Maintenance of Pure Culture in Lab

2
Growth and Maintenance of Culture

Vivek Negi
E-mail: viveknegi2006@gmail.com
INTRODUCTION
In molecular biology many microorganisms are involved in the experiment
and we need specialized techniques to handle them. Growth media must
have essential nutrients for proper growth and maintenance of culture.
Autoclaving of the media and discard are directly and indirectly involved
in good culture growth. While working with the microbes, issues related
to biosafety and biohazard must be attended for personal safety and
environmental health.
LEARNING OBJECTIVES
 Maintenance of microbial culture
 Autoclaving procedures
 Biohazard and biosafety
Maintenance of Microbial Culture

Agar Plates for Short-term Storage: Quadrant streak method must be
used to streak on rich agar Petri plate in sterile conditions to get well
isolated colonies. These plates can be stored in a refrigerator for about a
month. Plates should be carefully examined for any contamination and
if it is contaminated then it is discarded.
Spread plate/pour plate method: In spread plate methodology microbial
culture is evenly distributing over LB agar plate.
 On the basis of the microbe select and prepare particular agar
medium.
 To avoid condensation after pouring the plates, the autoclaved agar
is cooled in the range of 45°C and 50°C. In a 100 × 15 mm media
plate 15 to 20 mL of media is poured and the thickness of media is
0.3 cm approx.
 To avoid microbial contamination inoculum should be spread as
soon as possible.
 Changing of pipette tips must be avoided while preparing serial
dilutions to spread on the plate.
68 An Introduction to Biotechnology: A Genetic Manipulation Perspective
Creating Microbial glycerol stocks for long-term storage

 After inoculating the microbes for overnight in liquid culture, add
500 μL of the overnight culture to 500 μL of 40 per cent glycerol in
a 2 mL cryovial and gently mix.
 Store the glycerol stock tube at –80°C. The stock will be stable for
years until subjected to subsequent freeze and thaw cycles.
 Scrape off some of the frozen bacteria of the top. Do not let the
glycerol stock unthaw. Streak the microbes onto an LB agar plate
for overnight.
Protocol for lyophilization process

 Prepare the sterile solution—compound, mix, filter
 Fill into vials
 Partially insert a rubber closure onto the vials
 Aseptically load the vials into a freeze dry chamber
 Freeze every single solution in every vial below a pre-determined
critical temperature
 Use appropriate application of temperature and pressure to sublime
ice from the product
 Further apply temperature and pressure to remove the necessary
amount of bound water from the product
 Automatically stopper the vials, neutralize the chamber.
Tips for maintaining strains

 Streak the culture on agar plate as soon as you receive the plate.
 Refrigerate the plate only when the good visible growth of culture
is seen.
 Plates and slants are preferred over broth culture.
 Always start seed culture from single bacterial colony.
 Subculturing is preferred once in a week for maintaining broth
culture.
 If the strain looks contaminated, streak and maintain fresh culture.
 Restreak the cultures on slants/plates about once a month.
Autoclaving
An autoclave is used to sterilize materials to remove all forms of microbial
life with the aid of high temperature and pressure. Effective use of
autoclaving is must for proper sterilization of the materials and safe use
of autoclave.
Growth and Maintenance of Culture 69
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sterilliz
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spallan
B.C.E.,
Moses 1880s.,
s
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rate
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Fig. 2.1 Chronology of scientific advancement in autoclaving.
Historical significance (Fig. 2.1)

 The first sanitary code by Moses which included purification of
materials by fire and boiling water.
 Hippocrates used boiling water to clean surgical instruments.
 Lazzaro Spallanzani discovered the protocol to kill bacteria in sealed
glass flasks by heating it for 30 min.
 Pasteur had proposed boiling as the effective means of sterilization.
 Based on the Denis Papin invention of steam pressure cooker early
design of the first autoclave was devised by Charles Chamberland.
Charles Chamberland Papin steam cooker
Principle of autoclaving
 Steam penetrates materials in autoclave.
 Negative pressure is built due to condensation which draws in
additional steam.
 Moist heat coagulates the proteins which kill the bacteria.
Growth and Maintenance of Culture 71
Standard pressure and temperature of an autoclave

When the free-flowing steam at a temperature of 100°C at pressure of 1
atmosphere above the sea-level pressure is subjected under pressure of
15 pounds pressure per square inch, the temperature inside the autoclave
happens to rise up to 121°C, which is a common parameter employed
in the moist heat sterilization.
Table 2.1 Some Standard Temperatures/Pressures Employed
S. No. Temperature (°C) Pressure (psi)

1. 115 10
2. 121 15
3. 132 27
Psi = Pressure unit standing for pounds per square inch
Pre-Autoclave Procedures
(i) Polypropylene bags
They are autoclave or biohazard bags which are rigid, tear-resistant
and available in various sizes. They should not be fully filled and
some space should be left at the mouth region for passage of the
air. Indicator tape should be placed in an “X” pattern over the
biohazard bag.
(ii) Materials that can be autoclaved
• Plastic tubes, pipette tips and plates
• Glassware
• Surgical Instruments
• Biohazardous waste
(iii) Risk Management
Potential risks while operating autoclave
• Heat burns
• Steam burns
• Heat fluid scalds
• Hand and arm injuries due to door
• Body injury if there is any explosion
(iv) Health and Safety
• Trained person should be appointed as incharge of the autoclave.
• Autoclave should be operated only after wearing protective

clothing.
• Autoclave must be inspected annually and proper record be
maintained for its inspection, service and repairs.
(v) Personal Protective Equipment for Users
• Eye protection
• Heat-resistant gloves
• Lab coat, buttoned
• Closed-toed shoes
(vi) Loading Autoclave
• Place material in autoclave
• Do not overload
• The door should be firmly latched and closed
(vii) Steps to Autoclave
• Check that the water reservoir is filled.
• Set appropriate temperature for the cycle.
• Close and lock door.
• Pressure is achieved in 40 min approx. If autoclave is cold and
20 min approx. if in use.
• To STERILIZE turn on the autoclave.
• The autoclave should be turned to VENT when the cycle is
complete, and door must remain closed until pressure drops.
During venting temperature stays on for longer time and the
steam present in chamber prevents melting of plastics.
• Turn off the autoclave.
Unloading Autoclave
 Heat-insulating glove provides protection against burns.
 Before opening the door one must ensure that the pressure of
chamber is ‘8 psi’.
 While opening the door one should wear gloves and keep distance
from the door.
 Super-heated liquid in containers should not be opened to avoid
accidents.
 Autoclaved hot items should be placed in a room to cool the
temperature.
derived from those organisms; and allergens and toxins derived from
higher plants and animals.
Biosafety: The containment principles, technologies and practices that
are implemented to prevent unintentional exposure to pathogens and
toxins, or their accidental release.
FREQUENTLY ASKED QUESTIONS

1. What is the best way to preserve cultures for long?
2. Are there special requirements for reconstituting certain lyophilized
microorganisms?
3. What should we do with contaminated plates?
4. How long should we sterilize the liquids?
5. Why contamination issues are observed even after autoclaving?
REFERENCES
1. Thiel, T. (1999). Introduction to Bacteria in Science in the Real
World: Microbes in Action.
2. http://www.pasteur.fr/infosci/archives/chb0.html
3. http://www.astell.com/sterilisers
4. http://www.asu.edu/ehs/documents/autoclave-sop.pdf

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2

Growth and Maintenance of Culture

Maintenance of Microbial Culture

Creating Microbial glycerol stocks for long-term storage

Protocol for lyophilization process

Tips for maintaining strains

Fig. 2.1 Chronology of scientific advancement in autoclaving.

Historical significance (Fig. 2.1)

Charles Chamberland Papin steam cooker

Standard pressure and temperature of an autoclave

Table 2.1 Some Standard Temperatures/Pressures Employed

S. No. Temperature (°C) Pressure (psi)

• Autoclave should be operated only after wearing protective

FREQUENTLY ASKED QUESTIONS

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