Hathewaya histolytica (Weinberg and Séguin) Lawson and Rainey

1. Open vial according to enclosed instructions. 
2. Under anaerobic conditions, withdraw 0.5 mL of #2107 broth from a single test tube (5 to 6 mL), with a Pasteur or 1.0 mL pipette.  Rehydrate the entire pellet.
3. Aseptically transfer this aliquot back into the broth tube.  Mix well.
4. Use several drops of the suspension to inoculate a pre-reduced tube of #2107 broth and a pre-reduced Brucella blood plate (Anaerobe Systems, AS-111 or AS-141) and a #260 slant. An aerobic blood plate may also be streaked to check for purity.
5. Incubate the tubes and plate under anaerobic conditions at 37°C for 24 to 48 hours. Incubate the purity plate aerobically at 37°C. Turbid growth is evident in the broth at 24 to 48 hours.

ANAEROBIC CONDITIONS:   

Anaerobic conditions for transfer may be obtained by either of the following:

• Use of an anaerobic gas chamber, or                                        
• Placement of test tubes under a gassing cannula system connected to anaerobic gas.   

Anaerobic conditions for incubation may be obtained by any of the following:

• Loose screw caps on test tubes in anaerobic chamber,                       
• Loose screw caps on test tubes in an activated anaerobic gas pack jar, or   
• Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.   

ATCC recommends Brucella Blood Plates from Anaerobe Systems (AS-111 or AS-141).

Always use freshly prepared pre-reduced media or pre-reduced media that has been previously prepared but stored under anaerobic conditions.  Resazurin in the media is a color indicator for anaerobic conditions.  Observance of pink color in medium before use or during incubation shows anaerobic conditions have not been met and oxidation has occurred.  Medium should be discarded.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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