Lactophenol Cotton Blue (LPCB) Staining – Principle and Procedure

Lactophenol Cotton Blue (LPCB) staining is carried out by placing a small portion of fungal culture in a drop of staining solution on a clean microscope slide. A coverslip is then placed over the fungal material. The prepared LPCB mount is examined under a light microscope to observe the fungal hyphae and other microscopic structures.

The preparation of fungal material depends upon the type of specimen and the mounting method used. Fungi grown in culture may contain delicate microscopic structures, which can get disturbed during slide preparation. Different mounting methods are used depending upon the fungal structures to be examined. The fungal cultures should be handled under proper laboratory supervision.

What is Lactophenol Cotton Blue (LPCB) Staining?

Lactophenol Cotton Blue (LPCB) staining is a microscopic staining and wet-mounting technique used for the examination of fungal structures. It is commonly used in mycology laboratories to examine fungi grown in culture. The lactophenol cotton blue solution acts as both a stain and a mounting medium. It is used to observe fungal hyphae and reproductive structures such as conidia, conidiophores, sporangia and spores.

The fungal preparation made using lactophenol cotton blue is known as an LPCB wet mount. LPCB staining refers to the staining technique, while the LPCB wet mount is the prepared slide containing fungal material in the mounting medium. Lactophenol Cotton Blue is also abbreviated as LCB.

Objectives of Lactophenol Cotton Blue (LPCB) Staining

The following are the objectives of LPCB staining:

1. To stain the fungal structures for clear observation under the light microscope.
2. To preserve the shape and arrangement of fungal structures during microscopic examination.
3. It is used to examine the fungal hyphae and the presence or absence of septa.
4. To study the shape and arrangement of spores and spore-bearing structures such as conidiophores and sporangiophores.
5. It helps in the preliminary identification of fungi based on their microscopic morphology.

Principle of Lactophenol Cotton Blue (LPCB) Staining

In LPCB staining, the cotton blue dye has an affinity towards the chitin present in the fungal cell wall. The dye binds to the cell wall and gives it a blue colour. The stained hyphae and reproductive structures can be seen under the microscope. Their shape and arrangement are examined for fungal identification, as the intensity of the blue colour alone cannot determine the fungal species.

The LPCB solution also contains phenol, lactic acid and glycerol, which are used in mounting the fungal material. Phenol has a fungicidal action and kills the fungal cells. Lactic acid is used to preserve the fungal structures. It also helps in clearing the preparation. Glycerol retains moisture in the mount and prevents it from drying during microscopic examination.

Materials and Reagents Required for LPCB Staining

The following are the materials required for Lactophenol Cotton Blue (LPCB) staining.

Reagents and Composition of LPCB Staining Solution

The following is a formulation of LPCB staining solution:

1. Cotton blue (Aniline blue)– 0.05 g
2. Phenol crystals– 20 g
3. Glycerol– 40 mL
4. Lactic acid– 20 mL
5. Distilled water– 20 mL

These are the quantities of individual ingredients in one published formulation. They do not indicate the final volume of the prepared solution. Ready-made LPCB staining solution can also be used.

Laboratory Materials and Equipment

• Fungal Culture– A suitable fungal culture grown on a culture medium.
• Glass Slides– Clean microscope slides for preparing the fungal mount.
• Coverslips– Used to cover the fungal material on the slide.
• Mounting Needles– Used to tease and separate the fungal material.
• Inoculating Needle or Forceps– For transferring a small portion of fungal culture onto the slide.
• Dropper or Pipette– Used to add the LPCB staining solution.
• Compound Microscope– For examining the stained fungal structures.
• Staining Container– A clean glass container or dropper bottle for keeping the LPCB solution.
• Glass Beaker and Magnetic Stirrer– Required when preparing LPCB solution in the laboratory.
• Analytical Balance and Measuring Cylinder– For measuring the required quantities of chemicals.
• Glass Funnel and Filter Paper– Used during the preparation of LPCB solution.
• Reagent Bottle– For storing the prepared staining solution.
• Protective Equipment– Laboratory coat, chemical-resistant gloves and safety goggles. A suitable fume hood is required for handling phenol during solution preparation.

Precautions for LPCB Staining

The following are the precautions for LPCB staining:

• The slide and coverslip used for staining should be clean.
• A small amount of fungal material is taken for the preparation of a thin mount. Fungal structures may not be clearly seen in thick mounts.
• The fungal material should be teased gently using a mounting needle, as excessive teasing may damage the hyphae and spore-bearing structures.
• Air bubbles should not be present under the coverslip. Place the coverslip gently without applying excessive pressure.
• In preparations containing alcohol, the LPCB stain is added before the alcohol dries.
• Phenol is toxic and corrosive. Gloves, a laboratory coat and eye protection should be used while handling LPCB. Contact of the stain with the skin and eyes should be avoided.
• Handling of live mould cultures should be carried out in a suitable biological safety cabinet, without producing aerosols during transfer.
• Expired LPCB solution should not be used. The solution should also be free from microbial contamination, which may affect the staining.
• Used slides and staining materials should be discarded according to the laboratory procedures for biological and chemical waste. Stain containing phenol should not be poured into the drain.

Preparation of Lactophenol Cotton Blue Staining Solution

Lactophenol Cotton Blue (LPCB) staining solution is prepared using phenol, lactic acid, glycerol, cotton blue and distilled water.

Reagents Required

The following are the reagents required for the preparation of LPCB staining solution:

1. Phenol crystals – 20 g
2. Lactic acid – 20 mL
3. Glycerol – 40 mL
4. Cotton blue (Aniline blue) – 0.05 g
5. Distilled water – 20 mL

Procedure

The following are the steps involved in the preparation of LPCB staining solution:

1. Take 20 mL of distilled water in a clean glass beaker and add 0.05 g of cotton blue to it. Mix it until the dye dissolves.
2. Keep the cotton blue solution overnight. On the next day, filter it to remove the insoluble dye.
3. In another beaker, take 20 mL of lactic acid and add 20 g of phenol crystals.
4. Place the beaker on a magnetic stirrer until the phenol crystals are completely dissolved.
5. Add 40 mL of glycerol to the solution.
6. The filtered cotton blue solution is then added to the phenol, lactic acid and glycerol mixture. Mix it well.
7. Store the prepared LPCB stain in a tightly closed, labelled glass bottle at room temperature.

Precaution: Phenol is toxic and corrosive. The solution should be prepared in a chemical fume hood using suitable chemical-resistant gloves, safety goggles and a lab coat.

Procedure of Lactophenol Cotton Blue (LPCB) Staining

Lactophenol cotton blue staining can be carried out by the direct wet mount method or the adhesive tape mount method. The following are the steps involved in both methods:

Direct Wet Mount or Tease Mount Method

1. Select a suitable fungal culture approved for microscopic examination. The culture should be handled according to the laboratory biosafety procedures.
2. Take a clean and dry glass slide and a coverslip.
3. Add one drop of lactophenol cotton blue stain to the centre of the glass slide.
4. With the help of a sterile inoculating needle, take a small amount of fungal growth from the colony and place it in the drop of LPCB stain.
5. Gently tease the fungal material with a needle to separate and spread the fungal hyphae in the stain. Excessive teasing may damage the reproductive structures.
6. Place a coverslip slowly over the stained material from one side to prevent the formation of air bubbles.
7. The excess stain present at the edges of the coverslip is removed using blotting paper, if required.
8. Examine the slide first under the 10× objective of a light microscope. The hyphae, conidia, conidiophores and other fungal structures are then observed under the 40× objective.

Adhesive Tape Mount Method

The adhesive tape method is used for examining sporulating fungi. It helps to maintain the arrangement of conidia and conidiophores, which may get disturbed during the tease mount preparation.

1. Take a clean glass slide and add one drop of lactophenol cotton blue stain in the centre.
2. Cut a small piece of clear adhesive tape and hold it with forceps or a suitable applicator stick.
3. Gently touch the sticky side of the tape to the surface of the fungal colony. The fungal hyphae and reproductive structures get attached to the tape. The sporulating culture must be handled according to the laboratory biosafety procedure.
4. The tape is carefully removed from the colony without disturbing the attached fungal structures.
5. In some protocols, a drop of 95% alcohol is added to the tape for wetting before mounting. This step is not required in all preparations.
6. Place the tape over the drop of LPCB stain with the fungal material facing downwards.
7. Another drop of LPCB stain may be added over the tape if required by the method. A coverslip is then gently placed over the preparation and the excess stain is removed.
8. Examine the slide first under the 10× objective and then under the 40× objective to observe the arrangement of conidia, conidiophores and other fungal structures.

Results and Interpretation of LPCB Staining

Microscopic Appearance of Stained Fungal Structures

After LPCB staining, the fungal hyphae, spores and reproductive structures appear blue under the microscope. The cell walls may be stained light or dark blue. Some structures may remain faintly stained. The background usually appears pale blue.

The hyphae are examined for their shape, branching and the presence of septa. Septa are the cross walls that divide the hyphae into compartments. In some fungi, the hyphae are broad and have few or no septa.

The conidia may be present singly or in chains on the conidiophores. In other fungi, the sporangiospores are present within sporangia, which are borne on sporangiophores. The shape and arrangement of these structures are used for morphological identification.

The fungal structures may not be seen clearly when the mount is too thick. Air bubbles, overlapping hyphae and damaged reproductive structures can also affect the observation.

Examples of Fungi Observed by LPCB Staining

The following are some of the fungi and their microscopic appearance in LPCB staining:

1. Aspergillus – The hyphae are septate and branched. The conidiophores have a swollen terminal vesicle. Phialides are present on the vesicle and produce chains of conidia, forming a conidial head. In some species, metulae are also present between the vesicle and phialides.
2. Penicillium – It has septate hyphae with branched conidiophores. The phialides produce chains of conidia. These form a brush-like arrangement called a penicillus.
3. Mucor – The hyphae are broad and usually have few or no septa. The sporangiophores are simple or branched and bear rounded sporangia at their tips. The sporangia contain numerous sporangiospores. A columella is also present within the sporangium.

These are the common morphological characters used for preliminary identification of the respective fungal genera. The structures may differ among species, and LPCB staining alone cannot confirm the fungal species.

Applications of Lactophenol Cotton Blue (LPCB) Staining

The following are the applications of LPCB staining:

• Microscopic examination of fungi grown on culture media.
• It is used to study the branching of fungal hyphae and the presence or absence of septa.
• To examine the shape and arrangement of conidia and conidiophores.
• The sporangia and sporangiospores of fungi such as Mucor can be observed.
• It helps in the preliminary identification of fungi such as Aspergillus, Penicillium and Mucor based on their microscopic structures.
• The macroconidia and microconidia of dermatophytes are examined using LPCB stain.
• It is used in the preparation of wet mounts and adhesive tape mounts of fungal cultures.
• Comparing the microscopic structures of different fungal isolates.
• Used in mycology laboratory practicals for studying the morphology of filamentous fungi.

Limitations of Lactophenol Cotton Blue (LPCB) Staining

The following are the limitations of LPCB staining:

• It is used only for the preliminary identification of fungi. The fungal species cannot be confirmed by LPCB staining alone.
• Fungi having similar microscopic structures may be difficult to differentiate.
• The identification depends upon the presence of conidia, sporangia and other reproductive structures.
• Young or non-sporulating fungal cultures may not have the structures required for identification.
• The original arrangement of conidia and conidiophores may get disturbed during tease mount preparation.
• Some fungal structures may not stain uniformly, making them difficult to observe.
• Thick fungal mounts and overlapping hyphae can affect microscopic examination.
• It cannot be used to determine whether the fungal cells were living or dead before staining.
• LPCB staining does not provide information about the antifungal susceptibility of fungi.
• The presence of fungal structures in a clinical sample does not always indicate fungal infection. Other diagnostic methods may be required.
• Phenol present in LPCB is toxic and requires proper handling in the laboratory.

References

1. Barry, D. J., Chan, C., & Williams, G. A. (2007). Nitrocellulose as a general tool for fungal slide mounts. Journal of Clinical Microbiology, 45(3), 1074–1075. https://doi.org/10.1128/JCM.01609-06
2. Harris, J. L. (2000). Safe, low-distortion tape touch method for fungal slide mounts. Journal of Clinical Microbiology, 38(12), 4683–4684. https://doi.org/10.1128/JCM.38.12.4683-4684.2000
3. Larone, D. H., Westblade, L. F., Burd, E. M., Lockhart, S. R., Procop, G. W., & Walsh, T. J. (2023). Larone’s medically important fungi: A guide to identification (7th ed.). Wiley-ASM Press. https://doi.org/10.1002/9781683674436
4. Leck, A. (1999). Preparation of lactophenol cotton blue slide mounts. Community Eye Health, 12(30), 24. https://pmc.ncbi.nlm.nih.gov/articles/PMC1706009/
5. Rodríguez-Tudela, J. L., & Avilés, P. (1991). Improved adhesive method for microscopic examination of fungi in culture. Journal of Clinical Microbiology, 29(11), 2604–2605. https://doi.org/10.1128/JCM.29.11.2604-2605.1991
6. Thomas, P. A., Kuriakose, T., Kirupashanker, M. P., & Maharajan, V. S. (1991). Use of lactophenol cotton blue mounts of corneal scrapings as an aid to the diagnosis of mycotic keratitis. Diagnostic Microbiology and Infectious Disease, 14(3), 219–224. https://doi.org/10.1016/0732-8893(91)90035-E
7. Vyzantiadis, T.-A. A., Johnson, E. M., & Kibbler, C. C. (2012). From the patient to the clinical mycology laboratory: How can we optimise microscopy and culture methods for mould identification? Journal of Clinical Pathology, 65(6), 475–483. https://doi.org/10.1136/jclinpath-2011-200500