A comparison of two air samplers for recovery of indoor bioaerosols

Summary A preliminary study was performed using two sampling instruments for airborne bacteria and fungi collection. A Reuter Centrifugal Sampler (RCS) and the open-faced type membrane filter sampler (Sartorius MD8) were compared for evaluating their capability of viable particles recovery. 61 series of parallel samples were collected in the air of a microbiological laboratory. Bacteria and fungi per cubic metre of air were enumerated using appropriate culture media and reported in terms of colony forming units (CFU). Performances of the two instruments for fungi were comparable and significantly correlated, particularly when the Rose Bengal Agar (RBA) medium was used (geometric mean: 237 CFU/m³ for RCS and 247 CFU/m³ for MD8; correlation coefficient: 0.78). Bacterial counts from MD8 resulted consistently lower than those obtained from RCS. The observed high variability suggests the existence of selective collection efficiencies which tend to underestimate the actual occurrence of airborne microrganisms.     

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series : a depth prefilter over a 1·2 μm filter ; a 0·8 μm prefilter over a 0·45 μm filter ; and a 0·2 μm sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.     

Recovery of Cryptosporidium oocysts from small and large volume water samples using a compressed foam filter system

A novel filter system comprising open cell reticulated foam rings compressed between retaining plates and fitted into a filtration housing was evaluated for the recovery of oocysts of Cryptosporidium from water. Mean recoveries of 90·2% from seeded small and large volume (100–2000 l) tap water samples, and 88·8% from 10–20 l river water samples, were achieved. Following a simple potassium citrate flotation concentrate clean-up procedure, mean recoveries were 56·7% for the tap water samples and 60·9% for river water samples. This represents a marked improvement in capture and recovery of Cryptosporidium oocysts from water compared with conventional polypropylene wound cartridge filters and membrane filters.     

Determination of the collection efficiency of a microbial air sampler

The Biotest RCS Plus air sampler was compared with the well-established Casella slit sampler for the quantitative analysis of the microbiological content of air. The results of the tests reported here show that the RCS Plus sampling efficiency is similar to that of the Casella sampler over the range of particles likely to be encountered in the environment. For particles less than 4 μm down to the sub-micronic sizes the efficiency of sampling falls off only gradually so that the efficiency of sampling for 1·0 μm particles is only reduced to about 50%. The ability to pre-select 10 different volumes (from 1 to 1000 1) enables the samplers to be used for measuring a wide range of concentrations of airborne micro-organisms in a variety of locations.     

Genetic population structure of red snappers (Lutjanus malabaricus Bloch & Schneider, 1801 and Lutjanus erythropterus Bloch, 1790) in central and eastern Indonesia and northern Australia

The genetic population structure of red snapper Lutjanus malabaricus and Lutjanus erythropterus in eastern Indonesia and northern Australia was investigated by allozyme electrophoresis and sequence variation in the control region of mtDNA. Samples were collected from eight sites in Indonesia and four sites in northern Australia for both species. A total of 13 allozyme loci were scored. More variable loci were observed in L. malabaricus than in L. erythropterus . Sequence variation in the control region (left domain) of the mitochondrial genome was assessed by RFLP and direct sequencing. MtDNA haplotype diversity was high ( L. erythropterus , 0·95 and L. malabaricus , 0·97), as was intraspecific sequence divergence, ( L. erythropterus , 0·0–12·5% and L. malabaricus , 0·0–9·5%). The pattern of mtDNA haplotype frequencies grouped both species into two broad fisheries stocks with a genetic boundary either between Kupang and Sape ( L. malabaricus ) or between Kupang and Australian Timor Sea ( L. erythropertus ). The allozyme analyses revealed similar boundaries for L. erythropterus . Seven allozymes stocks compared to two mtDNA stocks of L. malabaricus including Ambon, which was not sampled with mtDNA, however, were reported. Possible reasons for differences in discrimination between the methods include: i) increased power of multiple allozyme loci over the single mtDNA locus, ii) insufficient gene sampling in the mtDNA control region and iii) relative evolutionary dynamics of nuclear (allozyme loci) and mitochondrial DNA in these taxa. Allozyme and haplotype data did not distinguish separate stocks among the four Australian locations nor the central Indonesian (Bali and Sape locations) for both L. malabaricus and L. erythropterus.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

Bdellovibrio bacteriovorus Parasitizing Rhizobium in Western Australia

S ummary . Bdellovibrio bacteriovorus strains which parasitize Rhizobium meliloti, R. trifolii, Agrobacterium tumefaciens and A. radiobacter have been found in Western Australian soils. Rhizobium lupini was not lysed by any strain. Some Bdellovibrio cells pass through 0·22 μm membrane filters, whilst the 0·45 μm filters allow several saprophytic soil bacteria to pass. The strength of the medium is shown to be of great importance for the detection or counting of Bdellovibrio cells. The Bdellovibrio strains are capable of destroying large numbers of rhizobia in the laboratory.     

The Preservation of Plant Pathogenic Bacteria

Some 160 cultures were preserved by freeze drying, under mineral oil and in soil. After storage for 5 years all freeze dried cultures were viable; most cultures of xanthomonads were viable under oil and in soil; pseudomonads survived well in soil but only moderately well under oil; soft-rotting Erwinia spp. survived poorly but storage under oil was better than in soil; other Erwinia spp. and most Corynebacterium spp. survived well in soil and under oil. The mean half lives in years ( h ) calculated for freeze dried cultures of groups of closely related bacteria were: Erwinia 'chrysanthemi group', 0·40; Erwinia 'carotovora group', 0·51; Pseudomonas 'syringae group', 0·50; Xanthomonas spp., 0·84 years. Estimated half lives for Corynebacterium spp. ranged from 1·8 to 6·5 years. There was no evidence that bacteria which had been in culture for more than 3 years before being freeze dried had a longer storage life than those freeze dried within 3 years of isolation. Cultures of the Pseudomonas 'syringae group'had a longer storage life when freeze dried by Greaves'method ( h = 0·73) than when freeze dried by Annear's method ( h =0·50). There appeared to be no general correlation between half life in storage and either the proportion of cells surviving the freeze drying process or the viable cell count immediately after freeze drying. Most of the variation in the results could be attributed to variation in viable cell count between different ampoules of the same batch of a culture.     

Hydrogen-dependent control of the continuous thermophilic anaerobic digestion process using membrane inlet mass spectrometry

The control of a thermophilic continuous anaerobic digestion system when subjected to potential inhibitory shock loadings was achieved through the regulation of dissolved H₂, monitored using membrane inlet mass spectrometry, by the controlled addition of carbon source. At a feed pump switching threshold equivalent to 1 μmol/1 H₂ a steady state rate of methanogenesis of approximately 40 μmol/1/min was obtained. Higher H₂ thresholds resulted in an inhibition of methanogenesis, but precise control of H₂ concentration was demonstrated with an oscillatory response of period 2·5–5·0 min.     

Comparative analysis of different TaqMan real-time RT-PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control

Aims:  The aim of this study was to compare the performance of four TaqMan RT-PCR assays with a commonly used nested RT-PCR and to include the Feline calicivirus (FCV) as an internal control.
Methods and Results:  RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3·2 × 10³ PFU of FCV. Detection results obtained on faecal and plasma samples were 35·6% and 4·9% with the nested RT-PCR assay, 8·0% and 0%, 0% and 0%, 13·8% and 0% and 36·8% and 3·9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30·11 and 30·43 for the detection of FCV with HEV contaminated samples and negative samples.
Conclusions:  The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control.
Significance and Impact of the Study:  FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.     

Comparative proximate body composition of Atlantic salmon with emphasis on parr from fluvial and lacustrine habitats

The nutritional status of Atlantic salmon Salmo salar parr was assessed by examining how proximate body composition and energy content (kJ g⁻¹) were associated with the habitat rearing origin of parr utilizing fluvial or lacustrine areas. Comparisons were made among sampling sites within the two habitats, as well as between the two major habitat types applying both fixed effect and random effect models. Mean protein concentration varied from 13·87 to 15·67% among fluvial sites, and from 14·96 to 16·38% among lacustrine sampling locations. Mean fat concentration ranged from 1·71 to 3·32% and from 2·22 to 4·29% among fluvial and lacustrine locations, respectively, with energy concentration, on average, c . 11% higher in lacustrine parr. Interpretation of results of comparisons between habitats was dependent upon the statistical model used. Benefits associated with lacustrine rearing, such as greater body size and nutritional status, have implications for overwintering survival in fresh water as well as subsequent survival during initial sea entrance as smolts.     

Nuclear abnormalities of marine fish erythrocytes

The aim of this study was to monitor erythrocyte nuclear abnormalities (NA) including micronuclei (MN) in cultured and wild sea bass Dicentrarchus labrax and wild mullet Mugil spp. Seasonal sampling was performed at seven locations along the eastern coast of the Adriatic Sea. The frequency of NA and MN was positively correlated to temperature (NA: P < 0·05, r = 0·11; MN P < 0·05, r = 0·10), and there was also a positive correlation between NA and MN frequency ( P < 0·001, r = 0·43). The lowest NA and MN values for both fish species were recorded in spring, while the highest were recorded in autumn. Significantly higher frequency of NA was seen in D. labrax compared to Mugil spp., while MN frequency was low in both species and not significantly different. There was no significant difference in NA and MN frequency between cultured and wild D. labrax sampled in the same month, and there was no difference between wild Mugil spp. sampled near or far from fish farms. In view of sampling sites, the highest values were detected in fishes from the Limski Channel, the lowest from the Janjina location.     

Circadian rhythms of demand-feeding and locomotor activity in rainbow trout

Under free-running conditions, most rainbow trout displayed circadian feeding rhythms, although the expression of circadian rhythmicity depended on the experimental condition: 16·7% of fish under constant dim light (LL dim), 66·1% under a 45 :45 min light-dark cycle (LD pulses), and 83·8% under constant light (LL). Under LD pulses, the period length of the free-running rhythms for feeding was significantly shorter (21·9 ± 0·7 h, n =8) than under LL (26·2 ± 0·3 h, n =10). Period length for locomotor activity under LL was 25·8 ± 0·6 h ( n =4). Under LD conditions, the daily demand-feeding profile was always confined to the light phase and chiefly composed of two main episodes, directly after lights on (light elicited) and in anticipation to lights off (endogenous). Contrasting to feeding, the diel locomotor activity profile varied remarkably: a diurnal activity pattern at the bottom, while a clearly nocturnal pattern at the surface. These results contribute to a better understanding of feeding and locomotor rhythms of rainbow trout, providing evidence for the existence of a biological clock involved in their circadian control. This finding contrasts with the previously recorded lack of an endogenous oscillator in the pineal organ driving the rhythmic secretion of melatonin, which suggests different locations from the pineal for the circadian pacemakers in this species.     

Extrinsic control parameters for ozone inactivation of Escherichia coli using a bubble column

Aims:  To investigate the effect of extrinsic control parameters for ozone inactivation of E. coli in a bubble column.
Methods and Results:  Ozone inactivation of Escherichia coli ATCC 25922 in Tryptic Soya Broth was examined. The parameters studied included temperature (ambient, 20, 25 and 30°C), exposure time (up to 30 min), gas flow rate (0·03, 0·06, 0·12, 0·25, 0·5 and 0·75 l min⁻¹) and concentration level (five different levels). The efficacy of ozone treatment was a function of the parameters investigated and optimum control parameters of flow rate (0·12 l min⁻¹), temperature (ambient) and ozone concentration (75  μ g ml⁻¹) resulted in a t _d5 (time required to achieve 5 log reduction) of 20 min.
Conclusions:  Optimum control parameters of gas flow rate, ozone concentration and temperature are reported for E. coli inactivation within a bubble column.
Significance and Impact of the Study:  In 2001, the FDA approved use of ozone as a direct additive to food and in 2004, issued guidelines for the use of ozone in liquid systems. However, these guidelines highlighted gaps in the literature for ozonation of liquid foods. This study provides useful information regarding optimum extrinsic control parameters for E. coli inactivation in liquid media using a bubble column to ensure microbiological safety.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

Phylogeography of the flathead mullet Mugil cephalus in the north-west Pacific as inferred from the mtDNA control region

The population genetic structure and historical demography of the flathead mullet Mugil cephalus were investigated using the mtDNA control region (CR) sequences (909–1015 bp) of 126 individuals collected from seven locations in the north-west Pacific between 2005 and 2007. Haplotype diversity ( h = 0·9333–1·000) and nucleotide diversity (π = 0·0046–0·1467) varied greatly among the sampling locations. Phylogenetic analysis of the CR sequences indicated that M. cephalus in the north-west Pacific belongs to two highly divergent lineages (lineages 1 and 2), with the inferred population structure being closely associated with the distribution of both lineages. Two populations were identified, one from the East China Sea and the other from the South China Sea. The former samples were obtained from Taiwan and Qingdao of north China and associated with lineage 1 haplotypes. The latter samples were collected from the Philippines, Pearl River of South China and two samples from Japan, all of which were associated with lineage 2. Japanese samples from Okinawa and Yokosuka had different degrees of mixing between lineages 1 and 2. Historical demographic variables in both populations indicated that Pleistocene glaciations had a strong impact on M. cephalus in the north-west Pacific, resulting in a recent demographic decline of the East China Sea population but in demographic equilibrium for the South China Sea population. Japan appears to be a contact zone between lineages 1 and 2, but it may also be indicative of coexistence between resident and migratory populations. Further global studies are required to clarify the taxonomic status of this cosmopolitan species.     

Techniques for live capture of deepwater fishes with special emphasis on the design and application of a low-cost hyperbaric chamber

A cost effective, simple, portable hyperbaric chamber was constructed from polyvinyl chloride to aid in the collection of adult rockfishes Sebastes sp. to hold as broodstock. This system was designed to recompress fishes quickly once brought to the surface on hook and line, and to allow for decompression over a period of days. The hyperbaric chamber is capable of continuous stable operation at <1 033 515 N m⁻² and can accommodate fishes up to 91·4 cm in length and 26·8 cm in diameter. Pressure in the chamber is maintained by a Goulds Booster pump that delivers continuous pressure and supplies sea water at a rate of 3·8 to 7·6 l min⁻¹ to as many as four chambers. The hyperbaric chamber operated very effectively and allowed successful decompression of 12 cowcod Sebastes levis captured at depths of 90·2 to 146·3 m.     

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.     

Study on the antimicrobial effect of silver-containing inner liners in refrigerators

Aims:  To investigate the effect of silver-based antimicrobial material incorporated in the inner liners of refrigerators on food safety and quality.
Methods and Results:  In the first stage, the bactericidal effect was tested in the laboratory. Silver-containing samples and control plates were inoculated with different bacterial suspensions and stored at various temperatures. After defined storage periods the bacterial reduction was calculated by comparing viable cell count on reference plates and on silver-containing plates. The reduction caused by the silver-containing material varied between 1·0 and 5·9 log₁₀ units, depending on bacterial strain, incubation time and temperature. In the second stage, food storage experiments have been carried out. Thus, perishable foods were stored in coated and untreated refrigerators. After certain time periods the products were analysed for their sensorial and microbiological characteristics. A clear drop in viable counts both on the refrigerator wall and on the food was demonstrated using the silver-based antimicrobial material.
Conclusions:  Silver prevents refrigerators from being a hot spot for contaminants that could be transferred upon contact with food.
Significance and Impact of the Study:  This study provides original results regarding the antimicrobial activity of silver-containing refrigerator surfaces.     

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.