How To Prepare, Sterilize, AND Test Culture Media

How To Prepare, Sterilize, AND Test
Culture Media
Preparation of Culture Media
Important precautions:
Prepare the media from dehydrated products in a
damp-free environment.
To prevent the risk of inhaling fine particles of
culture media powder, wear a dust mask while
handling media powder.
Wash the hands immediately after preparing media.
Once the ingredients are weighed, do not delay in making up
the medium.
Use completely clean dry glass wares.
The container in which the medium is prepared should have a
capacity of at least twice the volume of the medium being
Use distilled water, as the tape water contains substances
(chlorine) harmful to bacteria and can alter the pH of a
medium or cause a precipitate to form.
1. Follow exactly the manufacturer’s instructions for preparation i.e.
amount of media and volume to prepare.
2. Accurately weigh, add the media powder to the exact volume of
distilled water.
3. Heat the media and stir by placing media flask on the hot plate with
magnetic stirrer.
4. Do not shake vigorously, prevent overheating, boiling and foam
formation, which can damage the medium.
5. Autoclave a medium only when the ingredients are completely
Add distilled water
to make the right
volume. Heat & stir
(agar will burn if it is
not stirred) until all
of the ingredients go
into solution. When
the media boils, it is
ready for
Sterilization of culture media:
 The following methods are used to sterilize culture media:
1. Autoclaving.
2. Steaming at 100 0C.
3. Filtration.
This done in order to make the media
completely free of microorganism.
For this purpose we use Autoclave machine which
operates at following conditions i.e.
Temperature = 121oC
Time = 15 minutes and
Pressure = 15psi (Pounds per square inch).
Overheating effects
 Overheating is a common cause of darkening, precipitation, poor
gel strength and reduced bacteriological performance.
 All culture media should be in solution before
 Overheating effects will occur if agar media are allowed to gel in
bottles and are later steamed to melt the agar.
Preparation errors and possible causes
Preparation errors
Possible causes
Poor bacterial growth.
1. Prolonged and excessive heating, incomplete solution.
2. Inhibitory substances in water or containers.
3. Darkening and pH drift.
Turbidity, precipitation.
1. Poor quality water or containers.
2. Overheating or prolonged storage at 50°C.
3. pH value incorrect.
4. Incomplete solution.
Preparation errors
Possible causes
Soft gel.
1. Agar not in solution, poor mixing, prolonged storage at
2. Overheating at low pH values.
3. Error in weighing or overdilution with inoculum or media
4. pH too low for agar.
1. Overheating, incomplete solution or pH drift.
2. Presence of phosphate in addition of glucose or other sugars
and agar.
Dispensing of culture media:
 Dispensing means pouring of sterilized medium into suitable glass
 By using aseptic techniques, sterilized medium can be dispensed into
sterile Petri dishes or Screw capped test tubes.
Aseptic Techniques
 Aseptic technique refers to a procedure that is performed under sterile
conditions to prevent any contamination e.g. use of flame.
Dispensing sterile media into petri dishes
1. Arrange the sterile petri dishes on a level surface e.g. a table or
2. Mix the medium gently by rotating the flask or bottle. Avoid
forming or air bubble formation.
3. Sterilize the neck of the flask on a flame and pour 15–20 ml of
medium into each dish (90 – 100 mm diameter).
4. If air bubbles enter while pouring, rapidly flame the surface of
the medium before gelling occurs.
5. Rotate the petri dish on the surface of the bench to ensure
an even layer of agar.
6. When the medium has gelled and cooled, cover the plates
and seal them in plastic bags to prevent loss of moisture and
reduce the risk of contamination.
7. Do not leave the plates exposed to bright light especially
8. Store at 2–8 0C.
Dispensing and solidifying high protein media
 High protein media such as Dorset egg medium and Loeffler
serum medium are dispensed aseptically in screw-capped bottles
and solidified in a sloped position at a controlled temperature
(75–80 0C) for 1–2 hours.
 Solidification of protein media using heat to coagulate
the protein, is called inspissation.
Labeling and Storage:
1. All culture media must be clearly labeled.
2. Each batch of prepared medium should be given a number and
the date of its preparation.
3. Should be stored at an even temperature in a cool dry place
away from direct light.
4. Container tops must be tight-fitting.
5. Plates of culture media should be stored at 2-8oC, preferably in
sealed plastic bags.
Sterility Testing:
 The simplest way to test for contamination is to incubate 5% of the batch at
35–37oC overnight.
 Contamination by microorganisms capable of overnight growth, will be
shown by a turbidity in a fluid medium and growth on a solid medium.
Note: All media, even those that have been sterility tested at the time of
preparation, should always be checked visually immediately before being
inoculated, for any change in appearance that could indicate contamination.
 This is particularly important during the hot season and when the humidity
is high.