The certification of a reference material for the evaluation of methods for the enumeration of Bacillus cereus

A reference material containing Bacillus cereus was certified by the Community Bureau of Reference (BCR) for its number of colony-forming particles (cfp) in 0.1 ml reconstituted capsule solution. To this end, a batch of approximately 15,000 capsules was produced and tested for its homogeneity and stability. The variation in the number of cfp between capsules (homogeneity) was not found to be significantly different from a Poisson distribution. Stability was tested for extended periods at storage temperature (-20 degrees C) and at various higher temperatures up to 37 degrees C for 4 weeks to simulate transport conditions. Only at 37 degrees C did a small but significant decrease in the number of cfp occur. At -20 degrees C, no decrease in the number of cfp was observed over a period of about 4 years. For certification, 12 laboratories determined the number of cfp on two agars: Mannitol Egg-Yolk Polymyxin agar (MEYP, incubated at 30 degrees C) and Polymyxin pyruvate Egg-yolk Bromothymol blue Agar (PEMBA, incubated at 37 degrees C). The certified geometric mean value on MEYP after 24 h of incubation was 53.4 cfp 0.1 ml-1 of the reconstituted capsule solution (95% confidence interval 51.7-55.2) and on PEMBA, 55.0 (95% confidence interval 52.8-57.4). Based on these certified values, user tables were constructed specifying the 95% confidence limits when testing a smaller number of capsules, as would be done in individual laboratories. Based on the information on homogeneity, stability and the certification study, the BCR decided to certify the material as CRM 528.     

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC-MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.     

COMPARISON of ANALYTICAL SENSITIVITIES of THREE ENZYME IMMUNOASSAY PROCEDURES FOR the DETECTION of SALMONELLA LIPOPOLYSACCHARIDE

The analytical sensitivities of three different enzyme linked immunoassays (ELISA), two competitive and a capture format were assessed. the assay systems employed monoclonal antibodies to Salmonella lipopolysaccharide (LPS) outer core epitopes to detect crude LPS antigens from Salmonella typhimurium. the most sensitive ELISA was the capture procedure, being capable of detection 1.3 ng/ml of LPS. This technique, however also gave the greatest between-test variation and as a result, the lowest amount that could be detected with a 95% confidence limit was actually 12.8 ng/ml and it took the longest time to perform (3 h, 30 min). A competitive ELISA using limiting monoclonal antibody to compete between solid phase antigen and soluble antigen in the sample, ranked second in sensitivity, and can detect 2.8 and 3.8 ng/ml of LPS when tested with two different monoclonal antibodies. However, because of the slight between test variation, the actual sensitivities that could be detected with a 95% confidence limit were 3.1 and 4.6 ng/ml, respectively. This test takes approximately 1 h and 30 min to perform.
The classical type of competitive assay, employing a labelled antigen, was the least sensitive being capable of detecting 5.8 ng/ml if the LPS was conjugated with horseradish peroxidase and 16.0 ng/ml if alkaline phosphatase was used as a label. to account for the between-test variation, the sensitivities with a 95% confidence limit were 8.6 and 18.7 ng/ml for the respective assays, which take 2 h and 15 min to perform.
These sensitivities compare favorably with those published for similar assays, but all of the procedures were judged insufficiently sensitive for direct use on food samples to be tested for the presence of Salmonella species. However, the assays would be quite suitable for demonstration of Salmonella sp. after an enrichment procedure.     

The utilization of interphase cytogenetic analysis for the detection of mosaicism

This study describes a method for defining mosaic aneuploidy by interphase cytogenetics based on statistical limits established from control specimens. Fluorescence in situ hybridization (FISH) has been used to detect the number of copies of specific chromosomes in interphase nuclei from placental tissues of diploid controls and mosaic placentas. FISH was performed using probes D7Z1/D7Z2, D9Z1, D10Z1, and D18Z1, all purchased from Oncor, Inc. Statistical analysis of data obtained from diploid controls was used to determine the one-sided upper reference limit and corresponding 95% confidence interval for the proportion of cells with one and three signals for each of the probes used. The one-sided upper reference limits established the lower levels of monosomy and trisomy detectable using each of the four probes. These statistical parameters were then used to interpret the results obtained by FISH applied to the study of term placentas for the confirmation of prenatally diagnosed chromosomal mosaicism.     

Immunomagnetic separation and conventional culture procedure for detection of naturally occurring Salmonella in raw pork sausages and chicken meat

The aim of the study was to compare immunomagnetic separation (IMS) and conventional selective enrichment procedures using selenite cystine broth (SC) and Rappaport–Vassiliadis broth (RV) in 137 naturally contaminated food samples (69 raw pork sausages and 68 chicken meat). The utilization of SC or IMS appeared to be the most appropriate enrichment procedure: 15 out of 18 Salmonella -positive samples (83·3%) were detected by SC and 12 (66·7%) by IMS; RV yielded only seven positive isolations (38·9%). However, RV yielded the highest count of Salmonella colonies per plate and the lowest interference by competing organisms. IMS could become a reliable alternative to standard enrichment procedures and a combined IMS and selective enrichment broth could increase the chance of Salmonella recovery.     

Sickness absence as a global measure of health: evidence from mortality in the Whitehall II prospective cohort study

Objective To examine the association between sickness absence and mortality compared with associations between established health indicators and mortality.Design Prospective cohort study. Medical examination and questionnaire survey conducted in 1985-8; sickness absence records covered the period 1985-98.Setting 20 civil service departments in London.Participants 6895 male and 3413 female civil servants aged 35-55 years.Main outcome measure All cause mortality until the end of 1999.Results After adjustment for age and grade, men and women who had more than five medically certified absences (spells > 7 days) per 10 years had a mortality 4.8 (95% confidence interval 3.3 to 6.9) and 2.7 (1.5 to 4.9) times greater than those with no such absence. Poor self rated health, presence of longstanding illness, and a measure of common clinical conditions comprising diabetes, diagnosed heart disease, abnormalities on electrocardiogram, hypertension, and respiratory illness were all associated with mortality—relative rates between 1.3 and 1.9. In a multivariate model including all the above health indicators and additional health risk factors, medically certified sickness absence remained a significant predictor of mortality. No linear association existed between self certified absence (spells 1-7 days) and mortality, but the findings suggest that a small amount of self certified absence is protective.Conclusion Evidence linking sickness absence to mortality indicates that routinely collected sickness absence data could be used as a global measure of health differentials between employees. However, such approaches should focus on medically certified (or long term) absences rather than self certified absences.     

Prevalence and serotypes of Salmonella associated with goats at two Australian abattoirs

Aims:  This study was carried out to determine the prevalence and serotype of Salmonella in goats presented for slaughter.
Methods and Results:  A total of 121 goats were examined for the presence of Salmonella in matching rumen, faecal and carcass samples. Samples were analysed for the presence of Salmonella following the Australian Standard AS 1766.2.5-1991. Salmonella was isolated from 56 (46·3%) faecal samples, 55 (45·5%) rumen samples and 35 (28·9%) carcass samples. The dominant serotypes isolated were Salmonella serotype Saintpaul (31%), Salmonella serotype Typhimurium (13%) and Salmonella serotype Chester (11%).
Conclusions:  Salmonella was isolated from at least one of the three sample sites in 68% of animals. Carcase contamination with faeces, compared with rumen liquor, is a greater hazard for Salmonella contamination of goat carcases. Goat meat is a potential source of Salmonella serovars associated with human disease.
Significance and Impact of the Study:  Goat carcases contaminated with Salmonella during slaughter could be a source of food-borne disease if consumed raw or inadequately cooked, or may be a source of cross-contamination to other foods.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

Influence of the rumen anaerobic fungus Neocallimastix frontalis on the proteolytic activity of a defined mixture of rumen bacteria growing on a solid substrate

A grass + fishmeal ruminant feed was incubated for 7 d in a mineral salts medium with the non-proteolytic rumen bacteria Bacteroides succinogenes, Ruminococcus flavefaciens, Megasphaera elsdenii and proteolytic strains of Bacteroides ruminicola, Selenomonas ruminantium and Streptococcus bovis in the presence and absence of the anaerobic fungus Neocallitnastix frontalis . The fungus increased the dry matter digestion from 65·0 to 69·4%, and more than doubled the proteolytic activity of the culture filtrate. However, a greater difference was observed with the solid material, where the proteolytic activity increased from 0·71 to 6·89 mg ¹⁴C-casein hydrolysed/g/h, due mainly to EDTA-sensitive fungal protease.     

Elevated red myotomal muscle temperatures in the most basal tuna species, Allothunnus fallai

The present study tested the hypothesis that Allothunnus fallai can elevate its slow-oxidative red myotomal muscle (RM) temperature. Measurements on 30 A. fallai (750–850 mm fork length) captured by hook and line off the coast of southern New Zealand revealed that RM temperatures are elevated by mean ± s . d . 8·1 ± 1·3° C (range 6·7–10·0° C) above the mean ± s . d . ambient sea surface temperature 15·3 ± 0·8° C (range 14·3 to 16·4° C). These data provide evidence that the vascular modifications to the central circulation of A. fallai act as a counter-current heat exchanger and that RM heat conservation is a character state present in all extant tuna species.     

Comparison of four membrane filter methods for fecal coliform enumeration.

Four membrane filter methods fecal coliform enumeration were evaluated and compared in six laboratories based on determination of accuracy, specificity, upper counting limit, and recovery comparability. Recovery accuracy with pure cultures ranged from 89 to 100% for m-FC, mTEC (a procedure developed for thermotolerant Escherichia coli), and m-FC2 methods (the latter incorporating a 2-h, 35 degrees C resuscitation period), but was less than 60% for the MacConkey membrane broth method. These figures dropped by approximately 40 to 55% when the cultures were subjected to temperature (10 degrees C) stress. Close to 800 colonies were verified to determine specificity. False-positive colonies occurred most frequently with the m-FC2 method (18%), whereas false-negative colonies were most common on MacConkey membrane broth (26%). In counting range experiments using a variety of samples, the highest upper counting limit was 130 colonies per filter with the mTEC procedure. Recovery comparisons were based on over 130 samples including raw surface waters, raw sewage, and chlorinated and unchlorinated sewage effluents. In general, recoveries were significantly higher with the m-FC2 and mTEC methods; however, on m-FC2, growth of nontarget background organisms was also higher in most cases. Highest recoveries from chlorinated sewage effluents were obtained by the mTEC method, and the addition of a similar resuscitation period to the m-FC procedure (m-FC2) improved fecal coliform recovery from such samples. The best overall performance for fecal coliform enumeration was obtained with the mTEC method with high recovery and low levels of background colonies, good specificity and accuracy, and a high upper counting limit. This procedure also offers the advantage of enumerating E. coli within 24 h.     

Effect of selected antimicrobial compounds on the radiosensitization of Salmonella Typhi in ground beef

Aims:  In this study, we extended our previous work to determine the efficiency of antimicrobial compounds in increase of relative radiosensitivity of Salmonella Typhi in medium fat ground beef (23% fat) by testing 41 different essential oils (EOs), oleoresins and food sauces.
Methods and Results:  Ground beef samples inoculated with Salmonella Typhi (10⁶ CFU g⁻¹ ) were treated with each antimicrobial compound at a concentration of 0·5% (w/w). Then, the samples (25 g each) were packaged under air and irradiated in a ⁶⁰Co irradiator at doses from 0 to 1·75 kGy. Radiosensitivity was evaluated by calculating relative radiation sensitivity, defined as the ratio of radiation D ₁₀ value in the absence/presence of antimicrobial compound.
Conclusions:  Depending on the compound tested, the addition of antimicrobial compound decreased the D ₁₀ value of Salmonella Typhi, resulting in an increase of the radiation sensitivity up to more than four times. Among these antimicrobial compounds, Chinese cinnamon EO, clove EO and trans -cinnamaldehyde were most effective to increase the radiosensitivity of Salmonella Typhi in ground beef.
Significance and Impact of the Study:  These observations demonstrate that some active compounds can function as radiosensitizers of Salmonella Typhi.     

On the Use of the Upper Confidence Limit for the Variance from a Pilot Sample for Sample Size Determination

In a recent paper, Browne (1995) investigated the use of a pilot sample for sample size calculation. Monte Carlo simulations indicated that using a 100 · (1 — γ) per cent upper one-sided confidence limit on the population varíance σ² leads to a sample size that guarantees the planned power with a probability of at least 1 ? γ. The purpose of this paper is to get further insight into the results of Browne by analytical considerations. Furthermore, the expected power is investigated when applying the strategy and recommendations for the choice of the pilot sample size are given.     

A Rapid and Direct Plate Method for Enumerating Escherichia coli biotype I in Food

Direct enumeration of Escherichia coli biotype 1 in foods within 24 h has been achieved by a development of the method of Delaney, McCarthy & Grasso (1962) which is based on the production of indole at 44°. Indole positive E. coli growing at this temperature on a cellulose acetate membrane overlaying tryptone bile agar can be demonstrated. Characterization of 555 indole positive colonies from 843 samples of food showed that 95% were E. coli biotype 1 and a further 3·4% were 'faecal coliforms'. Anaerogenic and non-lactose fermenting E. coli are detected and the significance of these strains is discussed.     

Influence of salinity on early development in the spined loach

Spined loach Cobitis taenia developed successfully between 0·12 and 4·80‰ salinity. At 6·00‰, net production was strongly reduced, and development failed at or above 7·20‰. Below 0·12‰ S, net production became variable, indicating restrictive effects. In comparison with other primary freshwater fish species C. taenia has a low sensitivity to salinity. The upper limit for early development was equal to the highest salinity under which C. taenia adults are found along the Baltic coast. Therefore, salinity should not limit early development within the brackish habitats of spined loach.     

Effects of changing from full range of motion to partial range of motion on squat kinetics

It is commonplace for people involved in recreational weight training to limit squat depth to lift heavier loads. This study compares differences in movement kinetics when squatting in the full range of motion (FROM) vs. partial range of motion (PROM). Ten men with a 1-year minimum of resistance training attended 4 sessions each comprising 4 sets of squats following one of FROM for 10 repetitions (FROM10) at an intensity of 67% 1 repetition maximum (1RM) FROM squat, PROM for 10 repetitions (PROM10) at 67% 1RM PROM squat, FROM for 5 repetitions (FROM5) at 83% FROM squat or PROM for 5 repetitions (PROM5) at 83% 1RM PROM squat. Movement velocity was not specified. Squat kinetics data were collected using an optical encoder. Differences between conditions were analyzed by repeated-measures analysis of variance and expressed as mean differences and standardized (Cohen) effect sizes with 95% confidence limits. The PROM5 power was substantially more than the PROM10 (98 W, -21 to 217; mean, lower and upper 95% confidence limits), FROM5 (168 W, 47-289), and FROM10 (255 W, 145-365). The force produced during PROM5 was substantially more than PROM10 (372 N, 254-490), FROM5 (854 N, 731-977), and FROM10 (1,069 N, 911-1227). The peak velocity produced during FROM10 was substantially more than FROM5 (0.105 m·s(-1), 0.044-0.166), PROM10 (0.246 m·s(-1), 0.167-0.325), and PROM5 (0.305 m·s(-1), 0.228-0.382). The FROM5 was substantially more than FROM10 (86 J, 59-113), PROM5 (142 J, 90-194), and PROM10 (211 J, 165-257). Therefore, either range of motion can have practical implications in designing resistance training programs depending on if the training goal is related to power and force development, maximizing work output or speed. Moderate-load PROM training, common among recreational weight trainers, is unlikely to provide higher movement kinetics.     

Distribution and genotypic characterization of Salmonella serovars isolated from tropical seafood of Cochin, India

Aims:  To determine the distribution of Salmonella serovars in seafood and to examine the intraserovar genetic variations in Salmonella enterica subsp. enterica serovar Rissen and Salmonella Weltevreden by polymerase chain reaction (PCR)-ribotyping and enterobacterial repetitive intergenic consensus (ERIC)-PCR methods.
Method and Results:  A total of 417 seafood samples collected over 2003–2006 from fishing harbours and fish markets of Cochin (India) was studied for presence of Salmonella serovars. Seafood samples were analysed for the presence of Salmonella by Bacteriological Analytical Manual (BAM), U.S. Food & Drug Administration (USFDA) method. The study indicated that 23·2% of the seafood samples were positive for Salmonella and a total of 241 Salmonella isolates comprising of 27 different serovars were isolated from seafood. S. Weltevreden, Salmonella Rissen, Salmonella Typhimurium and Salmonella Derby were found to be the most predominant serovars in seafood. PCR-ribotypes and ERIC-PCR profiles showed multiple genotypic profiles for S. Rissen and S. Weltevreden in seafood and the level of discrimination indices obtained was at 0·974 for S. Rissen and 0·988 for S. Weltevreden, respectively.
Conclusion:  The study highlighted the major Salmonella serovars in the seafood of Cochin (India) and molecular fingerprinting pattern revealed genetic variation among S. Rissen and S. Weltevreden.
Significance and Impact of the Study:  Widespread occurrence of Salmonella contamination in seafood and multiple clones of S. Rissen and S. Weltevreden detected in seafood, thus, indicated the diverse routes of Salmonella contamination in seafood.     

Resistance of bacterial strains to dry conditions: use of anhydrous silica gel in a desiccation model system

The Viability of 18 bacterial strains desiccated on anhydrous silica gel and stored at a temperature of 22°C for at least 3 months was determined. According to their stability in the dried state, these strains could be classified into three typical groups. Group 1, containing Gram-positive strains and Salmonella serotypes, was marked by a very slow decrease of the concentration of culturable cells from day 14 on (respectively day 21 for Salmonella thompson . The rate of decrease expressed as regression coefficient (b) ranged from —0.000389 to —0.00521 log (cfp ml^-1) per d. The Group 2 strains Enterobacter cloacae and Escherichia coli did not reach a comparable slow decrease in the dry material within the indicated time period. Regression coefficients were respectively —0.04406 and —0.03412 log (cfp ml^-1) per d. The reciprocal values —(1/b) were respectively 23 d per log (cfp ml^-1) and 29 d per log (cfp ml^-1), indicating the time periods in which a reduction of 1 log unit of culturable cells occurred. Group 3 strains Pseudomonas aeruginosa, Aeromonas hydrophila and Aer. sobria were marked by a significant susceptibility to cell damage caused during desiccation and reconstitution. A high initial decrease (ID) of the concentration of culturable organisms seems to be a characteristic property of these bacterial strains: culturable organisms could not be detected after storage for 1 d ( Aer. hydrophila, Aer. sobria ) or 7 d ( Ps. aeruginosa ). The wide range of resistance of the different bacterial strains tested indicated that the silica gel model system is a suitable tool for microbiological challenge tests to investigate the survival of micro-organisms exposed to desiccation and their stability in dry materials.     

Reference material for the evaluation of a standard method for the detection of salmonellas in foods and feeding stuffs

To evaluate a standard salmonella isolation method a reference material consisting of 0.2 g spray-dried milk inoculated with Salmonella typhimurium and contained in gelatin capsules was prepared. The organisms were distributed homogeneously between capsules, and their numbers were stable for 120 d when the capsules were stored in dry conditions at 4 degrees C. Addition of these capsules with or without food samples to pre-enrichment broth gave low and reproducible levels of Salm. typhimurium contamination without altering the pre-enrichment and without influencing the other bacterial flora present. As a result of an interlaboratory trial, the reference material indicated that the food and/or its competitive flora may have a negative influence on the detection of salmonellas.     

Reference material for the evaluation of a standard method for the detection of salmonellas in foods and feeding stuffs

To evaluate a standard salmonella isolation method a reference material consisting of 0.2 g spray-dried milk inoculated with Salmonella typhimurium and contained in gelatin capsules was prepared. The organisms were distributed homogeneously between capsules, and their numbers were stable for 120 d when the capsules were stored in dry conditions at 4C. Addition of these capsules with or without food samples to pre-enrichment broth gave low and reproducible levels of Salm. typhimurium contamination without altering the pre-enrichment and without influencing the other bacterial flora present. As a result of an interlaboratory trial, the reference material indicated that the food and/or its competitive flora may have a negative influence on the detection of salmonellas.