the microbes of interest from a particular soil and climatic environment. A recent study by Balestra and Misaghi (1997) demonstrated that the efficiency of estimating bacterial diversity can be increased by using a set of media rather than a single medium. During the course of our previous study (Elliott and Des Jardin, 1998), it became apparent that some of the media being utilized for enumeration were inadequate for the study site’s environmental conditions, especially for fluorescent pseudomonads and actinomycetes. Thus, the goal of the current project was to compare various diluents and media for enumeration of seven groups of bacteria. This information would then be used to develop standard methods for future putting green studies examining bacterial populations of hybrid bermuda grass root systems. By developing a standard methodology, direct comparisons could be made among future studies. The seven bacterial groups selected for enumera-

tion are both general and specific in nature, but represent bacterial groups known to be associated with soil and plant roots (Hagedorn and Holt, 1975; Juhnke et al., 1987; Lambert et al., 1987, 1990; Miller et al., 1990; Mancino et al., 1993; Nijhuis et al., 1993; Xu et al., 1996). The three general groups of bacteria to be enumerated are total culturable bacteria, gram-positive bacteria and gram-negative bacteria. Specific gram-negative bacterial groups selected for enumeration include the fluorescent pseudomonads and Stenotrophomonas maltophilia (formerly known as Xanthomonas maltophilia). Actinomycetes, including Streptomyces spp. and Nocardia spp., are a sub-group of gram-positive bacteria that are common soil and rhizosphere inhabitants. Endospore forming, gram-positive bacteria, primarily Bacillus spp., are included in a group of bacteria described as heat tolerant based on the selection method. Isolates and strains within all of these specific bacterial groups have been examined or are currently being used for promotion of plant growth or disease control (Schippers et al., 1995; Cook et al., 1996).

2. Methods All media, chemicals and antibiotics were obtained from Difco Laboratories (Detroit, MI), Fisher Scientific (Pittsburgh, PA), or Sigma (St. Louis, MO).

2.1. Diluents The diluents evaluated included 0.1% sodium pyrophosphate (pH 7.0), phosphate-buffered saline (per liter, 8.5 g NaCl, 6.8 g KH 2 PO 4 and 11.4 g K 2 HPO 4 ), and a soil extract solution. The soil extract solution was prepared using a root-zone mix of sand and sphagnum peat (80 / 20, v / v). One kg root-zone mix was placed in 1 l deionized water, autoclaved 90 min, cooled and filtered through layers of cheesecloth. Deionized water was added to the filtrate to obtain a 1-l volume. Each diluent was also made with glycerol added (1% final volume) to obtain the six diluents compared within each group of media described below.

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2.2. Media Media used for comparison were dependent on the bacterial group to be enumerated. Broth media were solidified with 15.0 g Bacto agar per liter. Unless otherwise specified, media were supplemented with 100 mg ml 21 cycloheximide and 50 mg ml 21 nystatin to inhibit fungal growth. For total culturable aerobic bacterial enumeration, the media compared were solidified 10% tryptic soy broth (10% TSBA), solidified 10% Luria–Bertani broth (10% LBA) (Atlas, 1993), solidified 20% tryptone glucose extract broth (20% TGEA) (per liter: 0.6 g Bacto beef extract, 1.0 g Bacto tryptone, and 0.2 g Bacto dextrose) (Atlas, 1993), and each of these media amended with glycerol (1% final volume). Heat-tolerant bacteria (e.g., Bacillus spp.) were enumerated by placing dilution series tubes in an 808C water bath for 10 min. After cooling to room temperature, dilutions were plated on 10% TSBA. To compare enumeration of actinomycetes, the media included reduced arginine soluble salts (Herron and Wellington, 1990), Difco actinomyceteselective medium, 10% TSBA, humic acid vitamin agar (Hayakawa and Nonomura, 1987a) and soil extract agar (Norris et al., 1981). The latter was made using a root-zone mix of sand and sphagnum peat (80 / 20, v / v) as the soil. For fluorescent pseudomonads, the media compared included King’s B (Difco Pseudomonas agar F) (King et al., 1954), King’s B amended with (per liter) 75 000 units penicillin G, 25 mg novobiocin and 5 mg chloramphenicol (Simon et al., 1973), and S1 medium (Gould et al., 1985). Unamended S1 medium was also compared. Stenotrophomonas maltophilia was enumerated using a selective medium for this common bacterium (XMSM) (Juhnke and Des Jardin, 1989). Two media were compared for enumeration of gram-positive bacteria, Bacto azide blood agar base (Difco Laboratories, 1984) and methyl red agar (Hagedorn et al., 1989). The methyl red agar consisted of 10% TSBA amended with 20 g NaCl and 150 mg methyl red, per liter. Two media were compared for enumeration of gram-negative bacteria, solidified Bacto GN broth, Hajna (Difco Laboratories, 1984) and crystal violet agar (Gould et al.,

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1985). The latter medium consisted of 10% TSBA amended with 5 mg ml 21 crystal violet (C.I. 42555). To determine if the bacteria isolated on these four media were gram negative or positive, four representative colonies from each dilution series were selected and transferred to 10% TSBA. A total of 96 colonies were selected from each medium. After 24–48 h growth, Gram stain reactions were determined for each isolate.

2.3. Sampling procedures For each trial of each experiment, 20 cores (25 mm diameter 3 75 mm deep) were randomly obtained from a ‘Tifdwarf’ hybrid bermuda grass putting green. The top 12.5 mm of each core were removed to eliminate the leaf tissue and top half of the thatch layer. These soil and root cores were then mixed together very well. For each diluent, four replicate samples of 10 g each were placed in flasks with 90 ml of the diluent. Flasks were shaken for 30 min at 200 rpm with a rotary shaker placed at room temperature. Dilutions were made with the diluent and plated on the appropriate media for the bacterial group to be enumerated. All inoculated media were incubated at 288C. Fluorescent pseudomonads were counted after 24 and 48 h incubation using a short-wave UV lamp. Other bacterial groups were counted after 3, 7 and 14 days incubation.

2.4. Statistical analysis The SAS statistical package (SAS Institute, Cary, NC) was used for analysis of data. Waller–Duncan k-ratio t-test or LSD t-test were used for means comparison when appropriate.

3. Results and discussion

3.1. Diluents Six diluents, three formulae with or without glycerol, were evaluated in this study. Distilled or deionized water were not used as previous studies have shown they are not effective diluents (Olsen

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and Bakken, 1987; Lindahl, 1996). Sodium pyrophosphate is a common diluent in soil microbial studies (Miller et al., 1990; Trevors and Cook, 1992; Nijhuis et al., 1993; Lindahl, 1996). It has been noted to break down soil aggregates and dislodge cells when used in conjunction with rapid agitation (Trevors and Cook, 1992). Another diluent often used is phosphate-buffered saline (Juhnke et al., 1987; Hagedorn et al., 1989; Kloepper et al., 1991). Soil extract solution has also been compared with common diluents (Olsen and Bakken, 1987). It seemed logical to obtain a diluent from the soil source from which the microbes will be isolated. Glycerol was added to the diluents to aide in recovery of bacteria in a nutrient starved state since most bacteria can readily metabolize glycerol (Trevors and Cook, 1992). For six of the seven bacterial groups targeted for enumeration, sodium pyrophosphate with glycerol provided either the best enumeration or was not significantly different from other diluents in providing the best enumeration for these groups on their respective media. The only bacterial group which was negatively effected by this diluent was S. maltophilia. However, the best diluents for enumerating S. maltophilia, sodium pyrophosphate without glycerol and soil extract solution with glycerol, had negative effects on enumeration of other bacterial groups. Sodium pyrophosphate without glycerol decreased enumeration of the actinomycetes and soil extract solution with glycerol decreased enumeration of heat-tolerant bacteria.

3.2. Total aerobic bacteria The medium most often used for enumerating total, aerobic, heterotrophic, culturable bacteria is 10% TSBA (e.g., Martin, 1975; Gould et al., 1985; Juhnke et al., 1987; Hagedorn et al., 1989; Miller et al., 1990; Nijhuis et al., 1993). For comparison with this medium, 10% LBA and 20% TGEA were selected. The latter is a standard plate count medium at full strength (Difco Laboratories, 1984). These media were used at reduced strength because of the negative effect high concentrations may have on bacterial enumeration. These include detrimental effects of components in the media on sensitive bacteria or overgrowth by rapidly growing bacteria (Martin, 1975; Suwa and Hattori, 1984; Olsen and Bakken, 1987; Sugimoto et al., 1990). Glycerol was added to each of the three media, as a study by Trevors and Cook (1992) indicated potential benefits. There were significant differences between trials of this experiment (Table 1). The overall mean (per g soil and roots) for total culturable aerobic bacteria was log 10 9.0 CFU in the first trial, and the overall mean in the second trial was log 10 8.6 CFU. When the analysis was conducted separately for each trial, there were significant differences among diluents in both trials (P 5 0.022 for trial 1 and P , 0.0001 for trial 2), but significant differences among media were only observed in the second trial (P 5 0.0032). There were no significant differences in the interaction between diluents and media in either trial.

Table 1 Analysis of variance for enumeration of five different bacterial groups from a bermuda grass golf course putting green Source

Trials Replications Diluents a Media b Diluents 3 Media Error Total a

Total aerobic bacteria

Actinomycetes

Fluorescent pseudomonads

Gram-negative bacteria

df

Mean square

P.F

df

Mean square

P.F

df

Mean square

P.F

df

Mean square

P.F

df

Mean square

P.F

1 3 5 5

8.7777 0.0132 0.1514 0.0340

0.0001 0.5678 0.0001 0.1267

1 3 5 4

0.0100 0.2195 0.6076 105.0854

0.8378 0.4314 0.0290 0.0001

1 3 5 3

46.4822 0.6249 12.2147 59.9270

0.0001 0.8512 0.0002 0.0001

1 3 5 1

2.5938 0.0540 0.2208 47.5454

0.0001 0.5064 0.0109 0.0001

1 3 5 1

12.5737 0.0163 0.0638 21.6381

0.0001 0.6154 0.0472 0.0001

25 248 287

0.0213 0.0196

0.3545

20 206 239

0.5964 0.2382

0.0006

15 164 191

2.7745 2.3667

0.2981

5 80 95

0.2138 0.0688

0.0130

5 80 95

0.0976 0.0270

0.0054

The same six diluents were used for enumerating each bacterial group. Different, but appropriate, media were used for enumerating each bacterial group.

b

Gram-positive bacteria

M.L. Elliott, E. A. Des Jardin / Journal of Microbiological Methods 34 (1999) 193 – 202

For both trials, the majority of variation (46–50%) was associated with the diluent. In the first trial, sodium pyrophosphate with glycerol was not significantly better (P , 0.05) in enumerating total bacteria than sodium pyrophosphate without glycerol or phosphate-buffered saline without glycerol, but was significantly better than the other diluents. In the second trial, sodium pyrophosphate, with or without glycerol, was significantly better (P , 0.05) than the other four diluents in enumerating total bacteria. Also in this second trial, the media 10% LBA, with or without glycerol, 20% TGEA, and 10% TSBA were not significantly different (P , 0.05) from each other but were significantly different from the other two media.

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Nonomura (1987a) compared HAVA with seven other common actinomycete media, but HAVA had the highest percentage of actinomycetes and the largest populations. Based on these previous studies, RASS, SEA and HAVA were selected for comparison of actinomycete enumeration. Difco ASM was included for comparison as it is the only commercial actinomycete media available. A chitin-based medium was not selected for evaluation since that would exclude those actinomycetes that are incapable of utilizing chitin. No significant differences were observed between trials or among replications (Table 1). While there were significant differences among diluents across all media, among media across all diluents, and the interaction among diluents and media, 96% of the variation was associated with the media. The humic acid vitamin agar (HAVA) and reduced argininesoluble starch agar (RASS) media were not significantly different from each other but were significantly better than 10% TSBA, Difco actinomycete selective agar (ASM) and soil extract agar (SEA), in enumerating actinomycetes (Table 2). Only the diluent sodium pyrophosphate without glycerol was significantly different from any of the other five diluents across all media, and it resulted in fewer actinomycetes (Table 3). In the interaction among diluents and media, this diluent did not affect

3.3. Actinomycetes A previous study comparing media for enumeration of genetically transformed Streptomyces demonstrated that RASS was more effective than 10% TSBA, arginine glycerol salts agar, mineral salts starch casein agar, or tryptone yeast extract agar (Elliott et al., 1994). Another study examining general actinomycete populations demonstrated that SEA was as effective as the mineral salts starch casein agar in enumerating actinomycetes (Elliott and Des Jardin, 1998). A study by Hayakawa and

Table 2 Comparison of media across all six diluents for enumeration of five different bacterial groups from a bermuda grass golf course putting green Total aerobic bacteria Medium

a

10% TSBA 10% TSBA/glycerol 10% LBA 10% LBA/glycerol 20% TGEA 20% TGEA/glycerol P.F MSD/LSD a

Actinomycetes Log 10 CFU 8.8 8.8 8.8 8.8 8.9 8.8 0.1267 —

b

SE 0 0 0 0 0 0

c

Medium

d

RASS HAVA 10% TSBA ASM SEA

Fluorescent pseudomonads e

Log 10 CFU

SE

Medium

6.5 6.4 6.2 6.1 3.0

0 0.1 0.1 0 0

S1 S1 1 King’s B King’s B 1

a a b b c

0.0001 0.2

Gram-positive bacteria

Gram-negative bacteria

Log 10 CFU

SE

Medium

Log 10 CFU

SE

Medium

Log 10 CFU

SE

6.2 6.2 4.6 4.0

0.1 0.1 0.3 0.4

Methyl red Azide blood

6.6 a 5.2 b

0.1 0

Crystal violet GN, Hanja

7.1 a 6.2 b

0 0.1

a a b c

0.0001 0.6

0.0001 0.1

0.0001 0.1

TSBA, tryptic soy broth agar; LBA, Luria–bertani broth agar; TGEA, tryptone glucose extract agar. Glycerol added for a 1% total volume. Values are per g of soil and roots. Values are means from two independent trials with four replicates in each trial (n 5 8). Means in the same column followed by the same letter are not significantly different by Waller–Duncan k-ratio t-test (P , 0.05) or LSD t-test (P , 0.05). c SE, standard error d RASS, reduced arginine soluble starch agar; HAVA, humic acid vitamin agar; ASM, Difco actinomycete-selective medium; SEA, soil extract agar. e 1 , antifungal antibiotics were added to S1 medium and additional antibacterial antibiotics were added to King’s B medium. b

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actinomycete enumeration on RASS agar but did reduce enumeration on HAVA. Differences among media and diluents with regards to background contamination (i.e., non-actinomycete colonies) were also compared. There were no significant differences (P , 0.05) across all diluents among RASS, HAVA or ASM. HAVA was noted to have background bacteria that were very small, usually pinpoint in size, with few mucoid bacterial types present. However, a minimum of 5–7 days was required for the majority of actinomycetes to initiate growth on this medium, and final count could not be taken until after 14 days growth. While HAVA provided less background contamination than RASS, it was more difficult to prepare. The humic acid is prepared in a lengthy process that includes boiling urea and glucose in hydrochloric acid in a closed vessel for 5 h. RASS can be prepared from standard chemicals and media. Natural humic acid sources were not used in the preparation of HAVA. These sources may have negative effects on actinomycete enumeration, and quality of natural humic acids may vary from batch to batch (Hayakawa and Nonomura, 1987b).

3.4. Fluorescent pseudomonads Although King’s B medium is often used for general isolation and enumeration of fluorescent pseudomonads (e.g., Hagedorn et al., 1989), it was

not developed for those purposes but rather for diagnostic purposes (King et al., 1954). Since improvements had been made to King’s B medium for isolation and enumeration of this bacterial group, those media were included in this study (Sands and Rovira, 1970; Simon et al., 1973). There were significant but similar differences between trials of this experiment (Table 1). The overall mean (per g soil and roots) for fluorescent pseudomonads was log 10 4.8 CFU in the first trial, whereas the overall mean in the second trial was log 10 5.8 CFU. There were no significant differences among replications, but there were among diluents (P , 0.0001), among media (P , 0.0001), and in the interactions between diluents and media (P 5 0.001 for trial 1 and P 5 0.007 for trial 2). For both trials, the majority of variation (75–77%) was associated with the media. Both of the S1 media resulted in significantly greater enumeration of fluorescent pseudomonads than either of the King’s B media (Table 2). The S1 medium, with or without the antifungal antibiotics, also significantly reduced (P , 0.0001) the non-fluorescent pseudomonad background contamination in both trials. While there were significant differences among diluents across all media (Table 3), the interaction between either of the S1 media and the diluents in both trials did not result in significant differences. The addition of chloramphenicol, penicillin and novobiocin to King’s B medium did not increase

Table 3 Comparison of six diluents across selective and non-selective media for enumeration of seven bacterial groups from a bermuda grass golf course putting green Diluent

0.1% Na 4 P2 O 7 0.1% Na 4 P2 O 7 1 glycerol Phosphate-buffered saline (PBS) PBS 1 glycerol Soil extract solution Soil extract solution 1 glycerol P.F MSD

Log 10 CFU g 21 soil and roots Total bacteria

Actinomycetes

Fluorescent pseudomonads

Gramnegative bacteria

Grampositive bacteria

Heattolerant bacteria

Stenotrophomonas maltophili

8.9a a 0 b 8.9a 0 8.8b 0 8.8b 0 8.8b 0 8.8b 0 0.0001 0.1

5.4b 0.3 5.7a 0.2 5.8a 0.2 5.7a 0.2 5.6ab 0.2 5.6ab 0.2 0.0290 0.2

5.3b 0.4 5.4b 0.3 4.8bc 0.4 4.4c 0.4 5.4b 0.3 6.2a 0.1 0.0002 1.0

6.7a 0.2 6.6ab 0.2 6.6ab 0.2 6.5b 0.1 6.7a 0.2 6.7a 0.2 0.0472 0.1

6.0ab 0.2 5.9abc 0.2 6.1a 0.2 5.7c 0.2 5.8bc 0.2 5.9abc 0.2 0.0109 0.2

7.1ab 0 7.1ab 0 6.9b 0 7.0b 0 7.2a 0.1 7.0b 0 0.0196 0.2

5.5a 0.2 5.1b 0.3 5.2b 0.3 5.5a 0.1 5.5a 0.2 5.5a 0.2 0.0002 0.2

a Values are means from two independent trials with four replicates in each trial (n 5 8). Means in the same column followed by the same letter are not significantly different by Waller–Duncan k-ratio t-test (P , 0.05). b Values are the standard errors.

M.L. Elliott, E. A. Des Jardin / Journal of Microbiological Methods 34 (1999) 193 – 202

numbers of fluorescent pseudomonads isolated, nor did they reduce background contamination as reported previously (Simon et al., 1973). The S1 medium did substantially decrease the non-fluorescent pseudomonad background contamination. This may account for the increased numbers of fluorescent pseudomonads enumerated on S1 media compared to the King’s B media. The addition of antifungal antibiotics to S1 did not influence enumeration of fluorescent pseudomonads or background contamination.

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previously to enumerate gram-positive bacteria from the rhizosphere of cotton grown in a southern temperate climate (Kloepper et al., 1991). The methyl red inhibits bacilli and micrococci. The selective component in the azide blood agar base is sodium azide (Difco Laboratories, 1984). There were significant differences between trials of this experiment (Table 1). The overall mean (per g soil and roots) for gram-positive bacteria was log 10 5.8 CFU in the first trial, and the overall mean in the second trial was log 10 6.1 CFU. When the analysis was conducted separately for each trial, there were significant differences (P , 0.0001) among media in both trials. Significant differences among diluents (P 5 0.001) and the interaction between media and diluents (P 5 00.25) were only observed in the second trial. For both trials, the majority of variation (99%) was associated with the media. The methyl red agar did result in significantly greater (P , 0.05) enumeration of bacteria than the Bacto azide blood agar (Table 2). However, when Gram staining was conducted on 96 bacterial colonies from each medium, only 18% of the colonies on the methyl red agar were gram-positive bacteria, whereas 70% of the colonies on the azide blood agar were gram-positive bacteria. There were no significant differences (P , 0.05) between Bacto azide blood agar and the diluents in either trial. The overall mean comparison among diluents is provided in Table 3. In this study, the bacteriostatic rather than bactericidal effect of sodium azide was observed. Bacteria present 7 days after incubation were predominantly gram-positive bacteria. If the plates were incubated for 10 days, the additional bacteria that grew were predominantly gram-negative (unpub-

3.5. Heat-tolerant bacteria Bacteria which produce heat-resistant endopsores can be confirmed by heating old cultures at 70–808C for 10 min (Holt et al., 1994). This was used as a selection method for enumeration of endosporeforming bacteria, such as Bacillus and Amphibacillus, and heat-tolerant bacteria. Only one medium was evaluated, 10% TSBA, with the six different diluents to enumerate heat tolerant bacteria. There were significant differences among diluents but not between trials of this experiment (Table 4). There were no significant differences (P , 0.05) among sodium pyrophosphate, with or without glycerol, and soil extract solution without glycerol, but the latter diluent did have significantly greater counts than phosphate-buffered saline and soil extract solution with glycerol (Table 3).

3.6. Gram-positive bacteria To enumerate other gram-positive bacteria, the two media compared were methyl red agar and azide blood agar base. The methyl red agar had been used

Table 4 Analysis of variance for enumeration of two different bacterial groups from a bermuda grass golf course putting green Source

Trials Replications Diluents z Error Total z

Heat-tolerant bacteria

Stenotrophomonas maltophilia

df

Mean square

P.F

df

Mean square

P.F

1 3 5 38 47

0.0521 0.0147 0.0986 0.0319 —

0.2096 0.7107 0.0196 — —

1 3 5 38 47

16.6381 0.0534 0.3318 — —

0.0001 0.3921 0.0002 — —

The same six diluents were used for enumerating each bacterial group.

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lished results). Therefore, timing of the bacterial counts was as critical as the medium selected for enumeration.

treatment of dilutions, for use in future analysis of bacteria associated with the bermuda grass roots.

3.8. Stenotrophomonas maltophilia 3.7. Gram-negative bacteria Crystal violet is often added to media to inhibit growth of gram-positive bacteria (Difco Laboratories, 1984; Skipper et al., 1996). The Bacto GN Broth, Hajna was selected for comparison with crystal violet amended 10% TSBA. The sodium citrate and sodium desoxycholate in the GN medium are bactericidal to gram-positive bacteria and inhibit the development of coliforms (Difco Laboratories, 1984). There were significant differences between trials of this experiment (Table 1). The overall mean (per g soil and roots) for gram-negative bacteria was log 10 6.3 CFU in the first trial, and the overall mean in the second trial was log 10 7.0 CFU. When the analysis was conducted separately for each trial, there were significant differences among media (P , 0.0001) in both trials. A significant difference among diluents (P 5 0.0472) was only observed in the first trial. For both trials, the majority of variation (98–99%) was associated with the media. The crystal violet agar resulted in significantly greater (P , 0.05) enumeration of gram-negative bacteria than the GN Broth, Hajna, agar (Table 2). When Gram staining was conducted on 96 bacterial colonies from each medium, 95% of the colonies from the crystal violet agar and 87% from the GN broth, Hajna, agar were gram-negative bacteria. There were no significant differences (P , 0.05) between crystal violet agar and the diluents in either trial. The overall mean comparison among diluents is provided in Table 3. By using 10% TSBA with crystal violet, one can directly compare the gram-negative counts obtained with it to the total bacterial counts obtained on unamended 10% TSBA to obtain presumptive grampositive counts (Skipper et al., 1996). This would eliminate the need for using the azide blood agar base in studies. However, this technique may also underestimate the gram-positive bacterial population. It would also not permit easy selection of grampositive bacteria, except for those obtained with heat

Only one medium, XMSM, was evaluated with the six different diluents to enumerate S. maltophilia. There were significant differences between trials and among diluents (Table 4). The overall mean (per g soil and roots) for S. maltophilia in the first trial was log 10 4.8 CFU, whereas the overall mean in the second trial was log 10 6.0 CFU. In the first trial, sodium pyrophosphate without glycerol, phosphatebuffered saline with glycerol and soil extract solution with or without glycerol resulted in significantly greater (P , 0.05) enumeration of S. maltophilia than sodium pyrophosphate with glycerol or phosphate-buffered saline without glycerol. In the second trial, soil extract solution with glycerol resulted in significantly greater (P , 0.05) enumeration of S. maltophilia than sodium pyrophosphate with glycerol and phosphate-buffered saline with or without glycerol. The overall mean comparison among diluents is provided in Table 3. A new selective medium (VIA agar) has recently been developed (Kerr et al., 1996) but we were not aware of it when we began our study and have thus far had difficulty in obtaining the key component of the medium, the antibiotic imipenem. We look forward to comparing it with XMSM in the future, as VIA agar would certainly be easier to prepare. In this study, our goal was to develop a culturable method for examining general and specific bacterial populations of hybrid bermuda grass root systems grown on a putting green built to US Golf Association specifications. We believe this goal has been accomplished. Sodium pyrophosphate with glycerol was the best overall diluent in the study. Since there were no significant differences among the standard plate count media, 10% TSBA will be used for our future studies. It is simple to prepare and has been the most often used medium for enumerating total aerobic bacteria. Media that will be used for enumeration of specific bacterial groups include: S-1 for fluorescent pseudomonads, XMSM for S. maltophilia, azide blood agar base for gram-positive bacteria, crystal violet amended 10% TSBA for

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gram-negative bacteria and either RASS or HAVA for actinomycetes. By having a standard methodology, valid comparisons can be made among studies, even those completed at different geographic locations or in different years.

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