Principle:
Cetrimide is a quaternary ammonium, cationic detergent that is toxic to most bacteria, except Pseudomonas aeruginosa and a few other bacteria. Nitrogen and phosphorus are released from the bacterial cell and growth is inhibited when they come in contact with cetrimide. However, P. aeruginosa and a few other pseudomonads able to withstand this germicidal activity and hence growth is observed on the inoculated slant of agar medium containing cetrimide for a positive result. Magnesium chloride and potassium sulfate in the medium enhance the production of pyocyanin and pyoverdin (fluorescein) by P. aeruginosa. If the colonies of non-glucose-fermentative, gram-negative rods are isolated from the media containing cetrimide, the suggestive organism is P. aeruginosa.
Requirements:
i. Cetrimide agar: composition
- Gelatin —————————– 20.0 g/ltr
- K2SO4 ——————————- 10.0 g/ltr
- MgCl2 —————————— 1.4 g/ltr
- cetyltrimethylammonium bromide —- 0.3 g
- Agar ————————- 13.6 g
- Glycerin ——————– 10.0 ml
- Four milliliters of a 22.5% solution of cetrimide (0.9 g of hexadecyltrimethylammonium) can substitute for the cetyltrimethylammonium bromide.
- Final pH- 7.2
- Sterile inoculating loops or sticks
ii. UV light (360 nm)
iii. Positive control: P. aeruginosa ATCC 27853= growth; yellow-green to blue pigment
iv. Negative control: Escherichia coli ATCC 25922= inhibited
Procedure:
- Prepare a slanted cetrimide agar media in a test tube
- Pick a well isolated colony from cultured agar plate and streak the slant back and forth with inoculum loosely place the cap on the tube
- Incubate aerobically at 37 °C for up to 7 days.
- Observe for growth and pigment.
- If no pigment is visible, examine growth under UV light for the presence of fluorescein.
- If pigment is still not visible in UV light then incubate for 24 hrs in the dark to enhance pigment production.
Result interpretation:
- Positive result:
- Growth is observed with a yellow-green (fluorescein) to dark blue-green (pyocyanin) colored fluorescein
- P. aeruginosa grows on cetrimide agar and produces a blue-green (pyocyanin) pigment.
- Negative result: No growth is observed