Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

SST broth,a new serum free germ tube induction medium for identification of Candida albicans

Three serum free media viz, sucrose solution, starch solution and SST broth have been formulated. The objective of the present study was to evaluate these three different serum free media for induction of germ tubes by Candida albicans and to compare their efficacy with the pooled human serum. Out of 50 C. albicans isolates 47 (94 %) and 49 (98 %) produced germ tubes in pooled human serum and SST broth, respectively. Germ tube production was positive in 40 (80 %) and 36 (72 %) isolates, respectively in sucrose solution and starch solution. This study reports SST broth as a new stable and less expensive germ tube induction medium, which requires less time for preparation and can be used without any safety concerns. SST broth is found to be more effective than pooled human serum for induction of germ tubes by C. albicans isolates.     

Effect of suspension media on nonthermal inactivation of Escherichia coli

AIMS: To investigate the influence of suspension media on the survival of Escherichia coli M23 exposed to nonthermal, lethal stresses. METHODS AND RESULTS: Populations of E. coli M23 suspended in minimal medium (MM) or in different nutrient-rich broths were exposed to water activity 0.90 and/or pH 3.5 and inactivation was determined by culture-based enumeration. In response to the osmotic or acid challenges, E. coli M23 displayed enhanced survival in MM rather than in complex broth. That trend was reversed when populations were exposed to low water activity in combination with low pH. Comparison of microbial survival in three complex media indicated that even relatively small differences in composition influenced inactivation. In most media the combination of lethal stresses resulted in a synergism, which enhanced bacterial inactivation; however, an exception (tryptone soya broth) was observed. CONCLUSIONS: The suspension medium strongly influences the inactivation of E. coli M23 by osmotic and/or acid stresses. This should be considered when comparing studies of microbial survival that use different media and when broth-derived data are intended to represent specific environments (e.g. food matrices). SIGNIFICANCE AND IMPACT OF THE STUDY: The specific effects of synthetic media need to be appreciated when studying bacterial inactivation in conditions relevant to food-manufacturing regimes.     

Influence of medium composition on the growth and antigen expression of Helicobacter pylori

An evaluation of the ability of various solid and liquid media to support both growth and antigen expression, particularly lipopolysaccharide (LPS) expression, by Helicobacter pylori culture collection strains and clinical isolates was performed. Liquid-based basal media (brain heart infusion, Brucella broth, Mueller–Hinton broth and tryptone soya broth) supported the growth of strains, whereas solid basal media of the same formulation did not support growth. Optimal growth of all strains was obtained on solid and in liquid media containing blood. Supplemented solid media containing supplements other than blood supported growth but only to a small extent. In liquid media excluding blood, serum supplements enhanced growth and horse serum was found to be superior to fetal calf serum. In general, β-cyclodextrin did not increase growth. Mueller–Hinton broth or tryptone soya broth containing horse serum and a nitrogen source such as yeast extract or proteose peptone no. 3 were found to give optimal growth of H. pylori in a blood-free environment. Strains after cultivation in liquid media, irrespective of composition, maintained production of high-molecular weight (mol. wt) LPS with an O side chain independent of medium composition, whereas subculturing on solid media resulted in production of low-mol. wt LPS. Expression of proteins differed in liquid and on solid media, particularly proteins of 57 and 60 kDa, but qualitatively no differences were observed upon supplementation of basal media.     

Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption

The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss. In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared. Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB). Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility. O. mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria. Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A. hydrophila aroA vaccine.     

Influences of protectants,rehydration media and storage on the viability of freeze-dried <Emphasis Type="Italic">Oenococcus oeni</Emphasis> for malolactic fermentation

Malolactic fermentation (MLF) is an important process in wine production. To achieve successful MLF, expanding interest in ready-to-use Oenococcus oeni starter cultures has placed greater emphasis on developing starter production and preservation methods. In this study, influences of protectants, rehydration media and storage on the viability of O. oeni H-5 when subjected to freeze-drying were investigated. It was found that sodium glutamate (2.5%) was the best protectant, giving the cell viability 72.4%. Adding polysaccharides and disaccharides in suspension media also improved significantly the cell viability. Rehydration is an important step in recovery after freeze-drying. When freeze-dried O. oeni was rehydrated in GYM medium, the highest viability (87.1%) was obtained. Rehydration in the disaccharide solutions tested made the cell viability obviously decrease. After 6 months storage at 4°C, loss of viability occurred, the extent of which depended on protectants used, sodium glutamate again being the most effective.     

8种不同方法保藏病原菌效果的对比观察

采用８种不同的菌种保藏方法,对１８种常见病原菌的保藏时间及生物学特性的影响进行了对比观察。结果表明：冷冻干燥法保藏菌种时间最长（本实验室已保存１５年）;肉浸汤琼脂平板法保藏菌种时间最短（２～３月）。保藏时间由长到短依次为：冷冻干燥法＞半固体冷冻法≥半固体斜面加液体石蜡覆盖法＞半固体斜面法＞肉浸汤加液体石蜡覆盖法＞血琼脂平板法＞肉浸汤法＞肉浸汤琼脂平板法。且相同方法对不同菌种保藏时间不同。保藏时间在１年以内的菌种,其生物学特性无明显变异;而经冷冻干燥法保藏时间较长的白喉棒状杆菌、金黄色葡萄球菌、甲型副伤寒沙     

The growth and long term survival of Acholeplasma laidlawii in media products used in biopharmaceutical manufacturing

Acholeplasma laidlawii is a potential contaminant of bovine serum and has also been found as a contaminant in serum free cell culture media products. Anecdotal evidence of A. laidlawii contamination of tryptone soya broth circulated for a number of years before it was acknowledged that the organism could contaminate microbiological broth powders. The occasional occurrence of A. laidlawii in broth powders and possibly in powdered components of cell culture media as part of the normal bioburden poses a serious threat to routine pharmaceutical and biopharmaceutical operations where filtration is the sterilisation method of choice. Absence of visual evidence of contamination cannot be relied upon as there is variation with both organism strain and media product in the ability to produce turbidity. Strains of A. laidlawii which have been isolated from broth powders are not significantly different in temperature or media preferences from other strains. A. laidlawii is capable of growing to high titre at refrigeration and ambient temperatures in unsupplemented bacteriological sterility media or serum free cell culture media and can survive for prolonged periods in these products.     

Antifoam fouling and its reduction by surfactant precoat treatment of polysulphone ultrafilter

Summary Precoat treatment of polysulphone membranes with non-ionic surfactant BRIJ 58 was effective in decreasing antifoam fouling in ultrafiltration of five model process streams such as fermentation media, broth and yeast suspension. The mean flux increase of about 90 % was achieved by the surfactant pretreatment.     

Reproducibility of vitamin assays with mutants of Neurospora crassa maintained by freeze-drying.

Three mutant strains (ATCC 9277, ATCC 9278, ATCC 9683) of Neurospora crassa requiring choline, inositol, and p-aminobenzoic acid respectively for normal growth were deposited at the ATCC by G.W. Beadle 30 years ago and were preserved at various time intervals by freeze-drying. Each preservation batch yielded cultures that, when used for biological assays, exhibited the same biochemical properties as they originally possessed. The freeze-drying technique is shown to be applicable to preserving biological properties of sporulating fungi for bioassay over prolonged periods of time.     

Relationship between morphology,rheology and polygalacturonase production by Aspergillus sojae ATCC 20235 in submerged cultures

A full factorial statistical design, with the factors of, two taxonomically different strains, seven types of seed culture formulations (slants) and two types of fermentation media were used to investigate the effect of these parameters on the morphology and polygalacturonase production. The rheology of the final fermentation medium was analyzed and appropriate mathematical model was applied to calculate suspension viscosity. It was found that most fermentation broths showed non-Newtonian flow behavior. According to statistical analysis, factors of strain types and fermentation media and the interaction between them were found significant on the enzyme activity. The effect of seed culture formulations (slants) were found insignificant at the significance level of 1%. Interaction of slants with strain types and fermentation media were also found insignificant. Considering the morphology of the final culture, Aspergillus sojae with the desired pellet morphology in a complex media, inoculated with a seed culture prepared from molasses resulted in maximum polygalacturonase enzyme activity (0.2 U/ml) and lowest suspension viscosity with a broth rheology close to Newtonian flow behavior.     

Factors affecting viable cell counts of freeze-driedCryptococcus terricolus cells

Freeze-drying ofCryptococcus terricolus cells in distilled water resulted in a survival of only 0.1% of the cells. The viability could be increased to 16% by the use of a dextran-sucrose-sodium glutamate solution as suspending medium.For freeze-dried material with both low and high survival rates, and for five as well as for ten days old cultures, malt extract solution was the superior reconstitution medium. Less, but still distinct, protective action was found with a synthetic glucose-urea-salt solution.The viable cell counts of cells freeze-dried in dextran-sucrose-sodium glutamate solution were independent of the medium used for plating. When distilled water was used as a medium for freeze-drying, two to four times higher counts were obtained with malt extract agar than with synthetic glucose-urea-salt agar.Of the twenty different media tried for freeze-drying, sucrose solution gave the best protection. The viability was greatly influenced by the concentration used, maximum values being obtained when more than 10% of sucrose was added. The survival rate increased with the age of the cells until the fifth day, but was independent of the concentration of cells in the suspension. Under optimum conditions a survival rate of more than 80% was reached.     

Cryopreservation studies of Campylobacter

Seven strains of Campylobacter fetus ss. fetus, one of Campylobacter fetus ss. venerealis, and one of Campylobacter jejuni were preserved using a variety of cryopreservation methods. Organisms were frozen to -150 degrees C in a liquid nitrogen refrigerator, in the freezer compartment of a refrigerator (-20 degrees C), and in a mechanical freezer (-65 degrees C). In the latter two cases, viabilities of the organisms were compared after being frozen in Brucella Albimi broth and 10% glycerol. Viabilities were also examined after Campylobacter species were freeze-dried using rapid or slow cooling, using sucrose or skim milk as cryoprotective agents and in bulb-type vials on a manifold or batch vials. Preservation in liquid nitrogen resulted in no loss in viability after 4 years storage. When Campylobacter species were frozen at -20 degrees C, no cells were recovered after 1 month storage in Brucella Albimi broth or seven months in glycerol. A 6.5 log decrease in viability resulted after organisms were frozen at -65 degrees and subsequently stored at the same temperature for 2 years. In this case, glycerol had no protective advantage over Brucella Albimi broth. Postpreservation viability of organisms cooled slowly was two logs higher than those cooled rapidly prior to freeze-drying. When skim milk or sucrose were employed as cryoprotective agents during freeze-drying, equal viabilities resulted. Equivalent viabilities were also demonstrated when the bulb type or "batch" vials were utilized for freeze-drying. No significant differences were observed between the viabilities of the three species when a given cryopreservation method was employed.     

Effect of carbohydrates and related compounds on the long-term preservation of freeze-dried bacteria

A selection of bacteria were freeze-dried in horse serum containing various carbohydrates and related compounds. An accelerated storage test at elevated temperatures was used to determine the long-term viability of the dried organisms with the assumption that the results of accelerated storage reflect long-term viability. The results suggest that meso-inositol, non-reducing disaccharides and certain polyalcohols are the most suitable of the compounds tested for incorporation into suspending media for use in freeze-drying.     

Effect of various growth media upon survival during storage of freeze-dried Enterococcus faecalis and Enterococcus durans

AIMS: The effects of three different growth media (MRS, M17 and Lee's) on survival during freeze-drying and subsequent storage of six strains of Enterococcus faecalis and two strains of E. durans were investigated. METHODS AND RESULTS: Distinct Enterococcus spp. strains were grown on M17, MRS and Lee's broth, freeze-dried and stored at 20 degrees C in air under darkness. At regular intervals throughout storage, freeze-dried samples were rehydrated and then plated on M17 agar. CONCLUSIONS: A higher survival rate during storage of dried E. durans was obtained when growth occurred in MRS. The same effect was not observed, however, for the majority of E. faecalis strains, which clearly survived better in the dried state when this organism had been grown in M17 or Lee's medium. SIGNIFICANCE AND IMPACT OF STUDY: The survival of the dried Enterococcus spp. tested during storage was shown to be strain-specific and dependent on the growth medium.     

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.     

Comparison of media for recovery of total coliform bacteria from chemically treated water.

Five broth media and two solid media were compared for their ability to quantitatively recover total coliform bacteria from chemically treated water. M-Endo LES and mT7 media were used in the membrane filter technique. Lauryl tryptose broth, lactose broth, presence-absence broth, lactose broth with twice the amount of lactose, and lauryl tryptose broth with twice the amount of sodium lauryl sulfate were used in the fermentation tube procedure. The differences in recovery were not significant for the five broth media and M-Endo LES agar. The M-Endo LES and mT7 media were not significantly different; however, the five broth media did yield significantly higher counts than mT7.     

Comparison of media for recovery of total coliform bacteria from chemically treated water

Five broth media and two solid media were compared for their ability to quantitatively recover total coliform bacteria from chemically treated water. M-Endo LES and mT7 media were used in the membrane filter technique. Lauryl tryptose broth, lactose broth, presence-absence broth, lactose broth with twice the amount of lactose, and lauryl tryptose broth with twice the amount of sodium lauryl sulfate were used in the fermentation tube procedure. The differences in recovery were not significant for the five broth media and M-Endo LES agar. The M-Endo LES and mT7 media were not significantly different; however, the five broth media did yield significantly higher counts than mT7.     

Evaluation of a simple protein free medium that supports high levels of monoclonal antibody production

A simple protein free medium was formulated and tested in suspension culture using three hybridoma cell lines. The medium, referred to as CDSS (Chemically Defined Serum Substitutes), consisted of the basal medium DMEM:Ham F12, 1:1, with HEPES (D12H), plus pluronic F68, trace elements, ferric citrate, ascorbic acid, and ethanolamine. No protein or lipid components were added. All three cell lines were weaned off serum using CDSS and a commercially available protein free medium PFHM-II. Data shown here indicated that normally cells took 1–7 weeks to wean off serum and an additional 2–7 weeks to adapt to suspension culture. After adaptation the cells were able to grow well in suspension culture using both protein free media and in the main performed better than serum containing controls. The stability of the three hybridoma cells for antibody production following freeze/thaw procedures and long term subculturing was also tested. All three lines were frozen using our protein free CDSS medium (containing 0.75% bovine serum albumin and 10% dimethyl sulfoxide) in liquid nitrogen for up to one year. Cells thawed from these stocks recovered well and were able to maintain good growth and antibody production characteristics. One line was shown to grow using our protein free CDSS medium in suspension culture for 12 weeks without loss of antibody productivity.     

两株真菌降解菜籽饼中植酸的研究

利用选择性培养基从土壤中分离到两株能降解植酸的丝状真菌。这些菌株能利用肌醇作为唯一的碳源和能源而生长。在液态发酵中植酸的降解率分别为74．4％和95．0％;在固态发酵中植酸的降解率为40％左右。某些金属离子对菌株的降解率的提高具有一定的促进作用。对温度、pH和水分等影响因子也进行了初步的探讨。经初步鉴定,这两株菌株中有一株为拟青霉（Paecilomycessp）,另一株为青霉（Penicilliumsp．）,它们均不产黄曲霉素素。