DECARBOXYLASE AND DIHYDROLASE TESTS
Each substrate is sold separately..
K080 Arginine tablets - 50 ea
K1080 Arginine WEE-TABS - 28 ea
K380 Lysine tablets - 50 ea
K1380 Lysine WEE-TABS - 28 ea
K500 Ornithine tablets - 50 ea
K1500 ODC WEE-TABS - 28 ea.
K475 Ninhydrin in DMSO - 4.0 ml.
PRINCIPLE:
Decarboxylases attack specific amino acids and, by removing carboxyl groups, convert the amino acids to aliphatic amines forming CO2 as a second product. The decarboxylation of lysine (LDC) and ornithine (ODC), resulting in the formation of cadaverine and putrescine respectively, and the production of ammonia by arginine dihydrolase(ADH) create an alkaline pH shift which changes the color of the indicator present in the tablet. The tablets contain the respective amino acids in a mixture of salts correctly buffered for each test and a pH indicator which changes color as the reaction progresses.
INGREDIENTS / MSDS:
Each product contains the specific substrate with inert fillers. None of the tablets contain any materials known at this time to be hazardous.
STORAGE:
Store at room temperature.
MATERIALS REQUIRED:
All tests require fresh 24 hour growth on plated culture media. Consult the Manual of Clinical Microbiology for recommended media for the specimen. The following items are required but not provided: small glass tubes (e.g. 12 X 75) for large tablets ; inoculating loop; sterile pipette; distilled water, pH 7.0-7.2.
SETUP PROCEDURE:
To perform the tests, a heavy suspension of the organism is made in distilled water in a small tube and the tablet is added. For multiple tests, one suspension may be made in 1-2 mls of water then dispensed into the other tubes. Use 0.5 ml water with the WEE-TAB and 1.0 ml water with the regular tablets. The tube is then incubated at 37C. Use a glass tube if available. (See Discussion)
INTERPRETATION: ADH:
Arginine frequently reacts within 30 minutes with a positive red reaction from the phenol red indicator. WEE-TABS should be held 4-6 hours and regular tablets 12-24 hours to verify negative. Ninhydrin is not used to confirm the arginine reaction. If needed, the WEE-TABS may be held up to 24 hours and the regular tablets up to 48 hours.
INTERPRETATION:LDC / ODC REGULAR TABLETS:
Observe the LDC test at intervals up 24 hours. The brom cresol purple indicator in the LDC turns blue in a positive test. ODC has a phenol red indicator which turns pink. No change in color indicates a negative test. Positive reactions may be confirmed. (see Discussion)
INTERPRETATION:ODC / LDC WEE-TABS:
WEE-TABS should be observed at 4-6 hours. A red color indicates a positive ODC test. If the test is negative, a yellow color will develop due to the acidification of the dextrose present in the medium. The LDC WEE-TABS will have the characteristic blue color if positive and yellow negative.
DISCUSSION:
WEE-TAB decarboxylase test results are usually final at 4-6 hours and confirmation is optional, although we recommend doing the ninhydrin procedure. WEE-TABS are packed in plastic tubes which chloroform will melt. If confirming, drop the small test tube into a larger glass one before adding the reagent. All tests may be held up to 24 hours. If the test is still yellow at 24 hours, the results are negative and no confirmation is required. If the lysine or ornithine test has changed at 24 hours, the Ninhydrin procedure MUST be done to confirm a true positive result. Some organisms (such as non-fermentors) produce an alkaline reaction from deamination of the amino acid causing the appearance of a positive decarboxylase reaction. The decarboxylation should be verified through the use of the ninhydrin reaction. While the amino acids are soluble in water, the amines are readily extracted into chloroform. If a solution of ninhydrin in chloroform is added to an aqueous solution of cadaverine or putrescine in a strongly alkaline medium, the amines move into the chloroform. This is indicated by the appearance of a deep violet color.
NINHYDRIN PROCEDURE:
1) Prepare the Ninhydrin Reagent by adding 0.4 ml. of NINHYDRIN solution (K475) to 25 ml chloroform. The diluted reagent has the same expiration date as the original solution.
2) Add one drop of KOH followed by diluted Ninhydrin reagent in direct proportion to the test; 1 ml of reagent is used with the regular tablet, 0.5 ml with WEE-TABS. Allow the tube to stand without shaking for 10-15 minutes. Development of a purple color in the lower chloroform layer is a positive test. Disregard the color which develops on the upper aqueous phase upon addition of the 40% KOH solution.
QUALITY CONTROL:
Each lot of tablets or strips should be tested prior to use with organisms which produce known reactions such as those listed below. The Manual of Clinical Microbiology should be consulted for detailed reactions and identification charts. All finished tests should be discarded in a manner conforming with accepted laboratory procedures for biohazardous material.
EXPECTED REACTIONS:
Organism ARG ODC LDC
Enterobacter cloacae ATCC13047 + + -
Klebsiella pneumoniae ATCC33495 - - +
Serratia marcescens ATCC8100 - + +
Consult the Manual of Clinical Microbiology for a more complete list of reactions for identifying organisms.