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, i. e., 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 of sterile 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 detenninations 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-mm. 

 loop, from o.i cc., 0.5 cc., 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 d 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 



