The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus

Spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus were isolated on 4% (w/v) Tryptone Soya Agar containing 4 and 40 times the m.i.c. for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3% (w/v) Tryptone Soya Broth for 24 h at 37 degrees C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than the parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 45-83% that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacrylamide gel electrophoresis of the parent and mutant organisms of both strains.     

Method for Rapid Detection of Cyanogenic Bacteria

An agar plate method is described in which the production of hydrogen cyanide by as many as 50 microbial isolates per plate may be detected. Cyanide produced by the organisms reacts with copper(II) ethylacetoacetate and 4,4′-methylenebis-(N,N-dimethylaniline) in a paper disk suspended above the microbial colonies. Cell growth occurs in depressions in the agar surface, which allows separation of colonies and enhances sensitivity of hydrogen cyanide detection.     

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.     

Recruitment and Succession in a Tropical Benthic Community in Response to In-Situ Ocean Acidification

Ocean acidification is a pervasive threat to coral reef ecosystems, and our understanding of the ecological processes driving patterns in tropical benthic community development in conditions of acidification is limited. We deployed limestone recruitment tiles in low aragonite saturation (Ω_arag) waters during an in-situ field experiment at Puerto Morelos, Mexico, and compared them to tiles placed in control zones over a 14-month investigation. The early stages of succession showed relatively little difference in coverage of calcifying organisms between the low Ω_arag and control zones. However, after 14 months of development, tiles from the low Ω_arag zones had up to 70% less cover of calcifying organisms coincident with 42% more fleshy algae than the controls. The percent cover of biofilm and turf algae was also significantly greater in the low Ω_arag zones, while the number of key grazing taxa remained constant. We hypothesize that fleshy algae have a competitive edge over the primary calcified space holders, coralline algae, and that acidification leads to altered competitive dynamics between various taxa. We suggest that as acidification impacts reefs in the future, there will be a shift in community assemblages away from upright and crustose coralline algae toward more fleshy algae and turf, established in the early stages of succession.     

Development ofPuccinia striiformis West. on nutrient agar

Uredospores of a barley isolate ofPuccinia striiformis West. were germinated on glucose peptone agar and the development of colonies was observed with a microscope. A small number (0.1–1%) of germ tubes did not abort during the first 20 h of incubation and either differentiated an infection structure or continued slowly to elongate for several days. In 4 out of 9 experiments some of these sporelings produced colonies that reached maximum size (0.15–0.20 mm) after 3 weeks at 15° C.     

Diffusion in immobilized-cell agar layers: influence of microbial burden and cell morphology on the diffusion coefficients ofl-malic acid and glucose

Summary The diffusivities ofl-malic acid and glucose in an agar membrane entrapping small amounts ofEscherichia coli orRhodospirillum rubrum whole cells were measured using time lag (TL) and steady state (SS) methods. Diffusivities were overestimated by the SS method. For concentrations of immobilizedR. rubrum cells ranging between 10⁴ and 10⁹ organisms cm^–3 agar (20 ng-2 mg dry weight cm^–3 agar), the diffusion coefficient ofl-malic acid, determined by both methods, was related to the logarithm of the membrane cell content by a decreasing linear relationship. The diffusion coefficient of glucose obtained by TL analysis was not significantly affected by the presence in the membrane of 3 ng-0.3 mg dry wt.E. coli cm^–3 agar. However, values arising from the SS method decreased linearly as a function of the amount of immobilized organisms. Membranes containingR. rubrum cells offered higher diffusional resistance tol-malic acid and glucose than those loaded with the same amount ofE. coli cells.     

Spontaneous mutants of Microsporon gypseum resistant to griseofulvin

Summary 1) The spores of the microconidial mutant I–18 of the dermatophyteMicrosporon gypseum in agar medium with GF germinated and formed germ tubes deformated in a characteristic way. From 1µg GF/ml up with an increasing antibiotic concentration (expressed in logarithms) the munber of colonies grown (expressed in probits) decreased linearly.2) As a sensitivity measure of the spores the median efficient dose ED 50 was used which was determined by means of a graphic probit analysis. For the strain used this value was determined in the range between 1.35–1.95µg GF/ml in three independent experiments.3) From the smears of a thickened spore suspension (1.6–14.2 × 10⁷ viable spores) in medium containing a high GF concentration a very small, but as for the order a stable number of colonies grew, as found in eight independent experiments. On the medium containing 20µg GF/ml in average 61 colonies grew, on 40µg GF/ml 20 colonies, on 80µg GF/ml 3 colonies and on 160µg GF/ml 0.3 colony (expressed in 10⁷ viable spores tested).4) A part of these colonies were isolated and transferred 29 times on a medium without the antibiotic. Two isolates only show a permanently increased resistance to GF, viz. the strain D-29 which is 50 × more resistant and the strain N-53 which is 3.5 × more resistant than the wild strain I-18.     

The relationship between cell size and viability of soil bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (dwarf) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4m, about 10% for cells with diameters between 0.4 and 0.6m, and 30–40% for cells with diameters above 0.6m. Only 10–20% of the viable cells with diameters <0.4m increased their diameter to >0.4m prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.     

Heat-induced injury inListeria monocytogenes

Summary Heating ofListeria monocytogenes (Scott A strain) in potassium phosphate buffer (0.1 M, pH 7.2) at 52°C for 1 h led to injury, with the heat-injured cells failing to produce colonies on agar medium containing 5% NaCl. The detection of injury was based on the use of differential media: plating on tryptose phosphate broth+2% agar and 1% sodium pyruvate (TPBA+P) and on tryptose phosphate broth+2% agar and 5% NaCl (TPBA+S). Only non-injuredListeria formed colonies on TPBA+S whereas both heat-injured and non-injured cells formed colonies on TPBA+P. The bacterial count on TPBA+P minus that on TPBA+S represents the extent of heat injury. A large number of selective agars were tested and compared to TPBA+P for their ability to support repair and colony formation of heat-injuredL. monocytogenes. Media containing 0.025% phenylethanol, 0.0012–0.0025% acriflavin, 0.1–0.2% potassium tellurite, 0.001% polymyxin B sulfate, 5% NaCl or a combination of these ingredients were detrimental to the recovery of heat-injuredL. monocytogenes. Media currently in use forL. monocytogenes are not satisfactory for the recovery of injured cells.     

Succession of Indigenous Pseudomonas spp. and Actinomycetes on Barley Roots Affected by the Antagonistic Strain Pseudomonas fluorescens DR54 and the Fungicide Imazalil

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.     

Isolation of halophilic and halotolerant bacteria from a Japanese salt field and comparison of the partial 16S rRNA gene sequence of an extremely halophilic isolate with those of other extreme halophiles

Halophilic and halotolerant bacteria were isolated from soil samples of a Japanese salt field, an environment where salt concentrations vary annually. From 1 g of each of the five samples collected, over 1×10³ bacterial colonies (colony forming units (cfu)g^-1) grew on agar medium containing 2M Na⁺. In contrast, 0–4 bacterial colonies (cfu g^-1) were observed on agar medium containing 4M Na⁺. Two of the five samples contained numerous bacteria (10²–10³ cfu g^-1) capable of growth on a 2M Na⁺ alkaline (pH=9.5) medium, while few bacterial colonies were observed from the other three samples. Only one of the five samples was shown to contain bacteria capable of growth on a 4M Na⁺ alkaline medium. Most of the bacteria isolated on 4M Na⁺ agar were eubacteria, but one extreme halophile (TR-1, already described as Haloarcula japonica JCM7785) was also isolated. The 16S rRNA sequence of TR-1 was determined and shows high homology (94.4–98.5%) to Ha. marismortui and Ha. sinaiiensis. These results suggested that: 1) environments with seasonally varying salinity can harbour halotolerants as well as halophiles and, 2) closely related halophiles can be isolated from geographically distant habitats.     

Phenotypical divergences between populations of soil bacteria isolated on different media

Bacterial strains were randomly isolated from soil using three different media with glucose (TG), Tryptone Soya Broth (TTS), and succinate (TS) as carbon sources. Plate counts obtained were 12.0×10⁷, 4.5 ×10⁷, and 1.5×10⁷ g^–1 soil dry weight, respectively. The strains were characterized phenotypically by the API 20B test system. A cluster analysis of all isolates revealed 40 biotypes at 80% similarity, 23 in TG, 29 in TTS, and 27 in TS. Each of the 10 most common biotypes contained 10 to 2.5% of the isolates, and 17 biotypes contained one or two isolates. The common biotypes were unevenly distributed among the isolates from the different media. About 20% of the isolates from TG and TTS were unique for the particular medium, whereas among the isolates from TS, about 60% were unique. Thirty percent of the isolates belonged to biotypes that were common to all three populations. All media gave approximately the same high diversity measured as Shannon index and Equitability, indicating no direct correlation between plate count and diversity.     

Isolation of Rec? mutants from an F-prime merodiploid strain of Escherichia coli K-12

Summary This paper describes a method of screening mutagenised populations of an E. coli gal _A ^– gal _B ^– F-prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal⁺ papillae. With a suitable choice of gal ^–mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.     

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.     

Phenotypic expression of Vogesella indigofera upon exposure to hexavalent chromium,Cr6+

A eubacterium producing a blue pigment was isolated from a drinking water filter, and subsequently identified as Vogesella indigofera. This bacterium was further investigated for its morphological and biochemical characteristics after exposure to hexavalent chromium, Cr⁶⁺. The threshold Cr⁶⁺ concentration inhibiting the pigment production by V. indigofera was 200–300 g ml^–1 in liquid cultures of nutrient broth and 100–150 g ml^–1 on nutrient agar plates. The Cr⁶⁺ concentration preventing V. indigofera growth was 300–400 g ml^–1 in liquid cultures, but greater than 150 g ml^–1 on agar plates. Moreover, rugose colonies without the blue pigmentation were observed on agar plates amended with 150 (g Cr⁶⁺) ml^–1. The biochemical utilization profiles of the colonies without pigmentation did not differ from the original pigment-producing ones, indicating phenotypic plasticity of this bacterium. The difference of phenotypic expression of V. indigofera under various Cr⁶⁺ concentrations might have potential application as a pollution bioindicator for heavy metals.     

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37°C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of Arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair.In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37°C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/ or microconidia on casero medium and some reacted sexually with A. simii (a) or (–) mating type. Gelatin hydrolysis was variable.We suggest the following key tests to differentiate M. ferrugineum from T. soudanense: urease activity in urea broth; colony color on Lowenstein-Jensen medium; growth on lactose agar; growth at 37° C compared to room temperature; presence of reflexive branching on cornmeal Tween agar.     

A study of fungal spore poupulations in the atmosphere of Kuwait

The fungal air-spora of Kuwait was investigated using the Petri-dish trapping technique. During the period from 1st April, 1974 to 30th June, 1975, a total of 3685 colonies were recorded from 2 % malt agar plates. Fifty-five genera and 116 species were identified. Alternaria occupied the first place in the order of percentage incidence, being represented by 18.3% of the entire catch, followed by Aspergillus (17.1%), Penicillium (14%), Cladosporium (13.6%), Drechslera (13.3%), and Ulocladium (7.1%). The widest spectrum of species was displayed by Aspergillus (possessed 19 species) followed by Penicillium (17 species), Alternaria (6 species), Drechslera (5 species), and Ulocladium (4 species). The monthly total number of fungi showed marked seasonal periodicity with the greatest number of colonies occurring in March–April and November. The lowest counts were recorded in mid-summer (July) and mid-winter (January). Only 220 colonies and 17 species of thermophilous fungi were reported from plates incubated at 45 °C. The most common species were Aspergillus fumigatus and A. terreus.     

Tandem Coagulase/Thermonuclease Agar Method for the Detection of Staphylococcus aureus

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65°C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37°C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65°C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37°C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Asparagine and glutamine metabolism in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 achieved balanced growth when provided with either asparagine or glutamine as nitrogen source. Under these growth conditions R. acidophila synthesized a mixed amidase which exhibited similar activity (223–422 nmol/min·mg protein) against either nitrogen source. Determination of the free intracellular amino acid pools show that deamidation of asparagine and glutamine resulted in elevated levels of both aspartate and glutamate. Cell-free extracts of R. acidophila showed significant aminotransferase activity, particulary glutamine-oxaloacetate aminotransferase (89.7–209.3 nmol/min·mg protein), glycine oxaloacetate aminotransferase (135–227 nmol/min ·mg protein), alanine glyoxylate aminotransferase (66.3–163.2 nmol/min·mg protein) and serineglyoxylate aminotransferase (57.1–68.4 nmol/min ·mg protein). Short term labelling experiments using ¹⁴C-glyoxylate show that glycine plays an important role in amino nitrogen transfer in R. acidophila and that the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate were present in cell-free extracts. These data confirm that R. acidophila can satisfy all its' nitrogen requirements by transamination.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate—oxaloacetate aminotransferase - GPT glutamate-pyruvate aminotransferase - AGAT alanineglyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOGAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase - SGAT serineglyoxylate aminotransferase - INH isonicotinylhydrazide     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.