Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at -70 degrees C.

We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.     

The preservation of micro-organisms in biological specimens stored at-70 degrees C

The preservation of micro-organisms that may be found on the skin was studied by storage in liquid media at--70 degrees C. In the first part of the study the performance of 12 varieties of suspending media was evaluated with pure cultures of 17 species of micro-organisms maintained in the laboratory. After storage for 1 year the best medium (Oxoid Nutrient Broth with 15% glycerol) showed a mean survival for all organisms studied of 83.8%, with no significant differences between organisms. Even the worst medium (distilled water) permitted greater than 40% survival at 1 year. No changes in the characteristics of these micro-organisms were detected after 6 months storage in glycerol broth. In the second part of the study nose swabs were suspended in one representative medium (Bacto Nutrient Broth containing 7% glycerol). The mean percentage survival of staphylococci in these suspensions after 1 year's storage at - 70 degrees C was 75.4%. These results indicate that coagulase-negative coagulase-negative staphylococci in samples of skin flora may be stored under these conditions for long periods, greatly reducing the work-load in epidemiological studies of infection.     

Eight enrichment broths for the isolation of Campylobacter jejuni from inoculated suspensions and ground pork

Aims:  The efficiency of eight enrichment broths for the selective isolation of Campylobacter jejuni was compared to identify an optimal enrichment broth.
Methods and Results:  Brucella-FBP, Preston, Doyle and Roman, modified CCD (mCCD), Park and Sanders, Bolton, Hunt and Radle and Hunt broths were compared for their recovery of (i) Camp. jejuni in suspension, (ii) Camp. jejuni from inoculated ground pork, (iii) heat-injured Camp. jejuni (55°C for 20 min) in suspension and (iv) heat-injured Camp. jejuni from inoculated ground pork. Hunt broth and Bolton broth showed the highest and most rapid enrichment efficacy for the cell suspensions and ground pork, respectively. Preston, Park and Sanders and mCCD broths had relatively high enrichment efficiencies, while Brucella-FBP broth was significantly inferior to the other broths ( P  < 0·05).
Conclusions:  Cell recovery from the eight enrichment broths was dependent on the sample type and the state of the cells. The use of the appropriate broth is important for the rapid and efficacious enrichment of Camp. jejuni . In particular, heat-injured Camp. jejuni require a longer cultivation time and a suitable enrichment broth.
Significance and Impact of the Study:  The results from the present study provide information for selecting the most appropriate enrichment broth for Camp. jejuni and may contribute to improved detection methods for the organism.     

Evaluation of the effect of temperature and nutrients on the survival of Campylobacter spp. in water microcosms

Batch microcosms containing various water types (de-ionized and river water with or without sediment), incubated at a range of temperatures (5-37 degrees C), were used to facilitate a comparative evaluation of the significance of such variables and their interactions upon the collective and individual survival of four species of thermophilic Campylobacter. All variables significantly influenced (P < = 0.031) population decay rates. Minimal decay for the group was identified at low temperatures (5 degrees C) in river water, i.e. nutrient-containing microcosms. Collective decay rates within river water microcosms were significantly decreased (P = 0.03) from those observed in de-ionized water, particularly at environmental temperatures (5 and 15 degrees C). However, the increased nutrient levels observed in sediment-containing microcosms did not significantly (P = 0.41) reduce population decay rates. Overall, Camp. jejuni populations demonstrated the most resilience to the environmental stressors evaluated, with the exception of 15 degrees C where Camp. lari was the most persistent. Campylobacter coli and Camp. upsaliensis demonstrated comparable survival characteristics but were less resilient than Camp. jejuni and Camp. lari. These observations identify the suitability of water systems as a reservoir and medium for Campylobacter infection, and potentially identifies Camp. jejuni and Camp. lari as the main protagonists of water-mediated campylobacteriosis.     

Behaviour of Campylobacter jejuni in experimentally contaminated bottled natural mineral water

AIMS: The main objective of the present study was to estimate the survival of microaerophilic Campylobacter jejuni in filtered natural mineral water at 4 degrees C and 25 degrees C. The influence of the presence of biodegradable organic matter was tested, assuming that the bacterial contamination of a bottled natural mineral water could be associated with contamination by organic matter. METHODS AND RESULTS: Washed Campylobacter cultures were inoculated in natural mineral water and sterile natural mineral water, and incubated in the dark at 4 degrees C and 25 degrees C. The effect of temperature, the biodegradable organic matter added, incubation atmosphere and autochthonous microflora were tested on the cultivability of Camp. jejuni. CONCLUSIONS: The survival of Camp. jejuni in natural mineral water was better at 4 degrees C than at 25 degrees C, and the presence of organic matter led to a deceleration in the loss of cultivability and to the multiplication of Camp. jejuni in natural mineral water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that, in the event of dual contamination of a bottled natural mineral water (Campylobacter and biodegradable organic matter), the pathogen could survive (and even grow) for a relatively long time, especially at low temperature and in spite of the presence of oxygen.     

Skim milk enhances the preservation of thawed -80 degrees C bacterial stocks

The results from bacterial strain recovery efforts following hurricanes Katrina and Rita are reported. Over 90% of strains frozen in 10% skim milk were recovered whereas various recovery rates were observed for glycerol-stored stocks (56% and 94% of Escherichia coli, depending upon the laboratory). These observations led to a viability comparison of Streptococcus pyogenes, Campylobacter jejuni, Borrelia burgdorferi, Salmonella enterica subsp. Typhimurium, Pseudomonas aeruginosa and E. coli strains stored in glycerol or skim milk. In all bacteria examined, 10% skim milk resulted in significantly longer viability after thawing than 15% glycerol solutions currently used in most laboratories.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Improved preservation of glycogen in unfixed cyanobacteria embedded at -82 degrees C in nanoplast

A technique is presented for embedding unfixed cells at -82 degrees C in Nanoplast, a water-soluble melamine resin. By use of this procedure, improved ultrastructural preservation is demonstrated in cyanobacteria. Sections treated in this way are suitable for histochemical techniques such as Thiéry's silver staining method for glycogen.     

Inactivation of Campylobacter jejuni by high hydrostatic pressure

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of 相似文献   

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens isolated at an Irish poultry processing plant

AIMS: The antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens were determined in order to evaluate the level of antibiotic resistance of Campylobacter species in the Irish poultry industry. METHODS AND RESULTS: Seventy-eight Camp. jejuni and 22 Camp. coli strains were examined for susceptibility to eight antibiotics using the disc diffusion assay. The highest level of resistance of the Camp. jejuni isolates was recorded to ampicillin (35.9%), followed by 20.5% to tetracycline, 20.5% to naladixic acid, 17.9% to ciprofloxacin, 10.2% to erythromycin, 2.5% to streptomycin and 1.2% to kanamycin. Multidrug resistance to two or more antibiotics was seen for 30.7% of Camp. jejuni strains. Resistance of the Camp. coli isolates was shown to ampicillin (9%) and tetracycline (18.2%). CONCLUSIONS: The majority of Camp. jejuni strains were susceptible to antibiotics commonly used for human therapy. Camp. coli strains showed very low resistance levels and were susceptible to six of the eight antimicrobial agents studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Levels of Camp. jejuni and Camp. coli antimicrobial resistance in Irish poultry production was assessed to determine the current situation in Ireland. The prevalence of antibiotic resistance of Campylobacter strains isolated from broiler chickens was low.     

Extenders for preservation of canine and equine spermatozoa at 5 degrees C

Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of canine sperm retained their potential for progressive motility in CAP extender than in EYT, SM, CUE, EGB or BF-3 extenders. The SM extender was the best (P<0.05) for maintaining motility of equine sperm. The inclusion of 6% glycerol depressed (P<0.05) motility of canine sperm, but there was no effect (P>0.05) of glycerol concentration on the percentage of motile equine sperm. For both species, the interaction of glycerol level and extender was nonsignificant. CAP may be useful for storage of canine sperm at 5 degrees C and SM may be satisfactory for storage of equine sperm.     

The effects of UVB and temperature on the survival of natural populations and pure cultures of Campylobacter jejuni, Camp. coli, Camp. lari and urease-positive thermophilic campylobacters (UPTC) in surface waters

AIMS: To determine whether diurnal and seasonal variations in campylobacters in surface waters result from the effects of temperature and u.v. radiation, and whether natural populations of Campylobacter lari and urease-positive thermophilic campylobacters (UPTC) from birds survive better in surface waters than Camp. jejuni from sewage. METHODS AND RESULTS: Natural populations of Camp. lari and UPTC in sea water, and Camp. jejuni in river water, were exposed to artificial sunlight (equivalent to a sunny day in June). Both populations became non-culturable within 30 min, with T90s of 15 min and 25 min, respectively. Cultures of Camp. jejuni became non-culturable within 40 min and those of Camp. coli, Camp. lari and UPTC, within 60 min. In darkness, survival was temperature-dependent. Natural populations took 12 h at 37 degrees C and 5 days at 4 degrees C to become non-culturable in sea water, and slightly less in river water. Cultures of Camp. lari and UPTCs survived for significantly longer than Camp. jejuni and Camp. coli. Loss of culturability for all isolates was most rapid at 37 degrees C and slowest at 4 degrees C. Newly isolated strains from sea water and river water behaved in an almost identical manner to NCTC strains. CONCLUSION: Campylobacter lari and UPTCs survive for longer in surface waters than Camp. jejuni and Camp. coli, particularly in the dark. Low Campylobacter numbers in coastal waters in the summer, especially in the afternoon, are due to the combined effects of higher temperatures and higher levels of u.v. radiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter lari and UPTCs from birds predominate in bathing waters in Morecambe Bay because they are better able to survive; they also originate from closer to the shore than Camp. jejuni and Camp. coli in sewage effluent, which survive poorly and die before the incoming tide reaches the shore. The predominance of Camp. jejuni in river water results from its dominance of the inputs and not from its ability to survive.     

Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species-specific PCR assays

AIMS: To validate a phenotypic Campylobacter species identification method employed to identify campylobacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses. METHODS AND RESULTS: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum. It was also notable that 2-6% of the cultures were, in fact, mixed cultures. CONCLUSIONS: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum. SIGNIFICANCE AND IMPACT OF THE STUDY: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers.     

Conductivity values of tissue culture medium from 20 degrees C to 40 degrees C

Few studies are available that relate conductivity and temperature in solutions commonly used in tissue culture media. The purpose of this paper is to provide equations relating conductivity and temperature (in the range 20 degrees C-40 degrees C) for five solutions: 0.9% saline, MEM (Minimum Essential Media), horse serum, MEM with 1% horse serum, and MEM with 10% horse serum.     

A comparative study of preservation and storage of Haemophilus influenzae

The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20 degrees C and -70 degrees C. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20 degrees C and -70 degrees C from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20 degrees C. Initial concentration did not influence survival after storage at -70 degrees C. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.     

Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged starvation at low temperatures

AIMS: To evaluate the survival of Campylobacter jejuni relative to that of Escherichia coli in groundwater microcosms varying in nutrient composition. METHODS AND RESULTS: Studies were conducted in groundwater and deionized water incubated for up to 470 days at 4 degrees C. Samples were taken for culturable and total cell counts, nutrient and molecular analysis. Die-off in groundwater microcosms was between 2.5 and 13 times faster for C. jejuni than for E. coli. Campylobacter jejuni had the lowest decay rate and longest culturability in microcosms with higher dissolved organic carbon (4 mg l(-1)). Escherichia coli survival was the greatest when the total dissolved nitrogen (12.0 mg l(-1)) was high. The transition of C. jejuni to the coccoid stage was independent of culturability. CONCLUSION: The differences in the duration of survival and response to water nutrient composition between the two organisms suggest that E. coli may be present in the waters much longer and respond to water composition much differently than C. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from these studies would aid in the evaluation of the utility of E. coli as an indicator of C. jejuni. This study also provided new information about the effect of nutrient composition on C. jejuni viability.     

Cryosolvents useful for protein and enzyme studies below -100 degrees C

For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxy-ethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase-changes down to at least -160 degrees C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to -125 degrees C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Enhancing cell survival of atrazine degrading Rhodococcus erythropolis NI86/21 cells encapsulated in alginate beads

AIMS: To develop a method to produce beads with encapsulated Rhodococcus erythropolis NI86/21 with high cell density, extended shelf life, ease of handling and good atrazine degradation capabilities in both liquid and in agricultural soil. METHODS AND RESULTS: Our findings show that the supplementary recovery step in nutrient broth media shortly after cell encapsulation facilitates cell survival in both wet and dry beads upon extended storage at 4 degrees C. Air drying has little or no impact on encapsulated R. erythropolis cell's ability to degrade atrazine in liquid or soil. Bead storage for periods extending up to 12 months at 4 degrees C did not affect the capacity of R. erythropolis encapsulated cells to degrade atrazine in either BMN or nonsterile soil extracts. Bentonite-amended beads formulated with 1% skim milk and exposed to the supplementary growth step, outperformed all other bead formats. These beads provided adequate numbers of vigorous R. erythropolis cells in either liquid or soil media to degrade atrazine. CONCLUSIONS: Supplementary growth in nutrient broth media immediately following cell encapsulation greatly enhances R. erythropolis cells survival in both wet and dry beads upon extended storage at 4 degrees C. Wet and dried beads have similar capacity for atrazine degradation, and their usefulness and appeal in agronomic practise will only be known after bioassay evaluation and successful demonstration at field scale using incurred residues. SIGNIFICANCE AND IMPACT OF THE STUDY: R. erythropolis NI86/21 encapsulated cells have the potential to reduce residual atrazine in soil, thereby minimizing the likelihood of off-site transport to ground or river water and reduce the loss of crops because of phytotoxicity of residual herbicide. Owing to their ease of handling, storage and possible compatibilities with pre-existing mechanical equipment, dried bead formats are ideally suited for agricultural and remediational applications.     

Rapid construction of Campylobacter jejuni deletion mutants

AIMS: To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. METHODS AND RESULTS: We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. CONCLUSIONS: This method can be used for rapid construction of Camp. jejuni deletion mutants. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this method should facilitate functional characterization of various Camp. jejuni genes.