5 . There are many methods of taking 

 blood samples . K fish are very small, it is 

 best to sacrifice them and take blood from the 

 dorsal blood vessels after cutting off the caudal 

 peduncle . In larger fish blood can be taken 

 easily by cardiac puncture or from the aorta in 

 the roof of the mouth (Schiffman 1959). Another 

 way is to cut a gill arch or puncture dorsal 

 blood vessels in the caudal peduncle by a spring- 

 activated blood lancet. Most of the fish survive 

 such operations well . It is very important to 

 have the cut surfaces free from water, the 

 presence of which may dilute the blood sample 

 and cause hemolysis. If blood is taken from 



the caudal peduncle the vent must be covered 

 so that no excrement contaminates the blood 

 sample . If blood is taken with a syringe, the 

 needle and the syringe must be treated with an 

 effective anticoagulant, but must be free from 

 any Liquid which may dilute the blood sample, 

 particularly if the sample is very small. If 

 blood is collected by means of a syringe, a suf- 

 ficient number of treated and dry syringes with 

 needles should be on hand for the number of fish 

 to be examined at one time . 



6. If blood is collected directly to hemato- 

 crit capillaries, there are two sizes of commer- 

 cial capillaries treated with anticoagulant 

 available. The most frequently used capillaries 

 are 75 mm long with outside diameter of 1.2 to 

 1.4 mm. The other type for the Strumia tech- 

 nique is 32 mm long and 0.8 mm in diamter. 

 When fish are large enough the 75 mm capil- 

 laries are preferable. The 75 mm tubes re- 

 quire about 0.03 to 0.04 cub ml of blood and the 

 32 mm capillary about 0.01 cubic ml (or about 



10 ul). If blood is taken from the incision the 

 capillary is filled by holding it horizontally and 

 touching the drop of emerging blood. If blood 

 is collected by means of a syringe it can be col- 

 lected from the tip of the needle or the tip of 

 the syringe after the needle is removed. If 

 blood is collected in any vessel treated with an- 

 ticoagulant, it should be well stirred to keep it 

 homogeneous before filling the capillaries. 



Capillaries of 75 mm are available with- 

 out anticoagulant. These should be used when 

 blood is treated with anticoagulant in the syringe 

 or in any other way prior to filling the capillar- 

 ies. They may also be used if serum is required 

 instead of plasma . The hematocrit reading has 



no value whatsoever if blood is permitted to 

 coagulate during handling. 



7 . As soon as capillaries are filled with 

 blood, one end should be well closed. When sev- 

 eral capillaries are prepared they may be kept 

 in horizontal position until the last one is filled. 

 Capillaries may be closed either in flame or with 

 modeling clay; other methods of closing are not 

 as good. When closing in flame, capillaries 

 should not be filled more than 2/3 or 3/4 of their 

 length. Only the end which was not in contact 

 with blood and which is not soiled with blood can 

 be flame closed. The flame used should be hot 

 as possible. If no microbumer is available, a 

 20 or 22 gage hypodermic needle is an effective 

 substitute; a butane torch with a fine nozzle is 

 also satisfactory. It should take only a few 

 seconds to close the capillary. The part of the 

 capillary containing blood should not be heated 

 because the blood will break down. To prevent 

 blood from scorching, the capillary should be 

 held horizontally in the fingers in the place where 

 the blood ends . If heat penetrates too far, or the 

 flame is toolarge, or the capillary contains too 

 much blood, the capillary will be too hot to hold. 

 Until one becomes experienced it is advisable to 

 examine the fused end with a hand magnifier for 

 pinholes. 



If modeling clay is used it should not be 

 red but rather a color contrasting with blood. 

 A quarter-size disc, about 1/8 to 1/4-inch thick 

 should be prepared. It should be held in one hand; 

 with the other hand one should insert the capillary, 

 held horizontally, into the disc while rotating it 

 slowly . We have found that the only reliable way 

 of closing the 32 mm capillaries is a very hot 

 pinpoint flame. 



As soon as capillaries are sealed they 

 should be inserted vertically, seal down, into a 

 rack. Wooden racks with numbers are commer- 

 cially available. In this position capillaries with 

 blood can be kept for several hours at room tem- 

 perature (Guest and Siler, 1934) . Whenever work 

 is performed outdoors the capillaries should be 

 kept in shade and should not be allowed to freeze 

 even for the shortest time. 



8 . The most important step is centrifug- 

 ing. Any clinical centrifuge may be used, provided 

 that the tubes are protected from breakage during 



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