Characterization of the Repair of Injury Induced by Freezing Salmonella anatum

Fast freezing and slow thawing of Salmonella anatum cells suspended in water resulted in injury of more than 90% of the cells that survived the treatment. The injured cells failed to form colonies on the selective medium (xyloselysine-peptone-agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective (xylose-lysine-peptone-agar) plating medium. In Tryptic soy plus 0.3% yeast extract broth or minimal broth, most of the injured cells repaired within 1 to 2 hr at 25 C. Tryptic soy plus yeast extract broth supported repair to a greater extent than minimal broth. Phosphate or citrate at concentrations found in minimal broth supported repair of some cells. MgSO(4), when present with inorganic phosphate or citrate or both, increased the extent of repair. The repair process in the presence of phosphate was not prevented by actinomycin D, chloramphenicol, and D-cycloserine, but was prevented by cyanide and 2,4-dinitrophenol (only at pH 6). This suggested that the repair process might involve energy metabolism in the form of adenosine triphosphate. The freeze-injured cells were highly sensitive to lysozyme, whereas unfrozen fresh cells were not. In the presence of phosphate or minimal broth this sensitivity was greatly reduced. This suggested that, at least in some of the cells, the injury involved the lipopolysaccharide of the cell wall and adenosine triphosphate synthesis was required for repair.     

Enhancement of antibiotic activity against Staphylococcus aureus by exposure to hyperbaric oxygen

Growth of Staphylococcus aureus ATCC 6538P was studied in stationary broth cultures (11 mm deep) exposed to hyperbaric oxygen (100% O₂ at 3 atm absolute). The minimal inhibitory concentration (MIC) of the following antibiotics was determined after exposure to high-pressure oxygen (HPO) for 3, 6, and 12 hr: penicillin, streptomycin, tetracycline, oxytetracycline, kanamycin, and cephalothin. Logarithmic growth during exposure to HPO was retarded 60%. Air at 3 atm absolute did not retard growth. The longer the exposure of tube dilution tests to HPO, the lower the MIC. Regardless of the antibiotic used, MIC values relative to 100% for unexposed controls were similar for given exposures, and averaged 73% after 3 hr of exposure to HPO, 53% after 6 hr, and 34% after 12 hr. Similar enhancement with HPO and an iodophor suggests occurrence of a general phenomenon with antibacterial agents. Although HPO alone is primarily bacteriostatic, combined therapy with antibiotics and HPO may be useful against bacterial infections because the therapeutic effectiveness of a maximal dosage of antibiotic could be increased.     

Proceedings: Repair of injury in Salmonella anatum cells after freezing and thawing in milk

Fast freezing and slow thawing of Salmonella anatum cells in nonfat milk solids resulted in about 20% death and 50% injury of the cells surviving the treatment. Death was defined as the inability to form colonies on a nonselective plating medium [xylose-lysine-peptone agar (XLP)] after freezing and thawing. Injury was defined as the inability to form colonies on a selective plating medium (XLP with 0.2% sodium desoxycholate added). The injured cells repaired rapidly and within 2 hr at 25 °C, in the presence of 0.1% milk solids; all the injured cells regained the ability to form colonies on the selective medium. The treated cells showed a 1-hr extended lag phase of growth as compared to the unfrozen cells. Milk solids concentration in the freezing and repair menstrua influenced injury, repair of injury, and death. The repair process was affected by the pH and temperature of environment in which the injured cells were incubated. Maximum repair occurred at pH values between 6.0 and 7.4 and temperatures from 25 to 42 °C. The data suggested repair did not require the synthesis of protein, ribonucleic acid, or cell-wall mucopeptide but did require energy synthesis.     

Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp

Endoxylanase, for which the optimum temperature is 60 degrees C (optimum pH 7), is labile to heat. Because the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7. Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve. Among the various ions tested, orthophosphate anion (HPO(4)(2-)) was found to significantly improve not only the stability but the activity of xylanase. When K(2)HPO(4) concentration was increased from 50 mM to 1.2 M, the T(m )value of xylanase was increased from 60.0 degrees C to 74.5 degrees C. The affinity of xylanase on xylan also increased along with K(2)HPO(4) concentration. Thus, the xylanase activity at 0.6 M K(2)HPO(4) was 2.3-fold higher than that at 50 mM K(2)HPO(4), and 120.2-fold higher than that in 40 mM MOPS buffer. This enhanced activity in the presence of K(2)HPO(4 )probably takes place because the orthophosphate anion affects the binding and catalytic residues of endoxylanase.     

Activation of Na(+), K(+), Cl(-)-cotransport mediates intracellular Ca(2+) increase and apoptosis induced by Pinacidil in HepG2 human hepatoblastoma cells

The role of Na(+), K(+), Cl(-)-cotransport (NKCC) in apoptosis of HepG2 human hepatoblastoma cells was investigated. Pinacidil (Pin), an activator of ATP-sensitive K(+) (K(ATP)) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 cells. Pin increased intracellular K(+) concentration ([K(+)](i)). Bumetanide and furosemide, NKCC inhibitors, significantly inhibited the Pin-induced increased [K(+)](i) and apoptosis, whereas K(ATP) inhibitors (glibenclamide and tolbutamide) had no effects. The Pin-induced [K(+)](i) increase was significantly prevented by reducing extracellular Cl(-) concentration, and Pin also increased intracellular Na(+) concentration ([Na(+)](i)), further indicating that these effects of Pin may be due to NKCC activation. In addition, Pin induced a rapid and sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which was completely prevented by the NKCC inhibitors. Treatment with EGTA or BAPTA/AM markedly inhibited the Pin-induced apoptosis. Inhibitors of Na(+), Ca(2+)-exchanger, bepridil, and benzamil significantly prevented both [Ca(2+)](i) increase and apoptosis induced by Pin. Taken together, these results suggest that Pin increases [Na(+)](i) through NKCC activation, which leads to stimulation of reverse-mode of Na(+), Ca(2+) exchanger, resulting in [Ca(2+)](i) increase, and in turn, apoptosis. These results further suggest that NKCC may be a good target for induction of apoptosis in human hepatoma cells.     

L-asparaginase release from Escherichia coli cells with K2HPO4 and Triton X100

A method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied. It was found that a combination of K2HPO4 and Triton X100 was effective. The influences of K2HPO4 concentration, Triton concentration, E. coli cell concentration and pH on the release of enzyme and proteins were investigated in detail. Experimental results showed that 12.5% (w/v) K2HPO4, 2% (w/v) Triton X100 and 3 x 10(8) cells/mL made the amount of enzyme released over 70%. L-Asparaginase in K2HPO4 and Triton solution could remain stable at least for 24 h. The release effect of K2HPO4 and Triton X100 used simultaneously was better than that of K2HPO4 and Triton X100 used separately in succession. Electron microscopy indicated that the chemical treatment altered the surface structure of E. coli cells but did not break them. As the method does not produce a large amount of cell fragments and the amount of enzyme released is relatively high, it can be thought to be an valuable and economic method to release intracellular enzyme.     

Chemical oncogenesis in cultured mouse embryo cells in relation to the cell cycle.

Using the C3H/10T 1/2 CL8 line of mouse embryo fibroblasts and three different methods of obtaining cell cycle synchrony, namely arginine or isoleucine deficiency and release from postconfluence inhibition of growth, a sensitive phase for oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been found. This sensitive phase is located somewhere between the G1/S boundary and a point 4 hr prior to this marker. Methylation of cellular macromolecules by tritiated MNNG is not cycle-dependent in cells synchronized by arginine deficiency. The capacity of cells to repair DNA single strand breaks produced by MNNG was examined by alkaline sucrose sedimentation analysis in cells synchronized by arginine deficiency and treated with MNNG during phases of the cell cycle sensitive and insensitive to oncogenic transformation. Whereas DNA repair was found to be equally rapid in cells treated just before S phase (I), or just after commencement of DNA synthesis (III), transformation was maximal in I. By contrast, cells treated when blocked by arginine deficiency (II) repaired DNA slowly and were not sensitive to malignant transformation. Cells in I and II, which repaired DNA at very different rates, were equally sensitive to MNNG-induced lethality, while cells in III, which repaired DNA at the same rate as cells in I, suffered greater lethality. Thus, in this system it was concluded that there was no direct correlation between DNA repair, as measured by alkaline sucrose sedimentation analysis of prelabeled DNA, and malignant transformation or lethality produced by MNNG. In preliminary experiments malignant transformation induced by cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) has been found to occur mainly in S phase, indicating that diverse chemical oncogens may have different sites of action, or that activation of chemical oncogens is cell cycle-specific for some agents.     

Selection of a chemically defined medium for submerged cultivation of Streptomyces aureofaciens with high extracellular caseinolytic activity

A chemically defined medium was developed for the submerged cultivation of Streptomyces aureofaciens with a high secretion of caseinolytic activity. The medium composition is: 40 g/liter maltose; 1.640 g/liter L-leucine (0.0125M); 1.765 g/liter L-lysine (0.0125M); 6.976 g/liter K2HPO4 (0.04M); 4 g/liter CaCO3; 0.2 g/liter MgSO4.7H2O; 0.01 g/liter ZnSO4.7H2O; 0.01 g/liter FeSO4.7H2O: 0.01 g/liter MnSO4H2O, and 0.005 g/liter CoSO4.7H2O. Quantitative correlations were established between the concentrations of nutrients in the medium and the secretion of proteolytic activity. In this medium the secretion of proteolytic activity parallels growth, reaching a maximum after 70 hr at 30 degrees C in shaker cultures. The secretion appears to be an active process and to require aerobic conditions.     

Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.     

Effect of drug treatment on liver-slice function following 72-hour hypothermic perfusion

The viability of hypothermically perfused dog liver was evaluated with a tissue-slice technique. After being preserved for 72 hr, slices of liver were incubated at 30 degrees C for as long as 2 hr; then water content, K+/Na+ ratio, and ATP concentration were measured. Dog livers were assigned to the following experimental groups: Group 1 (no preservation; control); Group 2 (livers preserved for 72 hr); Group 3 (donor animals pretreated with 3.5 mg/kg of chlorpromazine (CPZ) and 20 mg/kg of methylprednisolone (MP), and livers preserved for 72 hr); Group 4 (livers pretreated with 2-deoxycoformycin (2-DOC), 50 mg/liter, and preserved for 72 hr); and Group 5 (combination of Group 3 and Group 4 treatments). Livers in Groups 2, 3, and 4 lost K+ during preservation, and the mean K+/Na+ ratio significantly decreased from a control value of 4.2 +/- 0.4 to 1.5-1.9 (P less than 0.05). Group 5 livers did not lose K+; mean K+/Na+ ratio was 3.9 +/- 0.5. Fresh livers (no preservation) rapidly reaccumulated K+ when the tissue slices were incubated for 2 hr at 30 degrees C; mean K+/Na+ ratio was 3.7 +/- 0.5. Tissue slices from Group 2 livers (72 hr preservation), and livers pretreated with CPZ-MP (Group 3) or pretreated with 2-DOC (Group 4) did not significantly reaccumulate K+ at 30 degrees C; mean K+/Na+ ratio was 1.7-2.1. Only slices prepared from liver pretreated with both CPZ-MP and 2-DOC reaccumulated K+; mean K+/Na+ ratio was 4.6 +/- 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex.

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.     

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

Purinergic receptors activate diverse signaling cascades and regulate the activity of cell volume-sensitive ion transporters. However, the effects of ATP and other agonists of P2 receptors on cell volume dynamics are only scarcely studied. In the present work, we used the recently developed dual-image surface reconstruction technique to explore the influence of purinergic agonists on cell volume in the C11-Madin-Darby canine kidney cell line resembling intercalated cells from kidney collecting ducts. Unexpectedly, we found that ATP and UTP triggered very robust (55-60%) cell shrinkage that lasted up to 2 h after agonist washout. Purinergic regulation of cell volume required increases in intracellular Ca(2+) and could be partially mimicked by the Ca(2+)-ionophore ionomycin or activation of protein kinase C by 4β-phorbol 12-myristate 13-acetate. Cell shrinkage was accompanied by strong reductions in intracellular K(+) and Cl(-) content measured using steady-state (86)Rb(+) and (36)Cl(-) distribution. Both shrinkage and ion efflux in ATP-treated cells were prevented by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the BK(Ca) channel inhibitors charybdotoxin, iberiotoxin, and paxilline. To evaluate the significance of cell-volume changes in purinergic signaling, we measured the impact of ATP on the expression of the immediate-early gene c-Fos. Thirty-minute treatment with ATP increased c-Fos immunoreactivity by approximately fivefold, an effect that was strongly inhibited by charybdotoxin and completely prevented by NPPB. Overall, our findings suggest that ATP-induced cell-volume changes are partially responsible for the physiological actions of purinergic agonists.     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.     

肝细胞生成素在睾丸组织中的相互作用蛋白

肝细胞生成素（HPO）具有复杂的生理功能,在睾丸中的高表达提示其在生殖活动中的重要性,而不仅局限于肝再生.构建了酵母表达载体pGBKT7-HPO,采用酵母双杂交系统,以HPO为诱饵蛋白,从人睾丸cDNA文库中寻找能够与HPO相互作用的蛋白质.经过筛选、验证阳性克隆,并进行PCR、测序和序列比对,得到4种相互作用蛋白质：NADH脱氢酶1、钠/钾ATP酶β3亚基、磷脂酶C δ1以及附睾分泌蛋白.提示HPO可能参与了细胞的蛋白质合成,能量代谢等.通过对候选蛋白的研究,为探讨HPO对睾丸组织细胞功能的调节机制提供了重要的线索.     

High phosphate (up to 600 mM) induces pseudohyphal development in five wild type Candida albicans

A method is described for the formation of nearly 100% pseudohyphae populations of wild-type Candida albicans A72. The method employs fungal growth at 37 degrees C (ca. 5x10(6) cells/ml) in a glucose-proline-N-acetyl-glucosamine medium supplemented with up to 600 mM phosphate (KH(2)PO(4)/K(2)HPO(4) 1:1) at pH 6.5. Four other strains of C. albicans (MEN, 10261, SG5314 and CAI-4) also formed pseudohyphae under these conditions, although the phosphate response profiles differed in the concentration required for each strain to form pseudohyphae.     

Fatty Acid Composition of Escherichia coli as a Possible Controlling Factor of the Minimal Growth Temperature.

Shaw, Maxwell K. (University of California, Davis), and John L. Ingraham. Fatty acid composition of Escherichia coli as a possible controlling factor of the minimal growth temperature. J. Bacteriol. 90:141-146. 1965.-If Escherichia coli ML30 is shifted from 37 to 10 C during exponential growth in glucose minimal medium, a 4.5-hr lag results. During this lag, the proportion of unsaturated fatty acids increases in the cellular lipids. However, the adjustment of the fatty acid composition does not appear to be prerequisite to growth at 10 C. If shifts are made to 10 C into minimal medium containing glucose after starvation for glucose at 37 C for 0.5 and 16 hr, the lag periods at 10 C are 4.5 and 6 hr, respectively. Withholding glucose during the lag periods does not affect the duration of the lag periods, but no change in fatty acid composition occurs if glucose is not present. Supplementing the medium with glucose after the lag period permits immediate growth at 10 C; however, the fatty acid composition is still typical of cells grown at 37 C. It is concluded that the fatty acid composition of cells does not determine the minimal temperature of growth.     

Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells.

ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.     

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.