the family Leguminosae have been used in 

 most work in this field. Extracts of many seeds 

 have been tested against many warm-blooded 

 vertebrates; however, tests for intraspecific 

 variability have been most extensively directed 

 toward the erythrocytes of man (Bird, 1951, 

 1952; Makela, 1957). To the authors' knowledge, 

 reports on the use of legume bark extracts 

 are limited to the work of Lau (see Makela, 

 1957), who found that Robinia pseudoacacia bark 

 extract agglutinated various mammalian eryth- 

 rocytes. 



This paper presented the results of a pre- 

 liminary search for reagents capable of dem- 

 onstrating blood types in Pacific salmon. Its 

 main purpose is to reveal the potentialities 

 of plant extracts as fish blood typing reagents. 

 Preliminary results of individual differences 

 shown by precipitation reactions of fish sera 

 with certain extracts are also discussed. 



METHODS AND MATERIALS 



Three methods were used to collect fish 

 blood samples. The usual technique was to 

 sever the caudal artery with a sharp knife or 

 razor. Where later use of the fish depended 

 on a minimum of visible mutilation, two other 

 techniques were used--the gill arches on one 

 side were severed and the blood drained 

 through the mouth, or the fish was bled by 

 cardiac puncture. Cells to be tested were 

 washed three times in a modified Alsever's 

 solution and brought to a 2 percent suspension. 

 Blood cells stored up to 15 days after bleeding 

 were used for testing, providing they were not 

 excessively hemolized. The majority of the 

 testing was done on material less than a 

 week after collection. 



Extracts of plant materials were made in 

 1 percent saline solution. Seeds were first 

 ground by mechanical grinder or mortar and 

 pestle. A paper cutter was found to be an 

 excellent tool for cutting bark. One gram of 

 seed or bark was mixed with either 4 or 9 cc. 

 of 1 percent saline, depending on whether the 

 extracts were to be tested for precipitating 

 properties or were to be used solely for ag- 

 glutination tests. In the latter case the 9 cc. 

 dilution was used. The suspensions were 



incubated for 2 hours at 37° C. and overnight 

 in the refrigerator, Centrifugation at 3,000 

 r.p.m. for 20 minutes yielded a supernatant 

 fluid essentially free of particulate matter. 

 This stock extract was used as a base for all 

 testing and dilutions. 



Agglutination tests were performed in 10x75 

 mm. tubes using 0.1 cc. of cell suspension 

 and an equal amount of stock extract or ex- 

 tract dilution. Precipitation tests utilized the 

 agar double diffusion technique of Ouchterlony 

 (1958). 



The extracts are quite stable when stored 

 at -30° C. The reactivity of the extracts was 

 not significantly altered by thawing and re- 

 freezing, although a few extracts lost potency 

 after several months of constant use. 



Comparable results were obtained using 

 both fresh and dried materials in this study. 

 Only green barks including the cambium layer 

 were collected. Fresh extracts were made, and 

 any bark remaining was dried and stored at 

 room temperature. Extracts made from barks 

 stored up to 8 months have shown little, if any, 

 loss of reactivity when compared with extracts 

 of freshly collected bark. 



A quantitative seasonal variation has been 

 noted in reactivity of certain barks. Extracts 

 made from bark collected in the late spring 

 and summer of Robinia pseudoacacia. Wistaria 

 floribunda,an(iCytisusscopariusharkshave shown 

 far weaker or negative reactions compared 

 to extracts made from the same plant in the 

 fall, winter, or early spring. It appears 

 advisable to collect bark when the plant is in 

 a dormant rather than actively growing state. 



STUDY OF PHYTOAGGLUTININS 



Many seed and bark extracts were tested 

 for agglutinins (table 2). Extracts were con- 

 sidered promising if they could be used to 

 detect individual variability or differences 

 among species. Although other promising ex- 

 tracts were found, only those listed in tables 

 3 through 8 were selected for more extensive 

 study. Only a single seed extract of those 

 originally screened is represented for each 



