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There is currently increased interest in the use of alternatives to autoclaved culture media, in order to maintain the properties of the media, while saving energy and time. In this study, we assess a new system for culture media preparation, using a conventional microwave with a water bath and a glass bottle with a rubber cap that allows depressurization. Sterilization, using the proposed system (1000 W, 3 to 20 min), was compared with autoclaving for the preparation of tryptone soy agar (TSA), tryptone soy broth (TSB), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). Microwave exposure for 7 min yielded sterile TSA plates. The productivity of both sterilization methods was assessed using the pour plate method, and significant increases in the growth of certain micro-organisms after using a microwave were observed for every culture medium, especially those that were sterilized by boiling (VRBG). The kinetics of microbial destruction showed that Escherichia coli and Bacillus subtilis spores were destroyed after 3 and 7 min in a microwave, respectively, while three decimal reductions were obtained for Geobacillus stearothermophilus spores after 15 min in an autoclave. This new sterilization method could be a feasible, rapid, and economical method to prepare microbiological media, with a quality similar to that obtained through autoclaving.

ABSTRACT Brucellosis, a re-emerging zoonotic infection, threatens animal welfare and public health with serious economic consequences. A definitive diagnosis requires Brucella isolation by culturing field specimens in specific media. This study aimed to (i) assess the effectivity of recommended Farrell’s médium (FM) and CITA medium (CM) for the isolation of four Brucella melitensis strains (16M, Rev1, and the 16MΔwzm and Rev1Δwzm in-frame deletion mutants) with variable susceptibility to polymyxins; (ii) develop a Brucella selective medium (BSM) suitable for these strains; (iii) test BSM, FM, and CM with other Brucella species; and (iv) develop an improved selective culture medium (BruSIC) for all brucellae, including B. abortus bv1. The four B. melitensis strains were strongly inhibited in FM and (except Rev1) CM. Since Rev1Δwzm’s CM inhibition was due to a synergistic effect of colistin and vancomycin, we formulated BSM with half the concentrations of both antibiotics, achieving a similar growth of B. melitensis to blood agar base (BAB) and an inhibition of contaminant microorganisms comparable to CM; CM performance was surpassed by BSM for the primary isolation of B. melitensis when tested in 1,789 real sheep samples. For other brucellae, BSM and CM were more inhibitory than FM for B. abortus bv1 when using plates immediately after preparation but not after ≥4 weeks of storage. To address this, we developed the improved solid medium BruSIC by replacing the calf serum in BSM with activated charcoal. BruSIC yielded faster colony growth than BSM and CM and similar CFU numbers than BAB (including for B. ovis in BAB-Serum) and inhibited accompanying microorganisms in sheep and cow samples as effectively as BSM. IMPORTANCE Farrell’s medium (FM) and CITA medium (CM), recommended for Brucella isolation in animal samples, are inhibitory for certain strains. A reformulated Brucella selective medium (BSM), containing half the CM vancomycin and colistin concentrations, improved the isolation of B. melitensis, but not Brucella abortus bv1. A novel Brucella selective culture medium (BruSIC), in which calf serum is replaced by activated charcoal, retains the selectivity and improves the productivity of BSM and CM. BruSIC allows the growth of all brucellae faster than in CM or BSM, and at CFU number equivalent to BAB supplemented by calf serum, including B. abortus bv1 and the serum-dependent Brucella ovis. Due to its performance and reduced cost, BruSIC represents an added-value alternative to the existing selective culture media for these bacteria.

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).

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.

ABSTRACT Glycine-vancomycin-polymyxin-cycloheximide agar (GVPC) is a recommended medium for the detection of Legionella spp. in water samples. However, its quality could be improved in terms of recovery of Legionella spp. and selectivity properties. Modifications were introduced in GVPC manufacture: autoclaving conditions (115°C, 15 min) and atmosphere during component-stirring (removal of oxygen and N2 injection). The use of softer autoclaving conditions (115°C, 15 min) improved the growth of Legionella anisa by the spiral method and Legionella pneumophila after membrane filtration. The medium manufactured with O2 removal and autoclaving for 15 min at 115°C allowed a faster growth of L. pneumophila (colonies visible at day 2) and a notable increase of L. anisa growth (colonies appearing at day 3, and statistically significant numbers of CFU at day 5). After 3 to 5 days of incubation, the improved media showed higher selectivity properties, particularly for Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 9027. A further improvement was achieved by the addition of N2 during ingredient stirring, leading to a statistically significant faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3. Selectivity properties were also enhanced, resulting in the complete inhibition of both E. faecalis strains and Escherichia coli and complete-partial inhibition of P. aeruginosa. Oxygen removal during GVPC manufacture using a vacuum pump system promotes the growth of L. pneumophila and L. anisa, and markedly inhibits the growth of E. coli, P. aeruginosa, and E. faecalis. IMPORTANCE 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. GVPC medium manufactured without oxygen improved the growth of Legionella pneumophila and Legionella anisa. Oxygen removal during GVPC manufacture also improved selectivity properties. A further improvement was achieved by the addition of N2 during ingredient stirring, leading to a faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3 and enhancement of selectivity properties. The introduction of the modified GVPC medium in routine practice can allow a better detection of Legionella spp. in water samples.