THE PRESERVATION OF BACTERIA BY DRYING ON CELLULOSE AND ALGINATE FIBRES

SUMMARY: Suspensions of Salmonella ndolo were dried in vacuo on both cellulose and calcium alginate fibres and recovered without loss. High recoveries were also obtained after the same organism had been dried on cellulose fibres and stored for a period of six months at room temperature.     

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.     

Comparison of Several Methods for Preserving Bacteriophages

A wide variety of bacteriophages were processed and stored under different conditions to compare methods for long-term preservation. Specimens were stored for 2 years at room temperature (24 to 28 C) and at 4 C as broth lysates in 50% glycerol, dried, and freeze-dried. Titers determined after processing indicated that, of the broth, glycerol, and freeze-dry methods, freeze-drying was most damaging to the phages tested, glycerol less damaging, and the broth method least damaging. After 2 years, titers of broth lysates were generally higher than those of glycerol or freeze-dried preparations. Dried preparations generally did not prove satisfactory. Preparations stored at 4 C showed better titers than those kept at room temperature. All titers declined with time regardless of the conditions of preservation.     

Effects of Various Gases on the Survival of Dried Bacteria During Storage

Salmonella newport and Pseudomonas fluorescens were dried together in papain digest broth and sucrose-glutamate, and stored in several gases at various water activities (a(w)) between 0.00 and 0.40 at 25 C for various periods up to 81 weeks. Both S. newport and P. fluorescens, dried in papain digest broth and stored in air, died rapidly if the conditions were very dry (0.00 a(w)) or moist (0.40 a(w)). Storage in carbon dioxide and argon gave greater survival than storage in air but lower survival than did storage in nitrogen or in vacuo. When the organisms were dried in a sucrose-glutamate mixture the differences between the gases were very small, and variations in residual water were less important. Of the inert gases, argon gave the best survival when the organisms were dried in papain digest broth, especially at 0.00 a(w); the survival in neon and krypton was lower and in xenon and helium it was much lower.     

THE RESISTANCE OF TWO STRAINS OF BACTERIUM COLI TYPE I TO DRYING UNDER ATMOSPHERIC CONDITIONS

SUMMARY: Suspensions of two strains of Bacterium coli type I were dried as thin films under atmospheric conditions and the numbers of organisms determined before and after drying. Three methods were used to grow the culture; in two the culture was grown in broth and in the other on agar slopes.
Strain 28.D.10 was less sensitive than strain NCTC 5934. After culturing in broth NCTC 5934 showed irregular daily fluctuations in sensitivity to drying; 28.D.10 became more sensitive for the first 2–3 weeks and thereafter less sensitive. Suspensions prepared from 18 hr plate cultures were more sensitive than from 24 hr plate cultures; a change from peptone water to Lemco broth for daily culturing slightly decreased the sensitivity to drying. With both strains suspensions prepared from broth cultures were more sensitive than those from agar slopes.
Continuous daily culturing of 28.D.10 on a solid medium as compared with broth decreased the sensitivity of suspensions and over a long period the culture appeared to be more stable. When strain NCTC 5934 was grown on a solid medium the suspension was as sensitive to drying as that obtained when broth was used but the daily fluctuation of results appeared to be less.
A decrease in the number of cells in the 24 hr broth culture of 28.D.10 coincided in time with an increase in the sensitivity to drying of the suspension prepared from the same culture.     

Effects of Moisture Content and Temperature on Storage of Metarhizium flavoviride Conidia

The effects of moisture content and temperature on the medium-term (3-4 months) storage of conidia of Metarhizium flavoviride were investigated. Conidia harvested after 24 days of culturing on rice showed greater tolerance to long storage than conidia from 12-day cultures. The moisture content of the conidia was of greatest importance; at harvest from the culture, conidial moisture contents could be 40%, while the optimal moisture content for storage was found to be 4-5%. Dried conidia stored in oil benefited from the addition of dried silica gel, as did conidia stored as powder. A range of mineral oils proved satisfactory for storage, and when dried silica gel was added to suspensions, germination levels were 79.8% after 105 days at 28-32 C. Dried conidia stored in oil maintained germination levels of up to 96 and 85% after 80 days at 10-14 C and 28-32 C respectively. Dried conidia stored as powder retained germination levels of 95% at 10-14 C, but only up to 27% at 28-32 C. In another experiment, dried conidia maintained greater than 90% germination over 128 days, with or without silica gel at 10 - 14 C or -15 - -18 C.     

Long-Term Preservation of Bordetella pertussis

Viability of Bordetella pertussis was preserved when glycerol broth suspensions were quick frozen and stored at -70 C for as long as 45 months.     

Bacteriophage formation without bacterial growth; the effect of niacin and yeast extract on phage formation and bacterial growth in the presence of penicillin

1. The addition of penicillin greatly increases the production of phage in bacterial suspensions containing 2.5 to 3.5 x 10(8) cells in 0.4 ml. broth plus 6.6 ml. Locke's solution. 2. Addition of niacin also greatly increases the formation of phage in the above system without the addition of penicillin. 3. The results indicate that niacin is necessary for phage production and that bacteria cannot utilize niacin in the presence of penicillin. 4. Staphylococcus muscae will grow in the synthetic medium of Fildes but do not form phage unless broth or yeast extract is added. 5. Phage formation requires the presence of one or more factors, besides niacin, present in broth and yeast extract which are not essential for bacterial growth. Penicillin does not prevent the utilization of the unknown substance or substances by the bacteria. 6. A solution containing biotin, guanine, adenine, beta-alanine, riboflavin, uracil, pyridoxamine, guanylic acid, adenylic acid, yeast nucleic acid, choline, p-aminobenzoic acid, a flavin component from liver, ribose, thymine, xanthine, folic acid, inositol, p-aminophenyl alanine, pantothentic acid and a strepogenin concentrate cannot replace broth or yeast extraction in increasing phage formation in the synthetic medium of Fildes. 7. The results indicate there is a continual competition between the bacteria and phage for certain essential building elements. 8. The results are discussed in relation to possible methods of control of virus diseases.     

Dried Escherichia diagnostic agents from live cultures

The possibility of using selected cultures of opportunistic Escherichia, freeze-dried in a saccharose-gelatine medium, as dried diagnostic reagents. In the agglutination test with Escherichia OK diagnostic sera, both fresh and stored for a period of up to 1 year, these dried diagnostic reagents prepared from live cultures showed the same degree of agglutination as live suspensions of similar Escherichia cultures.     

Preference for Dried Bonito Broth in Olfactory-Blocked or Taste Nerve-Sectioned Mice in the Two-Bottle Choice Test

We investigated how gustatory and olfactory information contributes to the preference for dried bonito broth in mice. In the two-bottle preference test, intact mice consumed dried bonito broth in preference to water or an amino acid-nucleotide (AN) solution containing the same concentration of amino acids and nucleotides as that in dried bonito broth. It was observed that mice with transected bilateral chorda tympani (CT) nerves, those with transected bilateral glossopharyngeal (GL) nerves, and those that were intranasally administered with zinc sulfate preferred dried bonito broth to water. Zinc sulfate was used to produce a temporary loss of olfaction. In the two-bottle preference test with dried bonito broth and an AN solution, the preference for the former was reduced in mice with transected bilateral GL nerves and in those with an olfactory blockade, but not in mice with transected bilateral CT nerves. These results suggest that dried bonito broth was preferred over the AN solution, and that simultaneous inputs from olfaction and the GL nerve contributed to this preference.     

Preference for dried bonito broth in olfactory-blocked or taste nerve-sectioned mice in the two-bottle choice test

We investigated how gustatory and olfactory information contributes to the preference for dried bonito broth in mice. In the two-bottle preference test, intact mice consumed dried bonito broth in preference to water or an amino acid-nucleotide (AN) solution containing the same concentration of amino acids and nucleotides as that in dried bonito broth. It was observed that mice with transected bilateral chorda tympani (CT) nerves, those with transected bilateral glossopharyngeal (GL) nerves, and those that were intranasally administered with zinc sulfate preferred dried bonito broth to water. Zinc sulfate was used to produce a temporary loss of olfaction. In the two-bottle preference test with dried bonito broth and an AN solution, the preference for the former was reduced in mice with transected bilateral GL nerves and in those with an olfactory blockade, but not in mice with transected bilateral CT nerves. These results suggest that dried bonito broth was preferred over the AN solution, and that simultaneous inputs from olfaction and the GL nerve contributed to this preference.     

Proceedings: Preservation and storage of microorganisms in the gas phase of liquid nitrogen

Methods are described which permit the rapid preparation and retrieval of suspensions of microorganisms stored in the gas space of a liquid nitrogen refrigerator.Inocula are currently used for assay plates, turbidimetric assays, bioautograph plates, and various fermentation processes. The most suitable suspending agents, 10% glycerol with 5% of either lactose, maltose, or raffinose, were devised by determining viabilities from alternate freezing and thawing of cell suspensions.Four milliliters each of concentrated cell suspensions were pipetted into cotton-plugged or plastic-capped glass tubes (13 × 100 mm). After rapid noncontrolled freezing in the nitrogen gas phase, the tubes were stored horizontally either in test-tube racks or in bulk containers.Frozen suspensions of vegetative cells of some algae, many genera of bacteria, as well as spores, vegetative cells, and fragmented mycelia of streptomycetes and fungi have been conveniently prepared from either agar or broth cultures. Sufficient inocula have been prepared to be used for periods of a few weeks to several years.     

A Bacterial Spore Test Piece for the Control of Ethylene Oxide Sterilization

Aluminium foil strips carrying varying numbers of spores of the Camp Detrick strain of Bacillus subtilis dried from water, 90% (v/v) methanol, 5 and 20% (v/v) serum, nutrient broth and isotonic saline were examined for their possible use as test pieces for the control of sterilization by ethylene oxide. Methanolic suspensions of these spores were found to be stable on storage, and foils carrying spores dried from methanol were the most reproducible and stable of those tested. The susceptibility of the test piece system could be controlled by varying the numbers of spores on each foil and the numbers of foils used per test.     

THE DETERMINATION OF ESCHERICHIA COLI I IN SEA WATER

SUMMARY: For the determination of Escherichia coli I in sea water lactose broth frequently gave higher presumptive and confirmed counts than MacConkey's broth. In the presumptive count there were 53 cases where lactose broth gave larger numbers than MacConkey's broth, with 11 equal counts, and only 25 cases with smaller counts ( P =0·00137). After confirmation the corresponding numbers of cases were 39, 10 and 27 ( P =0·134).
In the samples giving most probable numbers (MPN) of less than 100 E. coli /100 ml lactose broth was superior to MacConkey's broth ( P =0·021). At higher MPN values both media were satisfactory, but with highly polluted water MacConkey's broth might give better recoveries due to the suppression of high concentrations of non-coli-aerogenes bacteria.
Samples stored for 24 or 48 hr before testing gave higher presumptive recoveries when examined with lactose broth than with MacConkey's broth, the values for P being 0·028 and 0·0027 respectively.
It appears that lactose broth without inhibitory ingredients could be used with advantage in the examination of sea water.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Preservation of T2 Bacteriophage With Liquid Nitrogen

There have been few reports on the preservation of viruses at the temperature of liquid nitrogen (-196 C). In this study, factors affecting the survival of phage were observed. Phage lysates in broth did not lose titer after a storage period of 4 weeks in liquid nitrogen. The rate of freezing was not critical, but freezing in nitrogen vapor was not satisfactory. When the virus was partially purified and suspended in buffered saline solution, it rapidly lost titer. Of seven protective agents recommended in the literature, only gum acacia seemed to demonstrate significant protection of purified virus against loss of viability. The temperature of thawing of phage suspended in broth did not affect its titer after freezing and storing in liquid nitrogen.     

Further optimization of mouse spermatozoa evaporative drying techniques

It has been shown in the past that mouse spermatozoa could be dried under a stream of nitrogen gas at ambient temperature and stored at 4 °C or 22 °C for up to 3 months and was capable of generating live-born offspring. In previous desiccation work, dried sperm were stored in a vacuum-sealed plastic bag placed in a vacuum-packed Mylar bag. However, dried specimens stored in this way often lost moisture, particularly in samples stored at higher temperatures (22 °C) compared to lower temperatures (4 °C). The present report describes a method which minimizes this water loss from the dried sperm samples. Its use is described in a preliminary study on the effect of supplementing the trehalose with glycerol. The results have demonstrated that mouse sperm can be stored at 4 °C over saturated NaBr without the uptake of water which occurs when they are stored in Mylar packages. In addition, we were able to get some survival of sperm (9–15%) at room temperature storage after 3 months. The addition of glycerol to trehalose had little effect on the survival of dried mouse sperm stored over NaBr for 1 and 3 months.     

Stability of Suspensions of Influenza Virus Dried to Different Contents of Residual Moisture by Sublimation In Vacuo

After freezing, suspensions of influenza virus were dried by sublimation of water in vacuo to contents of residual moisture of 3.2, 2.1, 1.7, 1, or 0.4%. The stability of the several suspensions was determined by an accelerated storage test. Based on the times predicted for the dried preparations stored at different temperatures to lose 1 log of infectivity titer, the order of stability in relation to residual moistures was as follows: 1.7% > 2.1% > 1% > 3.2% > 0.4%.     

Use of polyvalent phage for reduction of streptomycetes on soil dilution plates.

An isolation method was developed in which prior to inoculation soil suspensions were exposed to suspensions of polyvalent phage isolated to Streptomyces spp. The phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates subsequent to inoculation, and this reduction was persistent after long incubation periods. The efficiency and applicability of the method developed were checked with different samples from a range of sources. The increased chances of development of other genera after the reduction of streptomycetes on soil dilution plates were assessed.     

虫药蜣螂对灵芝发酵物风味组成的影响

采用顶空-气相色谱-质谱联用对虫药蜣螂和添加蜣螂发酵后的灵芝发酵物的风味物质进行了测定,进而分析了添加蜣螂对灵芝发酵物风味组成的影响。结果表明,虫药蜣螂和灵芝发酵物(添加蜣螂)中分别含有40和30多种风味物质。添加蜣螂发酵后,灵芝发酵物跟对照相比新出现了5种风味物质,分别为苯甲醛、α-松油醇、苯乙醇、唑苯并噻和桃金娘烯醇。其中α-松油醇、苯乙醇和唑苯并噻是蜣螂中的风味成分,苯甲醛和桃金娘烯醇为新产生成分。所有5种风味物质均为食品香料物质。蜣螂中其它风味成分经灵芝发酵转化或降解后,未出现在灵芝发酵物中。因此,添加蜣螂发酵对灵芝发酵物的风味无不良影响。结果还提示灵芝细胞可生物转化或降解虫药蜣螂中的部分风味物质。