1. V. Gopal Krishna Bahagian Mikrobiologi Institute Sains Biol;ogi, Fak. Sains, U.M. 8 Feb. 2012

2. Types of preservation techniques Vegetative Silica gel impregnation with skim milk suspension Glycerol stocks Storage under liquid nitrogen Lyophilisation – freeze drying

3. Vegetative stocks Very useful for frequent usage Easy to prepare Fast to transfer Easy to store However, the duration of survival is rather short – days to months only, and Possibility of mutating (changing form)

4. Silica gel impregnation with skim milk suspension Technique that involves the storing of cultures in the form of 10% skim milk suspension absorbed by the granules of non-dyed silica gel granules Granules of silica gel are transferred to small test tubes, cotton plugged and dry-sterilised (180degrees Celcius for 2 hours) The tubes are immersed in ice cubes in a container to avoid heat build-up when the suspension is placed 2 mls of 10% sterilised skim milk (sterilised by wet sterilization – 121 deg. C for 20 minutes) is transferred to a slant of actively growing culture and the cells suspended.

5. Eight to ten drops of the suspended cells are dropped, one drop at a time (to avoid over-heat buid up), onto the ice-immersed tubes of silica granules. The granules are vortexed to break up the granules from aggulitinating and placed back in the ice for a short while The tubes are then stored in a dessicator for a week. At the end of the week, the granules are again vortexed, covered with parafilm and stored at 4deg. C until use. When required, asceptically one or two of the crystals are placed onto a growth medium.

6. Glycerol stock-cultures Cultures are ensured to be actively growing 20% glycerol in 2 ml. Bijou bottles or 1ml. Glycerol in 1.5ml. Microfuge tubes are wet-sterilised (121 deg. C for 20 minutes). The sterile glycerol is poured onto a slant of culture and the cells suspended in the glycerol asceptically. The suspended cells are then re-transfered to the Bijou bottle / Microfuge tube, labeled and stored at 20deg. C or lower. When need to use, the culture is quickly thawed under running water, a few drops transferred asceptically to the growth medium, and the balance kept back.

7. Storage under liquid nitrogen The culture to be stored is prepared as for the glycerol stock, but transferred to a microfuge tube, carefully labelled and transferred to a vertical microfuge holder. The whole holder is immersed into a tank of liquid nitrogen. Liquid nitrogen is replanished regularly in the tank When need to use, one of the tubes is carefully removed from the holder, thawed rapidly by placing under running water and part of the contents placed on a growth medium.

8. Lyophilisation – freeze drying This is the most difficult and the most expensive of the storage techniques but is known to be the most preferred of the storage techniques in view of the longitivity of the survival of the micro-organism. It is the most expensive technique due to the requirement of a special equipment called a freeze-dryer. The tubes too are special in nature

9. Process contd. It is nearly always,with regards to long term storage of cell suspensions, that contain greater than 10 to the power 8 cells ml–1 (Miyamoto-Shinohara et al., 2000; Costa et al., 2000). The reason for preserving high cell concentrations is on the premise that the majority of cells die during long term storage, but a sufficient number survive to enable the continuation of the strain. Bozoglu et al. (1987) suggest that survival of 0.1% of the original cell population is a sufficient number of survivors of freeze drying to allow continuation.

10. Lyophilisation - contd The tube used is as shown aside. A piece of absorbent filter paper is placed inside the tube. The neck is constricted by using flame to avoid the cotton wool to be place after the constriction from dropping into the culture portion – at the base. The cotton wool prevent cross contamination from other cultures, whilst drying as well as when the culture is reconstituted for growth.

11. The process continued The diagram aside shows the tube after it is wet sterilised. An aluminium foil is wrapped over the mouth of the tube to prevent the cotton from getting wet. A suspension of the culture to be lyophalised is made, as for the silica stock. About 2 mls. of the suspension is placed onto the filter paper asceptically, using a pasteur pippette. The tube then is kept at below -40 deg. C to freeze the sample Cotton plug Absorbent filter paper Constriction

12. The process continued The frozen cultures are then loaded onto a freeze dryer, which is pre-set at -40deg. C The drying process commences, where the liquid is directly converted into the gaseous state, by-passing the liquid phase. The freeze-dried samples are then paced in an holder of the lyophilised tube and sealed under vacuum. The tubes are then stored at 4deg. C until further use It must however be noted that the tube can be used only once. As such, several tubes of cultures are prepared at a time.

13. Summary points Among the five methods, the most preferred is the lyophilised form of storage, where 10%skim milk is the medium of storage The results from bacterial strain recovery efforts following hurricanes Katrina and Rita are reported. Over 90% of strains frozen in 10% skim milk were recovered whereas various recovery rates were observed for glycerol-stored stocks (56% and 94% of Escherichia coli, depending upon the laboratory).

14. References Cody L.W, 2008, Skim milk enhances the preservation of thawed 80 °C bacterial stocks Lech, K., Brent, R.,1993. Growth on Solid Media. In: Ausubel, F.M., Brent, R., Kingston, R.E.,Moore, D., Seidman, J.G., Smith, J.A., Struhl, K. (Eds.), Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY. 1.3.5.