Lactophenol Cotton Blue (LPCB) Stain: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses of LPCB Stain, Keynotes, and LPCB Stained Footages

Introduction of Lactophenol Cotton Blue (LPCB) Stain

LPCB stands for lactophenol cotton blue and Lactophenol Cotton Blue (LPCB) Stain is a combination of fixative, staining, and clearing agent. LPCB stain uses both as a mounting fluid and a stain and it is used for staining and microscopic identification of fungi. Its contents are lactic acid, phenol, cotton blue, and glycerol. Lactic acid helps in preserving the morphology of the fungal elements. Phenol acts as a disinfectant. Cotton blue stains the fungal elements as well as intestinal parasitic (cyst, ova, and oocyst) and non-parasitic structures (vegetable cells, mucus, muscle fibers, and other artifacts) while glycerol is a hygroscopic agent that prevents drying.

Principle of Lactophenol Cotton Blue (LPCB) Stain

Ingredients of lactophenol cotton blue (LPCB) stain-like lactic acid act as a clearing agent and aid in preserving the fungal structures. Similarly, phenol kills the organism and fixes it while glycerol prevents drying. Cotton blue stains the chitin in the cell wall of fungi and identification of filamentous fungi is made by their characteristic microscopic morphology e.g. shape, size, arrangement of spores, and hyphae providing color to the structure. It can be used alone or in conjunction with KOH.

Composition of Lactophenol Cotton Blue (LPCB) Stain

For 200 ml
Lactic acid: 40 ml
Phenol: 40 ml
Glycerol:80 ml
Cotton blue (Poirier blue or Aniline blue): 0.1 g
Distilled water: 40 ml

LPCB Stain Preparation

• Dissolve phenol in lactic acid, glycerol, and distilled water.
• Ultimately, add cotton blue and mix well.
• But this LPCB stain is prepared over two days.
• On the first day, dissolve the cotton blue in distilled water and leave it overnight to get rid of the insoluble dye.
• On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. Put on a magnetic stirrer until the phenol is dissolved or do manually.
• Add the glycerol.
• Filter the cotton blue and distilled water (D/W) solution into the phenol/glycerol/ lactic acid solution.
• Mix and store at room temperature.

Requirements for Lactophenol Cotton Blue (LPCB) Staining

• Compound light microscope
• LPCB stain
• Clean and grease-free microscopic slides
• Coverslip
• Dropper or bamboo sticks
• Fungal growth  in the medium

The procedure of LPCB Preparation

• Take a clean and grease-free glass slide.
• Put a large drop of LPCB with a Pasteur pipette or dropper.
• Transfer a small quantity of the culture to the drop.
• Tease the culture (in case of a mold) well with teasing needles
  so as to get a uniform spread.
• Put on a coverslip gently to avoid entrapment by air bubbles.
• Examine under low- (10 X) and high-power (40 X) objectives.
• Observe the morphological features carefully as shown below.

Observation

Fungal structures appear dark blue.

Results and interpretations of  Lactophenol Cotton Blue (LPCB) Stain

Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores

• Fungal spores, hyphae, and fruiting structures: Takes stained blue
• Background: stains pale blue.

Application of Lactophenol Cotton Blue (LPCB) Stain

• For staining and microscopic identification of fungi observing fungal spores, hyphae, and fruiting structures.
• It is also applicable in parasitology for the observation of Cysts of intestinal protozoa and ova take blue color while ova of helminths are stained deep blue.

Keynotes on LPCB Stain

1. LPCB wet mount is always examined at least 30 minutes after preparation.
2. A wet mount preparation should neither be too thick nor too thin.
3. In this preparation, both bile-stained and non-bile-stained helminthic eggs are stained blue.
4. LPCB kills the trophozoites of Entamoeba and Trichomonas, hence, can not be demonstrated by this.
5. In LPCB wet mount of stool phenol and lactic clear fecal debris.
6. In the LPCB wet mount of stool, glycerol provides a semi-permanent preparation. Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue. An additional advantage of this stain is that it can also detect blue-colored Cyclospora and Isospora oocyst.

Limitations of LPCB Stain

Even though LPCB stain is very useful has some demerits-

1. LPCB stain is only applicable for the presumptive identification method of fungi.
2. The ingredient of the LPCB solution may disrupt the original morphology of the fungi.
3. The stain can only be used to identify mature fungi and their structures and not the young vegetative forms of fungi.
4. A wet mount preparation should neither be too thick nor too thin.
5. Application of LPCB stain in Parasitology is not preferred because it kills the trophozoites of Entamoeba and Trichomonas.
6. This LPCB stain has an expiry date and thus can only use before expiry.

LPCB Stained Footages

Cryptococcus neoformans in LPCB Tease mount Microscopy

Geotrichum candidum in LPCB tease mount showing arthroconidia

Malassezia in LPCB tease mount preparatiion Microscopy

Aspergillus structures like septate hyphae, conidiophore or stipe, vesicle, Phialides, conidia, metulae, dichotomous branching (at an angle of approximately 45°) in LPCB Tease Mount Microscopy

Aspergillus fumigatus mycelium and conidia in LPCB-stained Microscopic Footage

Further Readings on  LPCB Stain

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
3. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmons, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Francisco 1996.
4. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
5. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
6. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/
8. https://mycology.adelaide.edu.au/laboratory/lacto/
9. http://himedialabs.com/TD/S016.pdf