is necessary to determine the amplitude of 

 such "normal" variation before examination of 

 blood can be used for detection of "abnormal" 

 or pathological conditions. Environmental con- 

 ditions at different hatcheries must have a 

 significant effect on the composition of fish 

 blood. Therefore for effective use of hemato- 

 logical examination of fish for management 

 purposes two sets of "normal" standards must 

 be established. One should be the general or 

 national standard with a wide range of values, 

 another should be for individual hatcheries. 



While microhematocrit cannot replace 

 complete hematological examination for research 

 purposes, it is the only hematological method 

 which is simple, accurate, relatively free from 

 observational and procedural errors, and fast 

 enough to be practical for a general use — ^^ It 

 is suggested that it be used as widely as possible 

 and that the findings be made easily accessible 

 so that in time general and local standards can 

 be established. 



Observations here presented show that 

 the commercial heparinized capillaries which 

 we have used gave results 7 to 18 percent higher 

 than when blood was treated with anticoagulants 

 before introduction into capillaries. Such differ- 

 ence has not been noticed with human blood 

 (table 3). This probably is due to much faster 

 coagulation of fish blood and the robe played by 

 erythrocytes in blood clotting in fishes, as 

 evident from the recent research and review of 

 this subject by Wolf (1959). Additional work is 

 needed and a study is in progress to develop a 

 treatment for microhematocrit capillaries 

 which will completely prevent clotting and gela- 



]J The microhematocrit method was used on a 

 large scale by fish hatchery biologists of the 

 Bureau of Sport Fisheries and Wildlife in the 

 central and eastern United States during 1959. 

 This method permitted detection of numerous 

 cases of anemia in rainbow trout due either to 

 malnutrition, or an infectious disease, or a 

 combination of both . Remedial nutritional 

 measures permitted the control of this anemia 

 and prevention of heavy losses to some degree . 

 As result of our recommendations many Federal 

 and State hatcheries are using the microhemato- 

 crit method in year around quality control of 

 fish. 



tion of erythrocytes and in this way obtain more 

 accurate hematocrit values with fish blood. 



After a microhematocrit is determined 

 there is about 20 ul of plasma left in each capil- 

 lary. This may be used for the ultra -micro 

 chemical tests described by Natelson, 1951. 



DESCRIPTION OF TECHNIQUE 



Performance of the hematocrit test is 

 very simple and rapid . Detailed information 

 given here about the technique and precautions 

 is based on the recommendations made by Mc 

 Govern et al_. (1955) and on our personal exper- 

 ience . 



1 . If the hematocrit is run asr a routine 

 periodic test of fish quality in the hatcheiry, it is 

 extremely important that fish selected for exam - 

 ination are a representative sample from the lot 

 or a population. Sampling techniques are de- 

 scribed by Hewitt and Burrows (1948). 



2 . If the hematocrit is run for diagnostic 

 purposes the sampling technique must be entirely 

 different from that used in evaluation of a lot or a 

 population. For diagnostic purposes fish should 

 not be sampled at random but only Individuals in 

 stress or showing abnormalities in appearance or 

 behaviour should be selected. It is also desirable 

 to examine for comparison several fish which 

 appear normal. 



3 . It has been shown by several authors 

 that the blood picture in fish may undergo pro- 

 found and rapid changes if fish are exposed to 

 partial asphyxia . Therefore it is important to 

 take the blood sample immediately after fish are 

 collected. If this is not possible, fish should be 

 kept under conditions which are as similar as 

 possible to those existing in the body of water in 

 which they were raised. 



4. Fish to be examined should be anesthe- 

 tized just before taking blood. The anesthetic 

 should be a rapidly acting one, so that no changes 

 in blood will occur while fish are being anesthe- 

 tized. We are using tricaine methanesulfonate 

 (M.S. 222) in concentrations from 1:2000 to 

 1:5000 which usually anesthetizes trout in less 

 than one minute. 



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