Evelyn B. Tilden

Preparation and properties of the endotoxins of Aspergillus fumigatus and Aspergillus flavus.

Endotoxins have been reported to occur inAspergillus fumigatus andA. flavus, but the data regarding them are incomplete. Optimal cultural conditions and methods of extraction are described here for the production of the toxins of both of these fungi. They are obtained from the well-washed mycelia; only minute amounts could be demonstrated in the concentrated culture fluids. Both are powerful nephrotoxins; a characteristic necrosis of the kidney cortex is evident grossly in mice, and histological study shows no difference between the effects on the kidney of the two toxins. However, the fumigatus extracts have strong hemolytic properties, especially for sheep erythrocytes, and marked dermonecrotic activity in rabbit skin. No hemolytic action has been demonstrated for the flavus extracts, which possess a powerful hemagglutinin for rabbit erythrocytes and only mild dermonecrotic properties. Immune sera have been prepared in rabbits for both toxins, and in each case these prevent all the observed effects of the toxin. Precipitin titers are closely correlated with neutralizing titers. Chemical differences are suggested by the different procedures applicable to concentration and purification of the toxins. The toxin fromA. fumigatus is precipitated by acetone and can be concentrated and purified to a considerable degree by acetone precipitation and dialysis. The flavus toxin has not been precipitated by any of the solvents tried but can be freed to some extent of inert substances by acid treatment and saturation with ammonium sulfate.

Partial purification and characterization of the endotoxin from Aspergillus fumigatus.

The endotoxin fromAspergillus fumigatus was studied and appears to be protein in character. Attempts to purify the material by fractional precipitation with acetone resulted in increase of hemolytic and toxic potencies. The hemolytic activity of the toxin is stable from pH 3.6 to 8.4. Electrophoretic analysis enabled the assignment of hemolytic activity to a definite electrophoretic component. Chromatography with DEAE cellulose using gradient elution effected increase in hemolytic activity. The bioassay for toxic activity is less quantitative than the assay for the hemolytic activity. Column electrophoresis confirmed the assignment of hemolytic potency to an electrophoretic component. Recovery of activity was low in this method. Both the hemolytic and toxic activities occur in the same electrophoretic component; however, there is evidence that they may be separate components.

The Effect of 2-Methyl-1,4-Naphthoquinone on the Incidence of Dental Caries

In 1943 it was suggested that the production of dental caries depends upon first, the rate of acid formation in the mouth, and second, the rate of neutralization of acid in the regions associated with the caries susceptible portions of the teeth (1). On this basis dental caries should be retarded by a decrease in the rate of acid formation or an increase in the rate of acid neutralization. Most of the successful methods of controlling caries have worked by decreasing the rate of acid formation. This is natural, because the rate of acid neutralization is controlled by many factors which at present cannot be materially changed (1). Probably the most successful experimental control of the rate of acid formation, with subsequent decrease in caries activity, has been accomplished by the reduction of fermentable sugars and carbohydrates in the diet (2, 3, 4), or by their elimination from the mouth through oral hygiene (5). These methods of caries control, however, depend entirely upon the cooperation of the patient. This cooperation cannot be expected of large masses of population. Another way of reducing the rate of acid formation is by interfering with the enzyme or co-enzyme systems which are necessary for acid production. Several agents may be used, some of which have been tried experimentally and some clinically. Iodoacetic acid has been tried experimentally with rats (6). It is probably too toxic for human use. A 30% urea solution (7), used as a mouthwash, acts as a protein denaturant, destroying the enzymes and hence preventing acid formation. A urea quinine mouth rinse has also proved successful (8). Ammoniacal silver nitrate reduced on the susceptible surfaces of the teeth leaves a deposit of colloidal metal which presumably retards fermentation (9). The enzyme inhibitor which has had the most wide-spread study and experimental use is the fluoride ion (10). This ion inhibits the hydrolysis of phosphoric esters and hence retards acid formation. Furthermore, the fluoride ion is absorbed by the dental enamel, forming a compound less soluble than unchanged enamel. Probably both actions are responsible for the success of fluorides in controlling or preventing caries. In 1942 (11, 12), it was found that synthetic vitaminK (2-methyl-1 ,4-naphthoquinone), when added in minute quantities to saliva-glucose mixtures, in vitro, would prevent the formation of significant amounts of acid. There was no evidence that its action as an enzyme inhibitor was closely associated with the vitamin activity. Preliminary experiments indicated that the material would

A Study of the Action of Antibiotics in Vitro on Microorganisms Isolated from Root Canals

Several reports have appeared on the treatment of infected root canals with penicillin. The earlier workers [Shaw, Sprawson and May (1), and Grossman (2, 3)] recognized that they had used too small a dose and explained unfavorable results on that basis. Buchbinder (4) used paper points which he calculated had soaked up 1,000 to 2,000 units of penicillin, or he injected oil containing 100,000 units/ml. directly into the canal with a syringe. In 33 of 36 cases treated, cultures from the canal became negative, though he was unable after 24 hours to demonstrate any penicillin in dressings which had originally contained 1,000 units. Stewart (5) forced penicillin into the canal with a syringe, using solutions containing 20,000 to 80,000 units in each milliliter and keeping as much of it as possible in the canal between sittings by packing with cotton and sealing with zinc oxide and eugenol. A hundred patients (112 teeth) were treated, and cultures from the canal were negative after 2 or 3 treatments in all but 2 cases. Stewart did not regard infiltrating above the apex to be necessary. In Benders series (6) of 53 cases, the canal was negative on culture in 70% of the cases after one treatment. The treatment was accomplished by flooding the canal by means of a syringe and needle with 25,000 to 50,000 units several times during one sitting. He used both brain heart infusion and sodium thioglycollate broth as culture media and added clarase to inactivate any penicillin remaining at the time the culture was made. Potkin (7) used paper points, adding just enough water to 100,000 units of penicillin to be completely absorbed by 200 paper points, hence each point represented a dose of 500 units. Unlike Buchbinder, Potkin found that the paper points still contained enough penicillin to inhibit Staphylococcus aureus after they had been in the canal 48 hours, even when the canal was putrescent. Potkin carried out a series of laboratory experiments in which he compared the inhibitory action of the paper points which were saturated with 500 units of penicillin and points which had absorbed 2 drops of various medicaments such as beechwood creosote and eugenol, using Staphylococcus aureus as test organism. From his photographs it appears that the penicillin point was as inhibitory

Complement Titer of the Blood in Allergic Conditions

Conclusion The amount of complement present in the blood of allergic patients of this series (18 cases) was similar to that in the control sera from persons coming to the general medical clinic.

Evaluation of Antimicrobial Agents. The Germicidal Activity of a Synthetic, Detergent, DC-12:

Dependence upon chemical methods for the destruction of microorganisms is regarded by the bacteriologist as justifiable only when the clean and certain method of sterilization by heat is impossible. No available chemical disinfectant is able in a reasonable time to accomplish what the autoclave so quickly and surely accomplishes, namely, the destruction of the spores of bacteria and fungi and of the organism of tuberculosis. Phenol, while effective against the 2 latter organisms (1, 2), is ineffective against bacterial spores (3). This limitation, indeed, is shared by most disinfectants, which often require a period of hours or even days to destroy spores. Saponated cresol (4), which is probably the most efficient disinfectant available at present, is used widely in hospitals and bacteriological laboratories but has not found favor with the dentists because of its odor. The compounds of mercury are now known to be bacteriostatic rather than bactericidal (4), and when they are washed away leave the bacteria alive and ready to multiply. Formalin (4), a time honored disinfectant, has recently been recommended for dental instruments by Tainter, Throndson, Beard and Wheatlake (5), who have found a means of disguising the penetrating odor. Phenol and cresol are perhaps the only disinfectants which are recognized as being active against the organism of tuberculosis. The standard test of the efficiency of a substance proposed as a disinfectant is still the comparison of its germicidal activity in vitro with that of phenol. The limitations of this test have become more and more evident in recent years, and it is well known that bacteriostatic substances such as mercurials may have high phenol coefficients. The present trend among research workers is toward practical tests which simulate actual conditions. We have chosen 2 such practical tests and have examined by these procedures such substances as are applicable to the disinfection of instruments, dental or surgical, for which heat sterilization is not practicable (6, 7). The present report is chiefly concerned with the evaluation of a quaternary ammonium salt, di methyl benzyl lauryl ammonium chloride.2 The germicidal activity of compounds of this type is presumed to be in the fatty acid (in this case the C-12) group. We shall refer to the substance as DC-12. The crystals are extremely soluble in water. The solution is colorless and has a slight aro-

Comparative Observations on Bacteriolytic and Hemolytic Titers of Certain Sera

The Bordet Gengou reaction was designed to test the hypothesis of the identity of bacteriolytic and hemolytic complement, 1 although Bordet recognized later that the identity could not be regarded as absolute, since he was able to make specific anticomplements for the sera of various animals. 2 Muir and Browning 3 suggested that the bactericidal action of normal serum might be due to some moiety of complement which they termed “bacteriophilic”. Gordon and his coworkers 4 have been unable to obtain a serum which is devoid of hemolytic complement and yet possesses bactericidal action, though the reverse might be true of sera absorbed with bacteria, unless absorption were continued too long or the number of bacteria used were overwhelming. A series of tests was made to determine which bacteria in the School collection could be depended upon to resist the bactericidal action of certain non-immune sera, and which bacteria were sensitive to it. The results with pooled human sera did not vary, but occasionally an organism was killed by some rabbit sera and not by others (e. g., B. enteritidis, B. typhosus Rawlins). Table I is a. resume of these tests. The technique used was as follows: The bacterial suspensions, from 18-hour cultures on agar slants, were made in saline containing 0.1% gelatin. Very few bacteria survived incubation in saline, but the addition of this very small amount of gelatin removed the toxicity of the saline. The first dilution was made to correspond with Tube 1 of the McFarland nephelometer, the final dilution was one ten-thousandth of the first. The number of bacteria on control plates without serum was 1,000 to 2,000 per cc., 1 cc. being the amount of suspension used in the tests. The mixtures of bacteria and serum were incubated for 4 hours in a waterbath at 37.5°C, and the entire mixture was plated by pouring.