Lactophenol Cotton Blue (LPCB) - Principle, Composition, Functions, Procedure, Uses, Disadvantages

Introduction to Lactophenol Cotton Blue (LPCB)

Lactophenol Cotton Blue (LPCB) is one of the most commonly used mounting mediums and clearing agents in the laboratory for the observation and identification of fungi. This clear blue liquid is also used as a staining agent.

Principle of Lactophenol Cotton Blue (LPCB)

Lactophenol Cotton Blue (LPCB) binds to chitin, which is also present in fungal spores and fungal cell walls.

In some situations, lethal agents such as 10% Potassium Hydroxide (KOH) can also be used as a clearing agent.

Composition of Lactophenol Cotton Blue (LPCB)

The composition of Lactophenol Cotton Blue (LPCB) is as follows:

• Lactic acid: 20 ml

• Cotton Blue (Poirrier’s Blue): 0.05 g

• Phenol Crystals / Phenol concentrate: 20 g / 20 ml

• Glycerol or Glycerine: 40 ml

• Distilled water / 1% aqueous solution: 20 ml / 2 ml

* 70% ethanol is placed on a microscope slide before using Lactophenol Cotton Blue (LPCB).

* Lactophenol Cotton Blue (LPCB) is prepared at least 2 days before use.

Function of Lactophenol Cotton Blue (LPCB)

Lactophenol Cotton Blue (LPCB) has many properties that make it ideal for its use as a mounting medium.

• LPCB contains lactic acid which functions as a clearing agent and helps preserve fungal structures.

• Phenol kills the fungus, making it safe to remove the slide from the biological safety cabinet (BSC).

  * Fungal spores are easily dispersed which can contaminate the laboratory or infect the microbiologist.

• The quick drying of the slide specimen is prevented by glycerol due to its hygroscopic property i.e. since it is viscous.

• The Aniline dye i.e. cotton blue present in the LPCB solution provides color to the fungal structures and hence enhances as well as contrasts the cellular structures.

Procedure to make Lactophenol Cotton Blue (LPCB)

Lactophenol Cotton Blue (LPCB) is sold commercially in various volumes and can be easily bought.

In case it must be manually produced, the procedure for producing Lactophenol Cotton Blue (LPCB) is as follows:

1. Dissolve the cotton blue in distilled water and leave it to rest overnight as this step is crucial in eliminating insoluble dye.

2. Heat the phenol crystal gently in the mixture of required volumetric proportions of lactic acid, glycerol, and water. Use gloves for protection and a magnetic stirrer for a homogenous mixture.

3. Add Cotton Blue (Poirrier’s Blue) to the concoction.

4. Mix well, let it cool, and store it at room temperature before it is ready to be used in the laboratory.

Procedure of LPCB preparation:

The procedure of the LPCB preparation test for fungal infection diagnosis is as follows.

1. Place 1-2 drops of Lactophenol Cotton Blue (LPCB) on a clean, grease-free slide.

   * Optional: Add one drop of 10-20% KOH solution to it

2. Place a small quantity of the specimen into the drop and tease the culture with the help of teasing needles.

3. Place glass cover on the mix gently to avoid entrapment of bubbles (press gently to remove air bubbles if present).

4. Use gauze/blotting paper to blot the excess solution

5. Place the slide on the microscopic stage and examine it with low-power (10x objective) and then under 40x objective for fungal bodies, budding yeast, or yeast with pseudohyphae.

6. The wet mount preparation of Lactophenol Cotton Blue (LPCB) should be examined at least 30 minutes after preparation.

Result, Interpretation of Lactophenol Cotton Blue (LPCB)

After Lactophenol Cotton Blue (LPCB), fungi appear as dark blue stained mycelium.

Advantages of Lactophenol Cotton Blue (LPCB)

1. Easy and cost-effective solution.

2. They are commonly used for the identification of fungi and their structures.

3. It stains both fungal cell walls and fungal spores.

4. It also stains cysts of intestinal protozoa while ova of helminths are stained deep blue. Also, both bile-stained and non-bile-stained helminthic eggs are stained blue.

5. In parasitic studies of stool samples, Lactophenol Cotton Blue (LPCB) clears fecal debris as well.

6. Lactophenol Cotton Blue (LPCB) can also detect blue-colored Cyclospora and Isospora oocysts.

Disadvantages of Lactophenol Cotton Blue (LPCB)

1. Lactophenol Cotton Blue (LPCB) is used as presumptive identification for fungi. Definitive identification is done by fungal culture, biochemical, or molecular methods.

2. It may disrupt the original morphology of fungi.

3. Identification is done if mature structures are present as a young vegetative state is not enough to determine the fungi involved.

4. The mounting media cannot be stored for a long period as it does not have a long shelf-life.

5. Although applicable, Lactophenol Cotton Blue (LPCB) is not used in parasitology as it kills trophozoites of Entamoeba and Trichomonas.