NITROCEFIN TEST FOR BETA-LACTAMASE PRODUCTION

The production of beta-lactamase enzymes by Gram negative bacteria including E. coli, P. aeruginosa and Klebsiella species is an important mechanism by which the organisms evade both in vivo and in vitro antimicrobial onslaught. Beta-lactamase production by resistant test isolates can be evaluated for beta-lactamase production using the Nitrocefin test sticks (Oxoid, UK).

The nitrocefin stick is impregnated with a solution of nitrocefin, phosphate buffer and dimethysulphoxide. One end of the stick is coloured black (for handling) while the other end of the stick (coloured red) serves as the test point. Test isolates for beta-lactamase detection should be brought to room temperature prior to carrying out the test. A well separated representative colony of the test isolates should be used for the test.

The test isolates used for nitrocefin test for beta-lactamase detection should be previously cultured on nutrient agar plates. The nitrocefin stick should be rotated several times around the representative colonies of the test isolate in order to pick up the cells; and because the reaction requires moisture to occur, one or two drops of distilled water should be added to the test culture before bringing the nitrocefin stick in contact with the test isolate.

The impregnated end of the nitrocefin stick (ladened with the test isolate) should be examined for 5-15 minutes for a change in colour production. A positive reaction is shown by the development of a pink-red colour while a negative reaction is indicated as no colour change.

Absence of a colour change indicates that the test organism did not produce beta-lactamase enzyme while the presence of a pink-red colour indicates that the test organism produced beta-lactamase enzyme phenotypically. The colour of the nitrocefin stick covered with the test isolate should be compared with an unused nitrocefin stick in order to ensure correct reading and interpretation of the test result.