o-tolidine = 3,3'-dimethyl-[1,1'-biphenyl]-4,4'-diamine = 3,3'-dimethylbenzidine
Reaction: Iron-containing hemoglobin has peroxidase activity, which releases oxygen from peroxide, which in turn oxidizes the orthotolidine.
Reagents:
(1) orthotolidine solution
(2) 3% hydrogen peroxide in a dark bottle
Orthotolidine solution (100 mL):
(1) 1.5 grams orthotolidine
(2) 40 mL ethanol
(3) 30 mL glacial acetic acid
(4) 30 mL of distilled water
Orthotolidine may be incorporated into a reagent strip, such as the Multistix (Ames).
A negative result is yellow. A positive result shows a green or blue color. The reaction should be read within 10 seconds.
It is a sensitive test, detecting blood a high dilution.
The o-tolidine test can be done in conjunction with the phenolphthalein test to increase the confidence that a stain is really blood.
False negative reactions may occur with:
(1) gastric acid
(2) high protein concentrations
(3) reducing substances
(4) high specific gravity
False positive results may occur with:
(1) oxidizing agents
(2) plant peroxidase (will release oxygen, rather than the hemoglobin): potato, radish, spinach, squash, tomato, turnip
False positive reactions do not occur on exposure to apple, cherry, grape or plum.
