S. pneumoniae serotype 3 (ATCC 6303) infection was performed as previously described (Puchta et al., 2014 (link)). Briefly, glycerol stock was thawed in tryptic soy broth at 37°C, and incubated statically at 37°C/5% CO2 until the culture reached mid-log phase (OD 600 nm = 0.45–0.55). Bacteria were washed twice in sterile PBS and resuspended at an appropriate volume to yield approximately 105 colony forming units (CFU) in 50 μl. Bacterial CFU were verified by culturing serial dilutions of the bacterial inoculate on Tryptone Soya Agar plates with 5% sheep blood (Fisher Scientific) at 37°C/5% CO2 for 18 h. Mice were anesthetized with an intraperitoneal injection of 12.5 mg/kg of ketamine and 1.25 mg/kg of xylazine (Webster Veterinary), and 50 ul of the prepared inoculum or sterile PBS was instilled intranasally. Mice were held vertically for 1 min to assist inoculum draining into the lungs. Blood CFU were determined by plating 10 ul of whole blood on Tryptone Soya Agar plates with 5% sheep blood (Fisher Scientific) at 37°C/5% CO2 for 18 h.
Tryptone soya agar plates
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Tryptone soya agar (TSA) plates are a general-purpose microbiological growth medium. They provide a nutrient-rich environment that supports the growth of a wide range of bacteria and fungi. The agar base is formulated to maintain cell viability and facilitate isolation of colonies.
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4 protocols using tryptone soya agar plates
1
Pneumococcal Serotype 3 Infection Model
2
Pneumococcal Serotype 3 Infection Model
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S. pneumoniae serotype 3 (ATCC 6303) infection was performed as previously described (Puchta et al., 2014 (link)). Briefly, glycerol stock was thawed in tryptic soy broth at 37°C, and incubated statically at 37°C/5% CO2 until the culture reached mid-log phase (OD 600 nm = 0.45–0.55). Bacteria were washed twice in sterile PBS and resuspended at an appropriate volume to yield approximately 105 colony forming units (CFU) in 50 µl. Bacterial CFU were verified by culturing serial dilutions of the bacterial inoculate on Tryptone Soya Agar plates with 5% sheep blood (Fisher Scientific) at 37°C/5% CO2 for 18 h. Mice were anesthetized with an intraperitoneal injection of 12.5 mg/kg of ketamine and 1.25 mg/kg of xylazine (Webster Veterinary), and 50 ul of the prepared inoculum or sterile PBS was instilled intranasally. Mice were held vertically for 1 min to assist inoculum draining into the lungs. Blood CFU were determined by plating 10 ul of whole blood on Tryptone Soya Agar plates with 5% sheep blood (Fisher Scientific) at 37°C/5% CO2 for 18 h.
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S. pneumoniae serotype 3 (ATCC 6303) infection was performed as previously described (Puchta et al., 2014 (link)). Briefly, glycerol stock was thawed in tryptic soy broth at 37°C, and incubated statically at 37°C/5% CO2 until the culture reached mid-log phase (OD 600 nm = 0.45–0.55). Bacteria were washed twice in sterile PBS and resuspended at an appropriate volume to yield approximately 105 colony forming units (CFU) in 50 µl. Bacterial CFU were verified by culturing serial dilutions of the bacterial inoculate on Tryptone Soya Agar plates with 5% sheep blood (Fisher Scientific) at 37°C/5% CO2 for 18 h. Mice were anesthetized with an intraperitoneal injection of 12.5 mg/kg of ketamine and 1.25 mg/kg of xylazine (Webster Veterinary), and 50 ul of the prepared inoculum or sterile PBS was instilled intranasally. Mice were held vertically for 1 min to assist inoculum draining into the lungs. Blood CFU were determined by plating 10 ul of whole blood on Tryptone Soya Agar plates with 5% sheep blood (Fisher Scientific) at 37°C/5% CO2 for 18 h.
3
Airborne Microbial Contamination Monitoring
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Monitoring of airborne contamination was conducted using a Surface Air System Super-180 sieve impactor active air sampler (Cherwell Laboratories, Bicester, UK). The air sampler was situated in the corner of the isolation room, approximately 1–1.5m above the ground, and sampled the air by actively drawing a pre-set volume of air through the sampler. Five-hundred-litre air samples were collected every 15 min over 10-h (08:00–18:00 h) and 24-h (08:00–08:00 h) periods on to non-selective tryptone soya agar (TSA) plates (Oxoid Ltd, Basingstoke, UK), favourable for environmental sampling. An activity log was compiled to record room activity that may correlate with peaks in air contamination. After sampling, TSA plates were incubated at 37°C for 48 h and enumerated. The total number of microbial cfu on each plate was corrected for the statistical probability of multiple particles passing through the same hole by referring to correction tables supplied with the equipment [13] . The probable count was then used to calculate the cfu/m3 sampled using the equation: where V is volume of air sampled, Pr is probable count, and X is cfu per 1 m3 of air.
4
Staphylococcus aureus Infection Model
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A -80°C frozen S. aureus N305 stock was thawed and grown for 6 h in brain heart infusion (BHI) medium (Oxoid) at 37°C and subcultured in the same conditions overnight. The inoculum was centrifuged for 15 min at 3220 × g and washed twice with 10 ml PBS. The number of bacteria was subsequently measured in the inoculum through measurement of OD600nm on a spectrophotometer. Based on these OD results the inoculum was diluted in PBS to the desired number of bacteria. The exact number of CFU of the inoculum was confirmed by plate counts on tryptone soya agar (TSA) plates (Oxoid). An average inoculum dose of 3.45 × 102 CFU/gland was used. All test compounds were injected into the glands at 4 h post-inoculation in a volume of 100 μL.
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