Lactophenol Cotton Blue (LPCB) Mounts

The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. The preparation has the following constituents;

Fig: Aspergillus spp in LPCB Mount
Fig: Aspergillus spp in LPCB Mount
  1. Phenol kills fungus.
  2. Lactic acid acts as a clearing agent and helps preserve the fungal structures.
  3. Cotton blue is an aniline dye that stains the chitin in the fungal cell walls which adds colour to the fungal preparation thereby enhancing and contrasting the structures.
  4. Glycerol is a viscous substance that prevents drying of the prepared slide specimen.

Lactophenol cotton blue solution is a mounting medium and staining agent used in the preparation of slides for microscopic examination of fungi. Fungal elements are stained intensely blue. This operation should always be performed under a biological safety hood and gloves should always be worn.


Standard Tease Mount

  1. Place a drop of 70% alcohol on a clean microscope slide.

    Material from cultures of filamentous fungi should be removed using a stiff inoculating wire, not the loop used for manipulations with bacteria or yeasts.
  2. Flame the wire by holding it upright in the hottest part of the Bunsen flame, just above the blue cone, until the whole length of the wire glows red hot.

    You must ensure that the inoculating wire has cooled before placing it in a fungal culture – it should have cooled sufficiently after approximately ten seconds.
  3. Remove the cap from the culture tube (but do not put it on the bench). Kill any contaminating microorganisms by flaming the neck of the tube.
  4. Remove a small amount of the culture.

    It is often useful to take a little of the agar medium together with the fungus. In any case, the material should be disturbed as little as possible when being transferred to the slide.
  5. Flame the neck of the tube once more and replace the cap.
  6. Immerse the fungal material in the drop of 70% alcohol.
    This drives out the air trapped between the hyphae.
  7. Tease out the material very gently with mounted needles*.
    Do not forget to sterilize the inoculating wire and the needles after use by heating to red heat in a Bunsen flame
  8. Before the alcohol dries out add one or at most two drops of the stain.
    A common fault is to add too much stain to the preparation.
  9. Holding the coverslip between your index finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.
  10. Lower the coverslip gently onto the slide, trying to avoid air bubbles.
    Your preparation is now ready for examination.

*Teasing the colony often disrupts the delicate fruiting structures of the filamentous molds, making it difficult in some instances to observe the characteristic spore arrangements or hyphal attachments necessary for definitive identification. In such cases, a transparency tape mount or microslide culture may be required.

Cellophane Tape Preparation

The cellophane or transparency tape method for microscopic examination of fungi is simple to perform, inexpensive and, rapid. It preserves the conidial arrangements of the more delicate filamentous molds and allows one to make an accurate identification.

  1. Place a drop of lactophenol aniline blue stain on a microscope slide.
  2. Press the sticky side of unfrosted, clear cellophane tape gently but firmly to the surface of the colony, picking up a portion of the aerial mycelium.
  3. Stick one end of the tape to the surface of the slide adjacent to the drop of stain.
  4. Stretch the tape over the stain, gently lowering it so that the mycelium becomes permeated with stain.
  5. Pull the tape taught and stick the opposite end to the glass, avoiding as much as possible the trapping of air bubbles.
  6. Your preparation is now ready for examination.


Mucor species in LPCB mount (Image source)
  1. Make the initial examination using a low-power objective lens. The thinner parts of the preparation, generally around the edges of the mounting material, will yield the best images.
  2. Switch to a higher power 40X objective for a more detailed examination of spores and other structures.

Composition of LPCB mount

Lactic acid20 mL
Glycerol (or glycerine)40 mL
Phenol crystals or
phenol concentrate
20 g
20 mL
Distilled water20 mL
Aniline blue or
1% aqueous solution (This is analogous to cotton blue.)
0.05 g
2 mL

Preparation of LPCB mount

LPCB can be purchased from commercial suppliers or prepared in-house by mixing the above-mentioned ingredients as mentioned in the steps below.

  1. Add 20 mL of lactic acid in a beaker.
  2. Add 40 mL of glycerol or glycerine.
  3. Add 20 mL of distilled water
  4. Add 22 g. of phenol crystals (22 ml of melted phenol).
  5. Add 0.05 g. aniline blue.
  6. Heat and stir the solution to dissolve the stain. Do not boil or go over 100°C.
  7. Mix well and let the solution cool. You can store the solution at room temperature and dispensed it with a pipette when needed.


  1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology). Lippincott Williams & Wilkins
  2. Parija, S. C., Shivaprakash, M. R., & Jayakeerthi, S. R. (2003). Evaluation of lacto-phenol cotton blue (LPCB) for detection of Cryptosporidium, Cyclospora and Isospora in the wet mount preparation of stool. Acta tropica, 85(3), 349–354. 
  3. Khubnani, H., Sivarajan, K., & Khubnani, A. H. (1998). Application of lactophenol cotton blue for identification and preservation of intestinal parasites in faecal wet mounts. Indian journal of pathology & microbiology, 41(2), 157–162.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

14 thoughts on “Lactophenol Cotton Blue (LPCB) Mounts

  1. Between thee wet/ tease mount method and the adhesive tape method, which one is more reliable? why?

    1. use phenol crystals (20gms) , cotton blue (0.05gms) , lactic acid (20ml) glycerol (40ml) and distilled water (20ml). Thoroughly mix them using a magnetic stirrer. You can also use 2-5ml of ethanol in the mixture if required.

  2. Hello,

    I have a question to the staining agent Lactophenol Cotton Blue used for a urine sample – does anything else other than fungal structures bind to LPCB, mucus threads or anything else? The reason I´m asking is that…
    a) …the long strands I`m seeing, lighting up in blue – if they pick up the dye, does this confirm their fungal origin?
    b) …i can see two different structures picking up the dye, long strands, but also much larger roundish/squarish cells that look a like epithelial cells. This confused me, as they are not supposed to pick up the dye? Can anyone help?

    1. Instead of alcohol, phenol is used for killing living organism in LPCB. I hope it was helpdul.

    1. Gomori methenamine silver (GMS) stain is preferred over Lactophenol cotton blue in the lung tissue or bronchoalveolar lavage (BAL) fluid.

  3. How long does the phenol cotton blue stain need to be in contact with a Fungus before it is safe to bring from under a hood to examine at microscopically?

  4. Could LPCB be used to identify fungi from fibers? I.e. the stain would colour the fungi cells and not the fibers?

    1. As the cotton blue present in the LPCB reacts with chitin present in the cell wall of fungi, it is helpful as primary differentiation. Further tests like differential staining methods and culture should be done for identification. I hope this helps you. Thank you for the comment.

We love to get your feedback. Share your queries or comments

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Recent Posts