D. REPORT OF DR. D. RIVAS

The following are the results of experiments conducted by me, during the past four months, on the germicidal action of chlorlyptus (chlorinated oil of eucalyptus, principal constituent C10H17OCl2) in vitro and in vivo, and comparison also with carbolic acid, oil of eucalyptus and dichloramine in test for irritation and toxicity.

Germicidal Action.—Based on the results obtained, chlorlyptus when used in a 5 per cent. paraffin oil solution was found to be a mild germicidal against typhoid B, strepto­coccus and staphylo­coccus when these organisms were suspended in ordinary bouillon culture or sterile salt solutions.

The germicidal action was found stronger when these micro-organisms were suspended in a sterile oily or lipoid substance, such as olive oil. The results of these experiments were not constant, owing probably to the imperfect suspension of the bacteria. Thus, while in some of the experiments chlorlyptus in 1 per cent. oil solution destroyed these micro-organisms, in other cases the same strength solution failed to give same result in same time.

The increased germicidal action of chlorlyptus on bacterial suspensions in olive oil may be accounted for by the fact that chlorlyptus is soluble in olive oil and not an admixture, as in the case of paraffin oil.

Chlorlyptus is not a coagulant, as are germicides of the phenol or hypochlorite types, and the germicidal action is therefore not strictly comparable.

The germicidal action of chlorlyptus oil solution, on pathogenic bacteria, on strepto­coccus and staphylo­coccus, suspended in pus, was found to be stronger than when these micro-organisms were suspended in ordinary bouillon culture or sterile salt solution. In one of the experiments, similar results were obtained when these micro-organisms were suspended in olive oil, chlorlyptus showing marked germicidal action.

Irritation and Toxicity.—The irritating action was found to be relatively mild in tests on laboratory animals. Thus, from 0.5 to 1 c.c. of chlorlyptus in paraffin oil 5 per cent. solution, injected into peritoneal or pleural cavities of guinea-pigs weighing 400 gm. was found to be without any appreciable disturbance in the health of the animal, and in some cases the injection of as much as 2 c.c. did not kill the animal.

Therapeutic Action.—Guinea-pigs were inoculated with purulent material containing strepto­coccus, staphylo­coccus and B. coli in peritoneal and pleural cavities respectively, and after six hours 1 c.c. of chlorlyptus 5 per cent. in paraffin oil solution was injected. Other infected animals were similarly treated twenty-four hours after inoculation, and another series forty-eight hours after inoculation. In some of these cases the animals died from shock but in a clearly defined series in which the injection of 1 c.c. of the chlorlyptus solution was made in the peritoneum of the guinea-pigs twenty-four hours after the inoculation, the animals lived. The control animal, inoculated with the purulent material and not treated with chlorlyptus oil solution, died.

In consideration that the injection of chlorlyptus oil solution [sic, referee] were made [? referee] in the peritoneal cavity this substance is apt to affect the vital organs in the abdominal cavity. It is my belief that in case of wall abscess of chronic inflammation, by limiting the action of chlorlyptus to the infected area, preventing at the same time the infection of the vital organs, chlorlyptus, because of its non-irritating quality, can be used effectively as an antiseptic.

CONCLUSIONS

1. Chlorlyptus is a mild and relatively nonirritating antiseptic of marked action on pus and suppuration.

2. When bacteria were suspended in olive oil or in pus, chlorlyptus showed marked germicidal action.

3. Chlorlyptus can be injected into the peritoneum or the pleural cavities of guinea-pigs in the proportion of 1 c.c. per 400 gm. of body weight without detriment to the animal.

4. Chlorlyptus in 5 per cent. oil solution (taking Clause 3 as comparison) can perhaps be injected in man as an antiseptic agent when there is a walled-in abscess in the peritoneum or pleural cavity where there is drainage, in the proportion of 0.5 to 1 c.c. per pound of body weight with good result.

REPORT ON THE GERMICIDAL ACTION OF CHLORLYPTUS ON PATHOGENIC BACTERIA IN VITRO AND IN VIVO

Experiment 1.—The germicidal action of eucalyptus oil.—Typhoid bacillus was destroyed in less than five minutes when exposed to the action of a 5 per cent. suspension of oil of eucalyptus. The exposure for four hours in a 5 per cent. suspension of chlorlyptus in paraffin oil was without effect on typhoid bacillus. It requires an exposure of two to four hours in a 10 per cent. suspension of chlorlyptus in paraffin oil to destroy typhoid bacillus.

Experiment 2.—Bacilliary action of chlorlyptus on the growth of pathogenic bacteria.—Typhoid and anthrax bacilli were selected for the experiment. Two series of five tubes each were made. The culture medium used was nutrient bouillon. Chlorlyptus was added in the following proportions: Tube 1, 1:10; Tube 2, 1:100; Tube 3, 1:1,000; Tube 4, 1:10,000, and Tube 5, 1:100,000. One series was inoculated with typhoid bacillus. All tubes were incubated for three days at 37 C.
Chlorlyptus inhibited the growth of typhoid bacillus when added to the bouillon in the proportions of 1:10. The growth of anthrax bacillus was inhibited by chlorlyptus when it was added in the proportions of 1:10, 1:100 and 1:1,000, as shown in the accompanying table. [The table was not submitted.—Ed.] In one instance the growth was markedly inhibited by chlorlyptus when added in the proportion of 1:10,000.

Experiment 3.—Germicidal action of chlorlyptus on typhoid bacillus.—Bouillon cultures of typhoid bacillus forty-eight hours old, and a suspension of forty-eight-hour agar cultures of typhoid bacillus in sterile salt solution were used for the experiment. Chlorlyptus was added in the proportion of 1:1,000; 1:1,500; 1:100; 2 per cent.; 3 per cent.; 4 per cent.; 5 per cent. and 10 per cent., respectively.
Inoculations were made in trypsinized peptone bouillon after the addition of chlorlyptus at different intervals, namely: at once, after five minutes, after ten minutes, after fifteen minutes, after thirty minutes, after one hour and after two hours, and tubes incubated at 37 C. for forty-eight hours.
Result: Growth was shown in all tubes except those in which chlorlyptus was added in the proportion of 10 per cent. and after the action of the antiseptic for two hours or longer.

Experiment 4.—Inhibitory action of chlorlyptus in the growth of typhoid bacillus.—Chlorlyptus was added to sterile bouillon in the proportion of 1:100, 1:1,000, 1:10,000 and 1:100,000, and incubated for forty-eight hours at 37 C. to eliminate any possible contamination of the bouillon during the manipulations. All tubes were found sterile and inoculated with typhoid bacillus.
Result: All tubes were found sterile again after being inoculated with typhoid bacillus and incubated at 37 C. for forty-eight hours, which shows chlorlyptus inhibited and the growth of typhoid bacillus in bouillon when this antiseptic was added in the proportions of 1:100 to 1:100,000.
Remarks: In another experiment made, chlorlyptus showed a weaker inhibitory action on the growth of typhoid bacillus.

Experiment 5.—Germicidal action of carbolic acid.—The technic was the same as that outlined in Experiment 1. except that carbolic acid was used instead of chlorlyptus.
Result: Carbolic acid showed a distinct germicidal action on typhoid bacillus in the proportions of 1 per cent. in ten minutes.

Experiment 6.—Action of nitrogen gas on the growth of typhoid bacillus in bouillon and nutrient agar when chlorlyptus was added to this culture medium.—Chlorlyptus was added to the bouillon in the proportions of 1:100, 1:1,000, 1:10,000 and 1:100,000, as outlined in Experiment 2; also to agar kept melted at 45 C. Tubes were inoculated with typhoid bacillus; plates were made of the inoculated agar tubes; all plates and tubes were incubated at 37 C. for forty-eight hours in an atmosphere of nitrogen gas.
Duplicate experiments were made with cultures of typhoid bacillus as above in bouillon and agar plates containing the same amount of chlorlyptus and incubated at 37 C. in ordinary atmosphere as control.
Result: Nitrogen gas did not show any appreciable increase of the germicidal action of typhoid bacillus when grown in medium containing chlorlyptus. Growth was about the same in cultures supplied with nitrogen gas as in those growing in ordinary atmosphere.

Experiment 7.—Germicidal action of chlorlyptus on pyogenic bacteria suspended in an oily medium.—Experiment with strepto­coccus: Cultures of strepto­coccus in blood agar three days old were suspended in olive oil (sterile), and chlorlyptus was added in the proportions of 1, 5 and 10 per cent. and inoculated in trypsinized bouillon at different intervals, namely: at once, after five minutes, after ten minutes, after fifteen minutes, after thirty minutes, and after one hour. Tubes were incubated at 37 C. for forty-eight hours.
Result: All tubes remained sterile. The germicidal action of chlorlyptus on strepto­coccus suspended in oil was almost at once and with certainty after five minutes when added in the proportion of 1, 5 and 10 per cent.

Experiment 8.—Germicidal action of chlorlyptus on staphylo­coccus, suspended in sterile olive oil.—The technic employed was the same as in Experiment 5, except that a culture of staphylo­coccus was used.
Result: All tubes remained sterile. The germicidal action of chlorlyptus was almost at once in the proportions of 1, 5 and 10 per cent.
Remarks: By repeating this experiment the result showed some variations. The discrepancy was probably due to an imperfect suspension of the micro-organism in the oil.

Experiment 9.—Germicidal action of carbolic acid on strepto­coccus suspended in olive oil.—The technic employed was the same as in Experiment 5, except that carbolic acid was used instead of chlorlyptus.
Result: The germicidal action of carbolic acid of strepto­coccus suspended in olive oil was almost at once in the proportions of 1, 5 and 10.

Experiment 10.—Germicidal action of chlorlyptus on staphylo­coccus.—The technic employed was the same as in Experiment 6 except that the carbolic acid was used instead of chlorlyptus.
Result: The germicidal action of carbolic acid on staphylo­coccus suspended in olive oil was almost at once, in proportions of 1, 5 and 10 per cent.

Experiment 11.—Germicidal action of chlorlyptus on pyogenic bacteria suspended in pus.—Chlorlyptus was added to sterile pus in the proportions of 1, 5 and 10 per cent., and then inoculated with staphylo­coccus and cultures were made in bouillon at once, after five minutes, after ten minutes, after fifteen minutes, after thirty minutes, after one hour and after two hours, respectively, and tubes incubated for forty-eight hours at 37 C.
Result: Growth was shown in all tubes except those inoculated from tubes in which chlorlyptus was added in the proportions of 10 per cent. after one hour.

Experiment 12.—Germicidal action of chlorlyptus on strepto­coccus suspended in sterile human blood serum.—Staphylo­coccus culture in agar forty-eight hours old was suspended in sterile human blood serum, and to the suspension chlorlyptus 5 per cent. in paraffin oil was added in the proportions of 1, 5 and 10 per cent. Inoculations were made at intervals, at once, after five minutes, after ten minutes, after fifteen minutes and after one hour in trypsinized bouillon. Tubes were incubated at 37 C. for forty-eight hours.
Result: Chlorlyptus showed inhibitory action on the growth of staphylo­coccus in the strength of 10 per cent., but did not produce complete sterilization. Similar results were shown with the 5 per cent., and in the 1 per cent. chlorlyptus did not show any inhibitory action at all.

Experiment 13.—Germicidal action of carbolic acid on staphylo­coccus suspended in human blood serum (sterile).—The technic employed was the same as in Experiment 10 except that carbolic acid was used instead of chlorlyptus.
Result: Carbolic acid produced a complete sterilization in the strength of 10 per cent. almost at once, and with certainty after five minutes. Similar results were produced with the 5 per cent. The 1 per cent. carbolic acid did not show any appreciable germicidal action on staphylo­coccus.

Experiment 14.—Toxic and irritant action of chlorlyptus.—Six normal guinea-pigs were used for the experiment. Guinea-Pig 1 was injected peritoneally with 1 c.c. of chlorlyptus, Guinea-Pig 2 with 2 c.c. of chlorlyptus, Guinea-Pig 3 with 3 c.c. of chlorlyptus, Guinea-Pig 4 with 4 c.c. and Guinea-Pig 5 with 5 c.c. 5 per cent. respectively. Guinea-Pig 6 was used as a control and not injected.
 Result: Guinea-Pigs 1 and 2 did not show any appreciable disturbance. Guinea-Pig 3 was sick for four days, after which it gradually recovered but it became sick again after one week and died ten days after the injection. Guinea-Pig 4 died over night. Guinea-Pig 5 died six hours after injection. Guinea-Pig 5 was injected at 11:30 with 5 c.c. chlorlyptus. Ten minutes after the injection it was lying relaxed, respiration and heart normal, conjunctive reflex present. One hour after the injection the animal seemed to present symptoms resembling those of narcosis: respiration and heart were normal. After four hours there was no change in the condition of the guinea-pig except that the respiration was irregular. Five and a half hours after it showed prostration with irregular respiration and heart action. Six hours after injection the animal was dead.
Autopsy: The peritoneum showed a congestion and a fibrinous exudation, amount of liquid increased, some part of which was probably chlorlyptus unabsorbed. Spleen about normal, liver congested, kidney about normal, suprarenal glands about normal, lungs normal, pleural cavity obtained no exudation, heart soft, flabby and congested.

Experiment 15.—Toxic and irritant action of chlorlyptus when injected into the pleural cavity.—Six normal guinea-pigs used for the experiment. Chlorlyptus was injected in the pleural cavity as follows: Guinea-Pig 1, 0.5 c.c.; Guinea-Pig 2, 1 c.c.; Guinea-Pig 3, 2 c.c.; Guinea-Pig 4, 3 c.c., and Guinea-Pig 5, 4 c.c. Guinea-Pig 6 was used as a control.
Result: Guinea-Pigs 1 and 2 recovered about four hours after injection. Guinea-Pig 3 died three days after and Guinea-Pigs 4 and 5 four and two hours after, respectively.
Conclusions: Guinea-pigs weighing on the average of 400 gm. may be injected peritoneally with one or two c.c. or intrapleurally with 0.5 to 1 c.c. of chlorlyptus without having fatal results from the injection.

Experiment 16.—Toxic and irritant action of eucalyptus oil.—Three normal guinea-pigs were used for the experiment. Guinea-Pig 1 was injected with 1 c.c. of oil of eucalyptus in the peritoneum, and Guinea-Pig 2 with 0.5 c.c. in the pleural cavity. Guinea-Pig 3 was used as a control.
Result: Guinea-Pig 1 died about three hours after injection, and Guinea-Pig 2 about two hours after the injection.
Autopsy: Both guinea-pigs showed marked congestion and a moderate degree of exudate in the peritoneum.

Experiment 17.—Toxic and virulent action of eucalyptus.—Three normal guinea-pigs were selected for the experiment, as in Experiment 16. The injection was made in the pleural cavity. Guinea-Pig 1 was injected with 0.5 c.c. and Guinea-Pig 2 with 1 c.c. of eucalyptus oil.
Result: Guinea-Pig 1 died the following day, and Guinea-Pig 2 one hour after the injection.

Experiment 18.—Toxic and irritant action of dichloramin-T, 0.5 per cent. in chlorcozane.—One guinea-pig was used for each experiment. Guinea-Pig 1 was injected with 0.5 c.c. and Guinea-Pig 2 with 1 c.c. of dichloramin-T peritoneally.
Result: Both animals became restless immediately after the injection, and died twelve hours after of acute hemorrhagic peritonitis.

Experiment 19.—Effect of chlorlyptus on staphylo­coccus suspended in salt solution and one of that solution injected into the peritoneum of the guinea-pig.—Three guinea-pigs were used for the experiment. Guinea-Pig 1 was injected with 0.5 c.c. of staphylo­coccus suspension as control. Guinea-Pig 2 was given the same, and immediately after received 1 c.c. of chlorlyptus. Guinea-Pig 3 was injected with the same amount, and chlorlyptus was injected twenty-four hours after injection.
Results: Guinea-Pig 1 was sick and weak with loss of appetite for some days, but gradually recovered. Guinea-Pig 2 died over night.
Autopsy: There was a large amount of exudate in the peritoneal cavity, irritation of the intestine, and other signs of acute inflammation. A moderate degree of congestion; spleen not enlarged; liver showed cloudy swelling and fibrinous exudate; lungs and heart about normal except for a moderate degree of congestion but no exudate. Guinea-Pig 3 was sick for some days, but recovered gradually one week after.

Experiment 20.—Effect of chlorlyptus in vivo on staphylo­coccus.—The experiment was conducted in the same way as in Experiment 17, but 2 c.c. were used instead of 1 c.c.
Result: Guinea-Pig 1 was injected with 2 c.c. staphylo­coccus suspension and died over night. Autopsy showed that the animal died of acute peritonitis. The peritoneum showed some fibrinous exudate and mesenteric vessels. Guinea-Pig 2 was injected with 2 c.c. of staphylo­coccus, and eighteen hours after was injected with 1 c.c. of chlorlyptus. The animal died two weeks after injection. Guinea-Pig 3 was injected with 2 c.c. staphylo­coccus suspension, and twenty-four hours after with 1 c.c. of chlorlyptus. The Guinea-Pig died ten days after. Autopsy revealed bronchopneumonia of the left lung and acute miliary abscess in the liver.

—(From The Journal A. M. A., Nov. 27, 1920, with additions.)