The observation of T. pallidum-like organisms reported by Collart, Borel, and Durel (1962, a, b, c, 1964) in the lymph nodes of both men and rabbits in seroreactive treated syphilis and the stimulating work of Smith and Israel (1967 a, b, 1968)

Serum tests for gonorrhoea, particularly the complement-fixation procedure using whole gonococcal cells as the antigen, have long been recognized to be of very limited value in diagnosis. For more efficient diagnosis and control of gonorrhoea there is clearly a need for a simple serological method which can be used as a screening test aimed particularly at the detection of the female carrier in circumstances in which discretion is required and in which routine genital examination is not practicable.In this context a simple "microprecipitin ring test", using a specific lipopolysaccharide antigen of gonococcal origin, has been devised. Preliminary trials have shown its possible usefulness (Chacko and Nair 1966a). The present report gives the results of a more extensive evaluation. The kinetics of the precipitin ring test were described by Nair and Chacko (1966).Material and Methods Preparation of the Antigen The specific antigen was prepared as follows:A 24-hour growth of a virulent strain of gonococcus (F62-type i, obtained through the courtesy of the VDRL, Atlanta, U.S.A.) grown in Chacko-Nair selective medium (I966b, i968) was emulsified in broth and o01 ml. aliquots were inoculated into the allantoic cavity of 8-day-old chick embryos through the air sac. Eggs were candled periodically and at the end of 48 hours' infection and growth of gonococci, they were kept in the refrigerator at 40C for 2 to 4 hours. The allantoic fluid was then aspirated under sterile conditions using a io-ml. syringe and an i8-gauge needle and the purity of the cultures was checked by Gram-staining and sub-culture.Next, the allantoic fluid was centrifuged lightly to remove all the macroscopic and fat particles. It was then pipetted into large centrifuge bottles and six *Received for publication September 12, 1967 tProfessor of Serology tAssistant Serologist volumes of absolute alcohol were added; the mixture was shaken thoroughly and kept at 5 to io0c for 24 to 48 hours. The contents were then centrifuged at 2,000 r.p.m. for one hour, the supernatant was discarded, and any excess alcohol burnt off. Half the original volume of double-distilled deionized water was added to the precipitate, which was then homogenized by using a 5-mi. pipette, was again centrifuged at 2,000 r.p.m. for one hour, and the supernatant watery extract pipetted out. An equal quantity of absolute alcohol was added and centrifuged at 2,000 r.p.m. for 30 minutes. To the supernatant, 6 volumes of absolute alcohol were again added, followed by a trace of sodium acetate and 4 to 8 ml. glacial acetic acid, the whole was mixed well and kept in the cold for 2 hours. The contents were centrifuged at 2,000 r.p.m. for 45 minutes and the deposit was dissolved in a volume of water one-eighth of the original volume of culture fluid taken. After further centrifugation to remove all the insoluble particles, the slightly turbid supernatant so obtained was titrated to obtain the exact units of antigen necessary for the test, as follows: Doubling dilutions of the original antigen pre...

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An egg-enriched medium made selective by the addition of Polymyxin B and Mycostatin, for the isolation of N. gonorrhoeae was described by Chacko and Nair (1966). The evaluation of this medium has since been extended to further cases ofN.gonorrhoeae and also to N. meningitidis. The results indicate the usefulness of the Chacko-Nair medium, not only in the diagnosis of gonorrhoea, but also in the isolation of N. meningitidis in culture. Methods and Material Preparation of the MediumThe basal medium was prepared as follows: 100 g. minced beef muscle free from fat were weighed into a 1,000-ml. Pyrex conical flask and 500 ml. distilled water were added and mixed with a Waring blender. 15 ml. 1/N. NaOH were added and the mixture was brought to a temperature of 75 to 80°C. for 5 minutes, preferably in a water bath. After cooling to 40°C., 0-20 g. Trypsin powder (1:250 Difco) was added and the mixture incubated at 37°C. for 6 hours for digestion to occur; 0 7 ml. glacial acetic acid was then added and the whole mixture was boiled for 10 minutes to arrest the digestion. It was then kept in a refrigerator overnight and filtered through gauze. Filtration was also carried out through filter paper to make it clear if found necessary. 500 ml. of 1 per cent. sodium chloride (AR) were prepared in a separate 1,000-ml. flask and well mixed with the above filtrate. 1 g. glucose and 5 g. disodiummono-hydrogen phosphate (AR) were added and dissolved. The pH was adjusted to 7-2-7-4, using a pH comparator and bromothymol blue as indicator. It was then distributed into 200 ml. quantities in conical flasks, 4 g. basic agar (Difco) being added to each flask. The agar was dissolved in a boiling water bath and autoclaved at 10 lb. pressure for 30 minutes. This stock of basal medium was preserved at room temperature until it was used. * Received for publication March 20, 1967. Just before use, 200 ml. of the basal medium were melted in a water bath and cooled to 55 to 50°C.; Polymyxin B sulphate (Pfizer) and Mycostatin (SquibbNystatin) were added to give a concentration of 30 units each per ml. of the medium and the contents were well mixed by rotating the flask. Enrichment of the Basal MediumFresh chicken eggs bought from the market were used for enrichment. The shell was cleaned with soap and water and dried. Tincture of iodine was applied over the air-sac portion, and the iodine was removed with 70 per cent. alcohol. The shell was pierced at the middle of the air-sac end, using the pointed edge of a small pair of sterile scissors, and was cut offto a diameter of 1 to 1 -5 cm. A sterile nichrome-loop was introduced into the egg and the white and yolk well mixed by rotating the loop inside, holding the egg near a flame to avoid the possibility of aerial contamination. To each 200 ml. of the melted basal medium in the flask kept at 55 to 50°C., the contents of a hen's egg were added directly and homogenized well by rotating the flask on a flat surface. This gave an approximate concentration of 10 per cent. enrichment of the medium with w...

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