Reliability of urinary 6-sulfatoxymelatonin as a biomarker in breast cancer

The objective of this study is to evaluate the effect of cryopreservation at different storage temperatures on urinary 6-sulfatoxymelatonin (aMT6s) concentration. Overnight urine from 28 postmenopausal women participating in the ORDET cohort study was filtered and separated into 6 mL aliquots. Urine samples were stored at -80 degrees C and at -30 degrees C for an average of 14 years. Urinary aMT6s concentration was assessed using a competitive immunoassay. Mean aMT6s values of samples stored at -30 degrees C were systematically lower than those of samples stored at -80 degrees C (10.7 ng/mL versus 15.8 ng/mL, p<0.001). Bland Altman plots showed disagreement between determinations at different storage temperatures at the highest levels of the metabolite concentration. The degree of agreement evaluated in terms of intraclass correlation coefficient was 0.68 (95% CI 0.41-0.84, p<0.0001). Pearson's correlation coefficient between aMT6s values of the two differently stored samples was 0.93 (p<0.001), while the Kendal tau coefficient for rank distribution was 0.73 (p<0.001). Our data suggest that storage temperatures might affect degradation of aMT6s during storage. However, individual characterization by melatonin levels does not seem to be affected by cryopreservation conditions.     

Importance of storage conditions for the stability of zinc- and cadmium-induced metallothionein

We examined the storage stability of metallothionein (MT), a cysteine-rich protein that has diagnostic potential as a cancer marker and in the assessment of Zn status and heavy-metal toxicity. MT was rapidly degraded in samples of rat whole liver at -20 degrees C or -70 degrees C. MT in supernatants from heat-treated rat liver homogenates stored as 1:5 dilutions of liver from Zn- or Cd-induced rats were stable (recovery >98%) for 100 d at temperatures of -70 degrees C and -196 degrees C but not at -20 degrees C, regardless of the presence of dithiothreitol (DTT) or argon. The variability of MT measurement by the 109Cd-hemoglobin affinity assay was however greatest in samples from Zn-induced rats stored without DTT. The integrity of the MT protein in supernatants of heat-treated homogenates stored for 100 d was demonstrated by Sephadex G-75 chromatography. When heat-treated supernatants were stored as dilute solutions (1:125 of liver), MT was unstable regardless of treatment or storage temperature. Our findings show that liver MT is stable for at least 4 mo as a supernatant of a heat-treated homogenate (1:5 dilution of liver) when stored at or below -70 degrees C and in the presence of DTT.     

Measurement of bisphenol A in human urine using liquid chromatography with multi-channel coulometric electrochemical detection

Smith-Lemli-Opitz syndrome (SLOS) patients have increased 7- and 8-dehydrocholesterol (DHC) concentrations. Using gas chromatography-mass spectrometry with selected ion monitoring we investigated whether storage time (24 h, 7 and 30 days, and 22 months at room temperature or at 4 degrees C) affected DHC concentrations in whole blood spots (WBSs) from SLOS patients and normal controls. Our results suggest that WBS sterol analysis can be used for SLOS screening and possibly related inborn errors of sterol metabolism with a 100% sensitivity and specificity on specimens stored for up to 30 days, either at room temperature or 4 degrees C. After 22 months of storage at both temperature SLOS samples can be indistinguishable from control samples. Therefore, great caution should be used to exclude SLOS by sterol analysis of WBSs stored for a long time.     

The influence of some sample handling factors on progesterone and testosterone analysis in goats

This study validated the use of commercially available radioimmunoassay kits for measuring the circulating progesterone and testosterone levels of goats. Progesterone and testosterone levels were then assayed in plasma which was collected from 23 does and 8 bucks. Collections from each animal were divided into three sodium fluoride-potassium oxalate (F/OX), one heparin, and one EDTA tubes and also into a tube without anticoagulant. Plasma from an F/OX tube was separated immediately from the blood cells by centrifugation. Serum or plasma was also separated after storage for 24 hours with F/OX, heparin or EDTA anticoagulant at 22 degrees C or with F/OX at 5 degrees C. A significant decline in assayable progesterone occurred in samples stored at 22 degrees C with each anticoagulant used and in the serum sample. Samples stored at 5 degrees C for 24 hours with F/OX anticoagulant contained concentrations of progesterone which did not differ significantly from those in samples where plasma was removed immediately. Assayable testosterone did not change with the anticoagulant used or vary with the storage temperature when F/OX tubes were stored at 5 degrees C and 22 degrees C for 24 hours. Results indicate that sample storage does influence levels of measured progesterone but not testosterone in goats. Progesterone assay is best done on plasma which is immediately separated from blood cells or on samples which are stored at 5 degrees C.     

Survival of Campylobacter jejuni inoculated into ground beef.

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent.     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

Soluble urokinase plasminogen activator receptor measurements: influence of sample handling

AIM: The influence of sample handling on soluble urokinase plasminogen activator receptor (suPAR) concentrations in serum and EDTA plasma was studied in 16 healthy premenopausal women. METHOD: Blood was collected in dry tubes and tubes containing EDTA and kept at 4 degrees C or 20 degrees C for 1, 3, 8, 24 or 72 hours before processing into serum or EDTA plasma. In addition, serum and EDTA plasma were frozen and thawed 1-8 times. All suPAR measurements were performed by ELISA. RESULTS: No significant differences were found between serum or EDTA plasma suPAR concentrations when whole blood samples were kept for 1, 3, 8 or 24 hours. Significantly higher suPAR levels were found in samples kept for 72 hours at 20 degrees C compared to samples processed into serum or EDTA plasma after short-term storage for no more than 24 hours after collection. No significant differences were observed when whole blood was kept at 4 degrees C for up to 72 hours. Repeated freezing and thawing had no significant effect on the serum and EDTA plasma suPAR levels. CONCLUSION: suPAR values in blood samples are dependent on the handling procedures of the samples. All samples of whole blood must be processed into EDTA plasma or serum within 24 hours if kept at 20 degrees C and within 72 hours if kept at 4 degrees C. However, repeated freezing/thawing cycles had no influence on suPAR values in the samples.     

Sample handling and stability of hepatocyte growth factor in blood samples

As regards clinical studies performed on hepatocyte growth factor (HGF) during recent years, we have aimed in the present study to investigate the eventual differences in sample handling of this cytokine that might influence the results of serum concentrations. Venous blood from patients with current infectious diseases and controls was used in different sub-studies. Compared with samples separated within one hour, no significant changes in serum HGF levels were observed when whole blood stayed 4, or 24h at 6 degrees C before or 6h in room temperature after separation but HGF levels were significantly higher (P<0.01) when whole blood was kept at room temperature 4 and 24h before separation. Serum HGF was stable up to 20 freeze-thaw cycles. The serum concentrations of HGF were significantly higher than levels in the plasma (19%; P<0.05). A significant increase in serum HGF levels (12%, P<0.05) was observed after shaking the whole blood sample to a visible haemolysis, although the HGF concentration in blood cells was around half of that in serum. HGF tolerated storage at -70 degrees C for at least 4 months. We conclude that standardized methods in sample handling are important in the study of HGF concentrations in blood samples.     

Changes of viability and composition of the Escherichia coli flora in faecal samples during long time storage

Long-time storage of faecal samples is necessary for investigations of intestinal microfloras. The aim of the present study was to evaluate how the viability and the composition of the Escherichia coli flora are affected in faecal samples during different storage conditions. Four fresh faecal samples (two from calves and two from infants) were divided into sub-samples and stored in four different ways: with and without addition of glycerol broth at -20 degrees C and at -70 degrees C. The viability and the phenotypic diversity of the E. coli flora in the sub-samples were evaluated after repeated thawings and after storage during 1 year. The samples stored for 1 year without thawing were also kept at room temperature for 5 days and subsequently analysed. According to phenotyping (PhP analysis) of 32 isolates per sample on day 0, all four samples contained two dominating strains of E. coli each, and between one and eight less common strains. Samples that were stored at -70 degrees C in glycerol broth showed equal or even higher bacterial numbers as the original samples, even after repeated thawings, whereas samples stored at -20 degrees C showed a considerably lower survival rate, also with addition of glycerol. Sub-samples containing glycerol broth that were kept at room temperature after storage for 1 year showed a clear increase in the number of viable cells as well as in diversity. The diversities in each sub-sample showed a tendency to decrease after several thawings as well as after storage. Generally, the E. coli populations in samples stored at -20 degrees C were less similar to the population of the original sample than that in samples stored at -70 degrees C. Samples that had been mixed with glycerol broth had an E. coli flora more similar to that in the original sample than those without glycerol broth. Furthermore, the sub-samples that were kept at room temperature after storage for 1 year generally were more similar to the original samples than if they were processed directly. We conclude that for long time storage of faecal samples, storage at -70 degrees C is preferable. If samples have to be thawed repeatedly, addition of glycerol is preferable both for samples stored at -70 degrees C and for samples stored at -20 degrees C. Our data also have indicated that when E. coli isolates from faecal samples are selected for, e.g. analysis of virulence factors, it is necessary to pick several isolates per sample in order to obtain at least one isolate representing the dominating strain(s).     

Survival of Escherichia coli O157 in abattoir waste products

AIMS: This study monitored survival and growth of Escherichia coli O157 in ovine and bovine abattoir waste. METHODS: Blood and gut contents were inoculated separately with cocktails of E. coli O157. Samples were stored aerophilically and microaerophilically at 5 degrees C, 15 degrees C and 30 degrees C to represent storage at different container depths and at extremes of UK ambient temperature. CONCLUSIONS: Results showed survival of E. coli O157 was irrespective of oxygen content with no significant differences observed between aerophilic and microaerophilic environments. Numbers of E. coli O157 in ovine and bovine gut contents showed no change when stored at 5 degrees C and increased 1-2 log(10) at 15 degrees C and 30 degrees C in 28 h. In ovine and bovine blood, irrespective of storage temperature, there was a 0.5-2 log(10) reduction or no change in numbers except in ovine blood stored at 30 degrees C where the fall in numbers was followed by a 3 log(10) increase. In aged (stored at 4 degrees C for 18 h before spiking) bovine blood there was no significant change in numbers at 5 degrees C while at 15 degrees C there was 2 log(10) rise after 48 h. At 30 degrees C there was an initial 1 log(10) decrease in numbers followed by a 1 log(10) rise over the following 40 h. SIGNIFICANCE AND IMPACT OF STUDY: Abattoir wastes may become contaminated from animals infected with Verocytotoxigenic E. coli O157 and in certain storage conditions these pathogens could significantly increase in numbers. There is need for care in abattoir waste disposal, not only for personnel subject to direct contact, but also in the prevention of cross contamination to adjacent land and water courses which could indirectly infect humans.     

Effect of blood storage on radiation-induced micronuclei in human lymphocytes.

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects (n=11), and from cancer patients (n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137Cs-irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22 degrees C) or in the refrigerator (5 degrees C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22 degrees C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5 degrees C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22 degrees C or at 5 degrees C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5 degrees C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.     

Effect of storage on the measurement of apolipoproteins A-I and A-II by radial immunodiffusion

We studied the effect of storage time and conditions on the measurement of apolipoprotein A-I and A-II by radial immunodiffusion. Purified A-I and A-II standards were stable for at least 6 months before any change in immunoreactivity was detected if stored at 4 degrees C at concentrations of 0.06-0.24 mg/ml for A-I and 0.016-0.064 mg/dl for A-II in 0.84 M tetramethylurea, 6.4 M urea, and 8 mM Tris-hydrocholoride, pH 8.0. Purified A-I (0.8-1.6 mg/ml) and A-II (0.5-1.0 mg/ml) were stable for 1 year if stored at -60 degrees C in 5 mM NH4HCO3 with or without 4.2 M tetramethylurea. Serum or plasma could be stored at 4 degrees C (under conditions where evaporation and bacterial growth were minimized) for at least 46 days or at -20 degrees C for up to 3 years without any change in A-I or A-II levels. For four serum samples stored at -20 degrees C for 2 to 3 years, the coefficient of variation of measurement ranged from 6.3 to 9.8% for A-I and from 6.7 to 10.6% for A-II. Samples stored at 4 degrees C had comparable apolipoprotein levels to those stored at -20 degrees C. However, apolipoprotein levels in serum samples were 3-5% higher than those obtained on plasma samples. We conclude that purified A-I or A-II and serum and plasma can be stored for long periods without any change in the measurement of the A-I or A-II by radial immunodiffusion.     

Ochratoxin A as the cause of spontaneous nephropathy in fattening pigs.

At a number of slaughters nephropathy and high ochratoxin A contents in kidneys have been observed in fattening pigs from two Swedish farms. In one herd the source of contamination was barley grown on the home farm and stored under such conditions that the growth of fungal species (Penicillium verrucosum var. verrucosum) producing ochratoxin A occurred, with the subsequent formation of the toxin. In this case high ochratoxin A levels in fattening pigs were found during a period of about 18 months. In the second herd, where compounded feed was used, it was impossible to locate the source of contamination. It was presumed that a consignment of feed was damaged by rain during storage at the farm. Ochratoxin A was found in fattening pigs from this herd for a period of about 2 months. Ochratoxin A appeared in the kidneys of all investigated pigs. In some animals the livers, whole blood, and plasma were analyzed, too. The livers contained somewhat lower amounts of ochratoxin A than the kidneys, whereas the content in whole blood and plasma, respectively, was 5 and 13 times greater. Kidneys spontaneously contaminated with ochratoxin A, when stored for 10 months at -70 degrees C, showed no systematic decrease in toxin content.     

Sampling and storage conditions of rainbow trout liver affects monooxygenase and conjugation enzymes

1. The effect of storage conditions of rainbow trout (Salmo gairdneri) liver on monooxygenase and conjugation enzyme activities was studied. Fish livers or whole fish were frozen and stored for various periods of time at -4, -20 or -80 degrees C. 2. Freezing the whole fish at -20 degrees C affected the biotransformation enzyme activities dramatically. The loss of monooxygenase activity exceeded up to one-tenth of the initial rate in 17 days. UDP-Glucuronosyltransferase activity increased 50%. Glutathione S-transferase appeared to be the most durable enzyme. 3. When the whole fish were stored in an ice-bath at -4 degrees C for up to 24 hr the activities measured decreased only half of that when frozen for 3 days. 4. When it is impossible to freeze the tissues studied in liquid nitrogen the activities are best preserved when whole, decapitated, bled fish are kept in an ice-bath for less than 24 hr.     

Effects of long-term storage at different temperatures on conidia of Botrytis cinerea Pers.: Fr.

Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (-80 degrees C, -20 degrees C, 4 degrees C and 21 degrees C). Germination tests performed during 3 years showed that viability at 21 degrees C was completely lost after 1 month. Conidia stored for 30 months at -80 degrees C, -20 degrees C and 4 degrees C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and respiration (O(2) consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at -80 degrees C was almost the same as for fresh conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at -80 degrees C has shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of B. cinerea are discussed.     

Time course of the age-related alterations in stored blood

The extent and time course of the impairments occurring in whole blood and erythrocyte cells stored under blood bank conditions were studied by monitoring the reduction of MAL-6 spin label added to the media containing whole blood or erythrocyte cells using electron spin resonance (ESR) technique. Impairments forming in the erythrocyte cells incubated for various times at 37 degrees C were also studied. Erythrocyte cells were found to undergo changes during the storage or incubation, leading to fast decay of MAL-6 spin labels signal height. The extent of the changes depends on storage or incubation time. However, the reduction in incubated or artificially aged erythrocyte (AAE) cells was faster than the reduction in whole blood (WB) and aged erythrocyte (AE) cells stored under blood bank conditions. Two exponential curves attributed to the liquid and cellular parts of a given samples were found to be described best in the reduction of MAL-6 spin label in WB, AE and AAE.     

Degradation of specific polyunsaturated fatty acids in red blood cells stored at -20 degrees C proceeds faster in patients with schizophrenia when compared with healthy controls

Deficits in red blood cell (RBC) polyunsaturated fatty acids (PUFAs) have been extensively reported in schizophrenia although reports are inconsistent. A possible explanation for this inconsistency is varying storage conditions of blood samples prior to analysis, especially freezer storage temperature. We conducted a prospective investigation of fatty acid degradation rates in RBCs from healthy control subjects when samples from each individual were stored at both -20 degrees C or -70 degrees C. Differences were detected between storage conditions. A second prospective study was conducted to investigate the effect of differential storage conditions on RBC membrane fatty acids from schizophrenic patients. We found that storage at -20 degrees C was associated with reduced levels of PUFAs. Comparison of decay rates suggest that schizophrenics decay approximately twice as rapidly as controls. Furthermore, this phenomenon appears to be specific for the longer chain PUFAs suggesting that an enzymatic process may be responsible, e.g. elevated phospholipase A(2) activity, as opposed to simple chemical oxidation.     

Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content.

The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.     

The effect of storage at different temperatures on the stability of Hepatitis C virus RNA in plasma samples.

One important issue related to Hepatitis C virus (HCV) RNA nucleic acid amplification testing (NAT) is the storage conditions of plasma samples in order to obtain reliable results. Many authors have reported that the storage conditions could affect the RNA stability and, hence, HCV RNA detection. We have studied HCV RNA stability in plasma samples after storage at different temperatures (-70, -20, 5 and 25 degrees C). Samples containing different HCV titres were stored and analysed by qualitative or quantitative NAT techniques at defined time points. At -20 degrees C, samples containing high HCV RNA titres were followed-up during approximately 2.6-2.7 years, samples with intermediate concentrations during approximately 1 year and samples with 100 International Units/millilitre (IU/ml) during 2.5 years. Independently of the HCV RNA concentration, the results show absence of decay in HCV RNA detectability. Samples stored at 25 degrees C maintain their HCV RNA titre during 14 days and samples at 5 degrees C were stable for at least 3 months.     

Impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 populations to undercooking and simulated exposure to gastric fluid

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75 degrees C) water followed by lactic acid (2%, 55 degrees C), vacuum packaged, stored at 4 (28 days) or 12 degrees C (16 days), and periodically transferred to aerobic storage (7 degrees C for 5 days). During storage, samples were exposed to sequential heat (55 degrees C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4 degrees C was lower than that of cells on nondecontaminated meat stored at 12 degrees C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12 degrees C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4 degrees C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12 degrees C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.