LPCB Stain: Introduction, Principle, Procedure, Result Interpretation

LPCB Stain: Introduction, Principle, Procedure, Result Interpretation and Its Applications

Introduction of LPCB stain

LPCB stain stands for lactophenol cotton blue and it is a combination of fixative, staining, and clearing agent. LPCB uses both as a mounting fluid and a stain. This is used for staining and microscopic identification of fungi. Its contents functions are as follows-

Lactic acid: It helps in preserving the morphology of the fungal elements.

Phenol: It acts as a disinfectant.

Cotton blue: It 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).

Glycerol: It is a hygroscopic agent that prevents drying.

Principle of LPCB stain

Ingredients of LPCB stain like lactic acid acts as a clearing agent and aids 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 such as shape, size, arrangement of spores, and hyphae providing color to the structure. It can be used alone or in conjunction with KOH.

Composition of LPCB stain

For 50 ml
Lactic acid : 10 ml
Phenol : 10 ml
Glycerol :20 ml
Cotton blue (Poirier blue or Aniline blue): 0.025 g
Distilled water : 10 ml

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

Requirements for test

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

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 of air bubbles.
Examine under low- (10 X) and high-power (40 X) objectives.
Observe the morphological features carefully as shown below.
Observation
Fungi appear as dark blue stained mycelium.
Results and interpretations
Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores. We concern with Aspergillus as shown below.
Fungal spores, hyphae, and fruiting structures: Takes stain blue
Background: stains pale blue.
Application of 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 Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue.
Various fungi and their structures ( yeast cells, budding yeast, hyphae, pseudohyphae, mycelium, spores) in LPCB preparation are as follows-

Aspergillus fumigatus Colony on SDA, LPCB tease mount under microscopy

Trichosporon on SDA and lactophenol cotton blue preparation under the microscope

Geotrichum growth on SDA and its fungal structures on lactophenol cotton blue preparation

Bipolaris growth on SDA and its structures on lactophenol cotton blue preparation

Syncephalastrum in lactophenol cotton blue preparation under the Microscope

Penicillium colonial morphology and its microscopic features in lactophenol cotton blue tease mount under a microscope

Fungus, Acremonium on SDA and lactophenol cotton blue preparation

Aspergilus flavus on Czapek Dox agar, Corn meal agar, and lactophenol cotton blue tease mount

Fusarium growth on SDA and its structures in lactophenol cotton blue preparation

Cryptococcus neoformans in lactophenol cotton blue tease mount

Candida albicans in LPCB tease mount

Cladosporium on SDA and its fungal structures on lactophenol cotton blue preparation

LPCB Mount of Curvularia species

Mucor in LPCB mount

Lactophenol cotton blue tease mount procedure and observation under the Microscope

Sporothrix schenckii under the microscope in lactophenol cotton blue preparation showing the following structures-conidia, conidiophores, and septate hyphae Conidia in clusters

Trichophyton mentagrophyte Isolated: features-
The helical pattern on lactophenol cotton blue Mount seen
Urease test-Positive
Hair perforation test-Positive

Arthroconidia of Trichosporon inkin – Long Cylindrical in Shape

Keynotes on LPCB stain

LPCB wet mount is always examined at least 30 minutes after preparation.
A wet mount preparation should neither be too thick or too thin.
In this preparation, both bile-stained and non-bile-stained helminthic eggs are stained blue.
LPCB kills the trophozoites of Entamoeba and Trichomonas, hence, can not be demonstrated by this.
In LPCB wet mount of stool phenol and lactic clear fecal debris.
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 being very useful has some shortcomings like-

It is only applicable for the presumptive identification method of fungi.
The ingredient of the LPCB solution may disrupt the original morphology of the fungi.
The stain can only be used to identify mature fungi and their structures and not the young vegetative forms of fungi.
A wet mount preparation should neither be too thick or too thin.
Application of LPCB stain in Parasitology is not preferred because it kills the trophozoites of Entamoeba and Trichomonas.
This LPCB stain has an expiry date and thus can only use before expiry.

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