affected by the ph of the medium, the dependence of the bacteriostasis by dyes

Transcription

1 THE BACTERICIDAL AND BACTERIOSTATIC ACTION OF CRYSTAL VIOLET C. E. HOFFMANN AND OTTO RAHN Bacteriological Laboratory, New York State College of Agriculture, Cornell University, Ithaca, N. Y. Received for publication September 30, 1943 In 1933, Ingraham verified the theory of Dubos (1929) that dyes produce bacteriostasis by poising the medium at a potential unsuitable for multiplication. This explained the long lag period produced by dyes and accounted for the fact that the rate of multiplication is not retarded by the dye after the potential is once adjusted again by some metabolic action of the bacteria. Apparently it contradicted Steam and Steam's theory (1924) that the dye combines chemically with certain proteins in the cell. As the oxidation-reduction potential is greatly affected by the ph of the medium, the dependence of the bacteriostasis by dyes upon the reaction of the medium was readily explained. In repeating some of Ingraham's experiments, we could not confirm her statement that all bacteria remained viable during the enforced lag period. In every experiment made with Escherichia coli and Streptococcus kactis, the number of cells decreased greatly, usually to less than 1% of the inoculum. Further studies made it clear that the bactericidal and the bacteriostatic effects are due to different chemical reactions. METHODS The dye used throughout was Crystal Violet number C.I. 681 of National Aniline and Chemical Co., with a total dye content of 90%. All dilutions were made from a 1:100 alcoholic stock dye solution. All media, unless otherwise stated, contained 0.75% tryptone, 0.5% glucose, and phosphate buffer to make a final buffer concentration of 1%. The inoculations were made from cultures 22 to 26 hours old except where otherwise stated. Two methods of cell counting were used, the plate count, which offers the only possibility of measuring the bactericidal effect, and turbidity measurements by means of a nephelometer. This method gave accurate and easily reproducible results. BACTERICIDAL ACTION The bactericidal effect of crystal violet was measured by plate counts. Figure 1 shows that with concentrations above 2.5 p.p.m., Streptococcus lactis is killed in logarithmic order, the death rate increasing proportionally to the dye. At 2.5 p.p.m., the rate decreases after 99.9% of the bacteria are dead. With 1 p.p.m. and less, the death rate is no longer dependent upon concentration, and the bacteria finally start multiplying. It seemed necessary to ascertain that the cells were really dead, not just prevented from multiplication by a coating of the dye. The dye could be removed completely by shaking with activated charcoal. To test the viability, broth 177

2 178 C. E. HOFFMANN AND OTTO RAHN with different concentrations of the dye was heavily inoculated with streptococci, and plate counts were made at the start and after certain intervals. After 5 hours, a portion of each flask was poured into a flask containing dry sterile charcoal. Plate counts were made immediately, and after 1.5, 3, 19 and 24 hours. Part of the remaining crystal violet culture was poured into charcoal after 24 hours, and counted 1, 6 and 24 hours later. The results are shown in table 1. In each case, charcoal- prevented further death, but it did not increase the plate count, and the bacteria had to undergo a lag phase before starting to multiply. There was no evidence that death by crystal violet was reversible. In the 1:10,000 dilution (omitted in table 1) all cells were dead by five hours. ' <-~~~~~~- 0j 7 05 ppmm.67 ppm~~5pp 1 10 \o0c304ogc2 wher thi randg2ae the deathrteimceaesaththconcentrations,c y w2itetimshr and taken from figure 1 where 99.9%O of the cells have been killed. This proportionality between concentration and death rate holds true only for high concentrations. Table 3 gives the death rates (K) of Streptococcus lactis in

3 BACTERIOSTATIC ACTION OF CRYSTAL VIOLET various dye concentrations taken from several experiments. calculated from the formula The death rates are K = 1 loinoculum t survivors While in higher concentrations, the death rate increases proportionally to the amount of dye, it is independent of the concentration and approximately con- TABLE 1 Effect of charcoal on the viability of cells of S. lactis All numbers indicate plate counts per ml. 179 TIME3 TIME CONTROL DILUTIONS O CRYSTAL VIOLET ~~NO C.V. - 1:50,000 1:100,000 1: 200,000 1:400,000 kours Start 29,000,000 21,000,000 24,050,000 25,400,000 28,500, ,000,000 17,500, ,500,000 12,500,000 5* 630,000,000 1, ,500 2,890,000 6,900, ,000, ,000 1,340,000 3,000, ,390,000, , , ,000 24t 1,070,000,000 Less than 100Less than 100Less than , ,000,000 Less than 100 Less than 100 Less than 10 14, ,000,000 Less than 100 Less than 100 Less than 10 8, ,000,000 Less than 100 Less than ,300 *Charcoal After 5 Hours *Charcoal 640,000, ,000 2,580,000 4,600,000 added 1.5 1,340,000,000 1, ,000 3,500,000 5,200, ,630,000,000 2, ,000 3,900,000 5,700, ,670,000,000 2,060,000,000 2,050,000, ,240,000,000 2,450,000,000 2,140,000,000 2,100,000,000 2,600,000,000 tcharcoal After 24 Hours tcharcoal 890,000,000 Less than 100 Less than 100 Less than ,000 added 1 1,090,000, Less than 100 2,000 21, ,000,000 Less than ,000 58, ,000,000 2,100 1,410,000,000 1,380,000,000 1,750,000,000 stant below 2 p.p.m. This independence is especially noticeable with E. coli which is far more tolerant to this dye. The logarithmic survivor curves are concave downwards as is the rule with dilute disinfectants (Rahn, 1943a). Furthermore, this death rate with dilute dyes is more rapid than the rate calculated from the concentration (table 3). It is constant because the bacteria die from another cause which kills them more qui6kly. The dye still destroys the bacteria, but that effect is completely overshadowed by another stronger lethal

4 180 C. E. HOFFMANN AND OITO RAHN effect. This effect is probably the result of the abnormal oxidation-reduction potential. This potential is independent of the dye concentration. It is not quite clear how an unfavorable potential can kill, but on the other hand, it is very dificult to explain a disinfection which is independent of the concentration of the disinfectant. Effect of ph. As the ph of the medium has a marked effect on the bacteriostatic action of crystal violet, an experiment was made to determine whether the TABLE 2 Death times and concentration exponent of S. lactis in crystal violet CRYSTAL VIOLET: ONE PART DIN CONCENTRATION DECATH TIME IN HOURS CONCENTRATION EXPONE.NT 50, , , , Average.0.86 TABLE 3 Death rates of Streptococcus lactis with crystal violet CRYSTAL VIOLET CONCENTRATIONS TIEc t 1: 1,500,000 1: 1,000, p.pm. I p.pm. 1:500,000 2 p.p.n. 1:400, ppdm. 1:200,000 S p.pm. 1: 100, p.p.m. 1:50, p.pm. 1:10, p.pm. * ~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~< i Average <4.25 Calculated death rate effect was also as great on the bactericidal action of the dye. Using Staphylococcu18 aureus and E. coli, Bittenbender and co-workers (1940) showed a very slight effect of ph on the dye action, but they stated that it was strongest at the lower acidities. To test this, dye dilutions in tryptone glucose broth with 1.25, 2.5, and 5.0 p.p.m. crystal violet were made. Each dilution was divided into three parts and the ph adjusted to 5.90, 6.90, and 7.60 with K2HP04 and KH2PO4 to a final phos-

5 BACTERIOSTATIC ACTION OF CRYSTAL VIOLET phate concentration of 1%. A 24-hour culture of Streptococcus lactts was diluted and added to the prepared flasks to give between one and ten million cells per ml. Plate counts were made at the start and at appropriate intervals. Table 4 shows only a slight effect of acidity on the death rate, but contrary to Bittenbender's results, the death rates in our experiments became higher with increased ph. 181 DYE CONCENTRATION TABLE 4 Effect of the ph on the bactericidal action of crystal violet Death rates of Streptococcus lactis ph S p.,.m (0.070) Downloaded from FIG. 2. SURVIVOR CURVES OF STREPTOCOCCUS LACTIS CELLS OF DIFFERENT AGES WITH CRYSTAL VIOLET Effect of the age of the cells. With all disinfectants and such agents as heat and cold, young cells are less resistant to the harmful effects than are the older cells. This is true also for crystal violet in strong solution. Figure 2 shows the effect upon cells of S. lactis, 6 hrs. 12 hrs. and 24 hrs. old. In 1:500,000 and higher dilutions, the cells of different ages died at the same rate. However, at 1:100,000 dilution (10 p.p.m.) the youngest cells were killed most rapidly and the older cells more slowly. This confirmed the previous observation that from about 4 p.p.m. up, the crystal violet acts strictly as a disinfectant for S. lactis. In on April 9, 2018 by guest

6 182 C. E. HOFFMANN AND OTTO RAHN similar tests with E. coli, only the former action was observed-that is, the cells all had the same death rate because the dye concentrations used were not high enough for the characteristic disinfectant action to take place. Crystal violet then acts as a disinfectant in a certain range and as a bacteriostatic agent in another, these ranges varying greatly with the test organism. These experiments on the true disinfection by crystal violet show this dye to behave like all other strong disinfectants. It kills proportionally to the dye concentration, it kills young cells more rapidly than old ones, and it is very little affected by changes in acidity. It may well be that it reacts by combining with certain vital compounds of the protoplasm as Steam and Steam (1924) assumed. In dilute solutions, however, crystal violet differs. It does not kill proportionally to the dye concentration; death is independent of the concentration, and more rapid than a calculation according to proportionality would indicate. Old cells are as sensitive as young ones, and an increased efficiency by increased ph becomes noticeable. This stage of dye action is the intermediate stage between bactericidal and bacteriostatic effect. BACTERIOSTATIC ACTION The bacteriostatic effect is characterized by a period of no visible development followed by multiplication at a rate which shows no retardation by the dye. This was explained by Dubos (1929) as due to an ability of the bacteria to counteract the unfavorable oxidation-reduction potential poised by the dye. Ingraham (1933) stated that crystal violet did not really change the potential of the medium, but held it at a point unfavorable for multiplication. Bacteria differ greatly in their sensitivity. Fusiform bacteria grow readily in 200 p.p.m. of crystal violet, (Slanetz and Rettger, 1933) while S. lactis is killed by 2 p.p.m. The length of the lag extension is directly proportional to the concentration of the dye (fig. 3) until a point is reached where the length of lag permits all cells to be killed before the potential can be changed by the bacteria. It is noteworthy that in this range, the dye concentration controls the length of the lag period, but not the death rate (table 3 and figure 1). This supports the theory that the extended lag is due to a poising effect at an unfavorable potential. As a change in dye concentration does not change the ph of the medium, increasing amounts of the dye in the bacteriostatic range would not change the factors leading to the death of the cells, but would greatly increase the difficulty of bringing the medium back to the correct potential for multiplication. The difference between this range and the strict disinfectant range has been emphasized before. Ingraham had observed that the length of the lag phase was-inversely proportional to the logarithm of the number of cells in the inoculum. Our experiments fully confirm Ingraham's observation. This holds true for the controls as well.! No satisfactory explanation has been yet proposed to explain this fact, unless we assume that the entire recovery process takes place within the cell (Rahn, 1943b).

7 BACTERIOSTATIC ACTION OF CRYSTAL VIOLET Young cells have a shorter lag phase than older cells. The question arose as to whether this would also apply to lag produced by the dyes, and figure FIG. 3. EFFECT OF ph ON THE LENGTH OF THE LAG PERIOD OF STREPTOCOCCUS LACMTi WITH CRYSTAL VIOLET Downloaded from on April 9, 2018 by guest HOURS J FIG. 4. GROWTH CURVES OF ESCHERICHIA COLI CELLS OF DIFFERENT AGES WITH 1:10-,000 CRYSTAL VIOLET shows that with E. coli, in every case the young cultures recovered first, with usually not too great a difference in length of lag between the 6 and 12 hour cultures, but with a marked increase in lag between these and the 24 hour

8 184 C. E. HOFFMANN AND OTTO RAHN cultures. About 99% of the cells died in each culture. Nevertheless, the youngest cultures initiated growth immediately, while the older cells, though the bactericidal action had ceased, were not able to initiate growth for a considerably longer time. With S. lactis, the same effect was observed except that the bactericidal effect did not stop until the cultures actually started the logarithmic growth phase. Recovery thus depends on the ability of the cell to bring the medium to a correct potential for multiplication. Old cells are more resistant to harmful conditions than young cells, but young cells have a greater rate of metabolism than older cells, which enables them to adjust the potential more rapidly. If the inhibiting effect of crystal violet were due to a poising action on the medium, oxygen should influence the length of the lag phase. Ingraham FIG w 0 I Control C Sealed) a. M (icontrol COpen) co 40 S._ i // *>Of.~tof 1:500 C,. 0 (~~~~~~~~~~~~~~~~~Open) 20.'~~~~~~~' ~~~~ ~CM. 1: No Growth~ ip20 4 ~~~5 6 HOURS GROWTH CURVES OF STREPTOCOCCUS LACTIS WITH AND WITHOUT OXYGEN IN THE PRESENCE OF CRYSTAL VIOLET found that if cells were allowed to multiply in crystal violet, and then were filtered off, the filtered medium was as toxic to a second inoculation as it had been to the first. It is quite possible that the filtering re-introduced oxygen and re-established the unfavorable potential. Two procedures were used to study the effect of oxygen. In one case, oxygen tension was increased by 'continuous aeration, and in the other case, oxygen tension was reduced by steam heating, rapid cooling, and sealing with vaspar. Aeration increased the length of lag for both E. coli and S. lactis. For E. coli, aeration,increq$sed the Jength of lag with 1.67 p.p.m. crystal violet from 9 to 19 hours, for Streptococcus lactis with 0.5 p.p.m. of dye from 19 to 27 hours. Figure 5 shows the effect of decreased oxygen tension on S. lactis. The length of lag isg appi~6lrnately halved by the removal of the oxygen. "'The dissolved oxygen may act either by poising the medium at an unfavorable

9 BACTERIOSTATIC ACTION OF CRYSTAL VIOLET ph, which is prolonged in proportion to the dye concentration, or it aids by forming an oxidized product with crystal violet which is a more powerful poising agent. This may be one of the reasons why strict anaerobes are relatively little affected by the dye. Steam and Steam, Ingraham, and others have shown that basic dyes increase in toxicity with an increase in ph. Several experiments were done on this point. At ph 5.1, S. lactis could grow in 1:500,000 crystal violet. At ph 8.1 the lowest dilution allowing growth was 1:1,250,000. Figure 3 shows the effect of ph on the length of the lag phase. From this it is evident that in any studies of the dye action, the ph has to be carefully controlled and standardized if comparable results are desired. SUMMARY To understand the effect of crystal violet upon bacteria, it is necessary to consider the bacteriostatic action separately from the bactericidal action. They are quite different. Above a certain concentration, the dye acts like any other disinfectant. The cells die in logarithmic order, and proportionately to the dye concentration (n = 0.86). The dye is more toxic to young than to old cells, and its toxicity increases only slightly with an increase in ph. The death rate is independent of the size of the inoculum. This strict disinfectant action is probably due to the combination of the dye with some indispensible cell constituents. At lower concentrations, the dye does not give a logarithmic survivor curve, and is not influenced by cell age or ph or the dye concentration. This unusual effect may be due to the unfavorable oxidation-reduction potential poised by the dye. In this range, cells usually overcome the dye action, and.multiply. The dye produces an abnormally long lag period which increases with the dye concentration and may become infinite. Once multiplication of the cells has started, it proceeds at a normal rate. The length of the lag phase is inversely proportional to the logarithm of the number of the inoculated cells. It increases with increased oxygen concentration and with increasing ph. Young cells recover more quickly than do old ones. The bacteriostatic effect of crystal violet is due to its property of poising the potential in a range unfavorable for cell multiplication. REFERENCES BITTENBENDER, W. A., DEGERING, E. F., TETRAULT, P. A., FEASLY, C. F., AND GWYNN, B. H Bactericidal properties of commercial antiseptics. Ind. Eng. Chem., 32, DUBOs, RENA 1929 The relation of the bacteriostatic action of certain dyes to oxidationreduction processes. J. Exptl. Med., 49, INGRAHAM, M. A The bacteriostatic action of gentian violet and its dependence on the oxidation-reduction potential. J. Bact., 26, RAHN, OTTO 1943a The problem of the logarithmic order of death in bacteria. Biodynamica, 4, (81-130), p

10 186 C. E. HOFFMANN AND OTTO RAHN RAHN, OTTO 1943b Death by chemical compounds. Biodynamica 4 (in print). SLANETZ, L. W., AND RETTGER, L. F A systematic study of the fusiform bacteria. J. Bact., 26, STEARN, A. E., AND STEARN, E. W Chemical mechanism of bacterial behavior. III. The problem of bacteriostasis. J. Bact., 9, STEARN, E. W., AND STEARN, A. E Conditions and reactions defining dye bacteriostasis. J. Bact., 11, STEARN, A. E Compound formation of crystal violet with nucleic acid and gelatin and its significance in dye bacteriostasis. J. Bact., 19,