ANIMAL TISSUES AND FLUIDS 31 



The addition of the sodium phosphate is not absolutely necessary. Cache states 

 that the addition of magnesium salts increases the value of the culture medium, 

 and advises the addition of 2 grams of magnesium phosphate per litre to ordinary 

 broth, in place of the sodium phosphate. Magnesium phosphate should be added 

 while the meat is macerating (Stage 1 above). 



5. The liquid is now strongly acid and must be neutralized, since bacteria 

 grow best in a neutral or slightly alkaline medium. 



Neutralization. To neutralize the medium, add normal soda solution to 

 the broth in small quantities at a time with a pipette, testing the reaction 

 at frequent intervals against litmus paper. When a drop of the broth placed 

 on a red litmus paper with the end of the stirring rod turns it slightly blue, 

 sufficient soda has been added. The reaction should be very slightly alkaline 

 to litmus, but acid to phenol-phthalein. 



Neutralization is the most difficult step in the preparation of broth. The amount 

 of alkali to be added varies considerably with different pieces of meat, and can 

 only be determined by trial. Add the soda solution very slowly stirring carefully 

 after each addition ; and as the neutral point is approached, test the broth after 

 the addition of each drop of alkali against both a red and a blue paper. A point 

 is ultimately reached when a drop of the liquid produces no change on either paper; 

 it is then sufficient to add a very small quantity of soda solution to attain the 

 requisite degree of alkalinity. According to Park and Williams, 7 c.c. per litre of 

 normal soda should be added to a neutral broth to obtain the most favourable 

 medium. 1 



[Eyre uses phenol-phthalein as the indicator and standardizes after Stage 3 

 before the addition of peptone and salt. 



[Technique. 1. Heat the merft extract in the steamer at 100 C. for 45 minutes. 



2. Measure 25 c.c. into a beaker and add about 0*5 c.c. of a 0-5 per cent, solution of 

 phenol-phthalein in 50 per cent, alcohol. 



3. Immerse the beaker in a water bath and raise to boiling point. 



4. Neutralize at the boiling point with deci-normal NaOH solution. 



[The reaction is expressed by stating the number of cubic centimetres of normal 

 alkali required to render one litre of the meat extract exactly neutral to phenol- 

 phthalein. 



[For the majority of organisms a medium which requires the addition of 10 c.c. 

 of normal alkali per litre of meat extract is found to be the best. In Eyre's scale 

 the reaction of such a medium is expressed as -f- 10 : + indicating that the medium 

 is acid and 10 that it is acid to the extent of 10 c.c. of normal alkali per litre.] 



6. Now pour the slightly alkaline broth into a glass flask, or better into 

 an enamelled vessel, and autoclave at 115-117 C. for 5 minutes. The 

 liquid becomes cloudy and deposits crystals of earthy phosphates. 



On taking out of the autoclave, filter while hot through a Chardin paper 

 moistened with water : the filtrate should be absolutely clear. The object 

 of this procedure is to remove any excess of earthy phosphates, and if omitted, 

 the broth is likely to become cloudy when sterilized. 



7. Add sufficient distilled water to the filtrate to make the total volume 

 up to 1 litre. 



8. This completes the preparation of the broth, which has now only to 

 be distributed into suitable vessels and sterilized. 



Sterilization. (A) If the broth is to be kept for future use, it may be 

 sterilized in a large flask the neck of which is either plugged with wool or 

 drawn out in the flame and sealed. The medium can thus be kept indefinitely, 

 and when required for use can be distributed into suitable vessels. 



(B) It is however usually more convenient to distribute the broth at once 

 into test-tubes or small flasks. 



1 Normal soda contains 40 grams of NaOH per litre of distilled water. 



