Tissue Staining: Types, Techniques, And Applications

Types of Staining

Types of staining are simple staining, differential staining, and special or structural straining. Simple staining entails immersing the sample in dye solution proceeded by rinsing and observations. This is a one-step approach of using only one dye, and it is utilized in studying morphology better and demonstrating the nature of the cellular contents of the exudates along with exploring the intracellular location of the bacteria. Basic stains like Gram safranin, Gram crystal violet or methylene blue are useful for staining most bacteria (Beech, Noimark, Page, Noor, Allan & Parkin, 2015).

In differential staining, two or more stains are used and let the cells be classified into different types or groups. Other than letting the observation of cell morphology or the shape, this type of staining frequently give more details about the characteristics of the cell wall such as its thickness. Its examples are Gram stain used for differentiating bacteria into two groups and the acid-fast stain which distinguishes species of Mycobacterium from other bacteria. Special staining isolates the particular part of microbes. Malachite green is utilized with heat to force the dye into the cells and provide the color. Safranin, a counterstain is therefore utilized in providing color to the non-spore forming microbes, and at the end of the process spores stain green, and other cells stain red (Ashikuzzaman, Shahriyar, Lijon, Rahman, Hassan & Asif, 2015).

The Nissl-staining technique is based on the interactions of critical dyes like cresyl violet, thionine, toluidine blue, methylene blue or any with the nucleic acid content of cells. This technique detects the nissl body in the cytoplasm of neurons on paraformaldehyde fixed frozen or vibratome tissue parts which are stained purple-blue. The organelles stained by Nissl are neurons and glial cells (García-Cabezas, John, Barbas & Zikopoulos, 2016).

Congo red is a different stain related to Alzheimer’s disease. Beta-amyloid plaques can be detected using Congo red staining, and their presence along with neurofibrillary tangles is vital for the diagnosis of Alzheimer disease (Ho, Troncoso, Knox, Stark & Eberhart, 2014). Symptoms in the early stages of Alzheimer are impairment of disorientation, declarative memory and loss of context. The primary brain structures which support these functions include the medial temporal lobe more so the hippocampal formation and adjacent cortex. Specifically, the CA1 hippocampal region is critical in spatial orientation, learning and distinct memory functions like the retrieval of remote episodic memory along with the strength of developed memories (Danielson, Zaremba, Kaifosh, Bowler, Ladow & Losonczy, 2016).

Simple Staining

In the histology world, Congo red stain is used to stain amyloid which is an exceptionally folded fibrillar protein which deposits in extracellular spaces in organs under particular pathological states (Ho, Troncoso, Knox, Stark & Eberhart, 2014). As this protein multiplies it increasingly restores the standard tissue components and finally leads to loss of function of essential organs and eventually death. Amyloid is same in structure to cellulose hence it behaves the same way in its chemical reactions. This is a linear molecule that permits amine and azo groups of the dye to generate hydrogen bonds with same hydroxyl radicals of the amyloid (Ho, Troncoso, Knox, Stark & Eberhart, 2014).

When it is explored in hematoxylin and eosin stained parts of tissue, amyloid emerges as an amorphous, glassy, eosinophilic substance. Because this can be confused with some other elements Congo red is required to recognize it. Moreover, when studied using frequent bright field microscopy, Congo red-stained amyloid emerges pale orange-red (Ho, Troncoso, Knox, Stark & Eberhart, 2014). Consequently, the bright field aspect alone is not diagnostic for amyloid since small deposits may be challenging to view. Therefore, Congo red-stained tissue sections should be surveyed under polarized light permitting the characteristic apple-green birefringence to be observed which is diagnostic for the amyloid presence. Congo red stain remains the standard gold test utilized by pathologists to discover amyloid in tissues of individuals with these states possibly the Alzheimer’s disease. Congo red stains amyloidosis, plant and fungi cell walls along with Gram-negative bacterial outer membrane (Costa et al., 2015).

Silver staining is related to Parkinson’s disease whereby the silver stained sections demonstrate all Lewy bodies along with Lewy neuritis (Saito et al., 2016). Large Lewy bodies can be detected with acidic dyes, but H&E routine stains are not adequately sensitive to discover all the Parkinson’s disease associated alterations. Presently, the presynaptic protein alpha-synucleic has been reported to be present in all kinds of Lewy bodies and Lewy neuritis, and immunocytochemical demonstration of alpha-synucleic is regarded as the gold standard, reliable recognition of the whole spectrum of the Parkinson’s disease-related cytoskeletal alterations. Silver stains mitochondria organelles.

Figure 1 Spherulites observed in Alzheimer’s hippocampal tissue stained with Congo red and hematoxylin

 a) Congo-red-stained spherulite giving apple-green birefringence under progressively crossed polarizers.(a-c) Typical of the senile plaques observed in Alzheimer’s disease. 

d) Spherulite structures (solid arrow-heads) in the same tissue section without either the strong affinity for Congo red.

e) apple-green birefringence under crossed polarizers. In all parts, the unstained spherulites were distributed in the granule cell layer (vertical arrow) of the dentate gyrus (DG) and a surrounding band in Ammon’s horn (CA) within the pyramidal cell layer.

Differential Staining

f) The hippocampal section with a lighter Congo red stain, hematoxylin-positive cells (vertical arrow) and the spherulites which also notably lack affinity for hematoxylin (solid arrow-heads) shown under partially crossed polarizers. 

g) The same region under fully crossed polarizers.

Levels of beta-amyloid are generally low in the cerebrospinal fluid of Alzheimer’s patients and cerebral amyloid angiopathic patients (Merlini, Wanner & Nitsch, 2016).

Figure 2 Nissl stained section

The nissl material which is uneven endoplasmic reticulum is darkly-stained by cresyl violet thus the phrase nissl stain (Flanagan, Sonnen, Keene, Hevner & Montine, 2018). Moreover, in nissl stained parts, the cell bodies of neurons that consist of a lot of irregular endoplasmic reticulum emerge as dark granules at low accolade. The granular and molecular layer patterns in the hippocampal formation are more transparent at higher accolade. Dendrites from the dark cell bodies fill the light violet areas, the neuropil where they form several synaptic networks with axons.

Figure 3 Silver stains. Microscopic findings in Parkinson’s disease with Alpha-synucleic immunohistochemistry.

1. A normal brainstem type Lewy body
2. a pale staining cortical type Lewy body.
3. Lewy neuritis in CA2 sector of hippocampus
4. Intraneuritic Lewy bodies in medulla.

Conclusion      

Tissue staining helps to highlight structures in biological tissues to observe. There are different staining types of which depend on the region being seen and the kind of disease associated with it. The association between the cell and organ functioning is reflected in tissue organization observed under the microscopy thus histology endorses the study of cell biology at every level. Also, histology is very essential in diagnosing a disease.

References

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Ashikuzzaman, M., Shahriyar, S., Lijon, M. B., Rahman, M. A., Hassan, M. M., & Asif, A. A. (2015). An investigation on heavy metal tolerance properties of bacteria isolated from textile effluent. Journal of Biodiversity and Environmental Sciences, 7(6), 62-71.

Beech, S. J., Noimark, S., Page, K., Noor, N., Allan, E., & Parkin, I. P. (2015). Incorporation of crystal violet, methylene blue, and safranin O into a copolymer emulsion; the development of a novel antimicrobial paint. RSC Advances, 5(33), 26364-26375.

Costa, T. R., Felisberto-Rodrigues, C., Meir, A., Prevost, M. S., Redzej, A., Trokter, M., & Waksman, G. (2015). Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nature Reviews Microbiology, 13(6), 343.

Danielson, N. B., Zaremba, J. D., Kaifosh, P., Bowler, J., Ladow, M., & Losonczy, A. (2016). Sublayer-specific coding dynamics during spatial navigation and learning in hippocampal area CA1. Neuron, 91(3), 652-665.

Flanagan, M., Sonnen, J. A., Keene, C. D., Hevner, R. F., & Montine, T. J. (2018). Molecular Basis of Diseases of the Nervous System. In Molecular Pathology (Second Edition)(pp. 651-690).

García-Cabezas, M. Á., John, Y. J., Barbas, H., & Zikopoulos, B. (2016). The distinction of neurons, glia and endothelial cells in the cerebral cortex: an algorithm based on cytological features. Frontiers in neuroanatomy, 10, 107.

Ho, C. Y., Troncoso, J. C., Knox, D., Stark, W., & Eberhart, C. G. (2014). Beta?Amyloid, Phospho?Tau and Alpha?Synuclein Deposits Similar to Those in the Brain Are Not Identified in the Eyes of  Alzheimer’s and Parkinson’s Disease Patients. Brain Pathology, 24(1), 25-32.

Merlini, M., Wanner, D., & Nitsch, R. M. (2016). Tau pathology-dependent remodeling of cerebral arteries precedes Alzheimer’s disease-related microvascular cerebral amyloid angiopathy. Acta neuropathologica, 131(5), 737-752.

Saito, Y., Akazawa-Ogawa, Y., Matsumura, A., Saigoh, K., Itoh, S., Sutou, K., … & Hara, Y. (2016). Oxidation and interaction of DJ-1 with 20S proteasome in the erythrocytes of early stage Parkinson’s disease patients. Scientific reports, 6, 30793.