This protocol describes the preparation of Group A streptococci and the isolation and fragmentation of 10S streptococcal cell wall peptidoglycan-polysaccharide polymers (SCW PG-PS) to be used for inducing SCW arthritis.
Streptococcus pyogenes, Group A, D58 strain, (ATCC #43202) Todd Hewett broth (Difco), pH 6.8 to 7.0, supplemented with 3 g dextrose/liter, autoclaved
0.006 M PBS, pH 7.0: dilute PBS, pH 7.0 (appendix2) with H2O (final NaCl concentration 0.006 M) Dry ice/ethanol bath 2% (w/v) SDS in 0.006 M PBS, pH 7.0 2 M potassium chloride 0.85% NaCl
Appropriately sized bacterial culture tubes and flasks Blood agar plates (Difco) 60°C water bath
Sonicator: Branson Sonifier 350 (VWR) or equivalent
Mid-speed centrifuge and rotors (e.g., Sorvall, RC-5B with SS-34 or GSA rotors) 0.45-pm filters
Additional reagents and equipment for measuring rhamnose concentrations in cell wall pellet (see Support Protocol 2)
CAUTION: Since Group A streptococci are human pathogens, precautions must be taken to avoid infecting oneself or others. Perform all sonications in a containment cabinet to prevent inhalation of aerosolized bacterial products.
Grow streptococcal culture
1. Inoculate 10 ml Todd Hewett broth with a thawed vial of Group A streptococci and grow overnight at 37°C without shaking.
2. Streak culture on blood agar plates and incubate 24 hr at 37°C. Examine for white colonies surrounded by areas of hemolysis to confirm purity of culture. Inoculate 10 ml of Todd Hewett broth with a single colony and culture overnight at 37°C without shaking.
3. Inoculate large Todd Hewett broth cultures with bacteria from small broth cultures using 1 ml of inoculum per liter.
The amount of broth inoculated for the large cultures depends on the experimental objectives. The bacteria may be grown in single large fermentation (750 to 1000 liters), or many small cultures (2 to 4 liters) may be pooled. Yields are usually 2 to 3 g bacteria per liter of broth.
4. Incubate culture, without aeration or stirring, at 37°C until late log-phase/early stationary-phase of growth (as demonstrated by a plot of OD600 versus time).
Animal Models for Autoimmune and
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