290 MANUAL OF MICROBIOLOGICAL METHODS 



enter the host plants through natural openings such as stomata, water 

 pores, and nectaries. For many simple tests, suspensions of bacteria 

 are merely sprayed on the surfaces of susceptible leaves, stems, flowers, 

 fruits, etc. For more exact tests, as for comparative virulence, one 

 suspends the growth from an agar culture in water, saline solution 

 (0.9 per cent NaCl), or a selected buffer (such as suitable mixtures of 

 dilute K2HPO4 and KH2PO4, and standardizes the concentration accord- 

 ing to a selected and measured turbidity. If the bacteria have been 

 grown in liquid culture, the entire culture may be used. This procedure, 

 however, is often unsatisfactory because, after spraying, secondary 

 organisms may grow in the nutrient medium. It is frequently better 

 to separate the bacteria from the medium by means of a centrifuge and 

 to resuspend the cells as with the growth from agar media. 



The number of bacteria in a suspension may be determined, for 

 example, (1) by a Breed count or by direct examination in a Petroff- 

 Hausser counting chamber or (2) by mixing a known volume of the 

 bacteria w^ith previously counted suspensions of yeast or red blood cells, 

 by making smears, and by determining the relative number of bacteria 

 and cells. Bacterial suspensions often are duplicated by comparing 

 their turbidity with that of a graded series of barium sulfate standards 

 (described by Riker and Riker, 1936). A common density for a bac- 

 terial suspension has the turbidity of a solution obtained by mixing 1 

 ml of a 1 per cent solution of barium chloride with 99 ml of dilute sulfuric 

 acid. Turbidity can be measured accurately and rapidly with a suitable 

 instrument. 



The prepared bacterial suspension is filtered through cheesecloth to 

 remove small pieces of agar or other materials which might clog the 

 spray nozzle and is placed in the spraying device. The plants are 

 sprayed so that good coverage is given, especially to the lower sides of 

 leaves which commonly have more stomata. A strong spray may force 

 the bacteria through the stomata into the leaves. The plants are placed 

 in an environment where they will not dry off for a number of hours. 



Certain additional precautions may be necessary for best results. 

 For example: (1) The relative humidity of the air surrounding the 

 host plant is maintained at saturation before as well as after inoculation. 

 This and suitable light help to provide wide-open stomata. The length 

 of time necessary varies with the host plant and the parasite. A satu- 

 rated atmosphere for 6 to 18 hr in both instances favors infection with 

 many leaf parasites. Various kinds of moist chambers, e.g., that 

 described by Keitt et al. (1937), can be used in the greenhouse. Small 

 outdoor plantings can be covered for a short time with a cloth tent 

 (Keitt, 1918), and water sprayed over the exterior. The amount of 

 mo-sture in the air apparently influences the intercellular humidity and, 

 correspondingly, the susceptibility of the host. (2) If the plant parts 



