34 CULTURE METHODS AND TECHNIQUE 



Fuller's procedure modified may then be a guide; this is as 

 follows: (i) Measure by a volumetric pipette or burette 5 cc. of 

 the culture medium, and dilute it with distilled water to 50 cc. 

 (2) Boil: for three minutes in a porcelain dish. (3) Add I cc. of the 

 stock solution of the indicator, phenolphthalein, and titrate by add- 

 ing the ^V P er cent caustic alkali from a burette. Stir constantly, 

 and a permanent faint pink coloration will indicate the first appear- 

 ance of alkalinity. Those inexperienced in the work should always 

 take two or even three samples of the culture liquid and compare 

 results as to the amounts of alkali employed. The data are then at 

 hand for neutralization or for making the medium correspond to a 

 desired reaction. If 6 cc. of the ^V normal alkali, for example, is 

 required to bring the fwo samples to the point of neutralization, 

 then the remaining 990 cc., assuming that we employ a liter of 

 culture medium, would require practically 100 times 6 cc., or 600 cc. 

 of J-Q normal, which is equivalent to 30 cc. of normal alkali. Ordi- 

 narily, the medium is not neutralized, but is left acid to the extent of 

 an omission of 10-15 cc. of normal alkali per liter. In the example 

 above, therefore, 15 or 20 cc. of normal alkali would be added. 

 A control titration may also be made. 



V. PRESENT METHOD OF ISOLATING ORGANISMS 



In making cultures with a view of isolating, or separating out 

 various microscopic organisms, the poured-plate (Petri dish) method 

 is now almost exclusively employed. Such cultures may be called 

 isolation or separation cultures, the use of the old term dilution 

 culture being less desirable. 



Materials needed. In order to make these isolation cultures, one 

 requires nutrient media and apparatus more or less as follows : 



Sterilized Petri dishes, of about 100 mm. diameter; test tubes 

 containing about 10 cc. of sterile agar agar ; a few very short test 

 tubes without agar ; a platinum needle ; a beaker, or tumbler, with 

 some cotton in it, to hold the tubes ; a thermometer ; and some 

 boiling water. The agar in the tubes is melted, either in the steam 

 sterilizer or in an open casserole of boiling water with cloth or 

 cotton at the bottom. In ordinary culture work, as well as in bac- 

 teriological work, three tubes, and consequently three Petri dishes, 

 constitute an isolation series. Three tubes of melted agar are 



