Journal of Applied Bacteriology 1977,43,183-187
An Examination of the Effect of Three Phenolic Disinfectants on Mycobacterium tuberculosis IDA K. HEGNA University of Oslo, Institute of Pharmacy, Department of Microbiology, Blindern, Oslo 3, Norway Received 8 April 1976 and accepted 23 June 1977 The effect of a phenolic disinfectant (o-phenylphenol 45% w/w) with linseed oil soap or with soya oil soap on Mycobacterium tuberculosis was determined by three methods. Neither the geometrical dilution test nor the modified capacity use-dilution test revealed any differences between the two disinfectants. However, paradoxically both methods proved that the highest concentration of the disinfectants tested (3.5% v/v) exhibited a very low germicidal effect on M. tuberculosis, whereas lower concentrations showed a much better effect. When determining the tuberculocidal effects of various concentrations of the two disinfectants at different exposure times, the higher concentrations showed very low erects, even after the longest exposure time. At concentrations of 2.0 and 1.0% (v/v), the disinfectants displayed the most rapid effects. In the present investigation the disinfectant with the linseed oil soap seemed to destroy the cells more quickly than that with the soya oil soap. The third disinfectant containing p-chloro-mcresol and o-benzyl-p-chlorophenol with a total of 9.2% (w/w) phenols in a detergent system, did not display, when employing the capacity use-dilution test, the same phenomenon in the concentrations used, but the experiment showed that the recommended use-dilution concentration ought to be doubled.
THE BACTERICIDAL and fungicidal effects of 45% (w/w) phenolic disinfectant in an aqueous solution with respectively linseed oil soap and soya oil soap, and another phenolic disinfectant containing 9 ~ 2 %(w/w) phenols in a detergent system, have been determined (Hegna 1977). As the bactericidal value of phenols is said to be influenced by the soaps (Berry & Stenlake 1942; Berry & Briggs 1956) a further examination of the effects on Mycobacterium tuberculosis of the same three phenolic disinfectants was undertaken. M. tuberculosis is considered to be more resistant to chemical disinfectants than other vegetative bacteria (Bergan & Lystad 1971; Croshaw 1971), and a human strain of this micro-organism was selected as a test organism in the present work.
Materials and Methods Test bacterium The human M . tuberculosis used in these tests was isolated at the National Institute of Public Health, Oslo, and received at our Department for experimental use. The strain was grown on Lowenstein-Jensen medium which is made by the National Institute of Public Health, Oslo, Norway, and corresponds to the Oxoid formula (Anon. 1973). The stock inoculum was made from 6-8-week-old cultures which were harvested and ground in a mortar with a small amount of nutrient broth to give an even suspension. The latter was diluted in saline to give a known density which was determined in an EEL colorimeter (with neutral filter). The optical density of the inoculum corresponded to a suspension of a strain of Escherichia coli with 36 x lo8 cells/ml. [I871
184
I. K. HEGNA
Yeast suspension The yeast suspension was prepared as described elsewhere (Hegna 1977). Disinfectants These were as described in the preceding paper (Hegna 1977). Test procedures Three different tests were done at room temperature (ca. 22°C). After the exposure time was ended, 0.025 ml of the test solution was transferred to 0-25 ml normal horse serum and mixed immediately. Platinum loops (4 mm id.) full of the serum were spread on Lowenstein-Jensen medium and incubated at 37°C in a nearly horizontal position. The results were recorded after 8-9 weeks. Controls with no disinfectant added were included in each test. Transfers from these were made in parallel with those from the tests. Test I The highest concentrations (made in sterile tap water) used of the disinfectants A and B were 5.0% (v/v) and of C 4.0% (v/v). Each disinfectant was diluted geometrically in saline with 1 ml in each test tube. The test inoculum was prepared by suspending 10 ml of the stock inoculum in 6 . 7 ml of the following: (1) sterile distilled water; (2) sterile 5% (dry w/v) yeast suspension, and (3) sterile 20% (v/v) horse serum. To each test tube one drop (0.025 ml) of the test inoculum was transferred with a calibrated Pasteur pipette. After exposure for 8 min the same method described above was followed.
Test 11 A modified Kelsey & Sykes method (Bergan & Lystad 197 1; Hegna 1977) was used. The initial concentrations (made in sterile tap water) of A and B were 3-5, 1.75 and 0.875% (v/v). The concentrations of C were 6.0, 4.0 and 2.0% (v/v). The test inoculum was prepared by suspending each 10 ml stock inoculum in 6-7 ml 5% (w/v) yeast suspension. After the exposure of the organism to the germicides samples were taken by the methods described above. Test 111 Concentrations of 5.0, 4.0, 3.0, 2.0, 1.0 and 0.5% (v/v) of disinfectants A and B were made in sterile tap water. The test inoculum was prepared as described under Test 11. One ml was added to each 3 ml test solution, the suspension was well shaken, and samples removed at predetermined time intervals and treated as described above.
Results The results from Test I (Table 1) did not reveal any differences between the tuberculocidal effects of disinfectant A and B. A 5% (w/v) yeast suspension reduced the antitubercular effect more than did 20% horse serum. With an 8 min exposure time, both disinfectant A and B displayed tuberculocidal effects at dilutions 4, and &, but not at 4 and the highest concentration (5% v/v). Disinfectant C did not exhibit any effect at the highest concentrations (4% v/v) whereas dilutions f and did. Yeast and serum greatly reduced the lethal effect of C.
t
EFFECTS OF PHENOLS ON M . TUBERCULOSIS
185
TABLE1 The effect of various dilutions of three disinfectants on Mycobacterium tuberculosis* Dilutions
* The disinfectants were: A, o-phenylphenol in a linseed oil soap; B, o-phenylphenol in soya oil soap; and C, p-chloro-m-cresol and o-benzyl-p-chlorophenol in a detergent system. t 1, Inoculum diluted in water; 2, in 5% yeast cell suspension; 3, in 20% serum. Initial concentrations (Yo):A and B. 5.0; C, 4.0. +, Growth; -, no growth.
The same phenomenon was demonstrated when employing the modified Kelsey & Sykes capacity use-dilution test. At the two highest initial concentrations of A and B, the results did not display any tuberculocidal effect after the first incremental addition, whereas after the second and the third incremental additions the lethal effect appeared (Table 2). Table 3 indicated the presence of a slight difference between the effects of disinfectant A and B. The latter required a longer time to kill the test organism. However, both disinfectants exhibited the most rapid tuberculocidal effect at 1a0 and 2.0%, whereas there was only a slow effect at 0.5%. With an inoculum containing 'young' (4-6-weekold) M . tuberculosis cells the same difference between disinfectant A and B was demonstrated and the strongest effect appeared at the concentrations 1.0 and 2.0%. Between the 6th and 9th weeks 1-4 colonies developed in the medium which previously had no growth. The control tubes and other cultures displaying a heavy to medium growth, were positive after 14 d incubation. TABLE2 The effect of three disinfectants on Mycobacterium tuberculosis in the capacity use dilution test under the 'dirty' condition"' Initial concentration
--* b
a
Disinfectant
A B C
8t
1 1 0
18
2 0 0
28
8
0 0 2
18
0 0
4 6
0
1
28
0 1 4
\
C
8
0
3
2 6
18
1 1
4
1 2 1
28 6 4 3
* Initial concentration (%) of A and B: a, 3.5; b, 1.75; c, 0.875; of C: a, 6.0; b = 4.0; c = 2.0. Number of positive subcultures of a total of 14. t Exposure time, min.
I. K. HEGNA
186
TABLE3 The eflect of some concentrations of two disinfectants* at various exposure times on Mycobacterium tuberculosis Time of exposure (min) Concentrations of disinfectant 5%
1,”
4%
IA
3%
;{
IB
2%
(B”
I%
I*
0.5%
IB
:[
A
An Examination of the Effect of Three Phenolic Disinfectants on Mycobacterium tuberculosis IDA K. HEGNA University of Oslo, Institute of Pharmacy, Department of Microbiology, Blindern, Oslo 3, Norway Received 8 April 1976 and accepted 23 June 1977 The effect of a phenolic disinfectant (o-phenylphenol 45% w/w) with linseed oil soap or with soya oil soap on Mycobacterium tuberculosis was determined by three methods. Neither the geometrical dilution test nor the modified capacity use-dilution test revealed any differences between the two disinfectants. However, paradoxically both methods proved that the highest concentration of the disinfectants tested (3.5% v/v) exhibited a very low germicidal effect on M. tuberculosis, whereas lower concentrations showed a much better effect. When determining the tuberculocidal effects of various concentrations of the two disinfectants at different exposure times, the higher concentrations showed very low erects, even after the longest exposure time. At concentrations of 2.0 and 1.0% (v/v), the disinfectants displayed the most rapid effects. In the present investigation the disinfectant with the linseed oil soap seemed to destroy the cells more quickly than that with the soya oil soap. The third disinfectant containing p-chloro-mcresol and o-benzyl-p-chlorophenol with a total of 9.2% (w/w) phenols in a detergent system, did not display, when employing the capacity use-dilution test, the same phenomenon in the concentrations used, but the experiment showed that the recommended use-dilution concentration ought to be doubled.
THE BACTERICIDAL and fungicidal effects of 45% (w/w) phenolic disinfectant in an aqueous solution with respectively linseed oil soap and soya oil soap, and another phenolic disinfectant containing 9 ~ 2 %(w/w) phenols in a detergent system, have been determined (Hegna 1977). As the bactericidal value of phenols is said to be influenced by the soaps (Berry & Stenlake 1942; Berry & Briggs 1956) a further examination of the effects on Mycobacterium tuberculosis of the same three phenolic disinfectants was undertaken. M. tuberculosis is considered to be more resistant to chemical disinfectants than other vegetative bacteria (Bergan & Lystad 1971; Croshaw 1971), and a human strain of this micro-organism was selected as a test organism in the present work.
Materials and Methods Test bacterium The human M . tuberculosis used in these tests was isolated at the National Institute of Public Health, Oslo, and received at our Department for experimental use. The strain was grown on Lowenstein-Jensen medium which is made by the National Institute of Public Health, Oslo, Norway, and corresponds to the Oxoid formula (Anon. 1973). The stock inoculum was made from 6-8-week-old cultures which were harvested and ground in a mortar with a small amount of nutrient broth to give an even suspension. The latter was diluted in saline to give a known density which was determined in an EEL colorimeter (with neutral filter). The optical density of the inoculum corresponded to a suspension of a strain of Escherichia coli with 36 x lo8 cells/ml. [I871
184
I. K. HEGNA
Yeast suspension The yeast suspension was prepared as described elsewhere (Hegna 1977). Disinfectants These were as described in the preceding paper (Hegna 1977). Test procedures Three different tests were done at room temperature (ca. 22°C). After the exposure time was ended, 0.025 ml of the test solution was transferred to 0-25 ml normal horse serum and mixed immediately. Platinum loops (4 mm id.) full of the serum were spread on Lowenstein-Jensen medium and incubated at 37°C in a nearly horizontal position. The results were recorded after 8-9 weeks. Controls with no disinfectant added were included in each test. Transfers from these were made in parallel with those from the tests. Test I The highest concentrations (made in sterile tap water) used of the disinfectants A and B were 5.0% (v/v) and of C 4.0% (v/v). Each disinfectant was diluted geometrically in saline with 1 ml in each test tube. The test inoculum was prepared by suspending 10 ml of the stock inoculum in 6 . 7 ml of the following: (1) sterile distilled water; (2) sterile 5% (dry w/v) yeast suspension, and (3) sterile 20% (v/v) horse serum. To each test tube one drop (0.025 ml) of the test inoculum was transferred with a calibrated Pasteur pipette. After exposure for 8 min the same method described above was followed.
Test 11 A modified Kelsey & Sykes method (Bergan & Lystad 197 1; Hegna 1977) was used. The initial concentrations (made in sterile tap water) of A and B were 3-5, 1.75 and 0.875% (v/v). The concentrations of C were 6.0, 4.0 and 2.0% (v/v). The test inoculum was prepared by suspending each 10 ml stock inoculum in 6-7 ml 5% (w/v) yeast suspension. After the exposure of the organism to the germicides samples were taken by the methods described above. Test 111 Concentrations of 5.0, 4.0, 3.0, 2.0, 1.0 and 0.5% (v/v) of disinfectants A and B were made in sterile tap water. The test inoculum was prepared as described under Test 11. One ml was added to each 3 ml test solution, the suspension was well shaken, and samples removed at predetermined time intervals and treated as described above.
Results The results from Test I (Table 1) did not reveal any differences between the tuberculocidal effects of disinfectant A and B. A 5% (w/v) yeast suspension reduced the antitubercular effect more than did 20% horse serum. With an 8 min exposure time, both disinfectant A and B displayed tuberculocidal effects at dilutions 4, and &, but not at 4 and the highest concentration (5% v/v). Disinfectant C did not exhibit any effect at the highest concentrations (4% v/v) whereas dilutions f and did. Yeast and serum greatly reduced the lethal effect of C.
t
EFFECTS OF PHENOLS ON M . TUBERCULOSIS
185
TABLE1 The effect of various dilutions of three disinfectants on Mycobacterium tuberculosis* Dilutions
* The disinfectants were: A, o-phenylphenol in a linseed oil soap; B, o-phenylphenol in soya oil soap; and C, p-chloro-m-cresol and o-benzyl-p-chlorophenol in a detergent system. t 1, Inoculum diluted in water; 2, in 5% yeast cell suspension; 3, in 20% serum. Initial concentrations (Yo):A and B. 5.0; C, 4.0. +, Growth; -, no growth.
The same phenomenon was demonstrated when employing the modified Kelsey & Sykes capacity use-dilution test. At the two highest initial concentrations of A and B, the results did not display any tuberculocidal effect after the first incremental addition, whereas after the second and the third incremental additions the lethal effect appeared (Table 2). Table 3 indicated the presence of a slight difference between the effects of disinfectant A and B. The latter required a longer time to kill the test organism. However, both disinfectants exhibited the most rapid tuberculocidal effect at 1a0 and 2.0%, whereas there was only a slow effect at 0.5%. With an inoculum containing 'young' (4-6-weekold) M . tuberculosis cells the same difference between disinfectant A and B was demonstrated and the strongest effect appeared at the concentrations 1.0 and 2.0%. Between the 6th and 9th weeks 1-4 colonies developed in the medium which previously had no growth. The control tubes and other cultures displaying a heavy to medium growth, were positive after 14 d incubation. TABLE2 The effect of three disinfectants on Mycobacterium tuberculosis in the capacity use dilution test under the 'dirty' condition"' Initial concentration
--* b
a
Disinfectant
A B C
8t
1 1 0
18
2 0 0
28
8
0 0 2
18
0 0
4 6
0
1
28
0 1 4
\
C
8
0
3
2 6
18
1 1
4
1 2 1
28 6 4 3
* Initial concentration (%) of A and B: a, 3.5; b, 1.75; c, 0.875; of C: a, 6.0; b = 4.0; c = 2.0. Number of positive subcultures of a total of 14. t Exposure time, min.
I. K. HEGNA
186
TABLE3 The eflect of some concentrations of two disinfectants* at various exposure times on Mycobacterium tuberculosis Time of exposure (min) Concentrations of disinfectant 5%
1,”
4%
IA
3%
;{
IB
2%
(B”
I%
I*
0.5%
IB
:[
A
