Propagation of Lyophilised Bacteria

Dr.T.V.Rao MD
The process of lyophilisation is actually an application of a physical phenomenon called sublimation: The transition of a substance from solid to a gaseous state, without first passing through the liquid phase. Application of a high vacuum, warming of the sample while under vacuum which causes water sublimation, driving off excess water through a drying phase, and finally sealing of the sample to prevent water uptake. Lyophilized units can be stored indefinitely at 4C in a refrigerator before opening them, and should be refrigerated if they are not likely to be opened within a few days after their receipt. There is no need to revive such material immediately upon receipt. Most freeze-dried cultures should show some initial signs of growth within 24-48 hours after rehydration and plating 1 Disinfect the ampule by wiping with 70% ethanol. 2. Wrap the scored area with the ethanol-dampened tissue to protect your fingers. The tissue should not be so wet that alcohol enters the ampule. 3. Bend and break the ampule at the narrow, pre-scored area (arrow). The alcohol dampened tissue provides good cushioning and protection against cuts for this step. 4. Aseptically add 0.20.5 ml of sterile water. 5. Using a sterile pipette, gently aspirate the contents several times to mix the suspension thoroughly. 6. Let the suspension to rest for 15-30 minutes. 7. Inoculate the suspension onto an appropriate medium, and incubate

Please Note
Each lyophilised microorganism is not used for more than four passages from the reference culture and which is authentic and traceable. Storage of Microorganisms Fastidious microorganisms have shorter survival periods than aerobic bacteria. They will need to be subcultured every few days. For example, Streptococcus pneumoniae and Neisseria gonorrhoea need to be subculture every third day. Store aerobic bacteria at 2-8C. Exceptions are a few species of Bacillus which remain viable for a longer period when stored at room temperature. Store microorganisms which require CO2 at room temperature in a candle jar or a container with a CO2 packet. Store yeast and fungus at room temperature. Store anaerobes in anaerobic conditions at 2-8C. Store Campylobacter at 35C in Microaerophilic conditions. If the resuscitated culture is stored in conditions other than the 2-8C (e.g. frozen), Microbiologists cannot guarantee the stated characteristics of the product. The common isolates of Staphylococcus, E.coli. Bacilli, Pseudomonas are less demanding in propagation. They can be subcultured on Nutrient agar, which is commonly available in all laboratories.

If the subcultures are worked longer and improperly preserved will attain mutations and many not express all the characters from original strains for academic or research use. After the fourth week, dispose of plates and start the process over with a new lyophilized pellet. *A microorganism may be used beyond expiry date if (one) the lyophilized pellet is grown before expiry date and (two) the microorganism is not used beyond week 4 of the maintenance program. All the Biohazard precautions to be followed like any other human pathogens using Biosafety hood if available *As per Reference: CLSI M22-A3 Vol. 24 No.19, Quality Control for Commercially Prepared Microbiological Culture Media: Approved Standard - Seventh Edition, April 2004. Email doctortvrao@gmail.com