Cold adaptation strategy in insects inhabiting central Yakutia

Cold hardiness in 20 insect species living in extremely cold climate of Yakutia has been investigated for the first time. It was shown that the Yakutian insects prefer to use the strategy of freeze tolerance according to which they produce special substances initiating the freezing of hemolymph at high subzero temperatures. The presence of ice-nucleating agents in the haemolymph of insects belonging to the phylogenetic group of Lepidopteran was shown. We postulate that Pieris rapae may shift between the different cold hardiness strategies when they move from moderately cold regions to a more severe environment.     

Isolation and identification of an ice-nucleating bacterium from the gills of the intertidal bivalve mollusc Geukensia demissa

In the fall, freeze tolerant intertidal invertebrates usually produce ice-nucleating proteins that are secreted into the hemolymph. These proteins help protect against freeze damage by insuring that ice formation is limited to extracellular spaces. Geukensia demissa, a freeze tolerant, salt marsh bivalve mollusc was examined for the presence of ice nucleating proteins. The ice-nucleating temperature (INT) of the hemolymph was not significantly different from artificial seawater of the same salinity indicating the lack of an ice nucleating protein in the hemolymph. The palial fluid did have an elevated INT, indicating the presence of an ice nucleator. The INT of the palial fluid was significantly reduced by boiling and filtration through a 0.45-&mgr;m filter. High INT was also observed in the seawater associated with the bivalves, and was demonstrated in water samples collected from salt marshes but not sand and pebble beaches. Moreover, the INT of water samples collected from a salt marsh decreased in the summer. All of these data suggest that the ice-nucleating agents in the hemolymph and the seawater are ice-nucleating bacteria. One species of ice-nucleating bacteria, Pseudomonas fulva was isolated from the gills of Geukensia. These bacteria could perform the same function as hemolymph ice-nucleating proteins by limiting ice formation to extracellular compartments.     

Freeze Concentration of Some Foodstuffs Using Ice Nucleation-active Bacterial Cells Entrapped in Calcium Alginate Gel

Cells of an ice nucleation-active strain of Ermnia ananas were entrapped in calcium alginate to prepare an ice-nucleating gel usable as ice nuclei for freeze concentration. The ice-nucleating gel was also adjusted as to specific gravity. When it was placed at a desired position in a liquid material such as egg white, ice formed at this position as the material was cooled. It was possible to put the ice- nucleating gel in liquid foodstuffs such as egg white and lemon juice before their temperatures reached subzero points. Application of this method produced freeze-concentrated foods whose properties were not significantly deteriorated.     

Climatic variability and the evolution of insect freeze tolerance

Insects may survive subzero temperatures by two general strategies: Freeze-tolerant insects withstand the formation of internal ice, while freeze-avoiding insects die upon freezing. While it is widely recognized that these represent alternative strategies to survive low temperatures, and mechanistic understanding of the physical and molecular process of cold tolerance are becoming well elucidated, the reasons why one strategy or the other is adopted remain unclear. Freeze avoidance is clearly basal within the arthropod lineages, and it seems that freeze tolerance has evolved convergently at least six times among the insects (in the Blattaria, Orthoptera, Coleoptera, Hymenoptera, Diptera and Lepidoptera). Of the pterygote insect species whose cold-tolerance strategy has been reported in the literature, 29% (69 of 241 species studied) of those in the Northern Hemisphere, whereas 85 % (11 of 13 species) in the Southern Hemisphere exhibit freeze tolerance. A randomization test indicates that this predominance of freeze tolerance in the Southern Hemisphere is too great to be due to chance, and there is no evidence of a recent publication bias in favour of new reports of freeze-tolerant species. We conclude from this that the specific nature of cold insect habitats in the Southern Hemisphere, which are characterized by oceanic influence and climate variability must lead to strong selection in favour of freeze tolerance in this hemisphere. We envisage two main scenarios where it would prove advantageous for insects to be freeze tolerant. In the first, characteristic of cold continental habitats of the Northern Hemisphere, freeze tolerance allows insects to survive very low temperatures for long periods of time, and to avoid desiccation. These responses tend to be strongly seasonal, and insects in these habitats are only freeze tolerant for the overwintering period. By contrast, in mild and unpredictable environments, characteristic of habitats influenced by the Southern Ocean, freeze tolerance allows insects which habitually have ice nucleators in their guts to survive summer cold snaps, and to take advantage of mild winter periods without the need for extensive seasonal cold hardening. Thus, we conclude that the climates of the two hemispheres have led to the parallel evolution of freeze tolerance for very different reasons, and that this hemispheric difference is symptomatic of many wide-scale disparities in Northern and Southern ecological processes.     

Host‐mediated shift in the cold tolerance of an invasive insect

While many insects cannot survive the formation of ice within their bodies, a few species can. On the evolutionary continuum from freeze‐intolerant (i.e., freeze‐avoidant) to freeze‐tolerant insects, intermediates likely exist that can withstand some ice formation, but not enough to be considered fully freeze tolerant. Theory suggests that freeze tolerance should be favored over freeze avoidance among individuals that have low relative fitness before exposure to cold. For phytophagous insects, numerous studies have shown that host (or nutrition) can affect fitness and cold‐tolerance strategy, respectively, but no research has investigated whether changes in fitness caused by different hosts of polyphagous species could lead to systematic changes in cold‐tolerance strategy. We tested this relationship with the invasive, polyphagous moth, Epiphyas postvittana (Walker). Host affected components of fitness, such as larval survivorship rates, pupal mass, and immature developmental times. Host species also caused a dramatic change in survival of late‐instar larvae after the onset of freezing—from less than 8% to nearly 80%. The degree of survival after the onset of freezing was inversely correlated with components of fitness in the absence of cold exposure. Our research is the first empirical evidence of an evolutionary mechanism that may drive changes in cold‐tolerance strategies. Additionally, characterizing the effects of host plants on insect cold tolerance will enhance forecasts of invasive species dynamics, especially under climate change.     

Physiological ecology of overwintering in hatchling turtles

Temperate species of turtles hatch from eggs in late summer. The hatchlings of some species leave their natal nest to hibernate elsewhere on land or under water, whereas others usually remain inside the nest until spring; thus, post-hatching behavior strongly influences the hibernation ecology and physiology of this age class. Little is known about the habitats of and environmental conditions affecting aquatic hibernators, although laboratory studies suggest that chronically hypoxic sites are inhospitable to hatchlings. Field biologists have long been intrigued by the environmental conditions survived by hatchlings using terrestrial hibernacula, especially nests that ultimately serve as winter refugia. Hatchlings are unable to feed, although as metabolism is greatly reduced in hibernation, they are not at risk of starvation. Dehydration and injury from cold are more formidable challenges. Differential tolerances to these stressors may explain variation in hatchling overwintering habits among turtle taxa. Much study has been devoted to the cold-hardiness adaptations exhibited by terrestrial hibernators. All tolerate a degree of chilling, but survival of frost exposure depends on either freeze avoidance through supercooling or freeze tolerance. Freeze avoidance is promoted by behavioral, anatomical, and physiological features that minimize risk of inoculation by ice and ice-nucleating agents. Freeze tolerance is promoted by a complex suite of molecular, biochemical, and physiological responses enabling certain organisms to survive the freezing and thawing of extracellular fluids. Some species apparently can switch between freeze avoidance or freeze tolerance, the mode utilized in a particular instance of chilling depending on prevailing physiological and environmental conditions.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Long-term reduction of cold hardiness following ingestion of ice-nucleating bacteria in the Colorado potato beetle,Leptinotarsa decemlineata

We investigated the effect of ingestion of ice-nucleating bacteria on the supercooling capacity and cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a freeze-intolerant species that overwinters as adults in shallow, terrestrial burrows. Ingestion of ice-nucleating bacteria (Enterobacter agglomerans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae), fed on slices of potato tuber, caused an abrupt decrease in supercooling capacity. No change occurred in the supercooling capacity of beetles fed Escherichia coli, as this species lacks ice-nucleating activity. Ingestion rates showed that tubers treated with different species were equally palatable. During diapause induction beetles evacuated food from their guts, but nevertheless retained sufficient ice-nucleating bacteria to diminish supercooling. Beetles fed P. fluorescens and P. putida exhibited reduced supercooling even after an 8-wk exposure to simulated winter conditions. Furthermore, P. fluorescens was isolated 10-wk post-ingestion from diapausing beetles. Our data suggest that ingested bacteria may be retained by insects during entry into diapause and that the cold hardiness of candidate crop pests, such as L. decemlineata, may be reduced by feeding them ice-nucleating bacteria prior to winter diapause.     

A simple moder for estimating the ice content of freezing ectotherms

1. 1.Although body ice content is an important variable affecting freeze tolerance, present calorimetric methods for its measurement necessarily require the termination of a freezing protocol.

2. 2.A simple iterative model, based on the colligative properties of solutions and requiring precise measurements of only equilibrium freezing point (of the unfrozen organism) and of core body temperature, allows estimation of the percentage of body water frozen at any time during a freezing episode.

3. 3.This model can also predict the lethal temperature for a freezing ectotherm, assuming that death occurs due to osmotic dehydration when 67% (of any other known lethal fraction) of the body water is frozen.

4. 4.The basic model is easily extended to evaluate the effects of variables such as: body mass, initial body water content, initial osmotic concentration, and test chamber microenvironment.

5. 5.This model is not intended to supplant existing more exact biophysical models of freezing kinetics. Rather it is proposed as a first approximation which is generally supported by published data and which should be of significant practical value for investigators of freeze tolerant organisms.

Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze‐drying microscopy (FDM) method for a variety of cell culture medium‐based solutions (with 0.05–0.8 M trehalose) that are important for long‐term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze‐drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water‐trehalose solutions was found to be similar to the glass transition temperature (T′_g ～ ?30°C) of the maximally freeze‐concentrated trehalose solution (～80 wt% trehalose) during the freezing step of freeze‐drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium‐based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as ?65°C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (?4 and ?7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze‐drying of living cells. We further confirmed the applicability of the collapse temperature measured with small‐scale (2 µL) samples using the FDM system to freeze‐drying of large‐scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze‐drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell‐based medicine. Biotechnol. Bioeng. 2010;106: 247–259. © 2010 Wiley Periodicals, Inc.     

Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40

Aims: The effects of different freeze‐drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated. Method and Results: Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze‐drying. The subsequent survival rates during storage and rehydration media of freeze‐dried biocontrol strains were also examined. Conclusions: The results indicated that cellobiose (5%) and d ‐galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze‐drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months. Significance and Impact of the study: This study showed that freeze‐drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale‐up possibilities and formulation development.     

Spirulina enhances the viability of Lactobacillus rhamnosus E/N after freeze-drying in a protective medium of sucrose and lactulose

Aims: Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E/N cells during lyophilization. Methods and Results: Spirulina, sucrose and lactulose were selected, on the basis of a Plackett‐Burman factorial design, as important protectants having the following protective effects on cell viability: 102·025, 36·885 and ?34·42, respectively. A full‐factorial central composite design was applied to determine optimal levels of three used agents. Conclusion: The optimal protective medium composition was determined to be: Spirulina 1·304% (w/v), lactulose 5·48% (w/v), and sucrose 13·04% (w/v) (Polish Patent P‐393189). The predictive value of cell viability in this medium was 89·619%, and experimental viability obtained during freeze‐drying was 87·5%. Significance and Impact of the Study: In this study, Spirulina was used for the first time as the protective agent in freeze‐drying medium, significantly increasing lactobacilli viability and giving synbiotic character of the final product.     

Phenotypic plasticity,but not adaptive tracking,underlies seasonal variation in post‐cold hardening freeze tolerance of Drosophila melanogaster

In temperate regions, an organism's ability to rapidly adapt to seasonally varying environments is essential for its survival. In response to seasonal changes in selection pressure caused by variation in temperature, humidity, and food availability, some organisms exhibit plastic changes in phenotype. In other cases, seasonal variation in selection pressure can rapidly increase the frequency of genotypes that offer survival or reproductive advantages under the current conditions. Little is known about the relative influences of plastic and genetic changes in short‐lived organisms experiencing seasonal environmental fluctuations. Cold hardening is a seasonally relevant plastic response in which exposure to cool, but nonlethal, temperatures significantly increases the organism's ability to later survive at freezing temperatures. In the present study, we demonstrate seasonal variation in cold hardening in Drosophila melanogaster and test the extent to which plasticity and adaptive tracking underlie that seasonal variation. We measured the post‐cold hardening freeze tolerance of flies from outdoor mesocosms over the summer, fall, and winter. We bred outdoor mesocosm‐caught flies for two generations in the laboratory and matched each outdoor cohort to an indoor control cohort of similar genetic background. We cold hardened all flies under controlled laboratory conditions and then measured their post‐cold hardening freeze tolerance. Comparing indoor and field‐caught flies and their laboratory‐reared G1 and G2 progeny allowed us to determine the roles of seasonal environmental plasticity, parental effects, and genetic changes on cold hardening. We also tested the relationship between cold hardening and other factors, including age, developmental density, food substrate, presence of antimicrobials, and supplementation with live yeast. We found strong plastic responses to a variety of field‐ and laboratory‐based environmental effects, but no evidence of seasonally varying parental or genetic effects on cold hardening. We therefore conclude that seasonal variation in post‐cold hardening freeze tolerance results from environmental influences and not genetic changes.     

Mechanisms underlying insect freeze tolerance

Freeze tolerance – the ability to survive internal ice formation – has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze‐tolerant insects accumulate many potentially protective molecules, there is no apparent ‘magic bullet’ – a molecule or class of molecules that appears to be necessary or sufficient to support this cold‐tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze‐tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze‐tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post‐thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold‐ and desiccation‐tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold‐tolerance strategy that has evolved multiple times, we suggest that a process‐focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis‐driven research to understand better how insects survive internal ice formation.     

Freezing and freeze-drying of the bacterium Rahnella aquatilis BNM 0523: study of protecting agents, rehydration media and freezing temperatures

Aims: The aim of the present study was to evaluate and compare freezing and freeze‐drying treatments for conserving Rahnella aquatilis (BNM 0523) with the goal to achieve an adequate commercial formulation of this biocontrol agent. Methods and Results: The effect of several protective agents, rehydration media and freezing temperatures on the viability and functional activity of the R. aquatilis was investigated. The storage stability at 3 months and 4 years was determined by checking the viability of the cells and their biocontrol capability against Botrytis cinerea by measuring the percentage of reduction of disease severity on apple. The best results were obtained by the freeze‐drying of the cells using a mixture of skimmed nonfat milk 10%, yeast extract 0·5% and glucose 1% as the protecting and rehydrating medium, and a quickly freezing (?70°C) before the freeze‐drying. In this case, the viability of the cells after 4 years was 98%, and their antagonistic ability showed a little decrease with respect fresh cells. Conclusions: The studies showed that R. aquatilis was resistant to freezing and freeze‐drying when it was used a mixture of cryoprotectants and that it was possible to obtain inoculums with high viability and good effectiveness for reduction of decay caused by B. cinerea. Significance and Impact of the study: This study is probably the first report about the resistance of R. aquatilis to freezing and freeze‐drying treatments and shows that these operations could be useful for obtaining a commercial formulation of this biocontrol agent.     

Effect of an Ice-Nucleating Activity Agent on Subzero Survival of Nematode Juveniles

Juveniles of five species of nematodes, Caenorhabditis elegans, Panagrellus redivivus, Pratylenchus agilis, Pristionchus pacificus, and Distolabrellus veechi, were added to solutions with (treatment) and without (control) a commercial ice-nucleating activity (INA) agent. Ten-microliter droplets of the solutions containing the juveniles were placed on glass microscope slides and transferred to a temperaturecontrolled freeze plate where the temperature was reduced to -6 to -8 °C. At this temperature, the droplets containing the INA agent froze while those without the agent remained liquid. After 2 minutes, the temperature of the plate was raised to 24 °C, and the slides were examined with a light microscope to determine the viability of the juveniles. The results showed that usually most juveniles (43% to 88%, depending on species) in solutions that did not contain the INA agent (controls) were active, indicating that the juveniles were capable of supercooling and were thereby protected from the subzero temperatures. Alternatively, less than 10% of the juveniles that had frozen for 2 minutes in solutions containing the INA agent remained viable, indicating that inoculative freezing of the solution was lethal to the supercooled juveniles. Our results suggest that, in geographical areas where winter temperatures may not be sufficiently low or sustained to freeze soil, the addition of an INA agent may help induce ice nucleation and thereby reduce the populations of nematode species that are unable to survive when the soil solution is frozen.     

Russian Wheat Aphid,Diuraphis noxia in the Czech Republic – cause of the significant population decrease

Diuraphis noxia is a newly established alien pest in the Czech Republic. There were two population explosions during the period 1993–2012. A successful overwintering of the anholocyclic populations of D. noxia was the cause of a population explosion in the oncoming growth season. Viviparous parthenogenetic females of the anholocyclic populations and diapausing eggs of holocyclic populations could overwinter at the same time. Analysis of the course of winter temperatures showed that a complete winter mortality of anholocyclic populations depended mainly on the duration of the freezing period, when temperature dropped below ?5°C, and on the intensity of the frost. We proposed the cumulative effective freeze (in minus degree days) as a key parameter for the aphid anholocyclic population's mortality during overwintering. This parameter equalled ?10.1 degree days for relatively warm winter period 2006–2007, whereas in the relatively cold winter period of 2009–2010, this parameter reached ?87.1 DD, which caused complete mortality of anholocyclic populations. A cumulative effective freeze could be used for short‐time prognosis of D. noxia population explosion.     

冰核真菌削弱赤拟谷盗抗寒力的初步研究

赤拟谷盗Tribolium castaneum是不耐结冰的害虫,在冬季它通过降低过冷却点以避免结冰造成的致命伤害。冰核活性细菌能显著提高昆虫的过冷却点,使之在较高的零下温度发生结冰。试验证明冰核活性真菌也能显著提高赤拟谷盗的过冷却点。对照组平均过冷却点为-14.9℃。用10 g/L的冰核真菌制剂喷洒虫体,风干后测定,平均过冷却点提高到-4.8℃。用0.1 g/L处理后至少在7天内过冷却点保持较高。这些结果表明冰核真菌可能成为一种在冬季使用的、防治不耐结冰害虫的促冻杀虫剂。     

Freezing induces a loss of freeze tolerance in an overwintering insect

Cold-hardy insects overwinter by one of two main strategies: freeze tolerance and freeze avoidance by supercooling. As a general model, many freeze-tolerant species overwinter in extreme climates, freeze above -10 degrees C via induction by ice-nucleating agents, and once frozen, can survive at temperatures of up to 40 degrees C or more below the initial freezing temperature or supercooling point (SCP). It has been assumed that the SCP of freeze-tolerant insects is unaffected by the freezing process and that the freeze-tolerant state is therefore retained in winter though successive freeze-thaw cycles of the body tissues and fluids. Studies on the freeze-tolerant larva of the hoverfly Syrphus ribesii reveal this assumption to be untrue. When a sample with a mean 'first freeze' SCP of -7.6 degrees C (range of -5 degrees C to -9.5 degrees C) were cooled, either to -10 degrees C or to their individual SCP, on five occasions, the mean SCP was significantly depressed, with some larvae subsequently freezing as low as -28 degrees C. Only larvae that froze at the same consistently high temperature above -10 degrees C were alive after being frozen five times. The wider occurrence of this phenomenon would require a fundamental reassessment of the dynamics and distinctions of the freeze-tolerant and freeze-avoiding strategies of insect overwintering.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.