LACTOPHENOL BLUE: CHARACTERISTICS, COMPOSITION, PREPARATION, USES - CHEMISTRY - 2025

The lactophenol blue or blue cotton is prepared with single dye properties. It is used in clinical laboratories to mainly color fungal structures such as hyphae, germ tubes and spores.

It helps in the presumptive or preliminary diagnosis of certain fungi, however, it is always advisable to reinforce the diagnosis with more specific tests, such as biochemical or serological tests.

Graphical representation of lactophenol blue stain, smear stained with lactophenol blue, microscope-mounted preparation. Source: public Domain Pictures.net/https://www.youtube.com/watch?v=P-kiFFZ7NvU /https://www.youtube.com/watch?v=wSZ77T5L6e8

Lactophenol blue is made up of lactic acid, aniline blue, glycerin, phenol, and deionized water. It can be prepared in the laboratory or it can be purchased ready-to-use. Once prepared it is ready to be used, under no circumstances should it be diluted, as this action will give terrible results.

The concentration of its components make it ideal for staining fungal structures, since phenol eliminates the accompanying bacterial microbiota, while lactic acid generates a protective film around the fungal structure. Finally, aniline blue has an affinity to adhere to the structures of the fungus.

Basis

With lactophenol blue, a simple staining of the fungal structures of a certain mycological culture is carried out. It is called simple because it uses only one colorant.

Lactophenol blue, apart from staining, also acts as a clarifier of the sample, thanks to the joint action of lactic acid and phenol, in addition, phenol behaves as a mordant and at the same time prevents the lysis of the microorganism by inhibiting the hydrolytic enzymes that may be present.

While lactic acid preserves the morphology of the structures of the fungus.

On the other hand, aniline blue has the property of adhering or binding to chitin-containing structures. The structures are stained light blue.

It should be noted that the cell wall of fungal microorganisms, especially molds, contain chitin, which is why this dye is ideal for staining these microorganisms.

characteristics

Lactophenol blue solution is a strongly acidic, slightly cloudy, aqueous dye with a light blue color. It remains stable if it is stored between 15 ° C and 25 ° C. The bottle must be kept properly closed. It is irritating and corrosive to skin and mucous membranes.

Composition

The formula to prepare one liter of lactophenol blue is as follows:

-250 g / L lactic acid

-1.0 g / L aniline blue or methyl blue

-500 g / L of glycerin

-250 g / L phenol

-250 ml of deionized or distilled water.

The phenol is dissolved in the previously warmed water. Lactic acid and glycerin are added and mixed very well. Finally, the aniline blue dissolves. It mixes again and is ready for use.

The portions to prepare 80 ml of lactophenol blue solution are:

-20 g phenol crystals

-20 ml of lactic acid

-40 ml of glycerin or glycerol

-20 ml distilled water.

Use

It is used to stain fungal structures and, through the morphological characteristics observed at the microscopic level, to be able to make a presumptive diagnosis of the fungus.

Process

Staining of fungal structures of cultures

- Take a pure culture of the fungus whose structures are to be observed.

- Preferably work in a laminar flow hood, or under the lighter, using a mask and gloves, since it is necessary to comply with biosafety standards, because most molds are easy to spread in the environment and therefore represent a danger for the operator.

- A drop of lactophenol blue is placed on a slide.

- Cut a small square of good quality adhesive tape and very carefully adhere it to the end of the cold sterilized platinum handle.

- The platinum handle is brought close to the most superficial part of the fungal colony and the culture is touched very carefully, exactly where the adhesive tape is located.

- Then it is taken to the slide and placed right on the drop of lactophenol blue, taking care that the tape is perfectly extended and lubricated with the dye. The platinum handle is very delicately removed.

- Another drop of lactophenol blue is placed on the tape and a coverslip on top. Do not apply too much pressure so as not to destroy the structures of the fungus, since they are extremely fragile. The colorant should be allowed to act for approximately 3 to 4 minutes.

- After this time, the preparation is ready to be observed under a microscope in 10X or 40X magnification. If 100X observation is required, immersion oil should be used.

- The structures of the fungus, in general, are stained blue, with the exception of the dematiaceous fungi that will retain their characteristic brown color of this type of fungus.

Direct biological specimen staining

If it is required to study a direct biological sample, such as sputum, pus, exudates, secretions, among others, these need to have a previous procedure for decontamination of the sample. NaOH solution is used for this.

In case of hair samples these can be dyed directly.

Several affected hairs (10 in total) are taken from the presumptive tinea capitis lesions (alopecic areas) and placed on a slide (3 to 4 hairs per slide). A few drops of lactophenol blue are placed on top and immediately covered with a coverslip.

Grasp the slide with forceps and heat gently over a burner. To do this, make 3 to 4 quick passes through the flame. Observe the microscope at 40X and look for spores outside or inside the hair (ectothrix or endothrix respectively). If necessary add immersion oil and observe at 100X.

Germ tube staining in the identification of

After incubating the presumptive culture of Candida albicans in human serum for 2 to 4 hours at 37 ° C, a portion of the solution is taken and stained with lactophenol blue. The preparation is observed under the microscope at 40X. The germ tube is present and the blastospores stain bright blue.

Lactophenol Safety Data Sheet

The National Fire Protection Association (NFPA), establishes the safety data sheet for chemical substances in terms of the risk that it represents according to various aspects, these are symbolized by a colored rhombus and a number ranging from 0- Four.

Health hazards are identified in blue, flammability in red, instability or reactivity in yellow, and any special hazard in white.

Health

The health risk posed by lactophenol blue according to the NFPA is level 3. This means that the substance is extremely dangerous when ingested or in direct contact with skin or mucosa.

It is a highly corrosive and irritating substance.

In contact with skin and mucous membranes it causes severe burns.

If ingested it can cause headache, confusion, lethargy, unconsciousness, respiratory arrest and even death.

Inflammability

According to the NFPA, Lactophenol Blue is rated Level 1 for flammability. This means that it burns only if it is heated above 94 ° C.

Instability

Listed according to the NFPA at level 0 (zero) in terms of reactivity. This means that it has no risk of reactivity, that is, it is a chemically stable substance.

Special hazards

It offers no additional special hazards.

References

1. SAR Laboratory. Lactophenol Blue. 2010. Available at: labsar.com.
2. Applied clinical chemistry laboratory. Lactophenol blue. 2015.Available at: cromakit.es
3. Merck Laboratories. Lactophenol blue microscopy in solution. 2017.Available at: Users / Equipo / Downloads
4. Emergency Card. Lactophenol blue solution for fungal staining. Universidad Pontificia Javeriana, Bogotá Colombia. 2014.Available at: javeriana.edu.co
5. González M, Diamont D, Gutiérrez B. Technique for staining fungal structures with vegetable dyes as a non-polluting alternative. Bioagro, 2011; 23 (1): 65-68. Available at: Redalyc.
6. Laboratory Himedia. Lactophenol Cotton Blue. 2017. Available from: himedialabs.com
7. Wanger A, Chavez V, Huang R, Wahed A, Actor J, Dasgupta A. Biochemical Tests and Staining Techniques for Microbial Identification. Science Direct, 2017; 61-73. Available from: Elsevier