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BETA-LACTAMASE IDENTIFICATION STICKS

Code: BR0066
Oxoid Identification Sticks, Beta-Lactamase are used for the detection of beta-lactamases produced by Gram-positive and Gram-negative bacteria. The tips of the sticks are impregnated with Nitrocefin, a chromogenic cephalosporin (Glaxo Research 87/312). The sticks are convenient to use and overcome the necessity for preparing fresh reagents daily.

Formula
One end of each stick is impregnated with a solution of Nitrocefin, phosphate buffer and dimethylsulphoxide. The opposite end is coloured black to identify the correct end for handling. The colour also identifies the product.

General Introduction and Intended Uses
Nitrocefin is the chromogenic cephalosporin developed by Glaxo Research Limited coded 87/312; 3-(2,4 dinitrostyrl) - 6R, 7R) -7-(2 thienylacetamido)-ceph-3-em-4 carboxylic acid, E-isomer. This compound exhibits a rapid distinctive colour change from yellow to red as the amide bond in the beta- lactam ring is hydrolysed by a beta-lactamase (E.C.3.5.2.6.); it is sensitive to hydrolysis by all known lactamases produced by Gram-positive and Gram-negative bacteria.

Demonstration of Beta-Lactamase Activity by Bacterial Cells
Nitrocefin hydrolysis has been found to be highly efficient in detecting beta-lactamase producing isolates of Neisseria gonorrhoeae,^1,2,3 Haemophilus influenzae,^2,4,5,6 staphylococci^5,6 and bacteroides^7,8,9,10,11. It should be emphasised that the efficiency of Nitrocefin tests in predicting the beta-lactam susceptibilities of other micro-organisms is at present unproven. In a positive reaction the beta-lactamase enzymes hydrolyse the amide bond in the beta-lactam ring. This is detected by a colour change on the end of the stick from yellow to pink/red.

Lactamase Induction
It should be noted that some organisms will not exhibit lactamase unless the enzyme has been induced by exposure to a beta-lactam antimicrobial. In such circumstances the organism should be tested from growth adjacent to beta-lactam antimicrobial discs or from agar containing beta-lactams.
Nitrocefin hydrolysis will give a rapid indication of beta-lactamase activity and the result so obtained will, in most cases, predict the outcome of susceptibility tests with these antimicrobials. However, it should not entirely replace conventional susceptibility testing as other factors also influence the results of such tests.

Technique
1. Remove the container from the freezer and allow it to reach room temperature.
2. Select a well separated representative colony from the primary isolation medium.
3. Remove one stick (colour coded black) from the container and holding the coloured end, touch the colony with the impregnated tip of the stick, rotate the stick, picking up a small mass of cells.
4. Place the inoculated tip of the stick between the lid and the base of the inverted plate.
5. The reaction requires moisture. The inoculated tip of the stick should be placed in the moisture condensate in the lid. If condensate is not available in the inverted lid add one or two drops of distilled water to the lid and moisten the tip of the stick.
6. Examine the reagent impregnated tip of the stick for up to five minutes and, if negative re-examine after fifteen minutes.
Note: Some staphylococci may take up to 1 hour before reaction shows a colour change.
7. A positive reaction is shown by the development of a pink/red colour. No colour change is observed with organisms that do not produce beta-lactamase. To ensure correct reading the colour of the stick should be compared to an unused stick.

Caution
Organisms producing pigmented colonies i.e. Staphylococcus citreus, may give false positive results. It is recommended therefore that when pigmented colonies are to be tested Nitrocefin in solution - code SR0112, should be used.

Quality Control
Reference strains should be tested to control the product at appropriate intervals.

Positive control:
Beta-lactamase producing staphylococcus

Negative controls:
Non beta-lactamase producing staphylococcus

Storage Temperature
Store below -10°C.

References
1. O’Callaghan C. H., Morris A., Kirby S. M. and Shingler A. H. (1972) Antimicrob. Ag. & Chemother. 1. 283-288.
2. Shannon K. and Phillips I. (1980) J. Antimicrob. Chemother. 6. 617-621.
3. Sng E. H., Yeo K. L., Rajan V. S. and Lim A. L. (1980) Br. J. Vener. Dis. 56.311-313.
4. Bell S. M. and Plowman D. (1980) Lancet i. 279.
5. Montgomery K., Raymundo L. and Drew W. L. (1979) J. Clin. Micro. (9) 205-207.
6. Lucas T. J. (1979) C. Clin. Pathol. 32. 1061-1065.
7. Gabay E. L., Sutter V. L. and Finegold S. M. (1981) J. Antimicrob. Chemother. 8. 413-416.
8. Timewell R., Taylor E. and Phillips I. (1981) J. Antimicrob. Chemother. 7. 137-146.
9. Bourgault A. M. and Rosenblatt J. E. (1979) J. Clin. Micro. 9. 654-656.
10. Lee D. T. F. and Rosenblatt J. E. (1983) Diagn. Microbial. Infect. Dis. 1. 173-175.
11. Markowitz S. M. (1980) Antimicrob. Ag. & Chemother. 17. 80-83.