Comparison of Honey and Alcoholic Fixatives in Tissues Using Different Types of Stains

Honey and Alcoholic Fixatives in Tissues


  • Shabana Nawaz Rashid Latif College of Physical Therapy, Lahore, Pakistan
  • AH Nagi Pathology department, University of Health Sciences, Lahore, Pakistan
  • Ayesha Bajwa Central Park College of Allied Health Sciences, Lahore, Pakistan
  • Aneela Amjad PSRD College of Rehabilitation Sciences, Lahore, Pakistan
  • Hafiz Mohammad Dawood Community Medicine, Sahara Medical College, Narowal, Pakistan
  • Maham Ashraf Fatima Memorial Hospital, Lahore, Pakistan



alcoholic fixatives, histopathology, honey fixatives, stains


Background: Formalin has been used as a standard fixative for various clinical routines globally. In 1987, after formaldehyde was used as a standard, various studies have been conducted to find a substitute for formalin due to its carcinogenic effects. Since then honey has been proven to be a safe alternative to formalin. Objective: Comparing the tissue fixation abilities of honey, Carnoy’s, Bouin’s and Zenker’s solution with the tissue fixation abilities of formalin using eosin, hematoxylin and Masson trichrome periodic acid Schiff is the objective of this study. Methods: This current study is a comparative model of the study conducted at an experimental research laboratory of the University of Health Sciences, Lahore, Pakistan. This study was designed to determine and explore more eco-friendly, more cost-effective, readily available and best substitutes for formalin fixatives, such as honey, Bouin’s solution, Carnoy’s solution and Zenker’s solution. 30 rats were allocated into five categories, Group 1, Group 2, Group 3, Group 4, and Group 5. Group 1 was the control group which consisted of 6 rats in which the fixative employed was Formalin. The other groups were the experimental groups. They all utilized the other fixatives, Bouin’s, Zenker’s and Carnoy’s solution and honey. This was followed by conventional dispensation and staining. The sections of the tissues were examined and assessed for nuclear details, cytoplasmic details and quality of staining under microscope. Each benchmark was rated on a scale of zero to three (where zero for poor and three for excellent). Results: The results of this current study demonstrated that the tissue that was fixated in honey, Carnoy’s, and Bouin’s solutions were comparable to the tissues fixated in formalin. The tissues that were fixated on Zenker’s solution showed only minute morphological changes among some tissues. Although this didn’t have an impact on accurate diagnostic supposition. Conclusion: Honey, Carnoy’s and Bouin’s solutions used for the fixation of tissues yielded good results. Hence this study concludes that they might be used efficaciously in daily routine histopathology laboratories as a substitute for formalin. 


Özkan N, Şalva E, Çakalağaoğlu F, Tüzüner B. Honey as a substitute for formalin? Biotechnic & Histochemistry 2012; 87(2): 148-53.

Paavilainen L, Edvinsson Å, Asplund A, et al. The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells. Journal of Histochemistry & Cytochemistry 2010; 58(3): 237-46. DOI: 10.1369/jhc.2009.954321

Sabarinath B, Sivapathasundharam B, Sathyakumar M. Fixative properties of honey in comparison with formalin. Journal of Histotechnology 2014; 37(1): 21-5.

Rosai J. Why microscopy will remain a cornerstone of surgical pathology. Laboratory investigation 2007; 87(5): 403-8.

Melville R, Lippmann M. Influence of data elements in OSHA air sampling database on occupational exposure levels. Applied Occupational and Environmental Hygiene 2001; 16(9): 884-99. DOI: 10.1080/10473220117850

Zanini C, Gerbaudo E, Ercole E, Vendramin A, Forni M. Evaluation of two commercial and three home-made fixatives for the substitution of formalin: a formaldehyde–free laboratory is possible. Environmental Health 2012; 11: 1-14. DOI:

Rutland CS. Histological and histochemical methods 4th edition. Journal of Anatomy 2008; 213(3): 356. DOI: 10.1111/j.1469-7580.2008.00957.x

Bancroft JD, Gamble M. Theory and practice of histological techniques: Elsevier health sciences; 2008.

Luz D, Ribeiro Jr U, Chassot C, De Salles Collet e Silva F, Cecconello I, Corbett CEP. Carnoy’s solution enhances lymph node detection: an anatomical dissection study in cadavers. Histopathology 2008; 53(6): 740-2.

Marx RE, Stern D. Oral and maxillofacial pathology: a rationale for diagnosis and treatment: Quintessence Publishing Company Hanover Park; 2012.

Avwioro G, Bankole J, Iyiola S, Avwioro T, Akinola G. One of the properties of honey in wound healing is prevention of autolysis. Der Pharmacia Lettre 2010; 2(3): 321-5.

Patil S, Premalatha B, Rao RS, Ganavi B. Revelation in the field of tissue preservation–A preliminary study on natural formalin substitutes. Journal of international oral health: JIOH 2013; 5(1): 31.

Muddana K, Muppala JNK, Dorankula SPR, Maloth AK, Kulkarni PG, Thadudari D. Honey and olive oil as bio-friendly substitutes for formalin and xylene in routine histopathology. Indian Journal of Dental Research 2017; 28(3): 286.

Li Y, Li Q, Zhu S, et al. The effect of strontium-substituted hydroxyapatite coating on implant fixation in ovariectomized rats. Biomaterials 2010; 31(34): 9006-14.

Gurcan MN, Boucheron LE, Can A, Madabhushi A, Rajpoot NM, Yener B. Histopathological image analysis: A review. IEEE reviews in biomedical engineering 2009; 2: 147-71. DOI: 10.1109/RBME.2009.2034865

Meloan SN, Puchtler H. “Harris hematoxylin,” what harris really wrote and the mechanism of hemalum stains. Journal of Histotechnology 1987; 10(4): 257-61.

Costa GM, Araujo SL, Xavier FAF, et al. picrosirius red and masson’s trichrome staining techniques as tools for detection of collagen fibers in the skin of dogs with endocrine dermatopathologies. Ciência Animal Brasileira 2019; 20.

Yamabayashi S. Periodic acid—Schiff—Alcian Blue: A method for the differential staining of glycoproteins. The Histochemical Journal 1987; 19: 565-71. DOI:

Gillespie JW, Best CJ, Bichsel VE, et al. Evaluation of non-formalin tissue fixation for molecular profiling studies. The American journal of pathology 2002; 160(2): 449-57. Get rights and content

Cox ML, Schray CL, Luster CN, et al. Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity. Experimental and molecular pathology 2006; 80(2): 183-91. Get rights and content

Williams JM, Duckworth CA, Vowell K, Burkitt MD, Pritchard DM. Intestinal preparation techniques for histological analysis in the mouse. Current protocols in mouse biology 2016; 6(2): 148-68.

Al-Maaini R, Bryant P. The effectiveness of honey as a substitute for formalin in the histological fixation of tissue. Journal of Histotechnology 2006; 29(3): 173-6.

Pereira MA, Dias AR, Faraj SF, et al. Carnoy's solution is an adequate tissue fixative for routine surgical pathology, preserving cell morphology and molecular integrity. Histopathology 2015; 66(3): 388-97.




How to Cite

Nawaz, S. ., Nagi, A., Bajwa, A. ., Amjad, A., Dawood, . H. M., & Ashraf, M. . (2023). Comparison of Honey and Alcoholic Fixatives in Tissues Using Different Types of Stains: Honey and Alcoholic Fixatives in Tissues. The Healer Journal of Physiotherapy and Rehabilitation Sciences, 3(8), 776–784.





Most read articles by the same author(s)