Benzidine test:

Benzidine test is a specific test used for the determination of blood (RBCs or haemoglobin) in urine. Kidney diseases or any injury to the urinary tract may result in leakage of blood in the urine. Blood (Haemoglobin) may occur in the urine as intact corpuscles (RBC) as in haematuria or free in the solution (Haemoglobin). Haemoglobinuria occurs in a certain condition in which the RBCs are haemolysed, and haemoglobin is liberated into the plasma. This haemoglobin is excreted by the kidneys and appears in the urine.

Haematuria can be recognized by the presence of red blood corpuscles under microscopic examination of the sediment, obtained by centrifugation of the urine.

Benzidine test principle:

H2O2 reduce benzidine solution. In this solution, the iron of haemoglobin gives blue colour complex.

Benzidine test Reagent:

The following chemicals are required to perform benzidine test;

• Benzidine reagent: 1. Benzidine dihydrochloride 2. Glacial acetic acid
• 3% hydrogen peroxide.

Benzidine test procedure:

• Take 3 ml of fresh benzidine reagent in a test tube.
• Add 2 ml of urine in it.
• And also add 1 ml of 3% hydrogen peroxide in it.

 

Observations:

A blue or green colour will develop if haemoglobin is present in the urine. This mean blood is present in urine.

Precautions:

To obtain a better result, follow the below steps.

• Before start performing experiment make sure to wash the apparatus before and then after the experiment.
• As always, carefully handle all the chemicals in the laboratory.
• Avoid urine touching with hands while doing the experiment.
• Use test tube holder for holding test tube.
• The test tube used should be clean neatly and free of any dirt and chemicals because we will not get the proper result then, so try to use a clean and clear test tube for a correct result.
• After experiment place the apparatus in their respective place.