UNIVERSITI TEKNOLOGI MARA

BIO 461
Microbiology

Experiment No: 8
Experiment Title: Culture Media
Lecturer’s Name: Ms Siti Saizah Mohd Said
Student’s Name: Fatin Binti Ahmad
Group members: 1) Fatin Nadia Binti Masron (2010264422)
2) Nur Syahidah Binti Husin (2010866154)
Student ID: 2010254504
Group: ASB2B1
Programme: Bachelor of Science (Hons.) Biology
Experiment Date: 21 February 2011
Experiment Submission: 7 March 2011
PROCEDURE
A. SOLID MEDIA
1. An amount of dehydrated nutrient agar was suspended in a fixed amount of
water.
2. The suspension was dissolved completely by boiling it.
3. 5 agar deeps were prepared by pipetting 10 mL of the melted nutrient agar.
4. 5 agar slants were prepared by pipetting 5 mL of the melted nutrient agar into 5
separate standard culture tubes.
5. The agar deeps and agar slants were placed into a rack.
6. The rack was auctoclaved at 15 lbs-pressure for 15 minutes at 121oC.
7. After being sterilized, the agar deeps were allowed to solidify in the upright
position whereas the agar slants were placed at 45o angle.
8. Using aseptic techniques, 5 mL of sterile defibrinated blood was added to 100 mL
of nutrient agar which was cooled to 50oC.
9. The organic mixture was poured into 4 different petri plates and allowed to cool
media side-up.
10. Two blood plates were immediately sealed with paraffin film. Remaining two
blood plates were left exposed to contamination for 5 minutes, and then sealed
with paraffin film.
11. All blood plates were incubated at 37oC.

B. FLUID MEDIA
1. A required amount of dehydrated nutrient broth was suspended in a fixed amount
of water.
2. The nutrient powder was dissolved by heating.
3. 6 mL of nutrient broth was pipetted into 5 different tubes.
4. The tubes were autoclaved together with the solid media.

C. THE FOLLOWING DAY

1. The media which was sterilized was compared with the media exposed to
contamination.
2. All observations were recorded.

CONCLUSION
Solid agar media can be used to isolate and count pure colonies. Fluid media however can
be used to cultivate pathogenic or non-pathogenic bacteria.

REFERENCES
1. http://www.biorigin.net/clientes/biorigin/inf/cultivoing.pdf
Retrieved on: 6 March 2011
2. http://www.suite101.com/content/macconkeys-agar-mac-bacterial-growth-medium-
a60936
Retrieved on: 6 March 2011
3. http://jb.asm.org/cgi/reprint/83/6/1217.pdf
Retrieved on: 6 March 2011
4. http://www.buzzle.com/articles/mannitol-salt-agar.html
Retrieved on: 6 March 2011
QUESTIONS
1. Why are the following ingredients incorporated into the culture medium?
a) Yeast extract
Yeast extract is rich in nitrogen sources. It is the constituent of amino acids,
nucleic acids and coenzymes.

b) Lactose
Lactose, together with a pH indicator, neutral red, is used in differential media. It
is used to differentiate between lactose fermenters and non-lactose fermenters.
In presence of lactose fermenters, the colonies will appear pink, whereas
colourless for non-lactose fermenters.

c) Bile salts
Bile salts are used in selective media to inhibit the growth of Gram positive
bacteria.

d) Crystal violet
Crystal violet is used in selective media to inhibit the growth of Gram positive
bacteria.

e) Phenol red
Phenol red is used as a dye in mannitol salt agar. The dye is used to differentiate
between species of staphylococci. Strains of staphylococci that produce organic
wastes that ferments the mannitol will change the colour of the medium to bright
yellow.

f) Sodium azide
Sodium azide is used to isolate facultative anaerobic bacteria from aerobic
bacteria.