Ureaplasma parvum Robertson et al.

1. Follow instructions as suggested for the culturing of Mollicutes:

PROCEDURES FOR PROPAGATING MOLLICUTES:

1. Open the thawed vial according to the enclosed instructions. 
2. Make serial dilutions by transferring the entire contents of the vial to a test tube containing 4.5 mL of appropriate broth. Repeat process by transferring 0.5 mL from the second to third tube, etc.
3. Use an uninoculated tube of broth to serve as a control
4. Incubate all tubes under the recommended conditions and appropriate temperature (see step 2). The time necessary for growth will vary from strain to strain. Growth in broth for this strain will generally be observed between 3 to 7 days. 
5. Depending on the medium used, growth will be indicated by increased turbidity, a color change, or both (see notes section). 
6. Inoculate a Trypticase Soy Agar with 5% Defibrinated Sheep Blood plate with 0.1 mL to check for contamination. Incubate plate at 37°C. No growth should occur on Trypticase Soy Agar with 5% Defibrinated Sheep Blood. 

2. Tubes may be incubated aerobically. The incubation temperature is 37°C. 

This strain requires an additional 5-10% horse serum (Thermo-Fisher, 16050-122) added to the broth for growth.

Ureaplasma strains grow very rapidly.  The indicator in the first tube will change from yellow to green within hours.  It is especially important to make serial dilutions of this strain. When alkaline conditions are reached (as indicated by the color change), the culture will rapidly die out unless refrigerated immediately (+4°C) or stored frozen at -80°C.  Refrigerated broth cultures may remain viable for periods up to 30 days.  No visible turbidity will be seen; therefore, the color change is the only indication of growth.  Transfer, freeze, or lyophilize the culture as soon as possible. 

Store vials at freezer temperatures until ready to use.

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