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;
- Phenol kills fungus.
- Lactic acid acts as a clearing agent and helps preserve the fungal structures.
- 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.
- 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.
Table of Contents
Standard Tease Mount
- 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.
- 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.
- 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.
- 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.
- Flame the neck of the tube once more and replace the cap.
- Immerse the fungal material in the drop of 70% alcohol.
This drives out the air trapped between the hyphae.
- 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
- 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.
- Holding the coverslip between your index finger and thumb, touch one edge of the drop of stain with the edge of the coverslip.
- 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.
- Place a drop of lactophenol aniline blue stain on a microscope slide.
- 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.
- Stick one end of the tape to the surface of the slide adjacent to the drop of stain.
- Stretch the tape over the stain, gently lowering it so that the mycelium becomes permeated with stain.
- Pull the tape taught and stick the opposite end to the glass, avoiding as much as possible the trapping of air bubbles.
- Your preparation is now ready for examination.
- 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.
- Switch to a higher power 40X objective for a more detailed examination of spores and other structures.
Composition of LPCB mount
|Lactic acid||20 mL|
|Glycerol (or glycerine)||40 mL|
|Phenol crystals or|
|Distilled water||20 mL|
|Aniline blue or|
1% aqueous solution (This is analogous to cotton blue.)
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.
- Add 20 mL of lactic acid in a beaker.
- Add 40 mL of glycerol or glycerine.
- Add 20 mL of distilled water
- Add 22 g. of phenol crystals (22 ml of melted phenol).
- Add 0.05 g. aniline blue.
- Heat and stir the solution to dissolve the stain. Do not boil or go over 100°C.
- Mix well and let the solution cool. You can store the solution at room temperature and dispensed it with a pipette when needed.