370 Transactions of the Society. 



them in pure culture apart from the meningococcus, and in every 

 apparently pure culture of the meningococcus these organisms have 

 always been found if diligently searched for. 



They can be demonstrated in incubated cerebrospinal fluid, 

 collected with every precaution against contamination ; and they 

 can be demonstrated, if searched for, in single colonies on serum- 

 agar plates, provided that these have been inoculated with in- 

 cubated cerebrospinal fluid. The best and most certain method, 

 in fact, of demonstrating their presence has been, in my hands, 

 by subculture from single colonies on serum-agar, itself inoculated 

 with incubated cerebrospinal fluid. This subculture, however, 

 must be into liquid media, an excellent medium being equal parts 

 of tested horse-serum and broth, the reaction of which to pheno- 

 phthalein may be + 10, -f 30, or even -f- 50. 



The technique I have mainly employed for the detection and 

 cultivation of the organism is as follows : — 



1. Cerebrospinal fluid from a suspected case of the disease is 

 discharged direct into a sterile glass flask, with a specially-ground 

 glass stopper provided with a sterile rubber cap sufficiently large to 

 come well down over the neck of the flask. In each case only 

 samples of fluid withdraw^n at first lumbar puncture have been 

 employed. 



2. On arrival at the laboratory the centrifuged deposit from a 

 small quantity of tlie fluid is examined. 



3. The remainder of the fluid is then incubated at 37*0° C. for 

 a period varying from forty-eight to seventy-six hours. 



4. A further volume of the fluid is now withdrawn from the 

 flask, and is centrifuged and examined. 



5. Of six cases in which prolonged examination was carried 

 out of films prepared from the deposit of the incubated fluid, the 

 organism has been found in three. 



6. Two or more serum-agar slopes, and sometimes, in addition, 

 three serum-agar plates, are now incubated ; the plates in series 

 with considerable volumes of the incubated cerebrospinal fluid. 



7. On the following day likely single colonies are picked off, 

 and are individually spread on to fresh serum-agar slopes to provide 

 a free confluent growth. 



8. On the following ^day the entire growth of each slope is 

 inoculated into serum-broth tubes, which are incubated for forty- 

 eight hours. 



9. From the serum-broth tubes fresli serum-agar and agar slopes 

 are inoculated for serological tests ; and lactose, saccharose, glucose, 

 mannite, and dulcite tubes are also inoculated from the same 

 source. 



10. Samples of the serum-broth tubes are at the same time, 

 and subsequently every day, centrifuged and the deposits examined. 



11. The serum employed in each case is thoroughly tested as 



