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[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

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.

Agar

Rapid identification of Enterobacteriaceae. II. Use of a beta-glucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples.

beta-glucuronidase activity is an exclusive characteristic of E. Coli and some shigellae among Enterobacteriaceae and Vibrionaceae. An agar medium (PGUA agar) which permits the detection of bacteria with beta-glucuronidase activity in mixed cultures was evaluated as a primary culture medium for clinical samples of urine. The medium was selective for enterobacteria and yielded significantly higher recoveries than MacConkey agar. Based on the examination of 3,460 urine samples, it was found that the use of the PGUA agar has several advantages over conventional methods: 1) 94% of all E. coli cultures could be identified on the basis of their appearance on the primary plates; 2) The use of the PGUA method did not result in any misidentidications as compared to 1% of cultured misidentified by the conventional procedure; 3) Approximately one-half of the urine samples which contained E. coli as the sole organism could be reported following the reading of primary culture plates; 4) The application of the PGUA medium resulted in a 46% reduction in the cost of media employed and a 67% reduction in the time required for the processing of urine samples.

Agar

Effect of agar percentage, agar thickness, and medium constituents on antibiotics assay by disc diffusion method.

The factors affecting the sensitivity of nafcillin, vancomycin, oleandomycin, chloramphenicol, methacycline and novobiocin assay, using the paper disc diffusion method, were studied. Agar concentration and pour volume were found to influence the assay sensitivity of antibiotics. The type of assay medium was effective e.g. brain heart infusion agar was the best medium for nafcillin and methacycline; synthetic amino acid medium for oleandomycin; Muller Hinton agar for vancomycin and chloramphenicol; and G & R medium for novobiovicin. All additives, including inorganic ions, sugar, organic acids, complexing agents, surface active agents and blood, decreased the activities of the studied antibiotics. Higher activities of vancomycin, methacycline were observed at pH = 5, of nafcillin, chloramphenicol, and novobiocin at pH = 6, and of oleandomycin at pH = 7.

Agar

Comparison of antibiotic-amended potato dextrose agar and acidified potato dextrose agar as growth substrates for fungi.

Fifteen fungal species, all isolated from food, were compared for their growth abilities on potato dextrose agar acidified to pH 3.5, and on nonacidified potato dextrose agar amended with 40 ppm chlortetracycline hydrochloride. Comparisons were made at 16, 21, 26, 32, and 37 degrees C. Of the 15 species, only Penicillium expansum exhibited better growth on the acidified medium than on the nonacidified antibiotic medium, while 9 species grew better on the nonacidified antibiotic medium. Five species grew equally well on either medium.

Agar

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.

Bacteriological Techniques

Clonal growth of Entamoeba histolytica and other species of Entamoeba in agar.

This is the first description of a method for growing axenized Entamoeba histolytica as colonies from single cells in agar suspension. Factors affecting the efficiency of colony formation included unknown characteristics of the amebal strain, age of cells used, and the type and concentration of agar employed. Colony formation was dependent upon filling deep vessels (culture tubes or tissue culture flasks) with agar, probably to maintain reduced oxygen tension, and upon solidifying the agar rapidly at 0 C. It was demonstrated in a study with the drug metronidazole that the agar colony method was useful for quantifying cell viability in drug testing. Eleven of 12 E. histolytica strains tested, as well as one Entamoeba terrapinae, one Entamoeba barreti, and 3 Entamoeba invadens strains formed colonies in agar suspension. E. histolytica-like amebae (Laredo type) and E. moshkovskii formed only tiny colonies in agar.

Agar

Evaluation of enrichment, storage, and age of blood agar medium in relation to its ability to support growth of anaerobic bacteria.

By measuring the colony size of a variety of anaerobic bacteria isolated from clinical specimens, an evaluation was made of the benefits derived from the addition of several enrichments to blood agar medium commonly used for the growth of anaerobes. Similar methods were used to study the effects of various storage conditions and age of the medium. The results were compared with those obtained on freshly prepared and enriched blood agar plates as well as commercially available blood agar plates. Freshly prepared and enriched blood agar was found to give substantially larger colonies than could be grown on commercially obtained blood agar plates when both were inoculated and incubated under identical conditions. Storage of plating media under CO2 for periods of up to 72 h had only a minor effect on the growth of the anaerobic bacteria studied, but longer periods of storage under CO2 resulted in a less efficient plating medium. Nonenriched brain heart infusion (BHI) was found to be a better basal medium than Trypticase soy agar (TSA) medium. Colony size on fully enriched BHI blood agar plates was greater than nonenriched BHI greater than nonenriched TSA greater than commercially prepared nonenriched TSA plates. The data suggest that freshness of the plates may be as important as using rich media.

Bacteroidaceae

Inability of the standardized disk agar-diffusion test to measure susceptibility of the fluorescent group of pseudomonads to gentamicin.

Four hundred thirteen clinical isolates of fluorescent pseudomonads consisting of 286 Pseudomonas aeruginosa and 127 organisms of the Pseudomonas fluorescent group were tested for susceptibilities to gentamicin by the broth-dilution method and by the standardized disk agar-diffusion test, using Mueller-Hinton agar from three sources. Different regression lines and significantly different mean inhibition zone diameters were found for the three Mueller-Hinton agars. Single zone-diameter breakpoints of either 13 or 16 mm for the standardized disk agar-diffusion test produced unacceptable rates of false-sensitive and false-resistant interpretations. When error rates were limited by using zone-diameter breakpoints determined by the method of error rate-bound analysis, the percentage of indeterminate interpretations became so large as to make the standardized disk agar-diffusion test impractical. It is concluded that the standardized disk agar-diffusion test should not be used for testing susceptibility of the fluorescent pseudomonads to gentamicin, and presumably other aminoglycosides.

Agar

Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila.

Charcoal-yeast extract agar is a new bacteriological medium that supports excellent growth of the Legionella pneumophila. It results from modifications made in an existing L. pneumophila medium, F-G agar. Yeast extract, instead of an acid hydrolysate of casein, serves as the protein source. Beef extractives and starch are not added. Activated charcoal (Norit A or Norit SG) is included at 0.20% (wt/vol). Comparison of charcoal-yeast extract and F-G agars showed that a greater number of colony-forming units of L. pneumophila was recovered from a standardized tissue inoculum on charcoal-yeast extract agar (4.35 x 10(6) colony-forming units) than on F-G agar (4.85 x 10(4) colony-forming units). Macroscopic colonies of L. pneumophila were visible on the new medium within 3 days, whereas 4 days of growth was required on F-G agar.

Agar

Reversal of the silver inhibition of microorganisms by agar.

Increasing use of silver in the treatment of water has necessitated an examination of microbiological methods for the measurement of silver inactivation of microorganisms. Three common agar media were tested for their ability to neutralize the bacteriostatic effects of silver. Results suggested that growth media differed in their neutralizing capacity; that is, the non-inhibitory media tryptone glucose agar and Trypticase soy agar showed more neutralizing capacity than eosin methylene blue agar. Furthermore, the neutralizing effect appeared to be a function of the soluble component of the media and not of the agar itself.

Agar

Clonal growth of Entamoeba in agar: some applications of this technique to the study of their cell biology.

A new technique for growing single Entamoeba trophozoites into colonies in agar has been developed. This method depended upon axenic cultivation and utilized Diamond's new TYI-S-33 medium. The present paper describes several different types of experiment which have utilized the agar technique. a) Cloning. The isolation of clones from agar way easy and successful. Clones of the HM-1 strain did not differ in virulence for newborn hamster liver or in colony morphology or colony forming efficiency. b) Viability measurements. Colony forming efficiency (CFE) was proportional to the number of viable cells in a culture. Pilot studies with the amebacides, metronidazole and emetine, showed that the agar method should be useful in drug testing. Colchicine at high concentration inhibited clonal growth in a non-specific manner. c) E. histolytica HM-1 and E. invadens cells were rapidly killed by exposure to 42 degree C but survived relatively well at 0 degree C. d) Hemolysis. In a preliminary experiment HM-1 colonies did not produce halo's of hemolysis when grown in agar containing sheep red blood cells.

Agar

Development of the agar disk method for the rapid selection of cephalosporin producers with improved yields.

To screen the abilities of mutant strains of Cephalosporium to produce cephalosporin C, colonies of the organism were grown on the surface of small (4-mm diameter) disks of agar medium. After incubation of the disks for periods of up to 5 days, the antibiotic contents of the disks were assayed by placing them on agar plates of the assay organism and determining the diameters of the inhibition zones. The amount of nitrogen source in the agar disk medium was used to control the amount of antibiotic produced in the disk and, thus, the sensitivity of screening. The relation of agar disk inhibition zone diameters to log shake-flask titers was linear with short incubation times (2 to 3 days) of the disks, but shifted towards a higher order with prolonged incubation (4 to 5 days). The optimum incubation time for the disks was 4 to 5 days, and then a 15% difference in zone diameters was significant with 10 disks per sample. The minimum difference between the shake-flask titers, which could be detected by the agar disk method with 10 disks per sample, was about 30% with 5 days of incubation for the disks. The results suggest that the shake-flask culture underestimated the degree of improvement in strain productivity.

Acremonium

In vitro culture of lymphocyte colonies in agar capillary tubes after PHA-stimulation.

Human peripheral lymphocytes were stimulated with phytohemagglutinin (PHA) in liquid culture, suspended in agar and incubated in glass capillary tubes. Compact colonies of lymphocytes were found growing along the tube bottom in a buffer film, while compact clusters and rare diffuse colonies were observed inside the agar. Several parameters affecting the clonal growth were studied and optimized: PHA-dose, agar concentration, gel length (volume), quantity and density of seeded cells per capillary and gel length. Colony yield mainly depends on the seeded-cell density with a sharp optimum at 2 X 10(5) cells/ml irrespective of gel length; higher cell densities reduce the colony yield, suggesting that colony growth is the result of both stimulatory and inhibitory factors produced by cooperating cells. Following the daily clonal growth was only possible with undisturbed tubes; the number of colonies steadily increased from day 2 until day 7. Densitometric colony scanning is possible, yet problematic. Colony yield (plating efficiency is 10--50-fold higher in agar capillaries than in the usual Petri dishes. An additional advantage is that the capillaries provide a basis for a simple and reliable assay system for determining regulatory factors of lymphocyte proliferation (including chalones).

Agar

Agar-coated dip-slides for the cultivation, storage and transport.

Dip-slides which are covered with suitable agar media (e.g. malt extract agar, potato dextrose agar, Czapek Dox agar) are well suited for the cultivation, storage and transport of moulds and phytopathogenic fungi. Most of the organisms develop equally well on dip-slides and in Petri dishes.

Agar

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).

Agar

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.

Agar