Separation of micro-organisms from meat and their rapid enumeration using a membrane filtration-epifluorescent microscopy technique

A new method of separating bacteria from beef mince has been developed, in which an alkaline protease, Alcalase 0.6 L, was used to degrade the meat proteins, leaving micro-organisms in suspension. The organisms were then counted, using a membrane filtration-epifluorescent microscopy technique. A correlation coefficient of 0.97 was obtained between this method and the standard plate count, indicating its suitability for use in quality control.     

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.     

Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat

The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed. Minced beef samples inoculated with L. monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth. Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide. The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure. The antibody used in this technique reacts with L. monocytogenes and L. innocua. The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1). There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e. plate counts on PALCAM. When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained. No false-negative or false-positive results were recorded for L. monocytogenes or L. innocua species using the SAIF technique.     

微生物在水环境中的存活和生长

在灭菌自来水模拟水体中,研究了7种细菌的存活和生长规律。Klebsiella pneumo-niae,Enterobacter aerogenes,Agrobacterium tumefatciens,在7天内平板计数降至0,而水体中镜检细菌总数(AODC)和活菌直接计数(DVC)结果无大变化,说明细菌已变成活的非可培养状态。Micrococcus,flavus 和 Streptococcus faecalis 的可培养菌数也可降至0。Pseudomonas sp.在48小时内由10~5降至10~2cfu/ml,随即升至10~6 cfu/ml 并持续到实验终了(41天)。Bacillus subtilis 在48小时平板计数降至10~2cfu/ml 并维持在该水平至实验结束(38天)。研究结果表明仅用涂布平板法检测多种细菌在水环境中的生存和分布是不合适的。     

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat

an immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca . This technique involved enrichment of the suspect sample at 30°C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1×10³ cfu ml⁻¹ was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua . The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM

Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat. Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC). However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively. The total bacterial abundance ranged from 1.1 to 3.2 x 10(6) ml(-1). As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria. Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria. Counts of filamentous bacteria made by AODC were only about half of those detected by TEM. In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates. Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4 x 10(5) ml(-1), respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids. Besides, cocci might easily be mistaken for large viral particles when counted by AODC. Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters.     

Use of the direct epifluorescent filter technique for the enumeration of bacterial spores

Heat treatment at 80°C for 10 min effectively destroyed all vegetative cells (except for Gram-positive cocci) and made easier the counting of bacterial spores, which stained orange, green or rarely transparent/black with a dull green halo, in the direct epifluorescent filter technique. The numbers of both orange- or green-staining spores were lower than the plate count. A variety of physiological conditions were used to investigate the relationship of the different staining patterns with germination status. It was concluded that orange-staining spores had germinated and their number agreed with the plate count after incubation in yeast glucose broth at 30°C for 4 h. This observation was unreliable, however, but it was found that a total spore count in the DEFT gave a good agreement with the plate count.     

Automated measurements of antilisterial activities of lactate and diacetate in ready-to-eat meat

Standard procedures to enumerate Listeria organisms rely on plating food samples on selective agar media. The procedures are labor-intensive, and because of the limited sensitivity, pre-enrichment step is required for the detection of low numbers of the pathogen. In the present study, an automated rapid optic procedure and the standard procedure were used to determine the behavior of the pathogen in ready-to-eat (RTE) meat and to test the effect of antilisterial agents. Listeria monocytogenes strain Scott A or a six-strain mixture of Listeria was studied using lactate (2.5%), diacetate (0.2%) and their combination in beef bologna and in sterile beef emulsion. Samples stored for up to 60 days at 5 and 10 degrees C were tested at time intervals during storage. Using the plate count method, each of the salts caused a delay in growth of the pathogen, and the salt combination was most effective causing listeriostatic effects and decline in growth of the pathogen at 5 degrees C. High negative correlation (r), ranging from 0.92 to 0.99, was obtained between the detection time (DT) recorded by the optic procedure (BioSys instrument) and cell numbers determined by the plate count procedure. The rapid (< 24 h) optic procedure was reliable in assessing the efficacy of antimicrobials and in rapid detection of low levels of listeriae that are undetectable by direct plating procedure.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

Collaborative trial of the direct epifluorescent filter technique (DEFT), a rapid method for counting bacteria in milk

The direct epifluorescent filter technique (DEFT) is a new rapid method which uses membrane filtration and epifluorescent microscopy for counting bacteria in milk. A collaborative trial of the DEFT was conducted between six laboratories. Each laboratory obtained a highly significant relationship between the DEFT count and plate count with a correlation coefficient generally > 0.9 but there were significant differences between these relationships. The repeatability of the DEFT, although ca 1·5 times worse than that of the plate count, was of a level acceptable in practice. Reproducibility of the DEFT was ca 3 times that of the plate count. This poor reproducibility was probably mainly due to counting errors. Possible reasons for this and ways of reducing counting errors are discussed.     

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.     

The influence of storing beef aerobically or in vacuum packs on the shelf life of mince

Aims: To investigate the influence of aerobic or vacuum pack storage of beef trimmings on the microbiology, colour and odour of subsequently produced mince. Methods and Results: Trimmings stored aerobically for 7 or 10 days and in vacuum packs for 7, 10, 14 or 22 days at 0 or 5°C were minced, stored aerobically at 0 or 5°C for up to 7 days and examined daily to determine Total viable, Pseudomonas, Lactic acid bacteria, Brochothrix thermosphacta, and Enterobacteriaceae counts, colour and odour. Mincing reduced counts, particularly of Pseudomonas, B. thermosphacta and Enterobacteriaceae, probably because of the action free radicals released from muscle and bacterial cells. Storage of vacuum‐packed trimmings for 22 days resulted in improved mince colour and inhibition of the growth of Pseudomonas. Conclusions: The shelf life of mince from trimmings is directly influenced by the trimmings storage conditions, and longer‐term vacuum storage of trimmings produced improvements in mince quality. Significance and Impact of the Study: There appears to be no scientific rationale for limiting the storage of vacuum packaging beef trimmings to 15 days, prior to mince production, as stated in EU 835/2004. This study identifies advantages in storing trimmings in vacuum packs for at least 21 days prior to mincing, in terms of improved mince quality.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

Quantification of Staphylococcus aureus and Escherichia coli in the liquid medium by fluorimetry and its use in phagocytosis assay

A fluorimetric technique was compared with the plate counting method for quantification of viable cells of Staphylococcus aureus and Escherichia coli in the liquid medium. The fluorimetric assay measures the release of fluorogenic 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl phosphate by the bacterial phosphatases. The increase in fluorescence was dependent on the size of bacterial inocula. Setting the fluorescence threshold at the middle of the logarithmic growth phase resulted in good linear relationship between bacterial counts and fluorescence ( r = 0·99 for both Staph. aureus and E. coli ). There was also an excellent correlation between the fluorimetric assay and the plate counting method in quantifying viable bacteria in saline ( r = 0·99). Both methods were further compared for evaluation of extracellular bacteria following phagocytosis. The fluorimetric technique, in general, gave a higher percentage of phagocytosis than the plate counting method with statistical significance for E. coli.     

Osteological development of the vertebral column and of the caudal complex in Dentex dentex

Osteological development of the vertebral column and caudal complex in common dentex was described under extensive larval rearing conditions. Generally, the cartilaginous bones developed prior to the membranous bones. The development of the axial skeleton began with the formation of the hypural 1, the neural arches 2 and 3, as well as the haemal arches 1–8 at 4·8, 4·9 and 5·0 mm total length ( L _T, measured in vivo ), respectively. By 7·5 mm L _T, all the cartilaginous elements were formed, except for the ventral ribs, which formed between the range of 8·4–18·0 mm L _T. The caudal lepidotrichia were the first membranous bones to appear (5·3 mm L _T) and attain their full meristic count (7·4 mm L _T), followed by the vertebral centra, which formed between 6·6 and 9·7 mm L _T. By 25·0 mm L _T, all the elements were fully ossified except for the ventral ribs. The developmental direction and order of all the elements were studied with respect to their formation and ossification. The results were discussed in the contexts of ichthyoplankton, ecology and aquaculture. Compared with other Sparidae species, common dentex followed a pattern of relatively rapid rate of osteological development.     

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.     

Survival ofLegionella pneumophila in the aquatic environment

Survival ofLegionella pneumophila SG 1 in seawater and river water was assessed using plate counts on buffered charcoal yeast extract agar amended with α-ketoglutarate (BCYEα) and [³H]thymidine-labeling. The [³H]thymidine-labeling method for assessing survival ofL. pneumophila in aquatic environments was compared with viable counts, direct fluorescent microscopy (DFA), and acridine orange direct counts (AODC). Protozoa were isolated from the samples employed in the study and identified by characteristic trophozite and cyst morphology. Selective filtration employing 2.0 μm Nucleopore filters was used to determine the effect of grazing on survival ofL. pneumophila in seawater and river water.Legionella viability as measured by plate counts (CFU/ml), declined to a greater extent than cell lysis, assessed by thymidine, DFA, and AODC counts, suggesting thatL. pneumophila survives in aquatic habitats to a greater extent than revealed through culturable counts.     

EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm²) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 10⁴, 1 × 10⁵ and 1 × 10⁶ CFU/cm², there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k-statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.