IMPROVED TECHNIQUES FOR THE BACTERIOLOGICAL EXAMINATION OF MOLLUSCAN SHELLFISH

SUMMARY: The main obstacles to the general acceptance of the Clegg & Sherwood (1947) method for the examination of molluscan shellfish are thought to be the difficulties of preparing the medium and of rolling the tubes satisfactorily. The use of Astell or McCartney bottles, or sealed plates, in place of the conventional 6x1 in. test tubes, is described.
The following simplified MacConkey medium is recommended in place of that of Clegg & Sherwood (all quantities % w/v): agar, 3·5; peptone, 1·5; lactose, 1·0; NaCl, 0·5; bile salt (Difco No. 3), 0·15 or Oxoid, qs.; neutral red, 0·003; distilled water to 100 ml, pH 7·2–7·4. With 5 ml of this medium inocula of 2 ml may be used. Oxoid MacConkey agar No. 3 (CM. 115a) is equally suitable.
A brief account is given of the use of this method and examples of the better performance of the new medium for the examination of molluscan shellfish.     

Neutralization efficacy of Dey-Engley medium in testing of contact lens disinfecting solutions

A quantitative assay for the demonstration of neutralizer efficacy was developed to monitor contact lens disinfecting solutions. Adequate neutralization of disinfecting agents is essential to the accurate determination of disinfecting activity with time. This method employed the recovery of small numbers of micro-organisms from neutralizing medium containing a disinfectant. A statistical estimation of significance between treatments demonstrated that Dey-Engley medium (DE; Difco) was generally effective when tested as an agar growth medium with several bacterial test organisms. DE medium from another vendor was less effective, underscoring the need for laboratory quality control and monitoring. DE agar (Difco) adequately neutralized all solutions tested at a 1:20 dilution. The solutions included those containing Dymed^TM (polyaminopropyl biguanide, 0·00005%), chlorhexidine (0·005%), Polyquad® (0·001%), chlorhexidine (0·005%) and thimerosal (BP, 0·001%), thimerosal (BP, 0·002%) and Tris(2-hydroxyethyl) tallow ammonium chloride (0·013%), and a solution preserved with 115 ppm benzalkonium chloride (BAK). A modification of this medium was developed which retained virtually all of the neutralizing efficacy for the solutions tested while allowing the use of automated testing procedures.     

An improved selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods

Columbia agar base containing 0·001% acriflavine, 1·5% lithium chloride, 0·25% phenyl ethanol, 0·05% aesculin. 0·05% ferrous salt, 1% mannitol, 2·5% egg yolk emulsion and 0·008% phenol red (ALPAMY), recovered Listeria monocytogenes and some strains of Listeria seeligeri quantitatively, but suppressed Listeria ivanovii and virtually all other bacteria common in fresh foods. When used with foods processed for safety, repair on non-selective buffered glucose tryptone soya peptone yeast extract catalase agar must precede the use of ALPAMY.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

METHODS FOR THE ISOLATION OF FAECAL STREPTOCOCCI (LANCEFIELD GROUP D) FROM BACON FACTORIES

SUMMARY: Two methods have been developed for isolating and enumerating group D faecal streptococci from localities such as bacon factories where they are heavily outnumbered by other organisms. The first depends on presumptive counts in a Lab-Lemco-peptone-glucose broth (pH 6·0) containing 0·1% of thallous acetate with confirmation by streaking on tetrazolium agar. The other method involves direct plating on tetrazolium-glucose agar (pH 6·0) containing 0·1% of thallous acetate. On the tetrazolium medium differentiation can be made between Streptococcus faecalis and its variants zymogenes and liquefaciens and the other group D organisms, Strep. faecium, Strep. durans and Strep. bovis .     

The preparation of ampicillin dextrin agar for the enumeration of Aeromonas in water

Introduction of ampicillin dextrin agar (ADA) has revealed problems in details of the preparation. The final pH of the medium varied substantially between different laboratories. Measuring temperature has a pronounced effect on the pH (0·7 units lower at 50°C than at 6°C). Addition of agar during medium preparation resulted in a fall in pH of 0·5 units. If poured plates were stored in the refrigerator, the pH was reduced by 0·1–0·4 units, in particular during the first day. Recovery of Aeromonas from pure cultures and naturally polluted samples was unaffected by variation in pH between 7·1 and 8·3 but colony differentiation was optimal at a higher pH. The use of ADA at a final pH of 7·8 ± 0·2 (at 25°C) is recommended. Different types of dextrin differed in respect of solubility, fermentability and colony differentiation. Optimal results were obtained with Difco 161 and Merck 3006.     

Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.     

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

The growth patterns of macrocolonies 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°C for 3 or 6 h (staphylococci) at contamination levels of above 10³ 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.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

Inactivation effect of X-ray treatments on Cronobacter species (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat milk*

Aims:  To determine the inactivation effect of X-ray treatments on Cronobacter ( E. sakazakii ) in tryptic soy broth (TSB), skim milk (0% fat), low-fat milk (1% and 2%) and whole-fat milk (3·5%).
Methods and Results:  X-rays were produced using the RS 2400 generator system (Rad Source Technologies Inc.). Cronobacter (in TSB), inoculated skim milk (0% fat), low-fat milk (1% and 2% fat) and whole-fat milk (3·5% fat) were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 2·0, 3·0, 4·0, 5·0 and 6·0 kGy X-ray doses. Surviving bacteria in the TSB and inoculated milk, before and after treatment, were enumerated using plating method onto trypticase soy agar. Greater than 7·0-log CFU reduction in Cronobacter population was observed with 4·0, 5·0, 6·0, 6·0 and 6·0 kGy X-ray in the TSB, skim milk, 1% fat milk, 2% fat milk and 3·5% fat milk, respectively.
Conclusions:  Treatment with X-rays significantly ( P  <   0·05) reduced Cronobacter to less than detectable limits (<1 log CFU ml⁻¹) in skim milk at 5·0 kGy and milk with 1% fat content and greater at 6·0 kGy dose levels. The D-value for Cronobacter in TSB was significantly ( P  <   0·05) lower than those in milk samples.
Significance and Impact of the Study:  Treatment with X-rays could be an effective and safe alternative technology to control pathogenic bacteria ( Cronobacter ) in the dairy industry.     

Habituation to normally lethal acidity by prior growth of Escherichia coli at a sub-lethal acid pH value

Both Col^- and ColV, I-K94⁺ strains of Escherichia coli , grown at pH 7–0, failed to grow after relatively short periods of exposure to pH 3·0 or 3·5. After growth in exposure medium initially at pH 5·0, both strains were almost unaffected by exposure to such acid pH values. Addition of catalase to nutrient agar only slightly increased plating efficiency after acid treatment and very slightly reduced the difference in survival, after acid treatment, between organisms grown from pH 5·0 and those grown from pH 7·0. Accordingly, acid resistance of organisms grown from pH 5·0 is not chiefly due to greater resistance to hydrogen peroxide already present in nutrient media.     

A monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol (DAS) was produced by a hybridoma, designated 2E5. It secreted antibody of the IgGl subclass and had a detection limit for DAS of 16 ng/ml with a direct enzyme immunoassay on a double antibody solid phase. The relative cross-reactivities with 3α-acetyl-DAS, diacetylverrucarol, neosolaniol, T-2 tetraol tetraacetate, fusarenon X, T-2 toxin, and HT-2 were 2224·5, 53·7, 13·9, 9·2, 6·4, 1·7, 0·6, and 0·35%, respectively.     

NIGHT BLUE AND VICTORIA BLUE AS INDICATORS IN LIPOLYSIS MEDIA

SUMMARY: Optimum conditions for the use of night blue and victoria blue as lipolysis indicators in fat emulsion agar medium required a dye strenth of 1: 15,000 in a medium of pH 8·0 containing 5% fat dispersed by hand shaking. Pour plates should contain 6 ml. and streak plates 10 ml. of the medium in a standard Petri dish. Incubation should be for 5 days at 30°. The pinkish-mauve medium with clear blue-zoned lipolytic colonies gave the same results as butter fat agar without dye but treated with CuSo₄, when tested with 962 pure cultures. The inhibitory powers of the dye were assessed and although strongly toxic in the aqueous phase to Gram-positive bacteria, victoria blue appears to have none to slight inhibitory power in the fat agar medium: night blue suppressed growth to about the same extent as tributyrin. The lipolytic flora of butter and to some extent milk shows a remarkable dominance of micorcocci. Organisms lipolytic on fat agar media are able to produce appreciable acid in a fat emulsion in a liquid medium.     

Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups

Viable counts of heterotropic soil bacteria were 3–5 times higher on low-nutrient agar media compared with a series of conventional agar media. Substantial amounts of monosaccharides and amino acids were present in solid media made from distilled water and agar powder, and a salt-solution agar medium (without organic substrates added) gave practically the same colony counts as the low nutrient soil extract agar medium. MPN values were comparable to or lower than plate counts. A search for slow-growing cells in the negative MPN tubes by fluorescence microscopical examination after 3 months incubation was negative.The viable counts were 2–4% of the total microscopical counts in different soils. Assuming that the colony-forming cells did not derive from the numerous dwarf cells present in soil, a calculated percent viability of the larger cells was about 10%. The ecological significance of the plate-counting technique is discussed.     

Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli

Escherichia coli K-12 was exposed to Quillaja saponaria saponins from various commercial firms (Sigma, Roth and Nor-feed) and to an extract of Yucca schidigera plant powder (DK Sarsaponin 30) at different concentrations (0·05–1·0% w/v). A concentration-dependent response was observed. Quillaja saponaria saponins from Sigma increased growth up to 0·1% (w/v) level, whereas Nor-feed and Roth saponins produced maximum growth at a much higher level (0·5 and 0·75%, w/v, respectively). These results suggest that quillaja saponins from various sources differ in their biological activity, although all three saponins had the same content of vanillin-sulphuric acid reactive moieties. The lyophilized water extract from the DK Sarsaponin powder showed maximum growth at 0·1% (w/v) level. The levels at which maximum growth was observed did not change on subjecting the quillaja or yucca saponins to heat treatment in an autoclave (121 °C for 30 min). All the saponins and the plant extract increased growth of Escherichia coli up to a certain concentration and thereafter decreased growth. In spite of the decreased growth at higher levels of saponins, it was higher compared to the control (without saponin) up to levels of 1% (w/v) for all saponins except Quillaja saponins from Sigma, for which the growth was lower at levels of 0·25% (w/v) and higher. Saponins have the potential to modulate microbial growth in natural and artificial fermenters.     

A blood-free enrichment medium for growing Campylobacter spp. under aerobic conditions

Detection limits for Campylobacter jejuni strains JH93 and ATCC 29428 in a new blood-free enrichment broth (BFEB) were investigated under aerobic conditions. Cultures of Camp. jejuni were inoculated into 50 ml BFEB containing 10% food homogenate in 50 ml screw-cap tubes. After 24 h enrichment under aerobic conditions, Camp. jejuni were isolated on four selective agar media. The least squares means of the detection limit 50% endpoint (DL₅₀) values were 0·4 (plain BFEB), 0·9 (crabmeat), 1·7 (mushroom), 1·7 (raw milk) and 2·1 (oyster) colony forming units (cfu) 5 g⁻¹ food. The efficiency of the BFEB was significantly affected ( P < 0·05) by food type and bacterial strain. Overall, the BFEB enrichment compared favourably with the existing US Food and Drug Administration method under modified atmosphere. In addition, the BFEB method did not require the use of blood, special equipment or Oxyrase^® to reduce oxygen tensions.     

Improved method for quantification of the bacteriocin nisin

Nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis , is used in some types of food preservation due to its inhibitory action on Grampositive bacteria and their spores. A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision. Several variables were evaluated. Results showed Micrococcus luteus as the most sensitive organism tested, a lower agar concentration (0·75% compared 1·5%) increased the sensitivity of the assay (21% improvement over standard method), and incorporation of 1% Na₂HPO₄ buffer into the bioassay agar made it possible to prevent false inhibitory zones from developing due to the low pH of the test solutions. This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method     

A monoclonal antibody-based enzyme immunoassay for the detection of T-2 toxin at picogram levels

Thirteen monoclonal antibodies reactive with HT-2 were prepared by using a HT-2 hemisuccinate coupled to human serum albumin as antigen for the immunization of BALB/c mice. In a competitive enzyme immunoassay on a double antibody solid phase using HT-2 hemisuccinate coupled to horseradish peroxidase as enzyme linked toxin all antibodies reacted much better with T-2 toxin and acetyl T-2 than with HT-2. Eleven antibodies showed almost the same sensitivity and specificity, and one of these, designated 3E2, is extensively described. Its cross-reactivities with HT-2, T-2 toxin, acetyl T-2, iso T-2, T-2 tetraol tetraacetate and T-2 triol were 1·0, 140·2, 161·2, 0·32, 0·14 and 0·016, respectively. Two other antibodies, designated 2A4 and 2A5, behaved quite differently. The cross-reactivities of antibody 2A4 with these toxins were: 1·0, 113·9, 374·4, 1·35, 0·34 and 0·023, respectively; for antibody 2A5 they were 1·0, 46·1, 155·4, 8·31, 0·9 and 0·08, respectively. All antibodies proved to be IgGl. By using the antibody 3E2 a highly sensitive and very specific enzymc immunoassay for the detection of T-2 toxin was developed. The detection limit for T-2 toxin was 5 pg/ml (0·25 pg/assay).     

Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood

The virulence factors of Vibrio vulnificus are not yet well understood. So far, many hydrolytic enzymes have been implicated in the pathogenesis of this micro-organism. The present research was carried out in order to study the presence of some of these enzymes in 133 V. vulnificus strains isolated from 45 seafood samples. The results showed that 100% of these strains were positive for the production of lecithinase and lipase (Tween-80), 99·2% for caseinolytic protease, 96·9% for DNase, 65·4% for mucinase and 46·6% for elastase. None of the strains was positive for the production of collagenase and 96% were haemolytic against sheep blood cells. In relation to colony morphology on brain heart infusion (BHI) agar and nutrient agar, 59·4% of strains showed opaque morphology on BHI agar and 57·9% on nutrient agar, 10·5% presented translucent morphology on both agars and 30·1 and 31·6% of strains showed a mixture of opaque and translucent morphology on BHI agar and nutrient agar, respectively. None of the translucent colonies was virulent to mice. Therefore, opacity was a useful marker for potential virulence. Of 45 food samples contaminated with V. vulnificus , 29 (64·4%) presented strains lethal to adult mice.