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Isolation of Shigellae. 8. Comparison of xylose lysine deoxycholate agar, hektoen enteric agar, Salmonella-Shigella agar, and eosin methylene blue agar with stool specimens.

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.

Aeromonas↗

Fastidious anaerobe agar compared with Wilkins-Chalgren agar, brain heart infusion agar, and brucella agar for susceptibility testing of Fusobacterium species.

Fastidious anaerobe agar supported the growth of 82 strains of fusobacteria better than brain heart infusion agar, brucella agar, and Wilkins-Chalgren agar. Fastidious anaerobe agar showed less hazing and fewer tailing endpoints with beta-lactam antibiotics. Whole-blood supplementation improved the performance of all media. Wilkins-Chalgren agar without blood failed to support the growth of 17% of the strains. All Fusobacterium ulcerans strains were resistant to clindamycin.

Agar↗

Comparison of blood agar, ampicillin blood agar, MacConkey-ampicillin-Tween agar, and modified cefsulodin-Irgasan-novobiocin agar for isolation of Aeromonas spp. from stool specimens.

The performance of four media for the isolation of Aeromonas strains from stool specimens, the importance of ampicillin-susceptible Aeromonas strains in the selection of culture media, and the usefulness of beta-hemolysis in screening blood-containing media for Aeromonas strains were evaluated in two phases. In the first phase, 36 of 1,672 stool specimens yielded Aeromonas isolates. Ninety-seven percent of the isolates were detected on blood agar containing 20 micrograms of ampicillin per ml (ABA), and 47% were detected on MacConkey agar containing 100 micrograms of ampicillin per ml and 1% Tween 80. In the second phase of the study, 43 of 1,924 stool specimens yielded Aeromonas isolates. Fifty-one percent of the isolates were detected on blood agar and on modified cefsulodin-Irgasan-novobiocin agar, and 84% were detected on ABA. The combination of ABA and modified cefsulodin-Irgasan-novobiocin agar provided 100% recovery of the Aeromonas isolates encountered. All of the Aeromonas isolates detected on blood agar were also detected on ABA, and 89% of the Aeromonas isolates detected on these media were beta-hemolytic. These results suggest that ABA is superior to the other media evaluated for the isolation of Aeromonas strains from stool specimens, but optimal recovery of the organism may require the use of more than one medium. The results also suggest that the occurrence of ampicillin-susceptible strains is not a limitation on the use of ABA, but at least 10% of Aeromonas isolates will be missed if beta-hemolysis is used to screen ABA plates for these organisms.

Aeromonas↗

[Effect of different grades of agar-agar on the nature of the test microbe growth inhibition zones in controlling antibiotic activity by means of agar diffusion].

The effect of various agar grades on the size and margin character of the inhibition growth zones in assay of antibiotic activity by the agar diffusion method was studied. It was shown that not all the agar grades could be used in antibiotic activity assay. Depending on the agar type the size of the inhibition growth zones produced by the same antibiotic concentration significantly varied. The variations in the size of the inhibition growth zones depended on the agar ability to bind antibiotics and were mainly defined by the agar purity. The agars with low content of nitrogen admixtures bound the antibiotics to a low extent. The commerical grades of the agars of the South-Sea and Korsakov Plants, the experimental grade of the TINRO agar with additional purification, as well as the agars imported from Argentina and France proved to be most useful for determination of the antibiotic activity by the agar diffusion method.

Agar↗

Morphological identification of Candida species on glucose agar, rice extract agar and corn meal agar with and without Tween-80.

A comparative study for the identification of 32 known strains of Candida species on the basis of morphology on glucose agar, rice extract agar and corn meal agar with and without Tween 80 revealed that when Tween 80 is incorporated in the media identification is possible for 96.8% of the species within 48 hours on rice extract agar and for 96.8% of the species within 48 hours on rice extract agar and for 90.6% of the species on glucose agar. The germ tubes and chlamydospores were also produced more on rice extract agar than on 0.1% glucose agar. Rice extract agar with Tween 80 can be used as single medium for morphologic identification of Candida species. The inoculated medium is first incubated at 37 degrees C for 3 hours and examined for germ tube formation and then incubated at 25 degrees C for 24 to 72 hours and examined for appearance of chlamydospores and mycelial morphology.

Agar↗

A comparison of blood agar supplemented with NAD with plain blood agar and chocolated blood agar in the isolation of Streptococcus pneumoniae and Haemophilus influenzae from sputum. Bacterial Methods Evaluation Group.

Streptococcus pneumoniae grows well and generally exhibits typical morphology on Columbia blood agar, whereas Haemophilus influenzae requires a more complex medium to meet its growth requirements - usually chocolated blood agar - on which S. pneumoniae is less easily recognisable. Therefore, a single medium that produces typical morphology of S. pneumoniae and facilitates the growth of H. influenzae would have considerable potential advantages. It has been claimed that blood agar supplemented with nicotinamide adenine dinucleotide (NAD) is such a medium. However, despite its routine use in several large diagnostic laboratories its performance has never been properly evaluated. In the present study, 1724 sputum samples were examined in four laboratories. The isolation rates of H. influenzae and S. pneumoniae on NAD-supplemented blood agar (SBA) were compared with those on a two-plate combination of plain blood (BA) and chocolated blood agar (CBA). The two-plate combination performed significantly better for both organisms; isolation rates for H. influenzae were increased from 8.16% on SBA to 11.07% on BA plus CBA and for S. pneumoniae from 4.18% to 4.68%. Isolation rates were also compared after incubation for 24 and 48 h. With the two-plate combination, isolation rates for H. influenzae and S. pneumoniae were increased by 0.98% and 0.16%, respectively, and for SBA by 0.57% and 0.32% after 48 h. However, despite this increase, SBA still performed less well than the two-plate combination.

Agar↗

Efficacy of brain heart infusion-egg albumen agar, yeast extract phosphate agar and peptone glucose agar media for isolation of Blastomyces dermatitidis from sputum.

The efficacy of brain heart infusion (BHI)-egg albumen agar, yeast extract phosphate agar and several modified peptone glucose agar media was evaluated for isolation of Blastomyces dermatitidis from sputum concomitantly seeded with the yeast form of the pathogen and Candida albicans. Based upon high per cent culture positivity of sputum, improved recovery (CFU/ml) of the seeded inoculum, faster growth rate of B. dermatitidis and low level of contamination, BHI-egg albumen agar, followed by yeast extract phosphate agar are recommended as the media of choice for the isolation of B. dermatitidis from contaminated clinical specimens.

Blastomyces↗

Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Enterococcus faecalis and E. faecium: comparison of Mueller-Hinton, Iso-Sensitest, and Wilkins-Chalgren agar media.

Forty-two isolates of Enterococcus faecalis and 56 isolates of Enterococcus faecium, including 8 vancomycin-resistant strains, were examined for comparative susceptibility to 27 antimicrobial drugs with the agar dilution method, employing Mueller-Hinton (MHA), Iso-Sensitest (ISTA), and Wilkins-Chalgren (WCA) agar. The Bauer-Kirby agar disk diffusion method was used to comparatively test 24 of the agents in parallel. The enterococci yielded better growth on ISTA and WCA. However, WCA completely antagonized co-trimoxazole and, though less, fosfomycin. Importantly, WCA slightly reduced the activities of teicoplanin (minimal inhibitory concentrations, MICs, raised up to twofold) and vancomycin (MICs raised two- to fourfold) against enterococci and staphylococcal quality control strains. Therefore, WCA was judged unsuitable for susceptibility testing of enterococci. For E. faecalis no discrepancies between agar dilution MICs and inhibition zone diameters were encountered with augmentin, ampicillin, ampicillin-sulbactam, chloramphenicol, mupirocin, oxacillin, teicoplanin, and co-trimoxazole. Overall, MHA yielded fewer very major (category I) and major (category II) discrepancies than ISTA. However, numerous minor (category III), slight (category IV), minimal (category V), and/or negligible (category VI) discrepancies were encountered with ciprofloxacin, doxycycline, erythromycin, fosfomycin, fusidic acid, meropenem, ofloxacin and rifampin. With respect to E. faecium, only cefotaxime, mupirocin, oxacillin, and teicoplanin yielded nondiscrepant results. Several very major (I) and major (II) discrepancies were observed with augmentin, ampicillin, ampicillin-sulbactam, doxycycline, fusidic acid, imipenem, and penicillin G. Minor discrepancies (categories III-VI) were particularly numerous with augmentin, chloramphenicol, ciprofloxacin, doxycycline, and piperacillin. The largest numbers of negligible (VI) discrepancies were noted with fosfomycin, fusidic acid, and ofloxacin. It is recommended to test one cephalosporin (cefuroxime or the like) in parallel for educational purposes and to exclude fosfomycin, fusidic acid, and rifampin from test batteries because of the wide scatter of test results. The large number of minimal (V) discrepancies of ciprofloxacin against E. faecalis, the numerous minor (III) and slight (IV) discrepancies of chloramphenicol against E. faecium, and the not insignificant number of very major (I) and minor (III) discrepancies observed with meropenem against isolates of E. faecalis necessitated proposals for new disk intermediate susceptibility criteria.

Agar↗

[Evaluation of three new culture media: brilliant-glycerol-lactose novobiocin-green agar, modified iron lysin agar and Rambach agar for the isolation of enteropathogenic E. coli, Salmonella sp. in acute gastroenteritis].

BACKGROUND: Acute gastroenteritis (AGE) is one of the most common diseases in children, particularly in those under the age of 5 years in whom a severe picture of hydroelectrolyte imbalance may be triggered accompanied, in most cases, by leukocyte response. The aim of this study was to evaluate three new culture media for the isolation of enteropathogens in infantile diarrhea. MATERIAL AND METHODS: From April to September, 1993, 170 samples of diarrhea stools from children up to 7 years of age attending pediatric hospitals in Bogota were studied. Three new culture media were used in the isolation of the enteropathogenic bacteria: BGLN, MILA agar, Rambach agar and a conventional agar medium S.S. Biochemical tests were performed to identify the isolated bacteria. RESULTS: A total of 98.5% of enteropathogenic E. coli was isolated in Rambach agar, which also presented excellent recovery rates of Salmonella sp. (100%) versus S.S> (64.3%) and BGLN (77.2%), respectively. The best selectivity for Shigella sp. was observed with BGLN with a 100% recovery rate. Out of the 170 samples 105 showed a leukocyte count of 70-75% and positive isolation for enteropathogenic bacteria. Six samples with the same leukocyte count did not present enteropathogenic bacteria, with the 59 remaining samples with a 20-25% PMN count being negative for enteropathogenic bacteria. CONCLUSIONS: The results suggest that the new culture media used in this study may have better recovery rates for enteropathogenic bacteria in acute gastroenteritis. Likewise, a correlation was observed between leukocyte count and isolation enteropathogenic bacteria.

Acute Disease↗

Agar diffusion, agar dilution, Etest, and agar screening test in the detection of methicillin resistance in staphylococci.

Methicillin resistant Staphylococcus is an important worldwide problem. Resistance is verified in strains harboring the mecA gene and laboratory methods used to detect resistance are object of constant investigation. In the present study, 99 clinical isolates of staphylococci (41 S. aureus, 33 S. epidermidis, 12 S. saprophyticus and 13 members of other species) were submitted to different phenotypic methods and conditions. Detection of the mecA gene by PCR was used as the reference method and detected 14/41, 10/33, and 10/25 isolates of S. aureus, S. epidermidis and other species, respectively. Results showed that, for S. aureus and S. epidermidis, agar diffusion, agar dilution, and the E test incubated during 24h at 35 degrees C correctly discriminated mecA positive from mecA negative isolates. For other species, all methods and conditions presented low specificity (ranging from 20% to 66.7%) and, particularly S. saprophyticus, may need molecular methods to correctly assess methicillin resistance.

Agar↗

Comparison of recovery of airborne microorganisms in a dairy cattle facility using selective agar and thin agar layer resuscitation media.

Thin agar layer (TAL) medium was developed at Kansas State University to improve the resuscitation of injured cells and has been shown to result in higher recovery than is obtained with selective media alone for cold-, heat-, salt-, and acid-injured cells. The experiment presented here was designed to determine the effectiveness of the TAL method for the recovery of possibly injured organisms from air. Eleven agar media were used for the experiment: tryptic soy agar (TSA), MacConkey sorbitol agar (MSA), TAL-MSA, Baird-Parker (BP) agar, TAL-BP agar, modified Oxford (MOX) agar, TAL-MOX agar, xylose lysine sodium desoxycholate (XLD) agar, TAL-XLD agar, Yersinia-selective (CIN) agar, and TAL-CIN agar. The TAL plates were prepared by pipetting 6 ml of selective agar into a BBL Rodac plate (65 by 15 mm). Selective agar was allowed to solidify, and then each plate was overlaid with 6 ml of TSA. Selective agar plates were prepared by pipetting 12 ml of agar into BBL Rodac plates and allowing the agar to solidify. Samples were taken at an indoor cattle facility at five separate locations with a BioScience SAS air-sampling instrument. For each plate, 60 liters of air was sampled. Three replications of the experiment were performed. The TAL method resulted in higher counts of microorganisms on all media tested. In addition, 175 isolates were selected randomly and identified in order to test the selectivity of TAL and the selective media for target organisms. The data obtained in this study show that the TAL resuscitation method is effective and necessary for the recovery of airborne organisms that may be injured.

Agar↗

Mueller-Hinton agar is superior to PDM blood agar for detection of methicillin-resistant Staphylococcus aureus.

The aim of this study was to compare the expression of oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) on Paper Disc Method agar supplemented with 5% defibrinated blood (PDM blood agar) and Mueller-Hinton agar supplemented with 2% NaCl (MH NaCl agar) using different susceptibility tests. Fifty mecA-containing isolates of S. aureus, exhibiting 46 different pulsed-field gel electrophoresis patterns, were comparatively tested using the E test, the single disk diffusion test, and the multipoint inoculation technique, under various culture conditions. The E test incubated at 35 degrees C for 24 h (breakpoint of resistance > or = 2.0 mg/L) detected 94% of the isolates on MH NaCl agar, compared with 28% for PDM blood agar (P < 0.05). The disk diffusion test (breakpoint < 10 mm in diameter) under these incubation conditions detected resistance in 100% of the isolates on MH NaCl agar and in 80% of the isolates on PDM blood agar (P < 0.05). The multipoint technique (breakpoint > or = 1 mg/L), applied at 35 degrees C for 24 h, detected 100% on MH NaCl agar and 46% on PDM blood agar (P < 0.05). Irrespective of the method of susceptibility testing evaluated, MH NaCl agar was superior to PDM blood agar for the detection of oxacillin resistance in mecA-containing S. aureus.

Agar↗

Comparison of the productivity of cefoperazone amphotericin teicoplanin (CAT) agar and modified charcoal cefoperazone deoxycholate (mCCD) agar for various strains of Campylobacter, Arcobacter and Helicobacter pullorum.

Cefoperazone amphotericin teicoplanin (CAT) agar was developed from cefoperazone deoxycholate (mCCD) agar by modification of the selective antibiotics in order to permit growth of strains of Campylobacter upsaliensis. In this study, 35 strains of Campylobacter and Arcobacter were tested for their ability to grow on CAT and mCCD media using the ecometric method. Six of these strains were also tested using the modified Miles-Misra method. Overall, nineteen strains out of the 35 tested grew better on CAT than on mCCD agar, although for eight strains, the difference was slight. These differences could not be attributed solely to poorer growth of C. upsaliensis on mCCD agar. No strain grew better on mCCD than CAT agar. Eight of the 35 strains tested did not grow on mCCD agar at all, however, only one strain failed to grow on CAT medium. The two methods of testing gave similar results, although the Miles-Misra method was found to be more sensitive and less prone to subjective interpretation. All four CNUPC (catalase negative, urease positive campylobacter-like) strains, one strain of C. sputorum biovar, fecalis, one of two Arcobacter cryaerophilus strains (incubated at 30 degrees C, aerobically) could be detected only using CAT agar. In addition, for some strains of A. butzleri, C. upsaliensis and C. hyointestinalis, CAT medium gave better growth scores than mCCD agar. The level of cefoperazone in mCCDA is inhibitory to some campylobacter strains, but suboptimal growth of Arcobacter strains is more probably due to synergistic interaction between deoxycholate and cefoperazone. CAT agar supports the growth of a wider variety of Campylobacter and Arcobacter species than mCCD agar.

Agar↗

[Differences in susceptibility of Helicobacter pylori to macrolide and other antibiotics in tests using blood agar and albumin agar].

Minimum inhibitory concentrations (MICs) of penicillin G, cefazolin, tetracycline, chloramphenicol, four aminoglycosides, and seven macrolides against Helicobacter pylori strains were determined on Brucella agar supplemented with bovine serum albumin fraction V (albumin agar) and compared with those on blood agar. MICs of the macrolides against H. pylori determined on albumin agar were markedly lower than those found with blood agar. The susceptibilities of 26 H. pylori strains to the macrolides were estimated from the MIC50 values determined on blood agar and albumin agar, which were 0.39 and < or = 0.05 micrograms/ml for erythromycin, midecamycin and rokitamycin, 3.13 and 0.10 micrograms/ml for clindamycin, 1.56 and < or = 0.05 micrograms/ml for josamycin, 1.56 and 0.10 micrograms/ml for kitasamycin, and 0.10 and < or = 0.05 micrograms/ml for roxithromycin, respectively. However, the MICs of other antibiotics against H. pylori and of all antibiotics against two reference Campylobacter strains differed minimally between values obtained on blood agar and albumin agar.

Agar↗

Effects of agars on determination of potency of polymyxin B sulfate by the agar plate diffusion method.

The effects of agars on the determination of the potency of polymyxin B sulfate by the agar plate diffusion method were investigated. The results showed that there were significant differences in the potency of polymyxin B sulfate between agars produced by different manufacturers. In order to investigate this phenomenon, two major components of polymyxin B sulfate, namely, the polymyxin B1 and B2 fractions, were isolated and purified by preparative high-pressure liquid chromatography, and the effects of agars on the potencies of these two components were individually examined. The results showed that there were significant differences in the relative potency of the B1 component versus that of the B2 component between the agar preparations produced by different manufacturers. On the basis of the relative potency of each of the components and the composition of the components, we developed a theoretical equation with which we could calculate the activity of polymyxin B sulfate on a certain agar. The values computed from the equation were consistent with those obtained from actual measurements. It was concluded that the effects of agars on the determination of polymyxin B sulfate activity will become evident only when the composition of the reference standard significantly differs from that of the samples. When the standard and samples have the same compositions, the sources of the agar preparation exert no effect on the determination of the potency by the agar plate diffusion method.

Agar↗