Showing papers on "Agar plate published in 2004"

New strategies for cultivation and detection of previously uncultured microbes

Abstract: An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O2 [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO2 (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO2. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms.

Abstract: Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 103 CFU of L. monocytogenes/ml and 105 CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log10 CFU of L. monocytogenes/cm2). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.

Atividade antimicrobiana "in vitro" de extrato alcóolico de própolis

Abstract: Propolis is a natural resin collected and modified by honeybees Since ancient times it has been used as a chemotherapeutic agent The propolis antibacterial activity was evaluated through bacterial inoculation on BHI agar plates with 5% of alcoholic propolis extract (10(6) bacteria mL-1) One hundred and sixty one bacterial strains were evaluated, as Gram positive bacteria (Staphylococcus sp, Streptococcus sp, Nocardia asteroides and Rhodococcus equi) as Gram negative (Escherichia coli, Salmonella sp, Proteus mirabilis and Pseudomonas aeruginosa) The strain was considered sensible to the propolis extract when no bacterial growth was evident on the plate after 72 hours at 37oC The control test used agar plates with 5% of ethanol (M2) and 5% of saline solution Propolis extract demonstrated antibacterial activity on 677% of the tested strains; 926% of Gram-positive and 425% of Gram-negative strains presented sensitivity The propolis extract showed effective antibacterial activity against the majority of tested strains

Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris.

Abstract: Phytases catalyze the release of phosphate from phyticacid. Phytase-producing microorganisms were selected byculturing the soil extracts on agar plates containing phyticacid. Two hundred colonies that exhibited potentialphytase activity were selected for further study. The colonyshowing the highest phytase activity was identified as

Comparison of the BBL CHROMagar Staph aureus agar medium to conventional media for detection of Staphylococcus aureus in respiratory samples

Abstract: Screening for Staphylococcus aureus has become routine in certain patient populations. This study is the first clinical evaluation of the BBL CHROMagar Staph aureus agar (CSA) medium (BD Diagnostics, Sparks, Md.) for detection of S. aureus in nasal surveillance cultures and in respiratory samples from cystic fibrosis (CF) patients. S. aureus colonies appear mauve on CSA. Other organisms are inhibited or produce a distinctly different colony color. S. aureus was identified from all media by slide coagulase, exogenous DNase, and mannitol fermentation assays. Susceptibility testing was performed using the agar dilution method. A total of 679 samples were evaluated. All samples were inoculated onto CSA. Nasal surveillance cultures were inoculated onto sheep blood agar (SBA) (BD Diagnostics), and samples from CF patients were inoculated onto mannitol salt agar (MSA) (BD Diagnostics). Of the 679 samples cultured, 200 organisms produced a mauve color on CSA (suspicious for S. aureus) and 180 were positive for S. aureus on SBA or MSA. Of 200 CSA-positive samples 191 were identified as S. aureus. Nine mauve colonies were slide coagulase negative and were subsequently identified as Staphylococcus lugdunensis (one), Staphylococcus epidermidis (three), Staphylococcus haemolyticus (one), and Corynebacterium species (four). CSA improved the ability to detect S. aureus by recovering 12 S. aureus isolates missed by conventional media. Of the 192 S. aureus isolates recovered, 122 were methicillin susceptible and 70 were methicillin resistant. Overall, the sensitivity and specificity of CSA in this study were 99.5 and 98%, respectively. There was no difference in the performance of the slide coagulase test or in susceptibility testing performed on S. aureus recovered from CSA compared to SBA or MSA. Our data support the use of CSA in place of standard culture media for detection of S. aureus in heavily contaminated respiratory samples.

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing Bacillus cereus.

Abstract: Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes. To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates. The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies. The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE. The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

Identification of Yersinia enterocolitica in minced meat: a comparative analysis of API 20E, Yersinia identification kit and a 16S rRNA-based PCR method

Abstract: The isolation and identification of Yersinia enterocolitica from minced meat on CIN agar medium is still one of the major problems in food microbiology because of the low selectivity of cefsulodin-irgasan-novobiocin (CIN) agar. A total of 198 minced meat samples were collected from commercial establishments (butcher shops and supermarkets) in seven German cities in order to investigate the sensitivity and specificity of three identification techniques suitable for the differentiation of Y. enterocolitica within the rich background flora on CIN agar plates. As expected isolation of Y. enterocolitica from minced meat on CIN agar medium after 72 h enrichment in peptone, sorbitol and bile salts (PSB) broth was difficult because all plates were abundantly covered with numerous 'typical'Yersinia-like colonies of bull's eye appearance as well as with atypical colonies. Based on the phenotype of the colonies it was possible to detect colonies showing Yersinia-like growth on CIN agar in 52 samples (26%). For identification of Y. enterocolitica the API 20E system (bioMerieux, Nurtingen, Germany), the Yersinia identification kit (Merlin, Bornheim-Hersel, Germany) and a 16S rRNA based PCR assay were compared. Only in one sample (0.5%) a Y. enterocolitica strain was detected by all methods. Of the three identification systems tested for routine laboratory diagnostics the API 20E system was found to be the most suitable tool to identify Y. enterocolitica colonies within the rich background flora from minced meat samples on CIN agar plates.

Neonatal intensive care unit: reservoirs of nosocomial pathogens.

Abstract: Improvement in the care and treatment of neonates had contributed to their increased survival. Nosocomial infection remains an important problem in intensive care units. Hospital wards had been shown to act as reservoirs of pathogenic microorganisms associated with infection. To assess the prevalence of pathogenic organisms in the environment of the neonatal unit, 92 swabs were randomly collected from cots, incubators and various equipments in the unit and were cultured on Blood agar and MacConkey agar plates. Air contamination was detected by exposing the same types of agar plates for 3 hours in several areas of the unit. After 48 hours incubation, isolates were identified biochemically. There is marked congestion in the unit. Ninety one percent of swabs yielded growth, with coagulase negative Staphylococcus being the predominant organism (44%), followed by Bacillus species (20%), E. coli (12.5%), and Klebsiella (8.5%), Pseudomonas species (3.6%) and moulds (3.6%). Sedimentation plates had colony counts of from 10 - 100 per plate and the majority of the cultures were polymicrobial cultures. The presence of various Gram-negative bacili including known neonatal pathogens (like E. Coli and Pseudomonas) especially on ward equipment and congestion in the ward has the potential to cause nosocomial infection.

Immunomagnetic separation of Escherichia coli O157:H7 from environmental and wastewater in South Africa

Abstract: Recreational and drinking water supplies polluted with sewage have become an important source of E. coli O157:H7 infection. Immunomagnetic separation (IMS) has been extensively used for the isolation of E. coli O157:H7 from food and stool samples but not for samples such as wastewater. In this study the IMS method was used in combination with the E. coli O157:H7 selective media, immunoassays, biochemical tests and PCR, to assess the prevalence of E. coli O157:H7 in selected sewage and environmental water in South Africa. Environmental and wastewater were seeded with E. coli O157:H7 to determine the sensitivity and selectivity of the enrichment-IMS-selective agar method. Naturally occurring E. coli O157:H7 organisms were recovered from selected samples by means of IMS. The IMS concentrates were plated on three selective E. coli O157:H7 media. E. coli O157:H7 was detected in seeded sewage and river water samples with numbers as low as 1.2 cfu·ml -1 . The IMS procedure was used to investigate the prevalence of E. coli O157:H7 in randomly selected sewage and river water samples in South Africa. A total of 91 sewage- and 40 river water samples were tested and 17.6% and 20% yielded suspected E. coli O157:H7 colonies on CT-SMAC agar medium respectively. PCR was used to confirm the presence of genes coding for Shiga toxin-1 (Stx1), Shiga toxin-2 (Stx2), enterocyte attaching and effacing genes (eaeA) and enterohaemolysin (hly). Standard immunoassay kits specific for the O157 and H7 antigen and a biochemical indole test were used for further E. coli O157:H7 confirmation. Three colonies from one sewage sample (1.1 % of all sewage samples) agglutinated with anti-E. coli O157 and H7 antiserum and contained the genes coding for Stx2, eaeA and hly. None of the colonies isolated from the river water samples were positive for E. coli O157:H7. CT-SMAC proved to have limited E. coli O157:H7 selective capabilities from samples such as sewage with high bacterial counts. Seeded sample experiments indicated that IMS is a suitable method for isolating E. coli O157:H7 from samples with high bacterial interference and low numbers of E. coli O157:H7. Evidence has been presented that the enrichment-IMS-selective agar procedure substantially increased the sensitivity of E. coli O157:H7 isolation compared to direct plating of test samples onto selective agar generally practised in the past.

Performance of selective and differential media in the primary isolation of yeasts from different biological samples.

Abstract: In view of the increase in yeast infections, especially polymicrobial ones, differential culture media have acquired increasing importance. The present study evaluated the Sabouraud chloramphenicol, Biggy agar, Pagano Levin agar and CHROMagar Candida media in terms of isolation, number of yeast colony forming units per plate, and inhibition of bacteria and filamentous fungi. To this end, we used 223 biological samples, including feces, and oral, vaginal and anal mucosae from 86 patients presenting or not symptoms of fungal infections. The four media did not differ significantly in terms of detection of yeast-positive cultures. The number of colony forming units per plate ranged from zero to 2.380, with a predominance of counts of 1 to 9 colonies per plate. No significant differences were observed among the four culture media in terms of number of colonies counted, for each kind of biological material. Fifteen species belonging to the genera Candida, Saccharomyces, Cryptococcus, Trichosporon and Rhodotorula were isolated, with C. albicans being the predominant species, followed by C. parapsilosis and R. rubra. CHROMagar Candida and Biggy agar were complementary in the isolation of the different species and favored a greater recovery of polymicrobial cultures. Pagano Levin agar isolated the smallest variety of species. Sabouraud chloramphenicol agar was the least effective in terms of bacterial inhibition and favored a greater development of filamentous fungi. The results suggest that more than one culture medium should be used for an adequate primary isolation.

Comparison of Agar and an Agitated, Thin-film, Liquid System for Micropropagation of Ornamental Elephant Ears

Abstract: Micropropagation of black-stemmed elephant ear (C. esculenta (L.) Schott 'Fon- tanesii')' and upright elephant ear (A. macrorrhizos G. Don) were compared in semi-solid agar media and agitated, liquid thin-fi lm bioreactor vessels at four explant densities (33, 100, 165, and 330 explants/L of media) using two growth regulator combinations: 1) 1 µM benzylaminopurine (BA)—growth medium, and 2) 3 µM BA plus 3 µM ancymidol—multipli- cation medium. The thin-fi lm liquid system outperformed agar culture for most measured responses. Some exceptions were relative dry weights at higher explant densities and multiplication rate of Colocasia. When the thin-fi lm liquid system was compared to agar culture, Alocasia explants produced their greatest biomass and had the least residual sugar at the highest explant density. Alocasia explants multiplied most rapidly and had the greatest relative dry weight on liquid media at the low explant densities. Alocasia plants were larger in growth medium than multiplication medium and larger in liquid medium than agar medium. When compared to agar, Colocasia in the thin-fi lm liquid system produced the greatest biomass at the highest explant density in growth medium, had the greatest relative dry weight at the lowest explant density, and used the most sugar at the highest explant density. Alocasia and Colocasia would likely produce greater fresh and dry weight at the highest explant density if additional sugar were supplied during thin-fi lm culture. Greater growth in thin-fi lm culture of Alocasia and Colocasia is due in part, to greater availability of sugar in liquid compared to agar medium.

Stability of Mueller-Hinton Agar Supplemented with Glucose and Methylene Blue for Disk Diffusion Testing of Fluconazole and Voriconazole

Abstract: The shelf life of Mueller-Hinton agar supplemented with 2% glucose and methylene blue (0.5 μg/ml) (MH-GMB) prepared in the laboratory to test disk diffusion of voriconazole and fluconazole was assessed using quality control (QC) strains of Candida albicans ATCC 90028, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019. MH-GMB agar plates were prepared as described in National Committee for Clinical Laboratory Standards document M44-P, and isolates were tested using 25-μg fluconazole disks and 1-μg voriconazole disks over a 36-day period. Zone diameters for fluconazole and voriconazole varied by no more than 4 mm over the study period, and 95 to 100% of results were within the established QC limits for the strains tested. Prepared MH-GMB agar plates provide acceptable performance for disk diffusion testing for at least 30 days when stored at 5°C.

Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures.

Abstract: BACKGROUND AND AIM Usually, bacteria are cryopreserved for short-term storage at low and ultra-low temperatures. There are no reports as to whether Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures as compared with other intestinal bacteria. A study was done on seven H. pylori strains and other intestinal bacteria to compare different temperatures for storage of organisms in saline solution. METHODS Seven H. pylori strains, specifically American Type Culture Collection (ATCC) strains 43504 and TN2GF4, and five strains isolated from the present patients were grown on a modified Skirrow's agar for H. pylori. Escherichia coli and Bacteroides distasonis, both representing isolates from the present patients, were grown on trypticase soy blood agar for E. coli, and EG agar for B. distasonis. Culture was for 4-5 days under microaerobic, aerobic and anaerobic conditions at 37 degrees C. Cells were harvested by scraping growth from the solid medium and into sterile saline. The cells were adjusted to concentrations of 109 viable cells/mL in saline and preserved at 4 degrees C, -20 degrees C, or -80 degrees C for 3 weeks before reculture under microaerobic, aerobic and anaerobic conditions at 37 degrees C for 7 days. After incubation, morphologically distinct colonies were counted, isolated, and identified by standard bacteriologic techniques. The H. pylori were morphologically analyzed by electronic microscopy before and after preservation. Mongolian gerbils were inoculated with the cryopreserved H. pylori to evaluate the bacterial infectivity. RESULTS Six of the seven H. pylori strains failed to culture after being preserved at 4 degrees C, -20 degrees C, or -80 degrees C. Only ATCC 43504 could be cultured after freezing at -80 degrees C. The number of H. pylori ATCC 43504 before preservation was 9.0 +/- 0.5 (log10 no. organisms/mL) and decreased to 5.7 +/- 0.6 after preservation. Morphologically, all H. pylori except ATCC 43504 strains transformed from a bacillary to a coccoid form after preservation. In addition, none of the H. pylori strains could infect Mongolian gerbils after preservation. Escherichia coli and B. distasonis were recovered. Titers before and after 4 degrees C, -20 degrees C, and -80 degrees C, respectively, were 9.1 +/- 0.2, 8.9 +/- 0.5, 8.6 +/- 0.3, and 8.7 +/- 0.3 for E. coli and 9.1 +/- 0.4, 8.7 +/- 0.6, 8.6 +/- 0.5, and 8.8 +/- 0.3 for B. distasonis. CONCLUSIONS Helicobacter pylori is a fragile bacteria for storage at low and ultra-low temperatures in comparison with other intestinal bacteria.

Phenotypic evaluation to differentiate Candida albicans from Candida dubliniensis

Abstract: This study evaluated the phenotypic tests used to differentiate Candida albicans from Candida dubliniensis. A total of 55 isolates from vaginal secretions, oral cavity and hemoculture were studied. They were originally identified as C. albicans, based on their morphological and physiological characteristics. These isolates were tested for colony color development on CHROMagar Candida medium, growth at 45 degrees C on Sabouraud Dextrose agar, lipolytic activity on Tween 80 Agar medium and colony morphology and chlamydoconidia formation on Staib agar medium. Of the 55 isolates studied, seven yielded one or more phenotypic characteristics suggestive of Candida dubliniensis. These isolates were tested by PCR with specific primers for Candida dubliniensis and API ID 32. The seven isolates were confirmed as Candida albicans. All of these finding indicate that DNA based tests should be used for definitive identification of Candida dubliniensis.

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated, Thin-Film Liquid System for Micropropagation

Abstract: Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.

Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping.

Abstract: The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces Isolates of Bacillus subtilis (two isolates), Pseudomonas spp (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D flagrans growth on agar plates When these isolates were added to cattle faeces containing D flagrans and nematode larvae of C oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D flagrans Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D flagrans to trap and kill developing larvae of C oncophora Increasing chlamydospore doses of D flagrans in faecal cultures resulted in higher nematode mortality Thus, no evidence of interspecific or intraspecific competition was observed The consequences of these findings are discussed

Study of Extracellular Plant Lactase

Abstract: Using a synthetic substrate, a simple and sensitive procedure for the determination of extracellular lactase was developed. The enzyme studied produced by the tested plant material (calli and roots of gherkin, pea, poppy, and Amsonia tabernaemontana Walt. seedlings) hydrolyzed the substrate (1-naphtyl-α-D-galactopyranoside) to α-D-galactose and 1-naphthol. By simultaneous azocoupling of 1-naphthol with Fast Blue BB, Fast Blue RR or Fast Blue B, violet-red, hardly water-soluble azo-dyes were produced. The evaluation of the intensity of dyed zones allowed the extracellular lactase activity to be assessed. No coloration of the agar medium was observed without inoculum, with heat-inactivated cells (10 min at 100°C) or in medium inoculated without substrate. On the agar plates with substrate and sterile gherkin, pea, poppy and amsonia seedlings, changes in coloration were observed indicating a release of lactase from the roots during germination. p-Nitrophenyl-β-D-galactopyranoside was used for the determination of the intra- and extracellular activity of the enzyme studied. The results showed a 37.9 % intracellular and 62.1 % extracellular distribution of lactase activity, when using Amsonia tabernaemontana Walt. as the plant material. The agar plate method described permitted rapid, simple, and specific detection of plant producers of extracellular lactase and proved to be perspectively useful in inhibitory and/or biotechnological studies.

Hydrodynamics of bacterial colonies: Phase diagrams

Abstract: We present numerical simulations of a recent hydrodynamic model describing the growth of bacterial colonies on agar plates. We show that this model is able to qualitatively reproduce experimentally observed phase diagrams, which relate a colony shape to the initial quantity of nutrients on the plate and the initial wetness of the agar. We also discuss the principal features resulting from the interplay between hydrodynamic motions and colony growth, as described by our model.

IN VITRO STUDIES TO CONTROL THE GROWTH OF MICROORGANISMS OF SPOILAGE AND SAFETY CONCERN IN HIGH-MOISTURE, HIGH-pH BAKERY PRODUCTS

Abstract: Initial agar plate studies were done to determine the effects of various levels (0 to 2,000 ppm) of potassium sorbate (KS) and sorbic hydroxamic acid (SHA) over a wide pH range (5 to 9) on the growth of microorganisms of spoilage and safety concern in high-moisture, high-pH bakery products. While growth of most microorganisms was inhibited for > 28 days on agar plates containing ∼1,000 ppm of KS at pH 5 and incubated at 30C, growth of all microorganisms occurred in plates at pH 7 and 9, regardless of the concentration of KS. SHA was equally effective at pH 5, however, it proved to be a more effective inhibitor against most microorganisms at higher pH (9). Subsequent agar plate studies were done with water-ethanol (WE) and mastic oil-ethanol (ME) emitters. While WE emitters failed to control the growth of all microorganisms under investigation, ME emitters controlled the growth of most microorganisms, with the exception of Listeria monocytogenes, for ∼12 to 28 days on agar plates packaged in high-gas-barrier Cryovac or metallized bags, respectively. Inhibition was not simply due to the levels of ethanol, which ranged from ∼1.2 to 2.8% v/v, but rather, the mastic volatiles in the package headspace. This study has demonstrated the potential of SHA and ME emitters to control the growth of several microorganisms of spoilage and safety concern in high-moisture, high-pH bakery products. However, the type of packaging material influenced the antimicrobial efficacy of this vapor-phase inhibitor.

Evaluation of Granada Agar Plate for Detection of Streptococcus agalactiae in Urine Specimens from Pregnant Women

Abstract: The Granada agar plate (GAP; Biomedics SL, Madrid, Spain) was evaluated for the detection of group B streptococci (GBS) in urine specimens from pregnant women submitted for testing for asymptomatic bacteriuria and was compared with blood agar (BA [Columbia agar with 5% sheep blood]; bioMerieux, Marcy l'Etoile, France). The GAP detected 103 out of 105 GBS, whereas BA detected only 50. Use of the GAP could be a good method for the detection of GBS in urine specimens from pregnant women.

Persistence of plasmid RP4 in Pseudomonas putida and loss of its expression of antibiotic resistance in a groundwater microcosm

Abstract: We examined the stability of plasmid RN and its expression of antibiotic-resistance genes in suspended and sorbed Pseudomonas putida in aquifer microcosms. Test tubes containing different proportions of sterilized aquifer soil and groundwater were inoculated with bacteria and incubated for up to 26 d. Serial dilutions were made to agar plates with or without antibiotics, to quantify the functional stability of the plasmid. The structural integrity of RN was examined by plasmid extraction, digestion with restriction enzymes, and agarose gel electrophoresis. The plasmid-borne resistance gene expression disappeared in 80-90% of the cells during day 1 of incubation in aquifer soil and then remained at that frequency throughout the experiment. The RP4 plasmid was present in cells without antibiotic-resistance gene expression, indicating that the observed loss of plasmid-encoded activity was most likely due to a reduction in expression of the resistance genes. The increased growth rate in groundwater amended with glucose and phosphate had no significant influence on plasmid loss or antibiotic-resistance expression, suggesting that plasmid loss and antibiotic-resistance expression were independent of the growth rate. Most of the reduction of resistance gene expression was associated with the presence of soil particles, and 70% of the resistance expression was retained in bacteria incubated for 1 d in groundwater alone. Bacteria sorbed to the soil particles had a lower frequency of expression of resistance genes than suspended bacteria, but the difference was not caused by sorbed inorganic or organic chemicals. Resistance gene expression was partly recovered in suspended bacteria after in vitro exposure to the antibiotics and after first isolating on agar without antibiotics and then replica plating to agar containing the antibiotics. (Less)

Experimental design for the microbiological four-plate test for the detection of sulphadimidine residues at the levels of concern

Abstract: The aim of the study was to evaluate the sensitivity of the reference four-plate test (FPT) for the detection of sulphadimidine residues. The test agar, trimethoprim (TMP) and Bacillus subtilis BGA as test organism were used in the study. After determining the minimum inhibiting concentration (MIC) for sulphadimidine, the potential effects of selected parameters (pH value of agar medium, TMP concentration, addition of dextrose to the agar medium and use of distilled or deionised water as solvents) on the increasing sensitivity of the method or on lowering of the MIC for sulphadimidine were studied. It was demonstrated that the sensitivity of FPT was 1 µg.ml -1 only. The addition of TMP to the agar medium at the concentration of 0.10 or 0.15 µg.ml -1 significantly increased (P<0.05) the sensitivity of the method from the viewpoint of detecting the presence of sulphadimidine residues at the level of the MRL (0.01 µg.ml -1 ). The using of deionised water significantly increased the diameters of inhibition zones (P<0.05). The different pH values of agar media had only a minor effect on the inhibition zone diameter. At pH 7.0 and 7.2, the MICs were equal.

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

Abstract: Aims: To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. Methods and Results: Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, −18 and −75°C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain–heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. Conclusions: Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. Significance and Impact of the Study: Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen.

Cloning grills: high throughput cloning for structural genomics.

Abstract: Cloning grills are aluminum grids designed to divide an agar plate into segments, thereby multiplying the number of E. coli cultures which can be streaked out on a single plate. The grills are autoclaved and placed in square petri dishes immediately after hot agar is poured. When the agar solidifies, the grill remains embedded in the media, and each of the 12 lanes accommodates the streaking out of a single culture. As the spacing of the grill lanes is the same as that of a 96-well plate, 12 cultures can be streaked at a time using a 12-channel pipette. This allows a plate of 96 cultures to be rapidly and accurately plated for colony isolation on only eight agar plates.

Isolation and antibiogram pattern of haemophilus influenzae isolated from bronchial washing of patients undergoing bronchoscopy

Abstract: Background – Haemophilus influenzae is the second most important causative agent of pneumonia in outpatients. The aim of this survey was to perform isolation, to identify biotype, serotype, and antimicrobial sensitivity pattern of Haemophilus influenzae in patients undergoing bronchoscopy. Methods – In this study 170 bronchial washing specimens were taken from patients 1 – 70 years old, and were cultured on blood agar, chocolate agar, cefsulodin chocolate agar, and Fildes media. The presence of encapsulated bacteria was identified by Fildes medium neqrosin and Congo red staining. Biotyping and serotyping were performed by serological methods. Results – The results showed that 14 (8.23%) cases were positive for H. influenzae, of which 78.5% were unencapsulated. The cases were identified as I, II, III, and V biotypes. A statistically significant relationship was found between unencapsulated H. influenzae and the age of the subjects (p = 0.0345), and their occupation, e.g., cotton-beater and mine workers ( p = 0.0196). From isolated bacteria, 85.7% were sensitive to chloramphenic ol and 71.42% to ampicillin and cefotaxime. Conclusion – Age and occupation are two risk factors for this bacterial respiratory, and the prevalent biotypes in the region were I, II, III and V. Fortunately, some antibiotics are too high degrees, effective on this agent.