Acholeplasma oculi Al-Aubaidi 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.
PROCEDURES FOR PROPAGATING MOLLICUTES:
a) Open the vial according to the enclosed instructions.
b) Using a Pasteur or 1.0 ml pipette, withdraw
approximately 0.5 to 1.0 ml from a tube containing
5.0 ml. Rehydrate the entire pellet.
c) Aseptically transfer this aliquot back into the tube. Mix well.
d) Make serial dilutions by transferring 0.5 ml from the
original tube to a tube containing 4.5 ml. Repeat process by transferring 0.5 ml from the second to a third tube, etc. Dilutions are important, not only for titration purposes, but also to keep culture in varying stages of growth. Many strains will die out rapidly once acid or alkaline conditions are reached. It is recommended to prepare several dilutions from the initial tube as the cryoprotectant used in the freeze‑drying process often inhibits growth.
e) Use an uninoculated tube of broth to serve as a control.
f) Plates may be inoculated to check colonial morphology. You can also spot each dilution on the surface of plate (4 or more/plate) to determine the number of colony-forming units. However, not all strains do well on solid medium.
g) Incubate all tubes and plates under the recommended conditions and appropriate temperature. The time necessary for growth will vary from strain to strain. Growth on plates generally requires additional incubation.
h) Depending on the medium used, growth will be indicated by increased turbidity, a color change, or both.
2. Tubes are incubated aerobically, plates are incubated under 5% CO2 or in a candle jar. The incubation temperature is 37oC. Growth will initiate in the first few tubes within 24 hours.
3. For long term storage of Acholeplasma sp., freeze‑drying or freezing is recommended. Liquid nitrogen storage is the best method. Optimally grown cells are centrifuged at 9000 rpm for 30 minutes, the supernatant poured off, and the packed cells resuspended in a smaller amount of #639 broth. To this, add an equal amount of sterile 20% glycerol as a cryoprotectant. This suspension is aliquoted into small plastic vials and stored at ‑70oC or below.
We have found that using a candle jar for CO2 conditions works better for those strains whose medium has an indicator present. CO2 incubators may lower the pH of the medium enough to cause a color change. This change may make it difficult to observe growth with those strains that show little turbidity.
Additional information on this culture is available on the ATCC web site at www.atcc.org.
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Al-Aubaidi JM. Orthographic error in the name Acholeplasma oculusi. Int. J. Syst. Bacteriol. 25: 221, 1975.
Al-Aubaidi JM, et al. Identification and characterization of Acholeplasma oculusi spec. nov. from the eyes of goats with keratoconjunctivitis. Cornell Vet. 63: 117-129, 1973. PubMed: 4631442
Skerman VB, et al. Approved lists of bacterial names. Int J Syst Bacteriol 30: 225-420, 1980.