Effects of Nutrition on Desiccation Tolerance and Virulence of Colletotrichum truncatum and Alternaria alternata Conidia

The promising mycoherbicides Colletotrichum truncatum and Alternaria alternata were grown respectively in liquid and solid semi-defined media. C. truncatum conidia produced in a medium with a C:N ratio of 5:1 showed higher desiccation tolerance (survival during storage) at 15% relative humidity and 25°C, greater germination on the host leaf and greater disease expression on Sesbania exaltata than those produced in media with C:N ratios of 15:1 or 40:1. Similar results were obtained with conidia of A. alternata produced on a medium with a C:N ratio of 15:1. Conidia washed with 0.9% (w/v) NaCl produced higher tolerance to desiccation, and greater disease incitement, than unwashed conidia of C. truncatum or conidia washed with water. In contrast, washing had no positive effect on desiccation tolerance in A. alternata .     

Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture

The surface hydrophobicity, viability and biocontrol ability of Penicillium oxalicum spores, produced either in aerial or submerged culture, were characterized. A phase distribution test showed that spores produced in both methods of culture were highly hydrophobic, but those produced in aerial culture were more hydrophobic. Spores stored fresh at either 4 or 25 degrees C retained a high viability (80%) after 27 weeks of storage, although aerial spores survived better. Freeze-drying severely affected viability, especially of submerged spores. Biocontrol ability against Fusarium oxysporum f. sp. lycopersici was studied in the growth chamber. Aerially- produced spores were more effective than submerged ones. Aerially-produced P. oxalicum spores appeared to have more advantages than those produced by submerged culture, in relation to both viability and efficacy. These results demonstrate that physiological changes occur depending on production conditions which significantly influences quality of the biocontrol agent.     

Tolypocladium cylindrosporum (Deuteromycotina:Moniliales), a fungal pathogen of the mosquito Aedes australis

A laboratory fermenter was used to produce up to 12 l of infective Tolypocladium cylindrosporum blastoconidia in Sabouraud dextrose broth. Two media derived from coconuts were also demonstrated as suitable alternative systems for the production of viable blastoconidia. T. cylindrosporum conidia when dried at 37 degrees C and stored at 4 degrees C retained their viability for 10 months, but, when stored at 25 degrees C, the conidia lost viability after 2 months and blastoconidia did not survive the drying process. Distilled water suspensions were a simple, economic technique for the long-term storage of spores at both 4 and 25 degrees C. The adsorption of conidia onto silica gel crystals was a very suitable technique for the storage of stock culture material at 4 degrees C. The virulence, production and storage capabilities of both spore types were examined.     

Solid Substrate Formulations of the Mycoherbicide Colletotrichum truncatum for Hemp Sesbania ( Sesbania exaltata ) Control

Colletotrichum truncatum was grown on kernels of eight different grains for 3 or 4 weeks at room temperature (22-24°C). Fresh preparations of conidia as well as fungus-infested corn and rice suspensions resulted in 100% mortality of hemp sesbania seedlings when sprayed postemergence with a 14 h dew period. Fresh preparations of mycelia and fungus-infested sorghum suspensions resulted in 90 and 65% mortality of hemp sesbania seedlings, respectively. Lower mortality ( ≤15%) occurred with the other ground fungus-infested grain suspensions. Fresh preparations of conidia, fungus-infested corn, rice and sorghum, and mycelia, when applied to soil pre-emergence, resulted in 100, 94, 100, 83 and 71% mortality of hemp sesbania seedlings 14 days after application, respectively. Lower mortality ( ≤23%) occurred with the other ground fungus-infested grain preparations. Freshly-prepared C. truncatum at 6.25, 12.5, 25 and 50 mg fungus-formulated rice cm -2 of soil surface, applied pre-emergence or at the time of planting, killed 97, 100, 100 and 100% of hemp sesbania, respectively. After storage at 22-24°C for 6 to 24 months, the rice formulation caused 67 to 93% mortality after 6 months, 39 to 81% after 12 months, and ≤2% after 24 months, respectively. When C. truncatum was refrigerated at 4-6°C, the rice formulation retained good efficacy through 24 months, and when frozen, for up to 8 years. C. truncatum formulated on rice stored under all the above conditions contained mainly sclerotia at 2.4 x 10 5 sclerotia g -1 . C. truncatum killed hemp sesbania seedlings with a single soil application through 4 plantings on the same soil. These results indicate that rice and possibly corn are excellent solid substrates for the formulation of C. truncatum . This is a simple and effective method for enhancing the activity of C. truncatum against hemp sesbania.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Production and Pathogenicity to Wheat of Pseudocercosporella herpotrichoides Conidia

Weekly estimates of numbers of Pseudocercosporella herpotrichoides conidia on naturally infected wheat straw, made from February to July 1982, showed there were most conidia (8.1 × 10⁶ per straw) in February and least (1.9 × 10⁴ per straw) at the end of June. The viability of these spores remained high throughout this period, with an average of 85 % germination after 24 h.
After removal of spores produced in the field, straws were incubated at 5, 10, 15, 20 or 25°C and subsequent sporulation assessed after 3 or 5 weeks. The optimum temperature for spore production was 5°C and very few spores were produced at 25°C. There was no difference in viability between spores produced at different temperatures.
Wheat seedlings placed amongst infected straw collected and retained spores on the upper and lower surfaces of all leaf blades and on outer leaf sheaths. Both naturally dispersed spores and spores sprayed on to plants were not removed by subsequent rainfall.
When wheat seedlings were inoculated between the coleoptile and outer leaf sheath with different numbers of P. herpotrichoides spores, lesion development was most rapid in seedlings inoculated with the greatest numbers of spores. However, after incubation for 12 weeks visible lesions were present on all plants inoculated with > c. 10 spores.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Liquid formulation of the biocontrol agent Candida sake by modifying water activity or adding protectants

AIMS: To evaluate the effect of modification of water activity (aw) and the addition of protective substances in the preservation medium of liquid formulations of the biocontrol agent Candida sake stored at 4 and 20 degrees C. METHODS AND RESULTS: The aw of the preservation medium of C. sake was modified from 0.72 to 0.95 by adding glycerol or polyethylene glycol (PEG). Moreover, several protectant substances at different concentrations were evaluated. Modification of lower aw-levels (0.721-0.901) with glycerol did not maintain the viability of the yeast cells. Higher aw-levels (0.93-0.95) with either glycerol or PEG improved the viability but not at acceptable viability levels. C. sake cells maintained viabilities >60% when sugars, such as trehalose, and polyols, such as glycerol and PEG were used as protectants in liquid formulations. Moreover, liquid formulations of C. sake stored at 4 degrees C showed higher number of viable counts than at 20 degrees C. When different sugars were tested, all of them, except 10% fructose, resulted in a viability higher than 50% of the C. sake formulations. Biocontrol of liquid formulation treatments was similar to fresh cells in controlling Penicillium expansum on wounded apples. CONCLUSIONS: Sugars such as lactose and trehalose could be considered as good protectants in order to obtain liquid formulations of C. sake cells as they maintain the viability >70% for 4 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF STUDY: This study shows that a suitable liquid formulation for commercial application can be produced with high viability and conservation of biocontrol efficacy. Moreover, if 10% lactose is the protectant used in the formulation, the economic costs would not be limiting for industrial production.     

Microscopic and Cytochemical Analysis of Extracellular Matrices and Endogenous Reserves of Conidia of Colletotrichum truncatum Harvested from Carbon- and Nitrogen-limited Cultures

The effects of nutrition during conidiation on extracellular matrices (ECM) and endogenous reserves of conidia of the promising mycoherbicide Colletotrichum truncatum have been examined. Transmission electron microscopy showed no ECM on the conidia, regardless of the C:N ratio (10:1, 30:1 or 80:1) of the medium on which they were produced. Cytochemical analysis using fluorochrome-labelled lectins revealed the presence of specific sugars in ECM around the germlings. Furthermore, ECM-containing amino groups (basic protein) were detected using colloidal gold. However, nutrition during conidiation had no effect on the patterns of lectin labelling or the pattern of colloidal gold staining of germlings. The relative amounts of trehalose, glycerol and mannitol in conidia produced in a liquid medium with a C:N of 10:1 were more than those obtained when the C:N was 30:1 or 80:1. Thus, internal carbohydrates such as trehalose and polyols may play an important role in viability of conidia during long-term storage.     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.     

Survival of Brucella abortus strain RB51 lyophilized and as liquid vaccine under different storage conditions.

Brucella abortus strain RB51 (SRB51) is a new cattle vaccine that is approved for use in the U.S. for prevention of brucellosis. At the present time, other countries are implementing or considering the use of SRB51 vaccine in their brucellosis control programs. In the current study, the effect of three stabilizing media, two fill volumes (1 and 3 ml), and three storage temperatures (-25, 4 and 25 degrees C) on the viability of lyophilized SRB51 over a 52 week period was determined. The effects of three concentrations of bacteria (5 x 10(8), 1 x 10(9), or 5 x 10(9) cfu/ml) and two storage temperatures (4 or 25 degrees C) on viability of liquid SRB51 vaccine were also determined. For lyophilized strain RB51 vaccine, fill volume did not influence viability (P> 0.05) during lyophilization. Although fill volume did not influence viability during storage in World Health Organization (WHO) media or media containing both WHO and Lactose Salt (LS) media, 1 ml fill volumes of SRB51 in LS media had greater (P< 0.05) viability when compared to 3 ml fill volumes. Lyophilized SRB51 vaccine stored at 25 degrees C had a more rapid decline in viability (P< 0.05) when compared to vaccine stored at -25 or 4 degrees C. With the exception of the 3-ml fill volumes of LS media, all three stabilizing media were similar in maintaining viability of SRB51 at -25 degrees C storage temperatures. However, when compared to WHO or WHO/LS media, stabilization in LS media was associated with a more rapid decline in viability during storage at 4 or 25 degrees C (P< 0.05). Initial SRB51 concentration in liquid vaccine did not influence (P> 0.05) viability during storage at 4 or 25 degrees C. When compared to liquid SRB51 vaccine stored at 25 degrees C, storage at 4 degrees C was associated with a slower decline in viability (P< 0.05) during 12 weeks of storage. Biochemical and morphological characteristics of SRB51 were stable under the storage conditions utilized in the present study. This study suggests that viability of SRB51 can be readily maintained during storage as a lyophilized or liquid brucellosis vaccine.     

Production of the fungal biocontrol agent Ulocladium atrum by submerged fermentation: accumulation of endogenous reserves and shelf-life studies

A method was developed for the induction of submerged conidiation of Ulocladium atrum Preuss (isolate 385) for the first time, using an oatmeal extract broth. Two inoculum types were produced by this process: spores and mycelial fragments. Spore production was stimulated by reducing the broth water potential (psi) to -2.1 MPa and adding 20 mM calcium chloride. In contrast, mycelial fragments were dominant at -7.0 MPa psi. Maximum total inoculum (mycelial fragments and conidia) yields were approximately 2 x 10(7) ml(-1) after 9 days incubation at 25 degrees C at 100 rpm. Biomass from liquid cultures responded to water-stress by accumulating increased concentrations of endogenous sugar alcohols (polyols), particularly glycerol. Long-term shelf-life studies showed that submerged inoculum from cultures subjected to an intermediate water-stress (-2.1 MPa psi) and containing enhanced levels of glycerol (> 300 mg g(-1) freeze-dried material) retained viability significantly better (P < 0.05) than that from unstressed cultures, when assessed on agar with fully available water. This level of viability was comparable to that of aerial U. atrum spores from a 4-week solid-substrate fermentation on oat grains. However, in contrast to aerial spores, the ability of submerged biomass to germinate in drier conditions declined significantly after 6 months.     

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.     

Water Activity and Other Factors that Affect the Viability of Colletotrichum truncatum Conidia in Wheat Flour-Kaolin Granules ('Pesta')

Optimization of shelf-life is critically important for biocontrol products containing living microorganisms. Conidia of Colletotrichum truncatum, a fungal pathogen of the weed, hemp sesbania (Sesbania exaltata), were produced in shake flasks (corn meal-soya flour medium) and on Emerson Yp Ss agar and formulated in wheat flour-kaolin granules ('Pesta'). The granules were conditioned at water activities of 0, 0.12, 0.33, 0.53 and 0.75 during storage at 25 C over desiccant or saturated salt solutions. The longest shelf-life (conidial inoculum viability) was found in samples in the water activity range 0-0.33, where the water was bound by the matrix and not readily available to the fungus. At a water activity of 0.12, granules were 100% viable (on water agar) for at least 24 weeks, and were 87% viable after 1 year. Sucrose (5% w/w) partially counteracted the detrimental effect of high water activity on the shelf-life of C. truncatum when incorporated in the granules.     

Stability of microsclerotial inoculum of Colletotrichum truncatum encapsulated in wheat flour–kaolin granules

Maintaining adequate viability of microorganisms in products for biocontrol is critically important for commercial reasons. Microsclerotia (MS) of the mycoherbicide agent, Colletotrichum truncatum, are its hardy, over-wintering fungal structures. Microsclerotial inoculum at 2, 7, and 23 MS/granule were matrix-encapsulated in wheat flour–kaolin granules (Pesta), in which the flour provided gluten for the matrix and a food base for the fungus. Pesta granules were dried to a water activity of 0.18–0.29. After storage for 52 weeks at 25°C, granules containing 7 and 23 MS were 100% viable and granules with 2 MS were 95% viable. Granules with 7 MS were 50% viable after 36 weeks at 35°C. Pesta granules (440 granules/g) with conidial inoculum at 3.3×105 c.f.u./g were less storage-stable than granules at the 2 MS/granule level. At all MS inoculum levels, granules stored for up to 2years produced 108 c.f.u./g in vitro when incubated on water agar. High water activity was detrimental to long-term viability. In the greenhouse, 7 MS/granule samples controlled 94% of hemp sesbania when incorporated into the soil pre-planting. The strategy of encapsulation of the naturally stable C. truncatum MS and drying to a favourable water activity led to excellent shelf-life for a live biocontrol agent.     

Evaluation on sperm quality of freshly ejaculated boar semen during in vitro storage under different temperatures

The purpose of this study was to assess the sperm quality of fresh ejaculated boar semen stored under different temperatures for up to 48 h in order to use the fresh semen efficiently. Spermatozoa were evaluated by 4 methods: Using trypan blue staining, the viability of spermatozoa stored at 39, 20, 15 and 4 degrees C for 48 h were 1.6, 46.9, 42.0 and 31.0%, respectively. Employing the hypoosmotic swelling test (HOST) showed 1.7%(39 degrees C), 28.7%(20 degrees C), 24.1%(15 degrees C), and 20.1%(4 degrees C) coiled-tail spermatozoa following 48 h storage. With Coomassie blue staining, the rates of acrosome-intact spermatozoa stored for 48 h were 4.5%(39 degrees C), 35.3%(20 degrees C), 55.7%(15 degrees C) and 22.8%(4 degrees C). Using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA), the percentages of acrosome-intact spermatozoa stored for 48 h were 4.3%(39 degrees C), 43.2%(20 degrees C), 17.3%(15 degrees C) and 14.8%(4 degrees C), respectively. The cytoplasmic droplets were found in 18.66% of the spermatozoa in fresh semen and were gradually shed during storage. The results of these 4 methods were highly correlated and could be used to characterized sperm-cell quality effectively. These findings indicated that both membrane integrity and viability of spermatozoa could be preserved well during in vitro storage at 20 degrees C and 15 degrees C for 24 to 48 h.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Stability of serum-free and purified baculovirus stocks under various storage conditions

In a context of large-scale production of baculoviruses in serum-free media for use as gene delivery vectors, the stability of these viruses has become an important factor. The development of robust processes heavily relies on baculovirus stock stability. In the present work, we studied over a period of 300 days the stability of baculovirus vectors produced in serum-free media stored at 4, -20, or -80 degrees C or in liquid nitrogen. The viral stocks investigated were either crude baculovirus supernatant, baculovirus supernatant concentrated 10 times and diafiltered against fresh serum-free media by tangential flow filtration, or baculovirus purified by size exclusion chromatography. The results showed that baculovirus supernatant and diafiltered concentrate stored at 4 degrees C underwent a progressive loss of infectivity after a period of 100 and 50 days of storage, respectively. Aggregation has been recognized as the probable mechanism for the loss of infectivity. Baculovirus stocks were unstable at -20 degrees C, whereas in liquid nitrogen they retained infectivity after successive freeze thaw cycles. Concentration and diafiltration of baculovirus supernatant prior to storing at -80 degrees C contributed to improving viral stock stability over time. Glycerol as well as DMSO and sucrose have proven to be equally effective as additives to maintain the purified baculovirus stability after storage at -80 degrees C or in liquid nitrogen.     

Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.