A METHOD FOR COUNTING THE TOTAL BACTERIA IN AMMONIATED HEVEA LATEX SYSTEMS

SUMMARY: A method has been developed for obtaining the total count of bacteria in ammoniated Hevea latex systems. This involves ultracentrifugation of latex or latex concentrate diluted in isotonic saline and the modified use of a normal phase contrast microscope to give a dark ground effect. Examples are given of the degree of bacterial contamination of field latex and concentrate. In these inhibitory ammoniated systems the total count may be high even if the viable count is low: in fact, in all the commercial latex concentrates examined the total count exceeded 10⁸ cells/ml. The value of interference and fluorescence microscopy has also been studied.     

A MEDIUM FOR THE CULTIVATION OF BACTERIA FROM HEVEA LATEX

SUMMARY: A medium has been devised for the culture of bacteria from fresh Hevea latex, ammoniated field latex and ammoniated latex concentrate. It is a modification of Kligler's iron agar, and can be used as a counting medium.     

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.     

Acceleration of yeast growth by addition of carbon to cultivation medium

Using a strain of Candida krusei IA-1 isolated from commercial Japanese sake lees, the effects of carbon on the lag phase and logarithmic growth phase were investigated in repeated batch cultivation. The lag phase was about 1 h shorter with the addition of 2%w/v of activated carbon to fresh medium. The free cell concentration was approximately 108 cells/ml in the presence, but only 105 cells/ml without addition. ©: Rapid Science Ltd. 1998     

The effect of ammoniation on the intake and nutritive value of straw

Studies were conducted to compare the increases in dry matter digestibility (DMD) in vitro and in vivo and to determine the metabolisable energy (ME) value of straw ammoniated at ambient temperature. Two stacks of straw sealed with polyethylene were allowed to react with 3% (w/w) anhydrous NH₃ for 30 and 56 days, respectively. Both DMD in vitro and nitrogen tests were carried out over an eight-week period subsequent to opening the stacks. Digestibility in vivo was measured with 12 wether lambs. The non-treated and ammoniated straws were given ad libitum, with a supplement of either ground barley or a lamb concentrate which contained 16% crude protein (CP).There was a mean increase of 15 percentage units in DMD in vitro for the ammoniated straw irrespective of whether it was treated for 30 or 56 days. The corresponding increase in mean DMD in vivo was 14.2 units. The CP content of the straw was increased from 3.1 to 7.6%. The increase in DMD in vitro and total N content was maintained throughout the sampling period. Approximately 58% of the anhydrous NH₃ added to the straw appeared to have been irreversibly “bound” to the straw. The ME values for the ammoniated straw were 6.78 and 7.49 MJ/kg when the straw was supplemented with either barley or the lamb concentrate, respectively. Straw ammoniation had a marked effect on intake. The overall increase in intake was 70% for the treated compared with the non-treated material.     

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.     

The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria

The growth and morphology of submerged bacterial colonies was investigated. Five separate colonial forms were recognized depending both on species and on agar concentration. These were (i) branched, dendritic structures seen only with Bacillus cereus ; (ii) lenticular colonies for all other species at high agar concentrations; (iii) small lobed to spherical colonies for non-motile organisms at low agar concentrations; (iv) and (v) large diffuse spherical colonies which can be further subdivided into 'snowball' or 'wispy' types for motile bacteria growing at agar concentrations below about 0·65% w/v. Viable count determinations suggested that agar concentration had little effect in the early stages of growth but that motile cells at low agar concentrations achieved higher cell numbers than did those in concentrations greater than 0·65% w/v. Transmission electron microscopy indicated that bacteria in lenticular colonies were tightly packed within lens-shaped splits in the agar whilst at low agar concentrations motile cells were well separated and appeared to move through the agar matrix.     

THE DETERMINATION OF COLI-AEROGENES ORGANISMS IN RAW MILK BY SURFACE SMEARS ON DRIED EOSIN METHYLENE BLUE AGAR PLATES

SUMMARY: The determination of the coli-aerogenes content of raw milk by smearing 0·1 or 0·2 ml of suitable dilutions on dried Levine's eosin methylene blue agar plates and incubating at 37° or 30°, was found too unselective as a routine technique. Only about two-thirds of the colonies showing the characteristic appearance of coli-aerogenes bacteria were confirmed as such by the formation of acid and gas in MacConkey's broth. Appreciable proportions of the numerous rose coloured colonies and of the very small dark colonies with metallic sheen, which are not considered to be coliaerogenes bacteria, formed acid and gas in MacConkey's broth.     

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-³H] leucine or [methyl-³H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum. Received 02 December 1998/ Accepted in revised form 25 April 1999     

The feeding value of ammoniated flax straw,wheat straw and wheat chaff for beef cattle

Two experiments were conducted to assess the feeding value of ammoniated and untreated flax straw, wheat straw and wheat chaff in comparison to a mixed bromegrass/alfalfa hay. Anhydrous ammonia was applied to the crop residues at the rate of 35 kg t⁻¹ dry matter. In the first experiment, the effect of ammoniation on crude protein, acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL), digestible organic matter in vitro and in vivo (DOM%), ADF and NDF digestibility of the crop residues was determined. In the second experiment, ammoniated flax straw, ammoniated wheat straw, ammoniated and untreated wheat chaff, each supplemented with barley, were compared to bromegrass/alfalfa hay as feed sources for wintering beef cows.Ammoniation increased the crude protein content of the crop residues ∼2-fold. Wheat straw DOM in vitro and in vivo was not increased by ammoniation. Ammoniation increased the DOM in vitro of wheat chaff from 36.3 to 46% and flax straw from 35.2 to 46.3%. The DOM in vivo increased from 53.3 to 63.4% (P < 0.05) for wheat chaff and from 33.9 to 58.4% (P < 0.05) for flax straw following ammoniation. Digestibility of ADF increased from 9.9 to 43.9% (P < 0.05) and of NDF from −0.6 to 37.9% (P < 0.05) in flax straw with ammoniation. Non-significant increases in ADF and NDF digestibility were observed for all other crop residues. Lignin content was not changed in the crop residues by ammoniation.In the winter feeding trial, young cows gained more weight than older cows (P < 0.05). Average daily gains of cows were greatest for hay followed by ammoniated flax straw, ammoniated chaff, untreated chaff and ammoniated wheat straw rations (P < 0.05). Increases in backfat in the younger cows was greatest with hay and ammoniated flax straw, followed by ammoniated chaff and ammoniated wheat straw (P < 0.05). Untreated chaff caused no increase in backfat thickness.Ammoniated flax straw (3.2 kg day⁻¹) given with barley (5.6 kg day⁻¹), is similar in feeding value to medium quality bromegrass/alfalfa hay. Furthermore, wheat chaff and ammoniated wheat chaff show good potential as alternatives to hay in winter feeding.     

Changes in microbial population during fermentation of tropical freshwater fish viscera

Freshwater fish viscera (FV) was homogenized, mixed with 10% (w/w of FV) molasses and 0, 2 or 4% salt and allowed to ferment at ambient temperature (26·2°C) under microaerophilic conditions. The results revealed a reduction in total viable count and the number of spores, coliforms, Escherichia coli , staphylococci and enterococci and an increase in yeasts and moulds and lactic acid bacteria during fermentation. Coliforms and E. coli were found to be absent after 6 d and enterococci on 8th day. The presence of salt resulted in a marginally lower number of all organisms except yeasts, moulds and lactic acid bacteria. Inclusion of either 0·5% propionic acid, 0·3% calcium propionate or 0·1% sorbic acid suppressed growth of yeasts and moulds with propionic acid being the most effective. The study indicated that a microbiologically stable product could be prepared by ensiling fish viscera with 10% molasses and 0·5% propionic acid.     

Interaction of Yeast Phosphoglyceromutase with Nucleotides

Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40 % O₂ were oxygen-resistant because when used as inocula for fresh 40 % O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40% O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

HYDROXYUREA EFFECTS IN THE C3H MOUSE II. MAMMARY TUMOR CELL KINETICS

The cellular kinetics of C3H mouse mammary tumors were studied following a single dose (3 mg/g body weight) of hydroxyurea (HU). This dose was large enough to cause a significant perturbation in the growth curves of these tumours. This was accomplished by labeling the cells with tritiated 5-iodo-2'-deoxyuridine and performing detailed autoradiographic analysis. This dose of HU caused a temporary inhibition in growth and completely inhibited DNA synthesis for 4–5 hr. The HU-killed cells (pyknotic and karyorrhectic) reach a maximum around 10–12 hr and are apparently all removed in about 1 day. Tumors from a fast-growing line (S102F) showed some evidence for cell synchrony upon recovery from HU inhibition but desynchronization occurred within one cell cycle. The cell generation time was not decreased during the acute recovery phase, but the growth fraction shifted from 0·6 to 1·0, and the data suggested that the normal flow of cells from the proliferating pool to the degenerate pool was temporarily interrupted. The cellular kinetic parameters have probably returned to normal by 48 hr after the HU injection.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology

Aim:  To evaluate the influence of water activity ( a _w), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising.
Methods and Results:  Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a _w (0·998, 0·982, 0·955, 0·928, 0·892) were combined with three incubation temperatures (10, 15, 20°C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a _w and temperature ( P <  0·0001) and a significant effect of pH ( P <  0·05). The radial growth rate and lag phase of P. vitreus were very sensitive to a _w reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a _w and growth became inhibited at a _w = 0·955. Optimal conditions for growth of P. vitreus were a _w = 0·998, 20°C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a _w than on temperature or pH under in vitro conditions.
Conclusions:  Low levels of a _w can prevent growth of P. vitreus , so wood moisture content should be adjusted accordingly.
Significance and Impact of the Study:  Implementation of these results should contribute towards the optimization and efficiency of bioincising.     

Analytical Studies on Regeneration of Protoplasts of Geotrichum candidum by Quantitative Thin-Layer-Agar Plating

A preparation of pure protoplasts of Geotrichum candidum became osmotically stable and colonies developed when the protoplasts were embedded in stabilizing thin-layer-agar and incubated with stabilizing basal medium. When growing protoplasts were exposed to distilled water and then reincubated with basal medium, the process of regeneration of protoplasts could be quantitatively demonstrated by counting colonies. The process was divided into three phases, lag, logarithmic, and stationary. Furthermore, the state of regeneration of protoplasts at each phase could be seen in detail by microscopic studies of protoplasts under similar growth conditions. In the lag phase, which lasted for 2 hr after inoculation, protoplasts were completely destroyed when placed in distilled water. During the logarithmic phase, from 2 to 5 hr after inoculation, protoplasts rapidly became osmotically stable and about 18% of them were growing. In the stationary phase, most protoplasts developed germ tubes within 2 hr. These results suggested that there are two main phases, although individual cells passed through three different conditions, osmotically labile, osmotically stable, and growing. No apparent structure of cell wall material could be detected by electron microscopy on the surface of the membrane of these osmotically stable cells.     

CHLOROPHYLL,NITROGEN, AND PHOTOSYNTHETIC PATTERNS DURING GROWTH AND SENESCENCE OF TWO BLUE-GREEN ALGAE1,2

A standardized, multiflask, batch culture system was developed to study the processes of algal senescence in Anacystis nidulans and Phormidium molle Gom, var. tenuior W. et G. West. Growth data over a 3-year period gave reproducible and comparable time-course curves. Although A. nidulans is unicellular and P. molle filamentous, the patterns of change with age were similar. Mean logarithmic doubling times and carbon yields were, respectively, 6.9 hr and 390 mg C/liter for A. nidulans and 7.2 hr and 710 mg C/liter for P. molle. Chlorophyll concentration and photo-synthetic capacity per unit carbon rose rapidly during the logarithmic phase to maximum levels in either late log phase (P. molle) or early linear phase (A. nidulans), then fell throughout the declining growth phase to low levels in the stationary phase. Nitrate was rapidly exhausted from the medium during the period of logarithmic growth and stoichiometrically converted to particulate organic form; very little subsequent fixation of molecular nitrogen occurred. The phycocyanins were rapidly destroyed during the logarithmic phase while the carotenoids remained relatively constant throughout the whole growth period and then slowly declined. Preliminary electron micrographs showed a progressive deterioration in cellular ultrastructure, especially a reduction in the number of photosynthetic thylakoids, commenting in the linear growth phase. Analysis of the results suggests that occurrence of linear growth kinetics and termination of culture growth were caused by exhaustion of nitrate. The observed decreases in chlorophylls, phycocyanins, and photosynthetic capacity during active culture growth show that senescence effects may not be, as assumed, restricted to the stationary phase of growth.     

THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.