Currently, GVPC is a recommended medium for the detection of
Legionella
spp. in water samples. However, recovery of
Legionella
spp. and selectivity properties can be improved.
In the era of rapid microbiological methods, there is a need to improve long-established culture techniques. Drawbacks of the pour plate method include having to melt each medium separately before seeding.
Chocolate agar (CA) is an enriched medium for the isolation and identification of fastidious bacteria. Defibrinated blood is used to manufacture CA, but this expensive product is not always affordable for companies in developing countries. Blood powder (BP) is potentially a cheaper alternative, although its pre-treatment using autoclaving can impair the quality of the media. Therefore, optimization of BP as a substitute for defibrinated blood for CA manufacture deserves further research. CA was manufactured with irradiated BP (dehydrated bovine blood powder) and its physical and microbiological characteristics were compared with those of conventional CA and CA prepared with autoclaved BP. Each medium was seeded with 20–200 CFU of target bacteria using the spiral pouring method. Finally, another medium was prepared using BP pre-treated by grinding and gamma irradiation and its performance assessed. Compared to conventional CA, the medium containing ground and irradiated BP provided a similar CFU count for both fastidious (Neisseria, Haemophilus, Campylobacter, and Streptococcus) and non-fastidious (Moraxella, Staphylococcus, Enterococcus, Klebsiella, and Pseudomonas) species, unlike the medium prepared with BP subjected only to irradiation, which provided a lower growth of fastidious species. Morphology and characteristics of all bacterial colonies were very similar in conventional CA and the new medium, the number of Pseudomonas CFU being higher in the latter. The medium prepared with ground plus irradiated vs. irradiated BP more closely resembled conventional CA, having a browner background. The new CA medium prepared with ground and gamma irradiation-sterilized BP has comparable productivity properties to conventional CA. Therefore, it could be a more practical and economical methodology to facilitate large-scale CA manufacture.
In the analysis of water samples, the type of filtration membrane material can influence the recovery of Legionella species, although this issue has been poorly investigated. Filtration membranes (0.45 µm) from different materials and manufacturers (numbered as 1, 2, 3, 4, and 5) were compared: mixed cellulose esters (MCEs), nitrocellulose (NC), and polyethersulfone (PES). After membrane filtration of samples, filters were placed directly onto GVPC agar and incubated at 36 ± 2 °C. The highest mean counts of colony-forming units and colony sizes for Legionella pneumophila and Legionella anisa were obtained with PES filters (p < 0.001). All membranes placed on GVPC agar totally inhibited Escherichia coli and Enterococcus faecalis ATCC 19443 and ATCC 29212, whereas only the PES filter from manufacturer 3 (3-PES) totally inhibited Pseudomonas aeruginosa. PES membrane performance also differed according to the manufacturer, with 3-PES providing the best productivity and selectivity. In real water samples, 3-PES also produced a higher Legionella recovery and better inhibition of interfering microorganisms. These results support the use of PES membranes in methods where the filter is placed directly on the culture media and not only in procedures where membrane filtration is followed by a washing step (according to ISO 11731:2017).
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