Methods for storing oxidase test reagents.

APPLIED MICROBIOLOGY, June 1969, p. 933-934 Copyright © 1969 American Society for Microbiology Vol. 17, No. 6 Printed in U.S.A. Methods for Storing Oxidase Test Reagents A. L. BARRY' AND K. L. BERNSOHN Departments of Microbiology and Medicine, University of California, Irvine, College of Medicine, Irvine, California 92650, and Los Angeles County General Hospital (Unit 11), Los Angeles, California 90033 Received for publication 18 March 1969 Oxidase test reagents are notoriously unstable, but they can be held ready for use if frozen or if reagent-impregnated filter paper is kept dry. The oxidase test is an important differential procedure in diagnostic bacteriology, especially useful for quickly confirming a tentative morphological identification of Pseudomonas aeruginosa or Neisseria species. This can be done as a filter paper spot test (5-8) or by flooding colonies with one of several reagents, with or without alphanaphthol (3, 4). When fresh solutions of the reagents are used, the test results are quite reliable, but variable results may be obtained if the solutions are not prepared frequently. This report describes two simple methods for storing the reagents to provide readily available material which will give reliable test results. Three stored reagents were compared with fresh solutions and reagent-impregnated, dried filter papers. Our studies confirmed the observation of Carpenter et al. (1) that the dried crystals of the oxalate salt of dimethyl-p-phenylenediamine had a longer shelf life than the monohydrochloride salt of this substance. Both had a longer shelf life than tetramethyl-p-phenylenedmine dihydrochloride, which lost significant activity after 1 year of storage. Once dissolved, the reagents tend to dis- color rapidly, but this process is retarded when 0.1% ascorbic acid is added (2). It is possible to overcome the auto-oxidation of these three reagents by freezing 1% aqueous solutions in 1to 2-ml samples suitable for regular use. Such samples (without ascorbic acid) had no loss of activity after more than 1 year of storage. A filter paper spot test is the most practical method for examining large numbers of individual colonies selected from agar plates without contaminating the culture by flooding the surface with the reagent. For this purpose, filter paper may be saturated with freshly prepared or recently thawed samples of the desired reagent. Such moist papers must be replaced after a few hours because the reagent becomes oxidized rapidly. Alternatively, reagent-impregnated filter paper can be dried and stored for prolonged periods of time I Present address: University of California at Davis, School of Medicine, Department of Medicine, Davis, 95616. with no loss of activity (6, 7). Small filter paper discs containing p-aminodimethylaniline are available from Difco and BBL. In our experience, these discs tend to darken greatly upon storage and this reduces their reliability. Filter paper strips which are said to contain both dimethyl-pphenylenediamine and alpha-naphthol are also available from the General Diagnostics Division of Warner-Chilcott Laboratories. Preliminary tests indicated that these strips are not as sensitive as the method of Kovacs (5), but the results are very useful as long as the strips are properly stored and appropriate controls are used. These strips are probably the most reliable commercial product available, but they are relatively expensive and inconvenient for testing large num. bers of colonies, since only one or two tests can be performed on each strip. Therefore, we devised a simple, inexpensive method for preparing sheets of reagent-impregnated, dried filter paper which can be held as long as 6 months with no loss of activity. A large number of colonies can be spotted onto one piece of filter paper (6 to 8 cm in diameter) which is kept at the workbench ready for use when needed. The following method has proven to be suitable for routine use in our clinical laboratory. (i) Saturate Whatman no. 1 filter papers with a 1% aqueous solution of dimethyl-p-phenylenediamine oxalate (other reagents were less satisfactory). (ii) Allow the papers to dry rapidly by hanging on strings or glass rods; contact with metal clips or tacks must be avoided. (iii) Place the dry papers in screw-cap jars with a generous supply of dessicant, as moisture darkens the reagent and obviates its value. With a clean nichrome or platinum loop, smear part of a colony onto the dry paper. The development of a red color within 10 sec indicates a positive reaction. One piece of filter paper may be held in a glass petri dish for several days or until it begins to darken. For routine use, these dried papers are simple and quick and provide reliable results. Occasionally it is worthwhile to confirm a nega- 933 934 NOTES tive test with the more sensitive method of Kovacs (5) or to examine mixed cultures by flooding the colonies with the oxalate salt. This is best accomplished with recently thawed (3 to 4 hr) samples of the frozen reagents. LITERATURE CITED 1. Carpenter, C. M., L. G. Suhrland, and M. Morrison. 1947. The oxalate salt of p-aminodimethylaniline, an improved reagent for the oxidase test. Science 105:649-650. 2. Cowan, S. T., and K. J. Steel. 1966. Manual for the identification of medical bacteria. Cambridge University Press, London. APPL. MICROBIOL. 3. Ewing, W. H., and J. G. Johnson. 1960. The differentiation of Aeromonas and C27 cultures from Enterobacterlaceae. Int. Bull. Bacteriol. Nomencl. Taxon. 10:223-230. 4. Gaby, W. L., and C. Hadley. 1957. Practical laboratory test for the identification of Pseudomonas aeruginosa. J. Bacteriol. 74:356-358. 5. Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178:703. 6. Leclerc, H., and H. Beerens. 1962. Une technique simple de mise en evidence de l'oxydase chez les bacteries. Ann. Inst. Pasteur Lille 13:187-192. 7. Rodgers, K. B. 1963. Oxidase reaction. Lancet 2:686. 8. Steel, K. J. 1961. The oxidase reaction as a taxonomic tool. J. Gen. Microbiol. 25:297-306.  (...truncated)