PLATING 



11 



the last portions will come through 

 well as low as 50° to 55° C. 



Keep the filtered agar hot in flowing 

 steam while preparing a second funnel 

 in the same way as the first. Then 

 filter as before. 



Plating. 



For miscellaneous milk' samples, the 

 character of which is not known, three 

 dilutions shall be made; 1 : 100, 1 : 

 1,000 and 1 : 10,000. Where the char- 

 acter of the milk is known, the number 

 of dilutions may be reduced. If the 

 milk is pasteurized, certified or known 

 to be fresh, and of high grade, the 

 1,000 and 10,000 dilutions may be 

 omitted. If the milk is known to be 

 old and of high bacterial count, the 

 100 and 1,000 dilutions may be omitted, 

 and dilutions in excess of 10,000 pre- 

 pared. In no case shall less than two 

 plates be made from each sample. 

 Where two satisfactory plates are obtain- 

 ed it is advisable to count both of them. 

 The water used for dilutions may be 

 placed in dilution bottles (99 cc, 

 49.5 and 9 cc. are convenient sizes) 

 and sterilized for one hour in an auto- 

 clave at 15 lbs. pressure. The bottles 

 should be marked so that it can be 

 determined that they have neither 

 gained nor lost water during or sub- 

 sequent to sterilization. Or, the water 

 may be sterilized in bulk, if kept in a 

 properly guarded container, and sub- 

 sequently measured directly into the 

 dilution bottles with sterilized pipettes. 

 The dilution bottles should, have 

 glass or cork stoppers, or some other 

 type of closing that makes shaking 

 possible. Cotton plugs are a less satis- 

 factory method of closure because a 

 small portion of the dilution water 

 will soak into the cotton'^. 



Straight sided pipettes graduated to 

 deliver 1 cc. are best. They may be 



either the two mark or the one mark 

 style. In either case, the errors of 

 measurement are caused more by faulty 

 calibration or by faulty manipulation 

 of the pipettes than by the particular 

 form of pipette used. In using two 

 mark pipettes, great care must be 

 taken to see that the quantities used 

 are exactly 1 cc, while many one 

 mark pipettes in use are calibrated to 

 contain 1 cc. rather than to deliver 

 1 cc. Breakage of tips of the latter 

 type of pipette also causes errors'*. 



In making dilutions the original 

 sample and each dilution bottle shall 

 be rapidly shaken 25 times, each shake 

 being an up and down excursion of 

 about one foot'* (entire shaking not to 

 take longer than about seven seconds). 

 After the final dilution fill a pipette to 

 the mark and allow contents to run 

 into an empty petri dish, the end of 

 the pipette touching the dish as the 

 liquid runs out. If the pipettes are of 

 the one mark style be sure that they 

 are so manipulated as to deliver a full 

 cubic centimeter. Use care to raise 

 the cover only as far as necessary to 

 insert the end of the pipette. 



Pipettes should be placed immediate- 

 ly in water after using to make sub- 

 sequent cleaning easier. 



The flasks (or test tubes) of agar 

 shall be melted in boiling water or 

 steam and after melting shall be 

 cooled to a temperature of between 

 40 and 45° C. before using. 



Pour about 10 cc. of the melted 

 agar in each inoculated petri dish, 

 and by a gentle rotary motion thor- 

 oughly mix the agar and the diluted 

 milk. As nearly as possible the same 

 amount of agar should be poured into 

 each petri dish so that the depth of 

 agar will be uniform in all. If desired 

 10 cc. may be measured out from the 

 flask with a sterile pipette. 



