Colony-forming Curve Research Articles

Phylogenetic identity, growth‐response time and rRNA operon copy number of soil bacteria indicate different stages of community succession

Bacterial community succession corresponds to changes in the phylogenetic identity, growth-response time and rRNA operon (rrn) copy number of culturable populations. To test this hypothesis, we compared the bacterial fractions culturable from the oxic zone of flooded, unplanted paddy soil microcosms after 1-day (early succession) and 70-day (late succession) incubation periods. The proportion of bacteria that was cultivable on solid media corresponded for early and late succession to 37-40% and 31-35% of total DAPI cell counts, which were 7.40 (+/-0.36) x 10(8) and 5.54 (+/-0.28) x 10(8) cells per gram of dry soil, respectively. In colony-forming curve analysis, late successional bacteria showed a significant delay in their growth response compared with those from early succession. A total of 59 early successional isolates grouped into 16 species-level clusters (SLC) plus three Bacilli-like SLC, while 66 late successional isolates formed 25 SLC plus five Bacilli-like SLC. Except Bacilli-like spp., isolates from early succession always belonged to different SLC than those from late succession. Betaproteobacteria and Gammaproteobacteria were typical of the early stage, while Alphaproteobacteria and Actinobacteria prevailed in late succession. Considering all SLC except those assigned to Bacilli, growth-response time and rrn copy number were significantly correlated with successional stage. Isolates of most early successional SLC (14 of 16) formed visible colonies within 1 (11 SLC) or 2 days (three SLC) and contained >or= 4 rrn copies. In contrast, isolates of late successional SLC (23 of 25) formed visible colonies within 2 days (four SLC) or, in most cases, only within 3-15 days (19 SLC) and contained <or= 2 rrn copies. Regardless of whether isolated from early or late succession, Bacilli-like isolates always showed a colony-forming time of 2 days and had 9-11 rrn copies.

Growth strategy of heterotrophic bacterial population along successional sequence on spoil of brown coal colliery substrate

The bacterial population of brown coal colliery spoil (Sokolov coal mining district, Czechia) was characterized by measuring viable bacterial biomass, the culturable to total cell ratio (C : T), colony-forming curve (CFC) analysis and species and/or biotype diversity. Bacterial representatives that differed in colony-forming growth (fast and/or slow growers) were used for growth-strategy investigation of heterotrophic bacteria. Spoil substrates from the surface (0-50 mm) and the mineral (100-150 mm) layers were sampled on 4 sites undergoing spontaneous succession corresponding to 1, 11, 21 and 43 years after deposition (initial, early, mid and late stages). The bacterial biomass of the surface layer increased during the initial and early stages with a maximum at mid stage and stabilized in the late stage while mineral layer biomass increased throughout the succession. The maxima of C : T ratios were at the early stage, minima at the late stage. Depending on the succession stage the C : T ratio was 1.5-2 times higher in the mineral than the surface layer of soil. An increase in the fraction of nonculturable bacteria was associated with the late succession stage. CFC analysis of the surface layer during a 3-d incubation revealed that the early-succession substrate contained more (75%) rapidly colonizing bacteria (opportunists, r-strategists) than successively older substrates. The culturable bacterial community of the mineral layer maintained a high genera and species richness of fast growers along the succession line in contrast to the surface layer community, where there was a maximum in the abundance of fast growers in the early stage. There was a balanced distribution of Gram-positive and Gram-negative representatives of fast growers in both layers. A markedly lower abundance of slow growers was observed in the mineral in contrast to the surface layer. Gram-positive species dominated the slow growers at the surface as well as in the mineral layers. The growth strategy of the heterotrophic bacterial population along four successional stages on spoil of brown coal colliery substrate in the surface layer displayed a trend indicative of a r-K continuum in contrast to the mineral layer, where an r-strategy persisted.

Colony-Forming Analysis of Bacterial Community Succession in Deglaciated Soils Indicates Pioneer Stress-Tolerant Opportunists

We investigated the response of bacterial communities inhabiting two deglaciated soils (10 and 100 years post-deglaciation) to two stimuli: (i) physical disruption (mixing), and (ii) disruption plus nutrient addition. PCR/DGGE analysis of 16S rRNA genes extracted from soil during a 168-h incubation period following the stimuli revealed that more bacterial phylotypes were stimulated in the 10-y soil than in the 100-y soil. In addition to 10-y and 100-y soils, two additional soils (46 and 70 y) were further differentiated using colony-forming curve (CFC) analysis during a 168-h incubation period, which revealed that younger soils contained a higher proportion of rapidly colonizing bacteria than successively older soils. "Eco-collections" of CFC isolates that represented colonies that formed "fast" (during the first 24 h) and "slow" (final 36 h) were harvested from 10-y and 100-y soils and differentiated according to response to three stress parameters: (i) tolerance to nutrient limitation, (ii) tolerance to temperature change, and (iii) resistance to antibiotics. The tested parameters distinguished "fast" from "slow" bacteria regardless of the age of the soil from which they were isolated. Specifically, eco-collections of "fast" bacteria exhibited greater nutrient- and temperature-stress tolerance as well as more frequent antibiotic resistance than "slow" bacteria. Further DGGE analysis showed that several eco-collection phylotype bands matched (electrophoretically) those of soil phylotypes enriched by mixing and nutrient stimulus. Overall, the results of this study indicated that the succession of colony-forming bacteria was differentiated by bacterial opportunism and temporal response to stimuli. Furthermore, although stress tolerance strategies are associated with opportunistic bacteria regardless of successional age, it appears that the proportion of opportunistic bacteria distinguishes early vs late succession forefield bacterial populations.

Incubation time and media requirements of culturable bacteria from different phylogenetic groups

Previously, time courses of the colony formation process of soil bacteria on agar plates were simulated by colony-forming curves (CFCs). Bacteria were isolated from the colonies that appeared during an incubation period corresponding to each component curve of the CFC, respectively, and they were placed into groups I, II, III or IV, which were based on the order of colonization, so that their physiological characteristics could be studied (Gorlach, K. et al. (1994) J. Gen. Appl. Microbiol. 40, 507–517). We report here on the results of phylogenetic analysis of the isolates from each CFC group. A wide variety of eubacteria were included in the collection. Strains closely related to Arthrobacter were those from CFC groups I or II, groups of faster growers, and strains in Proteobacteria α-subdivision were those from groups III or IV, which grew more slowly. In contrast, some phylogenetic groups, such as Firmicutes low G+C, Cytophaga/Flexibacter/Bacteroides and Proteobacteria β-divisions exhibited heterogeneity regarding CFC grouping. As for their requirement for media, isolates belonging to the Cytophaga/Flexibacter/Bacteroides division, the Proteobacteria α-subdivision, β-subdivision and the Firmicutes high G+C division, except for Arthrobacter-related strains, were oligotrophic bacteria. In contrast, those belonging to the Firmicutes low G+C division, the Proteobacteria γ-subdivision and the A. globiformis-related group in the Firmicutes high G+C division were not oligotrophic.

Advances in soil microbial ecology and the biodiversity.

Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection (EC). Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections. A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.

Effects of soil compaction on the microbial populations of melon and maize rhizoplane

Effects of soil compaction on the microbial populations of melon and maize rhizoplane were investigated in quantity and quality. The numbers of culturable bacteria and fluorescent pseudomonads on the rhizoplane were higher when plants were grown in more compacted soil and the relative increase was larger in fluorescent pseudomonads. Total bacterial counts, however, did not appear to be affected by soil compaction, resulting in the increase in the culturable bacteria among total counts in more compacted soil. The determination of extracellular enzymatic properties (pectinase, α-glucosidase, β-glucosidase and α-galactosidase) of each 100 isolates from bulk soil and root samples suggested that the microbial populations on the rhizoplane, especially when plants were grown in highly, compacted soil, were composed of high ratios of bacteria with abilities to utilize root exudates efficiently. The microbial community structure estimated from the colony forming curves of bulk soil and root samples suggested that the microbial populations on the rhizoplane, especially when plants were grown in compacted soil, were likely to be composed of more r-strategists which were defined as those who formed colonies within 2 days.

Enrichment culture and isolation of slow-growing bacteria

Enrichment containing large numbers of slow-growing bacteria was developed by repeated batch culture under high biomass concentrations (more than 10 000 mg biomass/l). The characteristics of slow-growing bacterial populations were elucidated by application of colony-forming-curve (CFC) analysis. The CFC were obtained by counting the number of visible colonies on agar plates at successive intervals. The enrichment consisted of several groups with different colony-forming rates and the slow-growing bacteria appeared on cell extract/agar plates after 7 days of incubation. It was found that large numbers of slow-growing bacteria survived under starvation conditions. One of the slow colony-forming bacteria, strain TI-X7, was tentatively identified as being of the genus Micrococcus. The enrichment contained a large amount of Micrococcus-like tetrad cells. The dialysate fractions in excess cell extract, permeable through dialysis tubing, were extremely effective for growth of strain TI-X7.

Analysis of arsenic-tolerant bacterial populations in a paddy field soil according to rates of development on solid media.

The colony-forming process of bacteria in a paddy field soil on 100-fold diluted nutrient broth (DNB) plates containing As(III) (250-1,000 ppm) was analyzed by the first-order reaction (FOR) model. The process on plates with 250 ppm As(III) was simulated by a superimposition of three FOR model curves, which are respectively referred to as component colony-forming curve (cCFC) I, II, or III. Bacterial isolates from the 250 ppm As(III) plates were divided into three groups according to cCFC, along which each respective bacterium produced its colony. The three cCFC groups showed different trends of physiological properties : (1) the range of t r , which is the waiting time for the appearance of the first colony of each single population in the presence or absence of As(III), (2) the range of the maximal tolerant As(III) concentration for each isolate and some of its taxonomic properties. Twelve out of 72 strains examined harbored one or more plasmids, suggesting location of As(III)-tolerant genes on the chromosomal DNA.

Comparative Study of Bacterial Populations in a Grassland Soil in 1987 and 1992.

Bacterial population in grassland soil (sampled in 1992) was studied in terms of component colony-forming curves (cCFCs) to be compared with that in soil sampled in 1987. The simulated colony-forming curve (CFC) pattern was essentially similar to that of the former study; that is, four cCFCs were observed during 163h of incubation time. Bacterial isolates were picked up from colonies appearing along each cCFC and clustered according to their morphological and physiological characteristics (biotypes). The histograms of the frequency distribution of tr value for the isolates corresponding to cCFC groups I, II and III showed a similar pattern as previously. The trend of appearing of oligotrophic bacteria among the isolates of cCFCs groups was also similar to that of the former study although the percent of oligotrophic bacteria differed; that is, oligotrophic bacteria were not found among isolates of cCFCs groups I and II, appeared at 30% in cCFC group III and increased to 68% in cCFC group IV; as the oligotrophic bacteria increased, the sensitivity to 1% NaCl also increased. The bacterial population of the grassland soil is considered to be comprised of at least four component groups (cCFC groups) through yearly variations, although the biotype composition of the respective groups varies either widely or narrowly.

Soil aggregates as microcosms of bacteria—protozoa biota

Forty-eight air-dried soil aggregates were separately incubated under alternately submerged and dry conditions at 20°C without adding exogenous nutrients. In the course of submersions, the population dynamics of bacteria and protozoa were followed in each aggregate. It was shown that every aggregate accommodated at least three major groups of bacteria as categorized on the basis of colony forming curve (CFC) analysis. The growth of these bacterial groups occurred in sequence during submersion. Three to fourteen protozoan groups from each aggregate were detected as active forms. Distribution patterns of these groups among the 48 aggregates differed. The number of groups which were active simultaneously in each aggregate were small and the composition of groups changed gradually in the course of submersion. Frequency of appearance of each group during submersion was different and most groups appeared sporadically. These observations suggest that, in spite of the microbial diversity among aggregates, only a few groups are active concurrently.

Examination of Bacterial Characteristics of Anaerobic Membrane Bioreactors in Three Pilot-Scale Plants for Treating Low-Strength Wastewater by Application of the Colony-Forming-Curve Analysis Method

Characteristic sludge ecosystems arising in anaerobic membrane bioreactors of three pilot-scale plants treating low-strength (less than 1 g of biological oxygen demand per liter) sewage or soybean-processing wastewater were examined by analysis of the colony-forming-curves (CFC) obtained by counting colonies at suitable intervals. The wastewaters, containing high amounts of suspended solids (SS) (SS/chemical oxygen demand ratio, 0.51 to 0.80), were treated by using two types of bioreactors: (i) a hydrolyzation reactor for solubilization and acidification of SS in wastewater and (ii) a methane fermentation reactor for producing methane. The colony counts for the two sewage treatment plants continued to increase even after 3 weeks of incubation, whereas those for soybean-processing wastewater reached an approximately constant level within 3 weeks of incubation. The CFCs were analyzed by correlating the rate of colony appearance on roll tubes with the physiological types of bacteria present in the bioreactors. It was found that there were large numbers of slow-colony-forming anaerobic bacteria within the bioreactors and that the viable populations consisted of a few groups with different growth rates. It is considered that the slow-growing colonies appearing after 10 days of incubation were the dominant microflora in the sewage treated by hydrolyzation reactors. In particular, highly concentrated sludge (30.0 g of mixed-liquor volatile SS per liter) retained by the membrane separation module contained a large number of such bacteria. Slow-growing colonies of these bacteria could be counted by using a sludge extract medium prepared from only the supernatant of autoclaved sludge. In addition, the highest colony counts were almost always obtained with the sludge extract medium, meaning that most of the anaerobic bacteria in these sludges have complex nutrient requirements for growth. This report also indicates the usefulness of application of the CFC analysis method to the study of bacterial populations of anaerobic treatment systems.

Evaluation of nutrient agars for the enumeration of viable aerobic heterotrophs in cooling water

The agar plate method is still the method of choice for quantifying viable bacteria in water, especially in routine laboratories. Eleven nutrient agar media were compared for colony forming ability by incubating at 25 and at 30°C. Appearance of colonies was recorded against time and colony forming curves were constructed. The highest counts after 5 days ( c. 75% of maximum count) were obtained on R3A agar incubation at 30°C. R3A agar at 30°C also allowed the most rapid colony development, with most of the colonies appearing between 2 and 5 days. Both plate count agar (PCA) and standard I Agar (Std I) performed poorly. PCA yielded 30% of the highest count after 5 days at 30 and at 25°C. Std I yielded 27% at 30°C and 23% at 25°C. The standard procedure of 48 h at 30°C on PCA yielded 14% of the final highest count, i.e. one log less. In contrast tc PCA and Std1, R3A agar contained magnesium and pyruvate, both of which were shown to increase the plate count. R3A also had a lower nitrogen content and nitrogen to carbon ratio. The highest count after 14 days was achieved on double strength R3A (R4A) at 20°C. R4A also showed the highest colony diversity. It was therefore seen to be the most suitable for population studies. However, colony development was slower. This makes it unsuitable for plate counts, whereas R3A lets most cells develop into colonies within the first 5 days of incubation.

Analysis of bacterial populations in a grassland soil according to rates of development on solid media

The process of colony formation by bacteria from grassland soil sampled in April, July and September was simulated by a colony-forming curve (CFC). The CFC was a super-imposition of several component curves (cCFC) given theoretically by the first order reaction (FOR) model [3,6]. The pattern of FOR model curves was not influenced by the time of sampling and four cCFCs were always recognized during an incubation period of 160 h. It was considered that the CFC describes an inherent property of the bacterial population of the field. Bacterial isolates were obtained from colonies produced in each of four cCFCs on agar plates. Isolates corresponding to one cCFC were classified as one group. The bacterial isolates were characterized by morphological and physiological tests and subsequently clustered. Few oligotrophic bacteria were obtained among bacteria which produced visible colonies within 63 h of incubation time. On the other hand, approx. 50% of bacteria which produced v colonies after 63 h were oligotrophic bacteria. The time required for the appearance of the first colony, tr of the FOR model, was very similar in the isolates belonging to one group. A close linear relationship was observed between tr value and doubling time of isolates.

Analysis of bacterial populations in a grassland soil according to rates of development on solid media

The process of colony formation by bacteria from grassland soil sampled in April, July and September was simulated by a colony-forming curve (CFC). The CFC was a super-imposition of several component curves (cCFC) given theoretically by the first order reaction (FOR) model [3,6]. The pattern of FOR model curves was not influenced by the time of sampling and four cCFCs were always recognized during an incubation period of 160 h. It was considered that the CFC describes an inherent property of the bacterial population of the field. Bacterial isolates were obtained from colonies produced in each of four cCFCs on agar plates. Isolates corresponding to one cCFC were classified as one group. The bacterial isolates were characterized by morphological and physiological tests and subsequently clustered. Few oligotrophic bacteria were obtained among bacteria which produced visible colonies within 63 h of incubation time. On the other hand, approx. 50% of bacteria which produced v colonies after 63 h were oligotrophic bacteria. The time required for the appearance of the first colony, t r of the FOR model, was very similar in the isolates belonging to one group. A close linear relationship was observed between t r value and doubling time of isolates.

Grouping of soil bacteria by analysis of colony formation on agar plates

The colony formation of soil bacteria was studied in relation to incubation time. The process was simulated by a colony-forming curve (CFC) which was a superposition of several component curves (cCFC) given theoretically by the first-order reaction (FOR) model. This model had been previously proposed to define the colony formation of cells of a single culture. Soil bacteria were divided into several groups by these cCFC, as different types of bacteria produced their own colonies. Bacteria belonging to a single group grew at a similar rate on the plating medium and each group was characterized by a different growth rate. Most copiotrophic bacteria were fast growers and most oligotrophic bacteria slow growers.

Analysis of Colony Forming Curves of Soil Bacteria

By observing of the colony formation of soil bacteria on a plate, we found that the colony forming curves of soil bacteria were remarkably different from those of pure cultured bacteria; the colony forming curves of the soil bacteria showed several tiers of curves: in the case of upland and paddy soil bacteria, the curves showed 2 and 3 tiers, respectively. We obtained the isolates from the colonies composing each tier of curve and studied the physiological properties by using the first order reaction (FOR) model. Each tier of the colony forming curve was found to be composed by the group having a similar growth rate. The ratio of the population size and the difference of growth rate of each group were analyzed by the superposition of FOR models. Parameter tt of the FOR model was considered to reflect directly the growth rate.

Detection of Proliferating Bacteria in Soil Populations by the Analysis of Colony-Forming Curves

Detection of Proliferating Bacteria in Soil Populations by the Analysis of Colony-Forming Curves