Showing papers on "Agar plate published in 1999"

An efficient microbiological growth medium for screening phosphate solubilizing microorganisms

Abstract: A novel defined microbiological growth medium, National Botanical Research Institute's phosphate growth medium (NBRIP), which is more efficient than Pikovskaya medium (PVK), was developed for screening phosphate solubilizing microorganisms. In plate assay the efficiency of NBRIP was comparable to PVK; however, in broth assay NBRIP consistently demonstrated about 3-fold higher efficiency compared to PVK. The results indicated that the criterion for isolation of phosphate solubilizers based on the formation of visible halo/zone on agar plates is not a reliable technique, as many isolates which did not show any clear zone on agar plates solubilized insoluble inorganic phosphates in liquid medium. It may be concluded that soil microbes should be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers.

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds.

Abstract: Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 degrees C. Plate counts declined from 3 x 10(6) to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or alpha-ketoglutaric acid, plate counts increased to 10(4)-10(5) CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H2O2, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells.

Evaluation of MRSA-Screen, a Simple Anti-PBP 2a Slide Latex Agglutination Kit, for Rapid Detection of Methicillin Resistance in Staphylococcus aureus

Abstract: The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

A Large Outbreak of Hemolytic Uremic Syndrome Caused by an Unusual Sorbitol-Fermenting Strain of Escherichia coli O157:H—

Abstract: Escherichia coli O157:H7 does not ferment sorbitol, a factor used to differentiate it from other E. coli. From December 1995 to March 1996, 28 children with hemolytic uremic syndrome in Bavaria, Germany, were identified; many had a sorbitol-fermenting (sf) E. coli O157: H- cultured. A case-control study showed a dose-response relationship between sausage consumption and illness. A second case-control study showed a relationship between mortadella and teewurst consumption and illness, particularly during December (mortadella odds ratio [OR], 10.5, P = .004; teewurst OR, 6.2, P = .02). Twelve sf 0157:H- were characterized to determine clonality and virulence traits. The strains possessed the Stx 2 , eae, and EHEC-hlyA genes but were nonhemolytic on blood agar plates. The O157:H- isolates belonged to phage type 88 and had identical pulsed-field gel electrophoresis patterns. This outbreak was caused by sf E. coli O157:H-, which is not detectable by culture on sorbitol MacConkey's agar. Consumption of two sausages, including a raw beef-containing sausage, was statistically related to illness.

Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis.

Abstract: Staib agar (Syn. Guizotia abyssinica creatinine agar) was evaluated for differentiation between the highly related yeast species Candida albicans and Candida dubliniensis. On these agar plates C. dubliniensis formed rough colonies due to mycelial growth and produced abundant chlamydospores whereas C. albicans grew only in smooth colonies and without chlamydospore formation. The rough colonies of C. dubliniensis could be readily distinguished from the smooth C. albicans colonies. These results demonstrate that, under certain growth conditions, mycelial growth with chlamydospore formation is a species-specific marker that can be used for the identification of C. dubliniensis.

Lacticin 3147 displays activity in buffer against Gram-positive bacterial pathogens which appear insensitive in standard plate assays

Abstract: Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147, which has been shown to be active against a range of food-borne bacteria. The reported inhibitory range for lacticin is extended to include methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, penicillin-resistant Pneumococcus, Propionibacterium acne and Streptococcus mutans. This extended host range is not obvious from traditional agar plate-based methods, but reductions in bacterial cell numbers by up to 6 log10 cfu ml−1 was observed after 2 h in time-kill curve studies conducted in broth, suggesting that the bacteriocin may have potential as a therapeutic agent in the treatment of human infections.

Antimicrobial effect of six endodontic sealers: an in vitro evaluation.

Abstract: The aim of this study was to determine the in vitro antimicrobial effect of six endodontic sealers after 2, 20 and 40 days. The sealers studied were Apexit, Endion, AH26, AH-Plus. Procosol and Ketac Endo. The microorganisms used were Candida albicans, Staphylococcus aureus, Streptococcus mutans and Actinomyces israelii. Petri dishes were filled with sterile agar and 0.1-ml wells were prepared and filled with the sealers. The agar plates were stored for 24 h at 37 degrees C. The samples were then removed, immersed in 4.5 ml of culture medium and divided into three groups. The samples in group 1 were stored for 2 days at 37 degrees C whereas the samples of groups 2 and 3 were stored at 4 degrees C for 20 and 40 days respectively. The samples were then removed and discarded, and 0.1 ml of the culture medium was seeded on the agar plates in order to perform colony forming unit counts. Apexit, Endion and AH-Plus produced slight inhibition on Streptococcus mutans at 20 days and on Actinomyces israelii at every time interval. No effect was found on Candida albicans and Staphylococcus aureus. Ketac Endo only produced an antimicrobial effect on Actinomyces israelii at 2 and 40 days. AH26 and Procosol showed antimicrobial effect at 40 days on Candida albicans, at 20 and 40 days on Streptococcus mutans and Staphylococcus aureus, and an effective inhibition on Actinomyces israelii at every time interval. Statistical analysis revealed both sealers and microorganisms to be significant factors affecting results in groups 2 and 3. In conclusion, the sealers evaluated in this study showed different inhibitory effects depending on time span. Overall, sealers containing cugenol and formaldehyde proved to be most effective against the microorganisms at the time intervals studied.

Agar underlay method for recovery of sublethally heat-injured bacteria.

Abstract: A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.

Evidence That Cryptococcus neoformans Is Melanized in Pigeon Excreta: Implications for Pathogenesis

Abstract: Cryptococcus neoformans has a laccase that catalyzes the synthesis of melanin in the presence of phenolic compounds (9, 17). Melanin synthesis by C. neoformans is associated with virulence (11, 14). Melanin can protect C. neoformans against antifungal compounds, oxidants, UV light, macrophages, and extremes in temperature (reviewed in reference 2). Since melanization confers reduced susceptibility to many insults, this trait may have originally been selected for environmental survival. Infections with C. neoformans are thought to occur by inhalation of aerosolized organisms from environmental sources, and the sizes of cryptococcal cells collected from pigeon excreta are compatible with aveolar deposition (10). Although pigeon excreta have not formally been considered to be a source of human infections, four independent DNA-typing studies have identified the same cryptococcal strains in collected pigeon excreta and clinical isolates (3–5, 18). Furthermore, there is considerable anecdotal evidence suggesting that humans were infected after exposure to avian excreta (reviewed in reference 2). In this study we present evidence that cryptococcal cells are melanized in pigeon excreta. (The data in this paper are from a thesis to be submitted by Angel Luis Rosas in partial fulfillment of the requirements for the degree of doctor of philosophy from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.) Samples of pigeon excreta were collected from areas adjacent to our institution. The presence of C. neoformans in the feces was initially investigated by a C. neoformans cell capture enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) to screen pigeon excreta slurries. The ELISA captures and immobilizes yeast cells and was performed as described previously (1). For IF, 50 μl of pigeon excreta supernatant was dried on poly-l-lysine-coated slides (Sigma Chemical Corp., St. Louis, Mo.). Blocking for nonspecific binding was done with 0.05% Tween 20 in 2% bovine serum albumin (BSA) for 1 h at 37°C. Fifty-microliter samples of the polysaccharide-binding immunoglobulin M (IgM) monoclonal antibody (MAb) 2D10 (1) or antimelanin IgM MAb 11B11 (13) (1:100 [vol/vol]) in 1% BSA were applied to the slides, which were incubated in a moisture chamber for 1 h at 37°C. After excess MAb was washed off, each slide was covered with 50 μl of a 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgM (Southern Biotech, Birmingham, Ala.) in 1% BSA for 1 h at room temperature. The slide was washed, a coverslip was placed over it with a mounting solution (50% glycerol, 50% phosphate-buffered saline [PBS]–0.1 M N-propyl gallate), and the slide was examined with an Olympus (Melville, N.Y.) AX70 microscope. Negative controls included slides with nonmelanized cells (ATCC 24067), NSO ascites fluid, or no primary MAb. IF with the MAb 2D10 and whole-cell capture ELISA demonstrated yeast cells that stained for cryptococcal polysaccharide in pigeon feces (Fig. ​(Fig.1A1A and B). IF with the melanin-binding MAb also stained yeast cells in the pigeon excreta supernatants (Fig. ​(Fig.1C1C and D). FIG. 1 Light and IF images of C. neoformans in pigeon excreta suspensions (magnification, ×1,000). (A and B) Images depict the binding of MAb 2D10 to a yeast cell. (C and D) Images depict the staining of the melanin-binding MAb 11B11 to a yeast cell. ... Excreta that demonstrated C. neoformans by capture ELISA and/or IF were plated with 50-μl aliquots on isolation agar (1 mM l-dopa, 29.4 mM KH2PO4, 10 mM MgSO4, 13 mM glycine, 15 mM glucose, and 6 μM thiamine [Sigma] with 0.1% biphenyl [Eastman Fine Chemicals, Eastman Kodak Company, Rochester, N.Y.], 0.025% vancomycin [Fugisawa USA, Inc., Deerfield, Ill.], 0.025% imipenem–cilastin [Merck and Co.], and 1.5% agar [Difco Laboratories, Detroit, Mich.]) (3) and incubated at 30°C. After a minimum of 6 days, darkly pigmented colonies were selected for further analysis. India ink suspensions of cells from colonies or after growth in Sabouraud dextrose medium (Difco) at 30°C were viewed with an Olympus AX70 microscope. Isolates from pigeon excreta melanized when they were grown in a defined chemical medium with l-dopa (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 μM thiamine, 1 mM l-dopa [Sigma]) at 30 or 37°C. Urease activity was measured with BBL urea agar slants (Becton Dickinson, Cockeysville, Md.). Selected isolates that were India ink positive, stained with MAb 2D10, melanized in l-dopa medium, grew at 37°C, and utilized urea were further identified as C. neoformans with the API 20C Clinical Yeast System (BioMerieux Vitek, Inc., Hazelwood, Mo.). Pigeon excreta that tested positive for C. neoformans were treated with enzymes, denaturant, and hot acid and examined by scanning electron microscopy for particles that resembled melanin “ghosts” (16). Briefly, dried pigeon excreta (250 ml) were dissolved in 1 M sorbitol–0.1 M sodium citrate (pH 5.0) and then treated with 10 mg of Novozym 234 (Calbiochem, La Jolla, Calif.) per ml for 1 h at 30°C. The debris was collected by centrifugation, suspended in 4 M guanidinium isothiocyanate for 2 h at room temperature, and then incubated in 6 M HCl for 1 h at 100°C. The denaturant- and acid-resistant material was dialyzed exhaustively against distilled water. Samples of the isolated material were prepared as previously described (7) and viewed with a model JSM-6400 electron microscope (JEOL, Tokyo, Japan). Spherical structures very similar to the melanin ghosts isolated from melanized cells (16) were observed in pigeon excreta that had been treated with enzyme, denaturant, and boiling acid. These particles contained buds and bud scars, consistent with an origin from melanized yeast (Fig. ​(Fig.2A2A and B). One particle had a hole in the surface, suggestive of a budding site where the daughter cell was sheared off during the melanin isolation. FIG. 2 Scanning electron micrographs of particles isolated following treatment with enzyme, detergent, and hot acid of pigeon excreta (A and B) or cryptococcal cells grown on excreta agar (C) (magnification, ×15,000). The structures are remarkably similar ... To examine whether pigeon excreta had precursors for melanization, C. neoformans was grown on pigeon excreta agar plates. Pigeon feces were suspended in a minimal amount of PBS and autoclaved. Agar plates were prepared with a defined chemical medium (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, and 3 μM thiamine [Sigma] with 2% Bacto-Agar [Difco]) with 5, 10, or 20% excreta. Positive control plates were made with 1 mM l-dopa, and negative control plates lacked a substrate. C. neoformans ATCC 24067 or Candida albicans SC5314 (gift of M. Ghannoun) was grown overnight in Sabouraud dextrose medium at 30°C with shaking and then collected and washed three times in PBS. Ten-day-old C. neoformans cells growing on 10% excreta plates were collected, subjected to the melanin ghost isolation protocol (16), and prepared for scanning electron microscopy. C. neoformans grown in medium without phenolic substrate completely solubilize after treatment with enzyme, detergent, and hot HCl (16). Colonies of C. neoformans grown on the defined agar plates with or without 10% excreta were selected and suspended in PBS, and the cells were observed in India ink suspensions. Measurements of total cell diameter were done with an Olympus AX70 microscope (magnification, ×1,000) with a grid (number of segments, 20) with resolutions to 0.1 μm. Colonies of strain 24067 on 5, 10, or 20% excreta plates turned brown by day 5 and became progressively darker over time. Colonies on the defined agar without phenolic substrate were white, whereas those on plates with l-dopa were black. C. albicans grown on the excreta plates did not become pigmented, suggesting that colony pigmentation was not due simply to the uptake of some type of pigment from the excreta agar. Treatment of C. neoformans cells from excreta agar by the melanin isolation protocol yielded particles that were very similar to melanin ghosts (Fig. ​(Fig.2C).2C). The diameters of the ghost-like structures isolated from C. neoformans grown on 10% excreta agar were similar to those of ghosts prepared from cells grown on the same agar (3.2 ± 0.77 μm versus 2.88 ± 0.92 μm; n = 20; P > 0.05 by Student’s t test). Similarly, the sizes of the particles visualized in pigeon feces were 2.91 ± 1.07 μm (n = 20; P > 0.05 by Student’s t test). We conclude that C. neoformans in pigeon excreta is melanized from the following evidence: (i) yeast cells in pigeon excreta stain with MAbs to C. neoformans polysaccharide and melanin; (ii) structures remarkably similar in size and appearance to melanin ghosts can be isolated both from pigeon feces contaminated with C. neoformans and from cryptococcal cells grown on excreta agar after treatment with enzyme, denaturant, and hot acid; and (iii) C. neoformans colonies on pigeon excreta agar plates are darkly pigmented. Melanization of C. neoformans in the environment has important implications for our understanding of cryptococcal ecology and pathogenesis. Melanin synthesis in pigeon excreta may potentially protect C. neoformans cells against environmental stresses such as UV light (15) and extremes in temperature (12). Human C. neoformans infection is acquired from the environment, and there is extensive circumstantial evidence implicating pigeon excreta as a potential reservoir for infection (reviewed in reference 2). Our finding suggests that the initial infection may involve melanized C. neoformans cells. Since there is evidence that the immune responses to melanized and nonmelanized cells are different (6), our results suggest that it is important to study the effect of melanization in initial infection.

Interactions between the bioactive glass S53P4 and the atrophic rhinitis-associated microorganism klebsiella ozaenae.

Abstract: In an aqueous environment, ions are released from a bioactive glass (BAG) and the pH rises in its vicinity. This may influence both growth and colonization of microorganisms. We studied the effects of the BAG S53P4 on the atrophic rhinitis-associated microorganism Klebsiella ozaenae. The glass was used in the form of granules or discs. Growth inhibition was studied using an agar plate test. Adhesion was studied by incubating bacterial suspension with the glass. The effect of the presence of the bacteria on the formation of the Si-rich layer on the bioactive glass was also analyzed. Furthermore, a follow up study of 19-74 months with ozena patients surgically treated with the BAG S53P4 was performed. The bioactive glass showed no clear growth inhibition of K. ozaenae in the agar plate test. K. ozaenae showed low adherence to the BAG S53P4. No growth of the microbe was seen on the glass during the 8 h incubations and the Si-rich layer was formed normally. The clinical follow-up study showed no infections of the implants and the symptoms of the patients were markedly reduced. Thus, the BAG S53P4 did not favor adhesion and colonization of K. ozaenae, in vitro, which is supported by the in vivo findings showing no BAG-associated infections or reinfections.

Evaluation of the Granada Agar Plate for Detection of Vaginal and Rectal Group B Streptococci in Pregnant Women

Abstract: Granada medium was evaluated for the detection of group B streptococci (GBS) in vaginal and rectal swabs compared with selective Columbia blood agar and selective Lim broth. From May 1996 to March 1998, 702 pregnant women (35 to 37 weeks of gestation) participated in this three-phase study; 103 (14.7%) of these women carried GBS. In the first phase of the experiment (n = 273 women), vaginorectal specimens were collected on the same swab; the sensitivities of Granada tube, selective Columbia blood agar, and Lim broth were 31.4, 94.3, and 74.3%, respectively. In the second and third phases (n = 429 women), vaginal and rectal specimens were collected separately; the sensitivities of Granada plate, selective Columbia blood agar, and Lim broth (subcultured at 4 h on selective Columbia agar in the second phase and at 18 to 24 h in Granada plate in the third phase) were 91.1, 83.9, and 75%, respectively, in the second phase and 88.5, 90.4, and 63.5%, respectively, in the third phase. There were no statistically significant differences in GBS recovery between the Granada agar plate and selective Columbia blood agar, but the Granada plate provided a clear advantage; the characteristic red-orange colonies produced overnight by GBS can be identified by the naked eye and is so specific that further identification is unnecessary. The use of the Granada tube and Lim broth did not result in increased isolation of GBS. In conclusion, the Granada agar plate is highly sensitive for detecting GBS in vaginal and rectal swabs from pregnant women and can provide results in 18 to 24 h.

Evaluation of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens

Abstract: The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three Vibrio spp. and one Aeromonas spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of Vibrio spp. and some Shigella spp. were required for detection. The growth rates of 125 enteric pathogens and 12 Yersinia spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12 Yersinia spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect Yersinia spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis.

ABC Medium, a New Chromogenic Agar for Selective Isolation of Salmonella spp.

Abstract: We describe a new chromogenic agar medium, ABC medium (alphabeta-chromogenic medium), which includes two substrates, 3, 4-cyclohexenoesculetin-beta-D-galactoside and 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside, to facilitate the selective isolation of Salmonella spp. This medium exploits the fact that Salmonella spp. may be distinguished from other members of the family Enterobacteriaceae by the presence of alpha-galactosidase activity in the absence of beta-galactosidase activity. A total of 1, 022 strains of Salmonella spp. and 300 other gram-negative strains were inoculated onto this medium. Of these, 1,019 (99.7%) strains of Salmonella spp. produced a characteristic green colony, whereas only 1 strain (0.33%) of non-Salmonella produced a green colony. A total of 283 stool samples were cultured onto desoxycholate citrate (DC) agar and ABC medium by direct inoculation and after selective enrichment in selenite broth. Overall, the sensitivity and specificity were superior for ABC medium (100 and 90.5%, respectively) than for DC agar (88 and 26.9%, respectively). We conclude that ABC medium offers a high degree of specificity for the detection of Salmonella spp. in stool samples.

Effect of pH, temperature and surface contact on the elaboration of fimbriae and flagella by Salmonella serotype Enteritidis.

Abstract: Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Flagella and fimbriae are putative virulence determinants of Salmonella spp. and ELISAs specific for the detection of flagella and SEF21, SEF14 and SEF17 fimbriae were used to assess the effect of temperature and pH upon their elaboration by isolates of Salmonella serotype Enteritidis in planktonic growth and on the surface of two-dimensional gradient agar plates. For three phage type 4 isolates of Enteritidis of comparative clinical provenance, similar phenotypes for the elaboration of these surface antigens were observed. SEFl4 fimbriae were elaborated in planktonic growth at 37"C, but not 20"C, at pH 4.77 and above but not at pH 4.04; whereas on agar gradient plates SEFl4 fimbriae were elaborated poorly but with best yields at pH 4.04. SEF17 fimbriae were elaborated in planktonic growth at 20"C, but not at 37"C, at pH 6.18 and above but not at pH 5.09 or below; whereas on agar gradient plates SEFl7 fimbriae were elaborated well even at pH 4.65. SEF21 fimbriae were expressed very poorly under all conditions tested. Planktonic growth at 37°C induced least flagella whereas growth at 20°C, and particularly surface growth at lower pH values, induced a 'hyper-flagellate' phenotype. Single colonies allowed to form on gradient agar plates were shown to generate different colonial morphologies which were dependent on initial pH. These results demonstrate that the physicochemical environment is an important determinant of bacterial response, especially the induction of putative virulence factors.

A novel chromogenic ester agar medium for detection of salmonellae

Abstract: A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter−1. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C8 fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.

Helicobacter pylori Can Be Induced To Assume the Morphology of Helicobacter heilmannii

Abstract: Cultures of Helicobacter pylori obtained from the American Type Culture Collection (strain 43504) were grown as isolated colonies or lawns on blood agar plates and in broth culture with constant shaking. Examination of bacterial growth with Gram-stained fixed preparation and differential interference contrast microscopy on wet preparations revealed that bacteria grown on blood agar plates had a morphology consistent with that normally reported for H. pylori whereas bacteria from broth cultures had the morphologic appearance of Helicobacter heilmannii. Bacteria harvested from blood agar plates assumed an H. heilmannii-like morphology when transferred to broth cultures, and bacteria from broth cultures grew with morphology typical of H. pylori when grown on blood agar plates. Analysis by PCR of bacteria isolated from blood agar plates and broth cultures indicated that a single strain of bacteria (H. pylori) was responsible for both morphologies.

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

Abstract: The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 02% P(3HB) The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth Four potent degrader isolates identified as species of Streptomyces were selected Degradation of P(3HB) by these isolates was studied for a period of 8 days The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent However, the isolates were equally efficient in degrading P(3HB) in liquid medium

Resuscitation from the viable but nonculturable state of Helicobacter pylori

Abstract: It is well known that the change of state from the culturable to not culturable on pathogenic organisms could easily occurred. These states of bacteria were so called viable but nonculturable (VNC). The coccoid form of H. pylori is seems to be in this state. The possibility of resuscitation of H. pylori was examined in vitro. The coccoid form bacteria was treated with Ammonium Sulfate and heat shock before culture, and cultured in Brucella broth containing, sodium pyruvate, laked human erythrocyte and serum. The growth of the rod spiral form was found in the coccoid form bacteria, and further the bacteria could grow on blood agar and Skirrow agar as well as the original strain. It was strongly suggested that the resuscitation from VNC state of cells to culturable form consists of two processes, stimulation and supplemented of appropriate nutrition.

Development of a new semiselective medium for isolating Xanthomonas campestris pv. manihotis from plant material and soil.

Abstract: An effective control for bacterial blight of cassava (Manihot esculenta), caused by Xanthomonas campestris pv. manihotis, requires the use of non-contaminated cuttings and seeds. Using classical agar plating techniques for screening planting material for contamination has not been very successful because of the lack of a reliable semiselective agar medium. The pathogen grows slowly on general plating media and is easily overgrown by saprophytic bacteria during isolation from diseased plants. In an effort to develop a semiselective medium, the utilization of several carbon and nitrogen sources was studied. Results of these tests provided information used to design a basal medium allowing good growth of the target organism while suppressing growth of several common saprophytes. Additional selectivity was achieved by incorporating three antibiotics into the basal medium. The new semiselective agar medium, designated cefazolin trehalose agar (CTA) medium, contained (per liter) 3.0 g of K2HPO4, 1.0 g ...

Comparison of direct and enrichment methods for the selective isolation of vancomycin-resistant enterococci from feces of pigs and poultry.

Abstract: Isolation results of vancomycin-resistant enterococci (VRE) of fecal samples from pigs and broiler and layer chickens obtained with two vancomycin-supplemented enrichment media, kanamycin aesculin azide (KAA) broth and Enterococcosel (ECC) broth, and three isolation media, KAA agar, ECC agar, and Slanetz and Bartley (SL) agar, were compared. Direct isolation on vancomycin-containing agar plates was not efficient in swine and layer chickens, which had only low numbers of VRE. In broilers chickens, the VRE content of the samples was high, and SL as well as ECC were found to perform better than KAA agar. The same three agar media were used as selective plating media after 1 and 2 days incubation of the samples in KAA and ECC enrichment broths. Sensitivities of the 12 different enrichment-plate combinations tested ranged from 0 to 81% in layer chickens and from 5 to 44% in samples from pigs. In the high prevalence type of samples from broilers, sensitivities still varied substantially from 52 to 78%. Incubating vancomycin-containing enrichment broths for 2 days compared with 1 day was favorable for the isolation of vancomycin-resistant Enterococcus faecalis, E. gallinarum, and E. casseliflavus but not for E. faecium and E. hirae/E. durans. ECC broth and ECC plates yielded the highest number of E. gallinarum and E. casseliflavus. In layer as well as in broiler chickens, ECC broth incubated for 2 days and plated on ECC agar was the most sensitive method. In pigs, however, KAA broth incubated for 2 days and plated on ECC medium yielded the highest number of VRE.

Single culture media in infectious keratitis.

Abstract: Purpose To evaluate the usefulness of the various culture media used in the traditional workup in infectious keratitis. Methods Microbiology data sheets from all corneal cultures performed at the University of California Davis Medical Center over a 1-year period were reviewed retrospectively. Results Bacterial cultures were sent in 76 cases. In 19 cases, culture specimens from ulcers were plated onto blood, chocolate, and inhibitory mold agar and were inoculated into an anaerobic medium. In 58 cases, blood and chocolate agar were sent. In 70% of cases, blood and chocolate agar provided identical information. Inhibitory mold agar was positive twice in 39 plates sent. A fungal pathogen had been identified on chocolate agar plates sent for these cases. Conclusion In the evaluation of infectious keratitis, plating onto chocolate agar or blood agar alone is a reasonable alternative to sending multiple cultures.

Heavy Metal Tolerance in Chromogenic and Non-Chromogenic Marine Bacteria from Arabian Gulf

Abstract: A simple method – direct agar diffusion assay – was optimised for rapid assessment of heavy metal toxicity to marine chromogenic and non-chromogenic bacteria. The procedure involved spotting of a 10 microliter test solution on the seeded agar plate and incubation of the plates at 30°C to accelerate bacterial growth. Under optimum conditions, test results were obtainable within 12–18 hr instead of 96 hr incubation time generally required for a marine bacterial assay by conventional agar plate methods. A range of sixteen heavy metals, each at 5 different concentrations was tested. Toxicity was demonstrated by the formation of a clear zone of growth inhibition around the point of application. Toxicity of tested chemicals could be easily demonstrated at concentrations as low as 0.1 μg per spot on the agar plate. A dose dependent relation between metal concentration (μg/spot) and the diameter of the clear zone on agar plate was observed, suggesting potential of this method as an easy and economical tool in quantitative toxicology studies.