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PubMed1978

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↗

PubMed1978

Disk agar diffusion susceptibility testing of yeasts.

A disk agar diffusion method was developed for testing the susceptibility of rapidly growing yeasts in vitro. A totally defined, completely synthetic agar culture medium (synthetic amino acid medium, fungal) and clinical isolates of Candida spp. and Torulopsis glabrata were used. Turbidimetric adjustment of cell suspensions resulted in standard, reproducible inocula, which gave sharp, clear zones of inhibition when applied by an agar overlay method. Optimal disk loads were determined for amphotericin B, amphotericin B methyl ester, 5-fluorocytosine, clotrimazole, and miconazole. Disk potencies were stable over a 2-month period when stored in a vacuum desiccator at -30 degrees C. Using an error ratebounded classification, the zones of inhibition were correlated with both broth dilution and agar dilution minimum inhibitory concentrations (MICs). With amphotericin B and amphotericin B methyl ester, all isolates were susceptible, yielding zone diameters which clustered within 5 mm. Overall correlations between zone diameters and broth dilution MICs with 5-fluorocytosine, miconazole, and clotrimazole were 97, 96, and 82% (excluding T. glabrata), respectively; correlations of zone diameters with agar dilution MICs were 96, 92, and 88%, respectively. Disk diffusion susceptibility testing of yeasts appears to be generally applicable. However, when results are equivocal, quantitative test methods should be used.

Amphotericin B↗

PubMed1977

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↗

PubMed1977

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↗

PubMed1976

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↗

PubMed1978

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↗

PubMed1979

Presumptive identification of group A, B, and D streptococci on agar plate media.

Several presumptive tests were evaluated for their effectiveness in differentiating streptococci. When the tests were combined into a battery and the resulting reactions were interpreted as patterns, the overall presumptive identification rate was at least 97%. We used the hemolytic reaction, susceptibility to bacitracin and sulfamethoxazole plus trimethoprim (1.25 micrograms plus 23.75 micrograms), and standard CAMP reactions on sheep blood Trypticase soy agar, and bile-esculin and 6.5% NaCl agar tolerance tests with incubation in candle extinction jars. Subsequently, 98.9% of the group A; 95.3% of the group B; 100% of the beta-hemolytic non-group A, B, or D; 92.3% of group D enterococcal; 100% of the group D non-enterococcal; and 92.8% of the viridans streptococci were presumptively identified. We then used the hemolytic reactions, susceptibility of bacitracin and sulfamethoxazole-plus-trimethoprim disks, CAMP disk reactions on sheep blood Trypticase soy agar and bile-esculin and 6.5% NaCl agar tolerance tests with incubation in normal atmosphere. Subsequently, 98.1% of the group A; 98.6% of the group B; 99.2% of the beta hemolytic non-group A, B, or D; 97.5% of the group D entercoccal; 97.6% of the group D non-entercoccal; and 92.4% of the viridans strains were presumptively identified.

Aerobiosis↗

PubMed1979

A method for establishing stable concentration gradients in agar suitable for studying chemotaxis on a solid surface.

A simple technique has been developed for establishing stable gradients of a substance in agar. The technique involves the creation of a spherically symmetric concentration profile in which concentration varies inversely with the distance from the source and is independent of the diffusion coefficient of the substance. It has been shown that the gradients established with this technique are stable for at least 190 h. and, on a theoretical basis, they can be kept stable for more than 1000 h. Time-variant gradients can also be established, if desired, using the same system and limiting either the source or the agar sink. It must be emphasized that a stable gradient cannot be obtained by using a shallow agar layer as a sink. The use of such conditions (e.g. the agar in a standard petri dish) can result only in time-variant gradients. The solution to the diffusion equation in a spherically symmetric system establishes the expected concentration profile, the basis for adjusting it, and the parameters that control the behavior of the system. Some useful applications for examining chemotaxis on a solid surface as well as possible further developments are discussed.

Agar↗

PubMed1979

Preparation and observation by SEM of hemopoietic cells cloned in soft agar.

A method is described to prepare clones of hemopoietic cells grown in soft agar for scanning electron microscopy (SEM). A critical modification of the otherwise quite standard SEM processing procedure for biological samples involved the use of silver micropore disks as an adherent substrate to support the highly labile, deformable agar slabs. This support allows maintenance of the normal flat pancake shape of the specimen through the thiocarbohydrazide osmium ligand binding steps, dehydration, and critical point drying. With this support and careful dissection of the surface agar with a fine steel needle using a stereomicroscope, selected areas and depths within the colony can be exposed and examined by SEM. Surface topography of cloned cells can be correlated with intracellular cytological features by excising areas of interest and directly embedding them in plastic for thin-section preparation and viewing by transmission electron microscopy (TEM). The dried-specimen-teasing method appears useful, because of the ease of preparation of the specimens, its reproducibility, and the degree of visibility and preservation of cell surface structures and intraclonal relationships. Our initial observations, using combined EM techniques, indicate that clonal cell topography is highly variable and that this variability appears to be related both to the relative age and proliferative status of the colony. Based on work to date, we suggest that topographical and spatial analysis, in vitro of cloned, agar-embedded hemopoietic stem cells is possible with simple modifications of conventional SEM preparative techniques.

Agar↗

PubMed1977

[The agar diffusion method round table discussion on the methods and indications for determining blood levels of aminoglycosides (author's transl)].

The present-day technique of determining the concentration of aminoglycosides in blood by agar diffusion is described including the apparatuses, set-up, and reliability tests. The results of comparative investigations showed a higher degree of conformity in Mueller-Hinton agar than in standard agar. Due to its accuracy the agar diffusion method is especially suited for the calculation of dosage recommendations and for the prediction of the maximal and minimal blood levels during treatment with aminoglycosides.

Agar↗

PubMed1977

[The results in our tuberculosis laboratory with penicillin blood agar medium].

A comparison of Löwenstein - Jensen and penicillin blood agar media, which can be prepared in small laboratories, is made in routine diagnostic cultivations of Mycobacterium tuberculosis. Tuberculosis. Tubercle bacilli grown in Löwenstein - Jensen medium were inoculated in Löwenstein - Jensen and penicillin blood agar media in numbers of 100.000, 10.000, 1000 and 100 bacilli and their growth was detected in each medium. It was found that the growth in both media was the same Inoculations were made from 153 sputum specimens to each medium and 5 positive growth were obtained. 3 out of positive cultures were grown on both media, the other 2 positives were grown only on penicillin blood agar. None of the media showed contaminations. These findings suggest that the penicillin blood agar medium has at least the same, or even better qualities than the Löwenstein - Jensen medium.

Agar↗

PubMed1979

Staining of granulopoietic agar colonies with a modified Papanicolaou technique.

The aceto-orcein method of staining agar cultured granulopoietic marrow cells has not been entirely satisfactory. In staining whole colonies the original colony structure is lost and the colony cells have blurred cytoplasm. A slight modification of the Papanicolaou technique used for staining vaginal smears improved the quality of stained granulopoietic agar preparations. Smeared Papanicolaou stained colony cells could be compared with smeared ordinary Giemsa stained blood and marrow cells. The Papanicolaou method enabled staining of agar colonies without manipulation of the original colony structure. The preparations of transferred intact colonies had cells with distinct cytoplasm and clear cellular outlines and a background without disturbing masses of stained agar.

Agar↗

PubMed1978

Microbial assays for mutagenicity: a modified liquid culture method compared with the agar plate system for precision and sensitivity.

A microbial assay system for mutagenicity was developed in which bacterial cells divided in liquid culture. The statistical and practical problems associated with dividing cells were avoided or reduced, whilst the advantages in precision and reliability resulting from the determination of mutation per colony-forming unit (survivor) and of separating the mutation and selection systems were retained. Seven mutagens, two of which required microsomal activation, were evaluated by this liquid-medium method and by the agar-plate method with two strains of Salmonella typhimurium to determine which assay system was the more sensitive. At highly mutagenic and/or very toxic concentrations of the test substance the liquid-medium assay was markedly more sensitive than the agar-plate assay, but at weakly mutagenic and less toxic concentrations the advantage of the liquid-medium test was reduced; however in only one case was the agar-plate assay obviously the more sensitive. There was a clear indication that the liquid-medium assay would be superior to the agar-plate assay for the detection of mutagenic agents active only at toxic concentrations, and also could be more easily and exactly quantified.

Animals↗

PubMed1977

The testing of the antibiotic sensitivity of bacteria on an agar medium: The problem of a double zone of inhibition.

When the sensitivity of Micrococcus luteus ATTC9341 to streptomycin, erythromycin, oleandomycin and spiramycin was tested by an agar diffusion method using antibiotic impregnated filter paper disks on unbuffered Penassay Seed Agar two zones of inhibition were observed around the disks after an incubation period of 24 hours at 30 degrees C. The pH of the M. luteus seeded Penassay Seed Agar was measured before and after 24 hours incubation at 30 degrees C and found to be 6.6 and 8.7, respectively. When the Penassay Seed Agar was buffered to pH 6.1 and the sensitivity of the microbe to the antibiotics was tested as before no double zones of inhibition could be observed. The phenomenon of the double zones of inhibition may possibly be due to the pH increase of the medium from a relatively low level to the optimum range of activities of the antibiotics during the incubation period.

Anti-Bacterial Agents↗

PubMed1979

Agar disk diffusion susceptibility characteristics of azlocillin, carbenicillin, mezlocillin, piperacillin, and ticarcillin.

The agar disk diffusion susceptibility of Enterobacteriaceae to mezlocillin and piperacillin was correlated with agar minimal inhibitory concentrations and compared with the susceptibility to carbenicillin. The agar disk susceptibility of Pseudomonas aeruginosa to azlocillin, mezlocillin, and piperacillin was correlated with agar minimal inhibitory concentrations and compared with the susceptibility to carbenicillin and ticarcillin. Criteria are offered for the zones of inhibition to provide information about resistant and susceptible isolates that correlate with known serum levels.

Carbenicillin↗

PubMed1977

Simplified 48-hour IMVic test: an agar plate method.

An agar plate method was developed for the performance of the IMVic (indole, methyl red, Voges-Proskauer, and citrate) tests in lieu of the conventional tubed liquid media. By modifying the composition of the media and adding agar, a single "X"-compartmented petri dish was prepared containing all four IMVic test media. Ease of performance and simplification of the test were achieved by inoculating all four media simultaneously from a single colony (single inoculum) on eosin-methylene blue agar. Tests with 87 cultures, representing 7 genera in the family Enterobacteriaceae, were completed with typical (correct) IMVic patterns for all cultures within 48 h. Parallel tests with conventional media showed that the agar plate method was superior, more sensitive, faster, and simpler to perform, and less time was required to identify Escherichia coli by 72 h.

Bacteriological Techniques↗

PubMed1975

[Agar tube method for the bioassay of antibiotics (author's transl)].

A simplified bioassay system for antibiotics has been presented. Glass tubes with open ends and pits are filled with agar medium containing test microbes by immersing vertically into the agar medium while it is warm. These agar tubes are inserted in test solutions and incubated. The growth-inhibitory zones appear in respective tubes due to upward diffusion of the antibiotic. The heights of such zones from the bottom of agar tubes are measured.

Amoxicillin↗

PubMed1978

Reisolation and growth conditions of bacillus agar-exedens.

Several agarolytic Bacillus strains have been isolated. The properties agree with those described by Wieringa (1941) for Bacillus agar-exedens. These strains are the first reisolates since the original cultures were lost. A second group of isolates is related to the agarolytic B. palustris var. gelaticus of Sickles and Shaw (1934). B. agar-exedens requires carbohydrates for growth. In mineral-glucose media growth is inhibited by peptone at pH values of about 7 or less. Under alkaline conditions no inhibition by peptone is observed. A method for the enrichment of B. agar-exedens is described.

Agar↗