The Effectiveness of Hot Hypochlorite Solutions to destroy M tuberculosis

Transcription

1 EFFECTIVENESS OF HOT HYPOCHLORITES OF LOW ALKALINITY IN DESTROYING MYCOBACTERIUM TUBERCULOSIS S. M. COSTIGAN General Laboratories, Division of Pennsylvania Salt Philadelphia, Pennsylvania Manufacturing Company, Received for publication February 21, 1936 The feeling expressed by some writers that sterilization by hypochlorites of milk plant apparatus, dairy equipment and restaurant dishes was not complete because of the inability of these products to destroy Mycobacterium tuberculosis stimulated a study to determine the effectiveness of hot hypochlorite solutions in killing this organism. Tilley (1920) found that Dakin's solution, 1:500 (10 parts of available chlorine per million) was ineffective in 10 minutes, while 1:200 dilution (25 parts of available chlorine per million) was effective in destroying M. tuberculosis. Whittaker (1928) showed that the calcium hypochlorite in concentrations up to 500 parts of available chlorine per million for 2 minute used to rinse pint milk bottles contaminated with sputum from tuberculous patients was not effective in destroying M. tuberculosis. This work was done at room temperature. Rice (1933) reported that a strongly alkaline hypochlorite, antiformin, 1 part of undiluted antiformin to 5 parts of sputum from tuberculous patients, destroyed this organism in 10 minutes. We found that when hypochlorite solutions of low alkalinity containing 50, 100, and 200 parts available chlorine per million are heated to a temperature ranging between 54 to 580C. for a period of three hours there is no loss in their available chlorine concentrations. Even boiling these solutions does not diminish their available chlorine. Solutions of the hypochlorite containing 50 and 200 parts of 57

2 58 B. M. COSTIGAN available chlorine per million and temperatures of 500, 550 and 60'C. with exposures of 2, 1, 21 and 5 minutes were chosen because at these temperatures and times, M. tuberculosis in water is not destroyed. ha addition to this, concentrations of 50 to 200 parts available chlorine per million of water are used as bactericides for dairy and food equipment. TECHNIC The organism used for the first series of inoculations was a 2 months' old culture of M. tuberculosis human strain No. 4777, obtained from the American Type Culture Collection. As it was impossible to get an even suspension of later cultures of No. 4777, a culture (A 14) for the second run was obtained from the Henry Phipps Institute, Philadelphia, and one for the third run, from Saranac Laboratory, Saranac Lake, New York. The growths were washed from the slants with sterile normal saline and placed in flasks containing glass beads. The suspension was shaken vigorously to break up the clumps, and then so diluted that 2 cc. added to the 20 cc. of disinfectant dilution would give a dosage of 20,000,000 organisms when 1 cc. of the final centrifuged mixture was injected into a guinea pig. The hypochlorite was heated in a water bath to the desired temperature and held for a few minutes before the suspension of organisms was added. At intervals of 2, 1, 21 and 5 minutes, 2 cc. of the suspension were withdrawn and 1 cc. was pipetted into each of two centrifuge tubes containing 5 cc. of sterile N/10 sodium thiosulphate to act as an antichlor. The suspension was centrifuged for 10 minutes and the supernatant removed, leaving 1 cc. in the tube which was injected into the left inguinal lymph gland region of a 250-gram guinea pig. Two animals, therefore, were injected for each time, temperature and concentration. It was believed desirable to determine the effect of the culture upon the rate of deterioration of the available chlorine of the solution. Four hundred million organisms were added to 20 cc. of the disinfectant dilution when it had reached the desired temperature. These tests were run in a manner similar to those from which the guinea pig inoculations were made. The solutions were titrated before and after the tests were made.

3 DESTRUCTION OF MYCOBACTERIUM TUBERCULOSIS 59 Control tests were made under identical conditions except that water instead of hypochlorite solutions was used. In later experiments, the procedure was the same except the organism suspension was filtered after shaking, and then counted. The animals were autopsied eight to twelve weeks after injection, and smears for acid fast bacilli were made from all organs showing lesions. In one small group of animals which were TABLE 1 Effectiveness of solutions containing 50 parts available chlorine per million parts of water in destroying Mycobacterium tuberculosis LEFTSIEO TEMPERATURE TIME INGUINAL LUNGS LIVER SPLEEN SNOCULATION LYMPH GLANDINUATO "C. minutes +~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Grouping of the results of 3 runs of the experiment. +, lesions with acid-fast bacilli; 4, lesions with acid-fast bacilli in 1 run negative in 2 runs;-, no lesions. tuberculin tested, the results of the tuberculin test supported the post mortem findings. The hypochlorite used was B-K1 which is essentially a solution of sodium hypochlorite containing 3.50 per cent, and is alkaline with calcium hydrate 0.12 per cent. At 200C. using the Leeds and Northrup glass electrode the ph of the 50 p.p.m. solution was found to be 8.35, and that of the 200 p.p.m. solution Manufactured by General Laboratories.

4 60n S. M. COSTIGAN RESULTS Table 1 shows the effectiveness of solutions containing 50 p.p.m. of available chlorine in destroying M. tuberculosis. At 50'C., this solution failed to kill in 2 minute. All animals showed tuberculosis with acid-fast bacilli in the left inguinal lymph glands. In one run the animals showed lesions and acid-fast bacilli in the lungs, liver and spleen with abscess formation at the site of inoculation. With 1-minute exposures, the animals in one TABLE 2 Effectiveness of solution. containing B00 parts available chlorine per million parts of water in destroying Mycobacterium tuberculosis LIE"T SITS 0F TEIMPEATURE TIM INGUINAL LUNGS LIVER SPLEEN INOCULATION LYMPH GLAND C. minutes 50 { -: Grouping of the results of 3 runs of the experiment. +, lesions with acid-fast bacilli; 1, lesions with acid-fast bacilli in 1 run negative in 2 runs; -, no lesions; *, lesions, no acid-fast bacilli. run showed tuberculosis with lesions and acid-fast bacilli in the left inguinal lymph glands while all others were negative. Animals receiving the organisms subjected to hypochlorite solutions of 50 parts available chlorine at 50'C. in times of 21 and 5 minutes did not show tuberculosis. When the solution was heated to 550C., exposures of 2 minute gave irregular results. In some runs characteristic lesions

5 DESTRUCTION OF MYCOBACTERIUM TUBERCULOSIS 61 developed with acid-fast bacilli in the inguinal lymph glands and spleen and at the site of inoculation, while in other runs the TEMPERATURE OC { TIME minutes i i i LEFT INGUINAL LYUTH GLAND TABLE 3 Water-control LUNGS LIVER SPLEEN SITE OF INOCULATION Grouping of the results of 3 runs of the experiment. +, lesions with acid-fast bacilli; L, lesions with acid-fast bacilli in 1 run negative in 2 runs; -, no lesions. TABLE 4 Deterioration of hypochlorite in 5 minutes after addition of organisms TEMPERATURE 50 P.P.M. 200 P.P.M. ".c {0J l0 Start End { Start End { Start End inoculated animals did not develop tuberculosis. Animals receiving organisms which had been exposed for 1, 2' and 5 minutes to the 50 parts available chlorine per million at 550C.

6 62 S. M. COSTIGAN were negative, indicating that the organisms had been killed. Likewise complete destruction even at 2 minute at 60'C. was obtained. Table 2 shows the effectiveness of solutions containing 200 p.p.m. of available chlorine in destroying M. tuberculosis. This solution gave irregular results when heated to 50'C. for 2 minute. In one run the animals receiving the organisms exposed for 1 minute developed lesions at the site of inoculation. No acidfast bacilli could be demonstrated nor could the disease be reproduced by injection of the emulsified lesions into susceptible animals. Whether this is the same phenomenon observed by Sabin and Anderson (1932) after the injection of the phosphatide fraction of these organisms cannot be said. All the remaining 500C. animals as well as all of the 55TC. and 60TC. were negative. Table 3 gives the findings in the control animals. All showed lesions with acid-fast bacilli in the left inguinal lymph glands, the region of inoculation. All of the 500C. animals showed lesions and acid-fast bacilli in the spleen as well as at the site of inoculation. In some runs, lesions with acid-fast bacilli were present in the lungs, liver and spleen in all animals while in some the lesions were absent. This was attributed to the differences of the virulence of the cultures used. Table 4 shows the effect of the number of organisms used in this experiment upon the deterioration of the hypochlorite solutions during a period of 5 minutes with the temperatures employed. CONCLUSIONS Heating suspensions of Mycobacterium tuberculosis (human strain) to 60TC. for 5 minutes does not destroy the organism. The hypochlorite solution of low alkalinity, containing 50 parts available chlorine per million parts of water heated to 500C., destroys Mycobacterium tuberculosis in 21 minutes; heated to 550C. it destroys it in 1 minute; and heated to 600C., it destroys it in 2 minute. The hypochlorite solution of low alkalinity containing 200 parts available chlorine per million parts of water heated to 500C. destroys Mycobacterium tuberculosis in 1 minute; heated to 550C. and 600C., it destroys it in 2 minute.

7 DESTRUCTION OF MYCOBACTERIUM TUBERCULOSIS 63 The author wishes to acknowledge the kind assistance given by Messrs. J. W. Yates and W. A. Hadfield in this work. REFERENCES RICE, J. W Tenth Annual Report, The Pennsylvania Association of Dairy and Milk Inspectors (1934). SABIN, F. R., AND ANDERSON, R. J Physiol. Rev., 12, 141. TILLEY, F. W Jour. Agr. Res., 20, TILLEY, F. W., MACDONALD, A. D., AND SCHAFFER, J. M Jour. Agr. Res., 42, WADE, E. M., ARCHBALD, R. W., AND WHITTAKER, H. W Jour. Bact., 15,