294 



< n\i' 1 1 i< 22 



i id Ri 22-3. Discrete clones, grow- 

 ing on agar nutrient in a petri dish, 

 obtained by plating a dilute culture 

 of bacteria. 



i IGURE 22-4. Separate bacterial 

 clones obtained by the streaking 

 method. {Courtesy of TV. E. Mele- 

 chen. ) 



handicap in detecting phenotypic changes 

 due to mutation. Mutants that change the 

 morphology of bacteria must be detected by 

 microscopic examination. Unfortunately, 

 individual bacteria show relatively few clear- 

 cut morphological variations — traits such as 

 si/e. shape, capsule, pigment, and the pres- 

 ence or absence of flagella. The only mu- 

 tants detected then are those which alTcct 

 the relatively few morphological traits to a 

 measurable degree, seriously limiting the de- 

 tection of mutants by examination of indi- 

 vidual bacteria. 



Nevertheless, if a suspected mutant is 

 found under the microscope, it is essential 

 to isolate it and. from the members of the 

 clone it produces, determine whether or not 

 the new trait appears in the offspring. One 

 of the several methods of isolating an indi- 

 vidual bacterium is the tedious though exact 

 procedure of removing a single individual 

 from a bacterial culture with a micromanip- 

 ulator and placing it in a fresh culture 

 medium. Single bacteria can also be ob- 

 tained by two indirect methods, less exact 

 but quicker. If the bacteria are growing 

 in a liquid medium, the culture can be suffi- 

 ciently diluted so that a sample of it contains 

 relatively few individuals. When this sample 

 is poured onto the surface of a petri dish 

 containing a nutrient agar medium, the in- 

 dividual cells are distributed on the medium 

 at random and usually so spaced that the 

 visible clone which each cell later produces 

 is discrete (Figure 22-3). As an alterna- 

 tive, a small amount of a broth culture can 

 be picked up in a sterile inoculating loop and 

 the bacteria spread by streaking the loop 

 across the surface of fresh nutrient agar 

 medium (Figure 22-4). At some places 

 on the medium, single bacteria will be de- 

 posited sufficiently far apart to give rise to 

 separate colonies. Which of these methods 

 is used depends upon the precision required. 



The study of the individual bacterium is 

 presently restricted to morphological varia- 



