News & Notes: Detection of Microorganisms with Overall Cellulolytic Activity

A modification is described of the plate method for the detection of microorganisms with overall cellulolytic activity, including those like Cytophaga, in which the activity is cell bound. Within a few days of incubation colonies of cellulose-degrading bacteria formed holes in discs of lens paper placed on freshly inoculated agar plates. Received: 14 June 1999 / Accepted: 20 July 1999     

Chromium(VI) and apparent phenotypic reversion in Salmonella TA100

After treatment with potassium chromate at concentrations causing ultramicroscopic cellular lesions, a significant proportion (up to 75%) of TA100 colonies fail to replicate on fresh minimal plates containing biotin. This suggests that chromium(VI) may not always induce his- reversion to his+ in Salmonella TA100. The terms 'false' or phenotypic reversion have been used to distinguish such instances from 'true' or genotypic reversion, where progeny his+ cells readily grow on biotin replica plates. Results of the present study indicate that the majority of chromate-exposed colonies, initially scored as his-, are identifiable as his+ after 24 h culture on nutrient agar. Moreover, chromate exerts a cytostatic effect on TA100 since early colony development is suppressed at high chromate concentrations. A gradual chemical reduction of chromium(VI) ions by normal media compounds is probably responsible for the re-emergence of colony growth during prolonged incubation of test plates. Thus, temporary growth inhibition at high chromate concentration appears to be responsible for most of the non-replicating colonies detected in mutagenicity assays of chromium(VI).     

Gentamicin as an antibiotic in dilution plates for the isolation of soil fungi

Summary Gentamicin is found quite satisfactory as an antibiotic in dilution plates for the isolation of soil fungi. An increase in number of fungal colonies is also noticed on media containing gentamicin.     

Efficiency of various selective media in determining Pythium population in soil

Of 15 selective media recommended for isolation and enumeration ofPythium spp. directly from soil, corn meal agar (CMA) supplemented with agar, sucrose, minor elements, thiamine, rose bengal, pimaricin, pentachloronitrobenzene and vancomycin (MPVM) was the most efficient. Streptomycin (30–50 ppm) and rose bengal (33–60 ppm) as used in certain tested media effectively suppressed development of bacteria and actinomycetes. However, these chemicals adversely affected germination of spores and mycelial growth and thereby the recovery ofPythium spp. from soil. Media containing pimaricin (5 to 100 ppm) were more effective than those with nystatin (40 ppm) in suppressing development of nonphycomycetous fungi on isolation plate. MPVM with pimaricin at 5 ppm was more efficient than that with 10 ppm of the antibiotic in recoveryingPythium from soil. However, there was no difference in recovery ofPythium by this medium containing rose bengal at 5 ppm or at 10 ppm, butPythium colonies were more dense and better delineated when the medium contained 10 ppm of rose bengal. CMA containing pimaricin (5–100 ppm) and vancomycin (200 ppm) permitted occasionally development of a large number ofMortierella and bacterial colonies from certain soils, that interfered with accurate determination of colonies of certainPythium spp. on the plates. Vancomycin at 300 ppm, as used in MPVM, substantially reduced development of bacterial colonies compared to 200 ppm of the antibiotic. Surface-soil dilution-plate was more effective than the soil-dilution-plate method in reducing bacteria andMortierella colonies on isolation plates without affecting recovery ofPythium. The importance of basal medium, complement of antimicrobial agents, and isolation methods for efficiency of selective medium in recovery ofPythium spp. directly from soil is discussed.     

Effect of selective media on loss of congo red binding inShigella flexneri

Summary The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.     

Role of contact in bacterial degradation of cellulose

Abstract Bacterial cells can adhere to cellulose fibres, but it is not known if cell-to-fibre contact is necessary for cellulose degradation. This problem was explored using aerobic cellulolytic bacteria, including known species and new isolates from soil. These were tested on plates containing Avicel, Solka floc, CF11 cellulose, carboxymethyl cellulose, or phosphoric acid-treated cellulose. Cellulose degradation was measured both by formation of clearing zones and by growth when cellulose was the only carbon source. The bacteria tested were either inoculated directly on the cellulose-containing agar, or separated from it by a pure agar layer or by membrane filters (not containing cellulose). Even when separated from the cellulose-containing agar all strains grew well. Clearing zones, best seen in phosphoric acid-treated cellulose, were larger under colonies separated from cellulose by an agar layer than under those in direct contact with cellulose. Such zones could also appear under filters. Our results show that bacterial degradation of cellulose does not depend on cell-to-fibre contact and suggest that when cellulose is at a greater distance from the cell, the removal of end products reduces catabolite repression of cellulose formation.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

Isolation of cellulolytic mesophilic clostridia from a municipal solid waste digestor

Ten obligately anaerobic, cellulolytic mesophilic bacteria were isolated from a municipal solid waste digestor used for biogas production. The isolates were rod-shaped, spore-forming bacteria in anaerobic conditions, and stained Gram-positive in young cultures, and hence were identified asClostridium. Small regular translucent and unpigmented colonies were observed on cellulose plates. The strains were gelatinase-negative, hydrolyzed esculin and starch, and fermented xylose and arabinose. The lecithinase, lipase, and indole tests were negative. The major fermentation products from cellulose included ethanol and acetate. The morphological and other biochemical characteristics indicated that these clostridia did not correspond to any previously described species. All the strains produced high activities of extracellular cellulases in cellulose media and degraded paper. Offprint requests to: L. Benoit.     

Morphogenesis in cell colonies grown from Convolvulus cell suspensions plated on synthetic media

Filtered cell suspensions of cultured callus tissue derived from the roots of Convolvulus arvensis L. were plated out on synthetic agar nutrient media in petri plates. Cell colonies which formed from the single cells or small cell groups in the suspension showed a considerable range of developmental patterns depending upon the physical and chemical environment to which they were exposed. Variation of the auxin and kinin concentrations and the nature and concentration of the source of reduced N compounds had the most profound effects on colony development. High auxin favored cell enlargement, high kinin favored the development of compact colonies composed of many small cells. Both auxin and kinin were required for cell colony formation. Cell differentiation responses which were observed but not subject to experimental control included formation of starch- and crystal-storing cells, differentiation of tracheary elements, formation of cellular filaments, and development of chlorophyllous tissue. Organ initiation was studied in cell colonies developed directly from plated cell suspensions and in cell colonies subcultured on various nutrient media. Bud initiation was produced repeatedly on media containing NAA at 10^-8 to 10^-6 m combined with kinetin at 10^-6 m . Root initiation was induced infrequently and unpredictably. Once roots had been formed from cell colonies derived from cell suspensions, the roots could be subcultured and induced to form buds; these in turn grew into whole plants. Subculture of young cell colonies to media containing different combinations of growth substances made possible a study of the effects of auxin and kinin on organization of primordia by the cell colonies. By following marked single cells plated on synthetic media, it was possible to produce single-cell clones which under proper nutrient conditions were induced to form buds. The value of the combined techniques of cell suspension culture and cell plating for the study of the physical and chemical factors influencing cell differentiation and organized development are pointed out.     

A STUDY OF SOIL BACTERIA DISSOLVING CERTAIN MINERAL PHOSPHATE FERTILIZERS AND RELATED COMPOUNDS

SUMMARY: Over a hundred isolates which produced haloes around their colonies on dilution plates containing calcium carbonate or dicalcium phosphate have been obtained in pure culture from the root region of the oat plant. Of these, more than 50% were pleomorphic, and this group had the highest proportion of isolates which could produce clear zones on agar media containing either calcium carbonate, dicalcium phosphate, tricalcium phosphate, freshly precipitated hydroxyapatite or basic slag. None of the isolates showed dissolving ability on agar media containing gafsa rock phosphate, variscite, strengite or taranakite. However, when an analytical method was used, 82% of the isolates tested proved able to release phosphate from gafsa rock phosphate, though to a much lesser extent than from dicalcium phosphate. None of the isolates tested by this method released phosphate from variscite, strengite or taranakite.
The nature of the organic acids produced from glucose by 26 of the isolates was also investigated. The majority produced mainly lactic acid, but a few also gave an acid with chromatographic properties similar to those of 2-keto-gluconic acid.     

Rapid method for selecting appropriate solid media for the enumeration of aerobic micro-organisms

A quick and cheap method for selecting appropriate solid culture media has been devised. It consists in the rapid picking of fragments of test colonies with the aid of a rubber strip in which pins are fixed in parallel, dispensing up to 8 colonies simultaneously in the wells of a Microtiter plate and streaking 4 strains at the same time on square Petri dishes containing the media under comparison. The approximate diameters of well-isolated colonies are measured with the aid of a series of calibrated spots. The results corresponded with those given by the spiral plate method used as a reference for colony count and diameter measure.     

Rapid method for selecting appropriate solid media for the enumeration of aerobic micro-organisms

A quick and cheap method for selecting appropriate solid culture media has been devised. It consists in the rapid picking of fragments of test colonies with the aid of a rubber strip in which pins are fixed in parallel, dispensing up to 8 colonies simultaneously in the wells of a Microtiter plate and streaking 4 strains at the same time on square Petri dishes containing the media under comparison. The approximate diameters of well-isolated colonies are measured with the aid of a series of calibrated spots. The results corresponded with those given by the spiral plate method used as a reference for colony count and diameter measure.     

Effects of medium composition on the recovery of bacteria from sea water

Water samples were collected from offshore and inshore localities at various depths off the Connecticut coast over a two-year period. Spread plates for bacterial counts at 20 °C were made on a variety of complex solid media. Counts on Difco-Marine Agar were controls in all cases with counts on test media related to these in ratio form. Initially, nine media were used and represented some from the literature as well as personal formulations. Differences between inshore and offshore samples were greatest with media containing the highest peptone concentrations. Two media containing the peptones Gelysate and Trypticase showed the highest overall counts. A second phase concerned a comparative study of these peptones varying in concentration from 0.1 to 10.0 g/l in a constant basal medium. None of the media invariably gave counts greater than the control, but peptone concentrations of 10.0 and 5.0 g/l resulted in the lowest comparative counts. Considering all samples, peptone levels of 0.1 and 1.0 g/l showed the highest counts. Counts for both inshore and offshore water samples decreased as peptone concentration increased. Qualitatively, high peptone media showed large, mucoid, confluent colonies which made the counting of smaller ones difficult. Pigmented colonies were more frequent on low peptone media. Bacteria were isolated from all media and from all stations; the percentage of various groups varied with peptone concentration and source of sample.Media containing three fish peptones in varying concentrations have also been investigated. None produced overall counts greater than Difco-Marine Agar and counts decreased with increasing peptone levels: there was a trend towards higher counts in offshore waters with fish extracts. Quantitative and qualitative aspects of the work are discussed.     

Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets

Abstract Samples of rumen ingesta from two rumen-fistulated dairy cows fed grass silage-based diets were examined for numbers and types of bacteria that developed colonies on rumen fluid-agar media designated to support the growth of (a) a wide range of species, (b) cellulolytic bacteria, (c) lactate-fermenting bacteria, (d) non-fermentative bacteria. The most numerous species was Bacteroides ruminicola followed by Butyrivibrio fibrisolvens . The most abundant cellulolytic species were Eubacterium cellulosolvens and Ruminococcus flavefaciens. Megasphaera elsdenii and Selenomonas ruminantium were important lactate fermenters but an unidentified bacterium that grew poorly on maintenance medium was by far the most numerous among bacteria isolated from lactate-containing medium. One strain remained sufficiently viable to show that it fermented lactate to propionate and acetate.     

Cloning and characterization of endosymbiotic algae isolated fromParamecium bursaria

Summary The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 10⁷ algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 10³ algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.     

盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

Ethanol production from cellulose by a coculture ofZymomonas mobilis and a clostridium

Zymomonas mobilis and a mesophilic cellulolytic clostridium (strain C7) were grown in coculture in a medium containing cellulose as fermentable substrate. The coculture was stable through at least ten serial transfers and produced markedly higher amounts of ethanol than monocultures of the cellulolytic clostridium. Glucose and cellobiose, derived from the breakdown of cellulose, accumulated in strain C7 monocultures, but not in cocultures. The molar ratio of ethanol to acetate was higher in cocultures than in monocultures of strain C7. The cellulolytic clostridium was relatively ethanol-tolerant, inasmuch as it grew and fermented cellulose in media containing up to 7 g of ethanol/100 ml. Cellulase (Avicelase) activity of strain C7 was inhibited by cellobiose, but not by glucose.     

Effect of pH on isolation and distribution of members of subdivision 1 of the phylum Acidobacteria occurring in soil

The pH strongly influenced the development of colonies by members of subdivision 1 of the phylum Acidobacteria on solid laboratory media. Significantly more colonies of this group formed at pH 5.5 than at pH 7.0. At pH 5.5, 7 to 8% of colonies that formed on plates that were incubated for 4 months were formed by subdivision 1 acidobacteria. These colonies were formed by bacteria that spanned almost the entire phylogenetic breadth of the subdivision, and there was considerable congruence between the diversity of this group as determined by the cultivation-based method and by surveying 16S rRNA genes in the same soil. Members of subdivision 1 acidobacteria therefore appear to be readily culturable. An analysis of published libraries of 16S rRNAs or 16S rRNA genes showed a very strong correlation between the abundance of subdivision 1 acidobacteria in soil bacterial communities and the soil pH. Subdivision 1 acidobacteria were most abundant in libraries from soils with pHs of <6, but rare or absent in libraries from soils with pHs of >6.5. This, together with the selective cultivation of members of the group on lower-pH media, indicates that growth of many members of subdivision 1 acidobacteria is favored by slightly to moderately acidic growth conditions.     

Optimization and characterization of extracellular cellulase produced by Bacillus pumilus MGB05 isolated from midgut of muga silkworm (Antheraea assamensis Helfer)

Mature larvae of Antheraea assamensis were collected from different locations of Assam to isolate the cellulolytic gut microflora. Altogether sixty cellulase degrading bacteria were isolated on agar plates containing microcrystalline cellulose as the sole carbon source. Among them, ten isolates showed hydrolyzing zone on agar plates containing carboxy methyl cellulose (CMC) after staining with Congo-red. Isolate MGB05 exhibited the highest CMCase activity (0.262?U/mL) at 72?h of incubation under submerged condition. FPase and β-glucosidase activity were 0.012?U/mL and 3.71?U/mL respectively. It showed maximum FPase (0.022?U/mL) activity on the 3rd day of incubation in the media containing wheat bran as a carbon source. β-glucosidase production was also found to be highest with wheat bran (20.03?U/mL) at 48?h of incubation. The optimum pH and temperature of FPase activity of MGB05 were found at 6.0 and 50?°C respectively while for β-glucosidase activity, it was maximum at pH?6.0 under 50?°C. In addition, metal ion Mg⁺⁺ and Ca⁺⁺ enhanced FPase activity up to 110.92% (0.026?U/mL) and 105.31% (0.025?U/mL) respectively. In-vitro antimicrobial bioassay of the most potent cellulolytic bacteria (MGB05) also showed high antimicrobial activity against Escherichia coli (2.9?cm) and Pseudomonas aeruginosa (3.0?cm). The isolate MGB05 has been identified based on 16S rDNA homology as Bacillus pumilus MGB05 with accession KP298708.2. Results encompass the prospective beneficial role of gut-microflora on digestion and disease resistance, which might be a potential probiotic component to enhance silk productivity.     

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining.

In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white.