Fibrobacter intestinalis Montgomery et al.
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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.
3. Aseptically transfer this aliquot back into the broth. Additional tubes may be inoculated with 0.5 ml each from the suspension. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC to check for aerobic contamination.
4. Incubate tubes under an anaerobic atmosphere at 37oC.
5. Within 48 hours, growth should be evident by turbidity and smooth sediment in the broth. No growth should occur on the agar plate incubated aerobically.
Anaerobic conditions for transfer may be obtained by any of the following:
· Use of an anaerobic gas chamber,
· Placement of test tubes under a gassing cannula system hooked to anaerobic gas, or
· Transfer from one stoppered anaerobic test tube to another using a pre-reduced syringe.
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.
Always use freshly prepared pre-reduced media or pre-reduced media that has been stored under anaerobic conditions.
Resazurin is a commonly used redox indicator that is pink when the redox potential is above -50 mV and colorless when the redox potential is below 110 mV, i.e. highly reducing. Most strict anaerobes require this low redox potential for growth, i.e. will not grow if resazurin is pink.
Pre-reduce syringes to be used for culture transfer by flushing the syringe with oxygen-free gas or by flushing with a reducing agent just prior to use.
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Montgomery L, et al. Transfer of Bacteroides succinogenes (Hungate) to Fibrobacter gen. nov. as Fibrobacter succinogenes comb. nov. and description of Fibrobacter intestinalis sp. nov.. Int. J. Syst. Bacteriol. 38: 430-435, 1988.