The rapid estimation of microbial contamination of raw meat by measurement of adenosine triphosphate (ATP)

Bacteria were separated from raw meat homogenate by a simple three-stage process. Centrifugation (10 s at 2000 g) removed coarse particles; stirring with the cation exchange resin Bio-Rex 70 removed smaller particles and filtration through 0.22 micron membranes removed soluble materials. By this process 70-80% of the microbial populations of meat homogenates were consistently isolated on the filters. A linear relationship was found between log10 microbial ATP and log10 colony count of meat over the range 10(5)-10(9) cfu/g. The value of ATP/cfu for meat samples was within the range previously reported for pure cultures. These data indicated that ATP extracted from the filters originated from bacteria in the meat samples. Several samples can be analysed simultaneously in an elapsed time of 20-25 min. The variability associated with estimates of both colony counts and ATP levels has been determined.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Effectiveness of a steam-vacuum sanitizer for reducing Escherichia coli O157:H7 inoculated to beef carcass surface tissue

A steam-vacuum sanitizer reduced aerobic plate counts associated with bovine faecal contamination from 5.5 log₁₀ cfu cm⁻² to 3.0 ± 0.21 log₁₀ cfu cm⁻² on beef carcass short plates. The same beef carcass short plates inoculated wiht 7.6 ± 0.09 log₁₀ cfu cm⁻² Escherichia coli O157: H7 in faeces, yielded an average residual level of E. coli O157: H7 of 2.1 ± 0.21 log₁₀ cfu cm⁻² after steam-vacuum treatments. This study demonstrates the effectiveness of a steam-vacuum sanitizer for removing E. coli O157: H7 from beef carcasses.     

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.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

Microbial load in poultry feed and detection of aflatoxin B1 using monoclonal antibody-based enzyme linked immunosorbent assay

Feed samples collected from different poultry farms and feed mills situated in Andaman and Nicobar islands in India were assessed for microflora and aflatoxin B₁ contamination. The bacterial counts ranged from 1.0 times 10⁷ to 8.8 times 10⁷ cfu/g of the feeds, while counts of fungi ranged from 1.0 times 10³ to 8.7 times 10³ cfu/g. The mycoflora comprised mainly of Aspergillus spp., A. flavus being most dominant. Aflatoxin B₁ was detected by monoclonal antibody-based enzyme linked immunosorbent assay technique and the content in different feed samples ranged from 5.5 to 90 ng/g.     

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.     

The effect of variation of thermal processing on the microbial spoilage of chub-packed luncheon meat

Process pasteurization values for reference temperature 70°C (P₇₀) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P₇₀-value and the time and temperature of cooking was derived. With minimal cooking (P₇₀= 40) the surviving microflora (10³/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P₇₀= 105), leaving a flora (10²/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P₇₀ between 40 and 90, a Streptococcus spoilage sequence occurred; with P₇₀ between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P₇₀ of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P₇₀= 75 was adequate to reduce the surviving microflora to the 10²/g level associated with current good manufacturing practice.     

A desk study of the relationship between temperature and hatching time for the eggs of five species of salmonid fishes

SUMMARY. Information on temperature (T°C) and time from fertilization to 50% hatch ( D days) for five species of salmonid fishes has been used to assess several mathematical models relating D and T . No single equation gave the best fit to all five data sets. The power law with temperature correction (α), log₁₀₁ D = log₁₀ a + b log₁₀ ( T - α) and the quadratic, log₁₀ D = log₁₀ a + bT + b ₁ T ² (where a, b, b ₁, and α are constants), each accounted for over 97 % of the variance of D and were good fits to the observed data points for all five species. There was little difference between the predictions obtained from these two equations within the range of observed temperatures. Therefore, the simpler power-law model is preferred. However, there were substantial within-species differences between values of D predicted from extrapolations of the two models from 2 or 3°C down to 0°C. When more data for low temperatures become available it will be possible to make a more objective choice of model.     

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.     

The populations, characterization and activity of suspended bacteria in the Welsh River Dee

In a 29 month study of bacterial populations at three sites on the Welsh River Dee, the aerobic heterotrophic bacteria increased from an average basal level of about 1.2 times 10⁴ colony-forming units (cfu)/ml near the source of the river, to 2 times 10⁵ cfu/ml in the lower reaches. The ratio, total bacterial cell count: viable count, decreased from 70 in the upland reaches to 10 in the lower parts of the river. There was no apparent seasonal variation in bacterial numbers but on occasions the bacterial populations in both upland and lowland reaches of the river increased simultaneously. Fluctuations in bacterial numbers over a 50-fold range were observed in this study. Bacterial isolates from both upland and lowland sites were predominated by two groupings of bacteria, the Pseudomonas—Agrobacterium—Alcaligenes group and the Flavobacterium—Cytophaga—Flexibacter group. Results suggested that the latter group may have been part of the autochthonous population. Seasonal variation in heterotrophic potential ( V _max) for acetate uptake was shown to occur over a 30-fold range in the lowland reaches of the River Dee. Peaks in activity at the lowland site occurred during the summer months, the range of V _max values for acetate ranged from 0.2 to 30 μg/1/h. Fluctuations in V _max values from the upland site were not seasonal but were instead linked to faecal pollution, V _max values from this site range from 0.04 to 3 μg/1/h.     

Note: Thermophilic campylobacters in dairy slurries on Lancashire farms: seasonal effects of storage and land application

Slurry storage tanks on Lancashire farms were sampled in May, June, October and November. There was a seasonal pattern in the number of thermophilic campylobacters. The average log₁₀ MPN per gram fresh weight (log₁₀ MPN gfw⁻¹) in stored samples in May and June was 0·78 ( S.D. 0·71) compared to 2·07 ( S.D. 0·70) in November and December. Campylobacters were readily detected in samples of mature slurry and composted bedding that farmers were about to put to land. The survival of campylobacters in slurry sprayed on land was studied in two seasons. In June, on farm A, stored slurry was mechanically aerated for 48 h and very low numbers of campylobacters (0·9 log₁₀ MPN gfw⁻¹) remained in the slurry before it was sprayed onto land. They became undetectable within 24 h once sprayed on land. In contrast, campylobacters in matured but unaerated slurry that was sprayed onto land on farm B in February were still detectable in samples taken 5 d after application to land, dropping from 2·11 log₁₀ MPN gfw⁻¹–1·37, a decline of only 0·74 log₁₀ MPN gfw⁻¹ in the first 5 d after application.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Comparison of conductimetric and traditional plating techniques for detecting yeasts in fruit juices

Frozen fruit juice concentrates containing an average microbial population of log₁₀ 1.54 cfu ml^-1 were examined by traditional plating techniques and direct and indirect conductimetry. The initial populations in diluted (1:4) concentrates increased to an average of log₁₀ 3.82 cfu ml^-1 during incubation at 25°C for 24 h. Incubation before plating and subjecting to conductimetric tests also facilitated the resuscitation of cells that may have been freeze-injured. Yeasts were recovered in equal numbers on acidified (pH 3.5) potato dextrose agar and dichloran rose bengal chloramphenicol agar (pH 5.6). Yeasts and bacteria were recovered on orange serum agar. Detection times determined by indirect conductimetry correlated fairly well ( r = -0.73) with populations (cfu ml^-1) detected on traditional plating media. Populations in diluted concentrates which were not incubated before examination were detected conductimetrically in an average of 48.9 h, whereas detection times for diluted concentrates incubated for 24 h at 25°C before testing were reduced to an average of 14.1 h. Examination by conventional (direct) conductimetry required an additional 10–20 h to reach changes in conductance of 5 μS h^-1.     

Routine metabolism of juvenile spot, Leiostomus xanthums (Lacépède), as a function of temperature, salinity and weight

Routine oxygen consumption rates of juvenile spot, Leiostomus xanthums , were measured over a range of temperatures, salinities and fish weights. As predicted, Q O₂ increased with temperature and decreased with body weight. However, Q O₂ decreased with decreasing salinity and did not show the expected minimum at isosmotic concentrations. The data are best described by the relationship: log₁₀ Q O₂ (mg O₂ g⁻¹ h⁻¹) = 0.129 log_lo salinity (%0) + 1.604 log₁₀ temperature (°C)-0.1401og₁₀(g)-2.767.     

A bacteriological profile of salad vegetables in Bangladesh with special reference to coliforms

Salad vegetables are often consumed fresh and thus can act as effective media for the transmission of associated pathogens. Fresh samples of cucumber, carrot and lettuce were collected from different markets in Dhaka metropolitan city, Bangladesh. Bacterial loads were found to be 7·1 times 10⁴ to 6·34 times 10⁸ colony forming unit (cfu)/100 g. Escherichia coli, Klebsiella, Enterobacter , and Serratia were amongst the coliforms (lactose fermenters) while Pseudomonas, Shigella and Acinetobacter were non-lactose fermenters associated with the samples.     

A note on estimation of food spoilage yeasts by measurement of adenosine triphosphate (ATP) after growth at various temperatures

The growth at 4, 10 and 15C of six psychrotrophic yeasts isolated from foods was compared using total viable counts and ATP measurement. A linear relationship ( r > 0.97) was obtained between log₁₀ (number of viable yeasts) and log₁₀ (ATP content) of cultures grown at these temperatures. This relationship was not temperature-dependent. The results are discussed and their significance for the rapid estimation of yeasts in foods is considered.     

Production and storage of Rhizobium leguminosarum cell concentrates for use as inoculants

Recovery of Rhizobium leguminosarum cells by centrifugation after growth in an industrial fermenter was 100-fold higher when cells were grown on yeast extract (5 g/1) as sole source of carbon and nitrogen when compared with the yields recovered when cells were grown in standard mannitol-yeast extract medium. Methods of storing concentrated suspensions of R. leguminosarum were investigated. Freeze-drying caused a marked decrease in viable cell numbers. Viable cell numbers of bacterial concentrates stored in peat decreased steadily from 10₁₁-10₁₂ cfu/g to 10₉ cfu/g or less during 26 weeks storage at room temperature or at 4°C. Cell concentrates stored in 40% glycerol at — 20°C maintained viable numbers higher than 10₁₁ cfu/ml during a 76 week storage period.     

The Effect of Temperature, Pressure and Chlorine Concentration of Spray Washing Water on Numbers of Bacteria on Lamb Carcases

Combined analysis of three experiments showed that when lamb carcases with initial bacterial numbers of between logi₁₀3.29 and 4.22/cm² were spray washed, statistically significant reductions in bacterial numbers of log₁₀O.5 were obtained when the spray wash water temperature was > 57°C, and reductions of log₁₀1.0 were obtained when the temperature was ≥ 80°C. Reductions at all temperatures were enhanced by log₁₀0.66 when the water contained 30 µg/ml chlorine, but increasing the concentration to 450 µg/ml reduced bacterial numbers only by a further log₁₀0–29. At highly contaminated sites increasing the duration of spraying from 30 to 120 s significantly increased the reductions obtained when water containing added chlorine was used. Reductions in bacterial numbers after spray washing with pressures of 3.5, 5.6. 7.7 kg/cm² were not significantly different.