INOCULATIONS WITH BACTERIA CAUSING PLANT DISEASE 289 



tration. Ways for testing the relative efficiency of several techniques 

 are considered in a later section. 



Soil ''inoculation." The introduction of large numbers of pathogenic 

 bacteria into the soil depends upon growing sufficient quantities in cul- 

 tures, either on agar or in liquid media. Special flasks, bottles, and other 

 containers having adequate flat surfaces are useful. Most plant patho- 

 gens are aerobic and need incubation under pronounced aerobic conditions 

 for the best growth. When agar is used, the surface growth is washed or 

 scraped off after sufficient growth has appeared, and a suspension is 

 made. When a liquid medium is employed, a satisfactory bacterial 

 count per cubic milliliter develops, with an organism like Agrobac- 

 terium tumefaciens (Smith and Town) Conn., with a medium less than 

 2 cm deep or with one well aerated by shaking or by some other means. 

 Satisfactory aeration may be secured in deep liquid cultures by bubbling 

 sterile air through a sintered glass or other aerator placed in the medium. 

 In large containers aeration can be improved by a few pounds of pressure. 

 This forces more air to dissolve in the liquid. Maintaining such pressure 

 also reduces contamination from leaky valves. Chemicals that poise 

 the oxidation-reduction potential may be helpful. The highest count of 

 active bacterial cells may occur somewhat before the maximum turbidity 

 is attained. Bacterial gum may cause considerable turbidity. Usually 

 the whole culture is employed for soil treatment, but one should avoid 

 adding too much extraneous matter with the inoculum. Such aerated 

 liquid cultures also work well with many fungi. 



Soil may be "inoculated" by pouring liquid suspensions on relatively 

 dry soil, by allowing the water to be absorbed long enough to avoid 

 puddling, and by mixing. The quantity of culture used for each plant 

 varies. One might begin with 1 part of culture to 10 parts of soil and 

 use a handful of this mixture about the roots of each plant. 



Inoculations through the soil are considerably more difficult than 

 those with various other methods. 



Seed inoculation. Perhaps the easiest way to infect a large population 

 is through treatment of the seed. Legume root nodule bacteria from a 

 fresh, active culture grown on agar are shaken into a water suspension 

 and are commonly spread on the seed just before planting. Many 

 commercial inocula are prepared by mixing the culture with some mois- 

 ture absorbing powder, such as autoclaved ground peat. Wood flour is 

 also absorbent and contains almost no bacteria. If the seed is drill- 

 sown, it is made only moist enough to "fix" the bacteria on the seed and 

 then dried sufficiently so as not to clog the drill. To secure uniform 

 results it is best to use plenty of bacterial culture. Fred, Baldwin, and 

 McCoy (1932) have reviewed this general subject. 



Spray inoculation. Spraying is the method most commonly used to 

 inoculate growing plants. It is particularly useful with bacteria that 



