Video:
https://www.youtube.com/watch?v=KHg_PyjQPwk
Introduction
Since microorganisms cannot be seen easily with the naked eyes,
they are usually grown on special nutrient preparations called
culture media (singular, medium). Growing microorganisms on
culture media enables microbiologists to study their structure and
metabolic properties, as well purifying and enumerating them. A
culture medium should contain all necessary nutrients in adequate
amounts for the growth of the microorganism.
Depending on the physical nature of the medium, three different
types of culture media can be used in Microbiology. Solid and semi-
solid media usually contain a thickening agent called agar, which
gives the media different degrees of consistency when poured into
Petri dishes. Liquid media do not contain agar. These different
media types are used in different situations and each has its own
advantages and disadvantages. Many types of media are used in
Microbiology and the choice of the media to use depends on the
specific objective that is sought. Thus, culture media could be
general-purpose, selective, differential, etc. Pre-prepared
commercial media are used mostly for routine laboratory activities
while more specialized media could also be compounded when
needed.
Experiment 1. Nutrient Agar Preparation
Nutrient agar is a general-purpose medium that is used as growth
medium for non-fastidious bacteria. It can be used to isolate bacteria
from the air, soil, water and various other sources. The constituents
include beef extract, peptone, sodium chloride and agar.
Purpose
To prepare nutrient agar medium for isolating bacteria from the
environment.
Materials required
Nutrient agar powder
Distilled water
Erlenmeyer flask
Measuring cylinder
Petri dish
Glass stirring rod
Bunsen burner
Cotton wool
Aluminum foil
Procedure
(For 250ml)
1. Measure 7 grams of nutrient agar powder into an
Erlenmeyer flask.
2. Pour 250 ml of distilled water and swirl at interval to
ensure proper dissolution of the medium.
3. Warm the mixture to near boiling point and swirl at
interval for complete dissolution.
4. Plug the Erlenmeyer flask with cotton wool to prevent
contamination.
5. Autoclave at 121 °C for 15 minutes.
6. After autoclaving, allow the medium to cool to about 45
°C.
7. Swirl to mix very well, then pour aseptically into plates
near a Bunsen burner flame.
8. Allow the plates to set before further use.
Result
1. Incubate your plates at 37 °C and record your
observations.
Questions
1. At what temperatures does agar melt and solidify?
2. What is the purpose of peptone in the medium?
3. Outline two reasons for the usage of agar as
solidifying agent in culture medium.
Further reading
1. Aneja K. R. (2014). Laboratory Manual of
Microbiology and Biotechnology. Medtech, New
Delhi. pp. 111-120.
2. Fawole, M.O. and Oso, B.A. (1988). Laboratory
Manual of Microbiology. Spectrum Books Limited,
Ibadan. pp. 11-14.
3. Tortora G.J., Funke, B.R and Case, C.L (2007).
Microbiology, An Introduction. Pearson ed., New
Jersey. pp. 168-173.
Experiment 2: Blood Agar Preparation
Blood agar is a culture medium consisting of blood (usually
animal blood) and nutrient agar. It is non-selective but
differential for species that exhibit haemolysis as one of their
main characteristics. The medium is used for isolation of
pathogenic bacteria from clinical samples like swabs, sputum,
urine, stool, etc. The main constituents include tryptone, beef
extract, sodium chloride, agar and sheep blood.
Purpose
For isolation of pathogenic bacteria from samples of clinical
origin.
Materials required
Nutrient agar powder
Sheep’s blood
Distilled water
Erlenmeyer flask
Cotton wool
Glass stirring rod
Bunsen burner
Cotton wool
Aluminum foil
Measuring cylinder
Procedure
(For 250ml)
1. Measure 7 grams of nutrient agar powder into an
Erlenmeyer flask.
2. Pour 250 ml of distilled water and swirl at interval to
allow dissolution of the medium.
3. Warm the mixture to near boiling point and swirl at
interval until it completely dissolves.
4. Plug the Erlenmeyer flask with cotton wool to prevent
contamination.
5. Autoclave at 121 °C for 15 minutes.
6. After autoclaving, allow the medium to cool to about 45
°C.
7. Aseptically add 5 ml blood into the medium.
8. Swirl it to mix very well, then pour aseptically into the
plates.
9. Allow the plate to set before further use.
Result
1. Incubate your plates for 24 hours at 37 °C and record
your observations.
Questions
1. Why do you need to pour distilled water unto the nutrient
agar inside the Erlenmeyer flask and not the other way?
2. Why is the blood not sterilized with the medium?
3. What is the medium’s colour before and after adding the
blood?
Further reading
1. Aneja K.R. (2014). Laboratory Manual of Microbiology
and Biotechnology. Medtech, New Delhi. pp. 111-120.
2. Fawole, M.O. and Oso, B.A. (1988). Laboratory Manual
of Microbiology. Spectrum Books Limited, Ibadan. pp.
11-14.
3. Tortora G.J., Funke, B.R and Case, C.L. (2007).
Microbiology, An Introduction. Pearson ed. New Jersey.
pp. 168-173.
Experiment 3: Preparation of Potato Dextrose Agar
(PDA)
Potato dextrose agar is used for the enumeration and isolation of
yeast and moulds from different samples. It offers excellent
foundation for the growth of fungal species. It can be easily
prepared in the laboratory. Its main constituents are Irish potato,
dextrose and agar.
Purpose
It is used for isolation of fungi including mold and yeast in the
Microbiology laboratory.
Materials required
Dextrose
Potatoes
Agar powder
Beaker
Erlenmeyer flask
Cotton wool
Distilled water
Scalpel
Aluminum foil
Gas stirring rod
Bunsen burner
Measuring cylinder
Procedure
(For 250ml)
1. Weigh 50 grams of potato.
2. Wash and cut them into small pieces.
3. Boil for about 30 minutes until they are soft.
4. Filter through cheesecloth, saving the effluent.
5. Add water to the broth to make exactly 250 ml inside the
Erlenmeyer flask.
6. Add 5 grams dextrose and 7 grams agar powder.
7. Stir occasionally and heat gently until the agar has melted.
8. Seal the Erlenmeyer flask with cotton plugs.
9. Autoclave the PDA at 121 °C for 15 minutes.
10. The medium is then cooled to 45 °C.
11. Add 5 ml streptomycin and swirl until it is well mixed.
12. Aseptically pour into plates near Bunsen burner flame.
13. Allow the plate to set before further use.
Result
1. Incubate your plates at ambient temperature for 7 hours
and record your observations.
Questions
1. Why is it necessary to add agar to the medium?
2. Incubate your plates at room temperature for 48-72 hours
and record your observation.
3. What is the essence of adding streptomycin to the
medium?
4. Why is the streptomycin not sterilized with the medium?
Further reading
1. Aneja K.R. (2014). Laboratory Manual of Microbiology and Biotechnology.
Medtech, New Delhi. pp. 111-120.
2. Atlas, R.M. (2004). Handbook of Microbiological Media, Third Edition. CRC Press
LLC, Florida. pp 1400.
3. Tortora G. J., Funke, B. R and Case, C. L (2007). Microbiology, An
Introduction. Pearson ed., New Jersey. pp. 168-173
