Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

The resistance of spores of resistant strains of Botrytis cinerea to quintozene, tecnazene and dicloran

Spores formed by strains of Botrytis cinerea resistant to quintozene, tecnazene, or dicloran did not always produce resistant colonies when grown on agar in the presence of these fungicides. Only about one half of spores produced by fungicide-vapour-resistant strains in the absence of fungicides gave resistant colonies whereas all spores produced by the same strains in the presence of fungicides formed resistant colonies on agar. Some spores produced by resistant strains that had developed on agar containing the fungicides were not viable, but those that were always gave resistant colonies on agar.     

Helper (OKT4) T cells from human T cell colonies produce potent colony-stimulating activity

Confluent T cell colonies were grown by culturing blood mononuclear cells in double agar layers containing autologous plasma and phytohemagglutinin (PHA) for one week (37 degrees C, 5% CO2). The plates were then overlaid with serum-free alpha medium which was harvested after 24 h. This medium was demonstrated to have colony-stimulating activity (CSA) of greater potency than conventionally prepared PHA-leukocyte conditioned medium, which was prepared by incubating cells from the same donors. Removal of OKT4-positive cells using a monoclonal antibody and complement abolished CSA production by cells from T cell colonies while the removal of OKT8-positive cells had no effect.     

A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington,D.C., buildings during the anthrax attack of October 2001

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.     

Detection of anthrax spores from the air by real-time PCR

AIMS: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure. METHODS AND RESULTS: One hundred litres of air were filtered through an air monitor device. After the membrane was suspended in PBS, spores of B. anthracis were added. The suspension was plated on Bacillus cereus selective agar (BCA) plates to detect B. anthracis colonies. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using a Light Cycler system and anthrax-specific primers. CONCLUSION: A single cell of B. anthracis was detected by real-time PCR within 1 h and was also isolated on a BCA plate within two d. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR and a Light Cycler provides a flexible and powerful tool to prevent epidemics.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.     

Ultrastructure of coccoid viable but non-culturable Vibrio cholerae

Morphology of viable but non-culturable Vibrio cholerae was monitored for 2 years by scanning and transmission electron microscopy. Morphological changes included very small coccoid forms, after extended incubation at 4 degrees C and room temperature, and sequential transformation from curved rods to irregular (approximately 1 microm) rods to approximately 0.8 microm coccoid cells and, ultimately, to tiny coccoid forms (0.07-0.4 microm). Irregular rod-shaped and coccoid cells were equally distributed in microcosms during the first 30-60 days of incubation at both temperatures, but only coccoid cells were observed after incubation for 60 days at 4 degrees C. When V. cholerae O1 and O139, maintained for 30-60 days at both temperatures, were heated to 45 degrees C for 60 s, after serial passage through 0.45 microm and 0.1 microm filters, and plating on Luria-Bertania (LB) agar, only cells larger than 1 microm yielded colonies on LB agar. Approximately 0.1% of heat-treated cultures were culturable. Cell division in the smallest coccoid cells was observed, yielding daughter cells of equal size, whereas other coccoid cells revealed bleb-like, cell wall evagination, followed by transfer of nuclear material. Coccoid cells of V. cholerae O1 and O139 incubated at 4 degrees C for more than 1 year remained substrate responsive and antigenic.     

Pyrolysis gas chromatography of Streptococcus faecalis: effect of cultural conditions on pyrochromatograms

Streptococcus faecalis 251 was cultured under a variety of different growth conditions, i.e. incubation for 24 or 70 h; at 22 degrees, 37 degrees or 45 degrees C; on blood agar or on MacConkey agar plates; aerobically or anaerobically. Replicate cultures were analysed by pyrolysis-gas liquid chromatography on columns of 7% Carbowax 2 M, TPA on Chromosorb G (AW-DMCS, 80-100 mesh) programmed from 40 degrees C up to 170 degrees C. Culture grown under identical conditions resulted in reproducible pyrochromatograms which were only slightly modified by change in temperature of growth from 37 degrees to 45 degrees C, or length of growth from 24 to 70 h, or growth on MacConkey agar instead of blood agar. Growth under anaerobic conditions resulted in a modified pyrochromatogram; while growth at only 22 degrees C resulted in a major change in pyrochromatogram.     

Phospholipase A-deficient mutants of Escherichia coli B

Phospholipase A-deficient mutants were isolated from Escherichia coli B/SM as follows. Replica plates were incubated to allow formation of colonies and then overlayed with soft agar containing washed sheep erythrocytes, lecithin Ca++, colistin, lysozyme and streptomycin. The mutant colonies were detected as colonies without hemolytic zones. Two or three cycles of treatment with mutagen and selection were necessary for their isolation. The mutants obtained could grow in a synthetic medium with glucose as the sole carbon source, and their phospholipid compositions were similar to that of the parent. They also gave the same agglutination titer as the parent with rabbit antiserum against the parent strain. They supported the growth of all members of the T-series of bacteriophages as effectively as the parent. Some hemolytic substance was produced from either lecithin or bacterial constituents when the mutants were infected with T even phages, but not with T odd phages. When the parent strain was infected with either T1 or T4, free fatty acids (FFA) were produced in the cell debris. Only a trace of FFA was formed in the debris of one of these mutants on infection with T4 and no FFA was formed on infection with T1.     

Colonial heterogeneity of Thiobacillus versutus.

In acetate-limited chemostat cultures started with single-colony cultures of Thiobacillus versutus, a mutant appeared after approximately 85 volume changes. The inhomogeneity of the culture was detected by the development of two different types of colonies on agar plates. When a pure culture of the mutant was grown in a chemostat, parent colonies appeared after almost the same period of time. Electron micrographs of the mutant grown on butyrate showed the presence of fibrils surrounding the cells. The cells of the parent strain were bald when grown under the same conditions. The growth kinetics of the parent and the mutant were investigated in batch cultures with a variety of substrates and were found to be identical. Major differences between the two strains were observed during growth on mannitol; the mutant attained a lower yield and excreted large amounts of extracellular polysaccharides.     

A Note on the Growth of Thiobacillus ferrooxidans on Solid Medium

An iron oxidizing strain of Thiobacillus ferrooxidans has been grown on solid medium using purified agar, carrageenan (Type 1) and agarose. This strain produces isolated and transferable colonies after 7 d incubation. Growth (increase in viable cells) by the direct plating method has been followed in relation to iron oxidation. Acidity, agar concentration and phosphate influenced colony development on solid medium.     

Effect of Controlled Atmosphere on Growth of Mold on Synthetic Media and Fruit

Growth of seven spoilage molds on agar plates at several temperatures in both controlled atmosphere (CA) and in air was studied. Each mold responded somewhat differently to CA at each temperature; however, there were some general tendencies. The lag phase was generally increased by CA and, in some cases, was substantially extended when incubation was just above the minimum growth temperature. The mycelial structure of molds seems to be different when grown in CA than when grown in air. With only two exceptions of 24 holding conditions, the maximum amount of mycelia was always less in CA than in air. Spore development varied with each mold at each temperature; generally, it was considerably less in CA than in air. CA storage of cherries above 34 F (1 C) did not retard mold infection to any extent; at 34 F, mold growth was inhibited and storage life was extended several days as compared to air storage. CA storage of strawberries at 34 F resulted in a mold-free product after 7 days of incubation, whereas the air-stored berries were slightly infected. However, when mishandled berries showing some mold growth were stored at 34 F, CA did not stop further mold growth.     

Video-supported Analysis of Beggiatoa Filament Growth, Breakage, and Movement

A marine Beggiatoa sp. was cultured in semi-solid agar with opposing oxygen-sulfide gradients. Growth pattern, breakage of filaments for multiplication, and movement directions of Beggiatoa filaments in the transparent agar were investigated by time-lapse video recording. The initial doubling time of cells was 15.7 +/- 1.3 h (mean +/- SD) at room temperature. Filaments grew up to an average length of 1.7 +/- 0.2 mm, but filaments of up to approximately 6 mm were also present. First breakages of filaments occurred approximately 19 h after inoculation, and time-lapse movies illustrated that a parent filament could break into several daughter filaments within a few hours. In >20% of the cases, filament breakage occurred at the tip of a former loop. As filament breakage is accomplished by the presence of sacrificial cells, loop formation and the presence of sacrificial cells must coincide. We hypothesize that sacrificial cells enhance the chance of loop formation by interrupting the communication between two parts of one filament. With communication interrupted, these two parts of one filament can randomly move toward each other forming the tip of a loop at the sacrificial cell.     

Colony growth of mouse bone marrow cells in agar contained in glass capillaries.

Mouse bone marrow cells were grown in semi-solid agar contained in glass capillary tubes. Several parameters affecting colony formation in the capillaries were studied. 10(4) cells in 100 mul incubation medium within one capillary produced 22 to 30 colonies of granulocytes and macrophages. Compared with the common petri dishes glass capillaries offer several advantages under the conditions used: 1. A twofold higher plating efficiency. 2. Applicability to optical scanning by light scattering and electronic counting, allowing automation and greatly improving sensitivity, statistical accuracy and reproducibility. Kinetics of colony growth can also be monitored. 3. Diminished risk of bacterial and fungal contamination. 4. A more than tenfold lower need for materials on similar statistical errors. Substituting methylcellulose for agar resulted in colonies of fibroblast-like cells adherent to glass surface. Glass non-adherent cells showed a threefold higher plating efficiency in agar.     

Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga neapolitana

Growth medium components and cultivation conditions for the extremely thermophilic bacterium Thermotoga neapolitana were optimized. A defined marine salts medium was formulated. Trace amounts of iron stimulated growth of T. neapolitana, while zinc inhibited growth at concentrations exceeding 11.1 μM. Other trace metals had no effect on its growth. Of the vitamins tested, only biotin was required for optimal growth. A defined mineral medium containing 5 g of carbohydrates per liter as the carbon source and 0.5 g of cysteine per liter as the sulfur source and reductant supported growth. Growth was stimulated by inclusion of vitamin-free Casamino Acids. Elemental sulfur, cystine, and dimethyl disulfide in the growth medium enhanced growth. Elemental sulfur and cystine relieved growth inhibition by hydrogen. T. neapolitana formed colonies in 2 days on plates of complex medium solidified with gellan gum and in 4 days on defined medium. The efficiency of plating was determined when growing cultures were sampled both aerobically and anaerobically and plated under aerobic and anaerobic conditions. Mean plating efficiencies were improved by sampling the growing cultures under strictly anaerobic conditions. Little or no improvement was obtained by inoculating plates inside an anaerobic chamber. Plating efficiencies of approximately 80% were obtained. Polycarbonate jars with aluminum lids withstood repeated incubation at 77°C without significant deterioration of the anaerobic seal and provided the most consistent results.     

Tuberculate spore formation by thirty-two strains of Histoplasma capsulatum

Summary 1. Thirty-two strains ofHistoplasma capsulatum were studied concerning their ability to form tuberculate spores and their conversion into the yeast phase.2. Nine strains did not produce tuberculate spores on Sabouraud's agar, on corn meal agar, on spent medium, on media with pH adjusted from 4.5 to 7.0 or on the first passage through hamsters.3. Tuberculate spore production did occur in these nine strains when Sabouraud's medium was enriched with phosphate, especially KH₂PO₄. In addition, all but two strains produced tuberculate spores after a second passage through hamsters.4. Growth on KH₂PO₄ enriched Sabouraud's agar led to a greater yield of yeast phase as compared to yeast phase resulting from colonies of the same strain grown on plain Sabouraud's agar. This may be due to the greater number of spores produced on the KH₂PO₄ enriched medium.5. A grinding technique of preparing inocula improved slightly the facility of obtaining the yeast phase over heavy inoculation with unground pieces of mold culture.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO THIRAM, ZIRAM, FERBAM, NABAM AND ZINEB

The concentrations of thiram preventing germination of spores of Botrytis cinerea in drops of a 1% solution of sucrose, and on the surface of a sucrose-nitrate agar have been determined. Thiram had much less effect on germination in the agar medium, even when a purified agar was used. There was no growth on sucrose-nitrate agar if the concentration of thiram exceeded 31 p.p.m. Attempts to obtain strains able to grow at higher concentrations were unsuccessful.
Similar results were obtained with ziram, nabam and zineb.
Ferbam also was more effective in preventing spore germination in spore drops than on agar media; this effect was obtained with ordinary and with purified agar.
On a sucrose-nitrate agar generally there was no growth if the concentration of ferbam exceeded 125 p.p.m., but in one of forty-eight plates containing 250 p.p.m. ferbam, five slowly growing colonies were produced, and from these colonies arose mycelium which grew and sporulated rapidly on 500 p.p.m. ferbam agar. Agar disk inocula were transferred from these cultures to agar containing higher concentrations of ferbam and in this way, and by repeating the process, a strain was obtained which grew slowly but continuously, and sporulated on agar containing 5000 p.p.m. ferbam. However, the poor solubility of this fungicide made it difficult to assess quantitatively the degree of adaptation.
A proportion of the spores from this strain germinated in drops containing about twice the concentration of ferbam which prevented germination of parent spores.
The resistance of the mycelium of the resistant strain was not lost after repeated subculture on fungicide-free agar. The resistant strain was as susceptible as the parent strain to thiram, ziram, nabam and zineb.
Attempts to obtain strains of Venturia inaequalis resistant to thiram, ferbam, ziram and zineb were unsuccessful.     

Rapid Screening for the Isolation of Mutants of Aspergillus nidulans with Increased Penicillin Yields

S ummary . After UV treatment conidia of a strain of Aspergillus nidulans were plated on an agar medium. The survivors gave rise to individual colonies which were inoculated separately on agar discs and incubated. The discs were then transferred to biological assay plates seeded with spores of a strain of Bacillus subtilis sensitive to penicillin. Using this primary screening method, mutants have been isolated which, when tested later in shake flask cultures, gave larger penicillin yields than the parent strains.     

APPLICATION OF A DOUBLE TUBE SYSTEM FOR ENUMERATION OF CLOSTRIDIUM TYROBUTYRICUM

Clostridium tyrobutyricum, a spore-forming, gram-positive, anaerobic bacterium, is considered to be the main organisms responsible for the late spoilage of cheese by gas formation. Most methods for detecting C. tyrobutyricum are based on spore germination and vegetative growth and take 4–7 days plus an identification step for confirmation. The purpose of this study was to develop a faster detection method using a Double Tube System. Because no selective medium is available for detection of C. tyrobutyricum, three media (Reinforced Clostridial, AC, and Tomato Juice) were compared using two strains of C. tyrobutyricum and one strain of C. sporogenes. Each 4 day-old test strain was inoculated on duplicated plates of each agar that were then placed in anaerobic jars or in the double-tube systems for 2–4 days at 30 or 37C. All three agars consistently supported growth of the test strains. Counts did not differ with incubation at 30 or 37C and were comparable using the conventional anaerobic jar or a Double Tube System. However, in the Double Tube System, colonies could be counted accurately at least 6 h earlier than on the plates in anaerobic jars.