The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

The use of automated tubidimetric data for the construction of kinetic models

Summary An automated tubidimetric instrument (Bioscreen) was used to observe the growth response ofListeria monocytogenes to combinations of temperature (15–30°C), hydrogen-ion (0.1–21.9 m) (equivalent pH 4.66–7.0) and NaCl concentration (0.5–9.5% w/v). Compared to traditional plate count techniques, the technique allowed many more data points to be captured and replicates to be used, with less expenditure of effort. Optical density curves were filtered (smoothed) to minimize the effect of signal noise and the mean signal from uninoculated wells was subtracted to minimize the effect of signal draft. A novel procedure for fitting growth curves to optical density data has been developed. The procedure involves the use of the logistic function and a calibration equation for fitting, in a single step, in the dimension of optical density. This approach allowed the four parameters of the logistic equation to be derived at each set of experimental conditions. A quadratic response surface was then fitted to the curve parameters using temperature, NaCl and hydrogen-ion concentration as three independent variables. Predicted time to 1000-fold increase in cell numbers compared well to predictions from predictive microbial growth equations generated in other laboratories using traditional plate counting. We propose that this technique should be further evaluated as a method for generating data for modeling the kinetics of microbial growth.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish.

Sterilized seawater was used to assess the effects of temperature and salinity on the survival of Vibrio vulnificus. In the temperature range of 13 to 22 degrees C, numbers of V. vulnificus increased during the 6-day incubation. Temperatures outside this range reduced the time of V. vulnificus survival in sterile 10-ppt seawater. At these restrictive temperatures, V. vulnificus numbers were reduced by 90% after 6 days of incubation. Incubation between 0.5 and 10.5 degrees C demonstrated that V. vulnificus survives poorly below 8.5 degrees C. At salinities between 5 and 25 ppt and at 14 degrees C, V. vulnificus numbers actually increased or remained unchanged after 6 days of incubation. At salinities of 30, 35, and 38 ppt, numbers of V. vulnificus decreased 58, 88, and 83%, respectively. V. vulnificus could not be recovered from deionized water, indicating lysis. When a rifampin-resistant strain of V. vulnificus was used to inoculate sterilized and unsterilized seawater (20 ppt, 20 degrees C), numbers increased in sterile seawater but decreased to undetectable levels in 14 days in the unsterilized seawater, indicating that biological factors may play a role in the survival of V. vulnificus in the environment. Since our studies demonstrated sensitivity to low temperatures, the survival of V. vulnificus in naturally contaminated oysters at temperatures of 0, 2, and 4 degrees C was also determined. Numbers of endogenous V. vulnificus in oyster shellstock increased by more than 100-fold in shellstock stored at 30 degrees C but were reduced approximately 10- and 100-fold after 14 days at 2 to 4 degrees C and 0 degrees C, respectively. We conclude that both biological and physicochemical factors are important to the survival of V. vulnificus in the environment and that temperature is critical to controlling its growth in oyster shellstock.     

Use of gradient plates to study combined effects of temperature, pH, and NaCl concentration on growth of Monascus ruber van Tieghem, an Ascomycetes fungus isolated from green table olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35 degrees C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30 degrees C but not at 35 degrees C. Growth was better at 30 and 25 degrees C as judged by the larger extent of the plates covered by mycelium compared with that at 35 degrees C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35 degrees C, whereas at 35 degrees C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Tissue culture of the deep-sea eel<Emphasis Type="Italic"> Simenchelys parasiticus</Emphasis> collected at 1,162 m

We successfully cultivated fin cells of the deep-sea eel Simenchelys parasiticus (collected at 1,162 m) in L-15 medium supplemented with fetal bovine serum (FBS) and additional NaCl. We found that the pectoral fin cells proliferated in L-15 medium enriched with 4 g/l of NaCl salt (pH 7.3) containing 10% FBS at 10 degrees C and 15 degrees C. No cells were attached to the plastic culture plates when Dulbecco's modified Eagle's medium (pH 7.8) or 0-2 g/l of NaCl was added to the medium or when incubation was carried out at 4 degrees C. The majority of the explant outgrowth cells were detached when temperature increased to higher than 15 degrees C. The rate of proliferation of the fin cells was extremely slow and was dependent on the FBS concentration. Cell growth was enhanced by approximately 2.2-fold, and doubling time decreased from 170 h to 77 h when the FBS concentration was increased from 10% to 20% (v/v). Our established deep-sea eel cells were passaged 16 times over a 1-year period under atmospheric pressure conditions.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5.8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15 degrees C in (1) aerobic conditions; (2) 30% CO2 + air; (3) 30% CO2 + N2; and (4) 100% CO2. When samples were held at 1 degree C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6 degrees C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1 degree C rather than 37 degrees C. In CO2 atmospheres growth of L. monocytogenes was inhibited on meat held at 6 degrees C, especially under 100% CO2. By contrast, storage at 15 degrees C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Inhibition of growth and uptake of amino acids in the yeast, Candida parapsilosis, by ambruticin

The antifungal antibiotic, ambruticin, inhibits growth of Candida parapsilosis and reduces its ability to take up amino acids. Increasing growth temperature from 30 degrees C to 39 degrees C leads to a 100-fold decrease in the minimum growth inhibitory concentration. Ambruticin is 20 times more effective at pH 5 than at pH 8 and exponentially growing cultures are much less susceptible than stationary phase cells. The activity of ambruticin is also dependent on the presence of certain exogenous nutrients. When acetate or succinate (10 mM) are included in the incubation medium, ambruticin has little effect on amino acid uptake. Glucose, mannose and glycerol do not decrease the efficacy of ambruticin. Ambruticin probably inhibits growth by reducing the utilization of exogenous and intracellular carbohydrates. This leads to a fall in energy production within the cell which can be monitored as a reduction in the activity of energy-dependent transport systems.     

The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

In vitro studies on optimal requirements for the growth of Spironucleus vortens, an intestinal parasite of the freshwater angelfish

Spironucleus vortens were cultivated in either an artificial medium at different temperatures, or in medium at various pH conditions or supplemented with different bile concentrations at 25 degrees C. Temperature, pH and bile requirements for the optimal growth of the parasite were determined. Parasites multiplied quickly at 28 and 31 degrees C and reached maximum numbers on Day 4 of cultivation, whereafter they did not survive. At 25 degrees C, parasites survived longer than those at 28 and 31 degrees C with no difference in multiplication rate during the exponential phase. The longest survival period was seen at 22 degrees C, although the growth rate of the parasite was not as high as those at 25 degrees C. At a higher temperature of 37 degrees C, no parasites were observed alive after the second day of cultivation. Optimal pH range for the parasite's growth was 6.5 to 7.5, with the highest cell number at pH 7.5. Parasites survived longest (15 d) at pH 6.0, although the maximum number of cells was lower than those at the optimal pH. Parasites were dead within 24 h at pH levels above 8.5 or below 5.5. All cultures supplemented with either bovine or fish bile yielded numbers of parasites lower than cultures with no bile. In addition, parasite growth was significantly suppressed in medium supplemented with higher concentrations of bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25 degrees C and pH 6.5 to 7.5 without supplementation with bile.     

The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica

The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype O : 9, two of serotype O : 3, and one each of serotypes O : 8 and O : 5, 27. In a culture medium acidified with HCl to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20 degrees, 10 degrees, 7 degrees and 4 degrees C for 21 d was in the ranges 4.18-4.36, 4.26-4.50, 4.36-4.83 and 4.42-4.80, respectively. The minimum pH for growth was also determined in media that contained 17, 33 and 50 mmol/l acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/l citric acid adjusted to pH values between 4.1 and 4.9 at intervals of approximately 0.2 unit, and 22, 44, and 111 mmol/l lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic greater than lactic greater than citric and the minimum inhibitory concentrations were greater at higher temperatures.     

Staphylococci in Competition: III. Influence of pH and Salt on Staphylococcal Growth in Mixed Populations

Previous results showed definite repressive effects on the growth of staphylococci in mixed cultures due to the competitive growth of psychrophilic saprophytes. This study was continued, and the influence of other environmental factors, pH and salt, on the competition between staphylococci and saprophytes was investigated. Initial pH values varied from 5 to 9. At the extremes of the pH range, staphylococci failed to grow, while the saprophytes grew under all of the conditions tested. At pH 5, the growth curves for the saprophytes were markedly altered from those obtained at neutral pH. The lag phases were greatly lengthened at and below 20 C, but normal numbers of saprophytes were reached in the stationary phase. At pH 6 and 8, staphylococcal growth showed the same inhibition observed at pH 7, at and below 20 C; normal multiplication was observed above this temperature, but with accelerated death phases. Thus, pH did not primarily effect staphylococcal growth through its influence on saprophyte growth and competition, but rather directly affected the growth of Staphylococcus cultures. Salt concentrations from 3.5 to 9.5% were investigated for influence on staphylococcal growth in mixed populations. Above 3.5% salt, staphylococcal inhibition at and above 20 C was not as marked as in the controls, although normal numbers were never reached. The saprophytes were increasingly inhibited, and their lag phases materially lengthened as salt concentration was increased. Salt acted directly on the Staphylococcus population and also, by repressing saprophyte growth, decreased competition, which allowed the staphylococci to grow.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Directed evolution of yeast pyruvate decarboxylase 1 for attenuated regulation and increased stability

Five generations of directed evolution resulted in yeast pyruvate decarboxylase 1 (Pdc1) variants with improved activity for 1 mM pyruvate at pH 7.5 in the presence of phosphate. The best variant, named 5LS30, contained the following mutations: A143T, T156A, Q367H, N396I, and K478R. In comparison with native Pdc1, 5LS30 had the substrate concentration required for half-saturation reduced by almost 3-fold at pH 7.5 and the phosphate inhibition reduced by 4-fold at pH 6.0. The apparent cooperativity for pyruvate displayed by 5LS30 was also reduced since it appeared to be activated by pyruvate more easily than the native enzyme. The temperature at which half of the Pdc1 activity was irreversibly lost in 5 min increased from 52.6 degrees C, seen with the native form, to 61.8 degrees C for 5LS30. Curiously, the optimal temperature for Pdc1 activity was found to be dependent upon pyruvate concentration. In 1 mM pyruvate, native Pdc1 performed optimally at 30 degrees C and 5LS30 at 40 degrees C, whereas in 25 mM pyruvate native activity peaked at 45 degrees C and 5LS30 at 55 degrees C. Two screening processes were developed for directed evolution of Pdc1 expressed in Escherichia coli: colony screening and culture screening. The latter proved to be an ideal method for isolating PCR-generated variants of the pdc1 gene with the desired phenotype. In this process, cultures were diluted and partitioned within 96-well plates such that each culture aliquot contained an average of two unique genotypes. This allowed rapid preparation of libraries for analysis of activity in crude lysates and can be applied to other directed evolution projects.     

Growth of the salt-tolerant yeast Zygosaccharomyces rouxii in microtiter plates: effects of NaCl,pH and temperature on growth and fusel alcohol production from branched-chain amino acids

Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z. rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25 degrees C. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of alpha-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30 degrees C. The results obtained under various incubation conditions with (deep-well) microtiter plates were validated in Erlenmeyer shake-flask cultures.     

Differential scanning calorimetric studies of a Bacillus halodurans alpha-amylase

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.     

Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa

The interaction of glycerol concentration from 0 to 16% and cooling velocity from 1 to 100 degrees C/min on freeze-thaw survival of ram spermatozoa was studied using a diluent based on 15% skim milk (450 mOs/kg water). Optimal spermatozoa survival (percentage motility and rating) was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min. Similar results were obtained with 8% glycerol at freezing rates of 5 to 30 degrees C/min. Although the ram spermatozoa tolerated several cooling velocities at each glycerol concentration, increasing the concentration of glycerol resulted in a downshift in the range of optimal cooling velocities. Glycerol concentrations above 8% were toxic and contributed greatly to the progressive decrease in spermatozoa survival. Comparison of the 15% skim milk diluent (450 mOs/kg water) with a 19% skim milk diluent (600 mOs/kg water) showed that optimal cryosurvival was obtained with 4 to 6% glycerol and freezing rates of 10 to 100 degrees C/min with both diluents.     

The viability of bifidobacteria introduced into kimchi

The viability of bifidobacteria in mul-kimchi, a type of kimchi with added water, was investigated under various conditions. When a mul-kimchi preparation was inoculated with five strains of Bifidobacterium at a concentration of 10(7) cfu ml-1, Bif. longum JK-2 showed the highest viability, maintaining a population of 10(6) cfu ml-1 after 1 week at 4 degrees C. The influence of NaCl concentration and initial pH on viability was further investigated in mul-kimchi inoculated with Bif. longum JK-2; NaCl concentrations greater than 3% (w/w) reduced viability considerably. In kimchi started with an initial pH of 6.5, the cells showed the highest survival. When mul-kimchi containing 2% NaCl (w/w) was inoculated with 10(8) cfu ml-1 Bif. longum JK-2, there was a 10-fold reduction in viability during 10 d of incubation at 4 degrees C. These results demonstrate acceptable levels of the organism in the product, suggesting the possible use of selected strains of bifidobacteria in commercial kimchi production.     

Characterization of type-II thyroxine 5'-deiodinase activity in rat Harderian gland

Thyroxine 5'-deiodinase activity was studied in male rat Harderian gland homogenates. The reaction rate was proportional to the tissue content in the homogenate and dependent on pH, with an optimum pH of 7.0, and temperature, between 4-37 degrees C. 5'-deiodinase activity was increased by dithiothreitol (DTT) in a dose-dependent manner, and inhibited moderately by propylthiouracil (PTU) and strongly by iopanoic acid (IA). Thyroidectomy enhanced the enzymatic activity (30-fold above the control value) but this increase is totally prevented by the in vivo iopanoic acid treatment. Thyroxine 5'-deiodinase activity was also dependent on T4 concentration (Km = 3.3 nM; Vmax = 10 fmol 125I-released/mg protein/h) and exhibited a nyctohemeral rhythmicity with a maximal activity at 03.00 h (4-fold above basal values) and minimal activity between 12.00-21.00 h.     

Sensitivity of Encephalitozoon cuniculi to various temperatures, disinfectants and drugs.

Spores of Encephalitozoon cuniculi were exposed to various temperature or to disinfectants, and their infectivity was then tested on monolayer cultures of canine kidney cells. The maximum survival time for spores suspended in medium 199 was 1 day at -20 degrees C, 98 days at 4 degrees C, 6 days at 22 degrees C, and 2 days at 37 degrees C. Only 2.5% survived 30 min at 56 degrees C. Boiling for 5 min or autoclaving at 120 degrees C for 10 min killed all spores. Dry spores survived less than a week at 4 degrees C but at least 4 weeks at 22 degrees C. Exposure for 30 min to recommended working concentrations of 9 of the 11 disinfectants tested killed all spores. The growth-inhibition effect of 7 antibiotics and chemotherapeutics was studied on canine kidney cell culture inoculated with E. cuniculi. None could completely inhibit growth. The most effective was chloroquine phosphate which, at a concentration of 12.5 mg per 1000 ml culture medium and during a test period of 8 weeks, reduced the harvest of E. cuniculi to 31% of that from inoculated, untreated cultures.     

Growth and survival of Escherichia coli O157:H7 under acidic conditions.

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.