Quarter-strength Ringer's Solution Research Articles

CLI: A new protocol for the isolation of Lactic Acid Bacteria from complex plant samples

Lactic Acid Bacteria (LAB) are predominantly probiotic microorganisms and the most are Generally Recognized As Safe (GRAS). LAB inhabit in the human gut ecosystem and are largely found in fermented foods and silage. In the last decades, LAB have also has been found in plant microbiota as a new class of microbes with probiotic activity to plants. For this reason, today the scientific interest in the study and isolation of LAB for agronomic application has increased. However, isolation protocols from complex samples such as plant tissues are scarce and inefficient.In this study, we developed a new protocol (CLI, Complex samples LAB Isolation) which yields purified LAB from plants. The sensitivity of CLI protocol was sufficient to isolate representative microorganisms of LAB genera (i.e. Leuconostoc, Lactococcus and Enterococcus).CLI protocol consists on five steps: i) sample preparation and pre-incubation in 1% sterile peptone at 30 °C for 24–48 h; ii) Sample homogenization in vortex by 10 min; iii) sample serial dilution in quarter-strength Ringer solution, iv) incubation in MRS agar plates with 0.2% of sorbic acid, with 1% of CaCO3, O2 < 15%, at pH 5.8 and 37 °C for 48 h.; v) Selection of single colonies with LAB morphology and CaCO3-solubilization halo. Our scientific contribution is that CLI protocol could be used for several complex samples and represents a useful method for further studies involving native LAB.

Effect of temperature and pH value on the UV-C sensitivity of Escherichia coli and Lactobacillus plantarum

The genetic disposition, the influencing factors of the environment as well as the development status affect the resilience of microorganisms and consequently may influence the inactivation efficiency of an UV-C pasteurization process. In this study, the effect of different growth conditions on the reduction rate of Escherichia coli DH5α and Lactobacillus plantarum BFE 5092 in Quarter-strength Ringer's solution (QSRS) was investigated. For E. coli DH5α, the temperatures below the optimum growth temperature led to higher UV-C resistant cells, whereas incubation at 45 °C facilitated the UV-C inactivation of E. coli DH5α. However, no considerably difference in UV-C sensitivity could be observed for L. plantarum BFE 5092 incubated at the different growth temperatures applied. In addition, L. plantarum BFE 5092, being more sensitive in stationary phase, was more resistant to UV-C than E. coli DH5α. No considerably effect on the inactivation efficiency could be observed for both species with regards to the initial pH value of the growth media. The effect of various temperatures and pH values on the inactivation behavior of L. plantarum BFE 5092 was also tested and no difference in reduction rates could be observed in the range of 2–40 °C and pH 4.5–7.

An investigation of the shelf-life (storage) of Bacillus isolates on seeds

Changes in bacterial population during storage of treated seeds of maize ( Zea mays L.), bean ( Phaseolus vulgaris L.), lettuce ( Lactuca sativa L.) and cucumber ( Cucumis sativus L.) coated with Bacillus sp. were investigated over a 12-month period. The isolates designated as B9, B11, B69, B77 and B81 were grown in Tryptone Soy Broth (TSB) synthetic medium for 72 h at 30 °C (100 rpm) in a shaker (model Sterilab). Cells were harvested at 10 000 rpm for 15 min (Beckman Centrifuge JA 20) and washed with sterile distilled water. Final viable cell concentration of pellets was determined by serial dilutions on Tryptone Soy Agar (TSA). The isolates were each applied to each seed lot using a seed sticker, Pelgel®, according to the manufacturer's (Nitragen [Lymphatec]) specifications. The seeds were coated with approximately 9 log 10 CFU/ml of the bacterium, air-dried in a laminar flow chamber and placed at ambient (shelf-life) temperature. For enumeration of attached cells, 1 g of each seed lot was placed in a 9 ml quarter-strength Ringer's solution, vortexed for 2 min, followed by plating of the serial dilutions on TSA. The initial bacterial populations, which adhered to the spermosphere of the seed samples, ranged between 6 and 7 log 10 CFU/g seed. There was an initial slight upward trend from month 1 to 5 by 1–2 log CFU/g for maize and bean. For maize, population stabilized between 6.91 and 7.54 log CFU/g in months 5 and 6. For bean, the highest population was obtained in month 4 between 6.52 and 7.95 log CFU/g. Populations stabilized in months 5 and 6 ranging from 6.23 to 6.98 log CFU/g. Thereafter, there was a downward trend from month 7. For cucumber, populations increased slightly up to month 4 in the order of magnitude of 1–2 before stabilizing between 6.11 and 6.78 log CFU/g in months 5 and 6. However, populations on lettuce decreased slightly up to month 2 before increasing slightly in months 3 and 4. Populations stabilized in months 5 and 6 ranging between 6.8 and 8.6. In all the samples, populations decreased from month 7 to the last sampling month. There was a 5% level of significant difference between the months for each of the seeds, but treatments within the seeds were not significant for maize, bean and cucumber.

Neutral density liquid formulations for nematode-based biopesticides.

Entomopathogenic nematodes are widely used as biological insecticides for agricultural and horticultural pests. In the vast majority of cases, commercial products contain nematodes partially dehydrated on to inert solid carriers. Nematode survival in these products is generally poor, they are difficult to handle and are not suitable for use with all nematode species. We have developed a non-viscous, non-adhesive and non-toxic liquid formulation for nematode storage and transport based on neutral density colloidal silica suspensions. Survival and virulence of nematodes stored in this formulation without aeration was superior to nematodes stored in aerated quarter strength Ringer's solution.

A New Race of Pseudomonas syringae pv. tabaci on Tobacco in Zimbabwe.

Two forms of Pseudomonas syringae pv. tabaci cause wildfire (Tox+) and angular leaf spot (Tox-) diseases of tobacco in Zimbabwe. Two races of the pathogen (races 0 and 1) occur in Zimbabwe (4). Two groups of cultivars are available: one with resistance to race 0 only, tbe second with resistance to races 0 and 1 (4). P. syringae pv. tabaci was first observed on cultivars with resistance to races 0 and 1 in 1993, and infection on these cultivars is now widespread. The bacteria isolated from the infected plants were gram-negative rods and produced fluorescent pigment on King's medium B. Levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity (LOPAT) tests indicated that the pathogen was a Group 1a pseudomonad (2). Pathogenicity and race designation for 58 isolates of the pathogen were determined on 8-week-old, greenhouse-raised tobacco plants. Inoculated plants were kept in controlled environment units (13/11 h photoperiod, 30 ± 2°C, relative humidity >75%) for 10 days. Pathogenicity was determined on cv. Kutsaga E1, which has no known resistance to any races of P. syringae pv. tabaci (4), and isolates from typical wildfire and angular leaf spot lesions were used for race designation by inoculating six plants of each indicator tobacco genotype. Genotypes included Nicotiana tabacum cv. Kutsaga E1 (susceptible to races 0, 1, and 2), which was used as the indicator genotype for race 0, N. longiflora (race 0 resistance) (1), N. tabacum cv. Kutsaga Mammoth 10 (resistance to race 0 derived from N. longiflora), N. rustica var. brasilea (resistance to races 0 and 1) (3,4), a breeding line, WZ 3-2-1-1, and cv. Kutsaga 35 (both resistant to races 0 and 1 with resistance derived from N. rustica var. brasilea). Six spots, three on either side of the midrib, were inoculated with an artist's airbrush (Aero-pro 251) operated at 250 kPa. The inoculum, (approximately 1 × 106 CFU/ml) suspended in a quarter-strength Ringer's solution was applied to one side of the midrib and Ringer's solution only to the other side, which served as control. Thirty-eight Tox+ isolates and 20 Tox- isolates were tested in a series of experiments in randomized complete blocks with four replications per treatment. Resistance to wildfire was characterized by localized chlorosis or whitish-tan hypersensitive lesions and susceptibility by necrotic lesions (>2 mm) surrounded by large chlorotic halos. Resistance to angular leaf spot was characterized by hypersensitive lesions or absence of symptoms and susceptibility by presence of symptoms (4). Sixty-six percent of the Tox+ isolates and 70% of the Tox- isolates successfully infected cultivars with known resistance to races 0 and 1, and were therefore designated race 2 (1). All other isolates were race 1. This is the first report of P. syringae pv. tabaci Tox+ and Tox- race 2 in Zimbabwe.

Oxygen uptake by porcine colon contents

Since the gut environment is generally assumed to be anaerobic, studies of the monogastric intestinal microflora have concentrated on the anaerobic microorganisms. However, the intestinal epithelium constitutes a very high surface area in relation to the volume of the gut contents, and all of this surface area is richly supplied with blood. Appreciable dissolved oxygen concentrations can be detected in the intestines of anaesthetised piglets (Hillman et al, 1993), although this is never detectable in the intestines of killed animals. It was considered important to investigate the persistence of intestinal oxygen after death, in order to determine the accuracy of postmortem measurements of this component of the intestinal environment. This report demonstrates the rapidity of removal of oxygen by porcine colon contents, and indicates that, since the oxygen supply to the intestine stops when the blood circulation stops, accurate measurements of dissolved oxygen can never be made in the intestines of killed animals.Colon contents were obtained from three freshly slaughtered 20 kg piglets and diluted to 20% (w/v) slurries in quarter-strength Ringer's solution (Unipath, UK). The slurries were transferred (25 ml) to 50 ml conical flasks, stirred by magnetic follower (approx 400 rpm) and surrounded by a water jacket to maintain a temperature of 39°C in the vessel contents. A polarographic oxygen electrode was inserted so that the membrane was submerged. Gas flow through the headspace was provided by compressed air and nitrogen, mixed via a pair of needle valves to provide a constant flow rate of 600 ml min-1. Calibration of the system was performed in sterile Ringer's solution using air and oxygen-free nitrogen: calibration was repeated between each experiment.

Oxygen uptake by porcine colon contents

Since the gut environment is generally assumed to be anaerobic, studies of the monogastric intestinal microflora have concentrated on the anaerobic microorganisms. However, the intestinal epithelium constitutes a very high surface area in relation to the volume of the gut contents, and all of this surface area is richly supplied with blood. Appreciable dissolved oxygen concentrations can be detected in the intestines of anaesthetised piglets (Hillman et al, 1993), although this is never detectable in the intestines of killed animals. It was considered important to investigate the persistence of intestinal oxygen after death, in order to determine the accuracy of postmortem measurements of this component of the intestinal environment. This report demonstrates the rapidity of removal of oxygen by porcine colon contents, and indicates that, since the oxygen supply to the intestine stops when the blood circulation stops, accurate measurements of dissolved oxygen can never be made in the intestines of killed animals.Colon contents were obtained from three freshly slaughtered 20 kg piglets and diluted to 20% (w/v) slurries in quarter-strength Ringer's solution (Unipath, UK). The slurries were transferred (25 ml) to 50 ml conical flasks, stirred by magnetic follower (approx 400 rpm) and surrounded by a water jacket to maintain a temperature of 39°C in the vessel contents. A polarographic oxygen electrode was inserted so that the membrane was submerged. Gas flow through the headspace was provided by compressed air and nitrogen, mixed via a pair of needle valves to provide a constant flow rate of 600 ml min-1. Calibration of the system was performed in sterile Ringer's solution using air and oxygen-free nitrogen: calibration was repeated between each experiment.

Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection.

The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10(8)-10(9) c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10(8)-10(9) c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous reserves and survival of blastospores of Beauveria bassiana harvested from carbon- and nitrogen-limited batch cultures

Beauveria bassiana was grown in submerged culture on modified Vogel's medium. Patterns of utilization of glucose and ammonium, and analysis of the elemental composition of stationary phase biomass showed that cultures initially containing 3·3 g (NH4)2SO4l−1 and 10 g glucose l−1 were carbon-limited, whilst cultures containing 0·8 g (NH4)2SO4l−1 and 10 g glucose l−1 were nitrogen-limited. Modal volume, wall thickness and endogenous reserves (total carbohydrate, glycogen and lipid) of blastospores harvested from the stationary phase of nitrogen-limited cultures were all greater than values observed for those from carbon-limited cultures. The longevity (ability to form germ tubes) of conidia and carbon-limited blastospores stored in quarter strength Ringer's solution was determined at 4°, 25° and 30 °C, and for both spore types, was inversely related to temperature. At any given temperature, conidia survived longer than blastospores, but at 25°, nitrogen-limited blastospores survived longer than carbon-limited blastospores. Endogenous reserves of blastospores from carbon- and nitrogen-limited cultures declined during storage at 25°, and were related biphasically to spore longevity.

ATP content of stored spores of Paecilomyces farinosus and the use of ATP as a criterion of spore viability

A study was made of the viability and ATP content of blastospores of Paecilomyces farinosus stored in quarter-strength Ringer's solution at 4 and 20 °C. Blastospore viability decreased with time at both temperatures. Loss of ATP by blastospores was approximately biphasic, with a sharp initial decrease being followed by a more gradual decline. Appreciable loss of blastospore viability was only observed after they had lost ca 30% of their initial ATP content.

Evaluation of the direct epifluorescent filter technique for assessing the hygienic condition of milking equipment.

The hygienic condition of 6 milking installations, 3 sanitized by circulation cleaning (CC) with chlorine-based chemicals and 3 by flushing with acidified boiling water (ABW), was tested using rinses of quarter strength Ringer's solution. The bacterial content of the rinses was determined using both colony counts and the direct epifluorescent filter technique (DEFT). A comparison of testing methods gave correlation coefficients between colony count and DEFT of 0.82 for plants using CC and 0.46 for plants using ABW. Five strains of bacteria belonging to different genera and commonly found on milking equipment were exposed to various degrees of heat and to various concentrations of chlorine. The effects of such treatments on the staining characteristics of the organisms were studied. It was observed that Staphylococcus aureus and Streptococcus lactis, although killed by heat treatment, stained a bright orange when treated with acridine orange dye. Pseudomonas fluorescens, Escherichia coli and vegetative cells of Bacillus cereus did not take up the orange stain after heat treatment, nor did any of the 5 strains stain orange after treatment with NaOCl. It is suggested that the DEFT is a useful and rapid means of counting bacteria in rinses of equipment where sterilization is due primarily to chlorination, but in the absence of a stain which can differentiate more accurately between dead and living organisms its use is limited where sterilization is carried out solely by heat.

Influence of Temperature and Dosage On Mortality of Seventh Instar Larvae of Galleria Mellonella (Insecta: Lepidoptera) Caused By Heterorhabditis Bacteriophora (Nematoda: Rhabditoidea) and Its Bacterial Associate Xenorhabdus Luminescens

The seventh instar larva of Galleria mellonella is killed by Heterorhabditis bacteriophora and its associated bacterium Xenorhabdus luminescens at temperatures from 12° to 28°. Whereas nematode development is inhibited at 12° and 30°, Xenorhabdus can grow and cause mortality from 12° to 33°. The length of the incubation period depends on the bacterial growth rate. Ninety-five percent of invasive juvenile nematodes survived more than 60 days in quarter strength Ringer's solution.