394 ELDON W. SANFORD 
In attempting to analyze the explanation and significance of 
the intratracheal fat, I soon found that if I bathed a cockroach 
in olive oil, the oil entered the spiracles readily and filled some 
of the tracheal tubes to a considerable extent. Schliiter has sug- 
gested, too, that some fat might enter the spiracles. This 
showed plainly that in work with tracheal tubes a new factor 
must be considered, namely, the sucking action of the tubes’ 
due to negative pressure.’ This factor was not used or elimi- 
nated in experiments by previous authors, and I now realized 
that I must always consider it in further experiments. 
Moreover, the fact that olive oil enters the spiracles affords an 
entirely new method of attacking the problem. ‘The oil enters 
during the bathing process because it is sucked in by the respira- 
tory movements which cause negative pressure within; oil is 
sucked in until pressure is equalized throughout the tubes, so 
that all air which is contained in the tubes is now at atmospheric 
pressure. The animal may now be bathed in the oil until it dies 
through suffocation due to blocking of the spiracles and therefore 
its breathing movements cease. Complete equalization of pres- 
sures throughout the tracheal system is then assured, and conse- 
quently there can be no subsequent sucking action. Knowledge 
of this process allows a new method of investigating whether or 
not fat is normally present in the tracheae after stained fat has 
-been eaten. For an animal can be bathed in oil which con- 
tains a stain of contrasted color to that of the stain which the 
food contained, this differently stained oil used for bathing will 
then enter the spiracles and equalize the pressures in the tubes, 
so all possible danger of sucking action in them during subse- 
quent dissection is obviated. As red oil was used in feeding, 
green oil would be ideal for the bathing process. However, no 
brilliant green stain for fat was available, so I used a green solu- 
tion which was found to enter the spiracles and tubes just as 
oil did. This was a mixture of equal parts of glycerin and 70 
per cent alcohol, and saturated with brilliant aniline green. By 
using this mixture to bathe until they died animals which had 
previously eaten red fat, it is evident that the following set of 
conditions would be given: green fluid in the main trunks, pre- 
