The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces

Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 times 10³ to 5 times 10⁷ colonies/cm² whilst DEFT enumerated populations above 3 times 10⁴ colonies/cm² and TVC above 3 times 10⁵ colonies/cm² (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 times 10⁵ colonies/cm² most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 times 10⁵ colonies/cm² than was TVC.     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep

Selective plating procedures were used to follow the fate of rifampicin-resistant mutant strains of the obligately anaerobic species Bacteroides multiacidus and Selenomonas ruminantium after their introduction at numbers around 10⁷/ml into the rumen of sheep. Bacteroides multiacidus strain F100 showed an initially rapid rate of loss (49%/h) but subsequently numbers declined more gradually approaching the limits of detection (10³/ml) after 100 h. Viable cell numbers also decreased in vitro upon addition of F100 cells to whole rumen contents, but remained stable upon addition to cell-free rumen fluid, suggesting protozoal predation. F100 cells were able to grow in vitro in whole rumen contents in the presence of an added utilizable substrate such as sorbitol, but addition of sorbitol to the rumen failed to enhance survival in vivo . In the case of S. ruminantium , introduced rif^R strains persisted in the rumen at levels around 10⁶ ml for at least 30 days.     

The distribution of some freshwater planktonic bacteria in two stratified eutrophic lakes

SUMMARY. Changes in bacterial populations and certain physical and chemical variables in Esthwaite Water between June and September 1975 were studied and compared with results obtained from 1972 to 1974 in the hypolimnia of Blelham Tarn and the Lund tubes. The counts of total bacteria ranged between 1 and 7 × 10⁶ml⁻¹ and were highest in the anoxic hypolimnion. The bacterial genera examined in more detail constituted only a small percentage of this count and included Ochrobium (10⁴ml⁻¹), Naumanniella (10³ml⁻¹), Leptothrix (10²ml⁻¹), Planctomyces (10³ml⁻¹), and Metallogenium (10²ml⁻¹). The iron bacteria appear to grow best in the oxycline where there was not only sufficient oxygen for aerobic growth but also a plentiful supply of reduced iron. Planctomyces numbers increased as the thermocline became depressed in September. The results from Blelham Tarn might be interpreted as further evidence of growth by iron bacteria in the absence of dissolved oxygen, but other explanations are possible. Examination of the results by multiple regression analysis showed that it was possible to explain a significant proportion of the bacterial variation (with the notable exception of the Planctomyces counts) in spite of considerable intercorrelation of the regressor variables.     

Rapid Brain Uptake of Manganese(II) Across the Blood-Brain Barrier

Abstract: ⁵⁴Mn²⁺ uptake into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique. Initial uptake from blood was linear with time (30 s to 6 min) and extrapolated to zero with an average transfer coefficient of ∼6 × 10^-5 ml/s/g for brain and ∼7 × 10^-3 ml/s/g for choroid plexus. Influx from physiologic saline was three- to fourfold more rapid and exceeded that predicted for passive diffusion by more than one order of magnitude. The lower uptake rate from blood could be explained by plasma protein binding as the free fraction of ⁵⁴Mn²⁺ in rat plasma was ≤30%. Purified albumin, transferrin, and α₂-macroglobulin were each found to bind ⁵⁴Mn²⁺ significantly and to restrict brain ⁵⁴Mn²⁺ influx. The results demonstrate that ⁵⁴Mn²⁺ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. The results support the hypothesis that Mn²⁺ transport across the blood-brain barrier is facilitated by either an active or a passive mechanism.     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

A CHEMILUMINESCENCE BIOSENSOR COUPLED WITH IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM

A chemiluminescence biosensor, using a fiber-optic-linked photometer and a data acquisition unit connected to a PC, was developed in conjunction with immunomagnetic separation for rapid detection of Salmonella Typhimurium. Magnetic microbeads coated with Anti-Salmonella antibodies and anti-Salmonella antibodies conjugated with horseradish peroxidase (HRP) were added to artificially-inoculated samples, and the immuno-reaction was completed in 60 min resulting in a sandwich complex. A magnetic field was applied to collect magnetic beads and the addition of luminol to HRP-conjugated antibodies resulted in a chemiluminescence reaction. The signal was collected through a fiber optic light guide, measured with a photometer, and recorded in the data acquisition unit. The minimum detection limit of the chemiluminescence biosensor for S. Typhimurium was 1.97 × 10³ CFU/mL and the range of the detectable signal was from 8.6 to 350 mV for cell numbers from 1.97 × 10³ to 1.97 × 10⁶ CFU/mL. Signal values for 10⁶ CFU/mL of S. Typhimurium were at least 97 and 394% higher than the corresponding values for S. enteritidis and four times the signal values for others including S. montevideo, S. california, S. heidlberg, and S. seftenberg, respectively. The biosensor response showed a significant difference (P < 0.05) between 10³ CFU/mL S. Typhimurium and 10⁶ CFU/mL of commonly-occurring bacteria in foods including Listeria monocytogenes, Pseudomonas aeruginosa, Citrobacter freundii, Campylobacter jejuni, Escherichia coli O157, and generic Escherichia coli. A regression equation, V = 0.0262 N ^5.7713, with R²= 0.9713 was obtained for the calibration curve over the detection range for S. Typhimurium. The whole procedure could be completed within 90 min.     

Mechanisms of cobalt uptake in plants: 60CO uptake and distribution in Chara

The mechanism of cobalt uptake was investigated using cells of the giant alga Chara corallina in which it is possible to resolve separately uptake by the cell wall and actual influx across the cell membrane. The absorption of ⁶⁰Co by Chara cells appeared to saturate within 2 h, but this was mainly due to rapid uptake into the cell wall which accounted for 87–92% of the total activity. Even after prolonged desorption most of the cell‐associated ⁶⁰Co was found on the cell wall. The intracellular distribution of absorbed ⁶⁰Co was investigated by fractionating the cell into cytoplasm and vacuole. It was shown that ⁶⁰Co influx to the vacuole occurs simultaneously with influx to the cytoplasm. The transported species appears to be Co²⁺ rather than the less charged Co(OH)⁺ or Co(OH)₂. ⁶⁰Co influx is pH dependent (optimum pH 7–9), and is sensitive to some other divalent metals. Influx from solutions containing 1 µ M ⁶⁰Co was inhibited by 5 µ M Cd²⁺, Cu²⁺, and Zn²⁺, but Mn²⁺ and Ni²⁺ had no significant effect. The sensitivity of Co uptake to N ‐ethyl maleimide (NEM) and cysteine suggests that the transport system involves direct binding of CO²⁺ to ‐SH groups.     

Post-processing microflora and the shelf stability of gari marketed in Port Harcourt

Gari was examined for its post-processing microbial content. Aerobic mesophilic bacteria and fungi were isolated from all samples. The total viable bacterial counts ranged from 2.0 × 10² to 8.0 × 10⁴ cfu/g. Fungal counts ranged from 1.0 × 10² to 1.5 × 10⁴ cfu/g. The total viable counts of fresh samples were much lower than those of market and packaged samples. Bacillus, Micrococcus and Proteus spp. were the bacteria isolated, Aspergillus niger, Aspergillus flavus and Penicillium spp. the fungi. Food borne parasites and pathogens such as Staph. aureus and Clostridium perfringens were not found. The gari samples were quite stable, having a shelf life of 3–6 months. The water activities of the samples ranged from 0.52 to 0.68. Based on the microbial counts of the samples, the critical upper limit for the safety of gari was set at 10⁴ cfu/g dry sample.     

Rapid and Sensitive Single-Step Radiochemical Assay for Catechol-O-Methyltransferase

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol- O -methyltransferase activity is described. The method is based on the conversion of catechol to [³H]guaiacol by catechol- O -methyltransferase in the presence of Mg²⁺, adenosine deaminase and S -adenosyl l -[methyl-³H]methionine. Incubation and direct extraction of [³H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol- O -methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.     

COMPARISON OF LUMINOL CHEMILUMINESCENCE WITH ATP BIOLUMINESCENCE FOR THE ESTIMATION OF TOTAL BACTERIAL LOAD IN PURE CULTURES

A rapid chemiluminescent assay of total bacterial load that is based on the oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as catalyzed by bacterial iron protoporphyrins is described and compared to the ATP bioluminescent assay of microbial biomass. An assay format that elicits linear light output response to a range of analyte concentrations of model compounds such as hematin and various heme-containing enzymes within the dynamic range of a BioOrbit 1251 luminometer is presented. When the assay was applied to eight pure bacterial cultures, the sensitivity was typically in the range of 10⁴-10⁵ cfu/ml, and was comparable to that obtained by the ATP assay. Similar levels of sensitivity can be derived from estimates of average values of 2.8 × 10^-18 mole of heme/cfu and 1 × 10^-19 mole of ATP/cfu. The potential of the luminal assay as an alternative rapid test for the estimation of total bacterial count in food and environmental samples is discussed.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

Size-dependent mortality of red sea bream, Pagrus major, juveniles released with fluorescent otolith-tags in News Bay, Japan

Totals of 2^.67 x 10⁵ and 7^.56 x 10⁵ juvenile red sea bream of three size groups (10, 20 and 40 mm t.l.) marked with a fluorescent substance in the otolith were released in News Bay, Oita Prefecture, Japan, in July 1987 and June 1988, respectively; the aim was to estimate growth and mortality of different developmental stages.
Of fish released in 1987 and 1988, 10 618 and 4413, respectively were recaptured during those two years. Released fish remained in the bay until the end of summer, and afterwards migrated out towards open waters. Fish of the 40-mm group released in 1987 grew to over 200 mm t.l. in one year. Mean growth rate for 19 days after release was higher in the 40-mm group (0^.87 mm day⁻¹) than in the 20-mm group (0^.74mm day⁻¹). Survival rates over 19 days were 59^.0 and 10^.1 % for 40-mm and 20-mm fish, respectively, in 1987, and those over 30 days were 69^.2, 3^.3 and 0^.0% for 40-mm, 20-mm and 10-mm fish, respectively, in 1988.
Cannibalism was indicated by the presence of marked otoliths for 20-mm fish in the stomachs of a few 40-mm individuals recaptured 2 days after release. Size-dependent growth and size-selective mortality were both noted in juvenile red sea bream, i.e. the relative size differential between larger and smaller individuals was maintained in the period between marking and recapture, and mortality was inversely proportional to size.