Remove Residual Chlorine before Microbial Analysis : Pharmaguideline

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Remove Residual Chlorine before Microbial Analysis

It is important to remove the residual chlorine from the water sample before the microbial analysis.
Raw water is chlorinated by adding sodium hypochlorite (NaOCl) to make its concentration 2-4 ppm. Chlorination of water is done to remove the microbial load from the water. It helps the water system to produce the purified water of specified microbial quality.

Mechanism of Action:

Water Sample
When sodium hypochlorite is dissolved in water, it produces hypochlorous acid and hypochlorite ion. These hypochlorite ions are known as “free chlorine”. This free chlorine is responsible for the chlorination of the microbes in water. These ions are highly active and penetrate the cell wall via lipids and destroy the enzymes in the cells.

All the free chlorine is not consumed in the chlorination. This remaining chlorine is known as "residual chlorine" which can interfere with the microbial analysis of the water. This may alter the actual results.

Sodium thiosulfate (Na2S2O3) can be used to neutralize or remove residual chlorine. It simply reacts and forms sodium hydrogen sulfate or sodium bisulfate which is an inactive salt.

Na2S2O3 + 4HOCl + H2O -----> 2NaHSO4 + 4HCl

Add 0.1 ml of 10% solution of sodium thiosulfate is added to the water sample bottle before sterilization. This can be prepared by dissolving 100 gm of sodium thiosulfate crystals in about 500 ml of water making up a final volume to 1000 ml. This solution can be stored under refrigeration for six months.

This neutralization of residual chlorine will help to determine the actual microbial load in the water sample and will not affect the microbial recovery.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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