The protective effect of some food ingredients on Staphylococcus aureus MF31

The upper limiting temperature of growth of Staphylococcus aureus MF31 in heart infusion broth (HI) was about 44°C but addition of monosodium glutamate (MSG) and soy sauce permitted the organism to grow above this temperature. This effect is similar to that of NaCl. Tomato ketchup, Worcestershire and HP sauces added to HI did not allow growth at the non-permissive temperature of 46°C but death was delayed. Staphylococcus aureus died in unsupplemented chicken meat slurry at 46°C but grew at 48°C in slurry supplemented with 5.8% NaCl and survived incubation for 18 h at 50°C in slurry supplemented with 5.8% NaCl and 5% MSG. Cultures grown at 37°C had a D ₆₀ value of 2 min in 50 mmol/l Tris (pH 7.2) buffer. Cultures grown at 46°C in HI containing 5.8% NaCl had a D ₆₀ value of 8 min in Tris buffer. Addition of 5.8% NaCl plus 5% MSG to the buffer increased the D ₆₀ by a factor of about 7 for both cultures. In storage experiments at room temperature, the culture grown at 37°C and at 46°C plus 5.8% NaCl died at about the same rate in salami. In milk powder, however, the count of 37°C culture decreased from 10⁹/g to 10⁶/g in 5 weeks while the count of 46°C culture remained unchanged. In cottage cheese, freeze-dried rice and macaroni, the 37°C cultures also died more rapidly. It is suggested that cultures grown at 46°C plus 5.8% NaCl may be suitable for experiments with artificially contaminated foods.     

Survival in foods of Staphylococcus aureus grown under optimal and stressed conditions and the effect of some food preservatives

Staphylococcus aureus was grown in a rich peptone medium which became alkaline with continued incubation. Cells were grown at 37 degrees C and in the same medium containing 1 M NaCl at 46 degrees C, a temperature at which this organism can grow only when protected by NaCl. Cells of these cultures are hereafter called 37 degrees C-cells and 46 degrees C-cells, respectively. The 37 degrees C-cells harvested when the pH was 7.1 to 7.7 had decimal reduction times (D60-value) of 1.8 to 3.1 min in 50 mM pH 7.2 Tris buffer. The D60 value of 46 degrees C-cells tested in the same way, harvested from cultures at pH 6.6 to 7.6, ranged from 5.3 to a maximum of 12.8 min. In milk, green beans, peas, or beef slurry, the D60-value of 46 degrees C-cells was about four times higher than that of 37 degrees C-cells. Length of survival after freeze-drying in skim-milk powder exposed to air was longest for the cells with the highest D-value. In freeze-dried peas and media acidified with acetic and lactic acids, 46 degrees C-cells survived longer than 37 degrees C-cells. However, the sensitivity of the two kinds of cells to potassium sorbate, sodium benzoate, and sodium propionate was essentially the same, but the 46 degrees C-cells were more resistant to butylated hydroxyanisole and sodium nitrite.     

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.     

Effect of lipid composition on the stability of cellular membranes during freeze-thawing of Lactobacillus acidophilus grown at different temperatures

Lactobacillus acidophilus CRL 640 grown at the optimal temperature of 37 degrees C (M37) appeared more sensitive to freeze-thawing than when it was grown at 25 degrees C (M25). In the first case, 87% of the cells died, in contrast to 33% for cells grown at 25 degrees C. All the surviving M37 cells showed sensitivity to NaCl. However, among the surviving M25 cells, only 85% were sensitive to NaCl. The rest of the cells were considered uninjured. Freeze-thawing in cells grown at 25 degrees C showed a liberation of nucleic acids and proteins. However, the leakage was higher in M37 cells after freeze-thawing. The greater fraction of damaged cells were observed in M25 culture after freeze-thawing. A relative increase of 81% in cardiolipid (CL), with respect to total phospholipids and 72% triglycosyldiglyceride (TGDG) with respect to the total glycolipids was observed in M37. In addition, a decrease of palmitoyl (C16:0), oleoyl (C18:0) fatty acids at CL, phosphatidylglycerol (PG), and diglicosyldiglyceride (DGDG) fractions and the increase of C19 cyc and C18:0, 10-OH fatty acids in neutral lipid, and CL fractions was also apparent. In M25 cells, the concentration of DGDG and PG was higher than in M37 cells. The difference in cryotolerance between the frozen cultures emphasizes the importance of selecting appropriate conditions of growth of microorganisms for use as dietary adjuncts.     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.     

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.     

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.     

Temperature-sensitive regulation of epidermal morphogenesis and the expression of cornified envelope precursors by EGF and TGFα

Epidermis reconstructed on de-epidermized dermis was used to investigate the effects of growth factors and culture temperature on epidermal morphogenesis and the expression of cornified envelope precursors. Cultures grown at 33°C or 37°C in the absence or presence of transforming growth factor alpha (TGFα), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), or insulin-like growth factor (IGF) show a similar morphology to that of native epidermis. Loricrin and SPRR2 are expressed in the stratum granulosum and SPRR3 is absent. Cultures grown in epidermal growth factor (EGF)-supplemented medium at 37°C have a normal morphology, whereas cultures grown at 33°C have a disorganized basal layer, no stratum granulosum, and nuclei are present in the stratum corneum. Loricrin is absent, and SPRR2 and SPRR3 expression extend into the spinous layers. Irrespective of the culture condition used, involucrin is aberrantly expressed in all suprabasal layers. EGF stimulated keratinocyte proliferation and migration to a greater degree than TGFα. Epidermis reconstructed on fibroblast-populated collagen gels at 33°C led to the same disturbances in keratinocyte differentiation as seen in cultures grown on de-epidermized dermis at 33°C in the presence of EGF, whereas parallel cultures grown at 37°C have a similar morphology to that of native epidermis.     

Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.     

Heat injury and repair in Campylobacter jejuni

A procedure for detecting and quantitating heat injury in Campylobacter jejuni was developed. Washed cells of C. jejuni A7455 were heated in potassium phosphate buffer (0.1 M, pH 7.3) at 46 degrees C. Samples were plated on brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate and on a medium containing brilliant green, bile, Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate. Colonies were counted after 5 days of incubation at 37 degrees C in an atmosphere containing 5% O2, 10% CO2, and 85% N2. After 45 min at 46 degrees C, there was virtually no killing and ca. two log cycles of injury. Cells grown at 42 degrees C were more susceptible to injury than cells grown at 37 degrees C. The addition to brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate of three different antibiotic mixtures used in the isolation of C. jejuni from foods or clinical specimens did not prevent recovery of heat-injured C. jejuni. Cells lost 260 nm of absorbing materials during heat injury. The addition of 5% NaCl or 40% sucrose to the heating buffer prevented leakage but did not prevent injury. Of the additional salts, sugars, and amino acids tested for protection, only NH4Cl, KCl, and LiCl2 prevented injury. Heat-injured C. jejuni repaired (regained dye and bile tolerance) in brucella broth supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate within 4 h. Increasing the NaCl in this medium to 1.25% inhibited repair, and increasing it to 2% was lethal. Heat-injured C. jejuni will repair at 42 degrees C but not at 5 degrees C.     

Heat injury and repair in Campylobacter jejuni.

A procedure for detecting and quantitating heat injury in Campylobacter jejuni was developed. Washed cells of C. jejuni A7455 were heated in potassium phosphate buffer (0.1 M, pH 7.3) at 46 degrees C. Samples were plated on brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate and on a medium containing brilliant green, bile, Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate. Colonies were counted after 5 days of incubation at 37 degrees C in an atmosphere containing 5% O2, 10% CO2, and 85% N2. After 45 min at 46 degrees C, there was virtually no killing and ca. two log cycles of injury. Cells grown at 42 degrees C were more susceptible to injury than cells grown at 37 degrees C. The addition to brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate of three different antibiotic mixtures used in the isolation of C. jejuni from foods or clinical specimens did not prevent recovery of heat-injured C. jejuni. Cells lost 260 nm of absorbing materials during heat injury. The addition of 5% NaCl or 40% sucrose to the heating buffer prevented leakage but did not prevent injury. Of the additional salts, sugars, and amino acids tested for protection, only NH4Cl, KCl, and LiCl2 prevented injury. Heat-injured C. jejuni repaired (regained dye and bile tolerance) in brucella broth supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate within 4 h. Increasing the NaCl in this medium to 1.25% inhibited repair, and increasing it to 2% was lethal. Heat-injured C. jejuni will repair at 42 degrees C but not at 5 degrees C.     

Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus

In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared. Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids. This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio. In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined. From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified. The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization. The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Factors Affecting the Resistance of Staphylococcus aureus to Hydrogen Peroxide Treatments in Milk

Staphylococcus aureus 196E was treated with 0.05% hydrogen peroxide in milk under varying conditions to determine the effects of treatment conditions and characteristics of the culture on bactericidal effectiveness of hydrogen peroxide. Time intervals required for 90 to 99.99% destruction of S. aureus decreased significantly as treatment temperatures increased from 37.8 to 57.2 C. Plots of survivor curves showed extended lags in destruction at 37.8 C, slight lags followed by logarithmic rates of destruction at 48.9 C, and logarithmic rates at 54.4 and 57.2 C except for trials in which there was very rapid initial destruction followed by logarithmic rates. S. aureus 196E was significantly more resistant to heat treatments at 54.4 C without added hydrogen peroxide than to treatment with 0.05% hydrogen peroxide at this temperature. Cultures grown at 37 C for 16 hr in milk were more resistant to hydrogen peroxide than were cultures grown at 35 C. Storage of cultures for 96 hr in milk at 4 C caused a decrease in the resistance of the culture. Numbers of staphylococci being treated had little effect on rates of destruction.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

Influences of incubation temperature and various saccharides on the production of organic acids and gases by gut microbes of rainbow trout Oncorhynchus mykiss in a micro-scale batch culture

We studied the influence of incubation temperature and additional saccharides on the metabolism of hindgut microbes of the rainbow trout Oncorhynchus mykiss in a 50 microl-scale batch culture system. Intestinal contents of rainbow trout reared at 15 degrees C were incubated with glucose, lactosucrose, sodium alginate or colloidal chitin (each 10 g/l) at 15 degrees C or 25 degrees C for 12 h. Levels of organic acids at 0 h and 12 h of incubation were quantified with HPLC. We also monitored gas release from these cultures during incubation. The main product was iso-butyric acid, except for the cultures with colloidal chitin where no net production of organic acids was observed. We detected higher levels of iso-butyric acid in cultures with lactosucrose than in the other cultures. Net production of this acid was less in cultures with colloidal chitin than in blank cultures. The volume of released gas was larger when incubated at 25 degrees C than at 15 degrees C. Cultures with colloidal chitin released more gas than blank cultures when they were incubated at 15 degrees C. Cultures with sodium alginate released less gas than blank cultures irrespective of incubation temperature. These results indicate that the hindgut microbes of this carnivorous fish mainly produce branched-chain fatty acids, very likely by microbial digestion of nitrogenous materials rather than saccharides. However, additional saccharides affected production of branched-chain fatty acids. The influence of incubation temperature in the present study also suggested that the environmental temperature of host fish should affect microbial digestion in the fish gut.     

Influence of Water Activity on the Growth of Clostridium perfringens

Each of four strains of Clostridium perfringens was grown in modified fluid thioglycolate medium which was adjusted to yield selected water activity (a(w)) levels. The adjustments to secure the desired a(w) levels were made with NaCl, KCl, or glucose. At each a(w) level, further modification was effected to produce four pH values. Cultures were incubated at either 37 or 46 C. The solute used to achieve the reduced a(w) levels appeared to have a definite effect on the magnitude of growth achieved, the rate of growth, and the limiting a(w) at which growth would occur. Use of glucose as the controlling solute permitted growth at the lowest a(w) level tested, 0.960, and yielded the greatest magnitude of growth as measured by turbidity values, at all of the a(w) levels investigated. Cultures grown in the medium with added KCl generally demonstrated the longest lag times and the least amount of growth. Regardless of specific solute used, as the a(w) level was lowered and the pH value decreased within each a(w) level, the rate and amount of growth were lessened. It appeared, however, that low pH values had less effect on inhibiting growth at low a(w) levels than at higher a(w) levels. Those cultures incubated at 46 C generally exhibited shorter lag periods than those at 37 C, although the maximal growth attained was somewhat less than that achieved at 37 C. The response to all of the investigated conditions was similar for each of the four strains tested.     

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.     

The attachment to, and invasion of HeLa cells by Salmonella typhimurium: the contribution of mannose-sensitive and mannose-resistant haemagglutinating activities

The association of the haemagglutinating activities of Salmonella typhimurium cultures with bacterial adhesion to HeLa cells, and the internalization of the bacteria by HeLa cells, was studied. Adhesion was not inhibited by alpha-methyl-D-mannoside (i.e. adhesion was mannose-resistant), and only four of the six strains tested produced type 1 fimbriae and the associated mannose-sensitive haemagglutinin (MSHA). The other two strains belonged to the non-fimbriate FIRN biogroup. Cultures of all six strains contained a mannose-resistant haemagglutinating (MRHA) activity when grown at 37 degrees C, but cultures of only one fimbriate and one non-fimbriate strain did so when grown at 18 degrees C. From the comparison of cultures grown at 18 degrees C and 37 degrees C, and of mutant strains with the phenotypes MRHA-negative/MSHA-positive, or MRHA-positive/MSHA-negative, it was concluded that the MRHA activity was responsible for the attachment of salmonellae to HeLa cells. Only bacterial adhesion that was resistant to mannose resulted in the internalization of the bacteria by the HeLa cells.     

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.