Showing papers on "Agar plate published in 1994"

Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85.

Abstract: Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis. We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P. medicaginis. We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide. Both antibiotics prevented disease of alfalfa seedlings caused by P. medicaginis. Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P. medicaginis, and antibiotic B caused swelling of the germ tubes. Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa. Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity. Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain. The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression. Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression. These results demonstrate that B. cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.

Production of a hemolytic factor by Candida albicans.

Abstract: Candida albicans exhibits hemolytic activity when grown on glucose-enriched blood agar. This activity is present on intact organisms, and it is secreted into the culture medium. Hemoglobin released from lysed erythrocytes can restore the transferrin-inhibited growth of C. albicans. We conclude that C. albicans expresses a hemolytic factor which allows it to acquire iron from host erythrocytes.

Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

Abstract: Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.

Comparison of respiratory activity and culturability during monochloramine disinfection of binary population biofilms.

Abstract: Biofilm bacteria challenged with monochloramine retained significant respiratory activity, even though they could not be cultured on agar plates. Microbial colony counts on agar media declined by approximately 99.9% after 1 h of disinfection, whereas the number of bacteria stained by a fluorescent redox dye experienced a 93% reduction. Integrated measures of biofilm respiratory activity, including net oxygen and glucose utilization rates, showed only a 10 to 15% reduction. In this biofilm system, measures of microbial respiratory activity and culturability yielded widely differing estimates of biocide efficacy.

Rapid identification of Candida albicans by using Albicans ID and fluoroplate agar plates.

Abstract: Two commercially available agar media, Albicans ID and Fluoroplate, that use a chromogenic or a fluorogenic substrate for the detection and identification of Candida albicans were evaluated. From 1,006 clinical samples containing 723 yeast strains, 352 C. albicans strains were detected with either of the two media. The sensitivity of each of the two media was 93.8% and the specificity was 98.6%, with five false-positive reactions for Candida tropicalis and no false-negative reactions.

Blood agar to detect virulence factors in tap water heterotrophic bacteria

Abstract: Cytolytic colonies were found in 57% of tap water samples, and up to 6% of samples were found to contain bacteria having three or more virulence factors. The factors evaluated were cytotoxicity, hemolysis, cell adherence, and cell invasiveness. Overall, 17% of the samples contained cytolytic colonies that were adherent and hemolytic. Among the media tested, tryptic soy agar with sheep blood (incubated at 35 degrees C for 48 h) was the best medium for the detection of cytolytic colonies. Of the colonies growing on this medium, 13% were cytolytic, whereas on medium R2A, less than 3% were cytolytic. Furthermore, when tryptic soy agar with blood was used, 24% of the samples contained colonies with at least three virulence factors whereas only 5% were positive with R2A. Routine monitoring by using tryptic soy agar with sheep blood is suggested as an appropriate procedure for the detection of bacteria with pathogenic potential in drinking water.

Oxygen requirements of Aspergillus species

Abstract: Summary The growth of 24 Aspergillus isolates at low oxygen tensions was assessed. Isolates selected included A. fumigatus (10), A. terreus (6), A. niger (6), A. nidulans (1) and A. flavus (1). Three different agar media were used—potato dextrose agar (PDA), pH 5.6; brain heart infusion (BHI), pH 7.4; and a specially developed medium (Hall's) containing resazurin—with oxygen concentrations of 0, 0.025, 01, 0.5 and 2.5%. The CO2 concentration was 5%. Agar plates were inoculated with 2 × 107 conidia/ml, loaded into jars and flushed with a special gas mixture at 37°C. The plates were inspected at intervals of 3, 5 and 10 days. On Hall's medium, none of the isolates grew at oxygen concentrations of 0 or 0.025%, but 21 (88%) of 24 grew at 0.1%. On PDA and BH1, all 14 isolates tested grew at oxygen concentrations of 0.5 and 2.5%. Three of these 14conidiated on PDA at oxygen 0.5% and 12 of 14conidiated on PDA at oxygen 2.5%. None grew without oxygen on these media. Thus, pathogenic Aspergillus spp. are capable of growth at low oxygen tensions, and this may have implications for pathogenicity and antifungal activity.

Extracellular proteolytic activity of Cryptococcus neoformans

Abstract: Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.

Agar Plate Method Using Lactobacillus plantarum for Biotin Determination in Serum and Urine

Abstract: An improved agar plate method of biotin bioassay using Lactobacillus plantarum ATCC 8014 and bromocresol purple was estab-lished to determine biotin levels in human serum and urine. Samples were treated with 4.5 N H2SO4 to liberate free biotin, autoclaved for 1 h and neutralized by 4.5N NaOH, then 10μl was added to wells in each plate. The biotin levels were measured in 190 serum and 59 urine samples, and the means were 2.7±0.53ng/ml and 12.4±5.56ng/mg of creatinine, re-spectively. The intra-assay coefficient varience (CV) were 3.2 (n=20) and 1.3% (n=23), respectively. The recovery of biotin added (10ng/ml) to serum was 110.7%, and to urine was 99.6%. These findings suggest that this assay is sufficiently accurate and reproducible for routine use in the clinical laboratory. The excretion of orally administered biotin was also demonstrated by the method.

Rapid detection of vancomycin-resistant enterococci.

Abstract: Campylobacter blood agar with clindamycin incubated in 6% CO2 served as a medium to both screen for vancomycin resistance and select for presumptive enterococci. Colonies that grew on the medium were specifically identified as enterococci within 30 min by the pyroglutamyl-beta-naphthylamide and rapid bile esculin tests. The combination of a selective medium plus rapid enzyme substrate tests offered an inexpensive means to enumerate vancomycin-resistant enterococci from specimens by using readily available reagents.

Expression and plasmid transfer of genes coding for the fimbrial antigen F107 in porcine Escherichia coli strains

Abstract: Summary Expression of the fimbrial antigen F107 of porcine E. coli strains on agar plates was achieved by microaerobic cultivation. For part of the strains of some types, addition of alizarin yellow and eosin to the agar medium proved to be necessary. Some of these strains reacted distinctly positive only when the small colonies growing between the larger ones on alizarin-yellow agar were tested. The fimbrial antigen of the Swiss strain 107/86 was provisionally designated the F107ab variant and that of the Hungarian strain 2134P and the Czech strain 8813, the F107ac variant. The F107 genetic determinants were found to be often linked with those encoding haemolysin production and are frequently carried by plasmids.

Isolation of Actinomycetes from Pine Litter Layers.

Abstract: Methods for the selective isolation of actinomycetes from pine litter layers were evaluated. Pretreatment of source material with a 0.05% of sodium dodecyl sulfate solution and the addition of 20 μg/ml of nalidixic acid to the agar media were effective in reducing bacterial growth. The addition of 200 μg/ml of benlate to the agar medium was effective in reducing fungal growth.By removing actinomycetes from the surface of pine needles with the surface sterilization method by 70% of ethanol and sodium hypochlorite solution containing 1% of chloride, we were able to isolate those actinomycetes which reside under the waxy layer of needles.Comparative studies of isolates obtained from pine litter layers showed both a qualitative and quantitative difference by layers.

Characterization of Hemolytic and Antifungal Substance, Cepalycin, from Pseudomonas cepacia

Abstract: Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106. A large amount of cepalycins were produced by growing the cells on 1% glycerin-nutrient agar medium covered with a cellophane membrane. The cell-washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl. Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II. The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption. From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane. Such an interaction may contribute to their hemolytic and antifungal activities.

Comparison of Gen-Probe Group A streptococcus Direct Test with culture for diagnosing streptococcal pharyngitis.

Abstract: The Group A Streptococcus Direct Test (GP-ST test; Gen-Probe, Inc., San Diego, Calif.) was compared with culture for the detection of Streptococcus pyogenes from throat swabs of 767 patients with pharyngitis. Swabs were tested by the GP-ST test after inoculating a 5% sheep blood agar (SBA) plate. SBA plates were incubated at 35 degrees C in room air for 72 h. SBA plates with no evidence of beta-hemolytic colonies after 18 to 24 h of incubation were subcultured by taking a swipe across the primary inoculum from the SBA plate to an agar selective for Streptococcus spp. In a low-prevalence (11.9%) population and in comparison with the number of positive cultures detected by the 72-h single-culture method (SBA plate method), the GP-ST test had a sensitivity of 88.6%, a specificity of 97.8%, a positive predictive value of 83.9%, and a negative predictive value of 98.5%. In comparison with the growth of any colonies of S. pyogenes on the 72-h SBA plates plus a subculture onto selective blood agar, the sensitivities and specificities were as follows: 72-h SBA plate method, 96.7 and 100%, respectively; GP-ST test, 85.7 and 97.8%, respectively. The GP-ST test is an easy-to-perform, reliable test for batch screening of throat swabs for S. pyogenes.

Heat resistance of different serovars of Listeria monocytogenes

Abstract: Twelve Listeria monocytogenes strains representing seven serovars were heat-treated in physiological saline by a glass capillary tube method. Five strains were treated at 58°, 60° and 62°C, three at 60°, 62° and 64°C and four at 60°C. Heat-treated bacteria were recovered on blood agar in two ways: (1) incubation at 37°C for 7 d; and (2) preincubation at 4°C for 5 d, followed by incubation at 37°C for 7 d. D and z values were determined. Better average recovery and higher D values were obtained when the preincubation procedure was used. The final evaluations of the heat resistance properties of the strains were therefore based on values for preincubated samples. D values recorded at 58°, 60°, 62° and 64°C for preincubated samples were 1.7–3.4, 0.72–3.1, 0.30–1.3 and 0.33–0.68 min, respectively. z values determined were 5.2–6.9°C. D values were compared statistically. Significant differences in heat resistance were noted both between serovars and between strains belonging to the same serovar.

A new selective enrichment procedure for isolating Pasteurella multocida from avian and environmental samples.

Abstract: A selective enrichment procedure, using two new selective media, was developed to isolate Pasteurella multocida from wild birds and environmental samples. These media were developed by testing 15 selective agents with six isolates of P. multocida from wild avian origin and seven other bacteria representing genera frequently found in environmental and avian samples. The resulting media--Pasteurella multocida selective enrichment broth and Pasteurella multocida selective agar--consisted of a blood agar medium at pH 10 containing gentamicin, potassium tellurite, and amphotericin B. Media were tested to determine: 1) selectivity when attempting isolation from pond water and avian carcasses, 2) sensitivity for detection of low numbers of P. multocida from pure and mixed cultures, 3) host range specificity of the media, and 4) performance compared with standard blood agar. With the new selective enrichment procedure, P. multocida was isolated from inoculated (60 organisms/ml) pond water 84% of the time, whereas when standard blood agar was used, the recovery rate was 0%.

Evaluation of methods for the detection of nasal carriage of Staphylococcus aureus

Abstract: In the present study we investigate the optimal methodology for determination of the nasal carriage rate of Staphylococcus aureus. Tests were performed on 91 healthy laboratory staff. The reproducibility of different sampling, transportation, storage and culture methods was examined. We compared sterile dry cotton wool swabs with sterile dry cotton wool swabs impregnated with charcoal and 5% blood agar plates with mannitol salt agar plates after different incubation periods. Finally, we investigated the detection rate for S. aureus following direct plating compared to storage in Stuart's transport medium for 7 days. There were no differences in isolation rates from the right or left nostril using either cotton or charcoal swabs. Charcoal swabs gave an increased isolation rate as compared to cotton swabs, and incubation in broth enrichment medium containing 6.5% NaCl also increased the isolation rate. Storage in Stuart's transport medium for 7 days gave an increase in isolation rate as compared to direct plating on blood agar. With mannitol salt agar plates the increase in isolation rate when incubation was performed for from 2 to 4, 2 to 7, and 4 to 7 days was 5.9%, 16.7%, and 11.5%, respectively. For the detection of S. aureus nasal carriers we find the use of charcoal swabs and Stuart's transport medium combined with cultivation on mannitol salt agar for 7 days to be the optimal method.

Regulation of bacterial gene expression in response to oxidative stress.

Abstract: Publisher Summary This chapter presents the approaches used to study the regulation of oxidative defense genes in Escherichia coli and Salmonella typhimurium . These general approaches should be useful in examining the response to oxidative stress in other organisms and in further elucidating the response in E. coli and S. typhimurium . The sensitivity of strains to oxidants has been assessed using several different approaches. The first evidence that E. coli cells possess a regulated defense response against oxidative stress came from growth curves of E. coli . AB11157 cultures grown in K medium to a density of 5 × 10 7 cells/ml were treated with 0, 10, and 30 μM hydrogen peroxide for 30 min. The cells were then challenged with a lethal dose of 5 mM hydrogen peroxide, and aliquots of cells were removed after 20, 40 and 60 rain, diluted, and spread on LB plates to determine the number of colony-forming units. The sensitivity or resistance of wild-type or mutant strains has also been determined by zone of inhibition assays. For these simple assays, 0.1 ml of an overnight culture is plated on an agar plate in 2 to 3 ml of soft agar and a disk impregnated with an oxidant is placed in the center of the agar. The zone of killing or inhibition surrounding the disk is then measured after 12 to 24 hr of growth. For this assay to be consistent the volume of the agar medium must be uniform.

Confirmation of Poly(1,3-dioxolan-2-one) Degrading Microorganisms in Environment

Abstract: Ecological biodegradability of aliphatic polycarbonates in different environments was estimated by the clear-zone method with an agar medium containing emulsified polycarbonates. It was found that poly(1,3-dioxolan-2-one) can be degraded by microorganisms in certain limited environments.

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

Abstract: Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium. Images

Evaluation of Methods for the Detection of Nasal Carriage of Staphylococcus Aureus

Abstract: In the present study we investigate the optimal methodology for determination of the nasal carriage rate of Staphylococcus aureus. Tests were performed on 91 healthy laboratory staff. The reproducibility of different sampling, transportation, storage and culture methods was examined. We compared sterile dry cotton wool swabs with sterile dry cotton wool swabs impregnated with charcoal and 5% blood agar plates with mannitol salt agar plates after different incubation periods. Finally, we investigated the detection rate for S. aureus following direct plating compared to storage in Stuart's transport medium for 7 days. There were no differences in isolation rates from the right or left nostril using either cotton or charcoal swabs. Charcoal swabs gave an increased isolation rate as compared to cotton swabs, and incubation in broth enrichment medium containing 6.5% NaCl also increased the isolation rate. Storage in Stuart's transport medium for 7 days gave an increase in isolation rate as compared to direct plating on blood agar. With mannitol salt agar plates the increase in isolation rate when incubation was performed for from 2 to 4, 2 to 7, and 4 to 7 days was 5.9%, 16.7%, and 11.5%, respectively. For the detection of S. aureus nasal carriers we find the use of charcoal swabs and Stuart's transport medium combined with cultivation on mannitol salt agar for 7 days to be the optimal method.

Novel screening assay for the detection of phenolic acid esterases.

Abstract: A novel plate assay method, developed for the screening of microorganisms or enzyme preparations for phenolic acid esterases, involves incorporating ethyl cinnamate into an agar medium. After inoculation and incubation, the plate is flooded with a pH-sensitive dye to reveal yellow zones around positive cultures against a blue background. A number of yeasts (Rhodotorula spp. and Candida spp.) and fungi (Penicillium sp. and Aspergillus sp.) gave positive results, while a number of commercial enzymes, particularly pectinases, also exhibited good phenolic acid esterase.

Occurrence and Properties of Copper-resistance in Plant Pathogenic Bacteria

Abstract: Copper-resistance was analyzed with 189 strains of plant pathogenic bacteria that belonged to the genera Pseudomonas, Xanthomonas, Erwinia, Agrobacterium, Clavibacter and Curtobacterium. For the assay, CuSO4 or copper compounds (copper fungicides) were incorporated into Bacto potato-dextrose agar (PDA). Bacteria that grew on PDA containing CuSO4 at concentrations of 1.25mM or more were arbitrarily categorized as copperresistant. The minimum inhibitory concentrations (MIC) of CuSO4 on the copper-PDA medium were one to two times greater than with casitone-yeast extract-glycerol agar medium, and 100 times greater than with aqueous solution. Copper-resistant strains were detected in many of the plant pathogenic bacteria, particularly in pseudomonads. The high MIC of CuSO4 were specifically found in members of the rRNA II subgroup of Pseudomonas such as P. cepacia and P. gladioli. Resistance to CuSO4 did not necessarily correlate with resistance to copper fungicides. In P. cepacia, P. gladioli, P. syringae pv. actinidiae, A. radiobacter and A. tumefaciens, copper- and streptomycin-resistances were concurrently found in the same strains. Copper-resistant strains of A. tumefaciens belonged to biovar 1 (all of 3 strains), biovar 2 (1 of 8 strains) and biovar undetermined (1 of 2 strains). A copper-resistant strain of A. radiobacter also belonged to biovar 1. Six strains of A. vitis tested were all copper-sensitive.

Evaluation of two new chromogenic media for detection of Salmonella in stools

Abstract: A clinical collaborative study was conducted to compare two new chromogenic agar media, Rambach agar and theSalmonella Detection and Identification Medium (SMID) (bioMerieux, France), with two conventional media, Salmonella-Shigella agar and Hektoen agar. Thirty-nineSalmonella strains involving 14 serotypes were isolated from 1,454 stool specimens. After enrichment in a selective broth, 100 % sensitivity was obtained with each medium. The SMID and Rambach agars are considerably more specific than the conventional media. Although SMID agar detects allSalmonella serotypes, it is not as specific as Rambach agar, which requires a complementary test (C8 esterase test) to detect all serotypes.

Culture of Helicobacter pylori under aerobic conditions on solid media

Abstract: The aim of the present study was to culture Helicobacter pylori under aerobic conditions and to investigate the characteristics of the organism when cultured aerobically. Most (22 of 23) of the Helicobacter pylori isolates grew under aerobic conditions, but with reduced viable cell counts. Blood agar was more suitable than chocolate agar. The morphological and enzymatic characteristics as well as the protein profiles of each organism were identical under aerobic and microaerophilic conditions. However, haemolysis of Helicobacter pylori was delayed under aerobic conditions. The MIC of metronidazole was slightly lower for some strains under aerobic conditions. These findings indicate that Helicobacter pylori is not only a microaerophilic organism but also adapts to aerobic conditions, which may have some important implications in microbiological and epidemiological studies.

Evaluation of media for determining hemolytic activity and that of API Listeria system for identifying strains of Listeria monocytogenes.

Abstract: Several media were used to evaluate the hemolytic activity of Listeria monocytogenes, and this property was used along with the API Listeria system to identify Listeria spp. All L. monocytogenes strains were identified correctly with this system, and blood agar base no. 2 and Columbia blood agar base supplemented with horse blood were suitable for detection of hemolytic activity.