Cat. no. Z51 Beta-Lactamase Test 20 tests/kit


Hardy Diagnostics Beta-Lactamase Test is used to detect the presence of the bacterial enzyme beta-lactamase by the rapid acidometric method.


Hardy Diagnostics Beta-Lactamase Test is a means of detecting the enzyme beta-lactamase, which confers penicillin resistance to various bacterial organisms by cleaving the beta-lactam ring of penicillins and cephalosporin antibiotics. A wide variety of bacteria produce this enzyme, including both gram-positive and gram-negative organisms. This acidometric method is recommended for use in testing beta-lactamase production by Neisseria gonorrhoeae, Haemophilus species and Staphylococcus species.


Each tube included in the kit contains the following in the reconstituted mixture, per liter of solvent:*

Penicillin 15.0gm
Sodium Chloride 5.0gm
Trisodium Citric Acid 1.5gm
Trisodium Phosphate 0.3gm
Phenol Red 18.0mg

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-30°C. Products should not be used if there are any signs of contamination, deterioration, if the expiration date has passed, or if the penicillin mixture turns yellow when the distilled water is added. Do not expose to excessive heat or moisture. Keep the cap tightly sealed with desiccant inside when not in use.



Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Using a pasteur pipette, add 0.1ml (approximately 5 drops) of distilled water (pH 6.8 to 7.2) to the Beta-Lactamase tube. Mix by gently tapping the bottom of the tube. Using a heavy inoculum (several colonies) of an overnight culture of organism, make a suspension in the Beta-Lactamase tube. Mix well, withdrawing the inoculation loop slowly and carefully to avoid creating an aerosol upon removal of the loop from the tube. All tests should be performed at room temperature (15-30°C).

Several hours incubation may be required (up to 24 hours) for bacteria in which the enzyme is inducible (Staphylococcus aureus strains). The amount of time required to produce sufficient enzyme varies for differing strains of S. aureus. Some strains produce smaller amounts of the enzyme or are slower inducers. Most beta-lactamase-positive strains of S. aureus, however, will produce sufficient enzyme to test positive in 10 to 60 minutes.

Production of a positive reaction usually occurs within two minutes for bacteria in which the enzyme is not inducible (Neisseria gonorrhoeae and Haemophilus influenzae). Mixed infections of both beta-lactamase producing and non-producing gonococci have been encountered. Therefore, when testing N. gonorrhoeae, an inoculum of at least 10 colonies of isolated organism should be used in order to insure a good sampling. When testing Neisseria species, a negative test should be held for 60 minutes before a final reading is made. When testing Haemophilus species, the tube should be held for 15 minutes before making a final reading.


A positive test is indicated by the mixture turning yellow in color within the time limits stated above. A negative reaction is indicated when no change in color occurs (mixture remains pink). Appearance of a slight reddish-orange color is also indicative of a negative reaction.


Some enteric bacteria produce the beta-lactamase enzyme as well, but the significance of this occurrence is not clear. These organisms should not be tested for beta-lactamase production.(6)

The production of beta-lactamase may be stimulated in S. aureus by growing the organism adjacent to a methicillin or oxacillin disk before testing.(2)

This acidometric method is not recommended for testing beta-lactamase activity of Moraxella spp., Enterococcus spp. or anaerobes. Consult listed reference for more information.(7)

This Beta-Lactamase Test will give a rapid indication of beta-lactamase activity for S. aureus, N. gonorrhoeae, and H. influenzae. These results will, in most cases, predict the outcome of susceptibility tests with the appropriate antibiotics. However, this product should not be used as a replacement for antimicrobial disk diffusion or MIC testing, as other factors influence the results of such tests.


Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Reaction
Haemophilus influenzae
ATCC ® 33533
Yellow color change observed:
Positive (Ampicillin-resistant)
Haemophilus influenzae
ATCC ® 49247
No color change (remains pink):
Negative (Ampicillin-sensitive)
Staphylococcus aureus
ATCC ® 43300
Yellow color change observed:
Positive (Penicillin-resistant)
Staphylococcus aureus
ATCC ® 25923
No color change (remains pink):
Negative (Penicillin-sensitive)

User Quality Control


Beta-Lactamase Test powder should appear white in color. The reconstituted reagent should appear pink in color. Do not use if the reconstituted, uninoculated reagent is yellow.

Beta-Lactamase Test
Beta-Lactamase Test showing positive (left) and negative (right) results.
LEFT: Ampicillin-resistant Haemophilus influenzae (ATCC ® 33533).
RIGHT: Ampicillin-sensitive Haemophilus influenzae (ATCC ® 49247).
Test was conducted using an overnight culture of
H. influenzae strains grown in elevated CO2 at 35°C.


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Jorgensen, J., et al. 1977. Antimicrob. Agents Chemother.; 1:1087-1088.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

7. Performance Standards for Antimicrobial Disk Susceptibility Tests, 6th ed., M2-A11, Vol. 33, No. 1. 2013. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

8. Thornsberry, C. and L.A. Kirven. 1974 Antimicrob. Agents Chemother.; 6:53-6:54

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