How to Detect Pneumocystis Carinii using Toluidine blue-0

The modified technique for the detection of Pneumocystis carinii of Gosey and his colleagues is highly recommended. This uses a glacial acid-sulphuric acid reagent to treat the smears prior to staining. (Toluidine blue-0). This sulphation stage helps to clear background material from the preparation, enabling the cysts to be seen more clearly.

Sulphation Reagent

Glacial acetic acid………………………………………………..45ml

Concentrated sulphuric acid………………………………………15ml

Caution: Both the above reagents are harmful and corrosive with irritating vapours, therefore handle them with great care, wearing protective gloves. When the sulphurie acid is added to the acetic acid, considerable heat is produced, therefore immerse the coplin jar in cold water, for safely procedures to follow when handling dangerous acids.  

• Pour the glacial acetic acid into a coplin jar (immersed in cold water)
• Slowly add the concentrated sulphuric acid and mix with a glass rod. Label and store at room temperature. In a sealed coplin jar, the sulphation reagent can be used for up to 1 week.

Toluidine blue-0 (TBO)…………………………………………….0.3g

Concentrated hypdrochloric acid…………………………………2.0ml

Absolute ethanol (or methanol)…………………………………..140ml

Distilled water………………………………………………………60ml

• Dissolve the TBO powder in the distilled water.
• With care, add the phydrochloric acid and mix.
• Add the ethanol or methanol and mix well. Label and store the stain at room temperature. It can be kept for up to 1 year.

Decolorizer

Use 95% v/v ethanol or 95% v/v methanol

                                                          Method

1. Immerse the methanol fixed smear and control smear in the sulphation reagent for 10 minutes, mixing the reagent with a glass rod after 5 minutes.
2. Remove the smear and wash them well in water (preferably running water) for 5 minutes. Allow to drain.
3. Cover the smear with the toluidine blue-0 stain for 3 minutes. Wash off with water.
4. Rapidly decolorize with 95% v/v ethanol. Wash with water. Allow the smear to air dry.
5. Add a drop of oil to the smears and cover with a cover glass. Examine with the 10x and 40x objectives.

Note: In the method of Gosey et al, the smear is dehydrated in 95% v/v ethanol and absolute ethanol, cleared, and mounted.