MacConkey and Glutamate Media in the Bacteriological Examination of Seawater

Confirmatory tests were made in 4807 positive tubes of MacConkey broth. It was found that when an equal volume of seawater was added to a double strength MacConkey broth only 88% of the tubes contained coliform organisms (12% false-positive reactions); when, however, the ratio of the volume of seawater to broth was 1/5 or less, coliform organisms were found in 93·5% of the positive tubes (6·5% false-positive reactions). Glutamate medium gives a higher rate of false reactions than MacConkey broth when an equal volume of seawater is added to double strength medium. When the volume ratio of seawater to glutamate medium is equal to or less than 1/5, then the minerals-modified lactose glutamate medium gives better results than MacConkey broth in the coliform and especially in the Escherichia coli count. The difference is statistically significant.
It is thus concluded that for the bacteriological examination of seawater the volume ratio of water to medium should be 1/5 or less and also that glutamate medium is the medium of choice.     

Bacteriological Examination of Seawater: Observations on Factors Affecting the Performance of Media

The performance of MacConkey broth and minerals modified glutamate lactose medium was examined in a series of experiments. It was found that the addition of seawater with or without antibacterial activity and the addition of 3·2% NaCl solution to double strength media all had similar harmful effects on their performance. Also a concentration of 3·2% NaCl in ordinary lactose broth caused a great decrease ( P =0·02) in the number of tubes containing coliform organisms. It is concluded that the high salt content of seawater interferes with lactose fermentation by coliforms. This interference was found to be so great that the number of tubes in which coliform organisms and Escherichia coli were detected, dropped from 2·5 to 3·5 times when an equal volume of seawater was added to double strength MacConkey and glutamate media. The results of the last experiment suggest that for best performance of the media, the volume ratio of seawater to medium should be equal or less than 1/10. Also glutamate medium was superior to MacConkey broth ( P <0·001), especially in the detection of E. coli .     

A New Medium for Bacillus thuringiensis Berliner

S ummary : A new solid medium for culturing Bacillus thuringiensis consists of groundnut cake, 10%; tamarind kernel powder, 1·5% and agar, 0·5%. The quantity of agar in the medium could be decreased from 1·5 to 0·5% by adding tamarind kernel powder. A spore yield (85% sporulation) of 3·2 g/l was obtained. Bacterial spores produced on the new solid medium were pathogenic to the larvae of the almond moth, Cadra cautella.     

A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157 : H7 in beef using direct epifluorescent microscopy and immunomagnetic separation

An antibody-direct epifluorescent filter technique (Ab-DEFT) detected 100% of the raw ground beef samples inoculated with Escherichia coli O157 : H7 cells (0·15 cells g⁻¹) and incubated in a prewarmed, modified buffered peptone water (mBPW) non-selective enrichment broth for 5 h at 42°C in an orbital shaking water bath (200 rev min⁻¹). Over 50% of the microscopic fields viewed were positive (1–10 fluorescent cells field⁻¹) in the Ab-DEFT. All positive screening results were confirmed within 24 h by subjecting 1 ml of the mBPW to the Dynabeads^® anti- E. coli O157 immunomagnetic separation procedure, followed by plating on MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indolyl-β- D -glucuronide. At this cell concentration, 41·7% of the inoculated samples were detected by the conventional method involving a 24-h selective enrichment. Exposure to viable cells before filtration was minimized by using a 0·58% formaldehyde concentration for 5 min at 50°C (killed >4·00 logs of E. coli O157 : H7 cells) without affecting cell fluorescence.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

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.     

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples

Aims:  This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples.
Methods and Results:  A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB . On direct culture, the sensitivity of the three media was 66·7%, 77·8% and 44·4% and after broth enrichment 66·7%, 83·3% and 77·8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively.
Conclusions:  All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter.
Significance and Impact of the Study:  This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.     

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.     

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.     

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.     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

Biotransformations of Paralytic Shellfish Toxins by Bacteria Isolated from Bivalve Molluscs

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.     

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.     

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.     

The effect of eucalyptus oil on growth and aflatoxin production by Aspergillus flavus

The effect of eucalyptus oil on growth and aflatoxin production by Aspergillus flavus was tested at three levels, viz . 0·05, 0·1 and 0·2 ml/50 ml SMKY medium. After 6 days of incubation on 0·05 and 0·1 ml supplemented SMKY medium, growth and toxin production were inhibited while at 0·2 ml concentration there was no growth. However, after 12 days of incubation toxin production was greater than the controls.     

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.     

KINETICS OF GROWTH OF HAEMOPOIETIC COLONY CELLS IN AGAR

Cell kinetic parameters of mouse granulocytic and mononuclear cells growing in colonies in agar cultures have been measured. Analysis of flash and continuous labelling studies with ³H-thymidine together with determinations of colony size, growth fraction and mitotic indices, gave the following values for the phases of the cell cycle: G₁= 6·3 1·6 hr, S = 5·8 ± 1·4 hr, G₂= 1·7 ± 0·1 hr and M = 0·7 ± 0·1 hr (42 ± 8 min). No difference in the cell cycle parameters of granulocytic and mononuclear cells were found in this study.
Colonies of different size from cultures of the same age group had similar labelling indices, indicating that the size of a colony is not a function of the rate of proliferation of cells in the colony. Rather, variation in colony size is probably representative of an initial delay in the onset of colony development.     

Biotransformations of paralytic shellfish toxins by bacteria isolated from bivalve molluscs

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.