NEGATIVE STAINING: RATIONALE, TECHNIQUE, ADVANTAGES AND DISADVANTAGES - BIOLOGY - 2025

The negative staining is a method of special coloring to highlight the presence of the capsule in some microorganisms, mainly Streptococcus pneumoniae, Klebsiella pneumoniae and Cryptococcus neoformans-, from clinical specimens or pure cultures.

The direct sample commonly used to apply negative staining is cerebrospinal fluid. This technique represents a rapid alternative for the presumptive diagnosis of meningitis, especially Cryptococcus neoformans.

A. Dental tartar stained with India ink, B. Cryptococcus neoformans var gatti weakly encapsulated as it occurs in nature, using nigrosin. C. Cryptococcus neoformans var gatti with a thick capsule as found in its parasitic form, using nigrosin. D. Cryptococcus neoformans with slightly diluted India ink. Gisell Quinde, from Wikimedia Commons / CDC, via Wikimedia Commons / Photo Credit: Content Providers (s): CDC / Dr. Leanor Haley, via Wikimedia Commons

Likewise, this stain can be applied on sputum and sterile liquids in general, as well as on strains obtained from young pure cultures. This technique uses nigrosine or India ink for its execution; therefore, it is a very simple and inexpensive methodology to apply that provides information of great diagnostic value in a short time.

In this sense, any laboratory is capable of performing this staining. Of course, the laboratory must have competent personnel capable of recognizing isolated or budding Cryptococcus neoformans yeasts and differentiating them from leukocytes and artifacts that the sample may present.

Basis

Nigrosine and India ink work in a similar way; therefore, either of the two substances can be used interchangeably.

This technique is called negative staining because it acts in a contrary way to the rest of the staining techniques. In this, what remains undyed is the structure that is being sought or that one wishes to see; that is, microorganisms.

Therefore, staining is based on staining the bottom of the smear a dark color. In this scenario, the capsulated structures will stand out in light or colorless color.

In general, the yeasts are refringent, surrounded by a clear halo that corresponds to the capsule. This occurs because India ink and nigrosin are substances unable to penetrate the polysaccharide that makes up the capsule of living microorganisms.

It is worth noting that other structures that may be present in the direct sample, such as leukocytes or red blood cells, do not penetrate either.

However, if the microorganisms are dead, the dye can penetrate inside them, so that this staining is also useful to evaluate the viability of the microorganisms.

Technique

materials

Nigrosine

Nigrosine owes its name to the black color it has. It is a synthetic substance that is obtained from the heating of the mixture of organic compounds -such as nitrobenzine, aniline and aniline hydrochlorite-, using a catalyst (iron or copper) in said reaction.

Chinese ink

Chinese ink is a substance used mainly by Asians for writing, making works of art, and monochromatic painting. It is very popular in Chinese culture.

It is obtained from squid ink mixed with pulverized charcoal, the product of burning low-resinous trees.

It is also possible to prepare it from the soot from the incineration of hydrocarbons (vegetable oils), together with a protein gelatin that gives it the right consistency to avoid the precipitation of carbon particles.

Specifications for sampling

- It does not require fasting.

- The CSF, sputum or sterile liquid sample must contain at least 1 ml of volume and must be transferred to the laboratory at room temperature immediately.

- CSF and sterile fluid samples must be taken by a specialized doctor.

- It can also be a pure culture of a suspicious strain linked to the aforementioned pathogens.

Execution of the technique with direct samples

- The samples must be centrifuged, then the supernatant is discarded and the sediment is taken.

- A drop of centrifuged material (sediment) and a drop of India ink or nigrosine are placed on a clean slide.

- It must be mixed well and covered with a coverslip sheet, allowing the drop to spread as a thin film without exceeding the edges.

- Subsequently, the preparation is mounted on the microscope.

- If the preparation is too dark, it can be diluted with water.

Execution of the technique with strains from culture

- A very small portion is taken from a young culture with a seeding needle and dissolved in a drop of India ink previously placed on a clean slide.

- A coverslip is placed on top.

- It is observed in the microscope at 10X and then at 40X.

You can also dissolve a portion of the colony in distilled water, and from there take a drop and mix it with the India ink. In this way, the preparation will not be so thick, making it possible to observe the structures in isolation; if there are crowds, it will not be well observed.

Another methodology is the following:

- Place a drop of the suspension culture on one end of the slide.

- Place a drop of nigrosine at the same end and mix.

- With the help of another slide, spread the sample as if making a hematological smear.

- Let dry and observe under the microscope.

Microscope observation

You must first focus with a 10X objective to have a wide view of the field. Subsequently, it should be sought if there are clear spaces; if there are, focus 40X to see details.

Advantage

- It is easy to execute.

- It is an inexpensive technique.

- This method does not require that the smear be fixed to heat or with chemicals; therefore, microorganisms are observed without distortions.

- The fresh preparation does not need to be dried, so it can be observed immediately, generating results quickly.

Disadvantage

Once assembled, fresh preparations must be observed immediately; if they are allowed to dry it is no longer possible to observe them and a new one must be installed.

Tissue staining with India ink

Another function that India ink can fulfill is in pathology laboratories. This is applied to surgically extracted tissue samples in order to mark the resection margins of the tumor.

The marked tissue is sprayed with acetic acid. This acts as a mordant and prevents ink from leaking out when the tissue is subjected to routine processing for the biopsy preparation.

The procedure consists of bathing the tissue in alcohol and xylene, and then soaking it in paraffin wax. This marking guides the pathologist when observing the tissue, indicating where the surgical resection margin or other point of interest is.

References

1. «Nigrosina». Wikipedia, The Free Encyclopedia. 11 Jul 2017, 22:46 UTC. 6 Jan 2019, 16:56: en.wikipedia.org
2. "Chinese ink". Wikipedia, The Free Encyclopedia. 1 Oct 2018, 03:08 UTC. 6 Jan 2019, 18:20: es.wikipedia.org/
3. Silva L, Silva C, Fernández N, Bueno C, Torres J, Rico M, Macías J and collaborators. (2006). Labor Personnel of the autonomous community of Extremadura. Specific agenda Volume IV. Editorial MAD. Seville-Spain, pp 211-212.
4. Silva M, García M, Corrales J, Ponce E. (2006). Specialist laboratory technician, Galician Health Service (SERGAS). Specific subject agenda volume 2. Editorial MAD. Seville-Spain, pp 79-80.
5. Forbes B, Sahm D, Weissfeld A. 2009. Bailey & Scott Microbiological Diagnosis. 12 ed. Argentina. Editorial Panamericana SA