Professional Documents
Culture Documents
10 Culture Media Preparation, Inoculation (2) (Autosaved)
10 Culture Media Preparation, Inoculation (2) (Autosaved)
10 Culture Media Preparation, Inoculation (2) (Autosaved)
testing).
Common ingredients of culture media
5. Mineral salts : are essential for growth and for metabolism of living
organism. E.g - SO4 - as a source of sulphur
Example:
Uses of agar
1. Solidify culture media
2. Provide calcium and organic ions for the bacteria
3. Used for adding heat sensitive ingredients such as blood and
serum while the agar is in gel phase at 45 – 50oC
4. The agar is very transparent so that growth of colonies is easily
visible.
B. Gelatin – is sometimes used as solidifying agent but it has many
disadvantages like:
The protein which are found in gelatin are susceptible to
microbial digestion
– Solid to gel transformation is very low which is 24oC, so it will
become changed to gel at 24oC & can not be used for bacteria
which need incubation above 25oC.
– 15% w/v gelatin is required to solidify the culture.
8. Accessory growth factors
• Include vit B (thiamine, niacin, and biotin).These provide the
necessary condition for growth
9. Other ingredients include, blood, serum, egg yolk etc depending
on the organisms need.
Types of culture media
1. Basic media
3. Selective media
5. Transport media
1. Basic Media
• These are simple media that will support the growth of micro
organisms that do not have special nutritional requirements.
- Nutrient broth
A. Solid media
• Are solidified by agar
• Are used mainly in Petri dishes as plate cultures, in bottles or
tubes as stab (deeps) or slope cultures.
• The purpose of culturing on solid medium is principally to isolate
discrete colonies of each organism present in the specimen. this
will enable pure cultures to be produced for identification and
sensitivity testing.
N.B: The colonial appearances and changes in solid media made
by colonies may provide valuable identification information
B. Semi solid media
• Are culture media prepared by adding small amount of agar (0.4 -
0.5% W/V) to a fluid medium.
• Semi solid media are used mainly as transport media, and for
motility and biochemical tests.
- alteration of pH and
Methods of sterilization
A. Autoclaving –
• The majority of culture media are sterilized by being autoclaved.
• This ensures the destruction of bacterial endospores as well as
vegetative cells.
• It is important to sterilize a medium at the correct temperature and for
the correct length of time as instructed in the method of preparation.
• Under sterilization and over sterilization will affect the quality of the
culture media .
• N.B. Most culture media are sterilized at a pressure of 15 lb/in 2 and
temperature of 121oC for 15 minutes.
B. Steaming at 100oC
• It is used to sterilize media which contain ingredients that would be
inactivated at temperature over 100oC.
the molten medium in to a small beaker or Petri dish and when it has
solidified, laying a narrow range pH paper on it’s surface.
• The colour of the paper is then compared against the pH colour chart
• Petri dishes
• Test tubes
• Bunsen burner
• Incubator
Depending on the characteristics and nutritional requirem ent,
bacteria can be inoculated on different culture media hence the
choice of culture media for inoculation of samples depends on:
1.The major pathogens to be isolated, their growth requirements, and
the features by which they are recognized.
2. Whether the specimens being cultured are from sterile sites or from
having normal microbial flora.
NB: Although a selective medium is usually more expensive than a
non selective one, it often avoids sub culturing , isolates a
pathogen more quickly, and makes it easier to differentiate and
interpret bacterial growth.
3. Cost, availability, and stability of different media in tropical
countries.
4. Training and experience of laboratory staff in preparing, using, and
quality controlling culture media.
Inoculation of culture media
NB: For sputum and stool samples since they have large number of
bacteria, prior to inoculation we have to dilute or homogenize the
samples.
2. Flame sterilize the loop, when cool or using a second sterile loop,
spread the inoculation systematically. This will ensure single colony
growth
Figure. Inoculation techniques.
Inoculation of Slopes
• To inoculate slopes (such as Dorset egg medium or TSI) use a
sterile straight wire to streak the inoculation down the center of
the slope and then spread the inoculation in a zigzag pattern as
shown in the figure.
Steel wool + acidified copper sulphate solution – this will coat the
iron and rapidly absorb oxygen when put in a sealed plastic bag.
NB. For isolating organism from sterile sites use culture media
which is non selective, and enriched.
Sites Having a Microbial Flora:
Interpretation requires patients’ clinical data to judge whether an
isolate is a pathogen which causes the patients illness.
The laboratory report should indicate those organisms for which
isolation technique has been performed.
For example when the pathogen have been isolated from feacal
specimen cultured on selective medium like SSA (salmonella
shigela agar). The report should state as ‘no salmonella or shigella
organism isolated’.
• Sites with normal flora include faeces (stool), sputum, skin, throat
and nose swabs, vagainal, cervical, and urethral swabs.
NB: For isolating organism from sites with normal flora selective
media are much better than general media.
Questions?
68 01/16/24