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Lyophilization Technique

Microorganisms can be preserved through three main methods: centrifugation, freeze drying (lyophilization), and cryogenic storage. Centrifugation involves centrifuging a culture and resuspending the pellet in a preservative solution. Freeze drying removes water from frozen microbes under low pressure. Cryogenic storage uses cryoprotectants like glycerol and DMSO before freezing and storing cultures in liquid nitrogen. All methods aim to maintain genetic stability during long term storage. Proper revival techniques rehydrate or thaw preserved cultures for regrowth.

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380 views13 pages

Lyophilization Technique

Microorganisms can be preserved through three main methods: centrifugation, freeze drying (lyophilization), and cryogenic storage. Centrifugation involves centrifuging a culture and resuspending the pellet in a preservative solution. Freeze drying removes water from frozen microbes under low pressure. Cryogenic storage uses cryoprotectants like glycerol and DMSO before freezing and storing cultures in liquid nitrogen. All methods aim to maintain genetic stability during long term storage. Proper revival techniques rehydrate or thaw preserved cultures for regrowth.

Uploaded by

Eva Pa'e O
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Who?

• Viruses, bacteria, fungi, nematodes, yeast, algae, and protozoa, that


are saprophytic, epiphytic, parasitic, antagonistic, and pathogenic

Why?
• To conserve and store the microorganism in order to maintain survival
and genetic stability

What?
• The life duration of a microbial isolate is affected by several factors
such as the microbial characters, composition, and pH of the medium,
aeration, relative humidity, and temperature of the storage.
How?
• Freeze by centrifugation
• Lyophilisation with freeze dry
• Cryogenic with liquid nitrogen

Centrifugation Freeze drying Cryogenic


Centrifugation
Cryo-tube
• Preservative : peptone 1%, skim
milk 1%, Na-glutamate 1%, and
horse-serum + peptone 10%.
Freeze drying
• Freeze drying involve the
removal of water from
products in the frozen state
at extremely low pressure
• The process can be used to
dry product which is
thermo labile and can be
rupture by heat drying
• Potential for long term
stability problem with
liposomal stability
Lyophilization
Microbe culture
Log phase

Centrifugation

Pellet + peptone

Centrifugation

Pellet + preservative solution like


mist desiccant, peptone 1%, skim
milk 1% or Na-glutamate 1%
0,1-0,3 mL into sterilized
ampoule
During revival process the ampoules are decapitate under sterile conditions
Freeze dryer and the dried pellets consisting of cells of the culture are transferred to a
suitable liquid medium and are allowed to dissolve in order to make a
suspension of cells. Then the cells are streaked on to agar plates. 
Revival
Lyophilization
Reagan:
Microbe culture • TSB
Log phase • Sucrose
• BSA
Centrifugation • Distillated water
Filter-sterilize through 0.2µm filter
Pellet + Reagan

Placed into
sterilized vial

Freeze on freezer

Freeze drying
Revival

Rehydrate freeze-dried with


0.3 mL broth medium

Mix well

Pour to 5—6 mL broth Growth medium were needed to


medium maximize the recovery cell

Streak to agar medium


Cryogenic
• Cryoprotectant : glycerol, dimethyl sulfoxide (DMSO), methanol,
saccharide, starch, dan polyvinyl pyrrolidone (PVP).
• Freezing on cryopreservation were done with gradually temperature,
first 0 C, then -40 C and finally at -196 C.
• Rapid cooling will made crystal on intracellular cell and made
unbalance of electrolyte, both will lead to cell lytic.
• During slow cooling, ice forms extracellularly, causing water to
osmotically leave cells, thereby dehydrating them
Cryopreservation
Microbe culture • Place the vials into a pre-cooled (4°C), controlled-rate freeze chamber and
Log phase place the chamber in a mechanical freezer at -70°C (or colder) for at least 24
hours.
Centrifugation • Alternately, use a pre-cooled (4°C) programmable freezer unit set to cool the
vials at -1°C per minute until a temperature below -40°C is achieved and
Pellet + peptone then set the temperature to abruptly drop to -130°C.

Centrifugation
Quickly transfer the vials to liquid
Pellet + preservative solution like nitrogen or a -130°C freezer
mist desiccant, peptone 1%, skim
milk 1% or Na-glutamate 1%

Add DMSO or glycerol

0,1-0,3 mL into sterilized cryotube


Video
Revival
• Remove the vial from the liquid
nitrogen freezer and thaw by gentle
agitation in a 37°C water bath (or a bath
set at the normal growth temperature
for that bacterial strain).
• Thaw the strain rapidly until all ice
crystals have melted (approximately 2
minutes).
• Unscrew the top of the vial and transfer
the entire contents to the prepared
growth medium.
Reference
Machmud, M. 2001. Teknik Penyimpanan dan Pemeliharaan Mikroba.
Buletin AgroBio 4(1):24-32.
ATCC. ATCC guide. 2015.
https://www.atcc.org/~/media/PDFs/Culture%20Guides/ATCC_Bacte
rial_Culture_Guide.ashx
Corning. 2013. Tips for effective preservation.
UGM. 2013. Pelatihan penyimpanan isolate.

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