Formalin performs better than ethanol for recovery of strongyle-type eggs using established and novel McMaster techniques

Formalin performs better than ethanol for recovery of strongyle-type eggs using established and novel McMaster techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Formalin performs better than ethanol for recovery of strongyle-type eggs using established and novel McMaster techniques Breanna Knight, Alan Lymbery, Amanda Ash This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9433494/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Gastrointestinal parasites (GI), including soil-transmitted helminths (STHs), continue to impose a considerable health burden in endemic regions, particularly in low- and middle-income countries. Reliable and cost-effective diagnostic tools are essential for parasite surveillance and control, yet preservation methods can influence diagnostic accuracy. Coproscopy remains the gold standard for morphological identification of GI parasites, however time constraints often require samples to be preserved for later analysis. Faecal samples are conventionally stored in 10% buffered formalin for microscopy, and a counterpart stored in ethanol if molecular analysis is required, however recent studies have suggested that ethanol may serve as a suitable preservative for both. This controlled study evaluated the suitability of 10% buffered formalin and 70% ethanol for preserving strongyle-type eggs over a three-month period and compared parasite recovery between two McMaster quantification techniques: the modified McMaster and McMaster 2 methods. Recovery of strongyle- type eggs was 4.7 to 8.5-fold higher in formalin-preserved samples than in ethanol-preserved counterparts. In addition, the morphological integrity of eggs was better maintained in formalin, with reduced damage and clearer visualisation of diagnostic features. These findings demonstrate that 10% buffered formalin performs better than 70% ethanol for both structural integrity and recovery of strongyle-type eggs for coproscopic quantification and identification. The McMaster2 method also yielded a higher number of strongyle-type eggs than the modified McMaster method, indicating slight improved quantification sensitivity. coproscopy strongyle-type eggs faecal preservation formalin vs ethanol McMaster Figures Figure 1 Figure 2 Key findings 10% buffered formalin significantly enhanced strongyle-egg recovery , yielding up to an 8.5-fold increase in strongyle type egg counts compared with 70% ethanol, confirming its superior performance for coproscopic quantification. Formalin-preserved samples maintained superior egg morphology , with minimal structural damage and clearer morphological diagnostic features, whereas ethanol caused deformation and desiccation which compromised identification accuracy. Ethanol was unsuitable as a universal preservative for floatation-based microscopy , producing reduced egg recovery and morphological distortion, despite its utility for molecular applications. The McMaster2 method demonstrated slightly higher egg recovery than the modified McMaster technique , suggesting improved sensitivity that may become significant with larger sample sizes. Formalin over ethanol remains the preferred fixative for microscopy-based GI parasite diagnostics , particularly in low-resource and field settings where reliable morphological identification and quantification of soil transmitted helminths are critical for surveillance and intervention monitoring. Introduction Gastrointestinal parasites, particularly soil-transmitted helminths (STHs), remain a major global health burden, especially in low-resource settings (Chen et al., 2024 ). Effective diagnostic tools are important for accurately identifying and quantifying parasitic infections, which in turn guides health interventions and the allocation of resources for control programs. Coproscopy techniques are the gold standard for morphological identification of gastrointestinal parasites and are widely used in large-scale surveys (Ash et al., 2017 , Kattula et al., 2014 ). The most commonly used of these is the Kato Katz technique, which allows for the detection and quantification of soil transmitted, however analysis must be conducted on fresh (unpreserved) samples and within 30 minutes for hookworm eggs which clear rapidly and become invisible (WHO, 2019 ). The logistics of collecting and analysing fresh faecal samples can be challenging, particularly during public health campaigns involving large sample numbers. In such cases, preserving faecal samples for later analysis is required. Formalin has been the preferred preservation medium for parasitological morphology due to its ability to minimize excessive tissue shrinkage and distortion of cellular structure of parasite eggs, and therefore allows for traditional faecal flotation methods to be employed (Thavarajah et al., 2012, Garcia et al., 1979 , Garcia and Shimizu, 1998 , Ash et al., 2017 , Kattula et al., 2014 ). These include the formalin-ethyl acetate concentration technique (FECT) which provides qualitative data (presence/absence) of parasite eggs/ova or modified McMaster method which provides quantitative data (faecal egg counts) (Lim et al., 2018 , Sayasone et al., 2015 ). However, formalin is not an effective preservation medium for molecular based diagnostic techniques such as PCR which requires sample preservation in ethanol to conserve DNA integrity (Srinivasan et al., 2002 ). Molecular assays can offer increased sensitivity for the detection and identification of parasite species and are therefore important components of parasite control where feasible (Stensvold and Nielsen, 2012 ). Recent research has suggested that ethanol is a suitable medium for both coproscopy (faecal floats) and molecular techniques, which if correct would greatly reduce the handling of large sample numbers during public health programs (Hass et al., 2024 , Jagła et al., 2013 ). This was contrary to longstanding original literature and warranted further investigation. Therefore, this study aimed to compare the recovery of strongyle eggs preserved in 70% ethanol or 10% formalin over increasing time periods. An ovine animal model was used as a proxy for the recovery of strongyle eggs in human faecal samples. Strongyle eggs in sheep are similar to human hookworm eggs and therefore allowed for a controlled comparison of preservation methods under conditions similar to those found in large-scale human diagnostic surveys, Additionally, this study compared the modified McMaster method with the novel McMaster2 method for the best accuracy in quantifying strongyle-type eggs stored in both mediums. Materials and Methods Sample collection and preparation Fresh sheep faeces were collected from the ground post-defecation from a flock of approximately 20 individuals. Immediately after collection, a small amount of water was added to the pooled faeces and thoroughly mixed into homogeny. Faeces from this mix were aliquoted into 48 sterile 10mL tubes and preserved in 10% buffered formalin (n = 24) or 70% ethanol (n = 24) at a ratio of 1:3 faeces to preservation medium. Samples were thoroughly mixed with preservative media to ensure eggs were preserved. Samples were collected in triplicate for each preservation medium and time point and were stored at ambient temperature out of direct sunlight. Recovery of strongyle eggs Faecal samples were assessed in triplicate using microscopy at the following time points: fresh, days one, three, five, seven, ten, 14, 30 and 90. At each time point, faecal samples were centrifuged and washed with water before analysis. Faecal egg counts were obtained through two McMaster methods to compare their performance in recovery of strongyle-type eggs in each preservation medium. The standard modified McMaster method (Whitlock, 1948 ) referred to here as McMaster1, was conducted only counting the top focal layer of each chamber, while the novel McMaster2 method used the entire depth of the McMaster slide, counting both the top and bottom focal layers of each chamber (Larkins et al., 2025 ). Faeces (2 g) were mixed thoroughly in 30 mL of saturated salt solution (specific gravity (s.g.) 1.2) and strained through a sieve thrice before 0.15 mL of the solution was loaded into each chamber of the McMaster slide. Loaded McMaster slides were left to sit for five minutes to allow the debris to settle before chambers were examined microscopically at 10 x magnification within 20 minutes. The egg per gram (EPG) of faeces was calculated as the number of strongyle-type eggs counted in both chambers multiplied by 50. Imaging of strongyle-type eggs recovered A zinc sulfate (s.g 1.18) centrifugation floatation method was conducted to concentrate parasite eggs for imaging to assess strongyle-type eggs for signs of deterioration (Zajac and Conboy, 2006 ). Remaining faeces (~ one gram) from the McMaster2 were used for imaging. Samples were screened and imaged using an Olympus BX50 microscope with an Olympus DP71 camera and captured using CellSens Standard 1.13 software. The first five to seven strongyle-type eggs visualised were imaged to prevent selection bias, except for the ethanol stored day 90 samples where only one egg was identified. Images of strongyle-type eggs for each time point were assigned a number and randomly selected for inclusion in morphological feature analysis. Statistical analysis Statistical analysis of significant difference between methods and storage medium at each individual time point was assessed using a two-way ANOVA with multiple comparisons, and the false discovery rate was controlled using the Benjamini, Krieger and Yekutieli method (Benjamini et al., 2006 ). The q value is an adjusted P value, and represents the tests called as significant that will be false positives. All graphing and statistical analysis were performed using GraphPad Prism version 8 for Windows, GraphPad Software, Boston, Massachusetts USA. Results Comparison of mediums on recovery of strongyle-type eggs Faeces stored in 10% formalin yielded a statistically significant increase in the recovery of strongyle-type eggs across all timepoints and both modified McMaster methods comparative to faeces stored in 70% ethanol (q value of 0.0002 to 0.017) (Fig. 1). The mean floatation efficiency for eggs recovered from 10% formalin storage was 68–70% across all time points, a 4.7 to 8.5-fold increase in recovery when compared with 70% ethanol which had a mean recovery rate of 8–15%. Comparison of McMaster1 and McMaster2 on recovery of strongyle-type egg Comparison between recovery rate of strongyle-type eggs between the McMaster1 and McMaster2 methods showed minimal statistical significance. While McMaster2 had a higher recovery of strongyle-type eggs in five of the nine time points for formalin (days 1, 7, 10, 14 and 30) and McMaster2 had a higher recovery rate of strongyle-type eggs in five of the eight time points available for ethanol (days 1, 3, 5, 7 and 14), only samples preserved in ethanol exhibited a statistical difference between McMaster1 and McMaster2, and only at a single time point (day 14, q = 0.038). McMaster2 could not be performed at time point 90 days for the ethanol preserved samples, due to the sample becoming too cloudy to view the bottom focal layer. Comparison of recovery of strongyle-type eggs at different timepoints The floatation efficiency of samples preserved in formalin was variable, with multiple spikes in recovery rates of strongyle-type eggs evident. The highest recovery rate occurred at day 7 (88%), followed by day 90 (77%) and the lowest recovery on day five (53%). The initial days after formalin fixing appeared to have the lowest recovery rates, compared with the later days, although there seemed to be large variations between timepoints. The floatation efficiency of samples preserved in ethanol was low, with a maximum of 30% recovery on day 1 (for McMaster2) and a minimum of 3.8% recovery at days 7 and 14 (McMaster1). The McMaster1 method was consistently below 10% recovery from day 5 through to the end of the experiment at day 90, while the McMaster2 method had a slightly increased recovery with only day 10 and 30 dropping below 10%. Gross morphology of strongyle-type eggs Samples imaged at each time point revealed changes of egg morphology in both formalin and ethanol fixed samples (Fig. 2). At time point 0, strongyle-type eggs were observed to have a thin, smooth ellipsoidal shaped shell with an embryo in the morula stage of development. Strongyle-type eggs from samples stored in 70% ethanol initially had no change in morphology from days 1–3, however severe degradation occurred in several areas of the shell, collapsing into the morula at day 10. Throughout the remaining timepoints, days 14–90, the shell continued to thin and lose its smooth ellipsoidal shape, becoming difficult to recognise. Strongyle-type eggs from samples stored in 10% formalin displayed a change in the shell after 24 hours, with wrinkles appearing to form. After 10 days the smooth ellipsoidal shape started to become irregular, with the shell starting to collapse inwards by day 14, however at day 90, the shell was less collapsed, although still not uniform. Discussion The recovery of strongyle-type eggs was up to 8.5 times higher in faeces preserved in 10% buffered formalin, compared with those preserved in 70% ethanol, using the modified McMaster quantification method. In addition to superior recovery, the morphological integrity of strongyle-type eggs was better maintained in formalin preserved samples. These findings demonstrate that 10% buffered formalin is a better medium for the preservation of faeces containing strongyle-type eggs than ethanol. Our findings align with earlier research reporting formalin’s superior performance over ethanol as a fixative for strongyle-type eggs (Foreyt, 1986 ),. Although formalin has long been considered the preferred preservation media for parasitological diagnostics (Nace et al., 1999 , Pietrzak-Johnston et al., 2000 , Bonita and Taira, 1996 , Carroll et al., 1983), ethanol is still considered an option by some (Jagła et al., 2013 , Hass et al., 2024 ). A study investigating preservation media on horse faecal samples suggested 40% ethanol performed better than 4% formalin, however it was noted that the samples were only immersed in the preservation media, not homogenised (Jagła et al., 2013 ). This lack of homogenisation would limit the media preserving eggs inside the sample, likely affecting the outcome. This in conjunction with relatively low concentration of media used (40% ethanol) limits the ability for direct comparison to results obtained here. More recently a publication has proposed ethanol as a universal preservation medium for both morphological and molecular analyses of monkey faecal samples (Hass et al., 2024 ). Interestingly, the authors noted that parasite eggs/larvae were actually better preserved in formalin than ethanol. They had also ascertained egg recovery through a sedimentation technique rather than floatation, again limiting direct comparison for egg recovery. Despite the lack of direct comparison with ethanol focussed papers, the results presented in this paper, together with previous evidence, indicate that ethanol is still unsuitable for floatation-based microscopy due to reduced recovery and compromised egg morphology (Naem et al., 2010 , Foreyt, 1986 ). These data reinforce that there is no “one size fits all” storage solution; formalin remains optimal for morphological examination, while ethanol may be preferable when downstream molecular analysis is required. Reliable egg recovery and quantification are essential for monitoring soil transmitted helminth (STH) burden and evaluating intervention strategies (Lim et al., 2018 ). Methods such as Kato Katz provide quantitative data through egg per gram counts but fresh faecal samples are required and analysis must be done in 30 minutes to detect hookworm eggs (strongyle-type eggs), The formalin-ethyl acetate concentration technique (FECT) can be performed on formalin fixed samples but provides qualitative data only. The modified McMaster technique as displayed here can be performed on formalin fixed samples and provides quantitative data. All these methods are low cost and practical for use in endemic regions where infrastructure and funding are often limited, but the ability to preserve samples for later analysis is an important consideration for large scale surveys. (Bradbury et al., 2022 , Larkins et al., 2025 ). The modified McMaster technique, commonly used in livestock diagnostics for quantifying strongyle type eggs, is a low resource option recommended by the World Health Organisation for the quantification of parasite eggs for human diagnostics (2019, WHO, 2019 ). This method typically counts eggs that float to the top of the chamber, excluding those that may not float, such as infertile eggs or those affected by the preservation medium (Whitlock, 1948 ). To address this, the McMaster2 method was developed during a large-scale STH survey to improve quantification accuracy by counting eggs in both the top and bottom focal layer (Larkins et al., 2025 ). The morphological integrity of the eggs was affected by both preservation mediums. In ethanol-preserved samples, the outer shell of the strongyle-type eggs exhibited evident desiccation and structural damage within 24 hours of exposure. Such degradation compromised the characteristic smooth, ellipsoidal shell which is a significant identifying feature of strongyle-type eggs. When these defining features are lost, accurate differentiation from faecal debris and artefacts within the floatation preparation becomes increasingly difficult. Consequently, severely degraded eggs may no longer be recognisable as strongyle-type, leading to potential underestimation of egg counts or under-diagnosis of infection in faecal microscopy. Formalin fixed eggs showed some degradation, predominantly wrinkling of the shell, but maintained their defining features of the thin ellipsoidal shell with distinct morula. This study provided the first direct controlled comparison of the modified McMaster1 and McMaster2 quantification methods on preserved faecal samples. Although there was a lack of statistical differences between the modified McMaster1 and McMaster2 methods, there was a slight difference with McMaster2 often recovering more strongyle type eggs. While it is well established that formalin is reactive towards protein and forms inter-molecular cross linkages (Vince et al., 1997 ) which increases the molecular weight, the strongyle-type eggs weren’t observed settling on the bottom focal layer when preserved in 10% formalin, but perhaps instead sitting somewhere between the top and bottom focal layers. There was a single timepoint where there was a statistical difference between McMaster and McMaster2 for the ethanol preserved samples, with McMaster2 being more successful for egg recovery. This weight change causing the eggs to settle on the bottom focal layer may have occurred as the ethanol altered the egg shell, causing the egg to become brittle and allowing the salt flotation solution to seep inside (Marquina et al., 2021 ). Together these observations that preservation-induced changes to egg density and structural integrity may subtly influence their distribution within the floatation medium, thereby affecting recovery efficiency depending on the McMaster quantification approach used. Conclusion This study determined that 10% buffered formalin performs better than 70% ethanol for preserving strongyle-type eggs for floatation-based quantification. Egg recovery was up to 8.5 times higher in formalin-preserved samples compared with ethanol, and morphological features essential for identification were better maintained. These findings confirm that formalin remains the preferred fixative for microscopy-based diagnostics, whereas ethanol is unsuitable for egg quantification due to reduced recovery and structural degradation. This study also represents the first direct, controlled comparison of the modified McMaster and McMaster2 techniques on preserved faecal samples. While no statistical differences were detected, McMaster2 consistently recovered slightly more eggs, suggesting a potential advantage in larger sample sizes. Together, these results reinforce the continued relevance of formalin in reliable, microscopy-based monitoring of soil-transmitted helminth infections, particularly in low-resource settings. Declarations Financial support This research received no specific grant from any funding agency, commercial or not-for-profit sectors. Competing interests The authors declare there are no conflicts of interest. Ethical standards Not applicable Author Contribution Author contributionsBK and AA conceived and designed the study. BK conducted data collection, BK and AL performed statistical analysis. BK and AA wrote the manuscript. Data Availability All data supporting the findings of this study are available within the paper. References Bench aids for the diagnosis of intestinal parasites second edition. In : MONTRESOR, A. (ed.). Geneve: World Health Organisation ASH A, OKELLO A, KHAMLOME B, INTHAVONG P, ALLEN J, THOMPSON RCA (2017) Controlling Taenia solium and soil transmitted helminths in a northern Lao PDR village: Impact of a triple dose albendazole regime. Acta Trop 174:171–178 BENJAMINI Y, KRIEGER AM, YEKUTIELI D (2006) Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93:491–507 BONITA R, TAIRA N (1996) Faecal examination of Fasciola eggs fixed with formalin solution using the beads technique. Vet Parasitol 67:269–273 BRADBURY RS, POTTERS SAPPSGH, FREAN IMATHISONBA, MEWARA J, SHEOREY A, TAMAROZZI H, CHIODINI FCOUTURIERMR, P., PRITT B (2022) Where Have All the Diagnostic Morphological Parasitologists Gone? J Clin Microbiol 60:e0098622 CARROLL MJ, COOK, J., TURNER JA (1983) Comparison of polyvinyl alcohol- and formalin-preserved fecal specimens in the formalin-ether sedimentation technique for parasitological examination. 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K., RANGANATHAN K (2012) Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol 16:400–405 VINCE DG, TBAKHI A, GADDIPATI A, COTHREN RM, CORNHILL, J. F., TUBBS RR (1997) Quantitative Comparison of Immunohistochemical Staining Intensity in Tissues Fixed in Formalin and Histochoice. Anal Cell Pathol 15:607965 WHITLOCK H (1948) Some modifications of the McMaster helminth egg-counting technique and apparatus. J Council Sci Industrial Res Australia 21:177–180 WHO (2019) Bench aids for the diagnosis of intestinal parasites, 2nd edn. World Health Organization;, Geneva ZAJAC AM, CONBOY GA (2006) Veterinary Clinical Parasitology Iowa. Blackwell Publishing Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9433494","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629866202,"identity":"f3ec862c-426a-4372-907e-3ed39eaf9066","order_by":0,"name":"Breanna 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15:54:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":518747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9433494/v1/33c3f163-1a95-4558-ad32-d71250df15fe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Formalin performs better than ethanol for recovery of strongyle-type eggs using established and novel McMaster techniques","fulltext":[{"header":"Key findings","content":"\u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e10% buffered formalin significantly enhanced strongyle-egg recovery\u003c/b\u003e, yielding up to an 8.5-fold increase in strongyle type egg counts compared with 70% ethanol, confirming its superior performance for coproscopic quantification.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFormalin-preserved samples maintained superior egg morphology\u003c/b\u003e, with minimal structural damage and clearer morphological diagnostic features, whereas ethanol caused deformation and desiccation which compromised identification accuracy.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eEthanol was unsuitable as a universal preservative for floatation-based microscopy\u003c/b\u003e, producing reduced egg recovery and morphological distortion, despite its utility for molecular applications.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eThe McMaster2 method demonstrated slightly higher egg recovery than the modified McMaster technique\u003c/b\u003e, suggesting improved sensitivity that may become significant with larger sample sizes.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFormalin over ethanol remains the preferred fixative for microscopy-based GI parasite diagnostics\u003c/b\u003e, particularly in low-resource and field settings where reliable morphological identification and quantification of soil transmitted helminths are critical for surveillance and intervention monitoring.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eGastrointestinal parasites, particularly soil-transmitted helminths (STHs), remain a major global health burden, especially in low-resource settings (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Effective diagnostic tools are important for accurately identifying and quantifying parasitic infections, which in turn guides health interventions and the allocation of resources for control programs. Coproscopy techniques are the gold standard for morphological identification of gastrointestinal parasites and are widely used in large-scale surveys (Ash et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kattula et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The most commonly used of these is the Kato Katz technique, which allows for the detection and quantification of soil transmitted, however analysis must be conducted on fresh (unpreserved) samples and within 30 minutes for hookworm eggs which clear rapidly and become invisible (WHO, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The logistics of collecting and analysing fresh faecal samples can be challenging, particularly during public health campaigns involving large sample numbers. In such cases, preserving faecal samples for later analysis is required.\u003c/p\u003e \u003cp\u003eFormalin has been the preferred preservation medium for parasitological morphology due to its ability to minimize excessive tissue shrinkage and distortion of cellular structure of parasite eggs, and therefore allows for traditional faecal flotation methods to be employed (Thavarajah et al., 2012, Garcia et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Garcia and Shimizu, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Ash et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kattula et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These include the formalin-ethyl acetate concentration technique (FECT) which provides qualitative data (presence/absence) of parasite eggs/ova or modified McMaster method which provides quantitative data (faecal egg counts) (Lim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Sayasone et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, formalin is not an effective preservation medium for molecular based diagnostic techniques such as PCR which requires sample preservation in ethanol to conserve DNA integrity (Srinivasan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Molecular assays can offer increased sensitivity for the detection and identification of parasite species and are therefore important components of parasite control where feasible (Stensvold and Nielsen, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent research has suggested that ethanol is a suitable medium for both coproscopy (faecal floats) and molecular techniques, which if correct would greatly reduce the handling of large sample numbers during public health programs (Hass et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Jagła et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This was contrary to longstanding original literature and warranted further investigation. Therefore, this study aimed to compare the recovery of strongyle eggs preserved in 70% ethanol or 10% formalin over increasing time periods. An ovine animal model was used as a proxy for the recovery of strongyle eggs in human faecal samples. Strongyle eggs in sheep are similar to human hookworm eggs and therefore allowed for a controlled comparison of preservation methods under conditions similar to those found in large-scale human diagnostic surveys, Additionally, this study compared the modified McMaster method with the novel McMaster2 method for the best accuracy in quantifying strongyle-type eggs stored in both mediums.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and preparation\u003c/h2\u003e \u003cp\u003eFresh sheep faeces were collected from the ground post-defecation from a flock of approximately 20 individuals. Immediately after collection, a small amount of water was added to the pooled faeces and thoroughly mixed into homogeny. Faeces from this mix were aliquoted into 48 sterile 10mL tubes and preserved in 10% buffered formalin (n\u0026thinsp;=\u0026thinsp;24) or 70% ethanol (n\u0026thinsp;=\u0026thinsp;24) at a ratio of 1:3 faeces to preservation medium. Samples were thoroughly mixed with preservative media to ensure eggs were preserved. Samples were collected in triplicate for each preservation medium and time point and were stored at ambient temperature out of direct sunlight.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRecovery of strongyle eggs\u003c/h3\u003e\n\u003cp\u003eFaecal samples were assessed in triplicate using microscopy at the following time points: fresh, days one, three, five, seven, ten, 14, 30 and 90. At each time point, faecal samples were centrifuged and washed with water before analysis. Faecal egg counts were obtained through two McMaster methods to compare their performance in recovery of strongyle-type eggs in each preservation medium. The standard modified McMaster method (Whitlock, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1948\u003c/span\u003e) referred to here as McMaster1, was conducted only counting the top focal layer of each chamber, while the novel McMaster2 method used the entire depth of the McMaster slide, counting both the top and bottom focal layers of each chamber (Larkins et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFaeces (2 g) were mixed thoroughly in 30 mL of saturated salt solution (specific gravity (s.g.) 1.2) and strained through a sieve thrice before 0.15 mL of the solution was loaded into each chamber of the McMaster slide. Loaded McMaster slides were left to sit for five minutes to allow the debris to settle before chambers were examined microscopically at 10 x magnification within 20 minutes. The egg per gram (EPG) of faeces was calculated as the number of strongyle-type eggs counted in both chambers multiplied by 50.\u003c/p\u003e\n\u003ch3\u003eImaging of strongyle-type eggs recovered\u003c/h3\u003e\n\u003cp\u003eA zinc sulfate (s.g 1.18) centrifugation floatation method was conducted to concentrate parasite eggs for imaging to assess strongyle-type eggs for signs of deterioration (Zajac and Conboy, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Remaining faeces (~\u0026thinsp;one gram) from the McMaster2 were used for imaging. Samples were screened and imaged using an Olympus BX50 microscope with an Olympus DP71 camera and captured using CellSens Standard 1.13 software. The first five to seven strongyle-type eggs visualised were imaged to prevent selection bias, except for the ethanol stored day 90 samples where only one egg was identified. Images of strongyle-type eggs for each time point were assigned a number and randomly selected for inclusion in morphological feature analysis.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of significant difference between methods and storage medium at each individual time point was assessed using a two-way ANOVA with multiple comparisons, and the false discovery rate was controlled using the Benjamini, Krieger and Yekutieli method (Benjamini et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The q value is an adjusted P value, and represents the tests called as significant that will be false positives. All graphing and statistical analysis were performed using GraphPad Prism version 8 for Windows, GraphPad Software, Boston, Massachusetts USA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eComparison of mediums on recovery of strongyle-type eggs\u003c/h2\u003e \u003cp\u003eFaeces stored in 10% formalin yielded a statistically significant increase in the recovery of strongyle-type eggs across all timepoints and both modified McMaster methods comparative to faeces stored in 70% ethanol (q value of 0.0002 to 0.017) (Fig.\u0026nbsp;1). The mean floatation efficiency for eggs recovered from 10% formalin storage was 68\u0026ndash;70% across all time points, a 4.7 to 8.5-fold increase in recovery when compared with 70% ethanol which had a mean recovery rate of 8\u0026ndash;15%.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison of McMaster1 and McMaster2 on recovery of strongyle-type egg\u003c/h3\u003e\n\u003cp\u003eComparison between recovery rate of strongyle-type eggs between the McMaster1 and McMaster2 methods showed minimal statistical significance. While McMaster2 had a higher recovery of strongyle-type eggs in five of the nine time points for formalin (days 1, 7, 10, 14 and 30) and McMaster2 had a higher recovery rate of strongyle-type eggs in five of the eight time points available for ethanol (days 1, 3, 5, 7 and 14), only samples preserved in ethanol exhibited a statistical difference between McMaster1 and McMaster2, and only at a single time point (day 14, q\u0026thinsp;=\u0026thinsp;0.038). McMaster2 could not be performed at time point 90 days for the ethanol preserved samples, due to the sample becoming too cloudy to view the bottom focal layer.\u003c/p\u003e\n\u003ch3\u003eComparison of recovery of strongyle-type eggs at different timepoints\u003c/h3\u003e\n\u003cp\u003eThe floatation efficiency of samples preserved in formalin was variable, with multiple spikes in recovery rates of strongyle-type eggs evident. The highest recovery rate occurred at day 7 (88%), followed by day 90 (77%) and the lowest recovery on day five (53%). The initial days after formalin fixing appeared to have the lowest recovery rates, compared with the later days, although there seemed to be large variations between timepoints.\u003c/p\u003e \u003cp\u003eThe floatation efficiency of samples preserved in ethanol was low, with a maximum of 30% recovery on day 1 (for McMaster2) and a minimum of 3.8% recovery at days 7 and 14 (McMaster1). The McMaster1 method was consistently below 10% recovery from day 5 through to the end of the experiment at day 90, while the McMaster2 method had a slightly increased recovery with only day 10 and 30 dropping below 10%.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGross morphology of strongyle-type eggs\u003c/h2\u003e \u003cp\u003eSamples imaged at each time point revealed changes of egg morphology in both formalin and ethanol fixed samples (Fig.\u0026nbsp;2). At time point 0, strongyle-type eggs were observed to have a thin, smooth ellipsoidal shaped shell with an embryo in the morula stage of development. Strongyle-type eggs from samples stored in 70% ethanol initially had no change in morphology from days 1\u0026ndash;3, however severe degradation occurred in several areas of the shell, collapsing into the morula at day 10. Throughout the remaining timepoints, days 14\u0026ndash;90, the shell continued to thin and lose its smooth ellipsoidal shape, becoming difficult to recognise. Strongyle-type eggs from samples stored in 10% formalin displayed a change in the shell after 24 hours, with wrinkles appearing to form. After 10 days the smooth ellipsoidal shape started to become irregular, with the shell starting to collapse inwards by day 14, however at day 90, the shell was less collapsed, although still not uniform.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe recovery of strongyle-type eggs was up to 8.5 times higher in faeces preserved in 10% buffered formalin, compared with those preserved in 70% ethanol, using the modified McMaster quantification method. In addition to superior recovery, the morphological integrity of strongyle-type eggs was better maintained in formalin preserved samples. These findings demonstrate that 10% buffered formalin is a better medium for the preservation of faeces containing strongyle-type eggs than ethanol.\u003c/p\u003e \u003cp\u003eOur findings align with earlier research reporting formalin\u0026rsquo;s superior performance over ethanol as a fixative for strongyle-type eggs (Foreyt, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1986\u003c/span\u003e),. Although formalin has long been considered the preferred preservation media for parasitological diagnostics (Nace et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Pietrzak-Johnston et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Bonita and Taira, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Carroll et al., 1983), ethanol is still considered an option by some (Jagła et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Hass et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A study investigating preservation media on horse faecal samples suggested 40% ethanol performed better than 4% formalin, however it was noted that the samples were only immersed in the preservation media, not homogenised (Jagła et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This lack of homogenisation would limit the media preserving eggs inside the sample, likely affecting the outcome. This in conjunction with relatively low concentration of media used (40% ethanol) limits the ability for direct comparison to results obtained here. More recently a publication has proposed ethanol as a universal preservation medium for both morphological and molecular analyses of monkey faecal samples (Hass et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Interestingly, the authors noted that parasite eggs/larvae were actually better preserved in formalin than ethanol. They had also ascertained egg recovery through a sedimentation technique rather than floatation, again limiting direct comparison for egg recovery. Despite the lack of direct comparison with ethanol focussed papers, the results presented in this paper, together with previous evidence, indicate that ethanol is still unsuitable for floatation-based microscopy due to reduced recovery and compromised egg morphology (Naem et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Foreyt, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). These data reinforce that there is no \u0026ldquo;one size fits all\u0026rdquo; storage solution; formalin remains optimal for morphological examination, while ethanol may be preferable when downstream molecular analysis is required.\u003c/p\u003e \u003cp\u003eReliable egg recovery and quantification are essential for monitoring soil transmitted helminth (STH) burden and evaluating intervention strategies (Lim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Methods such as Kato Katz provide quantitative data through egg per gram counts but fresh faecal samples are required and analysis must be done in 30 minutes to detect hookworm eggs (strongyle-type eggs), The formalin-ethyl acetate concentration technique (FECT) can be performed on formalin fixed samples but provides qualitative data only. The modified McMaster technique as displayed here can be performed on formalin fixed samples and provides quantitative data. All these methods are low cost and practical for use in endemic regions where infrastructure and funding are often limited, but the ability to preserve samples for later analysis is an important consideration for large scale surveys. (Bradbury et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Larkins et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe modified McMaster technique, commonly used in livestock diagnostics for quantifying strongyle type eggs, is a low resource option recommended by the World Health Organisation for the quantification of parasite eggs for human diagnostics (2019, WHO, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This method typically counts eggs that float to the top of the chamber, excluding those that may not float, such as infertile eggs or those affected by the preservation medium (Whitlock, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1948\u003c/span\u003e). To address this, the McMaster2 method was developed during a large-scale STH survey to improve quantification accuracy by counting eggs in both the top and bottom focal layer (Larkins et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe morphological integrity of the eggs was affected by both preservation mediums. In ethanol-preserved samples, the outer shell of the strongyle-type eggs exhibited evident desiccation and structural damage within 24 hours of exposure. Such degradation compromised the characteristic smooth, ellipsoidal shell which is a significant identifying feature of strongyle-type eggs. When these defining features are lost, accurate differentiation from faecal debris and artefacts within the floatation preparation becomes increasingly difficult. Consequently, severely degraded eggs may no longer be recognisable as strongyle-type, leading to potential underestimation of egg counts or under-diagnosis of infection in faecal microscopy. Formalin fixed eggs showed some degradation, predominantly wrinkling of the shell, but maintained their defining features of the thin ellipsoidal shell with distinct morula.\u003c/p\u003e \u003cp\u003eThis study provided the first direct controlled comparison of the modified McMaster1 and McMaster2 quantification methods on preserved faecal samples. Although there was a lack of statistical differences between the modified McMaster1 and McMaster2 methods, there was a slight difference with McMaster2 often recovering more strongyle type eggs. While it is well established that formalin is reactive towards protein and forms inter-molecular cross linkages (Vince et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) which increases the molecular weight, the strongyle-type eggs weren\u0026rsquo;t observed settling on the bottom focal layer when preserved in 10% formalin, but perhaps instead sitting somewhere between the top and bottom focal layers. There was a single timepoint where there was a statistical difference between McMaster and McMaster2 for the ethanol preserved samples, with McMaster2 being more successful for egg recovery. This weight change causing the eggs to settle on the bottom focal layer may have occurred as the ethanol altered the egg shell, causing the egg to become brittle and allowing the salt flotation solution to seep inside (Marquina et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Together these observations that preservation-induced changes to egg density and structural integrity may subtly influence their distribution within the floatation medium, thereby affecting recovery efficiency depending on the McMaster quantification approach used.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study determined that 10% buffered formalin performs better than 70% ethanol for preserving strongyle-type eggs for floatation-based quantification. Egg recovery was up to 8.5 times higher in formalin-preserved samples compared with ethanol, and morphological features essential for identification were better maintained. These findings confirm that formalin remains the preferred fixative for microscopy-based diagnostics, whereas ethanol is unsuitable for egg quantification due to reduced recovery and structural degradation. This study also represents the first direct, controlled comparison of the modified McMaster and McMaster2 techniques on preserved faecal samples. While no statistical differences were detected, McMaster2 consistently recovered slightly more eggs, suggesting a potential advantage in larger sample sizes. Together, these results reinforce the continued relevance of formalin in reliable, microscopy-based monitoring of soil-transmitted helminth infections, particularly in low-resource settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eFinancial support\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency, commercial or not-for-profit sectors.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare there are no conflicts of interest.\u003c/p\u003e \u003ch2\u003eEthical standards\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributionsBK and AA conceived and designed the study. BK conducted data collection, BK and AL performed statistical analysis. BK and AA wrote the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBench aids for the diagnosis of intestinal parasites second edition. \u003cem\u003eIn\u003c/em\u003e: MONTRESOR, A. (ed.). Geneve: World Health Organisation\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eASH A, OKELLO A, KHAMLOME B, INTHAVONG P, ALLEN J, THOMPSON RCA (2017) Controlling Taenia solium and soil transmitted helminths in a northern Lao PDR village: Impact of a triple dose albendazole regime. Acta Trop 174:171\u0026ndash;178\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBENJAMINI Y, KRIEGER AM, YEKUTIELI D (2006) Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93:491\u0026ndash;507\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBONITA R, TAIRA N (1996) Faecal examination of Fasciola eggs fixed with formalin solution using the beads technique. Vet Parasitol 67:269\u0026ndash;273\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBRADBURY RS, POTTERS SAPPSGH, FREAN IMATHISONBA, MEWARA J, SHEOREY A, TAMAROZZI H, CHIODINI FCOUTURIERMR, P., PRITT B (2022) Where Have All the Diagnostic Morphological Parasitologists Gone? J Clin Microbiol 60:e0098622\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCARROLL MJ, COOK, J., TURNER JA (1983) Comparison of polyvinyl alcohol- and formalin-preserved fecal specimens in the formalin-ether sedimentation technique for parasitological examination. J Clin Microbiol 18:1070\u0026ndash;1072\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHEN J, GONG Y, CHEN Q, LI S, ZHOU Y (2024) Global burden of soil-transmitted helminth infections, 1990\u0026ndash;2021. Infect Dis Poverty 13:77\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFOREYT WJ (1986) Recovery of nematode eggs and larvae in deer: evaluation of fecal preservation methods. J Am Vet Med Assoc 189:1065\u0026ndash;1067\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGARCIA LS, BREWER TC, BRUCKNER DA (1979) A comparison of the formalin-ether concentration and trichrome-stained smear methods for the recovery and identification of intestinal protozoa. Am J Med Technol 45:932\u0026ndash;935\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGARCIA LS, SHIMIZU RY (1998) Evaluation of intestinal protozoan morphology in human fecal specimens preserved in EcoFix: comparison of Wheatley's trichrome stain and EcoStain. J Clin Microbiol 36:1974\u0026ndash;1976\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHASS JK, HENRIQUEZ MC, HAMOU CHURCHERJ, H., MORALES, S. R., MELIN AD (2024) Assessing morphological preservation of gastrointestinal parasites from fecal samples of wild capuchin monkeys (Cebus imitator) stored in ethanol versus formalin. Sci Rep 14:3623\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJAGŁA E, ŚPIEWAK J, ZALEŚNY G, POPIOŁEK M (2013) Effect of Storage and Preservation of Horse Faecal Samples on the Detectability and Viability of Strongylid Nematode Eggs and Larvae. Bulletin- Veterinary Inst Pulawy 57:161\u0026ndash;165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKATTULA D, RAO SARKARR, AJJAMPUR SS, MINZ S, LEVECKE B, MULIYIL, J., KANG G (2014) Prevalence \u0026amp; risk factors for soil transmitted helminth infection among school children in south India. Indian J Med Res 139:76\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLARKINS A, KEOKHAMPHAVANH B, TAGGART KNIGHTB, INSISIENGMAY KKEATLEYS, B., ASH A (2025) Diagnostic sensitivity of formalin-fixed faecal microscopy for the detection of soil-transmitted helminths. Trans R Soc Trop Med Hyg 119:596\u0026ndash;605\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIM MD, BELIZARIO BROOKERSJ, GAY-ANDRIEU VYJR, LEVECKE FGILLEARDJ, VAN B, LIESHOUT L, MEDLEY GF, MEKONNEN Z, MIRAMS G, NJENGA SM, ODIERE MR, STUYVER RUDGEJW, VLAMINCK LVERCRUYSSEJ, J., WALSON JL (2018) Diagnostic tools for soil-transmitted helminths control and elimination programs: A pathway for diagnostic product development. PLoS Negl Trop Dis 12:e0006213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMARQUINA D, BUCZEK M, RONQUIST F, ŁUKASIK P (2021) The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies. PeerJ 9:e10799\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNACE EK, STEURER FJ, EBERHARD ML (1999) Evaluation of Streck tissue fixative, a nonformalin fixative for preservation of stool samples and subsequent parasitologic examination. J Clin Microbiol 37:4113\u0026ndash;4119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNAEM S, PAGAN C, NADLER SA (2010) Structural Restoration of Nematodes and Acanthocephalans Fixed in High Percentage Alcohol Using Dess Solution and Rehydration. 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Am J Pathol 161:1961\u0026ndash;1971\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSTENSVOLD CR, NIELSEN HV (2012) Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. J Clin Microbiol 50:540\u0026ndash;541\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTHAVARAJAH R, MUDIMBAIMANNAR, V. K., ELIZABETH, J., RAO, U. K., RANGANATHAN K (2012) Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol 16:400\u0026ndash;405\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVINCE DG, TBAKHI A, GADDIPATI A, COTHREN RM, CORNHILL, J. F., TUBBS RR (1997) Quantitative Comparison of Immunohistochemical Staining Intensity in Tissues Fixed in Formalin and Histochoice. Anal Cell Pathol 15:607965\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHITLOCK H (1948) Some modifications of the McMaster helminth egg-counting technique and apparatus. J Council Sci Industrial Res Australia 21:177\u0026ndash;180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO (2019) Bench aids for the diagnosis of intestinal parasites, 2nd edn. World Health Organization;, Geneva\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZAJAC AM, CONBOY GA (2006) \u003cem\u003eVeterinary Clinical Parasitology\u003c/em\u003e Iowa. Blackwell Publishing\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"coproscopy, strongyle-type eggs, faecal preservation, formalin vs ethanol, McMaster","lastPublishedDoi":"10.21203/rs.3.rs-9433494/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9433494/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastrointestinal parasites (GI), including soil-transmitted helminths (STHs), continue to impose a considerable health burden in endemic regions, particularly in low- and middle-income countries. Reliable and cost-effective diagnostic tools are essential for parasite surveillance and control, yet preservation methods can influence diagnostic accuracy. Coproscopy remains the gold standard for morphological identification of GI parasites, however time constraints often require samples to be preserved for later analysis. Faecal samples are conventionally stored in 10% buffered formalin for microscopy, and a counterpart stored in ethanol if molecular analysis is required, however recent studies have suggested that ethanol may serve as a suitable preservative for both.\u003c/p\u003e \u003cp\u003eThis controlled study evaluated the suitability of 10% buffered formalin and 70% ethanol for preserving strongyle-type eggs over a three-month period and compared parasite recovery between two McMaster quantification techniques: the modified McMaster and McMaster 2 methods. Recovery of strongyle- type eggs was 4.7 to 8.5-fold higher in formalin-preserved samples than in ethanol-preserved counterparts. In addition, the morphological integrity of eggs was better maintained in formalin, with reduced damage and clearer visualisation of diagnostic features.\u003c/p\u003e \u003cp\u003eThese findings demonstrate that 10% buffered formalin performs better than 70% ethanol for both structural integrity and recovery of strongyle-type eggs for coproscopic quantification and identification. The McMaster2 method also yielded a higher number of strongyle-type eggs than the modified McMaster method, indicating slight improved quantification sensitivity.\u003c/p\u003e","manuscriptTitle":"Formalin performs better than ethanol for recovery of strongyle-type eggs using established and novel McMaster techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 15:29:04","doi":"10.21203/rs.3.rs-9433494/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-26T22:02:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-24T13:48:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-24T13:11:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2026-04-16T05:24:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"63ec4da7-a5af-486c-b8f2-14fbb5862fe3","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T15:29:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 15:29:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9433494","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9433494","identity":"rs-9433494","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}