Occurrence and Thermoresistance of Spores of Psychrophilic and Psychrotrophic Aerobic Sporeformers in Soil and Foods

Spores of psychrotrophic (able to grow at 5°C) aerobic sporeformers occurred in soil in high numbers (2 × 10³-5 × 10⁶/g), whereas psychrophilic (able to grow at 0°C) spores were present at significantly lower levels (500–10⁵/g). Psychrotrophic spores were absent in herbs and spices: in pasteurized meals prepared industrially their numbers varied from <10 to 1000/g. For spores harvested from Trypticase Soy Agar (TSA), the heat resistance of the cold-tolerant sporeformers was low with D _90°C-values from 1–11 min. The recovery of heated psychrophilic spores on this medium at 5°C was equal to their recovery at 20°C. However, the recovery of heated psychrotrophic spores was lower at 5°C than at 20°C, whereas unheated spores gave the same counts at both temperatures. The heat resistance of naturally occurring spores of cold-tolerant sporeformers washed from soil was comparable with the resistance of spores formed on TSA.     

Effect of Time and Temperature in Assessing Microbial Contamination on Flat Surfaces

Experiments were performed to investigate incubation time and temperature effects for Rodac agar contact plates applied to bench top surfaces in industrial clean rooms. For studies of general microbial levels, incubation at 32 C for 43 hr was found suitable for Rodac plates containing 15.5 +/- 0.1 ml of Trypticase Soy Agar.     

Identification of nutritional components in trypticase responsible for recovery of Escherichia coli injured by freezing

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Identification of nutritional components in Trypticase responsible for recovery of Escherichia coli injured by freezing. J. Bacteriol. 91:1098-1104. 1966.-Freezing and storage of Escherichia coli at -20 C resulted in nonlethal or "metabolic" injury to a proportion of the surviving population. The injury was manifested as an increased nutritional requirement after freezing. Injured cells could not grow on a minimal agar medium, but could develop on Trypticase Soy Agar. The percentage of injured survivors varied among strains, but was little affected by altering the freezing menstruum. Trypticase was found to be the component in Trypticase Soy Agar responsible for the recovery of injured cells, and contained five closely related peptides that possessed most of the biological activity. Isolation of the peptides was accomplished by Sephadex gel chromatography, paper chromatography, and high-voltage paper electrophoresis. Hydrolysis of the peptides destroyed the ability to restore injured cells.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Microcount Method for Petrolatum-Based Topical Ointments Containing Waxes

A practical solvent system for the detection of microorganisms in topical ointments has been developed. The method involves dissolving 0.5 g of topical ointment in 50 ml of a solvent mixture (92 parts isopropyl myristate, 6 parts carbon disulfide, and 2 parts xylene) and filtering it through a 0.45-mum membrane filter. Residual solvent is then washed from the filter pad with 200 ml of sterile 0.5% Brain Heart Infusion broth containing 0.1% Tween 80. The filter pad is then removed and placed on a petri plate containing Trypticase Soy Agar medium. The petri plates thus prepared are then incubated at 37 C for 7 days, and the colonies produced are then counted. The toxicity of the solvent mixture was determined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella newington, and spores of Bacillus subtilis and was found to be less toxic than the heat-sterilized isopropyl myristate and comparable to the filter-sterilized isopropyl myristate.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

In Vitro Antibacterial Activity of Minocycline and Effect of Agar Medium Utilized in Its Susceptibility Testing

The in vitro activity of minocycline against 1,028 bacterial strains was determined in parallel in Mueller Hinton Agar and Trypticase Soy Agar. The broad antibacterial effect of minocycline against gram-positive cocci and gram-negative bacilli is confirmed. Minimal inhibitory concentrations for gram-positive bacteria in Mueller Hinton Agar were at least twofold less than in Trypticase Soy Agar. Minimal inhibitory concentrations for gram-negative bacilli in Mueller Hinton Agar were usually fourfold less than in Trypticase Soy Agar.     

Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

Variation in Plating Efficiency of Salmonellae on Eight Lots of Brilliant Green Agar

The plating efficiency of Salmonella anatum, S. cubana, S. dublin, S. tennessee, and S. typhimurium was determined for eight lots of Brilliant Green Agar made by two manufacturers. Washed cells were used as the inoculum and cultures were incubated at 41.5 C. All lots of Brilliant Green Agar were supplemented with 12 mg of sulfadiazine per 100 ml of medium. Of the eight lots of Brilliant Green Agar tested, average recovery of the test salmonellae in three did not differ from recoveries with Trypticase Soy Agar, which was used as a control to indicate the number of viable salmonellae in the test suspension capable of growth on a nonselective medium. Two lots of Brilliant Green Agar gave salmonellae recoveries with geometric means about 25% lower than, and significantly different from, those of the control agar. The remaining three lots of Brilliant Green Agar were generally unproductive.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Regression Curve Analysis of Cephalosporin Activity

Regression lines were calculated for cephalothin, cephalexin, and cephaloridine by relating zone diameters of inhibition to minimal inhibitory concentrations (MIC) obtained in Mueller-Hinton agar and in Trypticase Soy Agar. A regression line was calculated for cephaloglycin by obtaining MIC values in Trypticase Soy Agar at pH 6.6. Regression lines calculated from MIC values in Mueller-Hinton agar were practically superimposable on those based on MIC values in Trypticase Soy Agar. Organisms susceptible by disc testing to cephalothin were usually susceptible to cephalexin and cephaloridine.     

Effects of medium composition on the recovery of bacteria from sea water

Water samples were collected from offshore and inshore localities at various depths off the Connecticut coast over a two-year period. Spread plates for bacterial counts at 20 °C were made on a variety of complex solid media. Counts on Difco-Marine Agar were controls in all cases with counts on test media related to these in ratio form. Initially, nine media were used and represented some from the literature as well as personal formulations. Differences between inshore and offshore samples were greatest with media containing the highest peptone concentrations. Two media containing the peptones Gelysate and Trypticase showed the highest overall counts. A second phase concerned a comparative study of these peptones varying in concentration from 0.1 to 10.0 g/l in a constant basal medium. None of the media invariably gave counts greater than the control, but peptone concentrations of 10.0 and 5.0 g/l resulted in the lowest comparative counts. Considering all samples, peptone levels of 0.1 and 1.0 g/l showed the highest counts. Counts for both inshore and offshore water samples decreased as peptone concentration increased. Qualitatively, high peptone media showed large, mucoid, confluent colonies which made the counting of smaller ones difficult. Pigmented colonies were more frequent on low peptone media. Bacteria were isolated from all media and from all stations; the percentage of various groups varied with peptone concentration and source of sample.Media containing three fish peptones in varying concentrations have also been investigated. None produced overall counts greater than Difco-Marine Agar and counts decreased with increasing peptone levels: there was a trend towards higher counts in offshore waters with fish extracts. Quantitative and qualitative aspects of the work are discussed.     

Inhibition of Injured Escherichia coli by Several Selective Agents

A population of Escherichia coli ML30 cells was exposed to a quaternary ammonium compound, and injury to the cells was measured by a comparison of counts on Trypticase Soy Agar and Violet Red Bile Agar. Substantial injury could not be detected with a minimal medium. The ingredients of Violet Red Bile Agar were tested against damaged cells. The bile salts mixture alone in the medium prevented as many injured cells from growing as did any combination of the selective agents and inhibited as many injured bacteria as were inhibited by Violet Red Bile Agar itself. These dyes and salts were similarly assayed in minimal agar, and comparable results were obtained. Individual bile salts and other potential selective agents were added to the minimal medium, and the media were tested for inhibition of injured E. coli. Sodium deoxycholate was the bile salt most inhibitory to damaged E. coli cells.     

Thermal Injury and Recovery of Salmonella typhimurium and Its Effect on Enumeration Procedures

Exposure of Salmonella typhimurium 7136 to sublethal heating produced a temporary change in the tolerance of the organism to a particular stress medium. After sublethal heat treatment at 48 C for 30 min, greater than 90% of the viable population was unable to reproduce on Levine Eosin Methylene Blue Agar containing 2% NaCl. This sensitivity was dependent on the pH of the heating menstruum. In addition, the heated cells displayed a sensitivity to Brilliant Green Agar, Levine Eosin Methylene Blue Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. Unheated cells displayed a sensitivity to Brilliant Green Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. When the injured cells were placed in a suitable medium (Trypticase Soy Broth), they recovered and grew at a rate equal to that of normal cells. Recovery was also possible in Nutrient Broth, Lactose Broth, and Lauryl Tryptose Broth. Although recovery of the injured cell occurred in Tetrathionate Broth and Selenite F Broth, they were less than ideal growth media for the organism.     

Morphological Alterations in Bacillus megaterium as Produced by Aflatoxin B1

Morphologically abnormal cells were produced by Bacillus megaterium NRRL B-1368 in response to aflatoxin B(1). Filamentous forms were characterized by early granulation and unusually large and numerous deposits of poly-beta-hydroxybutyric acid within the cells. Pantoyl lactone was without effect as a reversing agent for the observed inhibition of cell septum formation. B. megaterium cells and spores produced on toxic (3.8 mug of aflatoxin B(1) per ml) and nontoxic Trypticase Soy Broth and Trypticase Soy Agar (TSA) were observed by using phase contrast and electron microscopy. Transfer of aberrant forms to nontoxic TSA yielded macrocolonies with daughter cells morphologically indistinguishable from untreated cells. Agar slide cultures of filamentous cells transferred to nontoxic TSA indicated that normal cells were formed. Electron photomicrographs showed a decreased number of mesosomes in filamentous cells as compared to control cells. There were no observable morphological differences in spores formed on toxic or nontoxic TSA.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.     

Development of a selective plating technique for the recovery of Escherichia coli O157: H7 after heat stress

The use of Sorbitol MacConkey Agar supplemented with 4-methylumbelliferyl β-D-glucuronide (MSMA), which is commonly used in the isolation of Escherichia coli O157: H7, has been shown to perform poorly when stressed cells of the pathogen are present. The incorporation of a resuscitation period (2 h at 25°C) on Trypticase Soy Agar (TSA) before overlay with MSMA was found to significantly ( P 0·01) improve recovery of heat-stressed (52°C/60 min) cells. Maximal recovery was, however, obtained by adding catalase (1000 U) to the TSA before overlaying with MSMA. This recovery protocol was shown not to result in the loss of the major known virulence factors of E. coli O157: H7 (genes encoding eae , VT1 and VT2).     

Ugan古河道胡杨可培养内生细菌的多样性

摘要：【目的】为了了解塔河废弃古河道胡杨可培养内生细菌的多样性。【方法】从2棵胡杨树干部抽出其内存液,采用三种不同的培养基对样品的内生细菌进行了分离纯化;对它们进行16S rDNA测定和系统进化分析。【结果】分离纯化不同表型的细菌62株,对它们的16S rDNA序列分析表明,62株菌分别属于四个大类群;厚壁菌门（Firmicutes）、放线菌门(Actinobacteria)、α-变形菌纲(Alpha Proteobacteria) 、γ-变形菌纲(Gamma Proteobacteria),18个属,32个种;芽孢杆菌属和假单胞菌属是胡杨可培养内生细菌的优势细菌种群,它们分别占已测种群的40.32%、16.13%。其中菌株KTH-63为葡萄球菌科的潜在的新属新种,它与最近源菌株的16S rDNA序列相似率为92.491%;9株菌KLH-21、KLH-1、KTH-8、KTH-14、KNA-26、KLH-18、KTH-20、KNA-3、KLH-25是潜在的新种（16S rDNA相似率为96.089 %-97.769 %）,胡杨树干内存液中潜在新种的发现率高达总分离检测菌株的16.13 % 。本研究获得的胡杨可培养内生细菌的群落结构数据给植物内生细菌新增了10个属,18个种。【结论】胡杨具有多样性极其丰富的可培养内生细菌菌种资源,土著新种的发现频率超出了预期,胡杨可培养内生细菌的群落结构极大地刷新了植物内生细菌的种群记录,极具进一步发掘的潜力。     

Liquid Nitrogen Preservation of Saccharomyces carlsbergensis and its Use in a Rapid Biological Assay of Vitamin B6 (Pyridoxine)

Growth medium as well as freezing menstruum greatly influenced the recovery of Saccharomyces carlsbergensis when it was quickly frozen in liquid nitrogen at - 196 C and quickly thawed at 40 C. Nearly 90% recovery in viability was obtained when S. carlsbergensis was grown in Trypticase Soy Broth and frozen in vitamin B(6) basal assay medium. The growth phase of S. carlsbergensis also influenced recovery after freezing. When S. carlsbergensis was grown in Trypticase Soy Broth and frozen in the broth at the logarithmic-growth phase, only 7% viability was retained; the recovery rate increased to 81% when the culture was frozen in the maximal stationary phase. To have the least possible lag period of growth after thawing, a technique called growth-phase conditioning was introduced. After 1 hr of growth-phase conditioning, S. carlsbergensis was clearly out of lag phase, and budding was observed. A vitamin B(6) microbiological assay with a 6-hr incubation period and with the use of liquid nitrogen-frozen S. carlsbergensis is described.     

A Method for the Rapid Isolation of Listeria monocytogenes from Infected Material

Two media, one for enrichment and the other for differentiation of Listeria monocytogenes , are described and a method is proposed for the selective isolation of this bacterium from material containing a mixed bacterial flora such as faeces, vaginal swabs etc. Addition of potassium dichromate, chromium trioxide, thionin, nalidixic acid and amphotericin B to Todd-Hewitt Broth (BBL) made a satisfactory enrichment broth in which good selective growth of L. monocytogenes was obtained without notable damage to cells. The differentiation agar was Trypticase Soy Agar (BBL) supplemented with gallocyanin, pyronin and nalidixic acid. On this medium L. monocytogenes colonies, when viewed by the Henry's oblique transillumination technique, were blue in contrast to colonies of other bacterial species which were pink or red. Trials with experimentally infected materials showed that L. monocytogenes could be recovered from faeces infected with as few as 20 L. monocytogenes cells/g. All common contaminants, with the exception of a few strains of Streptococcus faecalis , were inhibited.