Injuries of Cold Acclimatized Poplar Twigs Resulting from Enzyme Inactivation and Substrate Depression during Frozen Storage at Ambient Temperatures for a Long Period

Wintering twigs of poplar could be stored in frozen state forabout one year, but longer storage resulted in metabolic dysfunctionand death of the twigs. Analyses of enzyme activities revealedthat most of the activity of glucose-6-phosphate and 6-phosphogluconatedehydrogenases and glutathione reductase present normally inthe twigs disappeared. Another four enzyme activities assayedalso decreased to some extent. In the twigs, glucose 6-phosphatedecreased to a level of 10^–5 M and glutathione was presenttotally in the disulfide form. The extent of the decrease ofascorbate and ATP was considerably less than that of the abovetwo. Peroxide levels rose slightly, but lipid hydroperoxidelevels rose several fold. It is suggested that the reactions catalyzed by glucose-6-phosphatedehydrogenase and related ones are obligatory to the cells evenin the frozen state and that the activity to supply glucose6-phosphate, hence NADPH, is limited to a certain period oftime. Beyond this capacity, a decrease in the ability to removeperoxide may also occur, due to a shortage of NADPH supply,resulting in inactivation of some enzymes and the formationof lipid hydroperoxide in cellular membranes. (Received April 26, 1985; Accepted June 20, 1985)     

Increased Survival and Differentiation of Frozen Herbaceous Plant Organ Cultures through Cold Treatment

Cold treatment of donor carnation plants (Dianthus caryophyllus L.) at 4 C for 3 days or more resulted in a doubling in the percentage of excised, frozen shoot apices which survived freezing and a 6- to 7-fold increase in the percentage which formed leaf primordia or shoots. The optimal freezing parameters for both survival and differentiation were as follows: size of the shoot apex-two to three sets of leaf primordia; dimethylsulfoxide concentration in the freezing solution-5%; time in dimethylsulfoxide prior to freezing->30 minutes; average cooling rate-≥50 C/minute; initial warming rate-about 1450 C/minute. In general, the cells in the meristematic region of the shoot apex remained viable after freezing while those in the leaf primordia did not. Viability of the meristematic cells appears to be maintained by preventing the growth of intracellular ice crystals formed during rapid cooling by rapidly passing the tissue through the temperature zone in which lethal crystal growth occurs (mechanism of Luyet). Applications of this technique are discussed.     

Survival of Frozen Mycoplasmas

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.     

Adaptive Response to Cold Temperatures in Vibrio vulnificus

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35°C to 6°C conditions, it underwent transition to a non-culturable state. Cells adapted to 15°C prior to change to 6°C condition, however, remain viable and culturable. In addition, cultures adapted to 15°C were able to survive better upon freezing at −78°C compared with cultures frozen directly from 35°C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive “protective” proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2′-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with ³⁵S-methionine during exposure at 15°C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15°C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6°C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters. Received: 30 July 1998 / Accepted: 1 October 1998     

Survival of Virus in Chilled, Frozen, and Processed Oysters

Samples of whole and shucked Pacific and Olympia oysters, contaminated with 10(4)-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and - 17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly intervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 10(4) PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area.     

Inactivation of Ascaris Eggs in Source-Separated Urine and Feces by Ammonia at Ambient Temperatures

Sustainable management of toilet waste must prevent disease transmission but allow reuse of plant nutrients. Inactivation of uterus-derived Ascaris suum eggs was studied in relation to ammonia in source-separated urine without additives and in human feces to which urea had been added, in order to evaluate ammonia-based sanitation for production of safe fertilizers from human excreta. Urine was used concentrated or diluted 1:1 and 1:3 with tap water at 4, 14, 24, and 34°C. Fecal material, with and without ash, was treated with 1% or 2% (wt/wt) urea at 24 and 34°C. At 34°C eggs were inactivated in less than 10 days in urine and in amended feces. At 24°C only feces with 2% (wt/wt) urea or 1% (wt/wt) urea at high pH (10) inactivated all eggs within 1 month, and no inactivation was observed after 75 days in urine diluted 1:3 (18 ± 11 mM NH₃). At temperatures of ≥24°C, NH₃ proved to be an efficient sanitizing agent in urine and feces at concentrations of ≥60 mM. Treating fecal material at 34°C can give a 6-log₁₀ egg inactivation within 1 month, whereas at 24°C 6 months of treatment is necessary for the same level of egg inactivation. At temperatures of 14°C and below, inactivation rates were low, with viable eggs after 6 months even in concentrated urine.     

Male Weasels Decrease Activity and Energy Expenditure in Response to High Ambient Temperatures

The heat dissipation limit (HDL) hypothesis suggests that the capacity of endotherms to dissipate body heat may impose constraints on their energy expenditure. Specifically, this hypothesis predicts that endotherms should avoid the detrimental consequences of hyperthermia by lowering their energy expenditure and reducing their activity in response to high ambient temperatures (T_a). We used an extensive data set on the daily energy expenditure (DEE, n = 27) and the daily activity time (AT, n = 48) of male weasels (Mustela nivalis) during the spring and summer breeding season to test these predictions. We found that T_a was related in a “hump-shaped” (i.e. convex) manner to AT, DEE, resting metabolic rate (RMR) and metabolic scope (the ratio of DEE to RMR). These results support the HDL hypothesis because in response to warm T_as male weasels reduced their AT, DEE, and RMR. Although the activity and energy expenditure of large endotherms are most likely to be constrained in response to warm T_as because they are less able to dissipate heat, our results suggest that small endotherms may also experience constraints consistent with the HDL hypothesis.     

Survival of Campylobacter Jejuni/Coli in Ground Refrigerated and in Ground Frozen Beef Liver and in Frozen Broiler Carcasses

Survival of 5 strains of Campylobacter jejuni/coli in ground beef liver stored at 4° C and at –20° C was studied. After 6 days of storage at 4° C the beef liver was spoiled, which was indicated by APG log 7.25 and lactobacilli count log 7.0. During this storage Campylobacter counts decreased only slightly. After 12 weeks of storage at –20° C Campylobacter counts decreased by 2–3 logs in frozen ground beef liver. Survival of 4 strains of C. jejuni/coli on frozen broiler carcasses was also studied. Two inoculation levels, 103–104/g and 104–105/g were used. On frozen broiler carcasses Campylobacter counts decreased by 0.5–2.0 logs during 12 weeks at –20° C.     

Survival and Activity of Frozen Starter Cultures for Cheese Manufacture

A study has been conducted on the effect of freezing and storage in liquid nitrogen on 13 strains of lactic streptococci. Cultures were frozen in droplet form and collected in mesh bags. After rapid thawing, the activity of the frozen cultures was compared with a culture of the same organism of the age usually used in cheese-making. The activities of the test and control cultures were traced simultaneously by continuous recording of the pH changes in inoculated milks. Viable counts were performed before and after freezing in liquid nitrogen and after storage in liquid nitrogen. There was no decrease in viable count or loss in activity of the cultures due to freezing and storage. Frozen cultures of some strains showed a shorter lag period after inoculation of milk than control cultures. Frozen concentrated cheese-starter cultures behaved normally in the manufacture of Cheddar cheese.     

Effect of Cold Temperatures on the Viability of Chromobacterium violaceum

The effect of low, nonfreezing temperatures on the viability of five strains of Chromobacterium violaceum was studied. The viability of cultures grown at 30 C was determined after exposure to various diluents held at 0 to 2 C. A culture diluted at its growth temperature served as the control. Cells of strain N were most sensitive in the early part of the exponential phase of growth. Cells of strains 252 and 341 were most sensitive in the late exponential, early stationary phase of growth. Cells of strain 9 showed greatest loss of viability during the maximal stationary phase. Strain 69 was completely resistant throughout its growth cycle to cold injury. Cell viability was much greater in buffered salts solution than in distilled water, broth, or physiological saline, whether cultures were diluted at room temperature or in the cold. The proportion of cells surviving after exposure to cold, however, was the same regardless of the composition of the diluent. Loss of viability was progressive at 0 to 2 C and reached a maximum after 2 hr. There was no loss of viability of cells exposed to 20 C, but there was some loss at 12 C. Strain 341 cultivated at 15 C was much less sensitive to 0 to 2 C than when it was cultivated at 30 C. The composition of the growth medium seemed to have no effect on the survival of cells exposed to cold. The polyamines, spermine and trimethylenediamine, as well as erythritol and sucrose, exerted some protective action against the effects of cold but not uniformly for all strains studied.     

Ultrastructure of Bacteriomes and Their Sensitivity to Ambient Temperatures in Prostephanus truncatus (Horn)

In light microscopical sections of Prostephanus truncatus (Horn) (Col.: Bostrychidae) it was shown that both larvae and adults have a pair of bacteriomes dorsally located in the fat body parallel to the midgut. Bacteriome development was shown mainly to occur during larval stages. Bacteriome size was not found to be associated with body size in adults, but in larvae reared at 30°C bacteriome size increased progressively with body length. The shape of the bacteriomes varied from round to conic-oval, but a common feature was that they were larger and rounded in older larvae and females as compared to males, where they usually appeared more shrunken and slightly deformed. Electron microscopy of thin sections showed that the bacteriomes were composed of multinucleate syncytia surrounded by a layer of boundary cells. The syncytia harboured many small coccoid bacteroids. Typical eukaryotic organelles were found in the cytoplasm of the bacteriomes. These and other structural features were outlined. The effect of rearing temperatures at 30, 35 and 37°C on bacteriome development in larvae and adults was examined. The symbiotes could not be eliminated but a significant reduction of bacteriome size was found in females reared at 35°C and 37°C as compared to specimens grown at 30°C. A possible association of bacteriome size and reproduction was evaluated by transferring P. truncatus specimens reared at 35°C and 37°C to 30°C for two months and counting the number of offspring; their reproduction was compared with controls kept at 30°C throughout the experiment. Specimens from 35°C and 37°C had significantly lower reproduction rates than controls. The potential implications of heat sensitivity of bacteriomes of P.truncatus is discussed in an integrated pest management context.     

Survival of Vibrio parahaemolyticus in Oyster Shellstock at Two Different Storage Temperatures

In oyster shellstock harvested from Maryland waters Vibrio parahaemolyticus was found to be present, to survive storage for at least 3 weeks at 4 C, and to multiply after being held for 2 to 3 days at 35 C.     

Gross Damage Accumulation in Frozen Rabbit Liver Due to Mechanical Stress at Cryogenic Temperatures

The second phase of a pilot study dealing with the mechanical response of frozen biological tissues to external compressive load is presented. This stage deals with histological observations of the damage accompanying mechanically induced permanent deformation in frozen rabbit liver. No significant gross histological damage was observed in the liver samples due to either processing the tissue in the frozen state, due to slow cooling of the liver tissues down to −20°C, or due to rapid cooling of the samples down to −196°C. No histological changes were observed in tissue samples that were loaded within the elastic regime, that is, below the yield strength of the material. Therefore, it is concluded that histological changes due to mechanical stresses are associated with plastic (permanent) deformations. Histological observations indicate that linear cracks which appear to have no preferred orientation develop due to mechanical stress beyond the yield strength of the frozen tissue. These cracks accumulate until final failure of the frozen tissue, when the tissue sample collapses to rubble. Based on histological observations and concepts from solid mechanics, an interaction between crack formation and irregularities in the frozen medium is suggested. Significant sources for such irregularities, in an homogeneous tissue such as the liver, are blood vessels and bile ducts. These irregularities may either initiate crack formation or, on the other hand, may also arrest propagating cracks.     

Survival of Vibrio parahaemolyticus in Fish Homogenate During Storage at Low Temperatures

Fish homogenate inoculated with Japanese strains of Vibrio parahaemolyticus were either stored at 0.6 C or frozen and stored at -18 and -34 C. Greater survival of the organisms was obtained at 0.6 C than at the lower temperatures.     

Role of Suspending and Recovery Media in the Survival of Frozen Shigella sonnei

Shigella sonnei was frozen at -20 C in saline, nutrient broth, and milk, and plated, after thawing, upon synthetic medium, nutrient agar, and blood heart infusion agar. There was a difference in the numbers of cells recovered when the frozen and thawed cells were grown on different media. The synthetic medium was unable to recover cells injured by freezing or did so only poorly compared to the complex media. The addition of meat extract, peptone, or Casamino acids to the synthetic medium improved its ability to recover injured cells as measured by bacterial colony counts. This is suggestive of metabolic injury caused by the freezing processes since the cells which survived freezing required an enriched medium for growth. In this paper the term metabolic injury is used to express a change in the nutritional requirements of the organisms which resulted in an increase in growth factor requirements. Freezing the cells in saline resulted in greater injury compared to cells frozen in nutrient broth or milk; this suggested that these suspending agents possessed some protective quality. The metabolic injury increased with an increase in the length of time the cells were held in the frozen state.     

On the Fertility and Survival of Deep Frozen Boar Spermatozoa Thawed in Skim Milk

Satisfactory conception rates of deep frozen boar spermatozoa were obtained, with insemination by way of the cervix, after thawing the deep frozen spermatozoa in boar seminal plasma, both in preliminary trials (Crabo & Einarsson 1971, Crabo et al. 1972 b) and in a large field trial (Einarsson et al. 1972). Fertility with pellet frozen boar spermatozoa, thawed without dilution, was reported by Graham et al. (1971 a, b) and Pursel & Johnson (1971).     

Effect of Conditioning Treatments on the Survival of Radopholus similis at High Temperatures

Heat treatments are an environmentally safe method for eliminating quarantine pests from tropical foliage. Conditioning heat treatments can induce thermotolerance against subsequent and otherwise phytotoxic temperatures in tropical foliage, allowing heat treatments to be even more effective. However, if thermotolerance is also induced in nematodes of quarantine significance like Radopholus similis, heat treatments would be rendered ineffective. A lethal thermal death point (LT_99.9) was established for R. similis by recording mortality at 25 (control temperature), 43°C, 45°C, 47°C, or 49°C after a 0, 1-, 2-, 4-, 6-, 8-, 10-, 12-, or 15-minute exposure. In a second experiment, nematodes were conditioned at 35, 40, or 45°C for 0, 15, 30, 60, 120, and 180 minutes, allowed to rest for 3 hours, and then challenged at 47°C for 5 minutes. No nematodes survived the challenge heat treatment; rather, nematode mortality was hastened by the conditioning treatment itself. In a third experiment, R. similis inside anthurium roots were conditioned at 25°C or 40°C for 15 minutes and then treated at 45°C for up to 8 minutes. Mortality of conditioned and unconditioned nematodes was similar (P > 0.1). Conditioning treatments increase plant thermotolerance but do not induce thermotolerance in R. similis. Heat treatments have promise as disinfection protocols for quarantines.