Clostridium longisporum Hungate
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
2. Under anaerobic conditions, withdraw 0.5 ml of the recommended broth from a single test tube (5 to 6 ml) and rehydrate the vial contents.
Additional tubes of broth may be inoculated with 0.5 ml each from the suspension, and 0.1 ml may be inoculated onto a slant. Streak several blood plates to check for colonial morphology and purity.
Incubate one agar plate anaerobically for colony formation, and one aerobically as a purity check.
5. Within 24-48 hours, growth should be evident by turbidity in the broth tube with slight gas formation. The anaerobic plate will show colonies that are entire, smooth, glistening, low convex, and opaque. No growth should occur on agar plates incubated aerobically.
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 hooked 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.
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