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.