Lactophenol Cotton Blue Staining – Principle, Procedure, Result.

lactophenol cotton blue definition

Lactophenol Blue Solution or Lactophenol cotton blue is a wet mounting medium and staining agent which is used for the preparation of microscopic slides of fungi for examination. Fungal elements are stained intensely blue.

You might be thinking, why should we learn this technique or what is the purpose of this technique????

Well, it’s quite simple.

Fungi are a type of eukaryotic organism, and divide into two major groups that are yeast and molds.They contain chitin on their cell wall. They have a microscopic structure and that is why we can’t see them in naked eyes. We can see them under a microscope by staining with appropriate stains.

These fungal cells are useful as well as harmful to human beings because they produce many antibiotics,natural products as well as used in the industrial fermentation process. Fungal cells are harmful in the sense that they cause human diseases,produce toxic substances as well as harm important crops.

It is very important to examine the fungal species, so we can control them or we can produce drugs for fungal infection. Before examination of fungal cells under microscope we need to stain them, which is an important step.

The staining of fungal cells can be done by this Lactophenol Cotton Blue. These are some examples of fungi Rhizopus, Aspergillus, Penicillium, Fusarium, Candida, Mucor etc. Here we are going to stain fungal cells with the help of Lactophenol cotton blue method this staining technique is also called as mounting of fungus.

Although there are many criteria when identifying molds such as cultural characteristics, temperature tolerance, nutritional profiles, and various biochemical tests, modern classification schemes emphasize microscopic morphological features that are stable and exhibit minimal variation. The definitive identification of molds is based on the shape, method of production, and arrangements of spores (conidial ontogeny).

Preparation of Lactophenol Cotton Blue Solution

Ingredients required for 1 liter of Lactophenol Cotton Blue ;

Ingredients	Amount
Cotton Blue (Aniline Blue)	0.5gm
Phenol Crystals (C6H5O4)	200.0gm
Glycerol	400.0ml
Distilled water	200.0ml
Lactic acid (CH3CHOH COOH)	200.0ml

Methods for Lactophenol Cotton Blue Preparation:

It will take two days to prepare lactophenol blue. In preparation of LCB involve five important steps, such as Cotton Blue dissolution, phenol crystals dissolution, the addition of glycerol, Filtration, and storage.

1. Cotton Blue dissolution: On the first day, mix the Cotton Blue with distilled water and leave it overnight ((it will help to eliminate insoluble dye)) for proper dissolution.
2. Phenol Crystals Dissolution: On the second day, dissolve phenol crystals into lactic acid by using a magnetic stirrer until the phenol is dissolved (Follow proper safety instruction during dissolution of lactic acid and phenol crystal).
3. Addition of Glycerol: Add glycerol to this mixture of phenol and lactic acid, and make sure it dissolves properly.
4. Filtration: Filter the mixer solution of Cotton Blue and distilled water into the mixer of phenol, glycerol, and lactic acid solution. 
5. Storage: After the preparation of the mixer, store it at room temperature and protected from light for later uses.  Under these conditions, it has a shelf life of 52 weeks from the date of manufacture.

Aim of LPCB staining

Microscopic examination of fungal cells by using Lactophenol cotton blue.

Principle of Lactophenol cotton blue Staining

Lactophenol cotton blue is used in medical mycology for the examination of fungal cell structure, by preparing a wet mount. It is composed of two most important components such as methyl blue and lactophenol.

Methyl blue is a histological stain which stains collagen blue in tissue sections, and lactophenol is a mixer of four components such as phenol, lactic acid, and glycerol in water. Where phenol helps to kill any microorganisms, lactic acid preserves fungal structures, and glycerol inhibits the cellulolytic activity of the fungus.

When preparing the microscopic slide, methyl blue stains the chitin in the fungal cell walls, as a bright cerulean color, and lactophenol acts as a mountant.

Procedure of Lactophenol cotton blue Staining

Lactophenol cotton blue Staining can be performed by these two following methods;

A. Tease Mount Method:

1. Prepare an oil-free or Grease microscopic slide.
2. Place a drop of Lactophenol Blue on this oil-free slide.
3. Add the fungal specimen over this drop of Lactophenol Blue by using sterile inoculating needles.
4. With the help of two sterile dissecting needles, gently tease the fungus apart so that it is thinly spread out in the Lactophenol.
5. Place a clean coverslip edge on the mixer of Lactophenol and fungal specimen and then slowly lower it (Avoid trapping air bubbles under the coverslip) 
6. Then seal the edges of the coverslip using nail polish or paramount.
7. After all is done, Examine the slide under the microscope.

B. Scotch Tape Method:

1. Cut a strip of transparent tape and place ends between thumb and index finger with the sticky side out.
2. Make a loop extending outwards by closing the thumb and index finger. Open the culture plate with the opposite hand and press the tape against the colony of interest. 
3. Place a drop of Lactophenol Blue on a clean glass slide. 
4. Press tape against the Lactophenol.
5. Smooth the tape back on the slide by opening fingers and using gauze. 
6. Place another drop of Lactophenol Blue on top of the tape. 
7. Place a large and clean coverslip on top of the slide.
8. After all is done, Examine the slide under the microscope.

Result of Lactophenol Cotton Blue Staining

During microscopic observation of this slide, it reveals a blue color stained fungal spores, hyphae, and fruiting structures against the pale blue background. As you can see in this below picture;

Limitation

1. Wet mount or Lactophenol cotton blue staining slide can not be stored over a longer period of time.
2. Lactophenol cotton blue staining does not allow us to observe the early-stage differentiation of the fungal cell.
3. It may cause disruption of the fungal morphology.
4. Check expiry date before, use the solution sample.
5. Lactophenol Cotton Blue is useful in the recognition and presumptive identification of fungi.
6. The scotch tape mount is a temporary mount so, it will dissolve eventually
7. In tease mount, conidia or spores may be dislodged from the conidiogenous or sporogenous cells.
8. Once the specimen immersed in Lactophenol Blue Solutions it will be rendered safe for handling outside of the biological safety hood
9. Lactophenol without cotton blue is available and can be used for pigmented strains of molds. 

Application of Lactophenol Cotton Blue Staining

• Lactophenol Cotton Blue staining is a commonly used technique in microbiology for the identification and visualization of fungal elements. It is a simple and quick staining method that can be used to examine a variety of fungal specimens, including those obtained from clinical samples, environmental samples, and food products.
• The main application of Lactophenol Cotton Blue staining is in the diagnosis of fungal infections in humans and animals. Fungal infections are a significant cause of morbidity and mortality worldwide, and accurate and timely diagnosis is essential for appropriate treatment. Lactophenol Cotton Blue staining can be used to identify the specific fungal species causing an infection, which can guide treatment decisions.
• Lactophenol Cotton Blue staining is also used in environmental microbiology to identify and characterize fungal populations in soil, water, and air samples. This information can be used to understand the ecology of fungi and their role in various ecosystems.
• In the food industry, Lactophenol Cotton Blue staining can be used to detect and identify fungal contamination in food products. This is important for ensuring food safety and preventing spoilage.
• Overall, Lactophenol Cotton Blue staining is a versatile staining technique that has numerous applications in microbiology. Its ease of use and low cost make it a valuable tool for researchers and clinicians alike.

Quality Control

1. Appearance: The color of Lactophenol cotton blue solution should be Ink blue.
2. Visibility: It should be Clear, without any insoluble particles.
3. Microscopic Observation:  After staining the fungal cell, Fungal Spores and hyphae are observed under microscope using high power (40X) objective lens after staining with Lactophenol cotton blue.
4. Results: Fungal spores and hyphae will appear as pale to dark blue.

FAQ

Reference

• https://en.wikipedia.org/wiki/Methyl_blue
• https://en.wikipedia.org/wiki/Lactophenol_cotton_blue
• https://mycology.adelaide.edu.au/laboratory/lacto/
• https://laboratoryinfo.com/lactophenol-cotton-blue-lpcb/
• https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/1/61335dat.pdf
• http://www.generalmicroscience.com/microbial-laboratory-techniques/staining-fungus-using-lactophenol-cotton-blue/
• https://www.dalynn.com/dyn/ck_assets/files/tech/SL18.pdf
• https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/LactophenolCottonBlStn.htm
• http://www.himedialabs.com/TD/S016.pdf
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/
• https://microbenotes.com/lactophenol-cotton-blue-staining/
• http://www.biologycourses.co.uk/biomedical-science/biomedical-science-technique-lactophenol-cotton-blue-lpcb
• https://www.dalynn.com/dyn/ck_assets/files/tech/SL18.pdf