Combined Effects of Water Activity, Solute, and Temperature on the Growth of Vibrio parahaemolyticus

Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (a(w)) of 0.992 (+/-0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower a(w), respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a(w) to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a(w) with glycerol was less inhibitory to growth than similar a(w) reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a(w) levels lower than 0.992. Minimal a(w) for growth at 15, 21, 29, and 36 +/- 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a(w) was generally most tolerable at 29 C, whereas higher minimal a(w) for growth was required at 15 C. Solutes added to TSBS to achieve reduction in a(w), minimal a(w) for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a(w) for growth of V. parahaemolyticus depends upon the solute used to control a(w).     

Growth of heat-injured Vibrio parahaemolyticus in media supplemented with various cations.

Mid- to late logarithmic growth phase cells of Vibrio parahaemolyticus grown in tryptic soy broth (TSB) containing 0.5, 3.0, and 7.5% NaCl were heated for 8 min at 45 degrees C in 0.1 M phosphate buffer (pH 7.2) containing 3% NaCl. Colony formation on thiosulfate-citrate-bile salts-sucrose agar (TCBS) containing 2% NaCl was greatest for unheated cells that had been grown in 7.5% NaCl-TSB; cells grown in 0.5% NaCl-TSB formed a greater number of colonies on 1.0% NaCl-TCBS. Thermal injury was evident in heated cells, regardless of the NaCl concentration in TSB growth medium. The effects of Mg2+, K+, and Li+ added as chlorides to 0.5% NaCl-TSB on the growth of nonheated and heated V. parahaemolyticus were studied. Lower levels of Mg2+ and slightly higher levels of K+ were required to replace Na+ in TSB inoculated with thermally injured cells that had been originally grown in 3.0 and 7.5% NaCl-TSB. LiCl had an inhibitory effect on both nonheated and heated cells when present in the recovery medium (0.5% NaCl-TSB) at concentrations as low as 0.5%. Increased numbers of colonies were formed by heated cells plated in MgCl2-supplemented TCBS, regardless of the NaCl concentration in the original growth medium. Potassium had little, if any, effect on colony formation by nonheated V. parahaemolyticus recovered on TCBS and may have had a detrimental effect on heat-injured cells.     

Sublethal heat stress of Vibrio parahaemolyticus.

When Vibrio parahaemolyticsu ATCC 17802 was heated at 41 degrees C for 30 min in 100 mM phosphate-3% NaCl buffer (pH 7.0), the plate counts obtained when using Trypticase soy agar containing 0.25% added NaCl (0.25 TSAS) were nearly 99.9% higher than plate counts using Trypticase soy agar containing 5.5% added NaCl (5.5 TSAS). A similar result was obtained when cells of V. parahaemolyticus were grown in a glucose salts medium (GSM) and heated at 45 degrees C. The injured cells recovered salt tolerance within 3 h when placed in either 2.5 TSBS or GSM at 30 degrees C. The addition of chloramphenicol, actinomycin D, or nalidixic acid to 2.5 TSBS during recovery of cells grown in 2.5 TSBS indicated that recovery was dependent upon protein, ribonucleic acid (RNA, and deoxyribonucleic acid (DNA) synthesis. Penicillin did not inhibit the recovery process. Heat-injured, GSM-grown cells required RNA synthesis but not DNA synthesis during recovery in GSM. Chemical analyses showed that total cellular RNA decreased and total cellular DNA remained constant during heat injury. The addition of [6-3H]uracil, L-[U-14C]leucine, and [methyl-3H]thymidine to the recovery media confirmed the results of the antibiotic experiments.     

Relationships of Temperature and Sodium Chloride Concentration to the Survival of Vibrio parahaemolyticus in Broth and Fish Homogenate

The interaction of temperature and NaCl concentration in affecting the survival of three strains of Vibrio parahaemolyticus was studied in Trypticase soy broth and fish homogenate. Cells of V. parahaemolyticus suspended in Trypticase soy broth without NaCl were quite unstable and readily killed. The presence of NaCl appeared to be protective to the cells at 48 +/- 1 C, with the optimal concentration strain-dependent for the 3 to 12% range tested. Temperatures of 5 +/- 1, -5 +/- 1, and -18 +/- 1 C reduced the number of viable organisms per milliliter regardless of the NaCl concentration. In the presence of NaCl, viable cells, in numbers ranging up to 580 per ml, were still detected at the end of 30 days of storage. Similar results were obtained for cells suspended in fish homogenate, except that fish homogenate itself was protective as compared with Trypticase soy broth. This protection was significantly lower than that provided by NaCl in any amount tested.     

Method for the detection of injured Vibrio parahaemolyticus in seafoods.

The sensitivity of Vibrio parahaemolyticus cells to refrigeration and frozen storage and the development of a method for detecting injured and uninjured V. parahaemolyticus cells were studied. Cell suspensions in different kinds of seafood homogenates were either regrigerated (4 degrees C) or frozen (-20 degrees C), stored, and examined for cell survival during storage. V. parahaemolyticus cells were sensitive to both storage temperatures. Many cells died, and many survivors were sublethally injured. In general, refrigeration storage appeared to be more injurious than frozen storage. The initial recovery of the sublethally injured cells was highest in a nutritionally rich, nonselective liquid medium such as Trypticase soy broth, whereas maximum cell multiplication was observed in Trypticase soy broth containing 3% NaCl. The sublethally injured V. parahaemolyticus cells demonstrated sensitivity to the selective enrichment medium, glucose salt teepol broth. From these findings, a new method (designated as the "repair-detection" method) was developed for the isolation and enumeration of V. parahaemolyticus. Comparative studies between the recommended and the repair-detection methods showed that injured V. parahaemolyticus cells were present in commercial seafoods and that the repair-detection method was definitely more effective for the detection of total numbers of V. parahaemolyticus cells.     

Superoxide dismutase activity during recovery of thermally stressed Staphylococcus aureus MF-31.

Superoxide dismutase (SOD) activity was determined during the growth cycle of unheated and heat-injured cells of Staphylococcus aureus MF-31. SOd activity levels dropped in unheated cells during the lag phase, increased during logarithmic phase, and became constant in the stationary phase. Cells which were sublethally heated (52 degrees c, 20 min) in 100 mM phosphate buffer and subsequently allowed to recover in tryptic soy broth demonstrated an 85% decrease in SOD activity upon inoculation into recovery medium. As the injured cells repaired the heat-induced lesions and entered logarithmic growth, SOD levels rapidly increased. Heat-injured cells allowed to recover in tryptic soy broth plus 10% NaCl showed similar decreases in SOD activity levels. However, no subsequent increase was observed when specific activity was calculated based on milligrams of protein.     

Revival and Subsequent Isolation of Heat-Injured Bacteria by a Membrane Filter Technique

Cultures containing mixed flora from raw milk were heated at 62.8 C for 15, 20, 25, and 30 min. Dilutions were filtered through membrane filters, and the filters were incubated on Trypticase soy broth (TSB) and on TSB plus NaCl (TSBS). The TSB count indicated the total population which survived heating and included injured and uninjured cells. The colonies on TSBS indicated the uninjured cells and were marked by perforating the membrane near the colony. This membrane was then transferred to fresh TSB and incubated further. The injured organisms recovered and formed colonies which could be distinguished from previous colonies of uninjured organisms. Transfer counts on TSB were not substantially different from the initial TSB counts at 15, 20, 25, and 30 min of heating.     

Procedure for Isolation and Enumeration of Vibrio parahaemolyticus,

An evaluation of criteria used in the identification of Vibrio parahaemolyticus showed that cultural responses varied with respect to growth in broth with 10% NaCl, type of hemolysis, reactions in triple sugar-iron-agar, and serological reactions. With few or no exceptions, cultures were positive for cytochrome oxidase, utilized glucose fermentatively, were sensitive to pteridine (0/129) and novobiocin, and failed to grow in Trypticase soy broth (TSB) without NaCl. A procedure employing a direct plating technique, with or without prior enrichment, was designed for the isolation and enumeration of V. parahaemolyticus. The plating medium consisted of 2.0% peptone, 0.2% yeast extract, 1.0% corn starch, 7% NaCl, and 1.5% agar, with the pH adjusted to 8.0. The enrichment broth was TSB with 7% NaCl. Dilutions of food homogenates were either spread directly on the plates or inoculated into enrichment broth. TSB enrichments were incubated at 42 C for 18 hr. A loopful of the TSB tubes then was streaked onto the direct plating medium. Incubation of plates was at 42 C for 24 to 48 hr. Smooth, white to creamy, circular, amylase-positive colonies were then picked as suspect V. parahaemolyticus. Confirmation of gram-negative, fermentative, oxidase-positive, pleomorphic rods sensitive to pteridine 0/129 was made by a fluorescent-antibody technique. With this procedure, a satisfactory quantitative recovery of known V. parahaemolyticus from inoculated seafoods was made possible. V. parahaemolyticus was nto isolated from other salted foods.     

Heat-induced injury inListeria monocytogenes

Summary Heating ofListeria monocytogenes (Scott A strain) in potassium phosphate buffer (0.1 M, pH 7.2) at 52°C for 1 h led to injury, with the heat-injured cells failing to produce colonies on agar medium containing 5% NaCl. The detection of injury was based on the use of differential media: plating on tryptose phosphate broth+2% agar and 1% sodium pyruvate (TPBA+P) and on tryptose phosphate broth+2% agar and 5% NaCl (TPBA+S). Only non-injuredListeria formed colonies on TPBA+S whereas both heat-injured and non-injured cells formed colonies on TPBA+P. The bacterial count on TPBA+P minus that on TPBA+S represents the extent of heat injury. A large number of selective agars were tested and compared to TPBA+P for their ability to support repair and colony formation of heat-injuredL. monocytogenes. Media containing 0.025% phenylethanol, 0.0012–0.0025% acriflavin, 0.1–0.2% potassium tellurite, 0.001% polymyxin B sulfate, 5% NaCl or a combination of these ingredients were detrimental to the recovery of heat-injuredL. monocytogenes. Media currently in use forL. monocytogenes are not satisfactory for the recovery of injured cells.     

The immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus at different salinity levels

Addition of NaCl at 2.5% to 3.5% to tryptic soy broth (TSB) significantly increased the growth of Vibrio parahaemolyticus. Taiwan abalone Haliotis diversicolor supertexta held in 30 per thousand seawater were injected with V. parahaemolyticus grown in TSB containing NaCl at 0.5, 1.5, 2.5, 3.5 and 4.5% at a dose of 1.6 x 10(5)colony-forming units (cfu) abalone(-1). After 48 h, the cumulative mortality was significantly higher for the abalone challenged with V. parahaemolyticus grown in 2.5% than those grown in 0.5 and 1.5% NaCl. In other experiments, abalones held in 30 per thousand seawater were injected with TSB-grown V. parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then transferred to 20, 25, 30 and 35 per thousand seawater. All abalones held in 20 per thousand were killed in 48 h. The mortality of V. parahaemolyticus-injected abalone held in 30 per thousand was significantly lower over 24-120 h. Abalone held in 30 per thousand seawater and then transferred to 20, 25, 30 and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahemolyticus after 24 and 72 h. The THC increased directly related with salinity levels. Phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahaemolyticus decreased significantly for the abalone in 20, 25 and 35 per thousand. It is concluded that the abalone transferred from 30 per thousand to 20, 25 and 35 per thousand had reduced immune ability and decreased resistance against V. parahaemolyticus infection.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR.

The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n = 124), while no PCR amplicons were found in other vibrios or related genera (n = 50). High levels (10(6) to 10(10) CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10(8) V. parahaemolyticus cells or 10(9) E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35 degrees C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.     

Effect of Salt Concentration in the Recovery Medium on Heat-injured Streptococcus faecalis

Properties relating to the recovery of three heat-injured strains of Streptococcus faecalis were studied. All strains were cultured in all purpose plus Tween broth (APT) at 30 C for 24 hr before being subjected to heat in fresh APT broth. APT recovery medium containing various added amounts of NaCl, KCl, MgCl(2), or KCl and MgCl(2) was used to assess the effect of salts on the recovery of thermally injured S. faecalis. It was evident that, upon exposure to heat, S. faecalis cells became sensitive to increased salt concentrations. Analyses to determine the ribonucleic acid (RNA) content of heated cells showed a reduction of cellular RNA, but the per cent reduction was not directly proportional to the per cent reduction of the viable cells.     

Factors affecting inactivation of Moraxella-Acinetobacter cells in an irradiation process.

The effect of various stages of the irradiation processing of beef on the injury and inactivation of radiation-resistant Moraxella-Acinetobactor cells was studied. Moraxella-Acinetobacter cells were more resistant to heat inactivation and injury when heated in meat with salts (0.75% NaCl and 0.375% sodium tripolyphosphate) then in meat without salts. These salts had no effect on radiation resistance. Both radiation- and heat-injured cells were unable to form colonies at 30 degrees C in plate count agar containing 0.8% NaCl. Neither unstressed nor heat-stressed cells were able to multiply in minced beef incubated at 30 degrees C for 12 h. Only after the beef was diluted 1:10 with peptone water were the heat-injured cells able to repair and eventually multiply. Heated cells were more sensitive to radiation inactivation and injury than unheated cells. After repair, the cells regained their resistance to both NaCl and irradiation. Freezing and storage at -40 degrees C for 14 days had only a slight effect on either unstressed or heat-stressed cells.     

Eight enrichment broths for the isolation of Campylobacter jejuni from inoculated suspensions and ground pork

Aims:  The efficiency of eight enrichment broths for the selective isolation of Campylobacter jejuni was compared to identify an optimal enrichment broth.
Methods and Results:  Brucella-FBP, Preston, Doyle and Roman, modified CCD (mCCD), Park and Sanders, Bolton, Hunt and Radle and Hunt broths were compared for their recovery of (i) Camp. jejuni in suspension, (ii) Camp. jejuni from inoculated ground pork, (iii) heat-injured Camp. jejuni (55°C for 20 min) in suspension and (iv) heat-injured Camp. jejuni from inoculated ground pork. Hunt broth and Bolton broth showed the highest and most rapid enrichment efficacy for the cell suspensions and ground pork, respectively. Preston, Park and Sanders and mCCD broths had relatively high enrichment efficiencies, while Brucella-FBP broth was significantly inferior to the other broths ( P  < 0·05).
Conclusions:  Cell recovery from the eight enrichment broths was dependent on the sample type and the state of the cells. The use of the appropriate broth is important for the rapid and efficacious enrichment of Camp. jejuni . In particular, heat-injured Camp. jejuni require a longer cultivation time and a suitable enrichment broth.
Significance and Impact of the Study:  The results from the present study provide information for selecting the most appropriate enrichment broth for Camp. jejuni and may contribute to improved detection methods for the organism.     

Relationships between heat resistance and phospholipid fatty acid composition of Vibrio parahaemolyticus.

Vibrio parahaemolyticus was grown in tryptic soy broth (TSB) containing NaCl levels of 0.5, 3.0, and 7.5% (wt/vol). Cultures incubated at 21, 29, and 37 C were harvested in late exponential phases and thermal death times at 47 C (D47 c; time at 47 C required to reduce the viable population by 90%) were determined in phosphate buffer containing 0.5, 3.0, and 7.5% NaCl. At a given NaCl concentration in the growth medium, D47 c values increased with elevated incubation temperatures and with elevated levels of NaCl in the heating menstrua. Differences in thermal resistance of cells cultured at a particular temperature were greater between those grown in TSB containing 0.5 and 3.0% NaCl than between those grown in TSB containing 3.0 and 7.5% NaCl. D47c values ranged from 0.8 min (grown at 21 C in TSB with 0.5% NaCl) to 6.5 min (grown at 37 C in TSB with 7.5%, heated in 7.5% NaCl buffer). Methyl esters of major phospholipid fatty acids extracted from cells were quantitated. The ratio of saturated to unsaturated fatty acids in cells grown at a given NaCl concentration increased with elevated incubation temperature. At a particular growth temperature, however, saturated to unsaturated fatty acids ratios were lowest for cells grown in TSB containing 3.0% NaCl.     

A rapid twofold dilution method for microbial enumeration and resuscitation of uninjured and sublethally injured bacteria

AIMS: A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the twofold dilution method (2FD), was developed and evaluated. METHODS AND RESULTS: Following twofold serial dilution of samples in a 96 well microtiter plate, double strength selective broth or nonselective broth was added to each well. For resuscitation of heat-injured (55 degrees C for 10 min) coliforms, the selective broth was added to the wells after 3 h preresuscitation time in buffered peptone water. The results of the 2FD were compared to plating methods for total and coliform plate counts from mixed cultures and beef carcass surface tissue samples. CONCLUSION: The 2FD method results were not significantly different for uninjured cells (P > 0.05) from those obtained using Petrifilm and standard plating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R(2)=0.98 for total counts and R(2)=0.96 for coliforms from mixed cultures; R(2)=0.98 for total cell counts and R(2)=0.94 for coliforms from faeces inoculated beef carcasses). SIGNIFICANCE AND IMPACT OF THE STUDY: The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to conventional plating methods (P < 0.05).     

Recovery of chill-stressed Vibrio parahaemolyticus from oysters with enrichment broths supplemented with magnesium and iron salts.

The effects of magnesium and iron salts on the recovery and growth of chill-stressed cells of Vibrio parahaemolyticus were studied. Supplementation of glucose salt Teepol (GST) broth with 20 to 100 mM of Mg2+ significantly (P less than or equal to 0.05) increased the number of cells recovered from oyster homogenate stored at 3 degrees C. Populations detected with supplemented GST were comparable to those obtained with Horie arabinose ethyl violet (HAE) broth, with or without Mg2+. Recovery of V. parahaemolyticus from homogenates stored at -18 degrees C was also improved when enrichment broths supplemented with Mg2+ were used. Ferric iron (added as FeCl3) at 240 microM in GST and 240 or 960 microM in HAE significantly enhanced the extent of recovery of chilled cells. Ferrous iron was generally less effective. Teepol did not influence the growth of nonchilled cells, but significantly reduced the viable population in suspensions of chilled cells when used at a level of 0.4% in GST. The relatively high pH (9.0) of HAE caused a significant reduction in the number of viable, chill-stressed cells of V. parahaemolyticus. The overall results indicated that HAE broth is superior to GST for recovering V. parahaemolyticus from refrigerated and frozen oyster homogenates.     

Enrichment of Vibrio parahaemolyticus in a Simple Medium

A medium which contained 3% NaCl and 0.2% Teepol in 1/15 M phosphate buffer was prepared and was evaluated to be a useful enrichment medium for the isolation of Vibrio parahaemolyticus in marine specimens. Glucose salt Teepol broth produced a poorer result than direct culture.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.