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Culture Media Supplements

BROAD SPECTRUM BETA-LACTAMASE MIXTURE

Code: SR0113

Broad Spectrum Beta-Lactamase Mixture SR0113 is a mixture of beta-lactamase (E.C.3.5.2.6.) from Bacillus cereus1 569/ H9.
The enzymes are presented as a freeze-dried powder containing buffer and zinc salts. Each vial contains minima of 500 units beta-lactamase I and 50 units of beta-lactamase II. 1 unit of enzyme activity will hydrolyse 1 m mol of substrate per minute at pH 7.0 and at 25°C; beta-lactamase I is assayed using benzyl penicillin in the presence of EDTA, and beta- lactamase II using cephalosporin C in the presence of Zn2+.

Definition of Units of Enzyme Activity
The scientific literature describes a number of methods which are used to measure and define a unit of penicillinase of beta-lactamase activity2. Note that 1 IU of activity = 600 Levy units of activity.

Application
There are four major uses of this preparation of enzymes.
1. Inactivation of beta-lactam antibiotics in blood or other tissue samples prior to routine microbiological examination2,3,4.
2. Inactivation of beta-lactam antibiotics in blood and other tissue samples prior to the microbiological estimate of aminoglycosides or other non-lactam antibiotics4,5.
3. The inactivation of beta-lactam antibiotic preparations to enable sterility testing to be carried out before the administration of such preparations to patients undergoing therapy with immuno-suppressants, or who have a naturally low level of immunity6.
4. Assessment of the susceptibility of new beta-lactam antibiotics to inactivation by lactamase.

Methods
1 Blood Culture Procedures
Inject 5 ml of sterile distilled water into a vial of enzyme mixture and mix gently. Add 1 ml of this solution aseptically to the blood culture bottle, preferably before or immediately after inoculation with the blood sample (5-10 ml).

2 Microbiological Assay of Non-Lactam Antibiotics
1ml of the beta-lactamase enzyme solution should be added aseptically to 1 ml of blood sample or serum. This should be incubated at 30°C for a period of time depending on the beta-lactam antibiotic present. A minimum time would be 5 minutes and a maximum 60 minutes. After incubation, the blood or serum samples should be applied to wells in previously seeded antibiotic assay plates in the normal manner.

Stability of Reagents
Solutions of the enzyme will remain active for several days when stored at 4°C or several weeks when stored at minus 20°C.
Repeated freezing and thawing should be avoided. However, it is not advisable to store the solution for long periods because of the possibility of contamination.

References
1. Davis R. B., Abraham E. P. and Melling J. (1974) Biochem. J. 143. 115-127.
2. Waterworth P. M. (1973) J. Clin. Path. 26. 596-598.
3. Selwyn S. (1977) J. Antimicrob. Chemother. 3. 161-168.
4. Newson S. W. B. and Walshingham B. M. (1973) J. Med. Microbiol. 6. 59-66.
5. Sabath L. D., Casey J. I., Ruch P. A., Stumpf L. L. and Finland M. (1971) J. Lab. Clin. Med. 78. 457-463.
6. Code of Federal Regulations, Title 21, Part 436, Sec.436.20 U.S. Govt. Printing Office, Washington, D.C.

 
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