Note ________________________________ A NEW HEAT-STABLE ACID PHOSPHATASE TEST FOR MYCOBACTERIA,
Summary ________________ The heat-stable (70° C) acid phosphatase test performed by the method of Kind and King is a simple method for differentiating Mycobacterium kansasii, M. marinum, M. gastri, M. nonchromogenicum, and M. triviale from other slowly growing mycobacteria, and M. fortuitum from other rapidly growing acid-fast bacilli.
In a previous report, it was shown that the test for heat-stable acid phosphatase activity, performed on filter paper using the 11-naphthylphosphoric aciddiazonium o-dianisidine color reagent, was a rapid and simple method for the differential identification of mycobacteria (1). This test has been introduced by the Committee on Classification of Mycobacteria, The Japanese Society for Tuberculosis, as a tool for distinguishing mycobacteria (2). A number of investigators have communicated to us (1) the difficulty in obtaining the reagents commercially, and (2) the need for some other method of demonstrating heat-stable acid phosphatase activity using more readily available reagents. Accordingly, other methods for detection of this enzyme have been investigated. The ·procedure of Kind and King (3) for estimation of plasma phosphatase has provided results comparable to those reported earlier (1).
This report describes our experience with this new method for determining heat-stable acid phosphatase activity in a broad range of mycobacterial species. A total of 379 strains of mycobacteria, representing both slowly growing and rapidly growing species, was investigated. The species distributions of these strains are indicated in table I. Subcultures of all test organisms were grown on 1 per cent Ogawa egg medium (4); slow growers were incubated for 2 weeks and rapid growers for one
(Received in original form March 11, 1976 and in revised form May 13, 1976) 1 Requests for reprints should be addressed to Dr. H. Saito, Department of Microbiology and Immunology, Shimane Medical College,ll34 Ohtsu-cho, Izumo 693, Shimane Prefecture, Japan.
week. Mycobacterium marinum and M. chelonei subsp. chelonei were incubated at 33° C and all other cultures were grown at 37° C. The heat-stable acid phosphatase test was performed in the following manner. A loopful (3 mm inside diameter) of bacteria was scraped off and triturated against the inner wall of a test tube (15 X 150 mm) to prepare a homogeneous bacterial suspension in 0.5 ml of saline. The tubes first were heated quickly to 70° C in a beaker of water, then transferred to a 70° C water bath, held at this temperature for 30 min, and then cooled in running water. To the bacterial suspension was added 0.5 ml of 0.2M citrate buffer (pH 4.9) and 0.5 ml of O.OIM disodium phenylphosphate aqueous solution. The tubes were placed in a 37° C water bath for I hour. To the mixture was then added in order: 0.5 ml each of 0.5N NaOH, 0.5M NaHC0 3 , 0.6 per cent 4-amino-antipyrine, and 2.4 per cent potassium ferricyanide. A positive reaction was recorded when the reaction mixture developed a color ranging from faint pink to deep red. The disodium phenylphosphate solution with a few drops of added toluene was stable for one month at 4° C. When stored in brown bottles at 4o C the antipyrine and ferricyanide reagents were stable for a reasonable time, but the exact duration of full activity was not determined. The results of the test are summarized in table I. Among the slowly growing mycobacteria, none of the strains of M. tuberculosis complex (M. tuberculosis and M. bovis), or the scotochromogens (M. scrofulaceum and M. gordonae) yielded a positive reaction. Within the photochromogens, all strains of M. kansasii and M. marinum were positive, whereas all strains of M. simiae were negative. Within the nonphotochromogenic, niacin-negative mycobacteria, none of the strains of M. avium, M. intracellulare, M. xenopi, or M. terrae gave a positive reaction, whereas all strains of M. gastri, M. triviale, and, except for one strain, M. nonchromogenicum gave positive reactions. Among the rapidly growing mycobacteria, 24 of 30 strains of M. fortuitum gave positive reactions. With the exception of only one strain each of M. chelonei subsp. chelonei, M. rhodesiae, and M. flavescens, all other rapid growers were negative.
• • • The results of the present study are in good agreement with those obtained by the previous study per-
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 114, 1976
407
408
NOTE
TABLE 1 HEAT .STABLE ACID PHOSPHATASE OF MYCOBACTERIA
Species Slow growers Tuberculosis complex
Number of Strains
Acid Phosphatase Positive
Neg ative
Mycobactericum tuberculosis M. bovis
23 13
0 0
23 13
Phot
Summary ________________ The heat-stable (70° C) acid phosphatase test performed by the method of Kind and King is a simple method for differentiating Mycobacterium kansasii, M. marinum, M. gastri, M. nonchromogenicum, and M. triviale from other slowly growing mycobacteria, and M. fortuitum from other rapidly growing acid-fast bacilli.
In a previous report, it was shown that the test for heat-stable acid phosphatase activity, performed on filter paper using the 11-naphthylphosphoric aciddiazonium o-dianisidine color reagent, was a rapid and simple method for the differential identification of mycobacteria (1). This test has been introduced by the Committee on Classification of Mycobacteria, The Japanese Society for Tuberculosis, as a tool for distinguishing mycobacteria (2). A number of investigators have communicated to us (1) the difficulty in obtaining the reagents commercially, and (2) the need for some other method of demonstrating heat-stable acid phosphatase activity using more readily available reagents. Accordingly, other methods for detection of this enzyme have been investigated. The ·procedure of Kind and King (3) for estimation of plasma phosphatase has provided results comparable to those reported earlier (1).
This report describes our experience with this new method for determining heat-stable acid phosphatase activity in a broad range of mycobacterial species. A total of 379 strains of mycobacteria, representing both slowly growing and rapidly growing species, was investigated. The species distributions of these strains are indicated in table I. Subcultures of all test organisms were grown on 1 per cent Ogawa egg medium (4); slow growers were incubated for 2 weeks and rapid growers for one
(Received in original form March 11, 1976 and in revised form May 13, 1976) 1 Requests for reprints should be addressed to Dr. H. Saito, Department of Microbiology and Immunology, Shimane Medical College,ll34 Ohtsu-cho, Izumo 693, Shimane Prefecture, Japan.
week. Mycobacterium marinum and M. chelonei subsp. chelonei were incubated at 33° C and all other cultures were grown at 37° C. The heat-stable acid phosphatase test was performed in the following manner. A loopful (3 mm inside diameter) of bacteria was scraped off and triturated against the inner wall of a test tube (15 X 150 mm) to prepare a homogeneous bacterial suspension in 0.5 ml of saline. The tubes first were heated quickly to 70° C in a beaker of water, then transferred to a 70° C water bath, held at this temperature for 30 min, and then cooled in running water. To the bacterial suspension was added 0.5 ml of 0.2M citrate buffer (pH 4.9) and 0.5 ml of O.OIM disodium phenylphosphate aqueous solution. The tubes were placed in a 37° C water bath for I hour. To the mixture was then added in order: 0.5 ml each of 0.5N NaOH, 0.5M NaHC0 3 , 0.6 per cent 4-amino-antipyrine, and 2.4 per cent potassium ferricyanide. A positive reaction was recorded when the reaction mixture developed a color ranging from faint pink to deep red. The disodium phenylphosphate solution with a few drops of added toluene was stable for one month at 4° C. When stored in brown bottles at 4o C the antipyrine and ferricyanide reagents were stable for a reasonable time, but the exact duration of full activity was not determined. The results of the test are summarized in table I. Among the slowly growing mycobacteria, none of the strains of M. tuberculosis complex (M. tuberculosis and M. bovis), or the scotochromogens (M. scrofulaceum and M. gordonae) yielded a positive reaction. Within the photochromogens, all strains of M. kansasii and M. marinum were positive, whereas all strains of M. simiae were negative. Within the nonphotochromogenic, niacin-negative mycobacteria, none of the strains of M. avium, M. intracellulare, M. xenopi, or M. terrae gave a positive reaction, whereas all strains of M. gastri, M. triviale, and, except for one strain, M. nonchromogenicum gave positive reactions. Among the rapidly growing mycobacteria, 24 of 30 strains of M. fortuitum gave positive reactions. With the exception of only one strain each of M. chelonei subsp. chelonei, M. rhodesiae, and M. flavescens, all other rapid growers were negative.
• • • The results of the present study are in good agreement with those obtained by the previous study per-
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 114, 1976
407
408
NOTE
TABLE 1 HEAT .STABLE ACID PHOSPHATASE OF MYCOBACTERIA
Species Slow growers Tuberculosis complex
Number of Strains
Acid Phosphatase Positive
Neg ative
Mycobactericum tuberculosis M. bovis
23 13
0 0
23 13
Phot
