[Vor. 1 
52 ANNALS OF THE MISSOURI BOTANICAL GARDEN 
а possible relation of the characteristic salivary organism to 
the pollution of air was to be investigated, it was necessary 
to examine the outdoor air free from human contamination 
for the presence of micro-organisms closely allied to those 
characteristic of saliva. Ав particles shed from the skin may 
be present in the air, it was further necessary to examine those 
micro-organisms found on the skin which were closely allied 
to the ones characteristie of saliva. 
In examining the saliva for the type of micro-organism · 
most constantly present, 1.е., whether bacillus, coccus, or 
spirillum, the dilution method was used. It is reasonably 
safe to assume, after repeated trials, that the type of micro- 
organism which persists longest in continued dilutions is the 
type most abundant in the material examined. This is true 
provided the medium on which the organism is grown is approx- 
imately equally favorable for the development of all the types 
present. Тһе dilutions were carried out as follows: A sample 
of saliva was collected in a sterile test-tube and 1 cc. introduced 
into а second tube containing 9 сс. of sterile distilled water. 
The contents of the latter were then thoroughly mixed and 
1 ec. of the liquid introduced into a third tube likewise con- 
taining 9 cc. of sterile distilled water. This procedure was 
repeated until 6 dilutions had been effected. Obviously, 1 cc. 
quantities of each of the 6 successive dilutions contain re- 
1 1 1 1 . 
spectively 5, i0» 990, 10,000) 100,000) 2Nd 10006 CC. Of saliva. 
One plate each from dilutions 4, 5, and 6 was made, 1 cc. of 
the respective dilutions being introduced into 10сс. of nutrient 
+ 1 agar. After thorough mixing, the plates were incubated 
aérobically for 24 hours at 37°C. Тһе plate made from dilu- 
tion 5 produced 20 colonies, whereas the one from dilution 6 
showed no growth. From each of the 20 colonies a cover-glass 
preparation stained with gentian violet was made. Microscopic 
examination revealed the fact that each of the 20 colonies 
was composed of micro-organisms of the coccus type. Transfers 
were then made to agar slopes which were incubated at 37°C., 
for 24 hours. The cultures obtained in this manner were num- 
bered from 1 to 20 and kept at 20°C., as stock cultures. 
In examining the open air, sterile agar plates were exposed 
as indicated in table 1. 
