Survival or Growth of Escherichia coli O157:H7 in a Model System of Fresh Meat Decontamination Runoff Waste Fluids and Its Resistance to Subsequent Lactic Acid Stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 ± 0.1) or in water (pH 7.2 ± 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25°C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4°C and lowest at 25°C. The pathogen survived without growth in water washings at 4 and 10°C, while it grew by 0.8 to 2.7 log cycles at 15 and 25°C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10°C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25°C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15°C > 10°C > 4°C, while at 25°C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15°C may maintain a higher acid resistance than when acid habituated at 4°C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Population Dynamics of Soil Pseudomonads in the Rhizosphere of Potato (Solanum tuberosum L.)

Rhizosphere population dynamics of seven Pseudomonas fluorescens and Pseudomonas putida strains isolated from rhizospheres of various agricultural plants were studied on potato (Solanum tuberosum L.) in field soil under controlled environmental conditions. Rhizosphere populations of two strains (B10 and B4) were quantitatively related to initial seed piece inoculum levels when plants were grown at −0.3 bar matric potential. At a given inoculum level, rhizosphere populations of strain B4 were consistently greater than those of strain B10. In vivo growth curves on 4-cm root tip-proximal segments indicated that both strains grew at similar rates in the potato rhizosphere, but large populations of strain B10 were not maintained at 24°C after 7 h, whereas those of strain B4 were maintained for at least 40 h. Although both strains grew more rapidly in the rhizosphere at 24°C than at 12°C, their rhizosphere populations after seed piece inoculation were generally greater at 12 or 18°C, indicating that in vivo growth did not solely determine rhizosphere populations in these studies. In vitro osmotolerance of seven Pseudomonas strains (including strains B4 and B10) was correlated with their abilities to establish stable populations in the rhizosphere of potato. Stability of rhizosphere populations of the Pseudomonas strains studied here was maximized at low (i.e., 12°C) soil temperatures. These results indicate that Pseudomonas strains differ in their capacity to maintain stable rhizosphere populations in association with potato. This capacity, distinct from the ability to grow in the rhizosphere, may limit the establishment of rhizosphere populations under some environmental conditions.     

Behavior of Bacillus anthracis Strains Sterne and Ames K0610 in Sterile Raw Ground Beef

The behavior of Bacillus anthracis Sterne spores in sterile raw ground beef was measured at storage temperatures of 2 to 70°C, encompassing both bacterial growth and death. B. anthracis Sterne was weakly inactivated (−0.003 to −0.014 log₁₀ CFU/h) at storage temperatures of 2 to 16°C and at temperatures greater than and equal to 45°C. Growth was observed from 17 to 44°C. At these intermediate temperatures, B. anthracis Sterne displayed growth patterns with lag, growth, and stationary phases. The lag phase duration decreased with increasing temperature and ranged from approximately 3 to 53 h. The growth rate increased with increasing temperature from 0.011 to 0.496 log₁₀ CFU/h. Maximum population densities (MPDs) ranged from 5.9 to 7.9 log₁₀ CFU/g. In addition, the fate of B. anthracis Ames K0610 was measured at 10, 15, 25, 30, 35, 40, and 70°C to compare its behavior with that of Sterne. There were no significant differences between the Ames and Sterne strains for both growth rate and lag time. However, the Ames strain displayed an MPD that was 1.0 to 1.6 times higher than that of the Sterne strain at 30, 35, and 40°C. Ames K0610 spores were rapidly inactivated at temperatures greater than or equal to 45°C. The inability of B. anthracis to grow between 2 and 16°C, a relatively low growth rate, and inactivation at elevated temperatures would likely reduce the risk for recommended ground-beef handling and preparation procedures.     

Modelling the Growth Limits (Growth/No Growth Interface) of Escherichia coli as a Function of Temperature, pH, Lactic Acid Concentration, and Water Activity

The form of a previously developed Bělehrádek type of growth rate model was used to develop a probability model for defining the growth/no growth interface as a function of temperature (10 to 37°C), pH (pH 2.8 to 6.9), lactic acid concentration (0 to 500 mM), and water activity (0.955 to 0.999; NaCl was used as the humectant). Escherichia coli was unable to grow in broth in which the undissociated lactic acid concentration exceeded 11 mM or, with two exceptions, at a pH of 3.9 or less with no lactic acid present. Under experimental conditions at which the pH and the undissociated acid concentrations were the major growth-limiting factors, the growth/no growth interface was essentially independent of temperature at temperatures ranging from 15 to 37°C. The interface between conditions that allowed growth and conditions at which growth did not occur was abrupt. The inhibitory effect of combinations of water activity and pH varied with temperature. Predictions of the model for the growth/no growth interface were consistent with 95% of the experimental data set.     

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°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°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°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°C, whereas at 35°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.     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

Properties of Thermus ruber Strains Isolated from Icelandic Hot Springs and DNA:DNA Homology of Thermus ruber and Thermus aquaticus

Seventeen pink-pigmented strains of the genus Thermus were isolated from samples collected from thermal areas of Iceland. The strains were examined by using phenotypic characterization and DNA:DNA homology and were compared with recognized strains. Visually, the strains could be divided into three groups based on their pigmentation; however, spectroscopic studies of the pigments indicated little difference among them. Most strains required a vitamin supplement for growth and used fructose, maltose, mannose, or sucrose as the sole carbon source. In the presence of nitrate, two strains were able to grow under anaerobic conditions. The optimum growth temperature was 60°C; growth did not occur at 30 or 70°C.     

Effects of Incubation Temperature on Growth and Production of Exopolysaccharides by an Antarctic Sea Ice Bacterium Grown in Batch Culture

The sea ice microbial community plays a key role in the productivity of the Southern Ocean. Exopolysaccharide (EPS) is a major component of the exopolymer secreted by many marine bacteria to enhance survival and is abundant in sea ice brine channels, but little is known about its function there. This study investigated the effects of temperature on EPS production in batch culture by CAM025, a marine bacterium isolated from sea ice sampled from the Southern Ocean. Previous studies have shown that CAM025 is a member of the genus Pseudoalteromonas and therefore belongs to a group found to be abundant in sea ice by culture-dependent and -independent techniques. Batch cultures were grown at −2°C, 10°C, and 20°C, and cell number, optical density, pH, glucose concentration, and viscosity were monitored. The yield of EPS at −2°C and 10°C was 30 times higher than at 20°C, which is the optimum growth temperature for many psychrotolerant strains. EPS may have a cryoprotective role in brine channels of sea ice, where extremes of high salinity and low temperature impose pressures on microbial growth and survival. The EPS produced at −2°C and 10°C had a higher uronic acid content than that produced at 20°C. The availability of iron as a trace metal is of critical importance in the Southern Ocean, where it is known to limit primary production. EPS from strain CAM025 is polyanionic and may bind dissolved cations such at trace metals, and therefore the presence of bacterial EPS in the Antarctic marine environment may have important ecological implications.     

Determinants in Microbial Colonization of the Murine Gastrointestinal Tract: pH, Temperature, and Energy-Yielding Metabolism of Torulopsis pintolopesii

Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37°C and did not grow at 23 or 43°C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O₂ and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O₂. Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37°C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.     

Effects of Temperature, pH, and NaCl on Growth and Pectinolytic Activity of Pseudomonas marginalis

The interaction of temperature (4, 10, 18, and 30°C), pH (6, 7, and 8), and NaCl (0, 2.5, and 5%) and their effects on specific growth rate, lag phase, and pectinolytic enzymes of Pseudomonas marginalis were evaluated. Response surface methodology was adapted to describe the response of growth parameters to environmental changes. To obtain good conditions of storage, the combined action of salt and temperature is necessary. At 4°C with an NaCl concentration of 5% and a pH of 7, the lag time was 8 days and no growth was observed at 4°C with 5% NaCl and a pH of 6. In the absence of salt, P. marginalis could grow regardless of temperature and pH. Pectate lyase and pectin lyase were produced by P. marginalis, while pectin methyl esterase activity was not observed in our culture conditions. The enzyme production depended on temperature, pH, and salt concentration but also on the age of the culture. Pectinolytic enzymes were abundantly excreted during the stationary phase, and even at 4°C, after 2 weeks of storage, enzyme activities in supernatant culture were sufficient to damage vegetables. Both bacterial growth and enzymatic production have to be taken into account in order to estimate correctly the shelf life of vegetables.     

Growth of Candida ingens on Supernatant from Anaerobically Fermented Pig Waste: Effects of Temperature and pH

Candida ingens, a pellicle-forming yeast utilizing volatile fatty acids, grew over a pH range of 4.1 to 6.0 on nonsterile supernatants from anaerobically fermented pig wastes; growth was inconsistent between pH 4.1 and 4.6. When ambient temperature above the pellicle was 21°C and the temperature of the medium was 29 to 32°C, a pH range of 4.8 to 5.0 gave yields of 1.90 to 3.31 g of dry matter per liter, and 0.059 to 0.065 mol of volatile fatty acids was utilized per liter. There was no advantage in utilization of volatile fatty acids and yield of dry matter in keeping the pH constant during a 24-h growth period. C. ingens grew at pH 4.8 and 5.0 when both ambient and medium temperatures were 30°C. When ambient temperature was 10°C, maximum yield and utilization of volatile fatty acids occurred at a medium temperature of 28 to 30°C.     

Influence of pH and Temperature on Enumeration of Cellulose- and Hemicellulose-Degrading Thermophilic Anaerobes in Neutral and Alkaline Icelandic Hot Springs

Cellulose- and hemicellulose-degrading thermophilic anaerobes were enumerated in biomat samples of various temperatures from two different hot springs in the Hveragerǒi area of Iceland: one spring had a pH near 7, the second had a pH near 9. The most-probable-number technique was used for enumeration of bacteria in the samples, with media at many different temperatures (37 to 90°C) and two pH values (7 and 9). There were generally more xylan-degrading then cellulose-utilizing organisms in both environments. There was no growth at 80°C in the neutral spring or at 37°C in the alkaline spring. However, there were large numbers of both types of organisms in the alkaline spring at 80°C and in the neutral spring at 37°C. No cultures grew from the most-probable-number tubes inoculated with the Hveragerǒi samples and incubated at 90°C or with media at pH 9. However, xylan-degrading cultures at 70°C were enriched at pH 9 with samples from some other Icelandic hot springs.     

Iron Superoxide Dismutase Protects against Chilling Damage in the Cyanobacterium Synechococcus species PCC7942

A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB⁻, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB⁻ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB⁻ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB⁻ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB⁻ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Useful Host-Vector Systems in Bacillus stearothermophilus

We isolated a highly transformable thermophile, Bacillus stearothermophilus SIC1, which exhibited the following features. The growth temperature ranged from 45 to 65°C in L broth. The maximum cell concentration in 2L broth (2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.2) was determined as an optical density at 660 nm of 7.8, and the generation time was 11 min at 60°C. Strain SIC1 was a prototroph and was transformed by the protoplast procedure not only with repB plasmids (high-copy-number plasmids such as pTB913 and pUB110) but also with repA plasmids (low-copy-number plasmids such as pTB53). Transformation efficiencies with repB and repA plasmids were about 2 × 10⁶ to 5 × 10⁶ and 5 × 10⁴ transformants per μg of DNA, respectively. The transformant carrying plasmid pTB913Y/K could grow at 63°C in the presence of kanamycin. The regeneration frequency of protoplasts was 60%, and only 1 day was needed for regeneration at 55°C.     

Growth of Rhizobium japonicum Strains at Temperatures Above 27°C

A study was conducted to examine the growth responses of different Rhizobium japonicum strains to increasing temperatures, determine the degree of variability among strains in those responses, and identify temperature-related growth characteristics that could be used to select temperature-tolerant strains. Each of 42 strains was grown in liquid culture for 96 h at 19 incubation temperatures ranging from 27.4 to 54.1°C in a temperature gradient apparatus. Growth was estimated by measuring the change in optical density over time. Strains differed in their responses to increasing temperatures. Three characteristic temperatures were determined for each strain: the temperature giving the maximum optical density at 96 h (optimum temperature), the maximum temperature allowing a continuous increase in optical density during the 96-h period (maximum permissive temperature), and the maximum temperature allowing growth of the cultures after they were transferred to a uniform incubation temperature of 28°C (maximum survival temperature). The three characteristic temperatures varied among strains and had the following ranges: optimum temperature, from 27.4 to 35.2°C; maximum permissive temperature, from 29.8 to 38.0°C; and maximum survival temperature, from 33.7 to 48.7°C. Significant positive correlations were found between maximum permissive temperature and optimum temperature and between maximum permissive temperature and maximum survival temperature. Eight strains which had the highest maximum permissive temperature, optimum temperature, and maximum survival temperature were considered tolerant of high temperatures and were able to grow at temperatures higher than those previously reported for the most tolerant R. japonicum strains. The strains were of diverse geographical origin, but the response to high temperatures was not related to their origin. Evaluation of the temperature responses in pure culture may be useful in the search for R. japonicum strains better suited to environments in which high soil temperature is a limiting factor.     

Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.     

Characterization of a Protease from a Psychrotroph, Pseudomonas fluorescens 114

A psychrotrophic bacterium isolated from river sediment was identified as Pseudomonas fluorescens 114. It grew at 0°C and optimally at 20°C. The bacterium produced a protease with a molecular weight of 47,000, which was stable in the pH range of 5 to 9 and worked optimally between pH 6.5 and 10. Activity was optimal at 35°C and was lost immediately at 50°C and after 5 min at 45°C. At 0, 10, and 20°C, 24, 38, and 57% of optimal activity were observed, respectively.     

Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.     

Optimization of nutritional and environmental conditions for pyocyanin production by urine isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacteria involved in numerous diseases among which, are urinary tract infections (UTIs). The pyocyanin secreted as a virulence factor by this bacterium has many beneficial applications but its high cost remains an obstacle for its widespread use. In this study, a total of fifty urine isolates were identified as P. aeruginosa. All strains produced pyocyanin pigment with a range of 1.3–31 µg/ml. The highest producer clinical strain P21 and the standard strain PA14 were used in optimization of pyocyanin production. Among tested media, king’s A fluid medium resulted in the highest yield of pyocyanin pigment followed by nutrient broth. Growth at 37 °C was superior in pyocyanin production than growth at 30 °C. Both shaking and longer incubation periods (3–4 days) improved pyocyanin production. The pyocyanin yield was indifferent upon growth of P21 at both pH 7 and pH 8. In conclusion, the optimum conditions for pyocyanin production are to use King’s A fluid medium of pH 7 and incubate the inoculated medium at 37 °C with shaking at 200 rpm for a period of three to four days.