Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and 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.     

Minimum water activity requirements for the growth of Listeria monocytogenes

The ability of five strains of Listeria monocytogenes to initiate growth at five different temperatures in brain heart infusion (BHI) broth adjusted to various water activity ( a _w) values with either sodium chloride (NaCl), sucrose or glycerol was investigated. Glycerol was the least toxic of the three solutes studied, with three of five strains of L. monocytogenes capable of growing in BHI broth adjusted with glycerol to an a w value of 0.90 at 30 C, compared to a w minima of 0.93 and 0.92 in broth adjusted with sucrose and sodium chloride, respectively. The minimum a w value required for growth generally increased as the incubation temperature decreased. Listeria monocytogenes appeared to tolerate glycerol and NaCl best when growing at 30 and 15°C, respectively, while for sucrose, temperature did not appear to influence growth of the organism. Listeria monocytogenes is one of the few food-borne pathogens that can grow at an a w value below 0.93.     

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.     

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.     

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.     

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.     

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.     

Characterization of putrescine production in nongrowing Vibrio parahaemolyticus cells in response to external osmolality

Nongrowing Vibrio parahaemolyticus cells rapidly produced putrescine (Put) from added arginine when subjected to a low osmotic stress. This phenomenon was characterized in connection with a regulatory mechanism of the responsible enzymes, arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH). NaCl, KCl, LiCl, sucrose, and glycerol were used as solutes to prepare the resuspending media with various osmolalities. Regardless of whether the solutes were electrolytes or non-electrolytes, exposure of cells to low osmolality brought about instantaneous increases in both intra- and extracellular Put contents without significant changes in the contents of other polyamines. This acceleration in Put production was accompanied by no increases in the specific activities of ADC and AUH. On the other hand, when cells were exposed to the osmolality equivalent to 2 or 5% NaCl, all solutes except for glycerol did not cause a remarkable variation in the intracellular Put content, while the amount of Put in the medium varied depending on the solute used; sucrose and glycerol still greatly prompted Put production, as judged by high Put contents in the media, even at the osmolality equivalent to 5% NaCl. The cation efflux from cells, measured as the K+ release, was observed whenever the increase in Put production occurred. Furthermore, in vitro experiments showed that NaCl and KCl inhibited ADC to a similar extent, about 70% inhibition being observed at 200 mM. However, AUH was not affected by these compounds. These results suggest that the reduction in the concentrations of Na+ and K+ predominantly present in cells may cause the increase in activity of the preexisting ADC, which leads to the enhancement of Put production.     

不同渗透压调节剂对Candida krusei生理代谢的影响

比较了氯化钠、氯化钾、甘露醇存在的高渗环境下克鲁氏假丝酵母(Candida kru-sei)的生理代谢。3种渗透压调节剂对C.krusei生理代谢影响有显著差异。与甘露醇相比,氯化钠和氯化钾对细胞生长的影响更为显著,而氯化钾对细胞的毒性则又小于氯化钠。细胞对糖的消耗速率依次为甘露醇>氯化钾>氯化钠。甘油和海藻糖是C.krusei在高渗环境下的主要相容性溶质。氯化钠和氯化钾对甘油合成的促进作用明显高于甘露醇。在0.6mol/L氯化钠、氯化钾、甘露醇存在时,细胞甘油浓度较对照提高了74%、63%、57%;胞内甘油最大含量也分别达到对照的3.1,2.4和1.8倍。高渗环境下胞内海藻糖含量在发酵前期均有所降低,但发酵后期在0.6mol/L氯化钾和甘露醇存在时海藻糖迅速积累,其含量分别达对照的1.6和1.4倍。     

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.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

Radiation resistance and injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Phospholipid vesicle fusion and drug loading: temperature,solute and cholesterol effects,and, a rapid preparation for solute-loaded vesicles

The fusion of sonicated dipalmitoylphosphatidylcholine (DPPC) vesicles was studied by gel-exclusion chromatography as a function of temperature, permeable and impermeable solute concentration, and cholesterol content of the bilayer membrane. Fusion is faster at lower temperatures: there is no fusion at or above 35.5°C (0.10 M DPPC/0.1 M K₂SO₄/0.01 M Hepes buffer (pH 7.4)/0.02% NaN₃). There is about 10% fusion after 1 week at 30°C, and about 60% fusion after 2 days at 13–25°C. Between 13 and 8°C, the fusion product changes from 700-Å-diameter vesicles to the 950-Å vesicles previously reported by Wong et al. (Wong, M., Anthony, F.W., Tillack, T.W. and Thompson, T.E. (1982) Biochemistry 21, 4126–4132). At 1°C, fusion is about 90% complete after 1 day. Membrane-impermeable solutes (NaCl, trehalose and glucose) inhibit fusion in a manner reflecting the total particle concentration. There is no detectable fusion after 3 days (22°C) in either 1.0 M NaCl or 2.0 M sugar, the highest concentrations studied. A suggested explanation is that impermeable solutes osmotically inhibit the influx of solution that accompanies the fusion of vesicles to form a larger vesicle, and, could conceivably thereby inhibit the fusion reaction. By contrast, membrane-permeable solutes (glycerol, ethylene glycol, propylene glycol and ethanol) dramatically increase the fusion rate. 1.0 M ethanol causes 100% fusion in 15–30 min at 22°C. The simultaneous presence of 0.15 M NaCl entirely negates the fusion-promoting effect of 1.0 M ethanol. 1 mol% cholesterol completely inhibits fusion in 0.1 M KCl (20°C), and greatly slows it down either in 1.0 M ethanol at 20°C or in 0.1 M KCl at 4°C. A suggested mechanism is that cholesterol might concentrate in and stabilize bilayer lattice defect sites that are critical for the fusion reaction. The trapping efficiency of vesicles formed by the fast ethanol fusion conditions in the presence of the water-soluble markers, chromate and arsenazo III, ranged from 9.0 to 12.7% of the marker captured in the vesicles, corresponding to trapped volumes of 1.8 to 2.5 1/mol DPPC. Bromophenol blue gave anomalously high values of 67% and 13 1/mol DPPC, which presumably reflect binding, in addition to encapsulation.     

Production of extracellular polyols in Aureobasidium pullulans

Aureobasidium pullulans produced extracellularly considerable amounts of polyols in the media with sucrose, glucose, fructose and mannose as sole carbon source during the late exponential and stationary phase of growth. The maximum yield of polyol was about 23% in the 20%(w/v) sucrose medium, of which mannitol was the main polyol associated with minute quantities of glycerol. Stress solutes such as NaCl and KCl did not promote polyol production.     

Osmoregulatory responses of bacteria isolated from fresh or composted,olive-mill waste-waters

Halotolerant bacteria isolated from raw, olive-mill waste-waters (alpechin) and from composted alpechin could grow on solid medium containing up to 10% (w/v) NaCl. Most (70%) of these halotolerant isolates could also grow in liquid minimal medium with the same NaCl concentration and three isolates from this group were chosen for further study. When grown in tryptic soy broth (TSB), two isolates responded to lowering of water activity (a _w) by addition of NaCl or sucrose in the expected manner: by increasing the proportion of membrane anionic lipids diphosphatidylglycerol or phosphatidylglycerol. Some solute-specific differences were observed. In contrast, the third isolate did not alter its membrane phospholipid composition significantly in response to growth in NaCl, whereas in sucrose there was an increase in phosphatidylethanolamine. This response is contrary to the accepted interpretation of the function of such a _w-dependent changes, as being a mechanism for preserving the membrane lipid-bilayer phase. When all three isolates were grown in the presence of alpechin, there was a decrease in the proportion of phosphatidylglycerol and a rise in the level of phosphatidylethanolamine. Quantitative and qualitative differences in compatible solute composition were observed when the three isolates were grown in TSB with NaCl or sucrose added to lower a _w. The major compatible solutes in two of the isolates were proline and betaine, whereas in the third they were proline, betaine and ectoine; one isolate also contained some trehalose when NaCl but not sucrose was the osmolyte.     

Relationship Between Solute Permeability and Osmotic Remediability in a Galactose-Negative Strain of Saccharomyces cerevisiae

An osmotic remedial allele, gal 7-1, in the galactose pathway of Saccharomyces cerevisiae responds to either penetrating (ethylene glycol and diethylene glycol) or nonpenetrating (KCl, NaCl, and sorbitol) solutes in the growth medium. Extracts from cells grown under restrictive conditions gave no increase in enzyme activity (gal-1-phosphate, uridylyl transferase) when exposed to the penetrating solutes; thus protein synthesis or possibly polymer assembly is proposed as the critical step remedied by the addition of the solutes.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.