Tryptone soy agar

Ethanol, methanol, acetone, acetonitrile, Mueller Hinton broth (MHB), Mueller Hinton agar (MHA), Tryptone soy broth (TSB), Tryptone soy agar (TSA), Sabouraud dextrose broth (SDB), Sabouraud dextrose agar (SDA), Bovine serum albumin (BSA), Phosphate Buffer Saline (PBS), Triton-X 100, p-iodonitrotetrazolium chloride (INT), Thiazolyl Blue Tetrazolium Bromide (MTT), Amphotericin B, ciprofloxacin and Dimethyl sulphoxide (DMSO) were purchased from Sigma Aldrich, South Africa. Gentamicin was purchased from Virbac, New Zealand, Silica gel 60 from Merck, Germany, Minimal essential medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM) and foetal calf serum (FCS) from Highveld Biological, South Africa. Cell lines C3A human hepatocyte (ATCC No. CRL-10741) and human colon (Caucasian colon adenocarcinoma (Caco2)) (ATCC HTB 37) were purchased from America Type Culture Collection, while Vero African green Monkey kidney cells was obtained from the collection of the Department of Veterinary Tropical Diseases, University of Pretoria.

The vaccine strain B. abortus 19 (Shchelkovsky Biokombinat, Moscow oblast, Russia) and the virulent strain B. abortus 544 (obtained from our institute’s collection of microorganisms) were used in this study. The bacterial cells were cultured under aerobic conditions in tryptone soy agar (TSA; Sigma, St. Louis, MO, USA) at 37°C. All experiments with live Brucella were performed in biosafety level 3 facilities. Escherichia coli strain T7 (New England Biolabs, Frankfurt am Main, Deutschland, UK) was used to prepare the recombinant proteins. The E. coli cultures were routinely grown at 37°C in Luria-Bertani (LB) broth or agar which was supplemented, when required, with 100 μg/ml ampicillin.

The commercial strain of Lactiplantibacillus plantarum (International Collection Deposit Number: LMG P-21021, Commercial Code LP01) used in the serum was selected, amplified, and characterized by Probiotical Research S.R.L., Novara, Italy. The strain L. plantarum LP01 was isolated from a healthy human and not exposed to any genetic manipulation.
Two commercial reference strains were selected and amplified (Eurofins BactUp, Saint-Priest, France) DSM 1897 (ATCC6919) a phylotype IA strain of C. acnes isolated from acne-prone facial skin and CIP28.61 (ATCC12228), a strain of S. epidermidis of unknown origin (reference strain for FDA quality control tests). DSM 1897 (ATCC6919 or NCTC 737 (DSMZ)) strain: C. acnes type IA strain isolated from acne-prone facial skin, were grown at 37 °C in COS agar (Biomerieux, Lyon, France) anaerobically for 48–72 h.
Suspensions of OD₆₀₀ 0.5 to 1 were made and counted on solid culture medium. Correlations between OD₆₀₀ and CFU were used to determine the parameters for preparing the inoculum.
Tryptone soy agar was used as a classical isolation medium (Sigma-Aldrich, St Quentin Fallavier, France).

Bacteria Staphylococcus aureus ATCC 29213, Acinetobacter baumannii ATCC BAA 747, and Pseudomonas aeruginosa ATCC 15442, yeasts Candida parapsilosis CBS 8836 and Candida albicans ATCC 90028, and microscopic fungi Aspergillus flavus BTL G-33 and Aspergillus fumigatus BTL G-38 were used in assays. Microorganisms were stored at −70 °C in a freezer in the Laboratory of Biodeterioration Research of the Nature Research Centre (Vilnius, Lithuania). Bacteria for antimicrobial tests were grown on Tryptone Soy Agar (Merck, KGaA, Darmstadt, Germany), and yeasts and fungi were grown on Sabouraud dextrose agar (Merck, KGaA, Darmstadt, Germany). Inoculums were obtained from overnight bacterial cultures grown at 37 ± 1 °C. Yeasts were cultured for 2 days, and fungi were cultured for 4 days at 28 ± 1 °C. The optical density of the microorganism cell suspensions was measured with a spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 530 nm for yeasts and microscopic fungi and at 625 nm for bacteria. The resulting microorganism suspensions were vortexed for 15 s.

A similar volume of samples was primarily homogenized with 225 ml of Listeria enrichment broth (Merck, Germany). Incubation was carried out for 24 h at 37°C. Afterward, a milliliter of samples was transferred to Frazer broth (9 ml) (Merck, Germany). Incubation was carried out for 24 h at 37°C. PALCAM agar (Merck, Germany) and Oxford agar (Merck, Germany) media were used for other enrichment. Incubation was done for 48 h at 35°C. Three black colonies with black sunken were cultured on Tryptone Soy agar (Merck, Germany) containing yeast extract (0.6%). Incubation was carried out for 24 h at 37°C. Listeria monocytogenes was identified by biochemical tests [11 ].

Microbiological control was carried out by seeding the antigen mixture undiluted and 1/10 diluted in different culture media, such as Tryptone Soy Agar (TSA) (Merck, Darmstadt, Germany) for aerobic bacteria; Thioglycolate Broth (TGB) (Merck, Darmstadt, Germany) for strictly anaerobic, facultative anaerobic, and microaerophilic bacteria; and Potato Dextrose Agar (PDA) (Liofilchem, Roseto degli Abruzzi, TE, Italy) for fungi. TSA plates and TGB tubes were incubated for 7 days at 37 °C, and PDA plates were incubated at 25 °C for the same time. All cultures were performed in duplicates. The manipulation was carried out in a biosafety cabinet (AirScience, Fort Myers, FL, USA).