|Year : 2022 | Volume
| Issue : 1 | Page : 6-10
Utilization of processed honey and jaggery as an oral cyto-fixative
Kunal Sah1, BJ Janardhana Amaranath2, Sunira Chandra3, Saad Ahmad1
1 Department of Oral Pathology and Microbiology, Rama Dental College, Hospital and Research Centre, Rama University, Kanpur, India
2 Department of Periodontology, Faculty of Dental Sciences, Rama Dental College, Hospital and Research Centre, Rama University, Kanpur, India
3 Department of Oral Medicine and Radiology, Saraswati Dental College, Lucknow, India
|Date of Submission||10-Oct-2021|
|Date of Decision||12-Aug-2021|
|Date of Acceptance||10-Dec-2021|
|Date of Web Publication||31-Dec-2021|
B J Janardhana Amaranath
Department of Periodontology, Faculty of Dental Sciences, Rama Dental College, Hospital and Research Centre, Rama University, Kanpur
Source of Support: None, Conflict of Interest: None
Background: Ethanol is reasonably used as a cyto-fixative. Owing to its confinements, researchers are in search of natural and eco-friendly cytological fixative. The present study was undertaken to evaluate the efficacy of 20% honey and 20% jaggery as a fixative for oral exfoliative cytology. Materials and Methods: Three oral smears were obtained from each individual (n = 60) by gently scraping the buccal mucosa. One smear was fixed in 95% ethanol second smear in 20% processed honey and third smear in 20% jaggery and was stained with Papanicolaou stain. Two separate pathologists who were blinded for the fixative used evaluated the slides based on the five parameters (cell morphology, nuclear and cytoplasmic staining, clarity, and uniformity of staining). Results: In the present study, it was observed that 20% processed honey showed overall promising results followed by Group A (95% alcohol) and Group B (20% jaggery), but no statistically significant difference was observed between the groups. Conclusion: Honey in lower concentration is an excellent alternative to ethanol and jaggery as a fixative for oral exfoliative cytological samples.
Keywords: Cytology, ethanol, fixative, honey
|How to cite this article:|
Sah K, Janardhana Amaranath B J, Chandra S, Ahmad S. Utilization of processed honey and jaggery as an oral cyto-fixative. Indian J Dent Sci 2022;14:6-10
|How to cite this URL:|
Sah K, Janardhana Amaranath B J, Chandra S, Ahmad S. Utilization of processed honey and jaggery as an oral cyto-fixative. Indian J Dent Sci [serial online] 2022 [cited 2022 Dec 5];14:6-10. Available from: http://www.ijds.in/text.asp?2022/14/1/6/334520
| Introduction|| |
Fixation is an initial and important step in tissue processing for microscopic examination. The primary intention of fixation is to preserve the tissues in a life-like state, prevent bacterial putrefaction, prevent autolysis, and increase the refractive index of the tissue.
Ethanol (95%) is conventionally used as a fixative for exfoliative cytology but has its own drawbacks, i.e., subjected to pilferage, inflammable, expensive, evaporates easily, and requires license for its procurement, so the pathologists are in constant search of a natural and eco-friendly cytological fixative.
In recent years, natural sweeteners such as honey and jaggery have been experimentally used as cytological fixatives. Most of these studies have been done to assess tissue fixative capability of honey and jaggery compared with that of formalin., Literature search revealed that there are very sparse study wherein cyto-fixative capability of honey and jaggery is assessed compared with that of alcohol (ethanol). Hence, the present study is undertaken to assess the fixative ability of 20% honey and 20% jaggery compared with that of 95% ethanol which is a gold standard fixative in oral exfoliative cytology.
| Materials and Methods|| |
The study was carried out on 60 healthy subjects after taking written informed consent. Inclusion criteria consist of subjects with healthy/normal buccal mucosa free of any pathology. Before obtaining buccal smears, patients were asked to rinse their mouth with water. Three oral smears were collected from each student by gently scraping the buccal mucosa with the wooden spatula. Smear were fixed in 95% ethanol, 20% processed honey, and 20% jaggery. Smears were kept for a minimum duration of 15 min for fixation, and based on the fixative used, the following groups were considered:
- Group A: 95% ethanol (v/v): 95 mL of ethanol mixed with 5 mL of distilled water (n = 20)
- Group B: 20% aqueous honey solution (v/v): 20 mL honey (Dabur Honey, Dabur India Limited, Solan, India) was dissolved in 80 mL of distilled water (n = 20)
- Group C: 20% aqueous jaggery solution (w/v): 20 g of jaggery (obtained from the local market of Lucknow) was dissolved in 80 mL of distilled water. The solution thus obtained was filtered through a filter paper (n = 20).
Slides were treated with increasing grades of alcohol and stained with Harris hematoxylin for 2 min. The slides were then washed in running tap water for 8–10 min. Acid differentiation was done in acid alcohol followed by washing in running water. The slides were then dipped for 30 s each in 90% alcohol and 70% alcohol, respectively. The next step involved staining in OG 6 for 2 min followed by two changes in 90% alcohol. Slides were then stained with EA 36 for 2 min followed by increasing grades of alcohol. The slides were finally cleared and mounted in dextran polystyrene xylene.
All the slides were coded and were evaluated by two separate oral pathologists. In each side, a minimum of 20 epithelial cells without overlapping were considered for evaluation. Both the pathologists were blinded for the fixative used and evaluated the slide based on the five parameters [cell morphology, nuclear and cytoplasmic staining, clarity, and uniformity of staining, [Table 1]]. Any difference in opinion during evaluation was resolved on mutual consensus [Figure 1] and [Figure 2].
|Figure 1: Photomicrograph showing 20% honey-fixed smears (PAP stain, ×400)|
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|Figure 2: Photomicrograph showing 20% jiggery-fixed smears (PAP stain, ×400)|
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| Results|| |
Group A samples showed good nuclear staining followed by Groups B and C [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.31, [Table 2] and Kruskal–Wallis test: Chi-square value 3.69, P = 0.16, [Table 3]].
|Table 2: Staining features of each study group according to evaluation criteria|
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|Table 3: Comparison of evaluation criteria scores between the study groups|
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Group A and B samples showed good cytoplasmic staining as compared to Group C [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.25, [Table 2] and Kruskal–Wallis test: Chi square value 3.09, P = 0.21, [Table 3]].
Group A samples showed excellent cellular morphology followed by Groups B and C [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' Exact Test: P = 0.25, [Table 2] and Kruskal–Wallis test: Chi-square value 4.20, P = 0.41, [Table 3]].
Clarity of staining
Group B samples showed decent clarity of staining followed by Groups A and C [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.25, [Table 2] and Kruskal–Wallis test: Chi-square value 3.69, P = 0.16, [Table 3]].
Uniformity of staining
Group B samples showed decent uniformity of staining followed by Groups A and C [Graph 1]. However, the difference between these fixatives was not statistically significant [Fishers' exact test: P = 0.36, [Table 2] and Kruskal–Wallis test: Chi-square value 4.93, P = 0.41, [Table 3]].
| Discussion|| |
Literature search revealed that spare research has been undertaken to access the fixation ability of 20% processed honey and 20% jaggery in oral exfoliative cytology. Honey is basically a concentrated solution of dextrose and levulose, with 22 other more complex sugars in small amounts. It is produced from the flora by honey bee, and it contains several minerals, vitamins, ascorbic acid, hydrogen peroxide trace elements, and carbohydrates. Various studies have shown that the honey have antibacterial, acidic, antiautolysis, dehydrative, and tissue-hardening property. Sabarinath et al. compared the fixative ability of 10% honey with that of 10% formalin. Their results showed statistically significant differences between honey and formalin samples for both nuclear details and cytoplasmic staining. They concluded that the honey as a tissue fixative is easily available with no known toxicity and can be used as an alternative to formalin. Lalwani et al. evaluated the fixative properties of processed and unprocessed honey and compared it with formalin-fixed tissues. They suggested that both the processed honey and unprocessed honey can be used as an alternative for formalin. Singh et al. also analyzed the efficacy of ethanol and 20% unprocessed honey as a cyto-fixative and concluded that honey could be safely used as a substitute to ethanol. Ishaq et al. compared 95% alcohol and 20% honey fixative solutions in fine needle aspiration cytology. They observed that no statistically significant difference was observed between the fixative properties of honey and alcohol. They concluded that the honey can safely utilized as an alternative to the alcohol as a cyto-fixative.
Jaggery is an Indian sweetener produced by boiling sugarcane juice, till it solidifies. It is a natural mixture consisting of sugar and molasses. In past, various studies were undertaken to assess the fixation ability of jiggery; however, in most of the studies, comparison was done with 10% neutral-buffered formalin. In the present study, cyto-fixative ability of 20% honey and 20% jaggery was accessed and compared with 95% ethanol. Patil et al. studied the tissue fixation ability of 20% honey, 20% sugar syrup, and 30% jaggery syrup and compared with that of 10% buffered formalin using hematoxylin and eosin (H and E) stain. They observed that the tissue fixed with jaggery had good overall morphology, nuclear and cytoplasmic details, and staining quality with clearly discernible cellular outline as compared to other fixatives. Patil et al., in another study, evaluated the tissue fixative ability of 30% jaggery and 20% honey compared with 10% buffered formalin as a control over 6 months. They stained the tissues with H and E, periodic acid–Schiff, and Masson trichrome stains, and they observed that all the three fixatives demonstrated similar results, with jaggery-fixed tissues being comparable to formalin-fixed tissues. Pandiar et al. compared ethanol with that of 20% honey and 30% aqueous jaggery as a cyto-fixative and found no statistically significant difference between these fixatives. They concluded that these fixatives can be used as an alternative fixative for oral smears.
It was proposed by Patil et al. that the fructose present in honey and jaggery at acidic pH breaks down to aldehydes which cross-link with tissue amino acids similar to the action of formaldehyde which assists in fixation of the sample.
In the present study, we observed that 20% honey overall showed more promising results as compared to 95% ethanol and 20% jaggery as a cyto-fixative [Graph 1]. However, no statistically significant difference was observed when all the groups were compared [Table 2] and [Table 3]. The present study results are in conformation with previous studies.
| Conclusion|| |
Eco-friendly fixatives such as honey and jaggery are at par with that of 95% ethanol and can be utilized as a cyto-fixative for oral exfoliative cytology. Being highly economical and universally available, these can be easily employed in screening camp.
Medical Ethics Committee 14/03/16.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Bhattacharyya A, Gupta B, Singh A, Sah K, Gupta V. Probing natural substitute for formalin: Comparing honey, sugar, and jaggery syrup as fixatives. Natl J Maxillofac Surg 2018;9:14-21.
Ishaq R, Azmat H, Omair M, Sheikh AK, Tanwani AK. Comparison of honey with alcohol as a fixative in fine needle aspiration cytology. Int J Pathol 2017;15:15-8.
Pandiar D, Baranwal HC, Kumar S, Ganesan V, Sonkar PK, Chattopadhyay K. Use of jaggery and honey as adjunctive cytological fixatives to ethanol for oral smears. J Oral Maxillofac Pathol 2017;21:317.
] [Full text]
Sabarinath B, Sivapathasundharam B, Sathyakumar M. Fixative properties of honey in comparison with formalin. J Histotechnol 2013;37:21-5.
Lalwani V, Surekha R, Vanishree M, Koneru A, Hunasgi S, Ravikumar S. Honey as an alternative fixative for oral tissue: An evaluation of processed and unprocessed honey. J Oral Maxillofac Pathol 2015;19:342-7.
] [Full text]
Singh A, Hunasgi S, Koneru A, Vanishree M, Ramalu S, Manvikar V. Comparison of honey with ethanol as an oral cytological fixative: A pilot study. J Cytol 2015;32:113-7.
] [Full text]
Jagannadha Rao PV, Das M, Das SK. Jaggery – A traditional Indian sweetener. Indian J Tradit Knowl 2007;6:95-102.
Patil S, Premalatha B, Rao RS, Ganavi B. Revelation in the field of tissue preservation –
A preliminary study on natural formalin substitutes. J Int Oral Health 2013;5:31-8.
Patil S, Rao RS, Ganavi BS, Majumdar B. Natural sweeteners as fixatives in histopathology: A longitudinal study. J Nat Sci Biol Med 2015;6:67-70.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]