Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Comparison of coats and surface-dependent properties of Bacillus cereus T prepared in two sporulation environments

The surface or coat-associated properties of Bacillus cereus T spores produced from modified G medium (MGM) and fortified nutrient agar (FNA) were compared. The two populations appeared structurally similar by transmission electron microscopy. Spores prepared on FNA were more susceptible to ozone inactivation than MGM-prepared spores. When activated by heating for 15 min at 70–85°C, FNA-prepared spores were optimally activated at 85°C and did not become hydrophilic on heat activation while MGM spores were optimally activated at 70°C and became hydrophilic on activation. Susceptibility to removal of coat and outer membrane by chemical and enzymatic extraction treatments was measured by monitoring reduced ability to germinate in nutrients and acquired ability to germinate in the presence of lysozyme. Bacillus cereus T MGM-prepared spores germinated in lysozyme upon<1 h exposure to sodium dodecyl sulphate-dithiothreitol. FNA-prepared spores were lysozyme sensitive after > 2 h treatment. Thus, B. cereus T FNA spore coats and outer membranes were more resistant to these denaturing agents. Transmission electron micrographs revealed no change in appearance of extracted spores. Sporulation environment must be considered when laboratory-prepared spores are used to assess or predict the effect of control procedures on spores present in nature.     

Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide: II. Factors Affecting Rates of Inactivation by Phenolic Bactericides

Aqueous suspensions of Bacillus subtilis NCTC 8236 spores, surviving 150,000 or 300,000 rad of gamma irradiation under air from a cesium-137 source, exhibited an enhanced rate of inactivation, compared to nonirradiated spores, when heated with different phenolic bactericides. The apparent magnitude of the enhanced inactivation rate, observed from survival curves, increased progressively with the irradiation dose applied and diminished progressively as the severity of heat treatment with 0.2% chlorocresol was increased either by raising the temperature from 70 to 90 C or reducing the pH from 8 to 6. The enhanced inactivation rate was unaffected when the concentration of sodium chloride added to 0.2% chlorocresol was altered from 0.4 to 0.8%. The enhancement effect was also observed when the heat treatment was carried out with 0.5% phenol and 0.3% m-cresol.     

Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 10⁴. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85C for 10 min did not reduce the viable count by a factor of 10⁴. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum . These findings are relevant to the safety of minimally processed (e.g. sous-vide ) foods.     

Recovery of Spores of Bacillus stearothermophilus from Thermal Injury

Bacillus stearothermophilus grown in nutrient broth produced a product which promoted recovery from thermal injury of its spores. This phenomenon was observed with nutrient agar as the plating medium but not with a medium composed of Trypticase, Phytone, dextrose and phosphate (TPDP). Recovery of injured spores was greatest in such a medium if it contained starch or charcoal. Trypticase soy agar and dextrose tryptone agar were markedly inferior to TPDP medium.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

The effects of Korean propolis against foodborne pathogens and transmission electron microscopic examination

This study was performed to evaluate the effects of Korean propolis against foodborne pathogens and spores of Bacillus cereus and to investigate the antimicrobial activity against B. cereus structure by transmission electron microscopy (TEM). The antimicrobial effects of the Korean propolis were tested against foodborne pathogens including Gram-positive (B. cereus, Listeria monocytogenes and Staphylococcus aureus) and Gram-negative (Salmonella typhimurium, Escherichia coli and Pseudomonas fluorescence) bacteria by agar diffusion assay. Gram-positive bacteria were more sensitive than were Gram-negative bacteria. The vegetative cells of B. cereus were the most sensitive among the pathogens tested with minimum inhibitory concentration (MIC) of 0.036 mg/μl of propolis on agar medium. Based on MIC, sensitivity of vegetative cells of B. cereus and its spores was tested in a nutrient broth with different concentrations of propolis at 37°C. In liquid broth, treatment with 1.8 mg/ml propolis showed bactericidal effect against B. cereus. B. cereus vegetative cells exposed to 7.2mg/ml of propolis lost their viability within 20 min. Against spores of B. cereus, propolis inhibited germination of spores up to 30 hours, compared to control at higher concentration than vegetative cells yet acted sporostatically. The bactericidal and sporostatic action of propolis were dependent on the concentration of propolis used and treatment time. Electron microscopic investigation of propolis-treated B. cereus revealed substantial structural damage at the cellular level and irreversible cell membrane rupture at a number of locations with the apparent leakage of intracellular contents. The antimicrobial effect of propolis in this study suggests potential use of propolis in foods.     

Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells.

Morphological changes of Vibrio parahaemolyticus from rods to spheres took place after a culture was subjected to starvation at a wide range of temperatures. Scanning electron micrographs revealed that starved spherical cells gradually developed a rippled cell surface with blebs and an extracellular filamentous substance adhesive to the cell surface. Cells starved at a low temperature for certain intervals were counted by various bacterial enumeration methods, including plate count, direct viable count, and total cell count for both Kanagawa-positive and -negative strains. The results indicated that this species could reach the nonculturable stage in 50 to approximately 80 days during starvation at 3.5 degrees C. Kanagawa-negative strain 38C6 lost culturability more slowly than Kanagawa-positive strain 38C1 at low temperature. As detected by thiosulfate-citrate-bile salts-sucrose plate count, a high percentage of the surviving cells at 3.5 degrees C in starvation medium were possibly injured by the low temperature rather than by starvation. Both addition of nalidixic acid to the starved cultures and the most-probable-number method demonstrated that the cells recovered after a temperature upshift probably represented the regrowth of a few surviving cells. These surviving cells were capable of growth and multiplication with limited nutrients at an extraordinary rate when the temperature was upshifted.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

AIMS: To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. METHODS AND RESULTS: Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, -18 and -75 degrees C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain-heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. CONCLUSIONS: Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen.     

Recovery of Heated Clostridium perfringens Type A Spores on Selective Media

The enumeration of Clostridium perfringens spores on sulfite-polymyxin-sulfadiazine agar (SPS), tryptone-sulfite-neomycin agar (TSN), Shahidi-Ferguson-perfringens agar (SFP), tryptone-sulfite-cycloserine agar (TSC), and TSN lacking antibiotics (BASE) was studied. The spores were heated at 105 to 120 C by the capillary-tube method. The media were about equally efficient for the enumeration of heat-activated spores. Efficiency of the media for the recovery of spores surviving heat treatments at ultrahigh temperatures varied as follows: TSC >/= SFP > BASE > SPS > TSN. Greater recovery when survivors were enumerated on TSC or SFP was attributed to germination of injured spores by the lysozyme present in the egg yolk emulsion used in these media. Low recovery of survivors on TSN and SPS was due to both the absence of lysozyme and inhibition of injured spores by the selective agents of these media. Recovery of heated spores was reduced greatly by polymyxin, neomycin, and kanamycin, and slightly by sulfadiazine and D-cycloserine. The addition of lysozyme to SPS or TSN did not improve the percentage of heat-injured spores recovered because the selective agents of these media interfered with the action of lysozyme. The suitability of the selective media for the enumeration of survivors was greatly affected by the presence of certain foods.     

国家标准测定食品细菌总数培养基的改进研究

应用我国国家标准营养琼脂（GB4789 2－94简称NA）与美国食品药品管理局（FDA）标准平析以（简称SA）两种培养基对动植物食品中细菌总数进行检测对比,结果表明FDA标准平板比GＢ营养琼脂效果较好,前比后的检出率高出２３．９％,且菌落大而明显,为此,对这两种培养基进行优化筛选试验,并优选出Ｃ８培养基,扩大试验结果表明C8培养基的检出率较GB营养琼脂及FDA标准平板分别高出35．8％和9．5％。     

A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores     

Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia

The culturability of bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at 1/100 of its normal concentration (dilute nutrient broth [DNB]) as the growth medium. Three-tube most-probable-number serial dilution culture resulted in a mean viable count that was only 1.4% of the mean microscopically determined total cell count. Plate counts with DNB solidified with agar and with gellan gum resulted in viable counts that were 5.2 and 7.5% of the mean microscopically determined total cell count, respectively. Prior homogenization of the soil sample with an ultrasonic probe increased the viable count obtained by using DNB solidified with gellan gum to 14.1% of the mean microscopically determined cell count. A microscopic examination of the cell aggregates that remained after sonication revealed that the potential CFU count was only 70.4% of the total cell count, due to cells occurring as pairs or in clumps of three or more cells. Staining with SYTO 9 plus propidium iodide indicated that 91.3% of the cells in sonicated soil samples were potentially viable. Together, these findings suggest that the maximum achievable CFU count may be as low as 64.3% of the total cell count. Thirty isolates obtained from plate counting experiments performed with DNB as the growth medium were identified by comparative analysis of partial 16S rRNA gene sequences. A large proportion of these isolates represent the first known isolates of globally distributed groups of soil bacteria belonging to novel lineages within the divisions Actinobacteria, Acidobacteria, Proteobacteria, and Verrucomicrobia.     

Identification of a Germination System Involved in the Heat Injury of Bacillus Subtilis Spores

Bacillus subtilis A spores were injured by exposure to heat treatments of 110 to 132 C. Injury was demonstrated by the inability to form colonies on fortified nutrient agar (FNA) unless the medium was supplemented with CaCl(2) and Na(2) dipicolinate (CNA). A preliminary heat treatment fully heat-activated the spores, was not lethal, and did not prevent injury by subsequent secondary heat treatment. Exposure of heat-activated spores to 122 C reduced germination in FNA. The primary germination agents in FNA were identified, and a defined germination medium of glucose, NaCl, l-alanine, and sodium phosphate (GNAP) was developed. Germination of heat-activated spores in GNAP was equivalent to germination in FNA. Injury measured by colony formation on FNA and CNA was correlated to injury measured by reduced germination in both FNA and GNAP. Inactivation of the FNA and GNAP germination systems by secondary treatment exhibited similar kinetics. Therefore, injury expressed as the inability to form colonies on FNA involved alteration of the GNAP germination system.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

A new medium for the enumeration and subculture of bacteria from potable water

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new medium for the enumeration and subculture of bacteria from potable water.

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)