3.17.

11 Cetrimide Test

I. PRINCIPLE
Cetrimide is a quaternary ammonium, cat-
ionic detergent that is toxic to most bac-
teria, except Pseudomonas aeruginosa
and a few others (1, 2). When cetrimide is
in contact with bacteria, nitrogen and
phosphorus are released from the bacterial
cell. Organisms other than P. aeruginosa
and a few other pseudomonads are unable
to withstand this germicidal activity.
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 pyover-
din (fluorescein) by P. aeruginosa.

II. MICROORGANISMS TESTED Isolated colonies of non-glucose-fermentative, Gram-negative rods that are sugges-
tive of P. aeruginosa

III. MEDIA, REAGENTS, AND A. Medium 2. Four milliliters of a 22.5% solution

SUPPLIES 1. Agar slants containing the follow- of cetrimide (0.9 g of hexadecyltri-
ing ingredients per liter of de- methylammonium) can substitute
ionized water for the cetyltrimethylammonium
bromide.
pancreatic digest of
3. Final pH, 7.2
gelatin ..................... 20.0 g
4. Store at 2 to 8⬚C.
K2SO4 ......................... 10.0 g
B. Supplies
MgCl2 ...........................1.4 g
1. Sterile inoculating loops or sticks
cetyltrimethylammonium
2. Wood’s or UV light (360 nm) or
bromide .....................0.3 g
short-wavelength (254-nm) UV
agar ........................... 13.6 g
light (preferred)
glycerin ....................... 10.0 ml

IV. QUALITY CONTROL A. Perform QC on each new lot or shipment of media prior to putting it into use.
B. Inspect agar for evidence of freezing, contamination, cracks, dehydration, and
bubbles prior to storage and before use.
C. Organisms
1. P. aeruginosa ATCC 27853—growth; yellow-green to blue pigment
2. Escherichia coli ATCC 25922—inhibited

V. PROCEDURE A. Streak the slant back and forth with inoculum picked from the center of a well-
isolated colony.
B. Place cap loosely on tube.
C. Incubate aerobically at 35 to 37⬚C for up to 7 days.
D. Observe for growth and pigment.
E. If no pigment is visible, examine growth under UV light for the presence of
fluorescein.

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Cetrimide Test 3.17.11.2

V. PROCEDURE (continued) F. If negative for pigment at 24 h, incubate for additional days at 25⬚C in the dark
to enhance pigment production.

VI. INTERPRETATION A. Positive: growth. Optionally a yellow-green (fluorescein) to dark blue-green

(pyocyanin) color may be observed.
B. Negative: no growth

VII. REPORTING RESULTS A. P. aeruginosa is definitively identified if an oxidase-positive, Gram-negative

rod grows on cetrimide agar and produces a blue-green (pyocyanin) pigment.
B. Pseudomonas fluorescens and Pseudomonas putida may also grow and may
produce a fluorescent pigment on this medium (2) but are separated from P.
aeruginosa because they do not grow at 42⬚C.

VIII. LIMITATIONS A. Growth on this medium alone is not sufficient for identification of P. aeruginosa
to the species level, since other non-glucose-fermenting species (e.g., Achro-
mobacter xylosoxidans subsp. xylosoxidans and Alcaligenes faecalis) may grow.
Pigment must also be present.
B. Lack of growth on cetrimide agar does not rule out an identification of P. ae-
ruginosa.

REFERENCES 1. Lowburg EJL. 1955. The use of cetrimide

product in a selective medium for Pseudomo-
nas pyocyanea. J Clin Pathol 8:47–48.
2. Weyant RS, Moss CW, Weaver RE, Hollis
DG, Jordan JG, Cook EC, Daneshvar MI.
1996. Identification of Unusual Pathogenic
Gram-Negative Aerobic and Facultatively An-
aerobic Bacteria, 2nd ed, p 6. Williams &
Wilkins, Baltimore, MD.