A Note on the Diagnosis of Ratoon Stunting Disease of Sugarcane by Negative-Stain Electron Microscopy of the Associated Bacterium

After negatively staining with 1% (w/v) sodium phosphotungstate (pH 6·5) or 1% (w/v) ammonium molybdate (pH 6·5), the cell wall, cytoplasmic membrane and mesosomes of the RSD-associated bacterium obtained from the fibrovascular fluid of infected sugarcane were usually clearly displayed. The cells measured 0·19–0·39 μm (av. 0·27 μm) in width and 0·6–3·4 μm in length. Few mesosomes were visible and the cells were approx. 40% wider (0·27–0·52 μm, av. 0·38 μm) when stained with 1% (w/v) uranyl acetate (pH 3·0–4·2). Freezing and thawing the suspension before negative staining with sodium phosphotungstate did not greatly affect the size of the cells or resolution of the mesosomes. Glycine (0·25 M) as the suspending medium, fixation in 2% (w/w) glutaraldehyde, or placing wet instead of dry specimen grids in the electron microscope resulted in wider cells usually lacking mesosomes.     

A Note on the Survival of some Bacteria in Different Diluents

The best diluent for four bacterial species was 0·1% (w/v) peptone solution. Tap water containing 0·1% (w/v) sodium thiosulphate was less satisfactory but tap water, tap water treated with charcoal, quarter-strength Ringer's solution, 0·85% (w/v) sodium chloride solution and glass distilled water were all bactericidal to one or more of the test species.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.     

A Quantitative Evaluation of the Antifungal Properties of Glutaraldehyde

Fungistatic data were obtained from measurements of mycelial growth of several fungal species in the presence of acid or alkaline glutaraldehyde. Alkaline glutaraldehyde in concentrations > 0·1% (w/v) prevented growth of all species examined while 0·5% (w/v) acid glutaraldehyde was necessary to achieve this effect. Further fungistatic data were obtained using a Coulter Counter method to measure spore swelling. Fungicidal determinations resulted in a 99·99% reduction in viable count after 90 min contact with 0·5% (w/v) alkaline glutaraldehyde. A considerable drop in spore production was also observed after treatment with alkaline glutaraldehyde.     

Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli

Escherichia coli K-12 was exposed to Quillaja saponaria saponins from various commercial firms (Sigma, Roth and Nor-feed) and to an extract of Yucca schidigera plant powder (DK Sarsaponin 30) at different concentrations (0·05–1·0% w/v). A concentration-dependent response was observed. Quillaja saponaria saponins from Sigma increased growth up to 0·1% (w/v) level, whereas Nor-feed and Roth saponins produced maximum growth at a much higher level (0·5 and 0·75%, w/v, respectively). These results suggest that quillaja saponins from various sources differ in their biological activity, although all three saponins had the same content of vanillin-sulphuric acid reactive moieties. The lyophilized water extract from the DK Sarsaponin powder showed maximum growth at 0·1% (w/v) level. The levels at which maximum growth was observed did not change on subjecting the quillaja or yucca saponins to heat treatment in an autoclave (121 °C for 30 min). All the saponins and the plant extract increased growth of Escherichia coli up to a certain concentration and thereafter decreased growth. In spite of the decreased growth at higher levels of saponins, it was higher compared to the control (without saponin) up to levels of 1% (w/v) for all saponins except Quillaja saponins from Sigma, for which the growth was lower at levels of 0·25% (w/v) and higher. Saponins have the potential to modulate microbial growth in natural and artificial fermenters.     

A relatively small change in sodium chloride concentration has a strong effect on adhesion of ocular bacteria to contact lenses

Adhesion of bacteria to hydrogel lenses is thought to be an initial step of ocular colonization allowing evasion of normal host defences. The salt concentration of media is an important parameter controlling microbial adhesion. Salinity varies from 0·97% NaCl equivalents in the open eye to 0·89% in the closed eye state. In this study, the effect of sodium chloride in the concentration range of 0·8–1·0% (w/v) NaCl on adhesion of ocular bacteria to soft contact lenses was investigated using a static adhesion assay. Pseudomonas aeruginosa was found to adhere to lenses in significantly greater amounts than Serratia marcescens, Flavobacterium meningosepticum, Stenotrophomonas maltophilia and Staphylococcus intermedius . Increasing NaCl from 0·8% to 1·0% (w/v) increased adhesion of all bacteria tested. This adhesion was strong since the organisms could not be removed by washing in low ionic buffer. Adhesion of these organisms did not correlate with their cell surface properties as determined by bacterial adhesion to hydrocarbons (BATH) and retention on sepharose columns.     

Enhanced secretion and low temperature stabilization of a hyperthermostable and Ca2+-independent alpha-amylase of Geobacillus thermoleovorans by surfactants

AIMS: Selection of suitable surfactants for enhancing and stabilizing alpha-amylase of Geobacillus thermoleovorans. METHODS AND RESULTS: Geobacillus thermoleovorans was cultivated in shake flasks containing 50 ml of starch-yeast extract-tryptone (SYT) medium with/without surfactants. Titres of the enzyme in media were monitored. The enzyme was also preserved at 4 degrees C with/without surfactants and enzyme activities were determined. Among polyethylene glycol (PEGs) of different molecular weights, PEG 8000 (0.5%, w/v) caused a slight increase in the enzyme titre, while Tween-20, Tween-40 and Tween-60 (0.03%, w/v) exerted a significant stimulatory effect on enzyme secretion. In the presence of SDS, Tween-80 and cholic acid (0.03%, w/v), the enzyme production was nearly twofold higher than that in the control. The anionic (SDS, cholic acid) and non-ionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced alpha-amylase secretion. Furthermore, anionic surfactants exhibited stabilizing effect on the enzyme during preservation at 4 degrees C. CONCLUSIONS: PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide). Further, these surfactants were found to stabilize the enzyme at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The secretion of Ca2+-independent hyperthermostable alpha-amylase was enhanced in the presence of certain anionic and non-ionic detergents in the medium. Furthermore, the surfactants stabilized the enzyme during preservation at 4 degrees C. The use of this enzyme in starch hydrolysis eliminates the addition of Ca2+ in starch liquefaction and its subsequent removal by ion exchange from sugar syrups.     

Changes in the acidity and lactic acid content of 'Nono', a Nigerian cultured milk product

Changes in the pH value and lactic acid content of the uninoculated, cultured Nigerian whole milk product, 'Nono', during incubation at room temperature (27°± 2°C) for 120 h were monitored. The pH decreased from 6·78 ± 0·19 to 4·30 ± 0·11 while the lactic acid content increased from 0·32 ± 0·04% to 2·50 ± 0·02% (w/v). This was accompanied by souring and curdling of the milk particles.     

An Examination of the Effect of Three Phenolic Disinfectants on Mycobacterium tuberculosis

The effect of a phenolic disinfectant ( o -phenylphenol 45% w/w) with linseed oil soap or with soya oil soap on Mycobacterium tuberculosis was determined by three methods. Neither the geometrical dilution test nor the modified capacity use-dilution test revealed any differences between the two disinfectants. However, paradoxically both methods proved that the highest concentration of the disinfectants tested (3·5% v/v) exhibited a very low germicidal effect on M. tuberculosis , whereas lower concentrations showed a much better effect. When determining the tuberculocidal effects of various concentrations of the two disinfectants at different exposure times, the higher concentrations showed very low effects, even after the longest exposure time. At concentrations of 2·0 and 1·0% (v/v), the disinfectants displayed the most rapid effects. In the present investigation the disinfectant with the linseed oil soap seemed to destroy the cells more quickly than that with the soya oil soap. The third disinfectant containing p -chloro- m -cresol and o -benzyl- p -chlorophenol with a total of 9·2% (w/w) phenols in a detergent system, did not display, when employing the capacity use-dilution test, the same phenomenon in the concentrations used, but the experiment showed that the recommended use-dilution concentration ought to be doubled.     

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the "one variable at a time" and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone--1, yeast extract--0.5, sodium chloride-1, olive oil-1, Tween-80 1, manganese sulphate--5 mM, sucrose--1, pH-7. It was found that maximum production occurred in late log phase, i.e., after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3% (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.     

Influence of different rations and water temperatures on the growth rates of shortfinned eels and longfinned eels

Juvenile (12–152 g) shortfinned eels Anguilla australis and longfinned eels A. dieffenbachia caught in New Zealand streams were fed squid mantle Nototodarus spp. 4 days per week in laboratory experiments. A linear multiple regression equation showed the amount of food eaten (0–2·7% w day⁻¹) explained 77·7% of the variation in specific growth rates (–0·60 to +1·07% w day⁻¹) among individual eels, while previous growth rates, water temperature (10·0–20·6°C), and eel weight (12–152 g) explained a further 5·6, 1·4 and 0·8%, respectively. Growth in length ranged from –0·3 to +0·9 mm day⁻¹. Eels which were starved and then given high rations grew substantially faster than expected. Once growth rates were adjusted for differences in ration and other factors, there were no significant differences in growth rates between species or individual fish. Growth of shortfinned eels fed maximum rations of commercial eel food depended on fish size and water temperatures and ceased below 9·0°C. Growth rates in the wild were substantially less than the maximum possible, after seasonal changes in water temperatures were taken into account, indicating that food supplies and not low water temperatures were controlling growth rates in the wild.     

Adaptation of the tetrazolium method for testing the seed viability, and scanning electron microscopy study of some Western European orchids

A modified tetrazolium method was formulated for use with seeds of Western European orchids. The sequence of treatments which gave the highest percentage of coloured (i.e. viable) embryos was: (1) pretreatment in a solution of 5% (w/v) Ca(OCl)₂+ 1% (v/v) Tween-80, (2) soaking for 1 day in sterile water, (3) the classical tetrazolium test. The optimal duration of the pretreatment in Ca(OCl)₂+ Tween-80 depends upon the species, and to investigate the effect a scanning electron microscopy study was performed on the testa of 3 species. For a given species, the optimal pretreatment period was not affected by the year of harvest or the source of the seed lots.     

The kinetics of Bacillus subtilis spore inactivation on filter paper by u.v. light and u.v. light in combination with hydrogen peroxide

Inactivation of spores of Bacillus subtilis (ATCC 6633) on two different grades of cellulose filter paper (Whatman Grades 2 and 6), by ultraviolet light (u.v.), at an intensity of approximately 4·5 Wm⁻² and at fluences of up to 2 × 10³ Jm⁻², and u.v. in the presence of hydrogen peroxide, is described in terms of multi-target and single hit–single target kinetic expressions. Wet spores were inactivated at rates ranging from 6·7 to 10·6 higher than that of dry spores on both grades of filter paper. In addition, spore inactivation was up to 5·6 times more rapid on Grade 2 filter paper. Synergistic inactivation was seen to occur when spores were irradiated in the presence of 1% (w/v) hydrogen peroxide with rates up to 5·3 times higher than with treatment solely by u.v. The results obtained are discussed in general terms with particular reference to surface characteristics which might provide shielding to micro-organisms from incident u.v. light.     

Development and application of a simple filter paper imprinting technique for the detection and enumeration of colonies of ureolytic micro-organisms

A rapid, simple and inexpensive method has been devised to determine the proportion of colonies of ureolytic organisms in cultures of complex microbial populations. Spiral plated cultures displaying well separated colonies are tested for ureolytic micro-organisms by imprinting the colonies onto filter papers impregnated with a solution of 1 mol/l urea and phenol red (0·1% w/v) in 0·1 mol/l phosphate buffer (pH 6·8). A rapid colour change indicates ureolytic activity. The proportion of ureolytic colony-forming units in cultures of saliva specimens from 90 school children ranged from less than 1% to 40% (mean 9·9%± 7·7). Saliva and dental plaque specimens from 16 adult subjects were also tested and the occurrence of urease-positive organisms was substantially less in plaque (3·6%± 3·7, range 0·1–12) than saliva (18·7%± 13·8, range 1·3–51). The predominant ureolytic oral species was Streptococcus salivarius , 75 (54·7%) of 137 tested isolates being urease-positive.     

Interfering mechanism of sodium bicarbonate on spore germination of Bacillus stearothermophilus

Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO₃) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO₃ was attributed to the release of HCO⁻₃ and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO₃ was mainly due to bicarbonate. As NaHCO₃ increased from 0·3% to higher concentrations, the effect of HCO⁻₃ reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm⁻¹) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na⁺ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm⁻¹) and of amide II (1546 cm⁻¹) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO₃ in enhancing the sporicidal effect of glutaraldehyde is discussed.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Sensitization of thermally injured spores of Bacillus stearothermophilus to sodium benzoate and potassium sorbate

The effects of sodium benzoate and potassium sorbate added to the recovery medium, at different pH values (6·5, 6·0 and 5·0), on the recovery rates and heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Heated spores of strains 12980 and 7953 were inhibited by sorbate concentrations over 0·05%. Potassium sorbate at concentrations as low as 0·025%, and sodium benzoate at 0·1%, were very effective inhibitory agents for heat-damaged spores. Their effectiveness always increased at pH 5·0, at which no growth occurred, with sodium benzoate for strains 7953, 15951 and 15952, and with potassium sorbate for strains 15951 and 15952. Decimal reduction times, whenever recovery was possible, were not significantly ( P  > 0·05) affected. None of these compounds modified the z -values obtained for the spores of the four strains, which had a mean value of 7·53 ± 0·28.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

Optimization of a reconstituted skim milk based medium for enhanced CLA production by bifidobacteria

Aims:  To determine the effect of a range of supplements on the bioconversion of linoleic acid to conjugated linoleic acid (CLA) by Bifidobacterium breve NCIMB 702258 in reconstituted skim milk (RSM).
Results:  Seven supplements (yeast extract, casein hydrolysate, tryptone, l -cysteine hydrochloride, sodium acetate, sodium butyrate and sodium propionate) were identified as increasing the bioconversion of linoleic acid to c9 , t 11 CLA. Using these supplements, the percentage bioconversion of linoleic acid (0·35 mg ml^−l) to the c9 , t 11 CLA isomer was elevated from 15·5 ± 1·1% in 20% RSM (w/v) to 48·1 ± 2·2% in the supplemented RSM. Through additional supplementation of 20 mg m1⁻¹ inulin and optimization of inoculum and linoleic acid concentration, the percentage bioconversion to c9 , t 11 CLA was increased to 55·0 + 3·2%.
Conclusions:  Through supplementation, the concentration of CLA produced by bifidobacteria in RSM can be increased to levels comparable to those observed in the synthetic medium cys-MRS.
Significance and Impact of the Study:  The impact of 22 supplements on the production of the c9 , t 11 CLA isomer by the strain B. breve NCIMB 702258 in milk has been determined. The results provide an understanding of the factors, which influence CLA production by bifidobacteria in RSM.     

The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes , Flavimonas oryzihabitans , and three species of Pseudomonas . The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0·125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10³ to 10⁶ cfu ml⁻¹. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens . The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0·1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0·1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.