Showing papers on "Agar plate published in 2003"

Antimicrobial effect of 2% sodium hypochlorite and 2% chlorhexidine tested by different methods

Abstract: The objective of this study was to analyze the antimicrobial effect of 2% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) by agar diffusion test and by direct exposure test. Five microorganisms: Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aernginosa, Bacillus subtilis, Candida albicans, and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. For the agar diffusion test (ADT), 18 Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the microbial suspensions, using sterile swabs that were spread on the medium, obtaining growth injunction. Fifty-four paper disks (9 mm in diameter) were immersed in the experimental solutions for 1 min. Subsequently, three papers disks containing one of the substances were placed on the BHI agar surface in each agar plate. The plates were maintained for 1 h at room temperature, and then incubated at 37 degrees C for 48 h. The diameter of microbial inhibition was measured around the papers disks containing the substances. For the direct exposure test, 162#50 sterile absorbent paper points were immersed in the experimental suspensions for 5 min, and were then placed on Petri plates and covered with one of the irrigant solutions, or with sterile distilled water (control group). After intervals of 5, 1 0 and 30 min, the paper points were removed from contact with the solutions and individually immersed in 7 ml of Letheen Broth, followed by incubation at 37 degrees C for 48 h. Microbial growth was evaluated by turbidity of the culture medium. A 0.1 ml inoculum obtained from the Letheen Broth was transferred to 7 ml of BHI, and incubated at 37 degrees C for 48 h. Bacterial growth was again evaluated by turbidity of the culture medium. Gram stain of BHI cultures was used for verification of contamination and growth was determined by macroscopic and microscopic examination. The best performance of antimicrobial effectiveness of NaOCI was observed in the direct exposure test, and of CHX was observed in the agar diffusion test. The magnitude of antimicrobial effect was influenced by the experimental methods, biological indicators and exposure time.

Bioactive Packaging Materials from Edible Chitosan Polymer—Antimicrobial Activity Assessment on Dairy-Related Contaminants

Abstract: The inhibitory activity of chitosan-based edible coatings was assessed against 2 food pathogens (Staphylococcus aureus and Listeria monocytogenes) and 1 strain involved in food alteration (Pseudomonas aeruginosa) on model agar medium and on a real cheese food product. Colony counting and epifluorescence microscopy methods were conducted, and the results show a nonsignificant influence of the components of the food matrix in the protection of the microbial population against chitosan activity. Numeration on model agar medium showed 100% inhibition of the development of selected Gram-positive bacteria and 77% inhibition on Pseudomonas growth. Chitosan is thought to act through binding to the cytoplasmic membrane surface, and it is possible that the outer membrane protects the Gram-negative cells. Moreover, epifluorescence microscopic results showed a possible chitosan action during a short time duration on the synthesis of nucleic acids and especially on the relative proportion of RNA compared with DNA. This impact was followed by an adaptative mechanism of the cells. Edible chitosan coating could thus be used to increase the microbial lag phase while decreasing the maximum density of selected microorganisms and could have potential application for dairy products preservation.

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture.

Abstract: A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.

Detection of Other Microbial Species by Salmonella: Expression of the SdiA Regulon

Abstract: Salmonella, Escherichia, and Klebsiella do not encode any recognized type of N-acylhomoserine lactone (AHL) synthase, and consistent with this, they do not synthesize AHLs under any conditions tested. However, they do encode an AHL receptor of the LuxR family, named SdiA. MudJ fusions in four loci are known to respond to plasmid-encoded sdiA in Salmonella, but only the rck locus has been described. Here we report the location and sequence analysis of the remaining three loci. The srg-6::MudJ is within gtgA of the gifsy-2 prophage, and the srg-7::MudJ is within PSLT61 of the virulence plasmid. Both fusions are in the antisense orientation. The third fusion, srgE5::MudJ, is within a horizontally acquired gene of unknown function at 33.6 centisomes that we have named srgE. Previously, sdiA expressed from its natural position in the chromosome was demonstrated to activate a plasmid-based transcriptional fusion to the rck promoter in response to AHL production by other bacterial species. However, the MudJ fusions did not respond to chromosomal sdiA. Here we report that MudJ fusions to three of the four loci (not srg-6) are activated by AHL in an sdiA-dependent manner during growth in motility agar (0.25% agar) but not during growth in top agar (0.7% agar) or on agar plates (1.2% agar). In motility agar, the srgE promoter responds to sdiA at 30°C and higher while the rck and srg-7 promoters respond only at 37 or 42°C. Substantial AHL-independent SdiA activity was observed at 30°C but not at 37°C.

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

Abstract: A . M A C H U C A A N D A . M . F . M I L A G R E S . 2003. Aims: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. Methods and Results: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. Conclusions: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. Significance and Impact of the Study: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.

Survival of Campylobacter jejuni strains of different origin in drinking water.

Abstract: Aims: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. Methods and Results: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4°C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. Conclusions: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4°C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4°C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4°C. Significance and Impact of the Study: This study highlighted differences in culturability depending on culture conditions and on strain origin.

Evaluation of S. aureus ID, a New Chromogenic Agar Medium for Detection of Staphylococcus aureus

Abstract: S. aureus ID (bioMerieux, La Balme Les Grottes, France) is a new chromogenic agar medium designed to enable the isolation of staphylococci and the specific identification of Staphylococcus aureus. S. aureus produces green colonies on this medium due to production of α-glucosidase. To evaluate this medium, a total of 350 wound swabs were cultured onto S. aureus ID, CHROMagar Staph. aureus, and conventional media routinely used in our laboratory. After 18 to 20 h of incubation, 96.8% of strains formed green colonies on S. aureus ID compared with 91.1% of strains forming mauve colonies on CHROMagar Staph. aureus. A total of 94.3% of strains were recovered within 18 to 20 h with conventional media. The sensitivity was increased after 48 h of incubation to 98.7, 96.2, and 95.6% with S. aureus ID, CHROMagar Staph. aureus, and conventional media, respectively. A total of 97.4% of green colonies on S. aureus ID were confirmed as S. aureus compared with 94.4% of mauve colonies on CHROMagar Staph. aureus. We conclude that S. aureus ID is a highly sensitive and specific medium for the isolation and identification of S. aureus from wound swabs.

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species.

Abstract: The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37°C. The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar.

Chemically defined media for growth of Haemophilus influenzae strains

Abstract: A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are ≥ 109 CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and β-NAD).

Cultivatable bacteria in dentine after caries excavation using rose-bur or carisolv.

Abstract: To measure the amount of viable bacteria after excavation using conventional rose-bur or the chemo-mechanical Carisolv method, a total of 22 lesions were analyzed (one vital tooth per patient) in this open, controlled and randomized study. Two samples per lesion were taken under aseptic conditions using a rose-bur, one superficially in the caries lesion and one after completed excavation. In in vitro tests more material was collected from the hard caries free dentine as compared to the carious dentine. The samples were incubated on blood agar (aerobically and anaerobically), Rogosa (SL) agar and mitis salivarius (MS) agar. For blood agar (aerobic) both methods resulted in a significant decrease in CFU, for blood agar (anaerobic) and MS agar only the Carisolv method resulted in a significant decrease in CFU and for SL agar neither method resulted in a significant decrease in CFU. Comparing CFU before and after excavation, a considerable reduction of CFU was seen ranging from 10(1) to 10(4). Comparing the excavation methods, there were no significant differences, except in the case of blood agar (aerobic), which showed that Carisolv excavation was more effective in reducing CFU. This study indicated that bacterial sampling collected more material from hard dentine as compared from soft. Remaining bacteria after excavation were low in both groups. The Carisolv method seemed to remove bacteria at least up to and possibly beyond the extent of conventional drilling.

Development of a plating medium for selection of Helicobacter pylori from water samples.

Abstract: The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10(6) CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37 degrees C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.

Blood Agar and Mycobacterium tuberculosis: the End of a Dogma

Abstract: Incidental blood agar-based recovery of Mycobacterium tuberculosis led us to further investigate this routine medium for primary isolation and culture of M. tuberculosis. Fifteen respiratory tract and eight lymph node Ziehl-Neelsen-positive specimens were inoculated in parallel into tubes containing egg-based medium and 5% sheep blood agar. Colonies appeared sooner on this medium than on the egg-based medium, but this difference was not significant (P = 0.11, analysis of variance [ANOVA] test). Further experiments compared the growth of 38 respiratory and lymph node M. tuberculosis isolates when subcultured on the two media. After 6 days of incubation, 21 of 38 isolates had grown on blood agar, and the mean number of colonies was significantly greater on blood agar than on the egg-based medium (P < 0 0.001, ANOVA test). These results demonstrate that M. tuberculosis grows easily on blood agar within 1to 2 weeks, indicating that this basic medium is suitable for laboratory diagnosis of tuberculosis in addition to other media. Laboratories that routinely use prolonged incubations of blood plates, for example, for the recovery of Bartonella species, should consider the potential safety implications of encountering this highly infectious pathogen.

Laboratory assessment of the allelopathic effects of fine leaf fescues.

Abstract: Laboratory screening studies were conducted to evaluate the allelopathic potential of fine leaf fescues. Of the seven accessions selected from prior field evaluations for weed-suppressive ability, all inhibited root growth of large crabgrass and curly cress in laboratory assays. Grown in agar as a growth medium and in the presence of living fescue seedlings for 14 or 21 days, test species were sensitive depending on the fescue cultivars. Growth inhibition increased when fescue was grown for increasing periods of time in agar. Seedling fescues produced significant quantities of bioactive root exudates, which were released into the agar medium. Bioactive root exudates were extracted from living fescue roots by using methylene chloride. Shoot tissue was extracted in water and the aqueous extract was partitioned against hexane, ethyl acetate, and methylene chloride. Extracts were tested for inhibitory activity on seedling growth as measured by inhibition of curly cress germination and radicle elongation. Root exudates were more toxic (70% inhibition) than shoot extracts (up 40% inhibition), when formulated at 0.25 mg/ml concentration. Light microscopy and transmission electron microscopy were utilized in an attempt to identify the cellular location of production of secondary products contained in bioactive root exudates. Ultrastructural analysis indicated that the exudate is produced in actively dividing tips of fibrous root cells. The mode of release of these exudates into the environment remains unknown.

Antifungal activity of lactic acid bacteria

Abstract: Enrichment culture techniques produced more than 1200 isolates of lactic acid bacteria (LAB) that were screened for antifungal activity against the indicator mould Aspergillus fumigatus. Approximately 10% of the LAB were active, but only 4% had medium or strong activity in an agar plate assay. The majority of isolates with strong antifungal activity were Lactobacillus coryniformis strains, but Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified. Some of the isolates lost activity during storage but most maintained their fungal inhibitory effect. Large variations in sensitivity were observed between different moulds and yeasts. Antifungal cyclic dipeptides and phenyllactic acid were detected in culture filtrates from several of the LAB isolates. Lactobacillus coryniformis subsp. coryniformis strain Si3 produced an antifungal compound that lost activity when treated with proteinases. The antifungal peptide(s) was heat stable, with a size of approx. 3kDa and had maximum activity at pH 3.0 to 4.5. Addition of ethanol to the growth medium of strain Si3 prevented a decline in observed antifungal activity during the stationary phase. Glycerol addition to agar plates with L. coryniformis strains, overlaid with soft agar suspensions of yeast cells or fungal spores, strongly enhanced the antifungal effect. This was particularly true with spoilage moulds and yeasts, e.g. Penicillium roqueforti and Pichia anomala, not normally affected by the antifungal metabolites of L. coryniformis. Chemical and genetic data suggested that reuterin (3-hydroxypropionaldehyde) was the cause of this effect. The glycerol/diol dehydratase operon of L. coryniformis was partially elucidated and found to be similar to that Lactobacillus collinoides. Bioassay-guided isolation of new metabolites from LAB revealed that Lactobacillus plantarum MiLAB 14 produces hydroxylated fatty acids with strong antifungal effects. 3-Hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid and 3-hydroxy-5-cis-dodecenoic acid were characterized from the supernatant of MiLAB 14. The hydroxy fatty acids had total inhibitory effects in the range 10 to >100 µg ml-1 against several moulds and yeasts.

Evaluation of a Novel Medium for Screening Specimens from Hospitalized Patients To Detect Methicillin-Resistant Staphylococcus aureus

Abstract: A novel medium, Oxacillin Resistant Screening Agar (ORSA) medium, was evaluated for the screening of specimens for methicillin-resistant Staphylococcus aureus (MRSA) in the hospital setting. Screening swabs (swabs of the nose, throat, perineum, and infected sites) were inoculated onto the new ORSA medium and into an enrichment broth (Muller-Hinton broth supplemented with NaCl and oxacillin). After 24 h of incubation, the enrichment broth was subcultured onto one ORSA plate and one lipovitellin Chapman salt agar plate. The sensitivities for the detection of MRSA were calculated for each medium alone and for the media in combination. A low sensitivity (74%) was obtained when ORSA medium was used alone as a primary culture, whereas the sensitivity was 88% when a single selective enrichment broth was used. Among the 414 blue colonies observed on ORSA plates, only 47% were found to be MRSA, 40% were coagulase-negative staphylococci, 7% were Enterococcus species, and 2% were methicillin-sensitive S. aureus. The optimal incubation time for the ORSA plates was evaluated. On primary culture, 38% of the blue MRSA colonies were visible only after 48 h of incubation (no blue colonies were not seen after 24 h of incubation), whereas 94% of the colonies were already visible at 24 h when ORSA plates were used for subcultures. In conclusion, the advantage of the novel ORSA medium is the ease of recognition of mannitol-fermenting bacteria, but further identification tests are needed to confirm the identification of S. aureus. An enrichment broth is still needed to ensure a good sensitivity for the recovery of MRSA, and an incubation time of 48 h is required for primary culture on ORSA medium.

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Abstract: Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.

The influence of medium aeration on in vitro rooting of Australian plant microcuttings

Abstract: Media with different air filled porosity were compared with standard agar medium for root induction and root elongation for two Australian plants Grevillea thelemanniana and Verticordia plumosa×Chamelaucium uncinatum. Microcuttings from shoot cultures were pulsed for 7 days on a high auxin (40 μM IBA), agar-solidified medium in the dark. The rooting of the microcuttings was then compared on standard agar medium (M1, 1/2 MS, no hormones) and on three experimental treatments: – porous-agar medium (1/2 MS, no hormones, 30 g agar l−1, solidified then blended to provide aeration); – white sand, or white sand wet with M1 medium; and – a sterile propagation mix. The protocol using the propagation mix is referred to as IVS (In Vitro Soil). A separate experiment involved flushing the IVS soil profile with low or normal oxygen. The controls on M1 medium showed low and variable rooting percentages. The rate of root induction and the average total root length per microcutting at final harvest was significantly higher using the IVS protocol, porous-agar or white sand, while addition of agar medium to sand suppressed the percentage rooting and elongation as did flushing the air space in the IVS rooting medium with low oxygen. Other species tested on M1 medium and IVS including Pimelea physodes, Conospermum eatoniae, Verticordia grandis, and a Chamelaucium megalopetalum×C. uncinatum hybrid all showed a significant improvement on the IVS system. The IVS culture technique reduces plant-handling costs.

Prevalence of Strongyloides stercoralis infection in northeastern Thailand (agar plate culture detection).

Abstract: PEWPAN M INTAPAN, MD, MSc**, SOMSAK NILPAN, MSc***, SUPRANEE VEERAKUL, BSc*****, The prevalence of Strongyloides stercora/is infection was studied in the rural and urban popu- lations of 19 provinces of Northeastern Thailand. A total of 1233 fecal samples was collected from July to September 2002 and examined using agar plate culture method. The overall prevalence of S. sterco- ra/is was 23.5 per cent with the highest infection rate in Kalasin Province (61.0%), predominantly among 60 year olds and older (28.0%), and in males (32.8%). The factors associated with Strongyloides infection were sex (males) and age (the over 19 year-old age group). Conclusion : S. stercora/is infection remains highly prevalent among the population of north- eastern Thailand as confirmed by the agar plate culture method. The authors recommend that a pro- gram for effective strongyloidiasis control should have a justifiable priority. Key word : Strongyloides stercora/is, Northeastern Thailand, Agar Plate Culture

Quality Control Limits for Posaconazole Disk Susceptibility Tests on Mueller-Hinton Agar with Glucose and Methylene Blue

Abstract: An international collaborative study was performed in order to propose quality control limits for voriconazole disk diffusion tests on Mueller-Hinton agar with 2% glucose and 0.5 μg of methylene blue per ml. The supplement may be added to the agar before autoclaving, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates with 1-μg voriconazole disks generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (28 to 37 mm), Candida albicans ATCC 90028 (31 to 42 mm), and Candida krusei ATCC 6258 (16 to 25 mm). Candida tropicalis ATCC 750 was not useful for this purpose.

Starvation of Flavobacterium psychrophilum in broth, stream water and distilled water.

Abstract: Physical changes in Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS), were examined over a 19 wk period of starvation. Bacteria were maintained in either Cytophaga broth, filtered stream water, or filtered distilled water, or were maintained in broth after disinfection as a negative control for dead bacteria. Culturability and viability of the bacterium were assessed using colony-forming units (CFUs) and a commercially available live/dead kit. Antigenic profiles and general morphology of the bacterium were also examined using Western blot analysis and electron microscopy, respectively. The bacterium appeared to stop multiplying and became smaller and rounded when maintained in stream water. Its culturability declined until it was no longer possible to obtain colonies on agar plates at the end of the trial at 19 wk, and results from the live/dead kit did not correspond with the viability obtained as CFUs in culture. However, it was still possible to obtain growth of the bacterium after 36 wk with a resuscitation step in Cytophaga broth. Bacteria maintained in distilled water or treated with a disinfectant appeared non-viable using the live/dead kit and were unable to grow on agar 1 h after setting up the experiment; no morphological changes were observed in the bacteria maintained under these conditions. Bacteria maintained in broth were present as long, slim rods, some of which developed into 'ring' formations. Small differences were observed in the antigen profiles of the bacteria maintained under the different treatments, possibly due to a reduction in the size and metabolism of the bacteria. There was also a marked decline in the sensitivity of the PCR with bacteria maintained under the different treatments 14 wk from the onset of the study.

A xyloglucan from seeds of the native Brazilian species Hymenaea courbaril for micropropagation of Marubakaido and Jonagored apples

Abstract: Xyloglucan was extracted from seeds of Hymenaea courbaril and mixed with agar to prepare a solid culture medium used for micropropagation of the Marubakaido apple rootstock (Malus prunifolia Borkh) and cv. Jonagored (Malus domestica). The performance on gels created from a blend of 0.4%agar and 0.2% xyloglucan (w/v) was compared with that on media gelled with a standard concentration 0.6% (w/v) of agar. The growth of shoots and the multiplication rate were higher on the modified culture medium than on the agar-gelled medium. The occurrence of hyperhydric shoots was lower on the modified medium. In the absence of auxin, shoot rooting reached 70% (Marubakaido) and 66% (Jonagored) on the agar-xyloglucan medium and 6.7% and 10.4%, respectively, on the agar medium. When 0.25 µM indole-3-butyric acid (IBA) was added to both media, the modified medium gave better results in terms of rooting percentage and quality of roots than the agar-gelled medium.

Evaluation of a new agar medium containing cetrimide, kanamycin and nalidixic acid for isolation and enhancement of pigment production of Pseudomonas aeruginosa in clinical samples

Abstract: A new selective agar medium (CKNA), containing cetrimide 0.3 g/l, kanamycin sulfate 50 mg/l, and nalidixic acid 5 mg/l, was developed for the isolation of Pseudomonas aeruginosa. It was compared to Nalidixic Acid Cetrimide agar (NAC), Pseudomonas Aeruginosa Selective Agar (PASA) and Pseudosel(TM) agar (CET) using 1,148 clinical specimens. The sensitivities rates of P. aeruginosa with CKNA, NAC, PASA, and CET were 88.2%, 81.3%, 79.2%, and 84.0%, respectively. The specificities of CKNA, NAC, PASA, and CET were 99.2%, 98.4%, 99.2%, and 99.7%, respectively.

Isolation and some properties of cellulose-degrading Vibrio sp. LX-3 with agar-liquefying ability from soil

Abstract: A new bacterium, Vibrio sp. LX-3 was isolated from soil, which was a facultatively anaerobic and polarly flagellated Gram-negative rod. Sodium ions stimulated its growth but were not an absolute requirement. The isolate could digest both crystalline cellulose and agar. Carboxymethylcellulase was produced extracellularly when various celluloses were used as carbon sources, but no reducing sugars were found in the culture on cellulose over the incubation period. Only low agarase activity could be detected in the broth of agar although the strain LX-3 strongly liquefied agar.