How to Prepare Gram’s Iodine in the Laboratory for Gram staining

Iodine is one of the reagents used in Gram staining, a method that is among the most widely used in microbiological techniques of differentiating bacteria into Gram-positive and Gram-negative. 

When staining microorganisms under a microscope, proper preparation of iodine solution in the laboratory is important in order to achieve the right results.

This guide will take you through the essential steps on how to prepare Gram’s iodine digging deep on the materials needed, calculations and also the preparation process.

What is Gram's Iodine?

Gram iodine is a solution that consists of a mixture of the iodine crystals and potassium iodide that is dissolved in the distilled water at regulated amounts. The iodine dissolves well in water with the aid of the potassium iodide.

During gram staining, the stain is used as a mordant, which is used to fix the primary stain within bacterial cells. The procedure uses Crystal violet as the primary stain in the complex with iodine. This crystal violet-iodine complex gets entangled in the Gram-positive bacteria and it enables them to maintain the purple stain in the staining procedure.

Materials Required

The following laboratory materials will be required to prepare iodine solution to be used during the process of Gram staining:

1. Iodine crystals
2. Potassium iodide crystals (KI)
3. Distilled water
4. Analytical or digital weighing balance.
5. Conical Flask
6. Volumetric Flask
7. Mortar and Pestle
8. Funnel
9. Amber reagent bottle
10. Labelling tape and marker

Formulation of Grams Iodine Solution

NB: In our case, were going to prepare 1% Grams Iodine solution, 100 ml of the stain. That means dissolving 1g of pure iodine in 100 ml of the distilled water. Potassium iodide in this case is measured twice the amount of the Iodine used while still in the preparation of the stain. 

It is also advisable to crush the iodine crystals to increase the surface area at which the crystals will dissolve completely.

For 100 ml solution:

Iodine = 1 g

Potassium iodide = 2 g

Distilled water = 100 ml

Let’s say you want to prepare 1 L:

To prepare 1 liter, then just multiply the quantities by 10:

Iodine = 10 g

Potassium iodide = 20 g

Distilled water = 1000 ml

Procedure

1. Measure 2 grams of the Iodine crystals and transfer them into the conical flask
2. Using a Volumetric flask, measure 25 ml of distilled water and then dissolve the KI crystals. Mix Gently.
3. Measure 1g of the iodine crystals using the analytical balance and the put them in the motor.
4. Using the motor and the pestle, crush slowly the crystals to achieve the fine powder.
5. Transfer the fine powder into the conical flask and mix gently.
6. Measure and add 75 ml of the distilled water in to the solution. Mix to achieve the yellow orange or the brown colour of the stain.
7. Filter the solution using the funnel to achieve high purity stain.
8. Lastly, store the stain in amber reagent bottle for later use.
9. Label the bottle with the following details included:

·       Name: Iodine Solution

·       Concentration: 1%

·       Date of preparation

·       Prepared by: Include the date of preparation

Conclusion

Iodine preparation in the laboratory requires precise measurements of the iodine crystals, distilled water and the Potassium iodide. The crushing of the iodine is important as the crystals are quite stubborn in dissolving in water. Potassium iodide also plays a role dissolving the iodine crystals too.

Therefore, understanding these steps above is key in preparing a good stain for gram stain. We have a tutorial on what is gram stain and also on how to prepare the iodine solution. Watch the video to get a better understanding on the topic.