Effect of heat and acid decontamination treatments on the recovery of Legionella pneumophila from drinking water using two selective media.

Two different decontamination systems, heat and acid, and two isolation media, GVPC and MWY agar were tested for the recovery of Legionella pneumophila from drinking water. The samples were concentrated by filtration through 0.2 micron polyamide filter and the membranes were resuspended in the original water samples. The suspension was divided into three parts: the first was placed in a 50 degrees C water bath, the second was acidified in HCl-KCl solution and the third did not undergo any treatment. The isolation was made by means of media containing charcoal, yeast extract and glycine with cycloeximide (GVPC) or vancomycin, polimixin B, anysomicin and dyes (MWY). Heating at 50 degrees C for 30 minutes was seen to be the best decontamination system above all when used with GVPC agar. Moreover, with this pretreatment higher counts were obtained both on MWY and GVPC agar. The MWY agar produced the highest isolatin percentages and the highest counts.     

Comparison of polymerase chain reaction and conventional culture for the detection of legionellas in hospital water samples

A detection system for Legionella spp. based on the polymerase chain reaction (PCR) was used to assess the diagnostic value of PCR for the surveillance of contamination of man-made water systems by legionellas. A previously-published primer system was chosen to amplify a fragment of the 5S-ribosomal gene of Legionella spp. A total of 78 water samples from various sources were examined by PCR and culture on MWY Legionella selective agar. Fifty-seven of 78 water samples were positive by both test systems (73%), nine were positive by PCR only (11.5%), another nine were positive by culture but negative by PCR (11.5%), and three were negative by both techniques (3.8%). The PCR was inhibited when large amounts of rust were present in the samples. Culture failed to detect legionellas in samples that contained large numbers of other bacteria capable of overgrowing the legionellas. These results show that PCR is a rapid and sensitive technique for the detection of legionella contamination in water samples and that PCR and culture complement each other in monitoring of environmental water samples.     

Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems

Various sample pre-treatment techniques and different growth media for the isolation of Legionellae from hot water supplies in public buildings were compared. A total of 102 hot water samples from taps and showers was examined. The highest recovery frequency was obtained with the heat pre-treatment method and using the selective medium GVPC. However, the results differed according to the concentration of legionellas. In the case of low plate counts (< or =5000 cfu l(-1)), the heat pre-treatment technique gave a significantly higher percentage of positive samples compared with other techniques (P < 0.05). With increasing concentration, the differences between the procedures decreased until they became statistically not significant for concentrations above 50 000 cfu l(-1). The direct inoculum method allowed a significantly higher detection of concentrations (P < 0.001) compared with heat and acid decontamination methods, which brought about a 67-68% reduction in detectable Legionellae. Heat decontamination techniques show greater sensitivity and specificity. However, they underestimate the number of legionellas. In environmental surveillance programmes, this underestimate must be taken into consideration when assessing the health risk.     

Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems

E. LEONI AND P.P. LEGNANI. 2001 . Various sample pre-treatment techniques and different growth media for the isolation of Legionellae from hot water supplies in public buildings were compared. A total of 102 hot water samples from taps and showers was examined. The highest recovery frequency was obtained with the heat pre-treatment method and using the selective medium GVPC. However, the results differed according to the concentration of legionellas. In the case of low plate counts (≤5000 cfu l⁻¹), the heat pre-treatment technique gave a significantly higher percentage of positive samples compared with other techniques ( P  < 0·05). With increasing concentration, the differences between the procedures decreased until they became statistically not significant for concentrations above 50 000 cfu l⁻¹. The direct inoculum method allowed a significantly higher detection of concentrations ( P  < 0·001) compared with heat and acid decontamination methods, which brought about a 67–68% reduction in detectable Legionellae. Heat decontamination techniques show greater sensitivity and specificity. However, they underestimate the number of legionellas. In environmental surveillance programmes, this underestimate must be taken into consideration when assessing the health risk.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

Comparison of contamination and growth of Mycobacterium avium subsp. paratuberculosis on two different media

AIMS: The purpose of the study was to compare the growth of Mycobacterium avium subsp. paratuberculosis (Map) and the degree of contamination on Herrold's egg yolk medium (HEYM) and modified L?wenstein-Jensen medium (LJ). METHODS AND RESULTS: Culture of 2513 faecal samples from dairy cows was performed on each of the two media. The media were read after 5, 8 and 12 weeks of incubation. Overall, the proportion of contaminated samples was significantly higher on LJ (14.2%) than on HEYM (13.2%) after 12 weeks but the degree of contamination was slightly less on LJ. After 8 weeks of incubation, only 1.0% of the samples were Map positive in LJ with 4.9% on HEYM. After 12 weeks of incubation, 3.3% of the samples were Map positive in LJ whereas 6.9% were positive in HEYM. All suspect and culture positive samples were confirmed by IS900 PCR. CONCLUSIONS: HEYM supported growth of Map significantly better and faster than LJ, however it could not be determined conclusively which of the two media that provided the highest degree of decontamination when the incubation time was also included. SIGNIFICANCE AND IMPACT OF THE STUDY: HEYM should be the primary medium rather than LJ for detection of Map in cattle.     

Comparison of Methods for Isolation of Mycobacteria from Water

Twelve methods for the isolation of mycobacteria were compared by applying them in parallel to 26 samples of surface water and 109 samples of treated water. Each method was defined by a particular combination of decontamination method, growth medium, and incubation temperature. For the decontamination of surface water, we used cetylpyridinium chloride (CPC) (30 min, 0.05%), as well as sample preincubation in tryptic soy broth (TSB) followed by decontamination with a cocktail of NaOH, cycloheximide, and malachite green. Treated water was decontaminated with 0.005 and 0.05% CPC (30 min). After enrichment by filtration, all samples were incubated on Lowenstein-Jensen medium (LJ), Ogawa egg yolk medium (OEY), and Ogawa whole-egg medium containing ofloxacin and ethambutol (OEOE) at temperatures of 30 and 37(deg)C. The efficacy of each method was determined by calculating the positivity rate, negativity rate, contamination rate, mean number of mycobacterial colonies grown, and mean number of different mycobacterial strains isolated. The last value was determined by subjecting the isolates to PCR restriction analysis and mycolic acid thin-layer chromatography. Statistical analysis demonstrated that both the TSB method and 0.05% CPC were appropriate for the decontamination of surface water. Decontamination with 0.005% CPC was best for treated water. The results for incubation on LJ were at least equal to those for incubation on OEY and always superior to the results with OEOE. At an incubation temperature of 30(deg)C, all methods achieved higher yields than at 37(deg)C.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification of Aeromonas spp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril-ampicillin-dextrin-ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non-selective medium (TSA) did not show any significant difference ( P 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus , (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.     

Selective isolation of mycobacteria from soil: a statistical analysis approach

We compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: L?wenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 micrograms cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.     

Improving the success of culturing Helicobacter pylori from gastric biopsies

Factors influencing the successful isolation of Helicobacter pylori from human gastric biopsies were studied. Within 24 h, each of the gastric biopsies was inoculated onto chocolate blood agar media and incubated for up to 2 weeks. Among 63 (70%) culture positive cases in 90 patients, 58 (64%) cases were culture positive for both specimens, while five (6%) cases were culture positive in only one biopsy. Of the 63 positive cultures, 51 H. pylori strains (81%) grew on both media with and without antibiotics. Eight strains (13%) grew only on medium without antibiotics, while four isolates (6%) were obtained only from medium with antibiotics. These results support the previous histological observation of patchy colonization of H. pylori in the stomach. The success rate for culture of H. pylori from gastric biopsies increased when two biopsies were taken and inoculated on chocolate blood agar media with and without antibiotics.     

Comparison of culture media and chairside assays for enumerating mutans streptococci

AIM: This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples. METHODS AND RESULTS: When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Cariescreen SM) or on the plastic adherence assay (Dentocult SM Strip mutans). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91-97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay. CONCLUSIONS: Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration.     

Cultural and Biochemical Characteristics of Mycobacterium Paratuberculosis Isolated from Goats in Norway

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant. The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria. Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Fungal culture of musculoskeletal tissue: what’s the point?

There have not been any studies that review the prevalence of fungal isolates using selective media from samples of banked musculoskeletal tissue retrieved from living and cadaveric donors. A total of 2,036 swab and 2,621 biopsy samples of musculoskeletal tissue from tissue banks were received from the 1st August 2008 till 31st December 2010. Routine culture for fungi using selective media with a prolonged incubation period failed to demonstrate a greater prevalence of fungal isolates than by using non-selective culture media alone. Using selective culture fungi were recovered from only two Sabouraud agar plates (0.1%) but not from non-selective media. During the same period fungi were isolated from three graft samples cultured in non-selective broth media only (0.1%). There was no correlation of fungal isolates from selective or non-selective media inoculated at the same time nor from multiple graft samples collected from the same donor supporting the possibility of an exogenous source for fungal isolates rather than an endogenous source.     

Evidence of Legionella pneumophila in some arthropods and related natural aquatic habitats

Abstract Using direct fluorescent antibody (DFA) staining technique, Legionella pneumophila SG 1, 3 and 5 was evident in water samples collected from different localities of central Italian regions, Marche and Abruzzi; L. pneumophila SG 1 and 3 was also detected in aquatic stages of arthropods living in the Legionella -positive waters. Diptera, Coleoptera, Collembola and Isopoda were found to be positive for legionellas by DFA. Diptera, the most common in the waters surveyed, were represented by Chironomidae and Culicidae families, the latter being larval and pupal stages of genus Anopheles and Culex . Mosquito adults, emerged in laboratory from pupae collected in one sample of positive water, were also positive. The findings that aquatic arthropods harbor legionellas and whether they could be involved in the maintenance and dissemination of legionellas in nature are discussed.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30 degrees C under aerobic conditions, and specificity was high (i.e. confirmation rate usually greater than 90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/l.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30°C under aerobic conditions, and specificity was high (i.e. confirmation rate usually <90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/1.     

Performance of a new chromogenic plating medium for the isolation of Listeria monocytogenes from marine environments

AIMS: This study investigated the performance of a new chromogenic plating medium for the detection of Listeria monocytogenes from naturally contaminated samples obtained from marine environments in Morocco in comparison with the conventional plating media PALCAM and Oxford. METHODS: A total of 479 marine samples (sea water, sediment and mussels) were collected from 16 littoral sites in the region of Agadir (western centre of Morocco). They were examined for the presence of L. monocytogenes using a slight modification of the standardized French method (AFNOR V 08-055) for the detection of L. monocytogenes from food and three different isolation media: PALCAM, Oxford and a new chromogenic plating medium. RESULTS AND SIGNIFICANCE OF THE STUDY: The Oxford and the new chromogenic plating media were found relatively more efficient than the PALCAM medium for the isolation of L. monocytogenes (chi-square test, P < 0.05) from marine samples. However, the new chromogenic plating medium was significantly more selective for L. monocytogenes (P < 0.005) than the two other isolation media as 87.5% of the suspect colonies on this medium were indeed confirmed through identification of the isolates vs 12.7% for Oxford and only 3.8% for the PALCAM medium.