Nitrocefin Disks

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Detection of Beta-Lactamase
Description

Hardy Diagnostics Nitrocef Disks™ are intended for use in the rapid testing of isolated colonies of Neisseria gonorrhoeae, Moraxella (Branhamella) catarrhalis, Staphylococcus spp., Haemophilus influenzae, Enterococcus spp., and some anaerobic bacteria for the production of beta-lactamase.

SUMMARY

It has long been recognized that certain bacteria possess the ability to produce enzymes that inactivate beta-lactam antibiotics. Some hydrolyze penicillin class antimicrobics and are described as penicillinases. Others hydrolyze the cephalosporin class antimicrobics and are described as cephalosporinases. Some bacteria produce enzymes that hydrolyze both cephalosporins and penicillins.

Rapid beta-lactamase tests can yield clinically relevant information earlier than an MIC or disk diffusion test. Several clinical tests have been devised to detect beta-lactamases. These tests include the iodometric method, the acidometric method, and chromogenic substrates.(6) Iodometric methods are suitable for testing N. gonorrhoeae.(16) Acidometric methods produce acceptable results with Haemophilus spp., N. gonorrhoeae and staphylococci. Nitrocefin, a chromogenic cephalosporin, can be used to test Neisseria gonorrhoeae, Moraxella (Branhamella) catarrhalis, Staphylococcus spp., Haemophilus influenzae, Enterococcus spp., and some anaerobic bacteria, and has been found effective in detecting all known beta-lactamases. Nitrocefin is the only reliable test for detecting beta-lactamase producing Enterococcus spp. Nitrocef Disks™ are impregnated with nitrocefin, a chromogenic cephalosporin. As the amide bond in a beta-lactam ring is hydrolyzed by a beta-lactamase, nitrocefin changes color from yellow to red. Bacteria which produce beta-lactamase in significant amounts produce this yellow to red color change on the Nitrocef Disk™. These beta-lactamases are capable of inactivating "penicillinase-labile-penicillins", such as amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin.Nitrocef Disks™ are intended for the rapid testing of isolated colonies of Neisseria gonorrhoeae, Moraxella (Branhamella) catarrhalis, Staphylococcus spp., Haemophilus influenzae, Enterococcus spp., and anaerobic bacteria of the genera Bacteroides, Clostridium, Porphyromonas, Fusobacterium, and Prevotella. The beta-lactamase test is of little value for many organisms (e.g., Enterobacteriaceae) as organisms within a taxonomic group, or even a single strain, can produce a diversity of enzymes with different substrate specificities.

PROCEDURE

1. Using sterile forceps, remove a Nitrocef Disk™ from the vial and place it on an empty petri dish or microscopic slide. Immediately place the remaining unused disks into the freezer.

2. Prior to inoculation, allow the Nitrocef Disk™ to equilibrate to room temperature.

3. Moisten each disk with one drop of sterile deionized water. Alternatively, condensation on the lid of a petri dish can be used to hydrate the disk by transferring a drop with a sterile loop. Do not over saturate the disk, which could dilute the reagent.

Note: Water is critical to the development of the color reaction, if the disk begins to dry out it may be necessary to rehydrate the reaction area of the Nitrocef Disk™ with a small amount of water.

4. With a sterilized loop or applicator stick remove a well-isolated colony and spread it on the disk surface. An alternate procedure requires the use of sterile forceps to wipe a pre-moistened disk across a colony surface. Regardless of which method is used, ensure that the disk surface is exposed to the cell paste.

5. Observe the inoculated disk for the development of an orange/red color.

INTERPRETATION OF RESULTS

A positive beta-lactamase result is recorded when the Nitrocef Disk™ changes in color from its original yellow to orange or red. Most positive bacterial strains will produce a color change within 5 minutes. Some staphylococci, however, may take up to 60 minutes for a positive result.A positive beta-lactamase result predicts the following:

1. Resistance to penicillin, ampicillin and amoxicillin among Haemophilus spp., N. gonorrhoeae and M. catarrhalis.

2. Resistance to penicillin, as well as acylamino-, carboxy-, and uriedo-penicillins among staphylococci and enterococci.

A negative beta-lactamase result is recorded when the Nitrocef Disk™ remains yellow in color. A negative result does not rule out resistance due to other mechanisms.  

Cat No: HDZ7301

Size: 50 discs

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