Nimmy Francis I M.

Sc Microbiology

Storage Of Microorganisms
PURPOSE

LONG TERM STORAGE

SHORT TERM STORAGE

DEEP FREEZING

FREEZE DRYING

REFRIGERATION

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Short Term Storage
Microbial cultures are maintained in 1. Nutrient broth 2. Agar plates 3. Agar slants These cultures are preserved for further use in Refrigerator
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Nutrient Broth

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Agar slants However it is difficult to determine whether a culture growing on an agar slant is contaminated or not. 28-04-2012 6 .

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Agar deeps/ stabs  Cultures can be stored as stabs in small. flat- bottomed screw capped vials. but they will loss viability more quickly than frozen cultures 28-04-2012 9 .  Cultures stored in stabs are more resistant to drying and contamination.

Cultures streaked on agar slants or stab cultures may be viable over several months when stored at 4°C.  Plates have to be sealed to prevent their tendency to dry out.  Can be used for short-term storage. 28-04-2012 10 .

 The plates and tubes to be stored should be covered well with parafilms or screw caps.  Sealing the plates not only prevent molds from sneaking into the plates. 28-04-2012 11 . but it slows the agar from drying.

Overlaying With Mineral Oil The bacterial and fungal cultures can be covered over the fresh agar slants with sterile mineral oil.  The method is very simple and cell viability is high compared to frequent transfer and storage at low temperature.  28-04-2012 12 .  The oil must be above the tip of slanted surface.

 Some microorganisms have been preserved satisfactorily for more than 15-20 years by this method. Mineral oil covered cultures are preserved preferably at 0-50 C . 28-04-2012 13 .

28-04-2012 14 .Long Term Storage  Two principles  Desiccation  Low temperature storage  Deep Freezing and Freeze Drying are the two common long term storage methods.

 As water is converted into ice. 28-04-2012 15 . Freezing is a good way to store bacteria. the colder the storage temperature.  Ice can damage cells by dehydration caused by localized salt concentration. solutes accumulate in the residual free water and this high concentration of solutes can denaturate biomolecules. Generally. the longer the culture will retain viable cells.

28-04-2012 16 .  Cultures can be thawed and used up to several years later. which acts as an "antifreeze". A pure culture of bacteria is suspended in a liquid and quick-frozen (often with liquid nitrogen) at temperatures between -50°C and-95°C.  Sensitive microorganisms require the presence of glycerol (end concentration 15-20 %). or extra protein (skimmed milk powder) to protect them.

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Cryoprotectants  Glycerol  Skim milk  Dimethyl sulfoxide 28-04-2012 18 .

and then transfer the tube to -700C for long term storage and -200 C for short term storage 28-04-2012 19 .85 ml) are vortexes to ensure that the glycerol is evenly dispersed   Cultures are kept in labeled screw cap tubes Freeze the culture in dry ice or in liquid nitrogen.Glycerol storage of Bacterial cultures  Sterilized Glycerol (0.15 ml) added bacterial cultures (0.

2 ml of DMSO to the labeled tube  Use another sterile pipette and transfer 1 ml of broth culture to the tube with DMSO   Invert the tubes several times to mix Place the tube in the -200C freezer 28-04-2012 20 .Dimethyl Sulfoxide storage   Label sterile tubes with strain and the date Use a sterile pipette to transfer 0.

5 ml of an 18-24 hour pure broth culture into the skim milk media. dispense 0. suspend 3-4 well isolated colonies in the skim milk 28-04-2012 21 .  Freeze and maintain the culture preferably at 700C  To inoculate from a solid media.Skim Milk Storage  To inoculate from a broth .

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 The microbes survive in these powder like residue for several years and can be revived at any time by rehydration of the culture in a nutrient medium.  Bacterial strains ordered from strain collections are usually delivered in this form. 28-04-2012 23 .Lyophilization  A suspension of bacteria is quickly frozen and the water is removed by means of a high vacuum.

Schematic of a Laboratory Freeze Dryer with Shell Frozen Flasks Attached to a Manifold VACUUM GAUGE COOLING COILS ICE CONDENSER VAPOR CONDENSE S ON WALL AS ICE SHELL-FROZEN FLASKS VACUUM GAUGE VACUUM PUMP DRAIN .

Fundamental processing steps  Freezing  Vacuum  Sublimation  condensation 28-04-2012 25 .

 Freezing: ◦ The product exhibits the desired crystalline structure ◦ The product is frozen below its eutectic temperature  Vacuum : ◦ Enables the frozen solvent in the microorganism to vaporize without passing through the liquid phase 28-04-2012 26 .

 Heating ◦ Accelerate sublimation  Condensation ◦ The low temperature condenser plates remove the vapourized solvent from the vacuum chamber by converting it back to solid ◦ It completes separation process 28-04-2012 27 .

PHASE DIAGRAM 28-04-2012 28 .

Shell Freezer Freeze Dryer .

stabilizes the cells when water is ◦ Matrix agents. BSA . ◦ Mannitol. allows the entire sample to retain its shape during and processing..Freeze Drying Protocol For Bacteria Lyophilization medium ◦ Lyoprotectants. 28-04-2012 30 . Serum etc. removed.

5ml) to the labeled. If necessary.Protocol  Grow an overnight culture or lawn of the microorganism on LB or other appropriate nutrient agar plate  Add 4ml of Lyophilization buffer to the plate.  Quickly transfer the culture suspension (~1. the cells can be suspended using a sterile glass rod. sterilized vials 28-04-2012 31 .

 Carefully and aseptically place the vial caps loosely on top of the vials. Freeze the culture suspension inside the vials by placing the vials in a -200C freezer. so moisture can escape during the freeze drying process.  Apply vacuum to the chamber according to the preservation conditions. 28-04-2012 32 . and place the vials into a freeze drier chamber.

depending on the volume of each sample. tubes to completely  This may take anywhere from a couple hours to overnight. Allow the culture lyophilize/ dry out. Remove the samples from the freeze drier chamber and immediately seal the vials with the rubber caps Store the lyophilized culture collection at room temperature 28-04-2012 33   .

Storage In Sterile Soil  For preserving spore forming bacteria and fungi  Spore suspensions are added to sterile soil and the mixture is dried at room temperature and stored in a refrigerator  Bacterial cultures maintained by this method have been remained for 70-80 years 28-04-2012 34 .

cooled silica powder is mixed with a thick suspension of cells. heat sterilized. mixed and stored at low temperature  The basic principle in this technique is the quick desiccation at low temperature which allows the cell to remain viable for a long time 28-04-2012 35 .Storage in silica gel   Bacteria and yeast Finely powdered.

Anaerobic storage 28-04-2012 36 .

Candle Jar 28-04-2012 37 .

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THYOGLYCOLATE BROTH 28-04-2012 39 .

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semisolid. 28-04-2012 44 .  Transport media are formulated to maintain the viability of microorganisms. without significant increase in growth. non nutritive phosphate buffered media that provide a reduced environment.Transport media  Transport media are chemically defined.

 1948: Moffett.4  This modified medium is effective to maintain the viability of Salmonella and Shigella 28-04-2012 45 . Young and Stuart  Transport medium for gonococcal specimen Stuart. Toshach and Pastula  modified the medium and is known as Stuart’s Transport Medium   1964: Cary and Blair modified by adding inorganic phosphate for glycerophosphate and raising pH to 8.

Stuart Transport Medium Sodium Thioglycolate Sodium Glycerophosphate Calcium Chloride Methylene Blue Agar : 1.00 g : 10.00 g : 0.00 mg 28-04-2012 46 .10 g : 2.00 mg : 3.

 Label the tubes.Protocol  Obtain specimen with sterile swabs. Insert specimen swabs into the upper third of the medium in transport container.  Cut with sterile scissors or break off the protruding portion of the swab stick. 28-04-2012 47 . submit to laboratory with minimum delay. Tightly screw the lid on the bottle or vial.

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00 g : 1.00 g : 0.Sodium Thioglycolate Sodium Phosphate Basic Sodium Chloride Potassium Chloride Potassium Monophosphate Basic Magnesium Sulfate L Cysteine Resazurin Agar 28-04-2012 : 1.20 g : 0.00 g 49 .20 g : 0.15 g : 3.00 g : 1.00 mg : 4.10 g : 1.

10 mg of Streptomycin.000 U of Penicillin.  Poly vinyl alcohol based preservatives are used for the fixation of ova and parasites in clinical specimens.2 mg of Chloramphenicol – per ml of specimen to ensure the recovery of fungi.Preservatives  50. 0. 28-04-2012 50 .

.edu/~microbes  http://www.umsl. page no: 170  Prescott. page no: 862  www.Reference Introduction to Microbiology.anaerobesystems.. Harley and Klein's Microbiology. 7th ed. Tortora and Funke.com  28-04-2012 51 . 10th ed.

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