Preparation of Safranin Stain in the Laboratory

Safranin is a common biological stain within microbiology laboratories, particularly in staining, e.g. Gram staining and endospore staining. It is usually a counterstain that stains cells either pink or red, enabling laboratory technicians and scientists to differentiate between types of bacteria with the help of a microscope.

When you are in a microbiology lab, it is well-known that to make right microscopic observations and good results, it is essential to know how to prepare safranin stain correctly.

This manual describes how to prepare safranin stain in the lab, formula, calculations, materials and preparation process.

What Is Safranin?

Safranin is a simple stain, which attaches to the negatively charged bacteria cell components. It is mostly applied as an antstain in Gram staining where it turns Gram-negative bacteria pink/red following the decolorization process.

Safranin has common uses which include:

1. Gram staining (counterstain)
2. Endospore staining
3. Capsule staining
4. Plant tissue staining
5. Histological staining

The concentration of Safranin is standardized. 0.5% Safranin solution is typically made in the majority of microbiology laboratories. This means: 0.5 g Safranin in 100 mL solvent. The distilled water is normally used as the solvent.

In some cases, or protocols, a 0.1% Safranin concentration can be used. The dye content of the dye can range between 84% to 100%.

Materials Required

The laboratory preparation of safranin stain will require:

1.     Safranin powder (Dye content of 100%)

2.     Analytical balance

3.     Distilled water

4.     Conical Flask

5.     Volumetric Flask

6.     Glass stirring rod

7.     Filter paper

8.     Funnel

9.     Amber storage bottle

Formula and Calculation

In my case, I will be preparing 100ml of Safranin.

For 100 mL of Safranin Solution, 0.1% solution means: 0.1 Safranin in 100ml solvent

So, you need:

·       0.1 g Safranin powder

·       100 ml of the distilled water.

Preparation of Safranin Step-by-Step Procedure.

Step 1: Weigh Safranin Powder.

Weigh with an analytical balance: 0.1 grams of Safranin powder.

NB: Precision is essential to guarantee that there is staining strength.

Step 2: Dissolve the Safranin

Add around 70 mL of distilled water into a conical flask then add the safranin powder (weighed).

Stir thoroughly with a glass rod till the powder dissolves.

Note: The use of ethanol facilitates dissolution of the dye, as well as enhances stain penetration.

Step 3: Make Up the Final Volume(100ml)

Continue adding the distilled water until the solvent reaches a total volume of 100 ml.

Mix thoroughly.

Step 4: Filter the Solution

Use filter papers and funnel to filter the solution to eliminate any particles that do not dissolve.

Step 5: Store the Stain in amber bottles

Then, label the bottle(s) with:

·       Name: Safranin stain

·       Concentration: 0.1%

·       Date of preparation

·       Prepared by

Our procedural tutorial on the preparation of the safranin stain in the laboratory has a step by step guide in each of the steps. Watch and learn more about how the stain is prepared.

Conclusion

Safranin is the last counter stain during gram staining. It is important to note that the stain is easily contaminated and therefore you need to measure accurately in order to achieve the best results. For long term use the prepared stain can be stored in amber bottles for later use and should be labelled well.

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