Estimation of the Growth of the T1 Strain of Mycoplasma mycoides in Tryptose Broth by the Measurement of Lactate Dehydrogenase: I. Method

A highly significant correlation coefficient (r = 0.97, n = 18) was found between the concentration of lactate dehydrogenase measurable after the organisms had been disrupted and the concentration of colony-forming units during the logarithmic phase of growth of a broth culture of the T(1) strain of Mycoplasma mycoides var. mycoides. A concentration of 4.60 x 10(-7) milliunits of lactate dehydrogenase for each colony-forming unit was established. This relationship was used to convert the concentration of lactate dehydrogenase in the culture into an estimate of the concentration of viable mycoplasma. The lactate dehydrogenase was estimated by following the oxidation of reduced nicotinamide adenine dinucleotide, in the presence of pyruvate substrate, at 366 nm in a spectrophotometer. The nicotinamide adenine dinucleotide oxidase system probably contributed a small amount of enzyme activity to the test when lactate dehydrogenase was measured in this way. The method has been described and evaluated for the estimation of titers from 10(7) to 5 x 10(9) colony-forming units per ml.     

Effect of amphotericin B on the enzyme system of Candida albicans]

The mechanism of amphotericin B action was studied with the aid of cytochemical methods providing determination of the activity of the 4 main enzymes characterizing the cell energetics, i. e. succinate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase inside the cell. With an increase in the concentration of amphotericin B in the medium the activity of all the 4 enzymes decreased, the percentage of the inactive cells being regularly increased. Changes in the fermentative activity of C. albicans as dependent on the incubation time with the antibiotic were studied. Only the respiration activity decreased in 2 hours. As a result of a 4-hour exposure to the polyen in the cells of C. albicans the activity of the lactic acid fermentation, respiration through succinate dehydrogenase and activity of the pentose shunt decreased 1.5--2 times. In 24 hours of incubation the activity of the above decreased 80--90 per cent as compared to the activity of the initial culture.     

Factors Affecting the Viability of Mycoplasma mycoides in Bottled Contagious Bovine Pleuropneumonia Vaccine

S ummary . Some of the factors contributing to the loss of viability of V5 contagious bovine pleuropneumonia vaccine in BVFOS medium during storage were investigated. Results showed that: 1, some types of rubber stoppers were highly toxic to Mycoplasma mycoides ; 2, loss of viability was greater when the vaccine was stored in smaller bottles and was related to the proportionately greater air space; 3, the addition of glycerol to BVFOS medium resulted in slightly better survival during storage at 37° but decreased survival at 4°; 4, small bottles can be filled to higher levels, and the air space reduced, if the mouth of the bottle is protected during incubation by a metal cover instead of a cotton wool plug. It is recommended that the amount of air space should not exceed 40% of the volume of the bottle, the vaccine should be stoppered with non-toxic stoppers after incubation and the BVFOS medium should not be supplemented with glycerol. Vaccine prepared in this way should maintain a viable count of 10⁷ or more organisms/ml during storage at 4° for at least 12 weeks.     

Production of Types A and B Spores of Clostridium botulinum by the Biphasic Method: Effect on Spore Population, Radiation Resistance, and Toxigenicity

Spores of three strains each of type A and type B Clostridium botulinum were produced both by a biphasic (solid medium overlaid with an aqueous phase) and by a "conventional" (deep broth culture) procedure. Sporogenesis by the biphasic system was more rapid, convenient, and economical, and yielded as many or more heat-resistant (80 C, 10 min) spores per milliliter as by the conventional technique. Of several aqueous phases [thiamine-hydrochloride, yeast extract, (NH(4))(2)SO(4)] tested with strain 62A, the highest spore colony counts were obtained with 2.0% (NH(4))(2)SO(4). The six strains formed maximum spore numbers in 5 to 6 days of incubation. Spores produced by the two methods had essentially equal radiation resistances (D and lag values), and their subcultures gave similar toxin titers (LD(50) values).     

Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa

Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.     

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).     

The correlation method for rapid monitoring of Escherichia coli in foods

AIMS: A new rapid method was developed to rapidly monitor Escherichia coli counts in foods. MATERIALS AND RESULTS: One ml of modified selective broth with 4-methylumbelliferyl beta-D-glucuronide and 1 ml of food sample were mixed in a sterile test tube and incubated at 37 degrees C. The positive reaction (fluorescence under u.v. light) was monitored at regular 30 min intervals. The positive reaction times in test tubes were compared with actual E. coli numbers from tested samples. The growth of E. coli in test tubes (broth) was much faster than growth on agar. The first experiment was performed to evaluate the rapid correlation method using pure E. coli cultures. The correlation between E. coli counts by the conventional plating method and positive reaction (fluorescence production) times in test tubes was highly agreeable (r(2) = 0 x 95). In the case of low E. coli numbers, such as 2 x 0 log10 cfu ml(-1), the rapid correlation method detected their presence after 10 h incubation. When highly contaminated samples were assayed (8 log10 cfu ml(-1)), the rapid correlation method detected the presence of E. coli after 4 h incubation. In the ground beef experiment, the correlation between fluorescence production time and actual E. coli numbers was also strongly agreeable (r(2) = 0 x 92). CONCLUSIONS: From these results, it is obvious that the new rapid method can rapidly monitor E. coli counts in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the new method saved about 10-14 h incubation time compared to conventional plating methods. The rapid correlation method required much shorter incubation times compared to conventional plating methods for monitoring E. coli.     

Occurrence and properties of lactic dehydrogenases of fermentative mycoplasmas

Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(-)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(-)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.     

Extracellular protease production fromXenorhabdus nematophilus, a symbiotic bacterium of entomopathogenic nematodes

Effects of nutrients and growth temperature on the production of an extracellular protease fromXenorhabdus nematophilus, a symbiotic bacterium of entomopathogenic nematodes were studied for batch culture. Tryptone and fructose were most effective nitrogen and carbon sources to produce high level of the protease within 24–28 hr of incubation. The stability of protease was poor during the incubation of the bacteria. The protease activity increased in parallel with cell growth then decreased significantly for extended culture periods of the bacteria over 48 hr at 22–30°C. Autolysis of the protease was not a major cause for the decreased protease activity since no decrease in the protease activity was observed for the whole culture broth of the bacteria which was stored up to 80 hr at 4°C.     

Studies of lactate dehydrogenase of Mycoplasma mycoides var. mycoides.

Polyacrylamide gel electrophoresis and isoelectric focusing techniques have been used to compare NAD-dependent L(plus) lactate dehydrogenases (LDH) from ten different strains of Mycoplasma mycoides var. mycoides. The enzymes were not distinguished from one another, or from normal bovine LDH 1 by these methods. The kinetic behaviour of LDH form M. mycoides (T1 vaccine strain) suggested that the enzyme could readily reduce pyruvate or oxidize lactate in a manner which, in vertebrates, requires two different isoenzymes.     

Factors affecting the survival of Neisseria sicca

Cultures of Neisseria sicca incubated at 37 degrees C died rapidly (within 36 h) after growth ceased. Re-suspending cells in a brain heart infusion broth and storing at 4 degrees C greatly reduced the rate of decline in viability (decimal reduction time 6 days). An important factor in maintaining viability was apparently the presence of external energy source(s). Survival comparable to that in broth was obtained by incubation in Ringer's solution with pyruvate plus glucose (but not with pyruvate or glucose alone). Medium pH had little effect on survival in the range pH 7.0 to 8.5. Energy sources also promoted survival of cells in Ringer's solution or a buffered salts solution at 37 degrees C. Highest levels of survival (up to 30% at 24 h) were obtained with pyruvate, lactate, proline and glutamate. A number of other amino acids and the tricarboxylic acid cycle intermediates, isocitrate, oxoglutarate, succinate, fumarate, malate and oxaloacetate, enhanced survival to a lesser extent.     

Effect of pre-incubation pH on the growth characteristics of Aeromonas hydrophila at 5°C, as assessed by two methods

Two strains of Aeromonas hydrophila (the type strain ATCC 7966 and a food-derived strain JAH4) were pre-incubated at 5°C in Brain Heart Infusion (BHI) broth with pH adjusted to 6.0 or 7.0, and then incubated at the same temperature in BHI broth with pH adjusted to 6.0, 6.5, 7.0 and 7.5. Growth kinetics during incubation were determined by two methods: viable count (VC) and measurement of optical density (O.D.). Pre-incubation at different pH values did not significantly affect the maximum specific growth rates of the strains during incubation, but the lag phases were shorter after pre-incubation at pH 6.0 than at pH 7.0. The VC method was more sensitive than O.D. measurements for assessing lag phase.     

Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta

Confluent monolayers of bovine aortic endothelial cells were examined 2-72 h after exposure to 0.5-5.0 Gy of 60Co gamma-rays. Accumulation of prostacyclin [PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity. Platelet adherence to irradiated and control monolayers also was studied. There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium. These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy. Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM). However, by 72 h this increase was less significant. The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment. There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment. However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets.     

Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferating primary monolayer cultures

Parenchymal cells from normal adult rat liver, prepared with high yield (30 × 10⁶ cells/g liver) and viability index (>96%) by a non-perfusion method, were maintained in non-proliferating monolayer culture. Several metabolic functions were investigated for 7 days to evaluate functional integrity of the cultured hepatocytes. Leucine was linearly incorporated into protein for 4.5 h at each day of cultivation and the incorporation rate increased up to 2-fold after 3 days. Urea production was maintained at a rate of 0.5 μmoles/mg protein × h for at least 7 days, and its amount was enhanced 2-fold within 24 h by the addition of 3 mM NH₄Cl. Glucose was formed during the first days by the hepatocytes and was then taken up with increasing amount from the surrounding medium. Lactate consumption, on the other hand, was replaced by lactate production after one day of cultivation.Variations in enzyme levels of lactate dehydrogenase, arginase, glutamine synthetase and glucose-6-phosphatase were also studied during the whole culture period. Cell leakage, which was detected only in the case of lactate dehydrogenase (LDH), occurred through the 4th day along with a concomitant loss of intracellular LDH activity. After 4 days, however, the enzyme activity returned to the initial level. Arginase was maintained throughout the cultivation period and was stimulated 2- to 3-fold within 24 h by NH₄Cl. Glutamine synthetase declined within the first 4 h of cultivation and then remained in the hepatocytes with a transitory rise after 2 days. Its activity was also found to be inversely related to the concentration of glutamine in the culture medium up to 4 mM. Glucose-6-phosphatase gradually decreased during the cultivation period, the enzyme activity, however, was stimulated by glucagon within 24 h.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Repeated batch production of L-phenylalanine from phenylpyruvate and NH(4)Cl by immobilized cells of Nocardia opaca under hydrogen high pressure

Among various microbial cells examined under screening conditions, Nocardia opaca showed the highest activity for production of phenylalanine from phenylpyruvate. Here NH(4)Cl as well as amino acids were used as an amino donor for phenylalanine production. The phenylalanine production rate increased with increasing hydrogen pressure. The specific activity of phenylalanine dehydrogenase was increased by culturing N. opaca cells in nutrient broth containing 0.3% phenylalanine. As a result, the phenylalanine production rate increased from 0.69 to 4.4 mumol/min g dry cells. Immobilized cells were activated in nutrient broth containing ZnCl(2) before phenylalanine production. Phenylalanine dehydrogenase activity and cell number in the gel increased with increasing incubation time, and the maximum phenylalanine dehydrogenase activity was obtained at 36 h incubation. Then, phenylalanine was produced from phenylpyruvate, NH(4)Cl, and 100 atm H(2) with the activated immobilized cells. The rate of phenylalanine production was 0.24 mumol/min cm(3) gel. The conversion of phenylpyruvate to phenylalanine was 82%. Immobilized cells retained 76% of the initial phenylalanine production rate after 10 h reactions were repeated 11 times with two intervening reactivations.     

A strategy to prevent the occurrence of Lactobacillus strains using lactate-tolerant yeast Candida glabrata in bioethanol production

Contamination of Lactobacillus sp. in the fermentation broth of bioethanol production decreases ethanol production efficiency. Although the addition of lactate to the broth can effectively inhibit the growth of Lactobacillus sp., it also greatly reduces the fermentation ability of Saccharomyces cerevisiae. To overcome this conflict, lactate-tolerant yeast strains were screened. Candida glabrata strain NFRI 3164 was found to exhibit both higher levels of lactate tolerance and fermentation ability. Co-cultivation of C. glabrata was performed with Lactobacillus brevis and Lb. fermentum, which were reported as major contaminating bacteria during bioethanol production, in culture medium containing 2% lactate. Under these culture conditions, the growth of Lactobacillus strains was greatly inhibited, but the ethanol production of C. glabrata was not significantly affected. Our data show the possibility of designing an effective fuel ethanol production process that eliminates contamination by Lactobacillus strains through the combined use of lactate addition and C. glabrata.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Relationship between respiratory enzymes and survival of Escherichia coli under starvation stress in lake water

Survival, electron transport system (ETS) activity and the activity of NADH and succinate dehydrogenase of Escherichia coli ML30 were studied under starvation stress at different temperatures in a filtered-autoclaved lake water microcosm. ETS activity in E. coli declined rapidly at 30 degrees C but more slowly at 4 degrees and 15 degrees C over a 20 d starvation period. The decrease in ETS activity in E. coli only started after 6 d of incubation at 4 degrees C and 15 degrees C. Viability of E. coli, as determined by plate counts, declined faster at 37 degrees C than at the other temperatures and remained highest at 4 degrees C in filtered-autoclaved lake water. There was also a significant cell size reduction at 37 degrees C in filtered-autoclaved lake water but not at 4 degrees C. ETS activity after up to 16 d of starvation increased after the addition of nutrient broth to the filtered-autoclaved lake water at 15 degrees C and 30 degrees C suggesting that cells were still able to respond to nutrients, even after prolonged starvation. The response to the addition of nutrient broth, however, declined with the length of the starvation period. The activity of both succinate and NADH dehydrogenase declined over a 13 d starvation period. The loss of activity was fastest at 37 degrees C compared to lower incubation temperatures but even at 4 degrees C, a significant proportion of the activity was lost over the 13 d period.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.