Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contact Us Login 
An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 4  |  Page : 603
 

Exploring a safer alternative to eosin in soft tissue staining


Department of Oral Pathology and Microbiology, MGV's KBH Dental College, Nashik, Maharashtra, India

Date of Submission19-Jan-2022
Date of Decision28-Apr-2022
Date of Acceptance19-May-2022
Date of Web Publication22-Dec-2022

Correspondence Address:
Surabhi A Sarode
62, Om Sai Nagar, Old Kamptee Raod, Kalamna, Nagpur, Maharashtra
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.jomfp_27_22

Rights and Permissions

 

   Abstract 


Context: Hematoxylin–eosin (H&E) stain has stood the test of time as the standard stain for histologic examination of human tissues. Haematoxylin is a natural dye, on the contrary, its counterstain eosin is a synthetic dye which belongs to the xanthene group. Synthetic dyes are hazardous to human and animal health. With the increasing awareness of a green earth, it is advisable to use environment-friendly and biodegradable materials. Therefore, an attempt was made to develop as biofriendly substitute in the form of food colour as a counterstain for haematoxylin.
Aim: To assess the staining ability of food colouring agents in routine staining and to compare its staining efficacy with Eosin.
Settings and Design: Two food colours were obtained and stain was prepared by using 70% ethyl alcohol as counterstain for haematoxylin. Different tissue structures such as epithelium, keratin, collagen fibers, muscles, salivary glands, adipocytes, blood vessels, RBCs were observed and evaluated.
Methods and Material: Group A –10 slides stained with green food colour, Group B – 10 slides stained with tomato red food colour and Group C – 10 slides stained with conventional H and E. The stained sections were assessed and graded for nuclear staining, cytoplasmic staining, clarity, uniformity and crispness of staining.
Statistical Analysis Used: The non-parametric Kruskal–Wallis and Mann–Whitney U tests were performed for statistical analysis.
Results: There was no statistically significant difference between the three study groups with respect to all the parameters except crispness of staining. The crispness of Tomato Red and H and E was better compared to green food colour.
Conclusions: Food colouring agents can be used as a safe, biofriendly and inexpensive substitute to eosin in conventional soft tissue staining.


Keywords: Food colour, natural colour, synthetic dye


How to cite this article:
Sarode SA, Pradeep G L, Prakash N, Mahajan A, Mangle N. Exploring a safer alternative to eosin in soft tissue staining. J Oral Maxillofac Pathol 2022;26:603

How to cite this URL:
Sarode SA, Pradeep G L, Prakash N, Mahajan A, Mangle N. Exploring a safer alternative to eosin in soft tissue staining. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Jan 30];26:603. Available from: https://www.jomfp.in/text.asp?2022/26/4/603/364798





   Introduction Top


Biological stains have been used to visualize and identify tissue and cell components for over a century. Biological staining is the union between a colooured dye and a tissue substrate which resists simple washing. The early microscopists had available to them only the natural dyes extracted from plants and animals. Synthetic dyes are a product of the modern petrochemical industry.[1]

In the history of histology, a great shift and development in histologic stains were shaped by improved technologic development of microscopes and the establishment of the histologic stains factory (aniline dye) in 1856 in Germany which manufactured variety of new-histological stains.[2]

Stains are generally used to add colour to animal tissues, plant tissues, microbes and spores to make them optically distinct and technique is known as staining.[3] The most important and most used histological dye, haematoxylin is a natural dye produced from the logwood Haematoxylon campechianum and in combination with Eosin is used for the demonstration of general tissue structures.[4]

The use of colour additives dates back to the Ancient Egyptian period (1500 BC). As a result of the “colour revolution” thousands of new colouring compounds were synthesized for industrial applications. The artificial colours were widely available and used due to a range of advantages over the natural counterparts such as ease of production, low cost, high stability and better colouring properties.[5]

The use of non-allergic, non-toxic and eco-friendly stains has become a matter of significant importance due to the increased environmental awareness in order to avoid some hazardous synthetic ones.[3]

Nowadays, the use of colours as food additives in the Developed World is strictly controlled by legislation, since many of the synthetic compounds were shown to be toxic and harmful to humans. However, the legislation varies in different countries. The regulation in India permits eight synthetic colours (one of which is not legal in the EU) and 11 natural or nature identical additives, no inorganic dyes are permitted (The Prevention of Food Adulteration Rules, 1955).[5]

The present study was performed to assess the staining ability of food colouring agent as a counterstain for haematoxylin and to compare their staining efficacy with Eosin.


   Subjects and Methods Top


The present experimental study was conducted in the Department of Oral Pathology and Microbiology. Date of the approval from ethics committee - 13/08/2019.

10 g of prepared food colour was obtained [Figure 1] and [Figure 2]. This was dissolved in 50 ml of 70% ethyl alcohol. This solution was then filtered using filter paper [Figure 3] and [Figure 4]. Obtained solution was used as counterstain for haematoxylin.
Figure 1: Green food colour

Click here to view
Figure 2: Tomato red food colour

Click here to view
Figure 3: Prepared solution of green food colour

Click here to view
Figure 4: Prepared solution of tomato red food colour

Click here to view


Procedure

Archival wax blocks of previously diagnosed cases were taken. Three sets each comprising of 10 slides of 4 m thickness were prepared. All the slides were deparaffinized in xylene and rehydrated. The slides were divided into the following groups based on the cytoplasmic stain used-

Group A – 10 slides stained with green food colour

Group B – 10 slides stained with tomato red food colour

Group C – 10 slides stained with conventional H and E.

Following staining, all the sections were dehydrated in increasing grades of alcohol, cleared in xylene and mounted with the resinous mounting media-DPX.

Sections were observed using binocular microscope (Olympus CH20i) at ×100 and ×400 magnification. Stained sections were graded based on the following parameters as follows: Nuclear staining, Cytoplasmic staining, Clarity of staining, Uniformity of staining and Crispness of staining. The slides were scored as per [Table 1].
Table 1: Scoring criteria for stained slides

Click here to view


The scores were totalled. The slides were evaluated by two independent observers.

Statistical analysis

We subjected our results to statistical analysis. The non-parametric Kruskal–Wallis and Mann–Whitney U tests were performed to compare the staining efficacy amongst three groups. The P value obtained was statistically insignificant (p > 0.05). Results with 'p < 0.05' was considered to be statistically significant at 95% confidence interval.


   Results Top


Our objective was to check staining ability of food colours, on comparison between three study groups, we observed that the mean assessment ranks of soft tissue staining were comparable for all parameters and the differences found were not significant statistically. [Table 2], [Table 3], [Table 4], [Table 5] [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
Table 2: Comparison of staining efficacy among Group A and H&E

Click here to view
Table 3: Comparison of staining efficacy among Group B and H and E (inter-observer)

Click here to view
Table 4: Comparison of staining efficacy among Group A and Group B using Mann–Whitney Test

Click here to view
Table 5: Comparison of staining efficacy among three study groups using Kruskal-Wallis Test

Click here to view
Figure 5: Photomicrographs showing epithelium and connective tissue<

Click here to view
Figure 6: Photomicrographs showing skeletal muscle

Click here to view
Figure 7: Photomicrographs showing salivary glands

Click here to view
Figure 8: Photomicrographs showing adipose tissue

Click here to view
Figure 9: Photomicrographs showing keratin

Click here to view


Comparison between Group A and H and E revealed that the mean assessment ranks of soft tissue staining were comparable for all parameters of grading and the differences found in mean assessment ranks among these were not significant statistically [Table 2].

Assessment ranks of soft tissue staining were comparable for all parameters of grading and the differences found in mean assessment ranks among these were not significant statistically [Table 3].

Comparison between two food colour stains Group A and Group B revealed that the mean assessment ranks of soft tissue staining were comparable for all parameters of grading [Table 4].

Inter-group comparison between three study groups revealed that the mean assessment ranks of soft tissue staining were comparable for all parameters of grading [Table 5].

Comparable staining results among the groups were found. Food colouring agents stained cytoplasm adequately and the results were comparable with routine H and E [Figure 10].
Figure 10: Comparison between three study groups (Kruskal- Wallis)

Click here to view


Different tissue structures such as epithelium, connective tissue, skeletal muscles, salivary glands, adipose tissue and keratin were stained adequately using food colour as a counter stain for haematoxylin.

We also checked the longevity of staining and observed that there was no change in intensity of staining. All the parameters were reassessed by both the observers after 18 months and the scores were similar [Figure 11] and [Figure 12].
Figure 11: Longevity of staining for Group A

Click here to view
Figure 12: Longevity of staining for Group B

Click here to view



   Discussion Top


Man has always been interested in colours; the art of dyeing has a long past and many of the dyes go back into prehistory.[6] The stain is the marker, or the reagent used to generate the marker. Most biological stains were first manufactured as textile dyes, when each manufacturer gave the dye their own trade name.[7] The discovery of synthetic dyes in 1856 by William Henry Perkin gave way to a range of new synthetics that spread throughout the world.[8]

In the medical field, these stains are commonly used in anatomical, surgical and histopathology for the diagnosis of various diseases and to locate the tumour-free margins. The stains aid in the microscopic examination of cells, nucleic acids and proteins. The bonds between the stain and tissue substrates are mainly due to acid base reactions. Since every tissue is made of multiple structures, their staining properties also vary mandating the use of a combination of stains to prepare tissue sections of diagnostic quality.[9]

Most commonly used counterstain with haematoxylin is eosin. Eosin is a synthetic xanthene dye which is commercially available as Eosin Y, ethyl eosin, and Eosin B. Of these, 0.5 or 1% solution of Eosin Y, in distilled water or alcohol, is commonly used. Although it is very efficient, they are hazardous to human and animal health.[10]

Cellular structure differentiation and contrast are enhanced with the selective use of various natural and synthetic stains. There may be needed to apply a combination of these stains in some instances to demonstrate the presence of some tissue structures and/or cellular inclusions. The ability of a dye to stain-specific tissue structures is determined by certain factors. One of factors is the pH of the stain. Acidic structures would be stained by basic dyes while basic structures would be stained by acidic dyes.[11]

Natural dyes offer an important alternative as they are safer to use with no health hazards, have easy disposability, are biodegradable and can be used to make compost for agricultural purposes after they have been extracted.[12] Natural dyes find use in the colouring of textiles, drugs, cosmetics, etc., owing to their nontoxic effects, they are also used for colouring various food products.[13]

Food colours when used as a stain have an added advantage of being[12]

  • Non-toxic, non-flammable, non-hazardous
  • Economical
  • Easy disposal
  • Easy availability
  • Less technique sensitive[12]


The use of food colouring agents in India is regulated and restricted to the use of those that do not cause significant health risk. Food colours, besides imparting its green and pinkish colouration when used in combination with haematoxylin, resulted in contrast that was well appreciated. We observed quality staining with sufficient clarity and crispness with tomato red and green food colours. The results we got inspired us to try food colour as an alternative to eosin. Food colours stained epithelium, keratin, collagen, blood vessels, muscle fibres, adipose tissue and salivary glands suggesting its strong affinity to these structures and can be used as a novel staining method. The staining efficacy was similar/comparable among green food colour and H and E (conventional stain) for all assessment parameters (nuclear staining, cytoplasmic staining, clarity, uniformity and crispness of staining). Food colours are environment friendly and are less hazardous than waste disposal of used stain solutions.


   Conclusion Top


To the best of our knowledge, food colours have not been used for soft tissue staining. We observed quality staining with sufficient clarity and crispness with tomato red and green food colours. We found comparable staining results among the groups. Food colouring agents stained cytoplasm adequately and the results were comparable with routine H and E. The staining efficacy was similar/comparable among green food colour and H and E (conventional stain) for all assessment parameters (nuclear staining, cytoplasmic staining, clarity, uniformity and crispness of staining). Thus, this is an innovative study exploring the use of a safer alternative to eosin in conventional soft tissue staining.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th ed. Saint Louis, Missouri, U.S.A: Butterworth-Heinemann; 1985.  Back to cited text no. 1
    
2.
Alturkistani HA, Tashkandi FM, Mohammedsaleh ZM. Histological stains: A literature review and case study. Glob J Health Sci 2015;8:72-9.  Back to cited text no. 2
    
3.
Korade D, Sonwane L, Mahale D. Application aqueous plant extract as biological stains. Int J Sci Eng Res 2014;5:1586-9.  Back to cited text no. 3
    
4.
Bassey RB, Oremosu AA, Osinubi AA. Curcuma longa: Staining effect on histomorphology of the testes. Maced J Med Sci 2012;5:26-9.  Back to cited text no. 4
    
5.
Oplatowska-Stachowiak M, Elliott CT. Food colors: Existing and emerging food safety concerns. Crit Rev Food Sci Nutr 2017;57:524-48.  Back to cited text no. 5
    
6.
Siva R. Review article on status of natural dyes and dye-yielding plants in India. Curr Sci 2007;92:916-25.  Back to cited text no. 6
    
7.
Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 6th ed. Philadelphia, PA: Churchill Livingstone Elsevier Publications; 2008.  Back to cited text no. 7
    
8.
Bassey RB, Bakare AA, Osinubi AA, Oremosu AA. Staining properties of Lonchocarpus cyanescens on the testes. Sci Rep 2012;1:211.  Back to cited text no. 8
    
9.
Lavanya A, Sowmya SV, Rao RS, Augustine D, Haragannavar VC. Natural stain (Kumkum) formulated by the extract of Curcuma aromatica and slaked lime in histostaining of oral tissues: An observational study. J Oral Maxillofac Pathol 2021;25:88-96.  Back to cited text no. 9
  [Full text]  
10.
Mohandas R, Ramani P, Sherlin HJ, Sukumaran G, Ramasubramanian A, Don KR, et al. Organic stains used in histopathology-A systematic review. Drug Invent Today 2019;11:426-32.  Back to cited text no. 10
    
11.
Akinloye AJ, Illoh HC, Olagoke AO. Screening of some indigenous herbal dyes for use in plant histological staining. J For Res 2012;21:81-4.  Back to cited text no. 11
    
12.
Suryawanshi H, Naik R, Kumar P, Gupta R. Curcumalonga extract – Haldi: A safe, eco-friendly natural cytoplasmic stain. J Oral Maxillofac Pathol 2017;21:340-4.  Back to cited text no. 12
[PUBMED]  [Full text]  
13.
Kumar S, Singh NN, Singh A, Singh N, Sinha RK. Use of Curcuma longa L. extract to stain various tissue samples for histological studies. Ayu 2014;35:447-51.  Back to cited text no. 13
[PUBMED]  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
Print this article  Email this article
            

    

 
   Search
 
  
    Similar in PUBMED
    Search Pubmed for
    Search in Google Scholar for
  Related articles
    Article in PDF (2,912 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
   Subjects and Methods
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed192    
    Printed4    
    Emailed0    
    PDF Downloaded40    
    Comments [Add]    

Recommend this journal

Journal of Oral and Maxillofacial Pathology | Published by Wolters Kluwer - Medknow
Online since 15th Aug, 2007