WHAT IS SIMPLE STAINING? OUTSTANDING FEATURES - BIOLOGY - 2025

The single staining is a staining procedure quick and simple in which a single dye is used, so it is called simple. It is mainly used to determine the morphology and organization of the cells present in a sample.

Cells are naturally colorless, so it is necessary to make them visible in some way when viewed under the microscope.

Human cheek cells stained by simple methylene blue staining.

It is important to note that the dyes used in simple staining must be basic with a positive charge (cationic), so that they can spontaneously bind to the cell wall and cytoplasm.

These cellular structures are negatively charged. This is why the positively charged dye is attracted to cells and binds spontaneously to them. Thus, all cells present in a sample are quickly stained.

Dyes used in simple staining

There are several basic stains that can be used in the microbiology laboratory. The most used are:

- Methylene blue.

- Crystal violet.

- Malachite green.

- Basic fuchsin.

All of these dyes work well in bacteria because they have positively charged (cationic) color ions (chromophores).

The staining times for most of these stains are relatively short. They generally range from 30 seconds to 2 minutes, depending on the affinity of the dye.

It is important to bear in mind that before staining a sample by simple staining, it must be extended and fixed to the glass slide (slide); the extended and fixed sample is called a smear.

The 6 steps to simple staining

Step 1

Place the slide on a staining rack and apply the desired stain. Let the corresponding time act.

Normally simple staining takes a few seconds to a couple of minutes, depending on the stain used.

Observation

In this step it is important not to exceed the recommended time for the dye used, since crystals could form on the sheet, producing what are known as "artifacts" that distort the morphology of the cells.

Step 2

Thoroughly wash the smear from the slide with distilled water from a bottle, or also slowly flowing tap water, until the runoff becomes clear. This usually takes 5-10 seconds.

Observation

Do not apply the stream of water directly on the smear, to avoid that the force of the same damage the sample.

If you do not have distilled water, you can use tap water without problem as it will not affect the result of the staining.

Step 3

Blot the slide with absorbent paper towels in one direction and without rubbing. Make sure the underside of the slide is clean.

Step 4

Observe the stained smear under the microscope. Start with the most distant objectives to properly locate the area that you want to observe in more detail. Change the objective to get closer and closer to the sample.

Observation

For the use of the objective with a higher magnification (normally 100X), immersion oil should be used, as this helps the light to penetrate better and the image appears sharper. It is not necessary to use a coverslip.

Step 5

Finally, dispose of all samples in an appropriate container that is properly labeled "biohazard".

References

1. (2001). Microbiological Applications: Laboratory Manual in General Microbiology (8 ^th ed.). The McGraw-Hill Companies.
2. Harisha, S. (2006). An Introduction to Practical Biotechnology (1 ^st). Firewall Media.
3. Moyes, RB, Reynolds, J., & Breakwell, DP (2009). Preliminary staining of bacteria: Simple stains. Current Protocols in Microbiology, (SUPPL. 15), 1–5.
4. Pommerville, J. (2013). Alcamo's Fundamentals of Microbiology Laboratory (10 ^th). Jones & Bartlett Learning.
5. Prescott, H. (2002). Laboratory Exercises in Microbiology (5 ^th). The McGraw-Hill Companies.
6. Sumbali, G. & Mehrotra, R. (2009). Principles of Microbiology (1 ^st). Tata McGraw-Hill Education.