
==== Front
J Cytol
J Cytol
JCytol
J Cytol
Journal of Cytology
0970-9371
0974-5165
Wolters Kluwer - Medknow India

JCytol-41-150
10.4103/joc.joc_182_23
Original Article
Comparative Evaluation of Honey Versus Alcohol as a Fixative in Cytological Samples: An Institutional Study
Priyanka D.V.S.
Das Subhashish
Raju Kalyani
Soman Princy
Chaudhary Nikhil
Department of Pathology, SDUMC, Tamaka, Karnataka, India
Address for correspondence: Dr. Subhashish Das, Department of Pathology, SDUMC, Tamaka, Kolar - 563 101, Karnataka, India. E-mail: daspathology@gmail.com
Jul-Sep 2024
18 7 2024
41 3 150156
29 10 2023
07 4 2024
03 6 2024
Copyright: © 2024 Journal of Cytology | Indian Academy of Cytologists
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Introduction:

Fixation plays an important role in fixing the cells, making them amenable for evaluation. Ethanol (95%) has been used as a standard cytological fixative. Honey can be introduced as a natural fixative.

Objectives:

1. To study cytomorphological features of smears fixed in 20% unprocessed honey. 2. To study the efficacy of honey as a fixative when compared with ethanol in both hematoxylin and eosin (H and E) stain and Papanicolaou (PAP) stain for cytological samples.

Materials and Methods:

A cross-sectional study was done in the Department of Pathology for 2 months on 100 cytological samples consisting of body fluids (peritoneal and pleural), cervical smears, and FNACs. These smears were fixed separately in 95% ethanol and 20% unprocessed honey for 5 minutes and were stained with both PAP and H and E stain. The cytomorphologies of both the smears were compared based on five parameters, clarity of staining, uniformity of staining, overall morphology, nuclear details, and cytoplasmic details, and the final score was calculated by adding all the scores: Total Score ≤2, poor score; 3–4, good score; 5, excellent score.

Results:

Cervical smears fixed in honey showed similar features to ethanol-fixed smears. Fluid smears fixed in honey and stained with PAP yielded superior results compared to those stained with H and E, as shown by a kappa value of 0.259. However, neither of the smears demonstrated statistical significance. The FNAC samples stained with H and E and PAP exhibited nearly identical results, with a kappa value of 0.688 and 0.647, respectively. FNAC and PAP smears were the next two most successful cytological smear types, with cervical smears exhibiting the best results of the three.

Conclusion:

In honey-fixed smears, we can easily observe all cellular features with clarity. It enhances safety and the working environment in the laboratory. Honey can serve as an effective substitute in situations where resources are limited.

Alcohol
cytology fixatives
honey
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pmcINTRODUCTION

Accuracy of cytopathological diagnosis depends on multiple steps such as collection, proper fixation, staining, and quality control. Among these, fixation plays an important role in fixing the cells and making them amenable to evaluation and to reach an appropriate diagnosis.[1] Ninety-five percent (95%) ethyl alcohol (ethanol) has been used as a standard cytological fixative, which has addictive properties, whereas honey is a cheap and economical alternative as a fixative and it can be introduced in cytology as a natural fixative. Honey has low pH and contains several enzymes and also has antibacterial properties attributed to the presence of hydrogen peroxide and hence can possibly reduce contamination in the smears.[2] It is proposed that 20% aqueous honey can fix the oral smears satisfactorily in a mechanism akin to ethanol by coagulation and denaturation of proteins.[3456] In honey-fixed smears, we can clearly appreciate all cellular details such as nuclear staining, cytoplasmic staining, cellular morphology, clarity, and uniformity of the staining.[1] Using honey also improves the safety and work environment in the laboratory. So, honey can be used as a successful alternative in rural resource constraint setup, health camps, and public health service centers when alcohol fixatives are not available readily.[7] Fixation is an important step in cytopathological diagnosis. Numerous researches are conducted using honey as a cytological fixative in oral lesions, and also, numerous studies are published in Indian scientific literature regarding use of honey as a fixative in histopathological specimens instead of formalin.[45678] There is a paucity of studies in the Indian population that are conducted using honey as a fixative in cytological specimens using only Papanicolaou (PAP) stain. But, in the current research, honey as a fixative will be assessed for both hematoxylin (H and E) and eosin and PAP stain. As per published studies, almost no studies were done regarding use of honey as a fixative in cytological smears for both H and E and PAP stain.

OBJECTIVES

To study cytomorphological features of smears fixed in 20% unprocessed honey.

To study the efficacy of honey as a fixative when compared with ethanol in both H and E stain and PAP stain for cytological specimens.

MATERIALS AND METHODS

The present cross-sectional study was done in the cytology section of the Department of Pathology after obtaining ethical clearance on 21-08-2023 bearing ID No.SDUMC/KLR/IEC/303/2023-24. This study has been conducted for 2 months on 100 cytological samples consisting of various body fluids (peritoneal and pleural), conventional cervical smears, and fine-needle aspiration samples (breast, lymph node, thyroid, salivary gland, and abdominal wall deposits). The smears from all the cytological samples were fixed separately in 95% ethanol and 20% unprocessed natural honey (20 ml of Dabur organic natural honey was dissolved in 80 ml of distilled water) for 5 minutes and were stained with both PAP stain and H and E stain.

It is a double-blinded study where all the slides were labeled as A, B, C, and D, which were evaluated by two pathologists independently based on five parameters and scored accordingly (objectivity). In case of discrepancies, the slides were referred to a third pathologist.

The cytomorphological parameters of both the smears were compared based on five parameters, including clarity of staining, uniformity of staining, overall morphology, nuclear details, and cytoplasmic details, and the final score was calculated by adding scores from all the parameters.[2]

Clarity of staining: Score 0, obliteration of nucleus and cytoplasmic staining; Score 1, crisp and transparent staining.

Uniformity of staining: Score 0, different shades of color in individual cells; Score 1, homogeneous staining throughout the cells.

Overall morphology: Score 0, disintegrated cells with overlapping and folding; Score 1, absence of folds, overlapping, or nuclear swelling.

Nuclear details: Score 0, Nuclear granularity and disintegration; Score 1, Round nuclei with a smooth and clear nuclear membrane, nucleoli, and chromatin.

Cytoplasmic details: Score 0, disintegrated cytoplasmic membrane with obscured granular cytoplasm; Score 1, intact cytoplasmic membrane with a transparent/granular cytoplasm.

Total score/grade: score ≤2, poor; 3–4, good score; 5, excellent score.[2]

Analysis and statistical methods

The study was conducted using a cross-sectional analytical design. The study conducted by Priyadarshi et al.[2] determined that a sample size of 100 was needed for all cytological smears with a confidence interval of 95%, 81% of prevalence, and an absolute error of 8%. The collected data were encoded and inputted into a Microsoft Excel spreadsheet. Descriptive variables were represented as frequencies, percentages, mean, and standard deviation. Categorical variables were analyzed using Chi-square test as a test of significance for comparison between honey and ethanol. The data analysis was conducted using the SPSS 22 Version software. P value <0.05 was considered as statistically significant.

RESULTS

In the present study, a total of 100 cytological smears were collected, consisting of 41 cervical smears, 27 fluid smears, and 32 FNAC smears. This investigation included both H and E and PAP-stained fluid and FNAC smears that were treated independently using different fixatives. Smears were assessed based on the cellularity as adequate and inadequate as per the standard guidelines.

Table 1 presents the results of PAP-stained cervical smears that were fixed using both ethanol and honey. Out of the 41 smears, 17 (41.5%) exhibited an excellent score, while 22 (43.9%) showed a good score. The kappa value of 0.837 indicates perfect agreement with ethanol, and the P value of <0.001 suggests statistical significance.

Table 1 Comparison of conventional cervical smears using ethanol and honey as fixatives (41)

Parameters	Adequacy	95% Ethanol	Honey	Kappa Value	P	
		n	%	n	%			
Clarity of staining	Inadequate	18	43.9%	18	43.9%	0.802	<0.001	
	Adequate	23	56.1%	23	56.1%			
Uniformity of staining	Inadequate	5	12.2%	4	9.8%	0.875	<0.001	
	Adequate	36	87.8%	37	90.2%			
Overall morphology	Inadequate	10	24.4%	11	26.8%	0.936	<0.001	
	Adequate	31	75.6%	30	73.2%		
Nuclear details	Inadequate	2	4.9%	3	7.3%	0.788	<0.001	
	Adequate	39	95.1%	38	92.7%			
Cytoplasmic details	Inadequate	5	12.2%	5	12.2%	1.00	<0.001	
	Adequate	36	87.8%	36	87.8%			
Total Score	Excellent	15	36.6%	17	41.5%	0.837	<0.001	
	Good	22	53.7%	18	43.9%			
	Poor	4	9.8%	6	14.6%			

Table 2 presents a comparison of H and E-stained fluid smears fixed in ethanol and honey. Out of the 27 smears, 8 (29.6%) exhibited an excellent score and 16 (59.3%) showed a good score. The kappa value of 0.187 indicates that there is only a moderate level of agreement with ethanol, and this agreement is not statistically significant as the P value is greater than 0.05.

Table 2 Comparison of H&E-stained fluid smears using ethanol and honey as fixatives (27)

Parameters	Adequacy	95% Ethanol	Honey	Kappa value	P	
		n	%	n	%			
Clarity of staining	Inadequate	14	51.9%	17	63.0%	0.626	<0.001	
	Adequate	13	48.1%	10	37.0%			
Uniformity of staining	Inadequate	6	22.2%	3	11.1%	0.609	0.001	
	Adequate	21	77.8%	24	88.9%			
Overall morphology	Inadequate	4	14.8%	10	37.0%	0.275	0.088	
	Adequate	23	85.2%	17	63.0%			
Nuclear details	Inadequate	1	3.7%	7	25.9%	0.198	0.085	
	Adequate	26	96.3%	20	74.1%			
Cytoplasmic details	Inadequate	2	7.4%	9	33.3%	0.276	0.038	
	Adequate	25	92.6%	18	66.7%			
Total Score	Excellent	21	77.8%	8	29.6%	0.187	>0.05	
	Good	6	22.2%	16	59.3%			
	Poor	0	0%	3	11.1%			

Table 3 illustrates the relationship between PAP-stained fluid smears that were fixed in both ethanol and honey. Out of the 27 smears, 3 (11.1%) showed an excellent score and 23 (85.2%) showed a good score. The kappa value of 0.259 indicates a fair agreement with ethanol, but it is not statistically significant as the P value is greater than 0.05.

Table 3 Comparison of PAP-stained fluid smears using ethanol and honey as fixatives (27)

Parameters	Adequacy	95% Ethanol	Honey	Kappa value	P	
		n	%	n	%			
Clarity of staining	Inadequate	14	51.9%	16	59.3%	0.702	<0.001	
	Adequate	13	48.1%	11	40.7%			
Uniformity of staining	Inadequate	1	3.7%	5	18.5%	0.289	0.033	
	Adequate	26	96.3%	22	81.5%			
Overall morphology	Inadequate	4	14.8%	8	29.6%	0.377	0.031	
	Adequate	23	85.2%	19	70.4%			
Nuclear details	Inadequate	2	7.4%	6	22.2%	0.438	0.006	
	Adequate	25	92.6%	21	77.8%			
Cytoplasmic details	Inadequate	2	7.4%	6	22.2%	0.438	0.006	
	Adequate	25	92.6%	21	77.8%			
Total Score	Excellent	11	40.7%	3	11.1%	0.259	>0.05	
	Good	16	59.3%	23	85.2%			
	Poor	0	0%	1	3.7%			

Table 4 presents the correlation between H and E stained FNAC smears fixed in ethanol and honey. Out of the 32 smears, 11 (34.4%) showed an excellent score and 19 (59.4%) showed a good score. The kappa value of 0.688 indicates substantial agreement with ethanol. Additionally, a P value of <0.001 is statistically significant.

Table 4 Comparison of H&E-stained FNAC smears utilizing ethanol and honey as fixatives (32)

Parameters	Adequacy	95% Ethanol	Honey	Kappa value	P	
		n	%	n	%			
Clarity of staining	Inadequate	12	37.5%	12	37.5%	0.733	<0.001	
	Adequate	20	62.5%	20	62.5%			
Uniformity of staining	Inadequate	12	37.5%	9	28.1%	0.789	<0.001	
	Adequate	20	62.5%	23	71.9%			
Overall morphology	Inadequate	8	25.0%	9	28.1%	0.920	<0.001	
	Adequate	24	75.0%	23	71.9%			
Nuclear details	Inadequate	2	6.3%	4	12.5%	0.636	<0.001	
	Adequate	30	93.8%	28	87.5%			
Cytoplasmic details	Inadequate	3	9.4%	5	15.6%	0.717	<0.001	
	Adequate	29	90.6%	27	84.4%			
Total Score	Excellent	12	37.5%	11	34.4%	0.688	<0.001	
	Good	20	62.5%	19	59.4%			
	Poor	0	0%	2	6.3%			

Table 5 presents a comparison between PAP stained FNAC smears fixed in ethanol and honey. Out of the 32 smears, 15 (46.9%) showed an excellent score and 15 (46.9%) showed a good score. The kappa value of 0.647 indicates substantial agreement with ethanol, and a P value of <0.001 is statistically significant.

Table 5 Comparison of PAP-stained FNAC smears utilizing ethanol and honey as fixatives (32)

Parameters	Adequacy	95% Ethanol	Honey	Kappa value	P	
		n	%	n	%			
Clarity of staining	Inadequate	12	37.5%	12	37.5%	0.733	<0.001	
	Adequate	20	62.5%	20	62.5%			
Uniformity of staining	Inadequate	4	12.5%	1	3.1%	0.368	0.007	
	Adequate	28	87.5%	31	96.9%			
Overall morphology	Inadequate	3	9.4%	5	15.6%	0.717	<0.001	
	Adequate	29	90.6%	27	84.4%			
Nuclear details	Inadequate	2	6.3%	4	12.5%	0.636	<0.001	
	Adequate	30	93.8%	28	87.5%			
Cytoplasmic details	Inadequate	8	25.0%	9	28.1%	0.920	<0.001	
	Adequate	24	75.0%	23	71.9%			
Total Score	Excellent	15	46.9%	15	46.9%	0.647	<0.001	
	Good	17	53.1%	15	46.9%			
	Poor	0	0%	2	6.3%			

A statistically significant association was observed between cervical smears and fine needle aspiration cytology (H and E and PAP). The results of the fluid (H and E and PAP) smears did not show any statistically significant findings. Cervical smears that were fixed in honey had comparable characteristics to smears that were preserved in ethanol as shown in Images 1 and 2. Fluid smears fixed in honey and stained with PAP yielded superior outcomes compared to those stained with H and E, with a kappa value of 0.259 as portrayed in Images 3 and 4. However, neither of the smears exhibited statistical significance. The FNAC samples stained with H and E and PAP as depicted in Images 5-8 exhibited nearly identical findings, with a kappa value of 0.688 and 0.647, respectively. When comparing the three cytological smear types, cervical smears, followed by FNACs and PAP smears, yielded the best outcomes.

Image 1 (a) Shows cervical smear fixed in honey, stained with PAP stain, and (b) shows cervical smear fixed in ethanol in 100X magnification

Image 2 (a) Shows cervical smear fixed in honey, stained with PAP stain, and (b) shows cervical smear fixed in ethanol in 400X magnification. As we can see all the five parameters, score 5 was given

Image 3 (a) Shows fluid smear fixed in honey, stained with H and E, and (b) shows fluid smear fixed in ethanol in 400X magnification. Here, we cannot recognize any of the parameters except uniformity of staining, so score 1 was given

Image 4 (a) Shows fluid smear fixed in honey, stained with PAP stain, and (b) shows fluid smear fixed in ethanol in 400X magnification. Out of five parameters, only two parameters including clarity of staining and uniformity of staining can be seen, so score 2 was given

Image 5 (a) Shows breast FNAC smear fixed in honey, stained with H and E stain, and (b) shows FNAC smear fixed in ethanol in 400X magnification shows all the five parameters clearly, so score 5 was given

Image 6 (a) Shows breast FNAC smear fixed in honey, stained with PAP stain, and (b) shows FNAC smear fixed in ethanol in 400X magnification, where all the five parameters can be appreciated, so 5 was given

Image 7 (a) Shows breast FNAC smear fixed in honey, stained with H and E stain, and (b) shows FNAC smear fixed in ethanol in 400X magnification. Out of five parameters, only three parameters like clarity of staining and uniformity of stain cannot be appreciated, so score 3 was given

Image 8 (a) Shows Lymphnode FNAC smear fixed in honey, stained with H and E stain, and (b) shows FNAC smear fixed in ethanol in 400X magnification. Out of five parameters, nuclear staining in honey-fixed smear and background shows compromised results, so score 4 was given

DISCUSSION

The research described in Table 6 has assessed smears based on their cellularity, categorizing them as either adequate or inadequate based on defined standards and acquired outcomes.

Table 6 Outcomes of studies with others and their corresponding P values

Study	Smears used	P	
Priyadarshi et al.[2]	Fluid (F) + cervical smears (CS) + FNAC (FN) smears (300)	F: 0.777, CS: 0.465, FN: 0.877	
Ishaq R et al.[3]	FNAC sample (30)	> 0.05	
Singh A et al.[4]	Buccal smears (30)	0.47	
Khan et al.[7]	Buccal smears (200)	>0.05	
Nerune et al.[1]	Buccal mucosa (50)	>0.05	
Present Study	Fluid (F) [H&E, PAP] + Cervical smears (CS) [PAP] + FNAC (FN) smears [H&E, PAP]	F: >0.05,>0.05, CS: <0.001 FN:<0.001,<0.001	

Table 6 presents a comparative analysis of the findings from cytological smears in relation to previous research investigations. Priyadarshi et al.[2] conducted a study in which they collected and evaluated 300 cytological smears stained with PAP.

The results were derived from an analysis of five cytomorphological parameters. The kappa values obtained were 0.791, 0.834, and 0.885 for fluids, cervical smears, and FNACs, respectively. In comparison, the present investigation yielded kappa values of 0.259 for fluids and 0.647 for FNACs. The current study and a study by Priyadarshi et al.[2] observed similar findings in cervical smears.

Nevertheless, Ishaq et al.[3] conducted a study where they observed a statistically significant distinction (P = 0.006) just in the clarity of staining, which was ascribed to the viscous consistency of honey.

In their investigation, Singh et al.[4] discovered that honey-fixed smears exhibited a superior cell size and shape, while ethanol-fixed smears demonstrated greater staining clarity and uniformity. However, no significant statistical difference was identified between the two methods. The staining clarity did not reveal a significant P value, which was similar to the findings of the present investigation. The researchers have determined that 90% of smears fixed with ethanol and 80% of smears fixed with honey were suitable for examination. However, there was no significant statistical difference observed between the two fixatives.

Unlike the current investigation, a study conducted by Pandiar et al.[5] demonstrated contradictory findings regarding the purity of staining in honey-fixed smears. Nevertheless, there was no significant disparity observed in the statistical analysis.

In a study conducted by Khan et al.,[7] 200 buccal smears were collected and analyzed for five parameters. Among these parameters, nuclear staining, clarity of staining, and uniformity of staining showed statistically significant differences. The P values for nuclear staining, clarity of staining, and uniformity of staining were 0.008, <0.005, and <0.005, respectively. These findings are consistent with the current study, except for clarity of staining. The cytoplasmic staining and overall morphology did not exhibit any statistically significant difference between the two fixatives, as indicated by P values of 0.821 and 0.092, respectively. This finding contradicts the results of the current investigation.

In contrast, Nerune et al.[1] did not observe any significant variation in nuclear staining, cytoplasmic staining, maintained cell morphology, clarity of staining, nuclear staining, or uniformity of staining (P > 0.005).

In contrast, a study conducted by Sona and Preethamol[10] on buccal smears also found no statistically significant differences (P > 0.05) between the two fixatives in relation to the parameters listed above. Furthermore, it was discovered that the honey-fixed smears exhibited a more distinct background in comparison to the ethanol-fixed smears, which corresponds to the findings of the current investigation.

For many years, alcohol has been utilized as a fixative and is considered the standard fixative in cytology for preserving cellular morphology.[9] The presence of ethanol, with its addictive nature, combustible qualities, and inflammatory effects on the skin and eyes, necessitates the search for an alternative.[1] An alternative to alcohol, such as raw honey, which is easily accessible, less dangerous to living organisms and inexpensive and has fixative qualities similar to alcohol, can be used in cytology. Not only does it serve as an effective fixative but also it exhibits antibacterial properties and inhibits autolysis and putrefaction.[481112]

Honey has applications not just in cytology but also in histopathology. Multiple studies have indicated that natural bee honey may serve as a safer and environmentally friendly alternative to formalin as a fixative, given the health risks associated with formalin in routine histology.[1011121314151617181920]

Limitations

Honey has a shorter shelf life. When left for an extended duration, the presence of mold was seen. It is necessary to use refrigeration and regularly replace the fixative every 2 to 3 days in order to avoid the growth of mold. Fading of the slides was more and early in honey fixed smears when compared to ethanol-fixed smears.

CONCLUSION

All the cellular features are distinctly visible in honey-fixed smears. Honey enhances safety and the working conditions in the laboratory while also possessing antimicrobial characteristics. Honey can serve as a viable substitute in resource-limited rural settings like ours and in health camps and public health service centers where alcohol fixatives are not readily accessible.

Financial support and sponsorship

Nil.

Conflicts of interest

Nil.
==== Refs
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