Fatty Acid and Aliphatic Hydrocarbon Composition of Sarcina lutea Grown in Three Different Media

Sarcina lutea was grown in Trypticase Soy Broth, Nutrient Broth, and a chemically defined medium. Gas chromatographic analysis of lipid components demonstrated that the composition of the medium had an effect on the relative per cent composition of the aliphatic hydrocarbons and fatty acids present in the cells. The branched olefinic hydrocarbons from the organisms grown in Trypticase Soy Broth showed no predominance or only a slight predominance of odd-numbered carbon chains, whereas the hydrocarbons from cells grown in the other two media showed an obvious predominance of odd-numbered carbon chains. The monocarboxylic fatty acid content and distribution showed only minor differences, with all normal saturated fatty acids present in relatively small quantities for cells grown in Nutrient Broth and in a chemically defined medium.     

Haemolytic activity of Morganella morganii strains

Haemolytic activity on solid and liquid media of 103 Morganella morganii strains isolated from clinical sources was investigated. The ability to produce haemolysin was found in 42.7% of strains. All strains capable to produce haemolysin on blood agar media also revealed haemolytic activity in some liquid media. Haemolysins were found in the supernatants and filtrates of the cultures in peptone water but not in Brain Heart Infusion and Trypticase Soy Broth. The maximal titer of haemolysin was observed in the logarithmic phase of growth. Heating and incubation with trypsin led to complete loss of haemolytic activity.     

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.     

Coagulase Production by Injured Staphylococcus aureus MF-31 During Recovery

Suspensions of Staphylococcus aureus MF-31 injured by heat treatment at 54 C for 15 min produced coagulase during recovery in Trypticase Soy Broth. Coagulase also was produced by injured cells during recovery in a medium that did not support growth. Coagulase synthesis during recovery was independent of the molar strength of the buffer in which the cells were injured, the age of the cells, and the degree of injury. Return of salt tolerance and coagulase production required glucose, amino acids, and phosphate in the recovery medium. Vitamins stimulated coagulase production, but did not affect recovery. Although coagulase production was not necessary for repair of thermal injury to S. aureus MF-31, its detection was interpreted as an indicator of protein synthesis.     

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.     

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.     

Purification and characterization of a staphylolytic enzyme from Pseudomonas aeruginosa

A strain of Pseudomonas aeruginosa has been shown to produce an enzyme that lyses viable cells of Staphylococcus aureus. The maximal yield of the enzyme was obtained from shake flask cultures of P. aeruginosa which were grown for 18 to 22 hr at 37 C in Trypticase Soy Broth. A 333-fold purification of the enzyme was obtained by acetone precipitation of the culture liquor, followed by column chromatography on phosphonic acid cellulose and Bio-Gel P2. The staphylolytic enzyme exhibited maximal activity at 37 C in 0.01 m sodium phosphate (pH 8.5) and was stable at 37 C in the pH range of 7.5 to 9.5. The inhibition and stabilization of the enzyme by various organic and inorganic materials was investigated. Spheroplasts of S. aureus were formed by treating viable cells with the staphylolytic enzyme in 1 m sucrose or human serum.     

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.     

Liquid Nitrogen Freezing in Microbiological Assay Systems: I. Preservation of Lactobacillus leichmannii for Direct Use in the Vitamin B12 Assay

Suspensions of Lactobacillus leichmannii were stored in liquid nitrogen and were used as direct inocula in vitamin B₁₂ assays. Complete recovery of viable cells was obtained when the suspensions in basal B₁₂ medium were rapidly frozen by direct immersion into liquid nitrogen and rapidly thawed by agitating the suspensions in a water bath at 40 C. Greater than 90% destruction occurred when the suspensions were in saline. However, both suspensions were usable in the B₁₂ assay system. Assay results on a number of test materials indicated good correlation between freshly prepared suspensions and frozen suspensions in basal medium stored 3 months. Suspensions in saline stored for 1 year in liquid nitrogen showed no detectable difference from the first day after freezing. Suspensions frozen slowly at the rate of 1 degree per min from 4 to -40 C and subsequently immersed in liquid nitrogen had a longer lag period of growth and were not usable in the 18-hr assay incubation system. A major advantage of a stored inoculum for direct use in a microbiological assay is the reduced day-to-day variation in the inoculum.     

The vitamin requirements of Staphylococcus cohnii

In heat-resistance studies with spores of Clostridium sporogenes BC-2, an improved recovery medium was needed for severely heat-damaged spores as the used previously--Wynne medium in Miller-Prickett tubes--did not allow accurate counts of spores because of gas formation and disruption of agar. Initial test with pour plates of Viande-Leyure medium containing egg-yolk gave much increased counts for spores previously heated for 50 min at 112 degrees C; this increase was attributed to the presence of egg-yolk. Addition of egg-yolk to Reinforced Clostridial Agar, All-Culture Medium and Trypticase Soy Agar showed that Trypticase Soy Agar with egg-yolk was the best recovery medium. For the final formulation, the value of supplementation with cysteine-HCl and methylene blue was also shown. The resultant Egg-yolk Trypticase Soy Agar is conveniently prepared from BBL Trypticase Soy Agar (40 g) with the addition of 0.4 g/l cysteine-HCl, 4 mg/l of methylene blue and 2% Oxoid egg-yolk emulsion aseptically to the melted basal medium. For optimal spore counts, pour plates are incubated anaerobically for 5--7 d at 30 degrees C.     

Limitations of Thioglycolate Broth as a Sterility Test Medium for Materials Exposed to Gaseous Ethylene Oxide

Although ethylene oxide is a reliable sterilizer, the process may be limited by diffusion. Thus, situations may exist where microorganisms are protected from the sterilizing gas. It is possible that the exterior of a substance may be sterilized, whereas the interior is not. We investigated three general types of materials in which this limitation of diffusion could occur: the bore of glass and plastic tubing, the center of cotton balls, and plastic adhesive film/paper backing interface. These materials were contaminated as close to their geometric center as possible with Bacillus subtilis var. niger spores occluded in crystals of sodium chloride. After exposure of the contaminated materials (except aluminum foil) to ethylene oxide, thioglycolate broth (a standard sterility-test medium) indicated sterility, whereas Trypticase Soy Broth indicated nonsterility. It is likewise possible that aerobic microorganisms, surviving in or on material after exposure to dry heat or steam sterilization processes, would not be recovered by thioglycollate broth. Entrapped aerobic organisms will probably not grow out in the low oxygen tension zone of an anaerobic medium such as thioglycollate broth. It is recommended than an aerobic medium such as Trypticase Soy Broth be used concurrently with thioglycolate broth for sterility testing.     

Isolation and Purification of Staphylococcal Lipase

An extracellular lipase was isolated from the cell-free supernatant fluid of a 24-hr culture of Staphylococcus aureus grown in Trypticase Soy Broth at 37 C with continuous agitation. The purification was achieved by precipitation with alcohol followed by differential precipitation at pH 8.6 and 4.3. Subsequent purification with Sephadex G 200 and BioGel 300 yielded a preparation which showed a 350- to 450-fold increase in specific activity over the original cell-free supernatant fluid. The purified lipase was homogeneous over a BioGel 300 column and showed a single peak on electrophoresis in a Veronal buffer (pH 8.6, Γ/2 = 0.1). The electrophoretic mobility was -7.78 × 10^-5 cm² per v per sec.     

Effect of Available Water on Thermal Resistance of Three Nonsporeforming Species of Bacteria

Cells of Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus, previously grown in Trypticase Soy Broth (TSB) at a high level of available moisture (a(w) 0.994) and at low levels produced by addition of NaCl or glucose, were heated in neutral phosphate buffer, and in this buffer adjusted to low levels of available moisture by means of NaCl or glucose. Glucose in the heating medium was more protective than NaCl for E. coli and P. fluorescens, but hastened the thermal destruction of S. aureus. Added protection was given P. fluorescens during heating in glucose-buffer solution at a(w) 0.97 by previous growth in TSB adjusted to that a(w) value with glucose. Added protection was given E. coli during heating in NaCl-buffer solution at a(w) 0.98 by previous growth in TSB adjusted to that value with NaCl. With S. aureus, however, previous growth in TSB plus NaCl or glucose had little effect on heat resistance, but the solute in the heating medium had great influence, in that NaCl was very protective and glucose destructive. Opportunity may have been given during tempering of the cell suspension at 30 C in the heating medium prior to heating for the NaCl and glucose to diffuse into the staphylococcal cells.     

Sterility Test Method for Petrolatum-Based Ophthalmic Ointments

A sensitive sterility testing procedure for the detection of microbial contamination in petrolatum-based ointments is described. The method involves dissolving the ointment in filter-sterilized isopropyl myristate and filtering through a membrane filter. Improved sensitivity is obtained by blending the membrane in Trypticase Soy Broth before incubation. Filter-sterilized isopropyl myristate is shown to be less toxic to microorganisms than heat-sterilized isopropyl myristate. The isopropyl myristate method is more sensitive than the polyethylene glycol-ether method for the detection of microbial contamination.     

Spawn viability in edible mushrooms after freezing in liquid nitrogen without a cryoprotectant

Five strains of edible mushrooms (Lentinula boryana, Lentinula edodes, Pleurotus djamor, Pleurotus pulmonarius, and Volvariella volvacea) were studied. Spawn were prepared from sorghum seeds and then incubated for 14 days under optimum conditions for each species. Once covered by mycelia, the sorghum seeds were placed in polycarbonate vials for freezing in liquid nitrogen. The effect of adding a cryoprotective solution before freezing (either 10% glycerol v/v or 5% dimethylsulfoxide v/v) was evaluated as a function of mycelial growth and percent viability. Three main treatments were undertaken: (1) freezing with a glycerol or dimethylsulfoxide cryoprotectant, (2) freezing with water and (3) freezing without cryoprotectant or water. Samples were maintained frozen for a week, after which time they were thawed (10 min at 30 degrees C) and the seeds placed in Petri dishes with a culture medium. A recovery rate of 96.8% was obtained for the total number of samples summed over all strains and treatments. In contrast, 99.2% of the samples frozen without cryoprotectant were recovered. The recovery of frozen mycelia was delayed with respect to a control group, which was not frozen. However, no difference was observed in percent recovery and mycelial diameter when a new series of spawn was prepared from mycelia that had been previously frozen. Results obtained from this experiment demonstrate that an adequate recovery of mycelia can be obtained without using a cryoprotectant. This capacity might enable large quantities of commercial mushroom strains to be handled at reduced production costs. It is suggested that the mycelia survived freezing without cryoprotectants because they were embedded and protected within the sorghum seeds used to elaborate the spawn.     

New Medium for Blood Cultures

A new medium suitable for blood cultures is described. It contains dextrose, cysteine, iron, and magnesium, in a tris(hydroxymethyl)aminomethane buffer, and a mixture of peptones derived from animal tissues, casein, and yeast. In comparison with Trypticase Soy Broth, the growth rate constants of Staphylococcus aureus, Streptococcus (Viridans group), enterococcus, and Escherichia coli were higher in this medium, and growth appeared earlier in a significant number of clinical blood cultures.     

New Medium for Blood Cultures

A new medium suitable for blood cultures is described. It contains dextrose, cysteine, iron, and magnesium, in a tris(hydroxymethyl)aminomethane buffer, and a mixture of peptones derived from animal tissues, casein, and yeast. In comparison with Trypticase Soy Broth, the growth rate constants of Staphylococcus aureus, Streptococcus (Viridans group), enterococcus, and Escherichia coli were higher in this medium, and growth appeared earlier in a significant number of clinical blood cultures.     

Elution of Loosely Bound Acid Phosphatase from Staphylococcus aureus

Strains of Staphylococcus aureus from the International-Blair and the Seto-Wilson series of phage propagating strains were examined for acid phosphatase activity. This enzyme was found to occur in varying amounts in three different fractions: free (6 to 60%), loosely bound (25 to 82%), and firmly bound (0 to 46%). Propagating strain 3A, because of its high activity, was chosen for further study. The rate of enzyme production paralleled cell growth in Trypticase Soy Broth, but followed a biphasic pattern in a semisynthetic casein acid-hydrolysate medium with glyceryl phosphate. Maximal elution of acid phosphatase in the loosely bound fraction, presumably from the surface of cells, occurred in the alkaline pH range. From log-phase cells, elution was maximally effected with buffered 1.0 M KCl (pH 7.5), but stationary-phase cells required twice the concentration of KCl.     

Proceedings: Factors affecting repair of sublethal injury in frozen or freeze-dried bacteria

Conditions facilitating recovery of sublethally injured cells present in frozen or freeze-dried preparations of bacteria were investigated. Some cells of Streptococcus faecalis injured by freezing required RNA resynthesis for recovery, whereas the rest did not. Addition of 6% NaCl to control recovery medium was found to inhibit RNA synthesis completely in frozen-injured cells of S. faecalis, but not in intact ones. This seems to be a principal reason why 6% NaCl inhibits the growth of the injured cells.The recovery of viable cells of freeze-dried and stored Streptococcus thermophilus was greately increased by the addition of cysteine to the plating medium. The favorable effect of cystein was mainly due to its reducing activity. On the contrary, the addition to the plating medium of certain types of peptone, such as lactalbumin hydrolyzate, resulted in a considerable decrease of viability in frozen preparations of Vibrio metschnikovii.Thus, at present, it seems too early to try to discuss specifically the optimal composition of plating media to give the highest viability from frozen or freeze-dried preparations of bacteria.     

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.