302 DIFGO MANUAL 



the indicator from cerise alkaline reaction to reddish orange almost neutral 

 reaction. Reasonable care should be exercised to prevent overacidification as this 

 will tend to coagulate the plasma spontaneously. When the indicator color 

 change begins to turn from cerise to reddish orange, double check the pH by 

 removing a drop of the plasma aseptically and transferring it to a few drops of 

 distilled water to which indicator has been added on a spot plate or in a test tube. 

 The recheck of the reaction by the test tube or spot plate method is essential 

 because indicators do not always give true color changes in undiluted protein 

 solutions. Failure to adjust the reaction of Chicken Plasma may give prolonged 

 clotting times. For example, undiluted Chicken Plasma containing 0.002 per cent 

 Phenol Red may give a color reaction indicative of pH 7.2 whereas the true 

 reaction as determined by dilution in a test tube or spot plate may be pH 8.0 or 

 greater. Also, color reactions with Phenol Red in undiluted plasma indicative of 

 pH 7.2 reaction may actually be more acid by the test tube dilution and spot 

 plate methods. When the reaction has been adjusted to pH 7.2-7.4, stopper 

 tightly to prevent escape of carbon dioxide. Store in refrigerator at 2-6°C. if not 

 used immediately. Storage for periods longer than one week is not recommended. 



2. Transfer the reconstituted plasma containing Phenol Red to a clean, dry 

 sterile tube. Bubble sterile carbon dioxide or a mixture of sterile carbon dioxide 

 and nitrogen through the solution until the cerise color changes to reddish 

 orange. Recheck pH by test tube or spot plate as outlined above. Adjust final 

 reaction to pH 7.2-7.4. 



3. Same as method ^2 except blow sterile alveolar air through the plasma solu- 

 tion to give pH 7.2-7.4 using precautions stated above. 



It has been our observation that Embryo Extracts diluted with Balanced Salt 

 Solutions containing calcium in levels comparable with TC Balanced Salt Solu- 

 tion (Earle's BSS) give a faster clotting rate and a firmer coagulum with TG 

 Chicken Plasma than do extracts prepared with a lower calcium content. 



TG CHICK EMBRYO EXTRACT EEioo (B355) 



TC Chick Embryo Extract EE^qq is sterile desiccated whole, undiluted Chick 

 Embryo Extract certified for use in tissue culture procedures requiring embryo 

 extract. It is prepared from selected eleven-day old chick embryos, and is 

 packaged in 2 ml. quantities. 



TC Chick Embryo Extract EE^qq is reconstituted by adding, aseptically, 2.0 

 ml. of TC Reconstituting Fluid or TC Triple Distilled Water to the 2 ml. bottle. 

 When the contents of the bottle have uniformly combined with the water, they 

 are diluted further as desired with TC Balanced Salt Solution. A 20 per cent 

 Embryo Extract, EE20, has been found to be useful in tissue culture procedures. 

 This concentration is obtained by adding 4 volumes (8 ml.) of TC Balanced 

 Salt Solution to the reconstituted Embryo Extract EEjoq- The Reconstituting 

 Fluid and the Balanced Salt Solution may be added conveniently by means of 

 a sterile syringe and needle. 



The diluted Embryo Extract EE20, or other desired concentration, is allowed to 

 stand at 35-3 7 °C. for 30-60 minutes to obtain maximum solution of the nutrients. 

 Solution of the desiccated material is accelerated by gentle end-over-end rota- 

 tion of the bottle. Do not shake. A small portion of the desiccated Embryo Extract 

 may remain in suspension. This insoluble residue should be removed by centrifu- 

 gation of the bottle for 10 minutes at 2500-3000 rpm. The supernatant liquid is 

 now ready for use. Unused portions of the diluted Embryo Extract should be 

 stored in tightly stoppered containers in the refrigerator (2-6°C.). Storage of the 

 reconstituted TC Chick Embryo Extract for periods longer than one week, even 

 though refrigerated, is not recommended. 



It has been our observation that Embryo Extracts diluted with Balanced Salt 



