Biomedical subjects

R G Kroll

Publications and source records attributed to R G Kroll.

At least 37 records · Page 2Linked to original sources

PubMed1992

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods.

Lectins from Helix pomatia, Canavalia ensiformis, Agaricus bisporus and Triticum vulgaris agglutinated cultures of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. This agglutination was specific as it was inhibited (except with A. bisporus lectin) by the competing sugar substrates. The ability of three of these lectins, immobilized on a variety of supports, to separate these micro-organisms from pure cultures was investigated. Immobilization of the lectins on magnetic microspheres was the most effective method. Immobilized T. vulgaris lectin bound 87-100% of cells from cultures of L. monocytogenes, 80-100% of Staph. aureus, 33-45% of Salmonella spp. and 42-77% of E. coli. The A. bisporus lectin bound 31-63% of cells in cultures of L. monocytogenes, 83% of Staph. aureus but only 3-5% of the salmonella cells. Similarly H. pomatia lectin bound greater than 92% of Staph. aureus and 64% of L. monocytogenes cells but was poor at binding the Gram-negative organisms. This preference for binding Gram-positive organisms was confirmed when mixed cultures were studied. The T. vulgaris lectin was effective in removing L. monocytogenes (43%) and Staph. aureus (26%) from diluted milk and Salmonella (31-54%) from raw egg. Agaricus bisporus lectin removed L. monocytogenes from undiluted milk (10-47%) or ground beef (32-50%).

Animals↗

PubMed1992

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.

Alanine↗

PubMed1991

Increased sensitivity of an enzyme-amplified colorimetric immunoassay for protein A-bearing Staphylococcus aureus in foods.

A modification to the enzyme amplification system described by Self (1984), has been developed in which the addition of semicarbazide hydrochloride increased the sensitivity of detection of protein A-bearing Staphylococcus aureus in foods from 4-6 X 10(3) to 20 colony forming units (c.f.u.) g-1 or ml-1. This may be due to the removal of acetaldehyde produced as a by-product of the amplification cycle thereby permitting further cycling to proceed.

Animals↗

PubMed1991

Respiratory activity in Listeria monocytogenes.

Listeria monocytogenes possessed glucose oxidase and NADH oxidase activities in whole cells and lysed protoplasts respectively. The NADH oxidase activity sedimented with the membrane fraction and was inhibited by the respiratory inhibitors rotenone, 2-heptyl-4-hydroxy-quinoline-N-oxide and cyanide, suggesting the presence of a membrane associated respiratory chain.

Cell Membrane↗

PubMed1991

The adsorption of bacteria to immobilized lectins.

The agglutination of a selection of bacteria by some lectins was examined. The lectin from Codium fragile agglutinated seven strains of Salmonella typhimurium. The lectin from Helix pomatia agglutinated eight of 12 strains of Listeria monocytogenes and a further two strains gave a weak agglutination reaction. Helix pomatia lectin conjugated to magnetic microspheres enabled the adsorption of L. monocytogenes from suspension with subsequent elution by the competing ligand N-acetyl galactosamine. Affinity chromatography of a suspension of L. monocytogenes through a column of H. pomatia lectin immobilized on agarose, also adsorbed cells and enabled subsequent elution with N-acetyl galactosamine. The column technique enabled the more rapid adsorption of bacteria perhaps because of improved interactions between bacteria and immobilized lectin.

Adsorption↗

PubMed1991

The differential fluorescence of bacteria stained with acridine orange and the effects of heat.

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.

Acridine Orange↗

PubMed1990

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods.

A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 10(4) cfu/ml, and at ca. 10(5) cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperometric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.

Biosensing Techniques↗

PubMed1990

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique.

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred.

Animals↗

PubMed1990

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods.

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.

Enzyme-Linked Immunosorbent Assay↗

PubMed1990

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus.

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.

Animals↗

PubMed1990

Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods.

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese.

Colorimetry↗

PubMed1989

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods.

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.

Animals↗

PubMed1989

Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods.

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.

Animals↗

PubMed1988

Survival of some non-starter bacteria in naturally ripened and enzyme-accelerated Cheddar cheese.

Effects of the addition of a proteinase (Neutrase 1-5S) and a peptidase (aminopeptidase DP-102) as agents for accelerating the ripening of Cheddar cheese on the survival of some non-starter bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and a Salmonella sp.) were studied throughout a 4-month ripening period. The enzymes were found to have no significant effect on the survival of the Gram-positive bacteria but some significant effects were observed, at some stages of the ripening period, with the Gram-negative bacteria in that lower levels were recovered from cheeses treated with the enzyme system.

Aminopeptidases↗

PubMed1988

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique.

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.

Bacteria↗

PubMed1987

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

Heat treatment at 80 degrees 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 degrees 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.

Animals↗

PubMed1986

Prediction of the keeping quality of pasteurized milk by the detection of cytochrome c oxidase.

The keeping quality (KQ) of pasteurized milk samples stored at 5 degrees C and 10 degrees C was satisfactorily predicted after 18 h pre-incubation with 0.05% benzalkonium chloride at 20 degrees C, by estimating the numbers of Gram-negative psychrotrophic bacteria using the simple, cheap and rapid (5 min) assay of cytochrome c oxidase. Correlation coefficients for the relationship between cytochrome c oxidase activity at 20 degrees C and KQ at 5 degrees C or 10 degrees C of -0.89 and -0.84 respectively were obtained. The method correctly predicted the KQ of more than 89% of the samples of pasteurized milk. The assay was not satisfactory for use on samples after pre-incubation at 30 degrees C.

Animals↗

PubMed1986

Use of the direct epifluorescent filter technique for the enumeration of yeasts.

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.

Acridine Orange↗