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.     

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.     

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.     

Aerococcus viridans in the Hospital Environment

Aerococcus viridans has been described as an airborne organism prevalent in occupied rooms. It has also been described as an organism having many characteristics that might cause it to be confused with streptococci or staphylococci, and this may account for the fact that the presence of A. viridans has not been reported in the hospital environment or in clinical specimens. Swab specimens were taken from 47 objects in 11 different areas in a local hospital, cultured overnight in Trypticase Soy Broth, and streaked on blood-agar and on a selective serum agar containing potassium tellurite and crystal violet. Of 85 alpha-hemolytic cultures isolated, 11 proved to be typical A. viridans based on diagnostic tests that also were applied to a collection of gram-positive cocci, including authentic strains of A. viridans. These organisms are gram-positive cocci with a strong tendency toward tetrad formation in broth cultures. They are predominantly aerobic, have a very weak catalase activity, and lack porphyrin respiratory enzymes. Three similar cultures also were obtained from routine clinical specimens.     

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.     

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.     

Inactivation of the Bactericidal Activity of Human Serum by Liquoid (Sodium Polyanetholsulfonate

Four serum-sensitive strains of Escherichia coli were exposed to 10, 20, and 50% fresh, heat-inactivated, and fresh human serum to which had been added Liquoid at a final concentration of 0.05, 0.025, 0.0125 and 0.006%. It was found that 50% fresh serum (in nutrient, Mueller-Hinton, thioglycolate, or Trypticase Soy Broth) killed more than 10(4) organisms/ml within 3 min, whereas 20 and 10% fresh serum required up to 20 and 40 min, respectively, to kill a comparable number of organisms. To neutralize the activity of 50% fresh serum, 0.0125% Liquoid had to be added, whereas an 0.006% final concentration of Liquoid was sufficient to antagonize the activity of 10 and 20% serum. However, when exposing extremely small bacterial inocula to fresh serum, at least 0.025% Liquoid was necessary to abolish the serum-bactericidal activity of 20 and 50% fresh serum. Liquoid had to be added to 50% fresh serum within seconds to prevent the killing of the majority of test organisms derived from small inocula. It is recommended that blood samples drawn from septicemic or bacteremic patients be aseptically added to a suitable broth which contains at least 0.025% Liquoid in order to improve the chances of isolating pathogens present in small numbers.     

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.     

Effect of reducing agents on oxidation-reduction potential and the outgrowth of Clostridium botulinum type E spores.

Oxidation-reduction potential (Eh) levels were measured and standardized to pH (Eh7) for Trypticase soy broth containing various concentrations of reducing agents. Prereduced Trypticase soy broth with no added reducing agents exhibited a potential of -141 mV. Ascorbic acid at 0.2 to 0.005% and sodium thioglycolate at concentrations below 0.05% produced an Eh7 higher than the prereduced Trypticase soy broth containing no added reducing agents. The addition of cysteine hydrochloride,2-mercaptoethanol, and sodium formaldehyde sulfoxylate to prereduced Trypticase soy broth resulted in a reduction of Eh7 compared to the system without added reducing agents. The order of relative reducing intensity (from highest to lowest) for the reducing agents when comparing molar concentration was: sodium formaldehyde sulfoxylate,2-mercaptoethanol, cysteine hydrochloride, sodium thioglycolate, and ascorbic acid. Optimal growth of the test organism occurred at low Eh7 and low concentration of the reducing agents. A direct correlation existed between growth of the test organism and -Eh7 x -log concentration of the reducing agent.     

Effect of reducing agents on oxidation-reduction potential and the outgrowth of Clostridium botulinum type E spores.

Oxidation-reduction potential (Eh) levels were measured and standardized to pH (Eh7) for Trypticase soy broth containing various concentrations of reducing agents. Prereduced Trypticase soy broth with no added reducing agents exhibited a potential of -141 mV. Ascorbic acid at 0.2 to 0.005% and sodium thioglycolate at concentrations below 0.05% produced an Eh7 higher than the prereduced Trypticase soy broth containing no added reducing agents. The addition of cysteine hydrochloride,2-mercaptoethanol, and sodium formaldehyde sulfoxylate to prereduced Trypticase soy broth resulted in a reduction of Eh7 compared to the system without added reducing agents. The order of relative reducing intensity (from highest to lowest) for the reducing agents when comparing molar concentration was: sodium formaldehyde sulfoxylate,2-mercaptoethanol, cysteine hydrochloride, sodium thioglycolate, and ascorbic acid. Optimal growth of the test organism occurred at low Eh7 and low concentration of the reducing agents. A direct correlation existed between growth of the test organism and -Eh7 x -log concentration of the reducing agent.     

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.     

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.     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance

Background

We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs).

Recovery of Anaerobic Microorganisms from Clinical Specimens in Prereduced Media Versus Recovery by Routine Clinical Laboratory Methods

Prereduced anaerobically sterilized culture media, used with rigid adherence to the cultivation techniques described by Moore and his associates, were capable of recovering more than twice the number of anaerobic bacteria from clinical specimens than could be recovered by the conventional use of fluid thioglycolate medium and of blood-agar plates incubated anaerobically with hydrogen generation packets. No loss of clinical isolates was encountered with the more sensitive methods; however many of the isolates recovered only in prereduced media would not grow when placed into thioglycolate medium. A representative anaerobic isolate placed into aerobic transport broth was unable to survive beyond 30 min. Methods employing prereduced media were not difficult to master and were feasible for clinical laboratory use. Evidence implicating the gingival crevice flora as an important possible source of anaerobic bacteria that become involved in systemic infections was considered.     

Effect of Soy Proteins on the Growth of Clostridium perfringens

Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridium perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an E(h) of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N(2)-10% CO(2)) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease.     

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.     

Sporulation of Clostridium perfringens in a Modified Medium and Selected Foods

A modified sporulation medium for Clostridium perfringens was formulated in which a larger number of spores were produced than in SEC broth and in which spores of greater heat resistance were produced than in Ellner's medium when it was also used as the suspending medium. This modified medium consisted of 1.5% peptone; 3.0% Trypticase; 0.4% starch; 0.5% NaCl; and 0.02% MgSO(4). The addition of 0.1% sodium thioglycolate and 0.0001% thiamine hydrochloride was optional. The optimal temperature for sporulation of five strains was 37 C in comparison with 5, 22, and 46 C. Sporulation had occurred by 6 hr and was essentially complete after 20 hr at 37 C. Noyes veal broth without glucose also supported the formation of heat-resistant spores but in smaller numbers than did the modified medium. Very low numbers of spores, or none, were produced under the same conditions in pea or tuna slurries.     

Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens.

The influence of redox potential on dissimilatory nitrate reduction to ammonium was investigated on a marine bacterium, Pseudomonas putrefaciens. Nitrate was consumed (3.1 mmol liter-1), and ammonium was produced in cultures with glucose and without sodium thioglycolate. When sodium thioglycolate was added, nitrate was consumed at a lower rate (1.1 mmol liter-1), and no significant amounts of nitrite or ammonium were produced. No growth was detected in glucose media either with or without sodium thioglycolate. When grown on tryptic soy broth, the production of nitrous oxide paralleled growth. In the same medium, but with sodium thioglycolate, nitrous oxide was first produced during growth and then consumed. Acetylene caused the nitrous oxide to accumulate. These results and the mass balance calculations for different nitrogen components indicate that P. putrefaciens has the capacity to dissimilate nitrate to ammonium as well as to dinitrogen gas and nitrous oxide (denitrification). The dissimilatory pathway to ammonium dominates except when sodium thioglycolate is added to the medium.