A method for preserving saliva samples at ambient temperatures

To facilitate biochemical and biopharmaceutical studies when cold storage is unavailable, we assessed the stability of saliva samples containing preservatives stored at room temperature over a 1-year period. Two preservative mixtures were evaluated: sodium benzoate and citric acid (P1), and ethyl and propyl paraben (P2). Saliva samples were spiked with acetaminophen (APAP) or antipyrine (AP) and stored in preservative-coated vials and examined for concentrations of APAP, AP, melatonin, and cortisol at regular intervals as a function of preservative type and storage duration. Samples were stored at room temperature or at -20 degrees C (positive control) and analyzed periodically for APAP and AP by high-performance liquid chromatography and for melatonin and cortisol by radioimmunoassay. The effectiveness of the preservatives was determined by calculating the value of samples stored at room temperature in terms of percent of control (-20 degrees C) values. P1 effectively maintained the stability of APAP (100%) and AP (100%) for 360 days at room temperature; concentrations in samples at room temperature on day 360 were comparable to those on day 01. P1 also effectively maintained melatonin (100%) and cortisol (95%) concentrations for 180 days at room temperature. P2 preserved AP and cortisol in saliva for 60 days, but APAP for only 14 days.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Effect of formulation pH and storage temperatures on the preservative efficacy of some gases used as propellants in cosmetic aerosols

The effects of changes in formulation pH and storage temperature on the preservative activities of some aerosol propellants—butane, carbon dioxide, dimethylether and their combinations were investigated. A preservative challenge test method was used to determine the survival rates of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger at formulation pH levels 5·80, 7·28 and 8·10 and storage temperatures of 20°, 30° and 40°C.
A significant decrease in the pH of formulations was observed with no corresponding changes in the antimicrobial effectiveness when carbon dioxide was incorporated. Alterations in the antimicrobial profiles of these propellants due to changes in formulation pH were dependent on the propellant and the species of the micro-organism, especially when single propellants were used. Results also showed that the propellants exert antimicrobial activities against the various organisms at the three storage temperatures but there were significantly greater inhibitory activities at 40°C. With a combination of 10% butane/dimethylether (1:2) and 10 bar carbon dioxide there were no differences in the degree of microbial inhibition at the various formulation pH levels and storage temperatures. In most cases, the organisms were completely inactivated within 24 h. These findings showed that the combination of butane/dimethylether with carbon dioxide could be used to protect against microbial contamination and spoilage of formulations of different pH levels as well as those meant for storage at different temperatures.     

A note on shelf-life extension of British fresh sausage by vacuum packing

Vacuum packing of British fresh sausage in a low oxygen permeability film (Diolon) extended the product shelf-life at 6°C to more than 20 d compared with 9–14 d in conventional packs. After 10 d storage, counts of key spoilage organisms such as yeasts and Brochothrix thermosphacta were generally 2 log cycles lower in vacuum packs. Vacuum-packed sausages also displayed a slower rate of loss of free sulphite. Variations in pack permeability to SO₂ were not responsible for this. Losses of free SO₂ in stored sausages are largely due to the production of sulphite-binding agents by yeasts. Selective enumeration of these yeasts showed them to be inhibited by conditions of vacuum packing. The extension of shelf-life observed is ascribed to the reduction in growth rate of the spoilage flora in vacuum packs coupled with the consequent maintenance of inhibitory levels of sulphite for a longer period.     

The Behaviour of a Food Poisoning Strain of Clostridium welchii in Beef

S ummary : An inoculum of 10⁵ spores of Clostridium welchii F2985/50 in meat survived steaming at 100° for 5 h, the number being reduced sevenfold for every hour of steaming. They also survived for at least 6 months in frozen meat stored at -5° and -20°, whereas vegetative cells died more rapidly at -5° than at -20°. In beef stored for 13 days at 1°, 5°, 10° and 15° there was no multiplication but a slow destruction of vegetative cells, but there was little change in the spore count. Slow multiplication occurred at 20° but at 25° and 37° growth was rapid. Only about 3% of the spores germinated without prior heat shock, so the majority failed to germinate in raw meat stored at any temperature, but did so once the meat had been heated. In meat which had been heated and allowed to cool almost all of the spores had lost their heat resistance.
It was found that the minimal growth temperature was related to pH and medium, so that meat with a pH higher than that used in these experiments (pH 5°7–5°8) would probably have a lower minimal growth temperature for these organisms and would thus be more susceptible to spoilage.     

Fate of Aeromonas and Yersinia on modified-atmosphere-packaged (MAP) cod and trout

A.R. DAVIES AND A. SLADE. 1995. The growth/survival of Aeromonas spp. and Yersinia enterocolitica on cod and trout stored in two modified atmospheres at 0, 5 and 12°C was compared with that on aerobically stored fish. Both organisms grew on aerobically stored fish at all three temperatures. Growth on the modified-atmosphere-stored fish was never greater and was generally markedly less than on aerobically stored fish. This reduction in growth was greater in the higher carbon dioxide-containing atmosphere and at the lower temperature(s).     

The use of a transport medium (L15M15) for bulk tissue storage and retention of viability

Twenty-five human or mouse tissue samples, some up to 8×4×2 cm, were immersed in a special transport medium (TM), L15M15, up to 7 days before being processed or placed in tissue culture. To test the efficacy of this medium, we concurrently placed pieces of the same tissues in a sterile phosphate buffered solution (PBS). We also tested the preservative capabilities of TM and PBS at room temperature and with refrigeration. Differences between TM and PBS are demonstrated, which are more pronounced using room temperature up to 4 days time. The tissues stored in TM show fewer degenerative or autolytic changes than the same tissue stored in PBS under identical conditions. Using regrigeration further enhanced the preservative qualities of TM up to 4 days, but not PBS. There were no obvious differences between tissues stored in TM and PBS with refrigeration after 7 days. We conclude that transport medium L15M15 is a useful medium for preserving tissue viability, especially large tissue samples, up to 4 days, especially if refrigerated.     

Survival of enteric bacteria and coliphage MS2 in pure human urine

Aims:  The survival of Escherichia coli , Salmonella enterica serovar Typhimurium, Enterococcus faecalis and coliphage MS2 was studied in stored, fresh and diluted (1 : 1) human urine at 15 and 30°C.
Methods and Results:  Survival rate was studied by the plate count method. All the organisms showed rapid inactivation in stored urine, but they survived better in diluted and fresh urine. The high pH level and temperature were the major factors found to influence the survival of the micro-organisms with the survival rate being higher at 15°C than at 30°C.
Conclusions:  The destruction of all micro-organisms in stored urine required <1 week at 30°C. Thus, the storage of urine is a useful way to reduce the risk of contamination while using urine as a fertilizer.
Significance and Impact of the Study:  The urine fertilization is aimed for the developing countries and the high temperatures in these countries may hasten the destruction of micro-organisms in urine. On the contrary, a higher survival rate of these organisms in fresh and diluted urine is a public health concern because the dilution of urine with water is likely to happen during flushing.     

Model experiments to establish behaviour of Yersinia enterocolitica O:9 strains in various types of fresh dry sausage

In model experiments different kinds of raw sausages were inoculated with liquid cultures of virulent-plasmid-carrying clinical Yersinia (Y.) enterocolitica (e.) strains of the O:9 serotype, doses being between 10⁴ and 10⁵ cfu g^-1. The sausage samples were stored at 3–5° and 13–16°C. During the first 10 d of storage the Y.e. plate count was detected with Desoxycholate-Citrate-Lactose-Sucrose Agar every day, later on in addition to it with phosphate buffer-enrichment and with enrichment according to Schiemann (1982) in intervals of several days' duration. The pH and a _w values, the contents of salt and water were detected. The multitude of complexly acting factors and substances prevents obviously the proliferation of Y.e. in fresh dry sausages. Decay dynamics of Y.e. were found to be considerably affected by storage temperature. Cold storage, basically, had a conservation effect and thus delayed the dying process of model strains. Yersinia enterocolitica -contaminated fresh dry sausage may cause potential danger to consumers, because of relatively extended survival periods of the pathogen. Therefore, manufacturers are expected to observe most stringent hygienic rules of Good Manufacturing Practice.     

The Preservation of Bacteriophage by Drying

SUMMARY: Two bacteriophages were stored a t room temperature after being dried and recoveries of them were compared with those from broth suspensions which were stored at 4°. The T, phage of Escherichia coli was recovered without loss from both broth and dried material after 30 months. Some loss of C phage of Bacillus meguteriurn occurred on drying but recoveries from the desiccates were much higher than from the broth suspensions after prolonged storage.     

Bioactivity of stored luteinizing hormone-releasing hormone analogue (LHRHa) in sea bass, Lates calcarifer Bloch

The spawning induction activity of dissolved and pelleted (D-Ala⁶, Pro⁹ N ethylamide) luteinizing hormone-releasing hormone analogue (LHRHa) stored for various periods was assessed in mature female sea bass. The spawning response of mature fish was reduced significantly after injection of dissolved LHRHa (20 μg kg⁻¹) stored for more than 90 days in a refrigerator (4–10°C) or for more than 30 days at room temperature (28–30°C). Similar to fish administered fresh preparations of LHRHa, fish spawned successfully after injection of a solution of LHRHa previously frozen, subjected to alternate freezing and thawing, exposed to sunlight or implanted pelleted LHRHa (50 ng kg⁻¹) stored at room temperature for 30–120 days. Loss of hormone bioactivity after prolonged storage may have been due to bacterial growth in solubilized preparations. Injection or implantation of stored LHRHa did not influence egg production among treated sea bass. These results demonstrated the relatively prolonged shelf life of stored LHRHa.     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

Preservation of Mycoplasmas on Anhydrous Silica Gel

1. 26 strains of mycoplasmas were successfully preserved on anhydrous silica gel crystals for 80 weeks at +4°C.
2. There was a sharp decline in viability of the strains if they were stored at room temperature.
3. This preservation technique is simple to perform and requires only inexpensive equipment.     

The Survival of Starter Organisms in Concentrated Suspensions

S ummary : Two representative lactic acid bacteria were grown in a rich organic medium, the cells were harvested by centrifugation, and the organisms were suspended in skimmilk at concentrations of 25–55 × 10⁹/ml. The pH was adjusted to different values and the suspensions were stored, with and without the addition of 20% (v/v) glycerol, at 4° and - 20°.
Both organisms survived better at pH 7°0 than at pH 5°0. Glycerol protected the cells at the lower pH value but offered no benefit at neutrality. At 4° about half the cells died within 5 or 6 weeks. At - 20°, however, there was no appreciable loss of viability or acid producing ability up to 9 months at pH 7°0. Suspensions stored for 9 months at - 20° produced excellent cultured buttermilk within the normal incubation period from an inoculum comparable to that used for a fresh culture. Cells harvested early in the maximum stationary phase of the growth cycle were more active than older cells, and they survived better than older cells during storage in the frozen state.     

Preservation of the hyperthermophile Pyrococcus furiosus

Methods are evaluated for the preservation of the hyperthermophile Pyrococcus furiosus . The use of glass capillary tubes stored over liquid nitrogen with dimethyl sulphoxide appears to be the preferred method of preservation. Lyophilization resulted in loss of viability and storage at room temperature and +4°C resulted in considerable loss of viability within 4 weeks.     

Pressure- vs. heat-induced bacterial stress in cooked poultry sausages: a preliminary study

Vacuum-packaged poultry cooked sausages were pressure-treated at 500 MPa by combinations of time (5-45 min) and temperature (2-80 degrees C) and later stored at 6-8 degrees C for 12 we. Mesophile and psychrotrophe counts were determined 1 d, 3, 6, 9 and 12 we after treatment and compared with those of cooked sausages pasteurized at 80-85 degrees C for 40 min. Both pressure and heat treatments offer great possibilities for preservation. Sausages pressurized at 65 degrees C for 15 min showed mesophile numbers below 2 log cfu g(-1) throughout the chill storage. Pressurization, unlike heat treatment, causes a reversible bacterial stress. Thus, injured cells recovered during storage and, at 6 and 12 we, after a temperature abuse (room temperature for approx. 24 h), counts increased up to 6.5 - 7.5 log units. Psychrotrophes were more sensitive to both treatments; no growth was detected the day after (a lethality of more than 4 log units).     

Growth of Bacteria on Meat at Room Temperatures

The development of spoilage flora and the growth of individual psychrotrophs and pathogens on meat held at 20 or 30°C were studied. Under aerobic conditions psychrotrophic pseudomonads accounted for 60% of the spoilage flora at 20°C, but <20% at 30°C where they were displaced by species of Acinetobacter and Enterobacteriaceae which included both mesophilic and psychrotrophic strains. Mesophiles dominated the anaerobic spoilage flora at 30°C when clostridia were the major species, but at 20° the flora contained mesophiles and psychrotrophs in similar proportions and was dominated by Enterobacteriaceae. These results were largely predictable from the growth rate data for individual organisms.
Interactions between species occurred more frequently at 20°C than at 30°C. When pathogenic species were grown at 20 or 30°Cin competition with equal numbers of psychrotrophic spoilage organisms, no interactions were observed. When pathogens were grown in competition with high numbers of psychrotrophs, only Lactobacillus growing anaerobically inhibited Salmonella typhimurium and Escherichia coli , but other pathogens were inhibited to varying degrees depending on the competing species and the incubation conditions. In general, the degree of inhibition was greater at 20 than at 30°C and facultative organisms were more susceptible under anaerobic than aerobic conditions. It appears that the cumulative stresses of low pH, suboptimal temperatures and competition with large numbers of saprophytic organisms can inhibit many of the pathogens likely to be present on meat. The organisms least affected by the conditions on meat surfaces, Salmonella and Esch. coli , are likely to be the main hazards on meat of normal pH held at room temperatures.     

Properties of Pseudomonads Causing Spoilage of Vegetables Stored at Low Temperature

Twenty-four strains of pectolytic, fluorescent pseudomonads were isolated from soft rots of celery stored at 0.4-1°C and five strains were isolated from soft rots in cabbage stored at 1°C. When inoculated into the vegetable from which they were isolated these bacteria caused soft rot of wounded, but not of unwounded tissue. According to their biochemical reactions, the organisms were divided into three groups; Group 1 (15 strains) were identified with Pseudomonas fluorescens Biotype II (Doudoroff & Palleroni 1974) ( Ps. marginalis ); Group 2 (12 strains) and Group 3 (two strains) would be included in the 'Miscellaneous strains'of Ps. fluorescens described by the above authors. One strain biochemically representative of Group 1 showed a maximum growth rate at 27°C (doubling time, 0.88 h) and a doubling time at 0.2°C of 14.9 h. A strain representative of Group 2 showed a maximum growth rate at 29°C (doubling time 0.96 h) and a doubling time at 0.2°C of 16.6 h. Neither strain grew at 36°C. The temperature characteristics (calculated for the range 0.2-20.8°C) were 83 011 and 79 534 J/mol, respectively. The mean doubling time for the remaining Group 1 strains at 0.2°C was 17.6 h and for remaining Group 2 strains was 17.1 h.     

Direct Counting of Bacteria Preserved with Lugol Iodine Solution

Lugol iodine solution was compared with glutaraldehyde as a preservative for marine bacteria. Direct counts with the fluorochrome 4′,6-diamidino-2-phenylindole show no significant difference between the preservatives, but the use of Lugol solution has several advantages over glutaraldehyde, especially in the handling and storage of samples. Bacteria were counted in water samples that were preserved with Lugol iodine solution and stored at room temperature for 4 years. Microprotozoa were also counted in samples preserved with Lugol iodine solution by using the fluorochrome fluorescein isothiocyanate.     

A Note on the Microflora of Beef Muscle Stored in Nitrogen at 0°

Fresh beef stored in nitrogen at 0° was spoiled by Gram positive rods. Almost all the organisms isolated were Microbacterium spp., although in one experiment about 10% of the organisms isolated were Lactobacillus spp. The numbers and types of organisms isolated were similar on each of two media incubated under aerobic and anaerobic conditions.