Lactophenol Cotton Blue (LPCB) mounts: Principle and staining protocol

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 three components:

  1. Phenol:   kills any live organisms;
  2. Lactic acid : It  preserves fungal structures, and
  3. Cotton blue : It stains the chitin in the fungal cell walls.

    Fig: Aspergillus spp in LPCB Mount

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.

Preparation of lactophenol cotton blue (LPCB) slide mounts

  1. Place a drop of seventy percent alcohol on a clean microscope slide.
  2. Material from cultures of filamentous fungi should be removed using a stiff inoculating wire not the loop used for manipulations with bacteria or yeasts.
  3. 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.
  4. 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.
  5. Remove the cap from the tube but do not put it on the bench. Kill any contaminating microorganisms by flaming the neck of the tube.
  6. Remove a small amount of the culture. For fungal cultures, 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.
  7. Flame the neck of the tube once more and replace the cap.
  8. Immerse the fungal material in the drop of seventy percent alcohol. This drives out the air trapped between the hyphae.
  9. Tease out the material very gently with mounted needles.
  10. Do not forget to sterilise the inoculating wire and the needles after use by heating to red heat in a Bunsen flame
  11. Fungal structures are readily visualised after staining with a lactophenol cotton blue dye preparation.
  12. Before the alcohol dries out add one or at most two drops of the stain. A common fault is to add too much to the preparation. Holding the coverslip between your index finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.
  13. Lower the coverslip gently onto the slide, trying to avoid air bubbles. Your preparation is now ready for examination.
  14. Make the initial examination using a low power objective lens. The thinner parts of the preparation, generally around the edges of the mounted material, will yield the best images.
  15. Switch to a higher power 40X objective for more detailed examination of spores and other structures.