INTRODUCTION:

Oral mucosa exhibits a rapid turnover of cells and these exfoliated cells have a valuable role in diagnosis of certain local and systemic diseases. Oral cavity reflects the various events occurring in the body and this is reflected by cytomorphological and nucleomorphological variations in the exfoliated cells. Exfoliative cytology is based on the monitoring the exfoliated cells or cells flake off the mucosa whither through natural or artificial means. (1)

The history of exfoliative cytology dates back to 1860 when Bhale described the morphology of malignant cells in sputum of oropharyngeal carcinoma. The use of exfoliative cytology was restricted to gynaecological diagnosis. However Papianacolaou and Traut introduced new methods of staining and collection of specimens. The use of exfoliative cytology extended into oral cavity when comparative studies were conducted to study the cervical and oral cytology in menstrual cycles. (1)

Special stains are used as per requirements: Modified Ziehl Neelson (for acid fast bacilli), Gram staining (Bacteria), Mucicarmine (mucins), PAS (for glycogen, fungal wall, lipofuscin, etc), Oil red O (lipids), Perl’s Prussian blue (iron), modified Fouchet’s test (bilirubin), etc. Recently, immunocytochemistry is also being increasingly used in cytology specimens. Staining techniques used were; carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin .

There have been great changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques collectively referred to us histochemistry and have facilitated greatly in the study of organs and tissues.

Hematoxylin is a basic dye that is commonly used in this process and stains the nuclei giving it a bluish color while eosin (another stain dye used in histology) stains the cell’s nucleus giving it a pinkish stain (Victor, 2013). While these changes have taken place, there are old stain procedures that are still in use today and many others have been replaced with new immunalstaining or staining techniques (Sine, 2014). Some staining methods have been abandoned because the chemicals required have been medically proven to be toxic. Similarly, there have been great changes in workload requiring more advanced technics of staining. This present study indicate that, in the modern cytology a combination of different stain techniques are used to enhance the effectiveness of the staining process. In the modern cytology as a way of improving cytological stains, several stains have been modified and combined with other stains to improve their effectiveness.

MATERIALS AND METHODS:

There are total of 30 buccal smears were collected from ten healthy individual. these were stained by three different stains namely haematoxylin and eosin stain , light green stain and nuclear fast red stain .

Based on the stain they were categorized into three groups:-

1. Group A- Haematoxylin (nucleus stain) and Eosin (cytoplasm stain) – n = 10

2. Group B – Haematoxylin (nucleus) stain and light green (cytoplasm) – n = 10

3. Group C – nucleus fast red ( nucleus) stain and light green (cytoplasm) – n = 10

STAINING METHODS:

GROUP A:

CONVENTIONAL HEMATOXYLIN AND EOSIN STAINING:

METHOD:

· After fixation, the slides were washed in running water and immersed in Harris Hematoxylin for 8 minutes and again rinsed in running the tap water.

· Slides were immersed in ammonia and washed in running water and then immersed in acid alcohol for bluing and differentiation and washed under tap water.

· Slides were immersed in eosin for 2-3 dips and differentiated in grades of alcohol.

· Slides were air dried and then mounted with xylene.

GROUP B:

HEMATOXYLIN AND 1% LIGHT GREENSTAINING: (H and LG)

Method:

· After fixation, the slides were washed in running water and immersed in Harris Hematoxylin for 8 minutes and again rinsed in running the tap water.

· Slides were immersed in ammonia and washed in running water and then immersed in acid alcohol for bluing and differentiation and washed under tap water.

· Slides were immersed in 1%light green for 5minutes and differentiated in grades of alcohol.

· Slides were air dried and then mounted with xylene.

GROUP C:

NUCLEAR FAST RED AND 1%LIGHT GREENSTAINING:( LG and N):

Method:

· After fixation, the slides were washed in running water and immersed in nuclear fast red for 30 minutes and again rinsed in running the tap water.

· Slides were immersed in 1%light green for 5minutes and differentiated in grades of alcohol.

· Slides were air dried and then mounted with xylene.

RESULT:

Table 1:Comparison of nucleus staining between H&E, light green and nuclear fast red stain

Nucleus Staining	Group
	H and E		H and LG		LG and N
	N	%	N	%	N	%
Poor	0	0	0	0	0	0
Fair	0	10	1	10	1	10
Good	10	100	9	90	9	90
Total	10	100	10	100.0	129	100.0

Chi-Square Test	Value	p-Value
Fisher's Exact Test	10.010	<0.001

Table 2: Comparison of cytoplasm staining between H&E, light green and nuclear fast red stain

Cytoplasm Staining	Group
	H and E		H and LG		LG and N
	N	%	N	%	N	%
Poor	0	0	0	0	0	0
Fair	0	0	2	20	1	10
Good	10	100	8	80	9	90
Total	10	100	10	100	10	100

Chi-Square Test	Value	p-Value
Fisher's Exact Test	10.010	<0.001

Table 3: Comparison of background staining between H&E, light green and nuclear fast red stain

Background Staining	Group
	H and E		H and LG		LG and N
	N	%	N	%	N	%
Poor	0	0	0	0	0	0
Fair	0	0	1	10	0	0
Good	10	100	9	90	10	100
Total	10	100.0	10	100.0	10	100.0

Chi-Square Test	Value	p-Value
Fisher's Exact Test	10.010	<0.001

FIG 1-10X H&E STAIN

FIG 2-10X- H&LG

FIG 3-10X- LG&N

FIG – 4 –45X H&E

FIG -5-45X H&LG

FIG 6- 45X LG&N

Photomicrographs of the all three stains used in this present study, fig 1,4(H and E Stain) , Fig 2, 5(H &LG stain) , and fig 3,6 (LG&N Stain).

DISCUSSION:

Staining is used to highlight important features of the tissue as well as to enhance the tissue contrast.(2) Hematoxylin is a basic dye that is commonly used in this process and stains the nuclei giving it a bluish color While eosin stains the cell’s nucleus giving it a pinkish stain. There are other several staining technicques used for particular cells and components(3) combination of different stain techniques are used to enhance the effectiveness of the staining process. (1) There are several stains available to modified or combined with other stains to improve their effectiveness.

In this present study ,we combine two stains,found different color combinations of cellular features. The study showed that , adequate nuclear staining was noted in all the stains .Nuclear staining found in H&E (100%) whereas in light green(90%) and nuclear fast red stain (90% ) p value <0.001. Cytoplasm staining found in H&E stain (100%) , light green stain shows 80%and nuclear fast red stain showed (90%) , p value <0.001 . Background staining found almost similar result H&E(93%),light green stain (91%) and nuclear fast red stain (91%) p value <0.001, The result are summarised in Table 1,2,3.

The purpose of the staining is used to highlight important features of the tissue as well as to enhance the tissue contrast. There have been great changes in the techniques used for cytological staining through chemical, molecular biology assays and immunological techniques ,and have facilitated accurate diagnosis.

A variety of stains have been used for cytological evaluation, mostly commonly used stains are haematoxylin and eosin, leishman ,giemsa and PAP. Different types of stains used cytology to give effective result and good diagnostic accuracy, in terms of cellularity and morphological preservation of the cells.

There has been a rising need for efficient, accurate and less complex staining procedures required in touch imprint cytology to provide all cellular details and able to provide a reliable diagnosis .

CONCLUSION:

Several modifications have been made to improve their efficiency, There has been a rising need for efficient, accurate and less complex staining procedures is required . This study shows significant result ,Nuclear fast red used as nuclear fast red stain and light green stain used as cytoplasm stain cytoplasm , so these stain used as alternative of haematoxylin and eosin stain.

REFRENCES:

1. Anderson J. (2011). An introduction to Routine and special staining. Retrieved on August 18, 2014 from. Godwin, A. (2011).

2. Histochemical uses of haematoxylin-a review. Retrieved August 18, 2014 From www.arpapress.comHarris, T. J., & McCormick, F. (2010).

3. The molecular pathology of cancer. Nat Rev Clin Oncol, 7(5), 251-256.

Received on 27.08.2018 Modified on 29.09.2018

Research J. Pharm. and Tech 2019; 12(1): 79-82.

DOI: 10.5958/0974-360X.2019.00015.5