Code: SR0112

Reagents
SR0112 Vial of lyophilised Nitrocefin, containing 1mg Nitrocefin.
SR0112A Rehydration fluid. The vial contains 1.9ml of phosphate buffer (0.1M, pH 7.0) and 0.1ml of dimethylsulphoxide.

Directions
Reconstitute the contents of one vial of lyophilised Nitrocefin SR0112 by adding the entire contents (2ml) of one vial of rehydration fluid SR0112A. This yields a working Nitrocefin solution of 500mg/ml, (approx 10^-3 M) suitable for most applications.

Precautions
Nitrocefin, particularly in solution, is very light sensitive. The solution may be stored at -20°C for up to two weeks. INGESTION OR INHALATION, OR CONTACT WITH THE SKIN AND EYES SHOULD BE AVOIDED.

General Introduction and Intended Uses
Nitrocefin is the chromogenic cephalosporin developed by Glaxo Research Limited (code 87/312; 3-(2,4 dinitrostyrl) - (6R,7R-7-(2-thienylacetamido)-ceph-3-em-4-carboxylic acid, E-isomer)¹. This compound exhibits a rapid distinctive colour change from yellow (max at pH 7.0 = 390nm) to red (max at pH 7.0 = 486nm) 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. This characteristic reaction forms the basis of a number of methods suitable for diagnostic use.

Apart from its use in giving rapid indication of beta-lactamase potential, the reagent has been found extremely useful for the detection of beta-lactamase patterns from bacterial cell extracts on iso-electric focusing^2,3,4 and has been used in inhibition studies in development work on beta-lactamase resistant antibiotics⁵.

Description of Use
Demonstration of beta-lactamase activity in bacterial cells.
Nitrocefin degradation should be used to give a rapid indication of beta-lactam inactivating systems and the result so obtained will, in most cases, predict the outcome of susceptibility tests with beta-lactam anti-microbials. However, it should not entirely replace conventional susceptibility testing as other factors also influence the results of such tests, and on occasion intrinsic resistance to beta-lactam antimicrobials has not been correlated with production of beta-lactamase⁶. Nitrocefin degradation has been found to be highly efficient in detecting beta-lactamase producing isolates of Neisseria gonorrhoeae^7,8, Haemophilus influenzae^7,9,10,11 and staphylococci^10,11.

Excellent results have also been obtained with certain anaerobic bacteria, notably with Bacteroides species^13,14,15. It should be emphasised that the efficacy of the Nitrocefin tests in predicting the beta-lactam susceptibilities of other micro- organisms is at present unproven. Another chromogenic cephalosporin, PADAC (Hoechst-Roussel) was not as effective as Nitrocefin in detecting staphylococcal beta-lactamase¹².

Technique
Rehydrate the Nitrocefin as directed, and use this solution in the following ways:
1 Direct Plate Method¹
Add one drop of the Nitrocefin solution on to the surface of the colony. If the isolate is a high beta-lactamase producer then the colony and the surrounding area will quickly turn red. To detect a weak beta-lactamase producer the plate should then be incubated for 30 minutes before being reported as negative.
2 Slide Method¹
Add one drop of the Nitrocefin solution on to a clean glass slide. Using a sterile loop, pick one colony from the plate and emulsify into the Nitrocefin drop. Report as positive if the colour changes from yellow to red within 30 minutes (protect the slide from desiccation during the waiting period).
3 Broth Method¹
Add four drops of Nitrocefin solution to 1ml of the grown culture. Report as positive if the colour changes to red within 30 minutes.
4 Broken Cell Method¹
Sonicate 1ml of the culture in order to break open the cells. Add 4 drops of Nitrocefin solution. Report as positive if the colour changes to red within 30 minutes.
5 Paper Disc Spot Test¹⁰
A Whatman No.1 filter paper disc (diameter 7cm) is placed in a petri dish and impregnated with Nitrocefin solution (0-5ml). This impregnated paper is generally usable for one day, but should be kept away from light to avoid spontaneous degradation. An isolated colony is applied to the impregnated paper with a loop; a pink to red reaction developing within 15 minutes indicates beta-lactamase presence.

• Detection of beta-lactamase activity on gels
  Methods for preparing extracts containing the beta-lactamase activities from bacterial cells and the technique for analytical iso-electric focusing have been described by Matthew et al.² The developed gels are stained by applying Whatman No.54 paper impregnated with the Nitrocefin solution². Focused bands in the gel with beta-lactamase activity appear pink on a yellow background.
• Determination of beta-lactamase activity by spectrophotometric assay
  The working solution of Nitrocefin (500µg/ml) is diluted tenfold in buffer (0.1M phosphate; 1mM EDTA, pH 7.0). Spectrophotometric assays for beta-lactamase are carried out measuring changes in wavelength at 486nm. The molar extinction coefficient of Nitrocefin at this wavelength is 20,500. Test samples of the finished product for performance with control cultures.

References
1. O’Callaghan C. H., Morris A., Kirby S. M. and Shingler A. H. (1972) Antimicrob. Ag. & Chemother. 1. 283-288.
2. Mathew M., Harris A. M., Marshall M. J. and Ross G. W. (1975) J. Gen. Microbiol. 88. 169-178.
3. Sparks J. and Ross G. W. (1981) J. Med. Microbiol. 15. p. iv.
4. King A., Shannon K. and Phillips I. (1980) Antimicrob. Ag. & Chemother. 17. 165-169.
5. Guay R., Letarte R., Pechere J. C. and Roy B. (1980) IRCS Med. Science 8. 209.
6. Markowitz S. M. (1980) Antimicrob. Ag. & Chemother. 6. 80-83.
7. Shannon K. and Phillips I. (1980) J. Antimicrob. Chemother. 6. 617-621.
8. Sng E. H., Yeo K. L., Rajan V. S. and Lim A. L. (1980) Br. J. Vener. Dis. 56. 311-313.
9. Bell S. M. and Plowman D. (1980) Lancet i. 279.
10. Montgomery K., Raymundo L. and Drew W. L. (1979) J. Clin. Micro. 9. 205-207.
11. Lucas T. J. (1979) J. Clin. Pathol. 32. 1061-1065.
12. Anhalt J. P. and Nelson R. (1982) Antimicrob. Ag. & Chemother. 21. 993-994.
13. Gabay E. L., Sutter V. L. and Finegold S. M. (1981) J. Antimicrob. Chemother. 8. 413-416.
14. Timewell R., Taylor E. and Phillips I. (1981) J. Antimicrob. Chemother. 7. 137-146.
15. Bourgault A. M. and Rosenblatt J. E. (1979) J. Clin. Micro. 9. 654-656.