An investigation of indirect conductimetry for detection of some food-borne bacteria

Indirect conductimetry using a rapid automated bacterial impedance technique was investigated. Strains of Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. grown in Whitley Impedance broth all elicited indirect conductimetric changes. These indirect conductance responses were improved by the addition of 2 g/l glucose to the medium and resulted in maximum changes of 2340-4300 microS with associated maximum rates of change of 520-1210 microS/h. Furthermore, the indirect conductimetric assay detected growth of staphylococci, listeria and salmonella in media containing high concentrations of salts used as selective agents in culture media for the isolation of these organisms.     

A complete protocol using conductance for rapid detection of salmonellas in confectionery materials

Increased confidence in conductimetric detection of salmonellas was achieved by combining a bacteriophage-based test with use of a selenite cystine trimethylamine oxide dulcitol medium and a modified lysine decarboxylase broth. All 81 Salmonella isolates tested were detected and few of the 39 non-salmonellas gave false positives. Results from the screening of 43 inoculated product samples further support the use of this simple, rapid method for routine salmonella testing in the food industry.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Effect of Type of Enrichment and Duration of Incubation on Salmonella Recovery from Meat-and-Bone Meal

Twenty-five meat-and-bone meal samples were enriched with either selenite-cystine or tetrathionate and incubated for 1 and 2 days. Seven were previously found to be positive; of the other 18, 16 were positive for salmonella. The number of somatic serogroups per sample ranged from 1 to 11 with a mean of 3.8. Significantly more (P < 0.01) group C₁ salmonellae were isolated using tetrathionate than selenite, whereas significantly more of groups G, 35, and Difco poly-valent D were isolated from selenite than tetrathionate. Seventy-six percent of the presumptive colonies from Brilliant Green agar showed a positive lysine decarboxylase reaction, and there were no differences between media or times of incubation. Ninety-four per cent of the lysine decarboxylase-positive cultures showed a positive somatic antiserum response; again there were no differences between times or enrichments although there were significantly more total positive serogroups at 2 days than at 1 day from tetrathionate but not from selenite. There were indications that certain serogroups preferred either one or the other enrichment. There were no differences in total positive samples with the two enrichments although neither alone was sufficient to identify all positives. Several lactose-positive salmonellae were recovered.     

Antibiotic amendment for suppression of indigenous microflora in feed sources for an Escherichia coli auxotroph lysine assay

Growth responses of lysine auxotrophic mutants of Escherichia coli have been used as a measurement of bioavailable lysine in protein sources and animal feeds. Sterilizing feed samples by autoclaving to eliminate non-specific background growth of indigenous feed micro-organisms prior to conducting the bacterial assay may introduce chemical and physical alterations to the feeds, influencing the estimation of available feed lysine. In this study, an antibiotic- and antifungal-supplemented medium was constructed to support growth of an E. coli lysine auxotroph assay organism, and was tested for its ability to repress indigenous bacterial and fungal growth in feed samples. To determine which antibiotics to include, an ampicillin-sensitive E. coli lysine mutant strain (ATCC no. 23812) was screened for antibiotic resistance and transformed with a plasmid carrying an ampicillin resistance gene. Maximum optical density quantitative response of the E. coli auxotroph to lysine was not altered by the antibiotic medium amendments (ampicillin, novobiocin and cycloheximide). Indigenous microfloral growth in a variety of typical animal feeds was suppressed in the presence of the antistatic agents. The estimated lysine recovery was 91.6% and 98.1% when the medium was used in an assay of available lysine in a lysine-supplemented feed. This indicates that the antibiotic-amended basal medium can be used for the E. coli-determined lysine availability of a variety of animal feeds without prior sterilization of the feed sources.     

An improved protocol for the detection and rapid confirmation within 48 h of Salmonellas in confectionery products

A modified ornithine decarboxylase broth and a selenite cystine trimethlyamine oxide ribose medium were developed to improve selection and detection of Salmonellas. They were used with a third medium, lysine iron cystine neutral red broth (incubated conventionally) in a screen of 80 Salmonellas and 32 non-salmonellas which showed the combination of media to be specific and sensitive. Subsequent evaluation with 90 product samples (spiked and naturally contaminated) resulted in complete agreement by rapid and conventional methods. The detected wells in these experiments were also used to evaluate a latex slide agglutination test for Salmonellas. Carried out directly on the well contents, this was found to be a rapid, specific and sensitive test for confirmation of Salmonella presence. Further tests with 106 routine production samples gave complete agreement by both methods. Presumptive positives were obtained in <1.0–3·8% of the sample wells and subsequent modification to the Bactometer software has greatly reduced these figures in routine use. The method is recommended for Salmonella testing of foodstuffs with low background flora and high fat/low moisture content.     

Lysine Decarboxylase Activity in Broth and Agar Media

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.     

Improvements to a lysine medium for detection of salmonellas by elctrical conductance

Improvements in performance of a lysine conductance medium for the detection of salmonellas were achieved from a study of the effects of its various components. When sodium biselenite was included as an inhibitor for non-salmonella organisms conductance signals were depressed. The inclusion of sodium chloride reduced this toxicity and improved conductance responses. Increasing the pH to pH 7.0 prevented the medium becoming too acidic and inhibitory to salmonellas. The new medium detected 70-0% of salmonella-positive animal protein samples.     

Growth kinetics of mixed culture in salmonella enrichment media

R hodes , P., Q uesnel , L.B. & C ollard , P. 1985. Growth kinetics of mixed culture in salmonella enrichment media. Journal of Applied Bacteriology 59 , 231–237.
A technique for investigating the kinetics of salmonella enrichment is reported. Its use with four enrichment media (Rappaport' medium, Muller-Kauffmann tetra-thionate broth (MKT) tetrathionate broth and selenite F) is described and the effect of elevated temperature on the growth kinetics shown. Rappaport' medium at 37C and MKT at either 37C or 42C were far superior to selenite F and tetrathionate broth in their selective properties and, with the exception of Rappaport' medium, the use of elevated temperature increased the selectivity of the media.     

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10⁶ cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10⁷ to 1.8 × 10⁷ cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.     

Growth kinetics of mixed culture in salmonella enrichment media

A technique for investigating the kinetics of salmonella enrichment is reported. Its use with four enrichment media (Rappaport's medium, Muller-Kauffmann tetrathionate broth (MKT) tetrathionate broth and selenite F) is described and the effect of elevated temperature on the growth kinetics shown. Rappaport's medium at 37 degrees C and MKT at either 37 degrees C or 42 degrees C were far superior to selenite F and tetrathionate broth in their selective properties and, with the exception of Rappaport's medium, the use of elevated temperature increased the selectivity of the media.     

Effect of selenium compounds on selenium content, growth and 35S-cystine metabolism of skin fibroblasts from normal and cystinotic individuals.

Kidney samples from children with the inborn metabolic disease cystinosis contain 4 times more selenium (Se) than do kidney samples from normal individuals (p = 0.1). However, when cultured skin fibroblasts from cystinotic patients and normal control individuals are incubated in Se-D,L-methionine, Se-D,L-cystine, Se-cystamine X HCl, Se-urea, selenite or in medium without added selenium, only the cystinotic fibroblasts grown in Se-urea or selenite (SeO3=) contain more selenium than do the corresponding normal cells (p less than 0.05). In both types of cultured fibroblasts, the order of descending toxicity per ppm selenium is: Se-urea greater than Se-cystamine greater than Se-cystine greater than or equal to SeO3= much greater than Se-methionine. High (apparently toxic) concentrations of Se-urea and Se-cystamine lower the elevated intracellular free (nonprotein) cystine content of cystinotic fibroblasts to less than 60% of control values; at lower concentrations, these compounds raise the cystine content of these cells to over 140% of control values. Appropriate concentrations of SeO3=, Se-cystine and Se-methionine also elevate the free cystine content of the cystinotic cells. During a 75 minute incubation in 35S-cystine, the incorporation of 35S into the acid precipitable (protein) fraction of both cell types is significantly inhibited by Se-cystamine (approximately 55% control; p less than 0.05). The incorporation of 35S-cystine into glutathione is inhibited by Se-cystine (approximately 40% control) in both fibroblast types (p less than 0.05). In cystinotic cells, Se-cystamine significantly reduces incorporation of 35S-cystine into the cystine pool (40% control) as does SeO3= (67% control; p less than 0.05). Protein and glutathione synthesis in cystinotic fibroblasts are more strongly inhibited by Se-cystine and SeO3=, respectively, than in normal fibroblasts (p less than 0.05). These studies demonstrate that selenium compounds exhibit a different sequence of toxicity in fibroblasts than in the intact animal and that some previously unreported metabolic effects (i.e. inhibition of glutathione synthesis) may contribute to their toxicity.     

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

AIMS: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. METHODS AND RESULTS: Faeces samples collected from six adult bovines from different salmonella-negative herds were split into subpools and spiked with three strains of S. Dublin at a concentration level of c. 10 CFU g(-1) faeces. Each of the 18 strain-pools was divided into two sets of triplicates of four volumes of faecal matter (1, 5, 10 and 25 g). The two sets were pre-enriched with and without novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. CONCLUSIONS: Biological factors, such as faecal origin and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semi-solid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied more in sensitivity and rarely reached the same level of detection as MSRV in this experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that for MSRV-culture medium and xylose lysine decarboxylase agar as the indicative medium, the sensitivity of the faecal culture method may be improved by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin.     

Limitations of the Moeller lysine and ornithine decarboxylase tests.

A total of 40 fecal and environmental isolates, including 26 Escherichia coli strains, 9 members of the genus Klebsiella, and 5 members of the genus Enterobacter, were tested by enzyme assay for their endogenous and induced levels of lysine decarboxylase and ornithine decarboxylase when grown in Moeller decarboxylase medium. All of the coliforms examined had measurable lysine decarboxylase and ornithine decarboxylase activities whether or not they were positive in the Moeller test. In general, the Moeller lysine decarboxylase test reflected the inducibility of lysine decarboxylase whereas the Moeller ornithine decarboxylase test did not relect the inducibility of ornithine decarboxylase. Neither test measured the amount of intracellular enzyme; rather, they indicated whether the amount of polyamine liberated was sufficient to raise the pH of the culture medium above 7. Changing the growth conditions (i.e., the concentrations of glucose, lysine, and amino acids other than lysine) greatly influenced the lysine decarboxylase activity in coliforms. The limitations on the interpretation of the Moeller test results are discussed.     

Limitations of the Moeller lysine and ornithine decarboxylase tests.

A total of 40 fecal and environmental isolates, including 26 Escherichia coli strains, 9 members of the genus Klebsiella, and 5 members of the genus Enterobacter, were tested by enzyme assay for their endogenous and induced levels of lysine decarboxylase and ornithine decarboxylase when grown in Moeller decarboxylase medium. All of the coliforms examined had measurable lysine decarboxylase and ornithine decarboxylase activities whether or not they were positive in the Moeller test. In general, the Moeller lysine decarboxylase test reflected the inducibility of lysine decarboxylase whereas the Moeller ornithine decarboxylase test did not relect the inducibility of ornithine decarboxylase. Neither test measured the amount of intracellular enzyme; rather, they indicated whether the amount of polyamine liberated was sufficient to raise the pH of the culture medium above 7. Changing the growth conditions (i.e., the concentrations of glucose, lysine, and amino acids other than lysine) greatly influenced the lysine decarboxylase activity in coliforms. The limitations on the interpretation of the Moeller test results are discussed.     

An investigation of indirect conductimetry for detection of some food-borne bacteria

B olton , F.J. 1990. An investigation of indirect conductimetry for detection of some food-borne bacteria. Journal of Applied Bacteriology 69 , 655–661.
Indirect conductimetry using a rapid automated bacterial impedance technique was investigated. Strains of Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Salmonella spp. grown in Whitley Impedance broth all elicited indirect conductimetric changes. These indirect conductance responses were improved by the addition of 2 g/1 glucose to the medium and resulted in maximum changes of 2340–4300 μS with associated maximum rates of change of 520–1210 μS/h. Furthermore, the indirect conductimetric assay detected growth of staphylococci, listeria and salmonella in media containing high concentrations of salts used as selective agents in culture media for the isolation of these organisms.     

Comparative Studies on Enrichment And Selective Media for Isolation of Salmonellae from Faecal Specimens

Enrichment media (tetrathionate, selenite and Rapp ap ort broths) and selective media (desoxycholate citrate agar and brilliant green agar) were tested in different combinations to ascertain their capacity for isolation of salmonella bacteria. The material consisted of 299 samples of cattle faeces from two herds infected with salmonella (Table 1), and of 111 artificially contaminated samples of pig faeces (Table 3). The tetrathionate and selenite broths were equally useful for the material as a whole, whereas the results varied between different species of salmonella which is of great practical interest. The number of salmonella isolations was much lower when enrichment with Rappaport broth was used. The rate of salmonella isolations can often be increased by parallel enrichments with two different media. Of the selective agar media tested, brilliant green agar was superior to desoxycholate citrate agar.     

Benchmarking of commercially available CHO cell culture media for antibody production

In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.     

Quantitative detection of crystalline lysine supplementation in poultry feeds using a rapid bacterial bioluminescence assay

Lysine is an essential amino acid for both humans and animals; and it is usually the first or second limiting amino acid in most formulated diets. In order to estimate the lysine content in feeds and feed sources, rapid amino acid bioassays have been developed. The objective of this work is to assess a rapid assay for lysine supplementation in chicken feeds, using a luminescent Escherichia coli lysine-auxotrophic strain, to avoid prior thermal sterilization. An E. coli lysine auxotroph carrying a plasmid with lux genes was used as the test organism. The lysine assay was conducted using depleted auxotrophic cells in lysine samples. Luminescence was measured with a Dynex MLX luminometer after addition of the aldehyde substrate. Growth response (monitored as optical density at 600 nm) and light emission response of the assay E. coli strain were monitored to generate standard curves. Bioluminescent analysis of feed samples indicated that the method works well in the presence of a complex feed matrix. Comparison of both optical density and luminescent-based methods indicated that, when the assay takes place under optimal conditions, both methodologies correlated well ( r(2)=0.99). Except for the 0.64% lysine-supplemented feed, estimates for lysine based on the bacterial assay were over 80% (82-97%) of the theoretical values. Animal data showed that the bacterial bioluminescent method correlated well with the chick bioassay when diets with different levels of lysine supplementation were assayed for lysine bioavailability ( r(2)=0.97). Luminescent methodology coupled with a bacterial growth assay is a promising technique to assess lysine availability in supplemented animal feeds.