Comparison of four-hour and twenty-four-hour refrigerated storage of nonpotable water for fecal coliform analysis.

The problem of extending the storage time of water samples for fecal coliform analysis was addressed. Included in this report is a literature review of the storage problem. Twenty-eight samples were analyzed in replicate to determine the effect of 24-h storage of water samples at 4 degrees C. A new statistical approach to data analysis, coupled with the concept of practical acceptability, is presented. According to our results, many samples can successfully be stored at 4 degrees C for 24 h.     

Effect of coconut water and Braun-Collins solutions at different temperatures and incubation times on the morphology of goat preantral follicles preserved in vitro

Preservation of preantral follicles becomes very important to ensure follicle quality at the onset of cryopreservation or in vitro culture. However, for domestic animals, the ovarian donor of preantral follicles for in vitro studies is commonly encountered far away from reproduction laboratories. We investigated the effectiveness of coconut water and Braun-Collins solutions on the preservation of goat preantral follicles. At the slaughterhouse, the ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was immediately fixed (Control - Time 0). The other 18 fragments were randomly distributed into tubes containing 2 mL of coconut water or Braun-Collins solution at 4 degrees, 20 degrees or 39 degrees C and then stored for 4, 12 or 24 h. Histological analysis showed that the storage of ovarian fragments in coconut water and Braun-Collins solutions at 20 degrees or 39 degrees C for 12 or 24 h significantly reduced (P < 0.05) the percentage of morphologically normal preantral follicles when compared with the control. However, storage in coconut water at 20 degrees C for 4 h and in both solutions at 4 degrees C kept the percentage at control values. Ultrastructural analysis of follicles exposed to the stated conditions confirmed the integrity of preantral follicles stored at 4 degrees C in Braun-Collins and coconut water solutions for up to 12 and 24 h, respectively. Reduced cellular metabolism at 4 degrees C may explain why the best preservation of preantral follicles was at 4 degrees C, which may suggest a useful method for ovary transport in the future.     

Impact of different storage factors on the survivability of Campylobacter jejuni in turkey meat

Campylobacter jejuni is often prevalent in turkey and poultry, but the effects of storage temperatures and storage periods and the interruption of the cooling chain on its survival have not been evaluated so far. In this study, 700 samples of turkey meat were artificially contaminated by inoculating their surface with 10(3) CFU of C. jejuni per sample, wrapped in airtight cellophane bags, and stored under different chilling and freezing conditions for various storage periods; this was followed by analysis of the cultures. Subsequent to incubation at 25 degrees C for 48 h, C. jejuni was reisolated in only 7% of the samples. When the samples were stored under refrigerator conditions at 4 degrees C, the organism was reisolated in 42% of the samples after 1 week, and in 28% of the samples after 2 weeks. The recovery rates in the samples that had been stored frozen at -20 degrees C without interruption of the cooling chain were 68% after 2 weeks and 24% after 4 weeks. Different storage conditions were simulated in order to examine the impact of an interruption of the cooling chain on the survival of Campylobacter.     

Holding effects on coliform enumeration in drinking water samples

Standard procedures for analyzing drinking water stress the need to adhere to the time and temperature conditions recommended for holding samples collected for microbiological testing. The National Drinking Water Laboratory Certification Program requires compliance with these holding limits, but some investigators have reported difficulties in meeting them. Research was conducted by standard analytical methods to determine if changes occurred when samples were held at 5 and 22 degrees C and analyzed at 0, 24, 30, and 48 h. Samples were analyzed for coliforms by the membrane filter and fermentation-tube procedures and for heterotrophs by the pour plate method. A total of 17 treated water samples were collected from a large municipal distribution system from August to December 1981, and 12 samples were collected from January to May 1983. The samples were dosed with coliforms previously isolated from the water system, Enterobacter cloacae in 1981 and Citrobacter freundii in 1983. The coliform counts declined linearly over time, and the rates of decline were significant at both 5 and 22 degrees C. Within 24 h at 22 degrees C, levels of E. cloacae and C. freundii decreased by 47 and 26%, respectively, and at 5 degrees C, E. cloacae numbers declined by 23%. Results from these representative laboratory-grown coliforms reinforced those previously obtained with naturally occurring coliforms under the same experimental conditions. Significantly, some samples with initially unacceptable counts (greater than 4/100 ml) met the safe drinking water limits after storage at 24 h at 5 and 22 degrees C and would have been classified as satisfactory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of fluorescent in situ hybridization combined with flow cytometry for assessing interindividual variation in the composition of human fecal microflora during long-term storage of samples

This work was conducted to assess the accuracy of in situ hybridization to show differences in human microflora composition between volunteers and to optimize the storage of fecal samples to allow delayed analysis of gut microflora composition in humans. Fecal samples from 25 healthy subjects (14 women, 11 men aged 24-51) were collected. The samples were fixed in 4% Paraformaldehyde (PFA) solution at 4 degrees C overnight and stored at -70 degrees C. Twenty samples were analysed to quantify the variation due to interindividual differences in the composition of fecal microflora. The five remaining samples were stored either after PFA fixation or directly frozen at -70 degrees C and were monitored on a 12-month period. The fecal microflora was analysed by in situ hybridization combined with flow cytometry detection. Ribosomal RNA-targeted probes were used to assess the relative proportions of four phylogenetic groups: Clostridium coccoides-Eubacterium rectale (Erec 482), Bacteroides (Bac 303), Faecalibacterium prausnitzii (Fprau 645) and Bifidobacterium (Bif 164). Our results demonstrated that the method used is adapted to detect significant differences in fecal microflora composition in humans. Moreover, samples stored in PFA solution demonstrated a stable composition even after 8 months of storage. Conversely, frozen samples were less stable as the Bifidobacterium and C. coccoides-E. rectale groups showed significant differences after 2 months of storage. In conclusion, the fecal microflora composition can be analysed up to 8 months after 4% PFA fixation and storage at -70 degrees C. It represents an extended time compared with the 2-month period currently recommended. This will give more flexibility for applying this technology in epidemiological studies including a large number of samples.     

Evaluation of reactive oxygen metabolites in frozen serum samples. Effect of storage and repeated thawing

Measuring the free radical activity in serum samples from prospective studies is the best way to investigate the association between oxidative stress and human diseases. Prospective studies require the analysis of serum samples that have often been stored for a long time. Our study was designed to determine the effect of storage at -30 degrees C and -80 degrees C for two years on free radical activity. We analyzed the free radical activity by measuring circulating hydroperoxides in a pool of sera at baseline and after one day, one week, one month and 25 months of storage, using a photometric method (d-ROMs test). Measurements were performed in aliquots thawed only once at each time point and in aliquots frozen and thawed repeatedly over the study period. After two years we observed a small but statistically significant 4% decrease in the hydroperoxide concentration, which was substantially unaffected by storage temperatures and repeated freeze-thaw cycles. We also carried out the d-ROMs test in sera from ten apparently healthy volunteers at 2, 8, 24, and 48 hours after collection and storage at 4 degrees C and did not observe any significant variation. In conclusion, the d-ROMs test is a simple method suitable to evaluate the free radical activity in frozen serum samples after long-term storage.     

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa

Experiments were conducted to determine temperatures between 24 and 4 degrees C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25 x 10(6) spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (-0.7 degrees C/minute) from 24 degrees C to either 22, 20, 18 or 16 degrees C; then it was cooled slowly (-0.05 degrees C/minute) to a storage temperature of 4 degrees C. In Experiment 1B, rapid cooling proceeded from 24 degrees C to either 22, 19, 16, or 13 degrees C, and then slow cooling occurred to 4 degrees C. Initiating slow cooling at 22 or 20 degrees C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16 degrees C. Initiation of slow cooling at 22 or 19 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13 degrees C. In Experiment 2A, semen was cooled rapidly from 24 to 19 degrees C, and then cooled slowly to either 13, 10, 7 or 4 degrees C, at which point rapid cooling was resumed to 4 degrees C. Resuming the fast rate of cooling at 7 degrees C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13 degrees C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4 degrees C before fast cooling resumed to 4 degrees C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4 degrees C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19 degrees C, then cool slowly from 19 to 8 degrees C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0 degrees C. Storage at 6 or 4 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2 degrees C.     

Effect of ambient temperature storage on potable water coliform population estimations.

The effect of the length of time between sampling potable water and performing coliform analyses has been a long-standing controversial issue in environmental microbiology. The issue is of practical importance since reducing the sample-to-analysis time may substantially increase costs for water analysis programs. Randomly selected samples (from those routinely collected throughout the State of Wisconsin) were analyzed for total coliforms after being held at room temperature (20 +/- 2 degrees C) for 24 and 48 h. Differences in results for the two holding times were compared with differences predicted by probability calculations. The study showed that storage of the potable water for up to 48 h had little effect on the public health significance of most samples containing more than two coliforms per 100 ml.     

Effect of ambient temperature storage on potable water coliform population estimations

The effect of the length of time between sampling potable water and performing coliform analyses has been a long-standing controversial issue in environmental microbiology. The issue is of practical importance since reducing the sample-to-analysis time may substantially increase costs for water analysis programs. Randomly selected samples (from those routinely collected throughout the State of Wisconsin) were analyzed for total coliforms after being held at room temperature (20 +/- 2 degrees C) for 24 and 48 h. Differences in results for the two holding times were compared with differences predicted by probability calculations. The study showed that storage of the potable water for up to 48 h had little effect on the public health significance of most samples containing more than two coliforms per 100 ml.     

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.     

Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa

The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.     

Increased production of PGI2-like activity by frozen-thawed rat aorta.

The effects of storage conditions, temperature, and time on the ability of the rat thoracic aorta to produce a platelet aggregation inhibitor were investigated. Aortic fragments were incubated in Tris buffer, aliquots of which were then tested for their ability to inhibit ADP-induced human platelet aggregation. The incubation fluid of samples that had been soaked in Tris buffer at 4 degrees C for 24 hours contained no inhibitor activity, whereas the incubation fluid of similar samples that had been kept at 4 degrees C but not soaked in buffer contained comparable inhibitor activity as that of fresh samples. The incubation fluid of samples that had been kept at -20 degrees C or -80 degrees C contained greater inhibitor activity than that of fresh samples, and was maintained in -20 degrees C samples for 7 days, and -80 degrees samples for 28 days. The aortic inhibitor had similar properties as PGI2.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

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.     

Long-term storage of nuclear suspensions prepared from paraffin-embedded material for flow cytometric DNA analysis

Nuclear suspensions were prepared from archival material of cancer biopsies. DNA content analysis by flow cytometry was performed after storage at -80 degrees C for 24 h and 6 months, compared to 24 h storage at +4 degrees C. No significant variations in CV or DNA indices were observed. Repeated freezing and thawing did not reduce the DNA histogram quality.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.     

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.     

Effect of heat shock on browning-related enzymes in minimally processed iceberg lettuce and crude extracts

The effects of heat shock on PPO and POD activity in minimally processed Iceberg lettuce was examined during storage (10 days). The results were compared with the effect of temperature on crude extracts of these enzymes (in vitro analysis). Fresh-cut lettuce washed at 50 degrees C showed significantly lower PPO and POD activity throughout storage than lettuce washed at 4 degrees C and 25 degrees C. These results were consistent with a sensory analysis in which the panellists found the lowest browning scores in those samples treated at 50 degrees C.When PPO and POD were analysed in vitro, the samples treated at 50 degrees C showed a rapid loss of POD activity and a similar but slower loss of PPO activity in all tissues, while incubation at 4 degrees C and 25 degrees C showed no significant loss of activity. While heat shock did not lead to significant loss of activity it did repress the synthesis of PPO and POD during storage.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.