Study of the bacterial flora of a non-carbonated natural mineral water

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the prebottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9-12 months. The plate counts in R2A medium incubated at 22 degrees and 37 degrees C were low initially, increasing to 10(4)-10(5) cfu/ml within a few days of bottling. The number of bacteria recovered at 22 degrees C from PVC bottles was fairly constant during the storage period, but the population isolated at 37 degrees C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis. Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

M orais , P.V. & D a C osta , M.S. 1990. Alterations in the major heterotrophic bacterial populations isolated from a. still bottled mineral water. Journal of Applied Bacteriology 69 , 750–757.
The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9–12 months. The plate counts in R₂A medium incubated at 22 and 37C were low initially, increasing to 10⁴-10⁵ cfu/ml within a few days of bottling. The number of bacteria recovered at 22C from PVC bottles was fairly constant during the storage period, but the population isolated at 37C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis . Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Differentiation of four Aspergillus species and one Zygosaccharomyces with two electronic tongues based on different measurement techniques

Two electronic tongues based on different measurement techniques were applied to the discrimination of four molds and one yeast. Chosen microorganisms were different species of Aspergillus and yeast specie Zygosaccharomyces bailii, which are known as food contaminants. The electronic tongue developed in Link?ping University was based on voltammetry. Four working electrodes made of noble metals were used in a standard three-electrode configuration in this case. The St. Petersburg electronic tongue consisted of 27 potentiometric chemical sensors with enhanced cross-sensitivity. Sensors with chalcogenide glass and plasticized PVC membranes were used. Two sets of samples were measured using both electronic tongues. Firstly, broths were measured in which either one of the molds or the yeast grew until late logarithmic phase or border of the stationary phase. Broths inoculated by either one of molds or the yeast was measured at five different times during microorganism growth. Data were evaluated using principal component analysis (PCA), partial least square regression (PLS) and linear discriminant analysis (LDA). It was found that both measurement techniques could differentiate between fungi species. Merged data from both electronic tongues improved differentiation of the samples in selected cases.     

Effect of Vibrio alginolyticus on larval survival of the blue mussel Mytilus galloprovincialis

The effect of increasing concentrations of Vibrio alginolyticus on survival of Mytilus galloprovincialis larvae was studied in a 48 h static bioassay in 1 l glass bottles. Five bacterial densities were tested ranging from 10(2) to 10(6) bacteria ml(-1). Larval survival and normality (veliger larvae with the typical D-shape) were evaluated after 48 h. An inverse relationship between bacterial concentration and larval survival and normality was observed. In spite of high larval survival (79%) under conditions of high bacterial density (10(5) bacteria ml(-1)), the percent of normal larvae was 11%. Besides an irregular shape, abnormal larvae also presented velum reduction. Results from this study suggest that concentrations of V. alginolyticus lower than 10(3) bacteria ml(-1) should be maintained during M. galloprovincialis larval culture.     

Evaluation of blood culture bottles seeded with X-V factors for the detection of Neisseria meningitidis and Haemophilus influenzae

The purpose of this in vitro study was to determine the ability of seeded and not-seeded commercial pediatric blood culture bottles to support the growth of the most frequently responsible microorganisms for bacterial meningitides (Neisseria meningitidis, and Haemophilus influenzae). Tests have been carried out with an automated colorimetric pediatric blood culture system, BacTAlert, Organon Teknika. Bottles were inoculated with X-V factors and serial dilutions of the each bacterium in six times (10(1)-10(6) colony forming unit [CFU]/ml). The bottles, which were supplemented with X-V factors, proved to be effective and time to detection (TTD) was shorter than the un-seeded bottles (p0.05). Time difference between seeded and not-seeded bottles was getting greater at high dilutions of both bacteria. We consider that in presence of a few bacteria, the seeding of bottles with X-V factors is very critical obtaining N. meningitidis, and H. influenzae as the causative agents of meningitidis. The recovery rate of the microorganisms, which were isolated from cerebrospinal fluid by using the X-V factor-seeded blood culture bottles, is therefore higher than with the conventional culture methods.     

Survival of allochthonous bacteria in still mineral water bottled in polyvinyl chloride (PVC) and glass

The mortality of Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa, based on the culturability of these bacteria, was assessed in non-carbonated mineral water, bottled in polyvinyl chloride (PVC) containing the indigenous flora, sterile mineral water bottled in PVC, sterile mineral water in glass containers, and sterile tap water in glass containers. There was a general decrease in the culturability of these organisms in the four test waters, except that Ps. aeruginosa grew in sterile tap water. Escherichia coli and Kl. pneumoniae had the highest mortality rates under the conditions tested, while Ent. cloacae had a very low and constant mortality rate that would have resulted in the persistence of this organism in mineral water for a long period of time. After a sharp initial decrease in culturability, Ps. aeruginosa also had a very low mortality rate in mineral water bottled in PVC.     

A simple micro-growth assay for enumerating bacteria

A simple method for nonspecific determination of bacteria concentrations in a variety of liquid samples was developed. The assay was based on the time required for a sample grown in liquid media to reach a threshold turbidity. Samples were combined with media in a covered 96-well microwell plate and the turbidity was monitored in real time as the bacteria grew in a temperature-controlled plate reader. A significant problem with growth in microwells was condensation on the cover, which prevented accurate turbidity measurement. This problem was overcome by coating the cover with a small amount of surface-active agent. Salmonella and E. coli concentrations could be determined with a relative error of approximately 20% at levels from 10 to 10(6) cells/ml (eight replicates). An assay of 10 samples with standards required 10 min to set up and 20 min for data processing using a computer spreadsheet program. Growth time at 37 degrees C ranged from 4 h for samples at 10(7) cells/ml to 16 h for samples at 10 cells/ml.     

Biocorrosion and biodeterioration of antique and medieval glass

Over the past few years we have examined various antique and medieval glasses with regard to general biogenic damage, biopitting (crater erosion), bio‐crusts, and opalescent and white biogenic films. Experiments were carried out on pieces from Roman glass bottles excavated near Abu Tor, Sinai, some pieces of green and blue glass from Cologne Cathedral, some pieces from a little church in Evreux, glass samples from the fortress of the former Dukedom of Delmenhorst near Oldenburg, and some neolithic flint tools from the Negev Desert, Israel. Modern glass from a pigsty (19th century) additionally has been used for laboratory experiments on the attack of glass surfaces by fungi and bacteria. Some of the bacteria used in these experiments were isolated from the ancient pieces of glass. Biopitting with structures very similar to the biopitting of marble and limestone was found on almost all specimens. Lichens were not identified directly, but fungi and algae were observed in the pits as well as under the thin layers exfoliating from the Roman glass bottles. Initial steps of colonization and the potential for heavy‐metal accumulation by the isolated bacteria have been shown in laboratory experiments. A fractal dimension of diffusion‐limited disaggregation (DLD) is suggested as one possible explanation for the characteristic form and structure of the microbially induced and shaped biopitting patterns. A biopitting classification is suggested.     

The effect of summer and winter seasons on the survival of Salmonella typhimurium and indicator micro-organisms during the storage of solid fraction of pig slurry

AIMS: Investigations were carried out to observe the influence of winter/spring and summer periods on the survival of Salmonella typhimurium and indicator bacteria (psychrophilic, mesophilic, coliform and faecal coliform bacteria and faecal streptococci) in the solid fraction of pig slurry from agricultural wastewater treatment plant. METHODS AND RESULTS: Leather squares and PVC bottles with openings served as test carriers. They were inoculated with broth culture of Salm. typhimurium and introduced directly into the solid fraction. During the experiment, quantitative and qualitative examinations were carried out to determine the presence of Salm. typhimurium and observe the dynamics of indicator bacteria in the solid fraction. CONCLUSIONS: Salmonella typhimurium survived for 26 d in summer and for 85 d in winter/spring. The T90 values of indicator bacteria in summer ranged from 35.44 d (coliform bacteria) up to 100.29 d (mesophilic bacteria). The winter T90 values of indicator bacteria ranged from 74.58 d (faecal coliform bacteria) to 233.07 d (coliform bacteria). SIGNIFICANCE AND IMPACT OF THE STUDY: The present study demonstrated that it is necessary to pay increased attention to the manipulation of slurry solid fraction.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

Decomposition of Urea by the Larger Particulate Fraction and the Free Bacteria Fraction in a Pond Water

An aliquot of a water sample taken from an artificial pond on the campus of the Faculty of Science, Tokyo Metropolitan University, was filtered with Whatman GF/C glass fiber filters. Urea was added to the filtered and unfiltered waters, respectively to a final concentration of around 10 μg-at-N/l. These samples were kept in 10 liter glass bottles, which were kept on the surface of the pond with rope. Changes in the concentrations of urea, ammonium, nitrate, nitrite, and the number of bacteria were traced from 15 June through 13 July, 1973. The urea added to the unfiltered water decreased to about 1 μg-at-N/l within first three days. The decrement of the urea seemed to proceed as a first-order reaction with rate constant of 0.73 day⁻¹. On the other hand, the urea added in the filtered water kept nearly the initial concentration for 18 days. As the number of bacteria in the filtered and unfiltered water were not significantly different, decrease of the urea in the unfiltered water may be ascribable to the participate fraction removed by the filtration. The urea was not decomposed by free bacteria.     

Multiplication in liquid medium of Treponema sp. isolated from intestinal contents of swine

Treponema hyodysenteriae and Treponema innocens were multiplied by a simple culture method in liquid medium. TSB medium was prepared by the PRAS method in plasma bottles containing glass beads. Spirochaetes were injected through the rubber stopper and the bottles were incubated while revolving round their axes. The most abundant growth of spirochaetes in rotary culture was observed after 72 h incubation at 40 degrees C. whereas the highest number of viable cells in stationary culture was observed after 120 h. However, in the latter case the number of cells was lower than introduced at inoculation. Growth of the bacteria was stimulated by equine serum and 5% addition of rumen fluid. Optimal growth temperature was 40 degrees C.     

Suitability of poor medium in counting total viable airborne bacteria

The purpose of this study was to test the effect of incubation temperature and culture medium on viable counts of airborne bacteria. The incubation temperature had different effect on indoor and outdoor air bacteria. Indoor air bacteria grew as well at 20°C as 37°C, but less at 10°C. Outdoor air bacteria grew equally well at 10°C and 20°C, but less at 37°C. Both indoor and outdoor air bacteria grew differently on poor and rich media. The counts of both indoor and outdoor air bacteria were higher on poor R2A medium (low nutrient concentration) than on rich TYG and blood media (high nutrient concentration). The results indicate that a poor medium incubated at 20°C is adequate for counting viable airborne bacteria.     

Sterilization by Means of Hydrochloric Acid Vapour

As heat sterilization of glass bottles often results in breakage and of most plastic containers in deformation, we investigated a rapid low-temperature sterilization method as an alternative. Vapour evolving from hydrochloric acid was chosen because it does not leave toxic residues which might contaminate food packed in treated containers. Vapour evolving from 0.25 ml of 31% (w/w) hydrochloric acid reduced the number of viable spores of aerobic and anaerobic bacteria in bottles (300 ml) by a factor of at least 2500 and that from 0.01 ml of 37% (w/w) hydrochloric acid reduced the number of mould spores by a factor of over 40 000 within 5 min at 20°C.     

改良PCA培养基检测鸡肉胴体中的污染细菌

通过添加鸡肉浸液改良经典PCA培养基获得模拟鸡肉营养条件的近自然的CEA培养基,用于检测鸡肉中对营养要求苛刻的污染细菌。根据添加不同浓度的新鲜鸡肉浸液所获得菌落总数的变化获得最佳添加浓度,并利用生化特性及16SrRNA序列对仅生长于CEA培养基的细菌进行了鉴定。在CEA培养基上获得的细菌数量和多样性都显著高于PCA培养基(P0.05),且分离到3株无法在PCA培养基上生长的细菌,经鉴定其与Enterococcus faecalis、Rothia mucllaginosa,Staphylococcus saprophyticus subsp.saprophyticus的相似性分别为99%、96%、99%。E.faecalis因产生生物胺而被认为是肉品中的腐败菌,后两者则均是条件致病菌株,他们的存在对鸡肉产品的安全可能造成隐患。该方法中的近自然培养法能提高对微生物数量和种类的检测灵敏度,特别适于对营养要求苛刻的污染细菌的检出。     

Effect of Support Material on the Development of Microbial Fixed Films Converting Acetic Acid to Methane*

A study of the development of methanogenic fixed films on pieces of polyvinyl chloride plastic, etched glass and baked clay showed that support material markedly affected the rate of attachment and growth of bacteria converting acetic acid to methane. Film development, as indicated by the rate of acetate conversion to methane and carbon dioxide, was threefold faster on fired clay than on either PVC plastic or etched glass. Scanning electron micrographs showed that the film of bacteria attached to clay was thick and uniform, while the film attached to PVC plastic was thin although still uniform. Attachment to etched glass was spotty. The characteristics of clay which made it a superior support appeared to be its rough, porous surface which offered attachment sites to the micro-organisms and the presence of minerals in the clay, particularly iron which is known to stimulate methanogenesis and growth.     

Displacement of Enterococcus faecalis from hydrophobic and hydrophilic substrata by Lactobacillus and Streptococcus spp. as studied in a parallel plate flow chamber.

The displacement of Enterococcus faecalis 1131 from hydrophobic and hydrophilic substrata by isolates of Lactobacillus casei 36 and Streptococcus hyointestinalis KM1 was studied in a parallel plate flow chamber. The experiments were conducted with either 10 mM potassium phosphate buffer or human urine as the suspending fluid, and adhesion and displacement were measured by real-time in situ image analysis. The results showed that E. faecalis 1131 was displaced by lactobacilli (31%) and streptococci (74%) from fluorinated ethylene propylene in buffer and that displacement by lactobacilli was even more effective on a glass substratum in urine (54%). The passage of an air-liquid interface significantly impacted on adhesion, especially when the surface had been challenged with lactobacilli (up to 100% displacement) or streptococci (up to 94% displacement). These results showed that the parallel plate flow system with real-time in situ image analysis was effective for studying bacterial adhesion and that uropathogenic enterococci can be displaced by indigenous bacteria.     

Semi-Commercial-Scale Production of Japanese B Encephalitis Virus Vaccines from Tissue Culture

This study was undertaken to modify and develop procedures for tissue culture-inactivated Japanese B encephalitis (JBE) virus vaccine production in large quantities. Various types of glass bottles were tried and, considering many advantages, long cylindrical roller (CR) bottles were selected. Several variables were investigated including number and volume of trypsinized cells to be seeded, volume of growth medium required for optimum cell growth, amount of calf serum, and volume of harvest medium for a high-titer virus yield. A good confluent cell sheet in CR bottles was obtained within a week by increasing the calf serum from 4 to 10% and when such tissue in a CR bottle was inoculated with 45,000 viral mean tissue culture infective doses directly into the medium, the cytopathological effects (CPE) appeared on day 5. High-titer virus yields were obtained when the harvests were made at 4(+) CPE using medium 199 with 2% human albumin at pH 8.3 to 8.5. No appreciable gain in titer was found from such harvests by blending to release intracellular virions. The production methods finally adopted gave consistently good results, and several inactivated JBE virus vaccine lots with minimum immunizing doses, ranging from 0.005 to 0.017 ml, were prepared using a large number of CR bottles in a simulated commercial-scale production system.