Orthotolidine (o-tolidine) can be used to help identify a blood stain.


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.



(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.


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