How to preserve bacteria in Microbiology lab?

1. PRESERVATION OF BACTERIA
Dipendra Kumar Mandal (Clinical Microbiologist)
Lecturer
Manmohan Memorial Institute of Health Sciences,
Soalteemode, Kathmandu, Nepal

2. Introduction
• Bacterial species vary greatly in the ability of their cultures to
remain alive after the completion of growth(e.g. after 24 hr at 37°C)
• Problem maintaining them for various periods in a viable condition
• Necessary to eliminate genetic instability, protection against
contaminants and retain their original characters
• Most laboratories maintain a large collection of pure cultures,
frequently referred to as the stock culture collection
• Some species such as Neisseria are poorly viable and their cultures
usually die out within few days in routine conditions, must be
subcultured regularly( 2-4 days)

3. Introduction contd.
• Culture collections of microorganisms are valuable resources for and
scientific research in microbial diversity and evolution, patient
care management, epidemiological investigations, and educational
purposes
• Preserved individual strains of microorganisms serve as permanent
records of microorganisms’ unique phenotypic profiles and provide
the material for further genotypic characterizations
• Effective storage is defined by the ability to maintain an organism in
a viable state free of contamination and without changes in its
genotypic or phenotypic characteristics
• Microbial preservation methods have been evaluated extensively over
the past 50 years, and often, optimal methods for preservation depend
on a microorganism’s taxonomic classification

4. Maintenance Methods
• Pure culture- should be free from contaminants, kept pure
• Frequent way of erroneous results and conclusions if faulty or
contaminated cultures
• Stock cultures- organisms of interest maintained in laboratory as
reference
• Necessary to prevent contamination, retain viability and remain
true to type
• Two methods of maintenance of cultures
1. Agar Slants
2. Agar Stabs

5. Agar Slants
• Agar slants are a form of solid media
generated from the addition of a gelling
agent, such as agar, to a broth culture
• Inoculated and incubated until reasonable
growth – placed in a refrigerator after well
sealing done to prevent drying
• Hardy organisms(Spore formers, GPCs,
Yeasts and Fungi)- remain viable for weeks
to months
• Regular transfer to fresh slants necessary

6. Agar Stabs
• An inoculation technique used when
inoculating semi-solid medium for the
analysis of motility or oxygen usage, or
when inoculating certain types of solid
medium
• Inoculation is done with a straight needle
having a culture – deep all the way to the
bottom
• Culture grows as a thin zone along the
inoculation line
• Acid produced as waste is diluted along the
agar
• Preserve upto 2 weeks

8. Short-Term Preservation Methods
A. Frequency of transfer
B. Immersion in oil
C. Freezing at -20°C
D. Drying
E. Storage in distilled water
Long-Term Preservation Methods
1. Ultra low temperature freezing and
2. freeze-drying (lyophilization) are recommended for
long-term storage

9. Short-Term Preservation Methods
A) Direct Transfer to Subculture
• Simplest method for maintaining the short-term viability of
microorganisms : saved for more than 1 week
• Increases the likelihood of mutation with undesirable changes
• Maintenance Medium- support the survival of the microorganism but
minimize its metabolic processes and slow its rate of growth;
distilled water, tryptic soy broth, and nutrient broths with or without
cryopreservatives
• Transfer organisms into screw-top test tubes and to store them in an
organized location away from light and significant temperature
changes; storage at lower temperatures (5 to 8C)
• No set protocol for the frequency of transfer since storage conditions,
media used and types of microorganisms vary among laboratories

10. B) Immersion in Oil
• An alternative to capping tubes- add a layer of mineral oil to the top of
the specimen
• Many bacteria and fungi can be stored for periods of up to 2 to 3 years
by this method, and transfers are not needed as frequently
• Microorganisms are still metabolically active in this environment and
mutations can still occur
• Mineral oil should be medicinal-grade oil with specific gravity of 0.865
to 0.890 and heated to 170C for 1 to 2 h in an oven : sterilization
• An inoculum of 5 to 10 colonies of the microorganism should be placed
on an agar slant or in tubed broth media
• A layer of mineral oil at least 1 to 2 cm deep is added, and the agar must
not be exposed to air

11. C) Freezing at -20°C
• Refrigeration or freezing in ordinary freezers at -20°C may be
used to preserve microorganisms for periods longer than those
that can be accomplished by repeated transfers
• Viability may be maintained for as long as 1 to 2 years for
specific microorganisms, but overall, damage caused by ice
crystal formation and electrolyte fluctuations results in poor
long-term survival
• Modern self-defrosting freezers with freeze-thaw cycles must
be avoided because cyclic temperature fluctuation will destroy
the microorganism

12. D) Drying
• Molds and some spore-forming bacteria may be dried and stored for prolonged
periods
• Soil should be autoclaved for several hours on two successive days. A 1-ml
suspension of the microorganism is inoculated into the sterile glass tube, and the
tube is left open to air dry before being closed with a sterile stopper and sample
is stored in a refrigerator.
• Instead, commercial silica gel can be used in small cotton-plugged tubes after
being heated in an oven to 175C for 1.5 to 2 h, with moderately successful
recovery of fungi
• Gelatin discs: a thick suspension of bacteria is prepared and added to nutrient
gelatin . Drops of the bacterial suspension in gelatin are placed on sterile waxed
paper or on a plastic petridish and then dried off over P2O5 under vaccum
• Alternatively, a suspension of 10ˆ8 microorganisms can be inoculated onto
sterile filter paper strips or disks’. The paper is dried (over phosphorus
pentoxide in a desiccator under vacuum )in air or under a vacuum and is placed
in sterile vials. These vials can be stored in the refrigerator for up to 4 years,
and then single strips or disks can be removed as needed
• Commonly used for quality control organisms

13. L- drying:
• described by Lapage et al
• bacteria in small ampoules are dried from the liquid
state using a vacuum pump and desiccant and a
water bath to control the temperature

14. E) Storage in Distilled Water
• Most organisms grow poorly in distilled water, but some
survive for prolonged periods
• Many fungi and Pseudomonas sp. survive for several years in
distilled water at room temperature

15. Long-Term Preservation Methods
1. Ultra low temperature freezing and
2. freeze-drying (lyophilization) are recommended for long-
term storage
• Methods are less labor-intensive over time, require less
laboratory space (e.g., a cryovial versus broth or agar media),
and reduce the chances of mutation events
• Of course mutation still occur (e.g. mecA gene lost during long
term preservation at -80°c)
• Prolonged incubation requires-
i. Drying of the culture prevented by hermetic sealing
ii. Stored in a dark, either at RT or 4°C but not at 37°C
iii. Suitable preservation media

16. Ultra low-Temperature Freezing
• Maintained at temperatures of -70°C or lower for prolonged
periods using ultralow-temperature electric freezers and liquid
nitrogen storage units
• Presence of a cryopreservatives such as glycerol may reduce
the risk to microorganisms upon short exposure to higher
temperatures
• Storage Vials
– Plastic (polypropylene) or glass (borosilicate) tubes- able to withstand
very low temperatures and maintain a seal for their contents
• Cryoprotective Agents
– To protect microorganisms from damage during the freezing process,
during storage, and during thawing = are added to the culture
suspension

17. • Two types of cryoprotective agents
1. Those that enter the cell and protect the intracellular
environment and (e.g. Glycerol and dimethyl sulfoxide
(DMSO),
2. Others that protect the external milieu of the organism(e.g.
sucrose, lactose, glucose, mannitol, sorbitol, dextran,
polyvinylpyrrolidone, polyglycol, and skim milk )
• Universal cryoprotectant is DMSO 5% (V/V)
• Glycerol 10% (V/V) is the best preservative for bacteria
• Glycerol- autoclaving
• DMSO- filtering
• Skim milk 20% (Wt/V) in distilled water

18. • Preparation of Microorganisms for Freezing
– Cultures are allowed to mature to the late growth or
stationary phase before being harvested
– Broth specimens are centrifuged (low revolution) to create
a pellet of microorganisms and resuspended in 2 to 5 ml of
broth with the appropriate concentration of cryoprotectant
additive
– Agar – Scraping
– Volume of the aliquots to be frozen is typically 0.2 to 0.5 ml

19. • Freezing Method
– American Type Culture Collection (ATCC) recommends slow,
controlled-rate freezing at a rate of 1°C per min until the vials
cool to a temperature of at least -30°C, followed by more rapid
cooling until the final storage temperature is achieved
– When organisms are stored in liquid nitrogen, however, it is
still recommended that vials be placed initially in a -60°C
freezer for 1 h and then transferred into the liquid nitrogen
(-196°C)
– Small glass beads or plastic beads can also be added to storage
vials before freezing- culture suspension will coat the beads,
and then individual beads can be removed from storage for
reconstitution without thawing the entire sample

20. • Thawing
– Damage to microorganisms occurs as they are warmed from the
frozen state
– critical temperatures appear to be between -40 and -5°C (improves
recovery rates)
– Stored culture vials should be warmed rapidly in a 35°C water bath
until all ice has disappeared
– Once a vial is thawed, it should be opened and the organism should
be transferred to an appropriate growth medium immediately
– Great care must be exercised during the thawing phase since rapid
temperature changes and resulting air pressure changes inside vials
can cause the vials to explode
– So that protective clothing and eye wear must be worn during this
process

21. Freeze-Drying (Lyophilization)

23. Equipment

24. Schematic

26. History
 Freeze drying was first actively developed during WORLD
WAR II for transport of serum.
 After the war period the technology emerged into its
current popularity for the drying of many different
temperature sensitive materials like pharmaceuticals and
food materials.

27. • The sublimation occurs on the tip of
mount Everest due to:
low temperature
strong wind
low air pressure
sunlight which is suitable for
sublimation to occur.

28. Definition
Freeze drying is a stabilizing process in which a substance is
first frozen and then the quantity of the solvent is reduced, first
by sublimation (primary drying stage) and then desorption
(secondary drying stage) to values that will no longer support
biological activity or chemical reactions.
or
it is a process by which a solvent (usually water) is removed
from a frozen foodstuff or a frozen solution by sublimation of
the solvent and by desorption of the sorbed solvent (nonfrozen
solvent), generally under reduced pressure.

29. Introduction :
• Better preservation occurs with freeze-drying than with other
methods because freeze-drying reduces the risk of
intracellular ice crystallization that compromises viability
• Removal of water from the specimen effectively prevents this
damage
• Lyophilization is greatest with gram-positive bacteria (spore
formers in particular) and decreases with gram-negative
bacteria, but overall, the viability of bacteria can be
maintained for as long as 30 years

30. • Large numbers of vials of dried microorganisms can be stored
with limited space, and organisms can be easily transported
long distances at room temperature
• The process combines freezing and dehydration- Organisms
are initially frozen and then dried by lowering the atmospheric
pressure with a vacuum apparatus
• Specimens can be connected individually to the condenser
(manifold method) or can be placed (in a chamber) where they
are dehydrated in one larger air space
Contd.

31. Objectives of lyophilization process
• To preserve the biological activity of a product.
• To reduce the product weight to lower the transportation cost.
• To extend the shelf life or stability.
• To dry thermolabile materials.
• To eliminate the need for refrigerated storage.

32. Principle
 Lyophilization is carried out using a simple principle of physics
, sublimation. Sublimation is the transition of a substance from
the solid to the vapour state, without passing through an
intermediate liquid phase.
 Lyophilization is performed at temperature and pressure
conditions below the triple point (4.58 mm Hg, 0 ºC), to enable
sublimation of ice.
 The entire process is performed at low temperature and
pressure by applying vacuum, hence is suited for drying of
thermolabile compounds.
 The concentration gradient of water vapour between the drying
front and condenser is the driving force for removal of water
during lyophilization.

33. Triple point : triple point of a substance is the temperature and
pressure at which three phases (gas, liquid, and solid) of that substance
may coexist in thermodynamic equilibrium. The triple point of
pure water is at 0.01°C (273.16K, 32.01°F) and 4.58 mm (611.2Pa) of
mercury and is used to calibrate thermometers.

34. STEPS INVOLVED IN LYOPHILIZATION
FREEZING STAGE
PRIMARY DRYING STAGE
SECONDARY DRYING STAGE
PACKING

35. Freezing
• Freezing the product solution to a temperature below its
eutectic temperature.
• Decrease the shelf temperature to -50oc.
• Low temperature and low atmospheric pressure are
maintained.
• Formation of ice crystals occurs.
• The rate of ice crystallization defines the freezing process and
efficiency of primary drying.

36. Primary Drying (Sublimation)
• After freezing, the product is placed under vacuum. This
enables the frozen solvent in the product to vaporize without
passing through liquid phase, a process known as
SUBLIMATION.
• Heat is introduced from shelf to the product under graded
control by electrical resistance coils or circulating silicone.
• The temperature and pressure should be below the triple point
of water i.e., 0.0098°C and 4.58mmHg.
• The driving force is vapor pressure difference between the
evaporating surface and the condenser.
• Easily removes moisture up to 98% to 99%.

37. Secondary Drying (Desorption)
• Heat is applied to the frozen product to accelerate
sublimation
• The temperature is raised to 50°C – 60°C and vacuum is
lowered about 50mmHg.
• Bound water is removed.
• Rate of drying is low.
• It takes about 10-20 hrs.

38. Lyophilization Process

39. Packing
• Ampoules are sealed by either tip
sealing or pull sealing method
• Vials and bottles are sealed with
rubber closures and aluminum caps

40. • Storage Vials
– Glass vials are used for all freeze-dried specimens
– When freeze-drying is performed in a chamber, double glass
vials are used
– (chamber method) : an outer soft glass vial is added for
protection and preservation of the dehydrated specimen
– Silica gel granules are placed in the bottom of the outer vial
before the inner vial is inserted and cushioned with cotton
– Manifold method- a single glass vial is used
– storage vial must be sealed to maintain the vacuum and the
dry atmospheric condition
• Cryoprotective Agents
– Two most commonly used agents are
1. Skim milk for chamber lyophilization, and
2. sucrose for manifold lyophilization

41. • Skim milk is prepared by making a 20% (vol/vol) solution of
skim milk in distilled water.
• divided into 5-ml aliquots and autoclaved at 116°C with care
taken to prevent overheating and caramelizaton(nutty flavor)
of the solution.
• The preparation is then used in smaller volumes
• Sucrose is prepared in an initial mixture of 24% (vol/vol)
sucrose in water and added in equal volumes to the
microorganism suspension in growth medium to make a final
concentration of 12% (vol/vol).

42. Preparation of Microorganisms for Freezing
– As with simple freezing, maximum recovery of organisms is
achieved by using microorganisms in the late growth or
stationary phase from the growth of an inoculum in an
appropriate growth medium
– High concentrations of microorganisms are considered to be
important- ATCC concentration of at least 10ˆ8 CFU/ml but
Heckly suggests a concentration of 1010 CFU/ml or higher

43. Freeze-Drying Methods
chamber method: inner vials with the microorganism suspension are
placed in a single layer inside a stainless steel container
1. Container is placed in a low-temperature Freezer at
-60°C for 1 h and then transferred to a chamber containing dry ice
and ethyl Cellosolve and covered with a sealable vacuum top,
which is connected to a vacuum pump at a minimum of 30µm Hg
for 18 h
2. Outer vials are prepared by being heated in an oven overnight,
filled with silica gel granules and cotton, and placed in a dry
cabinet with <10% relative humidity
3. The freeze-dried inner vials are inserted into the outer vials, and
the outer vials are heat sealed

44. In the manifold method:
1. a rack of individual vials is used rather than a single
container. The rack is placed in a dry ice-ethyl Cellosolve
bath
2. After the freezing process, the vials are connected by
individual rubber tubes in sequence to the condenser
container filled with dry ice and ethyl Cellosolve and to the
vacuum pump

45. Freeze Dry Product Characteristics
• Sufficient strength
• Uniform color
• Sufficiently dry
• Sufficiently porous
• Sterile
• Free of pyrogens and particulates
• Chemically stable both in dry state and reconstitution

46. • Storage
– Storage at room temperature does not maintain viability and is
not recommended
– Storage at 4°C in an ordinary refrigerator is acceptable, but
survival may be improved at temperatures of -30 to -60°C
• Reconstitution
– The surface of the vial should be wiped with 70% alcohol, and
then the top of the glass vial can be scored and broken off or
punctured with a hot needle
– A small amount (0.1 to 0.4 ml) of growth medium is injected
into the vial with a needle and syringe or a Pasteur pipette, the
contents are stirred until the specimen is dissolved, and then the
entire contents are transferred with the same syringe or a pipette
to appropriate broth or agar media

47. Advantages of Lyophilization
• Removal of water at low
temperature
• Thermolabile materials can be
dried.
• Sterility can be maintained.
• Reconstitution is easy

48. Disadvantages of Lyophilization
• Many biological molecules are damaged by the stress
associated with freezing, freeze-drying, or both.
– E.g. the process of drying causes extensive damage to
molds, protozoa, and most viruses
– Hence, these microorganisms can not be stored by this
method
• The product is prone to oxidation, due to high porosity
and large surface area. Therefore the product should be
packed in vacuum or using inert gas
• Cost may be an issue, depending on the product

49. Common Lyophilized Products
• Bacteria
• Viruses
• Vaccines
• Plasma
• Pharmaceuticals – large and small molecules
• Fruit
• Coffee
• Flowers

50. Applications
• Microbiology, pharmaceutical and biotechnology – to
preserve microorganisms, increase the shelf life of products,
such as vaccines and other injectables
• Food industry
 to preserve food, very light weight.
 to produce essences or flavouring agents.
 freeze-dried fruits are produced.
 Culinary herbs are preserved.
 Instant coffee powder is prepared.
• Technical industries
 in chemical synthesis
 Formation of stable products.
• Others
 Flora & fauna preservation

55. Common procedures for preservation of microorganisms

57. FREEZE DRYING PLANTS AND EQUIPMENT
1. Pilot freeze drying
2. Industrial freeze drying
a. Tray and Pharmaceutical Freeze Dryers
b. Multibatch Freeze Dryers
c. Continuous Freeze Dryers
d. Tunnel Freeze Dryers
e. Vacuum-Spray Freeze Dryers

58. Pilot freeze drying

59. Batch freeze dryer

60. Continuous freeze dryer

61. Tunnel freeze dryer

62. Vacuum spray freeze dryer

63. ANY QUERIES?
Please…………..