BACILLUS TETANI 509 
unhesitatingly relied upon for isolation is the Barber method for 
separating single cells from cultures. It has been found, however, that 
the Barber method as ordinarily applied does not result in more than a 
minimal proportion of successful results, and this is due in part to the 
unfavorable effect of air (oxygen) upon the bacterial cell. For three years 
the procedure of isolating spores rather than vegetati\e rods has been 
followed^ with considerable success. By so doing the effects of exposure 
to oxygen have been circum^•ented, and if the precaution is taken to 
plant the single spores in small, deep tubes of liver-meat medium, 
which are immediately placed in a vacuum incubator^ for a few hours, 
a relatiAely high proportion of successful cultivations may be realized. 
On the average, about 35 per cent of growths may be obtained whereas 
less than 5 per cent are obtained if the vegetative cell is isolated. 
Cultures of anaerobic bacteria in fluid media, even those relatively 
poor in protein nitrogen are readily obtained by the use of test-tubes 
designed by Hall,^ or by the use of flasks, as shown in the outline figures 
on page 244. 
The method of procedure is as follows: The medium in the tube or 
flask is steamed for half an hour in the Arnold sterilizer,'* then removed, 
and the porcelain or glass ball is dropped into the neck of the vessel, 
(precaution being taken that the fluid reaches at least 2 cm. above the 
top of the ball), and the medium is cooled rapidly to incubator tempera- 
ture. Inoculation is readily accomplished by tilting the vessel until 
the ball drops to one side, thus aft'ording a passage to the underlying 
medium. A drop or two of infected material is discharged into the 
fluid below the constriction, the vessel is placed upright again, thus 
releasing the ball, which seats upon the constriction, and the customary 
incubation is practised. This simple device gives practically 100 per 
cent of successful growths, as has been shown by several thousand 
successful inoculations. Fermentation tubes, similarly constructed in 
that the opening is large enough to pass the ball, and constricted just 
below the bulb, also give excellent results. Ordinary fermentation 
tubes without the ball are unsatisfactory. The use of blood-agar plates, 
inoculated by the "spiral streak" method of Varney (page 238) com- 
bined with the Varney anaerobic jar has been found to be an eft'ective 
method of isolating pure cultures by the plate method. P^xperience 
has shown that at least one repurification by replating is desirable. 
BACILLUS TETANI. 
The infectious nature of tetanus was first demonstratefl clearly by 
inoculating rabbits subcutaneously with pus from a human case of the 
disease. This experiment, which reproduced the essential clinical 
1 Kendall, Cook and Ryan: Jour. Infec. Dis.. 1921, 29, 227. 
2 The De Freas vacuum oven is admirable for this purpose. 
3 University of California, Dept. Pathol., 1915, 2, 147. 
^ Carbohydrates are sterilized separately and added after the steaming process to 
avoid decomposition due to heat and OH ions. 
