14 BACTERIA IN RELATION TO PLANT DISEASES, 
and then dig under into the periphery of the diseased portion. If the tissues are 
rather dry the bacteria may be forced into the cavity by careful squeezing, or some 
drops (loops) of sterile water or beef-bouillon may be introduced into the cavity and 
stirred around before the bacteria are removed. If heat is inadmissible, the speci- 
men may be washed or soaked for a time (15 seconds to 60 minutes) in mercuric 
chloride water (1:1000) and the surface thus freed from many contaminating organ- 
isms. Carbolic acid (5 per cent in water) or lysol (5 per cent in water) may 
also be used for sterilizing surfaces. Of course these substances must be removed 
as far as possible before the surface is broken. ‘This may be done to some extent 
by swabbing with sterile absorbent cotton dipped into sterile water or by plunging 
into sterile water and shaking. ‘The disinfectants will be more certain to touch 
and sterilize every part of the surface if all adhering particles of air are driven off 
by first plunging into alcohol for a moment. 
In case of bacterial leaf-spots the writer generally obtains satisfactory cultures 
by cutting out the spot and plunging it for a few seconds (15 to 45) into 1:1000 
mercuric chloride water, then rinsing in sterile water for a few minutes, crushing and 
throwing into a tube of bouillon from which the plates may be poured in course of 
an hour, z. é., as soon as the bacteria from the interior of the spot have had time to 
diffuse into the bouillon. I frequently crush with a sterile glass rod, after throwing 
the material into a tube of bouillon, or else on a small sterile cover-glass which is 
then thrown into the bouillon. 
In cases where heat and chemical disinfectants are both inadmissible on 
account of danger of destroying the organisms within delicate tissues, as in thin 
leaves and other soft parts, the bacteria or fungus-spores accidentally lodged on 
the surface may be greatly reduced in number by gently rubbing all parts of the 
surface between the thumb and finger under distilled water and then washing them 
in three or four successive beakers of distilled sterile water, the fragments being 
transferred from one beaker to the other by means ofsterile forceps. Of course, the 
thumb and fingers must be well cleaned in advance by scrubbing and sometimes by 
the use of alcohol and corrosive sublimate, followed by sterile distilled water. When 
dry, these washed specimens may be scraped into, directly for plate cultures, or after 
the epidermis has been peeled off with cold sterile knives and forceps. 
Quantitative determinations may be made by grinding up a given quantity of 
the suspected plant tissue, e. g., a cubic centimeter or a gram, in a sterile mortar 
with clean sterile sand and 10 or 20 cc. of beef-broth or sterile water, and then 
making plates from carefully measured portions of the fluid, e. g., from one 2-inm. 
loop, from 0.1 ce., 0.5 ce., etc. A like number of check plates made from equal 
portions of healthy tissues ground under precisely similar conditions will soon 
demonstrate about how many colonies are to be expected per plate (and what kind) 
as the result of surface contamination or air-borne bacteria introduced during the 
process of grinding. 
The procedures described under c and @ should be repeated a number of times 
(the more the better) and always with uninoculated plants in abundance for compari- 
son. These control-plants or check-plants must remain healthy. If they also become 
