Media Fill of Anaerobic Roll Tubes

Media Fill of Anaerobic Roll Tubes

This a view of a technician filling anaerobic roll tubes with a cannula system that both dispenses an aliquot of aqueous media and gasses the tube with a gentle flow of O2 free CO2 at the same time.

Notice the agar has already been dispensed into each tube. The technician is wearing a chain-mail glove to protect the hand from cuts (in case the tube breaks) when tightening in the rubber stopper while tubing media. Next to the wall is a pH meter used to adjust pH during media preparation. A reservoir of freshly prepared peptone yeast extract medium, containing cysteine and resazurin oxygen indicator, is to the left of the pH meter. A foot pedal with cable attached to a wooden arm is pushed down to cause a Cornwall Syringe to draw a preset aliquot of medium from the reservoir and add it each tube as needed.

The next step is to secure the tubes in a large clamp assembly to prevent the stoppers from popping out during autoclaving. They are then autoclaved at 121oC for 25 minutes. Any tubes that are pink (resazurin indicator) after autoclaving indicate O2 is present and are discarded (37, 98, 124).


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Serial Dilutions of Anaerobes

Serial Dilutions of Anaerobes

Pictured is a set of roll tubes used in enumeration of an anaerobic microorganism in a broth culture. After the agar in the tubes has cooled to about 40 oC, a measured amount of the culture (usually 1 ml), to be enumerated, is added to a roll tube using the anaerobic culture apparatus. The Roll Tube is then stoppered and gently tipped back and forth for mixing, then slowly turned/rolled by hand under cool running water until it hardens as a thin layer all around the inside of the tube.

After appropriate incubation time and temperature, the tube is placed in the tube spinner and an ink felt marker is run up the side causing a spiral marking to allow counting of colonies. Then one simply rotates the tube slowly by hand, visually following the "track" or path and counts colonies, usually clicking a hand counter/adder. This gives an estimate of "colony forming units" (CFU), an indication of the population density of microorganism in the broth culture at the time of sampling. In the photo at right, three tubes containing Peptococcus constellatus are shown with increasing 10 fold dilution at each step. The first two tubes have colonies too numerous to count, but the tube farthest to the right is usable (37, 98, 124).



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Craig Bremmon
CEBTech Services
503 11 th Ave. SE, Suite 5
Aberdeen, SD 57401

bremm001@nvc.net
Phone: 605 226-3595
Mobile: 605 377-4062
Fax: 605 226-3595