METHODS 



Table 1 



Liquefaction 



Various methods of liquefying the liver 

 were tried including acid and enzymatic diges- 

 tion. Heating for periods long enough to break 

 down the liver tissue with "CI or H2SO4 de- 

 stroyed the bacilli (1). Pepsin and trypsin gave 

 only partial digestion (2). Treating with pep- 

 sin, decanting and treating the remaining un- 

 digested liver with trypsin gave about 90 percent 

 digestion, but was costly and time consuming 

 (3). Treating the livers with a 3 -percent solu- 

 tion of NaOH was found to give approximately 

 95 percent digestion . This had no deleterious 

 effects on the acid-fast bacilli as long as the 

 temperature was kept below 52°C. However, at 

 higher temperatures the bacilli began to disin- 

 tegrate . 



(1) 



Concentrations of HC1 and H2SO4 



tried: 3, 5 and 10 percent at 40° C . , 60° C. 

 and 100° C. for 10 min., 30 min. and 1 hr. 



(2) Concentrations of pepsin tried 

 0.25, 0.5 and 1.0 percent of 1:10,000 

 strength at 5ml . /gm . , 2ml . /gra, and 

 1.5ml./gm. for 20 min., 30 min. and 

 1 hr. at 1 percent HC1 and 52'C. 

 Concentrations of trypsin tried: 1, 5 and 

 10 percent of 1:250 strength at 5ml. /gm., 

 2ml. /gm. and 1.5 ml./gm. for 20 min., 

 30 min. and 1 hr. at pH 8.0-8.5 and40°C. 



(3) Concentration of pepsin used: 



0.5 percent of 1:10,000 strength at 1.5/ml. 



/gm. for 30 min. with 1 percent HC1 at 



52-C. 



Concentration of trypsin used: 10 percent 



of 1:250 strength at 1.5ml/gm. for 30 min. 



at pH 8.0-8.5 and 40°C. 



Laboratory procedure and preparation 



Fresh livers were inserted in water- 

 tight plastic sacks and frozen. The frozen liv- 

 ers were then weighed, the tare weight of the 

 plastic sacks subtracted and the sacked livers 

 placed in appropriate beakers (table 1). 



Wt . of liver 



0-55 grams 

 56-90 

 91-100 

 151-230 

 over -230 



Size of beaker 

 150 ml. 

 250 

 400 

 600 

 800 



If the sacks leaked after being frozen, 

 the livers were placed in an additional water- 

 tight sack before further processing. For 

 convenience, the weight of the liver was recorded 

 on the beaker. The beakers were filled with hot 

 tap water (45°C.-55°C.) without allowing the 

 water to enter the plastic sacks and placed into 

 a 45° C. water bath for a minimum of two hours. 

 Thawing of the ice crystals aids in the breakdown 

 of liver cells to prepare for final Liquefaction; the 

 faster the thawing, the more complete the break- 

 down of tissue . 



Digestion 



Water was poured out of the beakers and 

 following the preparation of a smear slide for 

 comparative purposes each liver was emptied 

 from the sack into a beaker . Three percent 

 NaOH was added in the quantity of 1.5 ml./gm. 

 of liver . The mixture was stirred and put back 

 in the water bath at 40° C . for approximately 16 

 hours after which 3 percent NaOH was added to 

 volume, the water bath turned up to 43 °C, and 

 the livers stirred about every half hour for 2 to 

 3 hours. 



Centrifugation 



A 33 ml . aliquot of each liquefied liver 

 was centrifuged for 30 minutes in a 55cc. hard 

 plastic or stainless steel centrifuge tube at 16,000 

 rpm., or 34,800 G's. The addition of 10 mis. of 

 a solution containing 9 percent Tween 80 in 95 

 percent ethyl alcohol before centrifuging inhibited 

 the formation of a liquid layer on top which traps 

 bacteria and would thus render the final bacterial 

 count unsatisfactory. 



If the centrifugation takes place in a cold 

 room, approximately 12 ml. of 9 percent alcoholic 

 Tween 80 should be used. 



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