The document discusses various methods for preserving bacterial and fungal strains. It describes preservation techniques such as serial transfer, preservation in distilled water, under oil, lyophilization, on silica gel, paper, beads and soil. It also discusses cryopreservation techniques like storing agar plugs or cell suspensions in liquid nitrogen. The goals of preservation are to maintain culture productivity, genetic purity and biochemical properties over long periods of storage and transportation. The document provides detailed protocols for various preservation methods.

1. THE PRESERVATION OF BACTERIAL AND FUNGAL STRAINS
Dr. Ishan Y. Pandya (PhD, Biotechnology)
E-mail: genomes.world37@gmail.com
Senior Faculty and Academic Head,
Guide: NEET Medical to CSIR-NET Competitive
Exams, and KVPY-IISC Exam

2. WHAT IS PRESERVATION?
Storing strains under suitable conditions
so that they can be used in future.

3. Object of preservation
Demonstration of specific
properties
Production strains
Comparison studies
Reference
Research

4. Goals of preservation
 Preservation of culture near its initial
productivity.
 Preserve genetic purity without loss
of biochemical properties.
 Easy transportation & handling.

5. METHODS
1. CULTURE PRESERVATION.
• SERIAL TRANSFER
• PRESERVATION IN D/W
• PRESERVATION UNDER OIL
• LYOPHILIZATION
• STORAGE OVER SILICA GEL
• PRESERVATION ON PAPER
• PRESERVATION ON BEADS
• PRESERVATION ON SOIL
• LIQUID DRYINNG.
2. CRYOPRESERVATION.
• FROZEN AGAR PLUGS
• PRESERVATION IN LIQ NITROGEN
• 2-STAGE FREEZING PROCESS

6. SERIAL TRANSFER
 Sub-culturing on regular schedule.
 First technique.
 Easy
 Disadvantages – contamination, loss
of genetic &phenotypic characters,
high labor cost, loss of productivity.

7. PRESERVATION IN D/W
 CESTELLANI introduced.
 Fungi preservation for 10 yrs.
 Long term storage –loss in virulence.

8. PROTOCOL
1. Fungal plugs are cut with sterile
polypropylene transfer tubes.
2. Transfer to cryovials containing ~2
ml sterile d/w.
3. Stored at RT.

9. PRESERVATION UNDER OIL
 For non sporulating strains since
limits oxygen.
PROTOCOL –
 Culture grown on N.agar slant.
 Autoclaved heavy mineral oil is
poured over the colony to a depth of
about 1 cm above the tip of slant.
 Culture stored at ~10’c.

10. LYOPHILIZATION
 Long term preservation.
 Freeze-drying.
 Plasmid bacteria can be preserved.
 Cryoprotective agents –skim milk
(15%w/v for agar slants) & (20%w/v
for pelleted broth cultures) OR
12%w/v sucrose.

11. PROTOCOL
1. Borosilicate ampoules precoated 55mm from bottom
loosely plugged with absorbent cotton are over sterilized.
2. The contents of slants are pooled and mixed with skim
milk.
3. Fill 0.2ml of the mixture in ampoules.
4. Cotton plugs are replaced,& the vials are refrigerated until
culture have been dispensed.
5. Cotton plug tops are burned off, the ampoule lips are
wiped clean of carbon residue,& remainder of plug is
recessed.
6. Vials loaded horizontally at -43’c .
7. Once vacuum is down to 100 millitorrs, temp is raised to
-1’c.
8. Vials are now placed in lidded metal container .
9. Nitrogen is flushed in the container &stored at -70’c.

12. STORAGE OVER SILICA GEL
-Neurospora
PROTOCOL –
1. Screw cap tubes ½ filled with activated
silica gel oven sterilized are cooled.
2. 0.5ml skim milk (10%v/v) suspension of
conidia / mycelium is dispensed into each
tube.
3. Tubes are quickly cooled.
4. Dried at 25’c.
5. Stored in closed container with desiccants.

13. PRESERVATION ON PAPER
 Spore forming fungi ,Actinomycetes,
Unicellular bacteria, Fruiting bodies of
myxobacteria – stored on pieces of
sterile filter paper at RT/ 6’c for 5-15
yrs.

14. PROTOCOL
1. Pieces of agar containing fruiting bodies are placed
on sterile filter paper in Petri dish.
2. Dried in desiccator under vacuum.
3. Stored at RT.
OR
1. Veg cells transferred to small filter discs on water
agar.
2. Incubate for the development of fruiting bodies.
3. Allow maturation for ~8 days.
4. Papers are placed into sterile containers.
5. Dried over silica gel in an evacuated desiccator.
6. Container is closed & stored.

15. PRESERVATION ON BEADS –
developed by LEDERBERG
 Protocol –on porcelain beads for bacteria.
1. Sterile beads are transferred to sterile
petri dish.
2. Inoculate 0.2-0.3ml cell suspension per
bead.
3. Beads are transferred to vial & closely
capped.
4. Dried in vacuum desiccator for 72-92 hr.
5. Stored at 25’c in closed metal cabinet.

16. PRESERVATION ON SOIL
 For soil borne sps.
 Upto 20 yrs.
 Effective method.

17. 2 -PROTOCOLS
1. Protocol for drying on soil.
a) Spore suspension is prepared from fungal culture.
b) Inoculate 1ml suspension per 5gm of soil taken in sterile
tubes.
c) Dried at 25’c & stored at RT.
2. Protocol for preserving actinomycetes on soil.
a) Take 100gm of potting soil in 250ml ehrlenmeyer tube.
b) Add 0.25% dry blood 7 autoclaved for 1 hr.
c) Add 20ml d/w.
d) Inoculate 2ml water suspension into flask & incubated.
e) Cover flask with Para film when mycelial growth becomes
evident.
f) Stored at 5’c.

18. LIQUID DRYING
 To avoid freezing damage..
 Effective for anaerobes.
 Preferred over lyophilization for
maintenance of 6 gm neg bacteria.

19. PROTOCOL
 FREEZE-DRIED DISC
1. Small glass vials filled with 0.1 ml 20%(w/v) skim milk containing
1.0%(w/v) neutral charcoal &5%(w/v) myo inositol.
2. Sterilize the vials at 115’c for 13min.
3. Frozen about -40’c for few hrs.
4. Freeze dried for 8-24 hr.
5. Results in disc of freeze dried carrier material.
 LIQUID DRYING PROCEDURE
 ~30 micro lit cell suspension + myo-inositol charcoal solution is
added to each vial with a freeze –dried disc of carrier material.
 Vials are subjected to liquid drying.
 First step drying is continued for 2hr at 1.5-4KPa at 20’c.
 Second step drying is continued for about 1 hr at 0.01-0.001KPa
at -20’c.
 Vacuum is replaced by sterile nitrogen or argon gas.
 Ampoules are transferred to soft glass tubes & sealed under
vacuum.

20. CRYOPRESERVATION
 Deep freezing of micro organism req
a cryoprotectant such as glycerol or
dimethyl sulfoxide (DMSO).
 WHEN STORED AT -70’C OR IN LIQ
NITROGEN AT -156-196’C.

21. 1.1. FROZEN AGAR PLUGS-fungi &FROZEN AGAR PLUGS-fungi &
actinomycetesactinomycetes
 PROTOCOL-
1. A 10/15%w/w solution of glycerol
prepared.
2. 2ml is suspended into borosilicate glass
vials .
3. Vials are autoclaved twice for 45 min each.
4. Culture plugs are cut & deposited into each
vial.
5. Frozen & stored at -70’c.

22. 2. PRESERVATION IN LIQ
NITROGEN
Protocol-
a) Straws used- polypropylene drinking
strews.
b) Cryoprotectant-10%(v/v) aq solution of
glycerol or DMSO.
 To store fungi-agar plugs removed via a
tungsten wire & introduced into the straws.
 For preservation of spore bearing
fungi/unicellular m.org-loop full of culture
suspended in cryoprotectant.
 Culture are dried in liq nitrogen.

23. PROTOCOL FOR FREEZING M.ORG
IN LIQ NITROGEN
 PREPARATION OF CULTURES ON
SLANTS.
1. Suspend growth from agar slants in
3-6ml of broth containing 10%
glycerol.
2. Dispend this suspension of spores
&mycelium in each screw cap glass
vials.
3. All vials are refrigerated for 30 min.

24. 3. 2-STAGE FREEZING PROCESS
 Slow cooling is followed by rapid
cooling to storage temp results in cell
content solidifying without ice
formation.
 Rapid thawing.
 Alcaligenes eutrophus.

25. PROTOCOL
 Dissolve the pellet in ice cold solution containing
polyvinyl ethanol(10%w/v) +glycerol(10%w/v) in 1:1
ratio.
 Keep suspension in ice bath for ~30min for
equilibration.
 0.5-1ml aliquot is dispensed into plastic cryovial &
tightly closed.
 Clamped onto labeled AI canes.
 Placed at -30’c ~1hr/ few min in gas phase of liq
nitrogen.
 Cans are placed into racks.
 Frozen rapidly at -80’c in liq nitrogen.

26. CULTURE PREPARATION BEFORE
FREEZING
 Suspending med-preserve viability
&biological activity.
a) 10% lactose solution in d/w is
diluted with equal vol of sterilized
calf serum.
b) Combination of supplements
Supplement of 10-20% skim milk +10%
trehalose +5% glutamate.

27. Books suggested:
• INDUSTRIAL MICROBIOLOGY –
Arnol.L.Demian and julian devies
• Comprehensive biotechnology- Moore
Moo Young