Exercise Itl 



CHEMICAL COMPONENTS OF CELLS (1) 17 



the whole business. That is a very important 

 part of getting ready to do an experiment. If 

 you can see in your mind's eye just what you'll 

 be doing and how you'll be doing it halfway 

 through — whether, for example, you'll be hold- 

 ing the test tube in your right or your left hand 

 at that moment — then you are ready to go to 

 work. So your first job after reading through 

 the above procedure and the further discussion 

 of the manipulations below is to make yourself 

 a schedule of just what you expect to do and 

 when. It might come out somewhat as follows: 



(1) Grind cells, extract with TCA, centrifuge. 



(2) Precipitate glycogen, extract and precipi- 

 tate nucleic acid. 



(3) Centrifuge both preparations. 



(4) Get both nucleic acid and glycogen sam- 

 ples ready to hydrolyze. Put into boiling water 

 bath together. 



(5) Prepare the protein hydrolysis. 



(6) Look at the yeast. 



(7) Neutralize the acid hydrolysates and start 

 the dialysis of glycogen. 



THE MANIPULATIONS 

 Centrlfuging 



In its most primitive form a centrifuge might 

 be a boy whirling a bucket of water in circles 

 around his head, which as you know can be 

 done without spilling any water. If the water 

 had small particles of sand suspended in it, this 

 motion would make them settle faster to the 

 bottom of the bucket. In its most complex 

 form, an ultracentrifuge spins quartz tubes in 

 an evacuated chamber at tens of thousands of 

 revolutions per minute, developing forces well 

 over 100,000 times gravity. Under these circum- 

 stances macromolecules, being somewhat denser 

 than water, are sedimented. Your centrifuge 

 operates in between these two extremes. Its 

 maximum rate is about 3000 revolutions per 

 minute (rpm), and you will always use it at its 

 top speed. 



A centrifuge is a potentially dangerous instru- 

 ment, and certain precautions must be observed 



even with such relatively slow types as you are 

 using: 



(1) Use only plastic tubes, and do not fill 

 higher than about i inch from the top. 



(2) Each tube in the centrifuge must be bal- 

 anced against another of the same weight just 

 across from it. In your experiment it will be 

 enough to have both these tubes contain the 

 same volume of solution, gauged by eye. In 

 faster centrifuges it is necessary to balance the 

 pairs of tubes, preferably with their cups, against 

 each other on a sensitive balance. 



(3) Place the centrifuge well away from the 

 edge of the work table, and be sure that it is 

 level. Otherwise it may creep off the table while 

 running. 



(4) Close the lid before starting the centrifuge 

 and leave it closed until the centrifuge has 

 stopped spinning. Let it stop by itself; do not 

 brake it by hand. 



(5) If the centrifuge begins to vibrate strongly 

 and clatter while running, stop it at once and 

 check the balance of the tubes. 



(6) Bring it to top speed gradually. 



(7) Since others will be sharing the instrument, 

 do your centrlfuging efficiently. Get everything 

 ready before occupying the instrument, and get 

 your tubes out of it immediately when the job 

 is finished. 



Handling reagents 



We have designed the fractionation scheme 

 to keep the use of strong acids and bases to a 

 minimum. You will nevertheless be using nor- 

 mal sulfuric and hydrochloric acids, and barium 

 and sodium hydroxides. Try not to spill, but 

 if you should spill any on the table or floor, 

 clean up immediately, using a fair amount of 

 water to dilute the acid or base. If any spills on 

 you, rinse at once with water, and tell the in- 

 structor. Any large spill of acid can be neutral- 

 ized by sprinkling it with sodium bicarbonate 

 (baking soda). 



The alcohol that we use to precipitate glyco- 

 gen and nucleic acid is flammable, and should 

 not be used close to any flame. If you or your 



