Slide 1: MAEER’S, MAHARASHTRA INSTITUTE OF PHARMACY, PUNE-38. PRESERVATION OF CULTURES Prof. S. A. Polshettiwar M. Pharm, D.I.T., PhD PRESERVATION OF CULTURES INTRODUCTION: INTRODUCTION To maintain isolated pure cultures for extended periods in a viable condition, without any genetic change is referred as Preservation . Slide 3: During preservation, most important factor is to stop the microbial growth or at least lower the growth rate. Due to this, toxic chemicals are not accumulated and hence viability of organism is not affected. The less number of generation reduces the mutation frequency. OBJECTIVES OF PRESERVATION: OBJECTIVES OF PRESERVATION To maintain isolated pure cultures for extended periods in a viable condition. To avoid the contamination To restrict Genetic change (Mutation) Preserved by all microbiology lab., fermentation industry and culture collection centre. Necessities of Preservation: Necessities of Preservation Academic Use Research Purpose Fermentation Industry Biotechnological Field List of Authentic collection centres: List of Authentic collection centres American Type Culture Collection( ATCC), U.S.A National Collection Of Industrial Bacteria ( NCIB), Scotland National Chemical Laboratory (NCL) Pune, India. Microbial type culture collection (MTCC), IMT, Chandigarh, India Significance of Authentic collection centres: Significance of Authentic collection centres To maintain isolated pure cultures for extended periods in a viable condition. The microbial species isolated & characterized by microbiologist are deposited in centre. It provide culture throughout the world to help microbiologist to obtain culture for various studies. Significance of Authentic collection centres: Significance of Authentic collection centres They also help to classify new isolated organism They provide industrially important organism to carry out the fermentation process. For vaccine preparation the specific strain of attenuated microorganism ie required, such demand is fulfilled by culture collection centre. METHODS OF PRESERVATION: Continuous metabolism ( metabolism is not completely stopped but continues in slow rate) Periodic transfer to fresh media ( Sub culturing) Storage in Sterile Soil Saline Suspension Oil Overlay METHODS OF PRESERVATION METHODS OF PRESERVATION: Suspended Metabolism Storage at low temperature Lyophilization or Freeze drying Storage in Silica Gel METHODS OF PRESERVATION Slide 11: 1.Periodic transfer to fresh media Microbes are preserved on agar slants & stored in a refrigerator. These cultures are periodically transferred to fresh media. Advantages:: Advantages: It is simple method any special apparatus are not required. Easy to recover the culture Disadvantages: Risk of contamination is more It may be possible to change in genetic & biochemical characteristic. 2. Storage at low temperature or Freezing : 2. Storage at low temperature or Freezing Freezing is a good way to store bacteria. Generally, the colder the storage temperature, the longer the culture will retain viable cells. Freezers can be split into three categories: laboratory, ultralow, and cryogenicCulture is maintained at 4 0 c in refrigerator or cold rooms or liquid nitrogen is provided for long term preservation of cultures. M.O. are prepared as a dense suspension in a medium containing a protective agent( Glycerol or DMSO), which prevents cell damage due to crystal formation. Then it is sealed into ampoules or vial & frozen at controlled rate -150 0 c then stored in liquid nitrogen refrigerator (-196 0 c). Slide 14: Laboratory freezers are those that can pull temperatures down to -20 to -40°C. These are single stage systems (one compressor) and often called general purpose freezers. Bacteria can be stored for moderate periods of time, e.g., 1 year, in general purpose freezers. It is best to use freezers without frost-free temperature cycling as this can wreak havoc on cells and other temperature sensitive biomolecules. General purpose freezers are inexpensive and found in most labs, thus they are readily available for storing cultures. The downside is that they are not sufficiently cold for long-term storage. Ultralow freezers are two stage systems (two compressors each having a different refrigerant) which pull down to around -86°C. Ultralow freezers are very prevalent, but space in them can sometimes be limited and competitive. Ultralow freezers also are much more expensive to purchase, run and maintain. The upside is that cells stored at -80°C tend to remain viable for several years. The lower temperature generated by ultralow freezers substantially reduces chemical reactions within the culture. However, molecular motion still occurs in frozen cells and thus the viability of the culture will decline. It is important to regularly monitor cultures to assess their level of viability. Cryogenic freezers are very cold and rely on liquid nitrogen or specialized mechanical systems to operate. For biological samples, cryogenic storage should be below -130°C. At this temperature, the molecular motion of water is halted and cells are trapped in a glass-like matrix. Bacteria stored in cryogenic freezers retain their viability for many years. In our laboratory bacterial and yeast cultures have been maintained at -140°C for 15 years without significant loss of viability. Storing cells in cryogenic freezers is the most effective and, as compared to freeze drying, the easiest method for long-term storage. The downside is cost and potential vulnerability of stocks to power outages, mechanical failures, and failed deliveries of liquid nitrogen. Additionally, tubes should never be stored in tanks submersed in liquid nitrogen. Screw cap tubes leak and will pull the nitrogen into the tube along with contaminants ( see link for more information ). Liquid nitrogen vapor phase freezers will effectively avoid this problem, but these freezers are very expensive (upwards of $10K) and require large volumes of liquid nitrogen. An alternative is mechanical cryogenic freezers that can go as low as -150°C, but these are also very expensive to purchase (about $20K). Both cryogenic freezers will cost several hundred dollars a month to operate Advantages:: Advantages: It has been successfully used for many species & cell remain viable for 10 to 30 yrs without changing their characteristic. Slide 16: 3.Storage in Sterile Soil This method is manly applied for the preservation of sporulating microorganism e.g. bacillus, streptomyces, aspirigilus, clostridium, penicillium species etc Storage at low temperature: Storage at low temperature 4. Oil Overly Method: 4. Oil Overly Method Method is similar to periodic transfer method, only difference is that it makes use of paraffin oil. Many bacterial species can preserved by covering the growth on the agar slant with sterile mineral oil or liquid paraffin. Oil must cover the slant completely. Oil reduces the loss of water & also slow down the exchange of gas within organism & its surrounding. : Advantages: It is cost effective method of preserving cultures of bacteria & fungi for 15 to 20 yrs but changes still occurs. It is highly recommended for anaerobic organisms Disadvantages: Some organism loose their sporulating ability Genetic & biochemical characteristic of microbes changes during periodic transfer of culture. Slide 20: A process used for preserving biological material, by removing the water from the sample, which involves first freezing the sample and then drying it, under a vacuum, at very low temperatures. Freeze drying can preserve different types of M.O. that would be killed by ordinary drying. A dense cell suspension is placed in small vials & frozen at -60 to -78 0 c . The vials are immediately connected to high vacuum. The ice present in the frozen suspension evaporates (sublimes) under vaccume. Result in dehydration of the bacteria with minimum of damage to delicate cell structures. The vials are then sealed off under vaccume & stored in a refrigerator. 5. Lyophilization or Freeze drying Method : Method Grow an overnight culture, or lawn, of the microorganism on LB or other appropriate nutrient agar plate. Prepare sterile crimp-cap vials by autoclaving ahead of time, with the caps (rubber stopper) placed loosely on top. Place paper labels printed with the culture identification inside the tubes prior to autoclaving. Alternatively, use tubes with caps designed for sterility . Add 4 mL lyophilization buffer to the plate. If necessary, the cells can be suspended using a sterile glass rod. Quickly transfer the culture suspension to the sterilized vials. Add approximately 1.5 mL per vial. Seal with the rubber cap. Freeze the culture suspension inside the vials by placing the vials in a -20 degrees Celcius freezer. Once the cultures are frozen, prepare the freeze drier by turning it on and allowing time for the appropriate temperature and vaccum conditions to stabilize. Of course, this is done according the manufacturer's instructions for the particular brand of freeze drier you are using. Carefully and asceptically place the vial caps loosely on top of the vials, so moisture can escape during the freeze drying process, and place the vials into a freeze drier chamber. Apply the vaccum to the chamber according to the manufacturer's instructions. Allow the culture time to completely lyophilize (dry out). This may take anywhere from a couple hours to overnight, depending on the volume of each sample and how many samples you have. Remove the samples from the freeze drier chamber according to the manufacturer's instructions, and immediately seal the vials with the rubber caps, then the crimp tops. Store the lyophilized culture collection at room temperature. Slide 22:
Advantages: Culture viable for more than 30 yrs Sub culturing is not required Maintain the culture without contamination. Remain Genetically Stable Minimal storage space required Easy
Employed for the preservation of sera, toxins, enzymes & other biologicals.
Slide 23: EXPECTED QUESTION Write the different technique used for preservation of cultures? Lyophilization cycle: variables: Lyophilization cycle: variables