Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups

Viable counts of heterotropic soil bacteria were 3–5 times higher on low-nutrient agar media compared with a series of conventional agar media. Substantial amounts of monosaccharides and amino acids were present in solid media made from distilled water and agar powder, and a salt-solution agar medium (without organic substrates added) gave practically the same colony counts as the low nutrient soil extract agar medium. MPN values were comparable to or lower than plate counts. A search for slow-growing cells in the negative MPN tubes by fluorescence microscopical examination after 3 months incubation was negative.The viable counts were 2–4% of the total microscopical counts in different soils. Assuming that the colony-forming cells did not derive from the numerous dwarf cells present in soil, a calculated percent viability of the larger cells was about 10%. The ecological significance of the plate-counting technique is discussed.     

Comparison of bacterial counts obtained from naturally contaminated foods by means of Stomacher and blender

Four Regional Health Protection Branch laboratories each compared aerobic colony counts obtained after "stomaching" and blending, for a minimum of 10 samples in each of the seven food groups: dry pastas; chocolate and cocoa powders; frozen entrees (macaroni and cheese, chow mein, chop suey, fried rice, seafood casseroles, and Salisbury steak); nonfat dry milk; shrimp and crabmeats; spices; and breakfast sausages. Overall, counts obtained after using the Stomacher were equivalent to or higher than counts obtained after using the blender in 73% of the comparisons (alpha = 0.05). Where differences existed, counts obtained after using the Stomacher tended to be higher than counts obtained after using the blender from milk powder and lower from sausage. Aerobic colony counts from these foods are not unacceptably biased when obtained by Stomacher.     

Enumeration and isolation of anaerobic microbiota of piggery wastes.

Media for enumeration of the microbiota of anaerobically stored piggery wastes were tested. Highest colony counts were obtained with 80 to 100% farm slurry supernatant included in the anaerobic roll tube media. Colony counts with these media numbered 2 X 10(9) to 12 X 10(9)/g (wet weight), which represents about 20% of the microscopic counts. Lower percentages of slurry supernatant in the media gave lower colony counts. Addition of glucose, cellobiose, and starch or of Trypticase to media with 20% slurry supernatant did not increase colony counts. Higher values were obtained when hemicellulose preparations were added to these media. Incubation at 25 degrees C gave the highest numbers. Incubation at 15 to 37 degrees C gave counts of about 70 and 10%, respectively, of those at 25 degrees C. Of the colonies picked for isolation, about 20% were obtained in pure culture. The isolates apparently belonged to the genera Peptococcus, Ruminococcus, Peptococcus, Ruminococcus, Pepostreptococcus, and Bacteroides.     

Enumeration and isolation of anaerobic microbiota of piggery wastes

Media for enumeration of the microbiota of anaerobically stored piggery wastes were tested. Highest colony counts were obtained with 80 to 100% farm slurry supernatant included in the anaerobic roll tube media. Colony counts with these media numbered 2 X 10(9) to 12 X 10(9)/g (wet weight), which represents about 20% of the microscopic counts. Lower percentages of slurry supernatant in the media gave lower colony counts. Addition of glucose, cellobiose, and starch or of Trypticase to media with 20% slurry supernatant did not increase colony counts. Higher values were obtained when hemicellulose preparations were added to these media. Incubation at 25 degrees C gave the highest numbers. Incubation at 15 to 37 degrees C gave counts of about 70 and 10%, respectively, of those at 25 degrees C. Of the colonies picked for isolation, about 20% were obtained in pure culture. The isolates apparently belonged to the genera Peptococcus, Ruminococcus, Peptococcus, Ruminococcus, Pepostreptococcus, and Bacteroides.     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria

Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.     

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations.

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria.     

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria.     

THE ESTIMATION OF SULPHATE-REDUCING BACTERIA (D. DESULPHURICANS)

SUMMARY: Sodium sulphide or cysteine stimulated the growth of sulphate-reducing bacteria; small populations often did not grow without such supplements. Ascorbic acid, glutathione or thiolacetic acid had similar properties but thiolacetic acid was sometimes inhibitory, Dilution counts in liquid media or colony counts in agar media did not bear any regular relation to the total count unless one of these supplements was present. With suitable precautions colony counts reaching 50 to 60% of the total count were obtained in media incorporating cysteine and a ferrous salt (as an indicator of sulphide formation).
Samples of natural origin containing sulphate-reducing bacteria gave greater viable counts in cysteine-iron media than in unsupplemented media. Blackend culture tubes with natural populations were sometimes due to cysteine-decomposing organisms; further examination of positive tubes was therefore necessary.     

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

Anaerobic Roll Tube Media for Nonselective Enumeration and Isolation of Bacteria in Human Feces

Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.     

A COMPARISON OF ROLL-TUBE AND PETRI DISH COLONY COUNTS ON RAW MILK

SUMMARY: A co-ordinated experiment was carried out at two centres, in which colony counts from raw milk samples determined by the roll-tube and Petri dish methods were compared. A bulk medium was standardized for both laboratories, as also were details of technique. Statistical analyses showed that roll-tube counts were generally lower than the corresponding Petri dish counts, but the difference varied considerably from milk to milk. The variation between replicate sub-samples was about the same for both methods. Experience and the results both indicate that roll-tubes were slightly more difficult to count than Petri dishes.     

Inhibitory effects of titanium (III) citrate on enumeration of bacteria from rumen contents.

Titanium citrate (TC) or L-cysteine-sodium sulfide was added as a reducing agent to buffers and agar media used for enumeration of bacteria from rumen contents of high-forage-fed steers. Approximately equal colony counts were found on TC and L-cysteine-sodium sulfide-reduced media with rumen contents taken 8 h postfeeding, when active bacterial growth was occurring. The colony counts on TC medium were only 56% of those with L-cysteine-sodium sulfide medium with rumen contents taken 1 h prefeeding when bacterial growth was minimal. When colonies from L-cysteine-sodium sulfide medium were transferred to TC medium and vice versa, almost all colonies grew. The data indicate that TC can be inhibitory to bacteria upon their initial isolation from natural habitats, particularly when growth rates are low in these habitats.     

Dyes as fungal inhibitors: effect on colony enumeration

The effects of three dyes on the colony enumeration of nine fungal strains (including members of the Deuteromycetes and Zygomycetes) in pure and mixed cultures were investigated. Using malt extract agar as basal and control medium, the following dyes and concentrations were assayed: auramine (25 ppm), gentian violet (5 ppm) and malachite green (1 ppm). The chemicals commonly used in commercial media dichloran (2 ppm) and rose bengal (50 ppm) were included in the study as reference mould-spreading inhibitors. Higher counts were usually obtained in the media containing dichloran, rose bengal or auramine, including the control medium in the absence of chemical when the mixed-conidium inocula did not include a spreading mould. Nevertheless in most cases no significant differences were observed between them. Malachite green (1 ppm) performed mainly as a strong inhibitor of spreading moulds, only allowing adequate colony development and recoveries of both Fusarium and Aspergillus strains tested.     

Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously “unculturable” organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

Isolation of Ureolytic Peptostreptococcus productus from Feces Using Defined Medium; Failure of Common Urease Tests

Colony counts of fecal samples from three persons, obtained by using a chemically defined anaerobic roll-tube medium (containing glucose, maltose, glycerol, minerals, hemin, B-vitamins, methionine, volatile fatty acids, sulfide, bicarbonate, agar, carbon dioxide (gas phase), and 1 mM NH(4) (+) as main nitrogen source), averaged 60% of the 8.8 x 10(10) bacteria per g obtained when 0.2% Trypticase and 0.05% yeast extract were added to the otherwise identical medium. When 0.2% vitamin-free Casitone replaced Trypticase and yeast extract, counts were 94% those of the more complex medium. When urea-nitrogen was added to the defined medium as the main nitrogen source in place of NH(4) (+), counts of relatively large colonies averaged 1.0 x 10(9) per g of feces from five persons-1.1% of counts on the medium containing Trypticase and yeast extract. All of the organisms from the large colonies in the urea roll tubes were morphologically similar, and all six representative strains isolated were identified as urease-forming Peptostreptococcus productus, a species not previously known to produce urease. Ureolytic strains of Selenomonas ruminantium and P. productus were negative for urease activity in three assay media when inocula were from media containing complex nitrogen sources. The study documents that P. productus is the most numerous ureolytic species so far found in human feces and suggests that NH(4) (+) and more complex organic nitrogen sources strongly repress its production of urease. The study also indicates the efficacy of chemically defined media for direct selective isolation of nutritional groups of bacteria from feces.     

Counting viable Azotobacter in vertisols

In counting Azotobacter in vertisols by the soil dilution and spread-plating method, mean colony counts/plate did not decrease in proportion to the dilution factor and consequently derived counts of Azobacter cells/g soil decreased with increasing dilution of the soil suspension. This non-proportionality phenomenon was analysed in several experiments with six soils. Conformity to the Poisson distribution for counts on parallel plates was measured by Fisher's index of dispersion (χ²), which proved too high (P<.05) at lower dilutions, indicating inaccurately low mean colony counts. At highest dilutions, proportional errors increased resulting in less precise estimates of means, because with a Poisson distribution the standard deviation is equal to the square root of the mean, and the multiplication factor for derived counts/g soil is greatest at highest dilutions. By varying both plate surface area and dilution factor, results indicated that the non-proportionality phenomenon is caused by crowding at lower dilutions increasing the probability of colony coincidence on the plates. Graphical analysis of results of several dilution series indicated that derived counts are best based on dilutions giving 10–40 colonies/9 cm diameter plate, which is best achieved from two-fold dilution series.     

Medium for the Enumeration and Isolation of Bacteria from a Swine Waste Digester

A habitat-simulating medium was developed for the enumeration and isolation of bacteria from a swine waste digester. A roll tube medium with growth factors for strict anaerobes from previously studied anaerobic ecosystems was used to evaluate the effects of deletion, addition, or level of digester fluid, digester fluid treated with acid or base, rumen fluid, fecal extract, anaerobic pit extract, tissue extract, carbohydrates, peptones, short-chain fatty acids, minerals, vitamins, N and P sources, reducing and solidifying agents, buffers, and gases on colony counts. Decreasing the agar concentration from 2.5 to 1.0% increased the counts twofold. Blending increased the counts 1.7-fold. With a medium (174) containing digester fluid, peptones, minerals, cysteine, sodium carbonate, and agar, colony counts were 60% of the microscopic count and improved yields 2.5 to 20 times those obtained with media previously used for digesters or developed for other anaerobic ecosystems. Colony counts continued to increase for up to 4 weeks of incubation. Medium 174 permits the enumeration of total, methanogenic, and, with deletion of reducing agent, aerotolerant bacteria. The results suggest that the predominant bacteria grow slowly and have requirements different from those of bacteria from other ecosystems.     

The Spleen Colony Technique. Iii. Comparison of the Overlap Effect and of Errors In Cfu-S Determination and the [3H]-Thymidine Suicide Data For Several Strains of Mice

A mathematical model of errors of the spleen colony technique is applied to data obtained from four mouse strains and F₁ hybrids. the variance of the colony counts was close to the Poisson distribution in inbred mice and F¹ hybrids. However, it should be checked regularly. the magnitude of the error in CFU-s determination and of the estimations of the S phase fraction was derived, and is presented relative to the mean colony counts for all mouse strains studied. the optimum spleen colony counts are generally higher than those which are commonly used. However, the utilization of the optimum spleen colony counts requires a correction for the effect of colony overlap.     

Medium Without Rumen Fluid for Nonselective Enumeration and Isolation of Rumen Bacteria

Colony counts which approximated those in a habitat-simulating, rumen fluid-agar medium (RFM) were obtained in medium 10, a medium identical to the RFM except for the replacement of rumen fluid with 1.5 x 10(-6)m hemin, 0.2% Trypticase, 0.05% yeast extract, and a 6.6 x 10(-2)m volatile fatty acid mixture qualitatively and quantitatively similar to that in rumen fluid. Single deletion of Trypticase, yeast extract, or the volatile fatty acid mixture from medium 10 significantly reduced colony counts. Colony counts were also reduced when medium 10 was modified to contain higher concentrations of Trypticase or volatile fatty acids. Significant differences were found between colony counts obtained from diluted rumen contents of animals fed a cracked corn-urea diet, and the colony counts obtained from animals fed either a cracked corn-soyean oil meal or an alfalfa hay-grain diet. Qualitative differences were found between the predominant bacterial strains isolated from rumen contents of animals fed cracked corn diets and strains isolated from animals fed alfalfa hay-grain. Regardless of differences in the predominant flora associated with diet, medium 10 and the RFM supported growth of similar bacterial populations. The results show that medium 10 is suitable for enumeration and isolation of many predominant rumen bacteria.