Showing papers on "Agar plate published in 1978"

Chapter XI Laboratory Diagnosis, Serology and Epidemiology of Streptococcus pneumoniae

Abstract: Publisher Summary This chapter discusses the diagnosis, serology, and epidemiology of Streptococcus pneumonia that is a species of the genus Streptococcus of the family Streptococcaceae. Pneumococci are gram-positive, capsulated, lanceolate diplococci often arranged in short, straight chains; typically, they grow diffusely in serum broth and give rise to smooth colonies surrounded by narrow zones of α-haemolysis on blood agar. They are facultative anaerobes and some strains are carbon dioxide dependent upon isolation from clinical specimens. They are killed by heat at 60°C for 30 minutes, easily lysed, soluble in bile, sensitive to optochin, and many types are virulent for mice. Fluid specimens and swabs are inoculated onto blood agar and into serum broth. The cultures are incubated at 37°C. On blood agar, typical pneumococcal colonies may be observed as round, flat, smooth, translucent, often with a central pitting, and with a greenish discoloration of the surrounding medium. If an isolated strain of gram-positive diplococci grows with typical colonies on blood agar and diffusely in serum broth, the final diagnosis of S. pneumonia becomes a matter of differentiating between this and other species of α-haemolytic streptococci.

Evaluation of a micromethod for determination of Streptococcus mutans and Lactobacillus infection.

Abstract: A micromethod was developed for quantitative estimation in saliva of Streptococcus mutans and lactobacilli. With a semiautomatic pipette, 25 microliter of diluted saliva was spotted on the surface of an agar plate containing a selective medium. This volume gave a spot with a diameter of about 10 mm in which separate colonies could be counted. The results obtained with the spotting technique showed excellent agreement with those obtained with conventional agar plating. The method is convenient and results in a substantial saving of culture media.

New medium for selection and presumptive identification of the Bacteroides fragilis group.

Abstract: A medium, Bacteroides fragilis bile-esculin (BBE) agar, was designed for the selection and, presumptive identification of the B. fragilis group. BBE agar contains bile, esculin, ferric ammonium citrate, hemin, and gentamicin in a Trypticase soy agar base. Growth in the presence of 20% bile and esculin hydrolysis, detected by blackening of the medium, provide presumptive evidence for the identification of the B. fragilis group. In addition to stimulating the growth of many strains of the B. fragilis group, hemin provides the option of testing isolates for catalase production. Gentamicin and bile prevent the growth of most organisms other than the esculin-positive bacteroides that can tolerate bile. Of 160 clinical isolates of the B. fragilis group tested on BBE agar, 159 grew well on the medium and 157 blackened it. Other anaerobes, Enterobacteriaceae, and enterococci either failed to grow on BBE agar or did not produce the characteristic morphology and blackening associated with isolates of the B. fragilis group. In a clinical laboratory trial, 687 specimens from patients were inoculated onto BBE agar plates. The B. fragilis group was recovered from 81 (11.8%) of these specimens in 24 to 48 h. Use of BBE agar in the clinical laboratory enables earlier recovery and identification of this important pathogen.

Microbiological Methods and Fluorescent Microscopy for the Direct Demonstration of Mycoplasma Infection of Cell Cultures

Abstract: Our principal experience with immunofluorescence is in its application to the identification of mycoplasma colonies growing on agar medium (5). The immunofluorescent identification results of mycoplasmas isolated from cell cultures over an 11-year period are summarized herein. Most recently we have used immunofluorescence to identify M. hyorhinis in infected cell cultures. These results will be presented with special reference to auxotrophic variants of M. hyorhinis. The auxotrophs are strains that are noncultivable on media that support the growth of the type strain of M. hyorhinis (BTS-7) and other similar strains. The term “noncultivable mycoplasma” (7) is used in this sense to describe a strain (DBS 1050 = ATCC 29052) that grew in cell cultures but not on agar medium. This specific connotation is obscured by the current use of the term in reference to apparent isolation failures.

Reversal of the silver inhibition of microorganisms by agar.

Abstract: 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.

Pectolytic enzymes in Rhizobium.

Abstract: A sensitive pectin agar plate assay was used to demonstrate low levels of pectolytic enzymes in infective and noninfective strains of Rhizobium. The possible relation of this characteristic to legume infection is discussed.

Chapter IX Biotyping and Serotyping of Pasteurella haemolytica

Abstract: Publisher Summary This chapter presents the procedure for the biotyping and serotyping of Pasteurella haemolytica . The genus Pasteurella consists of pleomorphic, gram-negative, nonmotile rods, characterized by a fermentative carbohydrate metabolism. Being oxidase positive, they are readily differentiated from members of Enterobacteriaceae , to which the same description applies. They grow poorly or not at all on the usual selective plating media for enteric organisms. In triple-sugar-iron agar (TSI), they produce acidity throughout, without gas or hydrogen sulfide formation, a reaction not typical of Enterobacteriaceae , except certain yersinias, which are further distinguishable from Pasteurella species by being motile and growing freely on MacConkey agar. P. haemolytica is most readily distinguished from P. multocida by its failure to produce indole and its production of a usually narrow haemolytic zone, sometimes no bigger than the colony itself, on bovine or ovine blood agar. Typing, both in the cultural and serological sense has served as a useful tool in the search for an understanding of diseases related to P. haemolytica .

Stability of minocycline, doxycycline, and tetracycline stored in agar plates and microdilution trays

Abstract: When the agar dilution method is used for performing antimicrobic susceptibility tests, Mueller-Hinton agar plates may be prepared with varying concentrations of the drugs to be studied. With most antimicrobics, the plates can be prepared in advance and stored in the refrigerator for as long as four weeks. The present study documents the instability of minocycline when stored under refrigeration in agar plates. Doxycycline and tetracycline also lost activity but at a slower rate. Degradative changes occurred most rapidly at the lower concentrations; at 16 μg/ml or greater, the drugs were relatively stable. When diluted in Mueller-Hinton broth and stored at −60°C in microdilution trays, the three tetracyclines could be held up to six weeks with no loss of bioactivity. Concentrated stock solutions were kept at −60°C for six months with no loss of potency.

Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts.

Abstract: Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.

Membrane filter procedure for enumeration of Candida albicans in natural waters.

Abstract: A membrane filter procedure is described for the enumeration of Candida albicans in natural waters. Several hundred milliliters of sample can be examined by filtration through 1.2-micrometer membranes. Selectivity is achieved by the use of a defined (yeast-nitrogen base plus maltos-) agar medium inclusion of the antimicrobial agents chloramphenicol and cycloheximide, and incubation at 37 degrees C. C. albicans colonies are differentiated primarily through color by use of a bismuth salt indicator system. Average recovery of various strains of C. albicans stressed in seawater at 4 degrees C was 82%, compared with those of spread plate controls on a noninhibitory medium. With river water and raw sewage, 90% of typical C. albicans colonies were confirmed as such in a simplified germ tube test. Atypical colonies verified as C. albicans were infrequent (3%). C. tropicalis and Torulopsis candida were the most common false-positive colonies.

Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Abstract: Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.

Effect of Oxygen Concentration, Temperature and Combined Nitrogen on the Morphology and Nitrogenase Activity of Rhizobium sp. Strain 32H1 in Agar Culture

Abstract: Summary: Cultures of Rhizobium sp. strain 32H1 grown in a layer of soft agar on top of a layer of harder agar developed maximum nitrogenase activity [80 to 90 nmol acetylene reduced h−1 (mg protein)−1] after 12 d in air at 28 °C. Electron microscopy of sections of cores of the soft agar layer showed differences in the morphology of rhizobia in colonies growing at the surface, in the middle or at the bottom of the layer. The properties of rhizobia growing in the colonies in the middle of the soft agar layer suggested that only these contained nitrogenase. These rhizobia were present as a distinct band of cells passing through individual colonies at a constant depth in the agar. They were larger than normal vegetative Rhizobium, were pleiomorphic and were similar in morphology to the nitrogen-fixing bacteroids formed by strain 32H1 in cowpea root nodules. Their location within the soft agar layer changed with the O2 concentration in the atmosphere above the agar during growth, and they were not found in cultures showing little or no nitrogenase activity. Loss of nitrogenase activity occurred when cultures were grown at 33 °C, in a O2/Ar (1:4, v/v) atmosphere, or when the concentration of combined nitrogen in the agar medium was increased from 2 to 20 mM-N.

Time- and media-saving testing and identification of microorganisms by multipoint inoculation on undivided agar plates

Abstract: Motility and various biochemical activities of isolates of bacteria and yeasts were tested on undivided agar plates by using a simple, manually operated multipoint inoculation apparatus that allowed the analysis of 25 isolates per 9-cm-diameter petri plate. Fermentation of all 17 carbohydrates tested as well as 13 other biochemical activities commonly used for identification of bacteria were readily demonstrated by the multipoint inoculation plate method, and the results agreed very well with those of conventional tube tests. In addition to speedy inoculation and low cost of materials, the multipoint inoculation plate method offers several other advantages when compared with conventional tube tests or with some of the manufactured test kits currently available for recognizing members of the family Enterobacteriaceae.

Development and evaluation of a potency index screen for detecting mutants of Penicillium chrysogenum having increased penicillin yield.

Abstract: U.v.-treated conidia of an industrial strain of Penicillium chrysogenum were spread on a growth-limiting agar medium. Colonies arising from the survivors were surrounded with a spore suspension of Bacillus subtilis to which penicillinase had been added. After appropriate incubation, discrete zones of bacterial inhibition, with sizes limited by the penicillinase, appeared around each colony. Using the criterion of potency index (diameter of inhibition zone divided by diameter of colony) strains were selected that subsequently gave improved penicillin production in shake-flasks. The technique also ranked correctly four industrial strains in order of their known penicillin-producing capacity. Employing three operators, 5000 isolates could be screened in each experiment and approx. 15000 strains could be screened in a month.

Significance of appropriate techniques and media for isolation and identification of Ureaplasma urealyticum from clinical specimens.

Abstract: Controversy over the association of Ureaplasma urealyticum with reproductive failure may be due to methods used to isolate the microorganism. U. urealyticum isolations from clinical material should be done simultaneously in broth and on Shepard's differential agar medium (A7) containing manganese sulfate. Urine sediments result in a 9% (P = 0.0002) higher rate of isolation than than cervical and urethral swabs. Primary isolations may not display standard textbook morphology. Isolated colonies may be present, but brown streaks in cervical mucus or a coalescent haze around epithelial cells in urine sediment may also be seen in areas of concentrated growth. The broth and agar media used, method of incubation, type of specimen, and method of storing specimens before culture are all factors which influence the recovery of U. urealyticum.

Effect of leaf extracts of Aloe arborescens Mill subsp. natalensis Berger on growth of Trichophyton mentagrophytes

Abstract: The antifungal activity of a lyophilized powder containing aloe leaf homogenate (whole-leaf powder) against Trichophyton mentagrophytes was investigated. The minimal inhibitory concentration was 25 mg/ml by the agar dilution method, using Sabouraud glucose agar medium. At subinhibitory concentrations, the powder exerted its main effect on colony growth by prolongation of the lag phase and inhibition of growth rate. Homogenates of fresh whole leaf were filtered through Whatman GF/A paper, and the filtrate was dialyzed and concentrated by molecular filtration using an Amicon hollow-fiber dialyzer concentrator DC-2, and a powder containing components with molecular weights higher than 10,000 (high-molecular-weight component powder) was prepared by lyophilization. The minimal inhibitory concentrations against three strains of T. mentagrophytes were all 10 mg/ml. The inhibitory activity was fungicidal and was lost by heating at 100 degrees C for 30 min. Both the whole-leaf powder and the high-molecular-weight component powder induced various morphological abnormalities in spores and hyphae by the inhibition of spore germination and development of hyphae.

Development of the Agar Disk Method for the Rapid Selection of Cephalosporin Producers with Improved Yields

Abstract: 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.

Growth of clinical isolates of anaerobic bacteria on agar media: effects of media composition, storage conditions, and reduction under anaerobic conditions.

Abstract: The quantitative growth, the colony size, and the rate of growth of 47 clinical anaerobic isolates were compared on five different media, namely Brucella agar, brain heart infusion agar, Columbia agar, Schaedler agar, and tryptic soy agar. There was no significant difference in the quantitative growth of the anaerobes inoculated onto the five media. Although no single medium was superior for the growth of all isolates, 12 of 22 isolates, inoculated onto media stored for 4 weeks or less, grew best on Schaedler agar. The effects of supplementation of the media with reducing agents and reduction of the media before use were also analyzed and were found to be affected by the composition and length of storage of the media, as well as the bacteria tested.

Recovery of sublethally heat-injured Salmonella typhimurium on supplemented plating media.

Abstract: The efficacy of 32 additives to Levine eosin-methylene blue-salts agar medium (EMBS) for the recovery of sublethally heat-injured Salmonella typhimurium was evaluated. In order of decreasing effectiveness, lactate, mannitol, and alpha-glycerophosphate mediated 90% or more recovery of injured cells; similar levels of recovery were obtained on EMBS supplemented with 1% (wt/vol) tryptic soy broth, protease peptone, or plate count agar. Other additives showed little or no capacity for repair or strongly inhibited heated and nonheated cell suspensions. Conditions of growth and storage before heat treatment were also found to markedly affect susceptibility to heat injury.

Evidence for Production of Inhibitor by the Vesicular-Arbuscular-Mycorrhizal Fungus Gigaspora Margarita

Abstract: Incubation of spores of Gigaspora margarita on agar medium containing activated charcoal resulted in an increased rate of hyphal growth. Circular patterns simulated by hyphal growth from groups of ...

Method for Rapid Detection of Group B Streptococci by Coagglutination

Abstract: A quick and reliable technique for the identification of group B streptococci has been developed. The method requires no elaborate equipment or expensive reagents and can be used to detect the group B organisms in mixed broth cultures or to identify suspect colonies selected from agar plates. The method is a coagglutination technique in which 1 drop of specifically sensitized protein A-containing Staphylococcus aureus is mixed with 1 drop of supernatant of an actively growing culture. The soluble group-specific carbohydrate substance of the group B streptococci reacts with the staph particles to produce agglutination that is macroscopically readable. One colony of group B streptococci taken from an agar plate and inoculated into Todd-Hewitt broth will give a positive reaction within 6 h of incubation; with a larger inoculum, the positive reaction occurs within a shorter period. The method was applied for detection of group B streptococci in mixed broth cultures. In laboratory studies involving random mixtures of organisms, 59.3% of positive cultures were detected within the first 8 h of incubation, and 71.7% were found within 24 h. In clinical studies with mixed broth cultures grown directly from vaginal swabs, 78.6% of the positive cultures were detected within the first 8 h of incubation, and 92.9% were found within 24 h.

Detection of Algal Lysing Biological Agents in Lakes by the Soft-agar Overlayer Technique

Abstract: The detection of biological agents responsible for the deterioration of the blooms of blue-green algae in lakes was attempted by the soft-agar overlayer technique. One ml of algal suspension and a measured volume of sample were added into about 2ml of melted 0.8% agar cooled to 45°C-48°C, and the mixture was poured over the surface of a hardened layer of modified Detmer medium containing 1.5% agar. After the upper layer solidified the plate was incubated at 25°C to 30°C in the light of 3000 lux to 5000 lux. After 10 days incubation, plaques on agar plates were counted. Most of the agents detected by this technique were amoebae, bacteria and fungi. Especially, development of large plaques was accompanied by amoebae.

Quantitative Assessment of Bactericidal Activities of β-Lactam Antibiotics by Agar Plate Method

Abstract: Quantitative bactericidal activities of β-lactam antibiotics were determined by the agar plate method. Broth cultures, of which the colony-forming units were counted before the study, were inoculated on antibiotic-containing agar plates, utilizing a 10 −3 , 10 −2 , or 10 −1 dilution or undiluted culture plated with each 0.001-ml calibrated loop. These plates were incubated at 37°C overnight, and the minimal drug concentration at which no bacterial growth was observed on the plates was defined as minimal inhibitory concentration. After this procedure, the agar surface was treated with β-lactamase spray to inactivate the antibiotic. These plates were incubated again at 37°C overnight. The minimal drug concentration at which no evidence of bacterial growth was visible on the plates (resulting in a 100% kill) was defined as minimal bactericidal concentration. The lowest concentration which reduced the number of colony-forming units to 1/1,000 that in the original inoculum (resulting in a 99.9% kill) was defined as minimal lethal concentration. When compared for Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis , alpha-hemolytic streptococcus (non-enterococcal), beta-hemolytic streptococcus, and enterococcus, the minimal bactericidal concentrations were generally several fold higher than the minimal inhibitory concentrations. Minimal lethal concentrations were virtually the same as minimal inhibitory concentrations for gram-negative strains; however, for some gram-positive strains, minimal lethal concentrations were higher than minimal inhibitory concentrations.

Acidometric Agar Plate Method for Ampicillin Susceptibility Testing of Haemophilus influenzae

Abstract: The need for an accurate and rapid method of testing ampicillin susceptibility of Haemophilus influenzae, especially strains isolated from patients with meningitis and septicemia, is indisputable Various methods have been employed for this purpose Each has advantages and disadvantages This report describes a modification of the capillary acidometric procedure in which an agar plate is substituted for a tube All beta-lactamase results obtained by this modified technique correlated with minimal inhibitory concentrations determined in liquid media and the chromogenic cephalosporin substrate method This modified acidometric agar procedure is a simple, inexpensive, accurate, and rapid way to determine H influenzae susceptibility to ampicillin

Evaluation of an automated agar plate streaker.

Abstract: An automated agar plate streaker was evaluated. The Autostreaker mechanizes the agar plate streaking process by providing storage for plates, labeling and streaking one or more plates for either isolation or quantitation, and stacking in one of several racks for subsequent incubation. Results showed the Autostreaker to produce agar plates with well-separated colonies and accurate colony counts. A total of 1,930 clinical specimens were processed either in parallel with manual methods or solely by the Autostreaker. Technologist acceptance of machine-streaked plates was outstanding.

Colony formation by subpopulations of human T lymphocytes. I. Effects of phytohaemagglutinin and lymphocytosis-promoting factor from Bordetella pertussis.

Abstract: A method is described for the growth in semi-solid agar medium of human lymphocyte colonies in response to stimulation by lymphocytosis-promoting factor (LPF) derived from Bordetella pertussis. Colony formation was dependent on (a) a liquid pre-culture step with LPF prior to agar seeding, (b) presence of LPF in the agar medium, (c) a cell density in the agar culture of more than 5000 cells per ml. Optimal colony formation was obtained with 30 μl LPF preparation in the liquid pre-culture step and 20 μl LPF preparation in the agar medium. Moreover, colony development improved after addition of 5 × 10−5M 2-mercaptoethanol and 0·6% human AB serum. The frequency of LPF-induced T-lymphocyte colonies in ten normal adult donors was 5450±1800 per 106 mononuclear blood cells. In comparison, the frequency of phytohaemagglutinin (PHA)-induced colonies was 20,000±2500 per 106 mononuclear cells plated directly in the agar medium. Lymphocytes seeded in agar medium with LPF started to divide within 2–3 days of culture and formed colonies of 30 to 200 cells on day 7 of culture. Cultures became moribund at day 8. The colony cells were negative for surface immunoglobulin and approximately 75% formed rosettes with sheep red blood cells (SRBC). No synergistic effect between LPF and PHA on colony formation was observed when PHA and LPF were used in the first and second step of culture respectively. Similarly, addition of LPF did not influence the growth of PHA-induced colonies. When mononuclear cells were depleted of monocytes prior to agar culture addition of supernate factor(s) from cultures of adherent blood mononuclear cells (AC-CM) was necessary to ensure optimal development of colonies. Mononuclear cells were separated by various rosette-depletion techniques. Colony-forming cells were recovered in the sheep red blood cell-rosetting fraction (E-RFC) of human blood lymphocytes indicating that these precursor cells are themselves T lymphocytes. E-RFCs were separated into Fc(γ)-receptor-positive and negative subpopulations. The frequency of colony-forming cells in the Fc(γ)-receptor-negative T-lymphocyte population was two to ten times higher than that of the Fc(γ)-positive T-cell population. Co-culture of equal numbers of Fc-positive and Fc-negative T lymphocytes reduced the frequency of T colonies by 20 to 95%. Mononuclear cells were separated by passage through Ig-anti-Ig-coated columns. Attempts to grow LPF-stimulated colonies from passaged lymphocytes failed, suggesting that the colony-forming cells or cells necessary for colony formation were trapped in the column. In contrast, PHA-induced colony formation was enriched in cells which had passed through Ig-anti-Ig-coated columns.