Tryptic soy agar plate

Sg strains TX20005, TX20008, TX20030, TX20031, and TX20034 were kindly provided by B. E. Murray, University of Texas Health Science Center, Houston, TX. They were isolated from the blood of endocarditis patients^{29 (link)}. ATCC 43143 was purchased from ATCC. Sg strains and Lactococcus lactis MG1363 were grown at 37 °C in brain-heart infusion (BHI) broth with shaking or on BHI or Tryptic Soy agar plates (Difco Laboratories, Sparks, MD), as previously described^{10 (link)}. Stationary phase bacteria were harvested after 16 hours of culturing. Exponential phase bacteria were obtained by diluting overnight culture into fresh media (1:100) and cultured for 6 hours.
Human colon cancer cells lines HCT116 and HT29 were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA).

Staphylococci strains were recovered from primus isolates taken from patients at INPer. They were conserved frozen (−80  °C in an ILSHIN ultra-freezer). Characterization of staphylococci was carried out by streaking the cultures on tryptic soy agar plates (BD Diagnostic Systems, Germany). Plates were incubated for 18–24 h at 37 ° C to obtain isolated colonies. Individual colonies were used to inoculate tryptic soy broth and grown overnight after which genomic DNA was extracted (see below). Identification of Staphylococcus species was carried out using the VITEK^®2 equipment (bioMérieux SA, 376 Chemin de l‘ Ome. France). Briefly, staphylococci colonies were resuspended in 0.45% saline solution and adjusted to a turbidity of 0.5 in a McFarland nephelometer. The bacterial suspension was transferred into the VITEK^®2 GP ID card testing 64 biochemical properties for gram-positive bacteria, and eight specific tests for SE species (phosphatase and urease production, growth in 6% NaCl, resistance to novobiocin and polymyxin B, arginine hydrolysis, catalase, D-mannitol fermentation). Additional growth characteristics, biochemical (D-mannitol fermentation, coagulase, and catalase), and molecular tests (PCR detection of coa and mecA; see below) were performed at the arrival of the staphylococci collection to the Pathogenicity Laboratory at Faculty of Medicine, UNAM.

For bacteriological diagnosis, liver, and kidney samples from dead goslings were first inoculated onto tryptic soy agar plates (BD Science, MD, USA) containing 2% fetal calf serum, and incubated at 37 °C under an atmosphere with 5% CO₂ for 48 h. Then the spleen, liver, and kidney tissue were pooled and tested for the presence of goose parvovirus^{28 (link)}, goose hemorrhagic polyomavirus^{29 (link)}, AstV^{21 (link)}, reovirus^{30 (link)}, and Tembusu virus^{31 (link)}, respectively.

The Difco/BBL Gram stain kit (BD, Franklin Lakes, NJ) was used for performing the Gram stain procedure. The kit consists of 4 bottles of 1X solutions of crystal violet, Gram’s iodine, acetone-alcohol and safranin. The microscope slides, for use in this study, were prepared using fresh cultures of Staphylococcus aureus strain 6538 (a Gram-positive coccus; ATCC, Manassas, VA) and Klebsiella pneumoniae strain 13882 (a Gram-negative bacillus; ATCC) that had been maintained and grown for 24 h at 37°C on tryptic soy agar plates (BD). These BSL-2 microbes were selected because they are medically important pathogens and relevant to the participants. Smears of each bacterium were made onto separate slides and these were heat-fixed just prior to their use by the study participants. The kit reagents and the microscope slide preparations were examined before being used in the study to ensure their performance accuracy and to provide the expected staining outcomes. The Gram stain procedure was performed following the protocol described in a standard microbiology laboratory manual [11 ].