Margaret Pittman

Influence of antigens on release of free fatty acids from Arlacel A (mannide monooleate).

Summary With use of the modified method of Dumcombe for assay of FFA, it was found that water-in-oil emulsified tetanus toxoids, cholera and typhoid vaccines which induced abscesses in human beings contained more FFA than did emulsified products not inducing abscesses. The purer the parent tetanus toxoid, the lower was the free fatty acid content of the emulsified antigen. C. tetani culture filtrates, cholera vaccine, one typhoid vaccine and several types of allergenic extracts promoted the release of FFA from the emulsifier, Arlacel A. Preliminary data suggest that the hydrolytic component of C. tetani filtrates is an enzyme. The relationship of FFA released by the interaction of antigens on the emulsifying agent of the oil adjuvant to untoward reactivity remains to be determined.

Sensitivity of mice to histamine during respiratory infection by Hemophilus pertussis.

Summary Mice inoculated intranasally with a culture of H. pertussis developed lung infections from which more than half of the mice survived. The average time of death ranged from 16 to 23 days. They developed a high degree of sensitivity to histamine comparable to that induced by pertussis vaccine, but it was slower in developing and it persisted much longer. A high percentage of the mice that died from histamine shock showed gross lung lesions and H. pertussis was recovered from the lungs of the majority. Most of the mice that survived this infection were resistant to an intracerebral inoculation of H. pertussis.

Sterility testing: detection of fungi and yeasts in the presence of preservatives☆

Abstract Thirty-four strains of fungi and yeasts were studied for growth in six media in the presence of Merthiolate. All strains were killed in a 1:500,000 dilution; 1:5,000,000 was the maximum concentration that permitted recovery of all strains in Fluid Thioglycollate Medium (FTM). Fluid Sabouraud Medium (FSM) containing either sodium thioglycollate or sodium hydrosulphite was almost as effective. FTM with sodium hydrosulphite substituted for sodium thioglycollate and Bonnel-Raby medium were inferior to FTM, also to FSM. Rate of initiation of growth was reduced in the low concentration of 1:7,000,000 with maximum recovery at 21 days, whereas maximum recovery occurred in 7–10 days in the six control media which were similar in promoting growth. Phenylmercuric borate, phenol, benzethonium chloride and parabens were also fungicidal. Results are discussed relative to the discontinued use of FSM in the United States in official sterility testing and for the use of FTM in the test of mercurial-preserved biologies incubated at 20–25 °C.

The “S” and “R” Forms of Hemophilus Influenzae

During the course of an experiment with a culture of Hemophilus influenzae, transplants were made on a plate of Levinthals solid transparent medium. It was observed that the culture contained 2 types of colonies. Well defined small transparent colonies resembling the textbook description of the typical Pfeiffer bacillus colony were found but in addition there were larger, slightly opaque iridescent mucoid smooth colonies. Transplants were made from the transparent colonies on Levinthal agar and all the colonies were found to be identical with the mother colony. The same was true of transplants from the opaque iridescent colonies. It was thus possible to separate 2 cultures each of which produced typical colonies. Since the largest transparent colonies were found to have a rough surface the strain producing these colonies has been called an “R” strain; and since the iridescent colonies from the other culture had a smooth surface this strain has been called an “S” strain. This culture and 2 others from which “S” and “R” forms were subsequently isolated had been seeded many times on blood or chocolate agar and the variation in the colonies had not been noticed. Yet the difference in the colony appearance was marked when transplants were made on the transparent agar plates. Very striking was the difference when the plate cultures were held before a 100 watt electric light bulb or in the sunshine. The morphology of the bacteria from the 2 strains has also been found to be dissimilar and characteristic for each type. This is best noticed if the stained preparation is made from a solid medium culture which is not older than 24 hours. The bacteria from the “S” culture appear as small coccobacilli that vary little in size.

Some quantitative aspects of passive anaphylaxis in pertussis-vaccinated mice.

Summary 1. Pertussis vaccine significantly increased the susceptibility of mice to passive anaphylaxis. Two lots of anti-bovine-serum-albumin rabbit serum containing 0.816 and 2.52 mg antibody N per ml, respectively, were used. With each, the response was graded in relation to amount of antibody injected and reproducible titrations of the SD50 were obtained. The SD50 values for the respective sera were 4.06 ± .27 and 7.9 ± 1.03 mg N/ kg, respectively. Significance of the difference was not definitely established. In non-vaccinated mice, even with several fold increases in antibody, the highest incidence of shock, was around 50% and frequently the response was irregularly graded. 2. Within a range of dosage of pertussis vaccine that varied as much as 16 fold and which significantly affected the histamine LD50, the passive anaphylactic SD50 values of the sera were not significantly altered. 3. Comparable anaphylactic sensitivity was observed when a serum was; administered either 48, 24, or 6 hours before the antigen. Besides the theoretical significance, the findings indicate that mice treated with pertussis vaccine may be useful in anaphylaxis studies.

Determination of the bacterial content of cholera vaccine.

International and national opacity reference preparations are employed to determine the concentration of vibrios in freshly harvested cultures of Vibrio cholerae for cholera vaccine. The measurements have been the basis for calculating the number of vibrios per milliliter given on the labels of the vaccines. This study showed that suspensions of Inaba 35A3 (In35A3) visually equal to 10 opacity units of the U.S.A. Turbidity Reference (1947) had higher nephelometric measured units and colony-forming units than a visually equivalent suspension of Ogawa 41 (Og41). Maximum differences were 13·3 vs 6·7 neph u and 2·4 vs 0·23 CFU, respectively. Cultivation on alkaline nutrient agar, pH 8·5, or on casamino acid agar, pH 7·4, had very little influence on unitage. However, growth of In35A3 at 40–41 °C significantly decreased the neph u as compared with 35–38 °C grown culture. Inaba V86 (InV86) had optical properties similar to Og41 and both InV86 and Og41 were less affected by the higher growth temperature than In35A3. Nitrogen assays were not sufficiently extensive to determine the relationship of nitrogen content to visual and nephelometric unitage or to CFU. The differences in the optical properties emphasizes the need to establish for each vaccine production strain the relationship between optical readings (visual or instrument) and a constant baseline of reference. Potency is the final criterion.

A Proposed Mouse Protection Unit for Anti-Meningococcus Serum.

The use of mucin for the enhancement of the infectivity of meningococci in the mouse has now been studied for over 5 years. 1 The first use of this technic in the titration of the protective action of sera was reported over 3 years ago. 2 Subsequently, other laboratories have reported the titration of protective antibodies in antimeningococcus serum and in each case the details of technic employed have varied slightly or markedly. 3 4 5 6 7 8 9 In order that values obtained in the titration of antimeningococcus sera in various laboratories may be comparable, that figures obtained by the use of the test may have a wide application, and that sera used for therapeusis may be studied on a basis which allows quantitative comparison, it has seemed advisable to set up and distribute a tentative standard polyvalent serum as a control and to assign to this serum certain values so that other sera may be referred to it quantitatively. It has also seemed desirable that a uniform technic for performing the mouse protection test be used. In order to obtain a polyvalent serum and data for this purpose, a serum (of which ample amounts were available) was selected because it resembled closely the older control serum M 18 in its protective capacity and other immunological reactions, This has been designated M 19. Repeated estimations of antibody nitrogen for Group I∗ were carried out with this serum and it was determined that the serum contained 0.65 mg of antispecific polysaccharide nitrogen 10 per cubic centimeter. It has been shown 8 that the Group I protective power of antimeningococcus serum probably parallels the type-specific antibody nitrogen content. This has also been found to be the case with antipneumococcus sera. In the latter case 1.00 mg of antibody nitrogen is found to be equivalent to approximately 1000 protective units, a convenient figure.