Method For Viability Assessment Research Articles

An Overview of the Current State of Cell Viability Assessment Methods Using OECD Classification.

Over the past century, numerous methods for assessing cell viability have been developed, and there are many different ways to categorize these methods accordingly. We have chosen to use the Organisation for Economic Co-operation and Development (OECD) classification due to its regulatory importance. The OECD categorizes these methods into four groups: non-invasive cell structure damage, invasive cell structure damage, cell growth, and cellular metabolism. Despite the variety of cell viability methods available, they can all be categorized within these four groups, except for two novel methods based on the cell membrane potential, which we added to the list. Each method operates on different principles and has its own advantages and disadvantages, making it essential for researchers to choose the method that best fits their experimental design. This review aims to assist researchers in making this decision by describing these methods regarding their potential use and providing direct references to the cell viability assessment methods. Additionally, we use the OECD classification to facilitate potential regulatory use and to highlight the need for adding a new category to their list.

Standardized Protocol for Resazurin-Based Viability Assays on A549 Cell Line for Improving Cytotoxicity Data Reliability.

The A549 cell line has become a cornerstone in biomedical research, particularly in cancer studies and serves as a critical tool in cytotoxicity studies and drug screening where it is used to evaluate the impact of pharmaceutical compounds on cellular viability. One of the most widely adopted methods for viability assessment, which is also used in evaluating drug cytotoxicity, is the resazurin-based assay. This assay exploits the ability of living cells to convert resazurin into fluorescent resorufin, providing a reliable indicator of metabolic activity. By measuring this conversion, cell viability can be estimated. Resazurin assay is extensively used for evaluating cytotoxic effects on various cell lines, including A549 cells, thereby bridging the gap between in vitro experimentation and drug development. However, frequent data inconsistencies in pre-clinical drug screening highlight the critical need for standardization to ensure reliability and reproducibility. This manuscript addresses these challenges by describing the optimization of resazurin-based viability assays for A549 cells in both 2D cultures and 3D fibrin gel models. By optimizing this test, the study aims to enhance the reliability of cytotoxicity results and introduces a new standard operating procedure, thus providing consistent results with minimal measurement uncertainty. This standardization is crucial for advancing drug screening and ensuring robust research findings.

366.3: Integrated organ viability assessment method for hypothermic and subnormothermic machine perfusion for liver transplantation.

366.3: Integrated organ viability assessment method for hypothermic and subnormothermic machine perfusion for liver transplantation.

Methods for assessing the viability of cells cultured in vitro in 2D and 3D structures

Over the past decades, cell viability tests have been an essential research tool in cell biology, tissue engineering, and regenerative medicine. Assessment of cell viability is mandatory in the production and quality control of cell products for biomedical applications.
 Methods of viability assessment can be broadly classified according to the underlying mechanism and how the results obtained are evaluated. This article presents variants of the most commonly used tests and protocols for assessing cell viability. Their advantages and disadvantages are presented, which should be considered when planning experiments, e.g., when developing cell preparations for regenerative medicine.
 The authors point out the main factors influencing the choice of viability assessment method: efficiency, speed, safety, reproducibility, sample integrity, compatibility with biomaterial, and cell line type. Finally, the authors discuss separately cell viability tests that can be applied not only to 2D cell structures but also to 3D cell structures, which have recently become widespread due to more accurate modeling of biological processes.

The influence of pre-treatment on germination of three species of Catasetum (Orchidaceae)

Abstract: Asymbiotic germination techniques are successful in species of the Catasetum genus. The diverse composition of the culture media can be critical; however, viable seeds are required for asymbiotic germination. The reliable method for viability assessment is the tetrazolium test. A decisive step in the efficiency of the tetrazolium test is the seeds preconditioning with sucrose solution, which has the potential to improve germination. Therefore, this work aimed to evaluate the seed germination of three species of the genus Catasetum in culture media, preconditioned or not with sucrose solution. Seeds of the species Catasetum osculatum, Ctsm. galeritum and Ctsm. complanatum were assessed by tetrazolium test and germinated in two different media, pretreated or not with 10% sucrose solution. The statistical analysis showed that the use of sucrose pretreatment significantly increased the germination rates of the species. We conclude that the pretreatment with 10% sucrose for 24 hours, regardless of the species and the culture medium, significantly increases the germination of Ctsm. complanatum, Ctsm. galeritum and Ctsm. osculatum seeds.

The Algorithm for Using Transit-time Flow Measurement and High-resolution Epicardial Ultrasound for Intraoperative Grafts Assessment during Coronary Artery Bypass Surgery.

Aim To develop an algorithm for using ultrasonic flowmetry (USF) and epicardial ultrasonic scanning (EpiUSS) for intraoperative assessment of anatomic and functional viability of conduits.Material and methods For viability assessment of 460 coronary grafts in 150 patients who were operated at the Bakulev National Medical Research Center for Cardiovascular Surgery (2018-2021 г.), markers of graft failure were analyzed using the USF and EpiUSS data confirmed by results of graft angiography. According to RОС analysis, the Qmean and PI values indicative of the graft failure were determined. A CHAID decision tree was developed for assessing the prognostic significance of the analyzed parameters. Based on this prognostic model, an algorithm was developed for intraoperative diagnosis of anatomic and functional graft viability during coronary bypass surgery.Results The Qmean ≤20.5 ml/min values were associated with an increased relative risk (RR) of detecting graft failure (RR, 8.2; 95 % confidence interval, CI, 4.4-15.2). The developed model shows a high accuracy of predicting the graft failure (AUC = 0.906±0.03). The RR of graft failure at PI ≥2.65 was 3.3 (95 % CI, 2.17-5.08). The prognostic model for PI (AUC = 0.745±0.042) was sufficiently accurate with respect of possible graft failure. Nodes of high and low risk for graft failure were determined in the developed decision tree. The obtained model was characterized by high sensitivity and specificity (100 and 84.3 %, respectively).Conclusion The combined use of USF and EpiUSS allows a highly accurate assessment of both morphological and functional characteristics of graft flow. The developed algorithm for the intraoperative diagnosis of anatomic and functional graft viability can be recommended for clinical use.

Flow cytometry-based viability staining: an at-line tool for bioprocess monitoring of Sulfolobus acidocaldarius

Determination of the viability, ratio of dead and live cell populations, of Sulfolobus acidocaldarius is still being done by tedious and material-intensive plating assays that can only provide time-lagged results. Although S. acidocaldarius, an extremophilic Archaeon thriving at 75 °C and pH 3.0, and related species harbor great potential for the exploitation as production hosts and biocatalysts in biotechnological applications, no industrial processes have been established yet. One hindrance is that during development and scaling of industrial bioprocesses timely monitoring of the impact of process parameters on the cultivated organism is crucial—a task that cannot be fulfilled by traditional plating assays. As alternative, flow cytometry (FCM) promises a fast and reliable method for viability assessment via the use of fluorescent dyes. In this study, commercially available fluorescent dyes applicable in S. acidocaldarius were identified. The dyes, fluorescein diacetate and concanavalin A conjugated with rhodamine, were discovered to be suitable for viability determination via FCM. For showing the applicability of the developed at-line tool for bioprocess monitoring, a chemostat cultivation on a defined growth medium at 75 °C, pH 3.0 was conducted. Over the timeframe of 800 h, this developed FCM method was compared to the plating assay by monitoring the change in viability upon controlled pH shifts. Both methods detected an impact on the viability at pH values of 2.0 and 1.5 when compared to pH 3.0. A logarithmic relationship between the viability observed via plating assay and via FCM was observed.

Assessing Viability and Stress Tolerance of Probiotics-A Review.

The interest in probiotics has increased rapidly the latest years together with the global market for probiotic products. Consequently, establishing reliable microbiological methods for assuring the presence of a certain number of viable microorganisms in probiotic products has become increasingly important. To assure adequate numbers of viable cells, authorities are enquiring for information on viability rates within a certain shelf-life in colony forming units (CFU). This information is obtained from plate count enumeration, a method that enables detection of bacterial cells based on their ability to replicate. Although performing plate count enumeration is one manner of assessing viability, cells can still be viable without possessing the ability to replicate. Thus, to properly assess probiotic viability, further analysis of a broader group of characteristics using several types of methods is proposed. In addition to viability, it is crucial to identify how well the cells in a probiotic product can survive in the gastrointestinal tract (GIT) and thus be able to mediate the desired health benefit while passing through the human body. A broad spectrum of different assay designs for assessing probiotic gastric tolerance have been used in research and quality control. However, the absence of any consensus on how to assess these qualities makes it difficult to compare between laboratories and to translate the results into in vivo tolerance. This review presents and discusses the complexity of assuring that a probiotic is suitable for beneficial consumption. It summarizes the information that can be subtracted from the currently available methods for assessment of viability and stress tolerance of a probiotic, hereby altogether defined as “activity.” Strengths and limitations of the different methods are presented together with favorable method combinations. Finally, the importance of choosing a set of analyses that reveals the necessary aspects of probiotic activity for a certain product or application is emphasized.

Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores.

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

The Correlation of Chronological Age and Micro Ribonucleic Acid-135b Expression in Spent Culture Media of In vitro Fertilisation Patient.

ABSTRACTBackground:The lack of accuracy in embryo viability assessment methods still remains a challenge to increase the in vitro fertilisation (IVF) success rate. The chronological age and micro ribonucleic acid (RNA)-135b influence the quality of the embryo since microRNA-135b expresses stably in the spent culture media. Therefore, microRNA-135b has the potential to become a non-invasive biomarker of IVF embryo quality.Aims:(1) The aim of this study is to determine the chronological age and microRNA-135b expression distribution of IVF patients. (2) to determine the correlation between chronological age and microRNA-135b expression in spent culture media of IVF patients.Study Setting and Design:An observational study was conducted in Yasmin IVF clinic.Materials and Methods:The chronological age data were collected from the medical records and 31 spent culture media samples from 11 IVF patients were taken on day 5 of embryo culture. We also collected the basal media sample as the control group. The microRNA-135b expression was analysed using quantitative real-time polymerase chain reaction (qPCR) analysis.Statistical Analysis Used:The data analysis was performed using IBM SPSS Statistics 25.Results:The chronological age and microRNA-135b expression were distributed abnormally. There was a significant positive correlation with moderate statistical power between chronological age and microRNA-135b expression in spent culture media. MicroRNA-135b expression increased 4.9-fold in spent culture media than basal media of IVF.Conclusions:The increase of chronological age is followed by the rise of microRNA-135b expression in spent culture media of IVF patients. The microRNA-135b is a potential biomarker to predict IVF embryo quality.

Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts.

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

FDG-PET/CT: novel method for viability assessment of livers perfused ex vivo.

Ex vivo liver machine perfusion is a promising option to rescue marginal liver grafts mitigating the donated organ shortage. Recently, a novel liver perfusion machine that can keep injured liver grafts alive for 1 week ex vivo was developed and reported in Nature Biotechnology. However, liver viability assessment ex vivo is an unsolved issue and the value of 18F-fluorodeoxyglucose (FDG)-PET/CT for such purpose was explored. Discarded two human and six porcine liver grafts underwent FDG-PET/CT for viability assessment after 1 week of ex vivo perfusion. PET parameters [standardized uptake value (SUV)max, SUVmean, SUVpeak and total lesion glycolysis] were compared between hepatic lobes and between porcine and human livers. The prevalence of FDG-negative organ parts was recorded. The estimated effective radiation dose for PET/CT was calculated. All organs were viable with essentially homogeneous FDG uptake. Of note, viability was preserved in contact areas disclosing the absence of pressure necrosis. Four porcine and two human organs had small superficial FDG-negative areas confirmed as biopsy sites. Total lesion glycolysis was significantly higher in the right hepatic lobe (P = 0.012), while there was no significant difference of SUVmax, SUVmean and SUVpeak between hepatic lobes. There was no significant difference in FDG uptake parameters between porcine and human organs. The estimated effective radiation dose was 1.99 ± 1.67 mSv per organ. This study demonstrates the feasibility of FDG-PET/CT for viability assessment of ex vivo perfused liver grafts after 1 week.

Evaluation of the vital viability and their application in fungal spores’ disinfection with flow cytometry

More attention was focused on fungi contamination in drinking water. Most researches about the inactivation of fungal spores has been conducted on disinfection efficiency and the leakage of intracellular substances. However, the specific structural damage of fungal spores treated by different disinfectants is poorly studied. In this study, the viability assessment methods of esterase activities and intracellular reactive oxygen species (ROS) were optimized, and the effects of chlorine-based disinfectants on fungal spores were evaluated by flow cytometry (FCM) and plating. The optimal staining conditions for esterase activity detection were as follows: fungal spores (106 cells/mL) were stained with 10 μM carboxyfluorescein diacetate and 50 mM ethylene diamine tetraacetic acid at 33 °C for 10 min (in dark). The optimal staining conditions for intracellular ROS detection were as follows: dihydroethidium (the final concentration of 2 μg/mL) was added into fungal suspensions (106 cells/mL), and then samples were incubated at 35 °C for 20 min (in dark). The cell culturability, membrane integrity, esterase activities, and intracellular ROS were examined to reveal the structural damage of fungal spores and underlying inactivation mechanisms. Disinfectants would cause the loss of the cell viability via five main steps: altered the morphology of fungal spores; increased the intracellular ROS levels; decreased the culturability, esterase activities and membrane integrity, thus leading to the irreversible death. It is appropriate to assess the effects of disinfectants on fungal spores and investigate their inactivation mechanisms using FCM.

Heat demand mapping and district heating assessment in data-pour areas

Buildings represent 40% of the European Union's energy consumption and 36% of its greenhouse gas emissions making it obvious that the decarbonisation of the European Union depends on the sustainable provision of heat in its cities. District heating presents it self as a clear solution to this issue. It is capable of supplying waste and renewable heat from where it is available to where it is needed and can provide a powerful driver for the integration of renewables in the electrical system through the flexibility that power to heat technologies can provide. Due to the inability of long-distance transport of heat, spatial planning and GIS mapping has proven to be a very important tool in heat planning. This usually requires a lot of highly detailed data which is often not available. The research presented in this paper is tackling this issue trough a heat demand mapping and district heating viability assessment method using mostly public databases. The developed method consists of three key steps: assessment of the aggregated heating demand, bottom up mapping used for validation and top down mapping of the entire observed area. The result of the mapping is used in the assessment of the district heating potential based on the difference between the price and levelized cost of heat as well as the assumed cost of the distribution infrastructure. The method has been implemented on the case of Croatia showing a significant potential for the economic utilization of district heating.

A Cell Viability Evaluation Method Based on Respiratory Thermodynamic Feature Detected by Microscopic Infrared Thermal Imaging Sensor

Cell viability is an important indicator while screening drugs. An inaccurate evaluation of cell viability can cause large errors in anti-tumor dose experiments, and this becomes very unfavorable for cancer treatment. Generally, cell viability refers to the ratio of the number of live cells to the total number of cells. However, this evaluation method does not consider the effects of differences in metabolic abilities between different living cells. In this paper, there is a new cell viability evaluation method based on respiratory thermodynamic feature that includes the respiratory intensity, proliferation rate, and heat released by cells. These three parameters can be directly measured by a microscopic infrared thermal imaging sensor, which is fast and non-invasive and does not require consumables. The three parameters were simultaneously measured based on a micro-infrared thermal imaging sensor and fitted to the mathematical model. Finally, the method was verified by comparing it with traditional c ounting method and comparing the amount of omethoate with traditional counting method. The results indicated that omethoate is 12.36% lower than in the traditional counting method with the same level of complete cell inactivation. Therefore, this method is more accurate than the conventional cell viability assessment method, and the dosage is more precise when the uniform effect is achieved, which provides a basis for precise doses in tumor treatment and can reduce side effects in the human body. This method has a significant effect on the manufacture of cell activity detecting sensors. <fig orientation="portrait" position="float" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <graphic orientation="portrait" position="float" xlink:href="yang7-2946033.eps"/> </fig>

Use of standard test organisms for sound validation of UV-based ballast water treatment systems

To challenge ballast water treatment system (BWTS) efficacy for organisms in the size-class 10–50 μm, intake concentration during tests must reach certain minimum requirements. Often, natural concentrations are too low to meet intake requirements and standard test organisms (STOs) are added. We tested the robustness of Tetraselmis suecica and Odontella sp. to a range of UV-treatments to explore fluences needed to meet the IMO discharge standard (<10 org. ml−1) evaluated using two viability assessment methods. To meet discharge standards, fluences of >1000 mJ cm−2 were required using vital stain whereas 135–500 mJ cm−2 were needed using regrowth assays. Besides, results suggest that T. suecica and Odontella sp. were at least as robust as natural algae towards UV-treatments. We suggest the advantageous use of these species as STOs in test water to support intake water requirements and to obtain more conservative validation of UV-based BWTS to ensure more environmental protective procedures.

Development of a novel automated analytical method for viability assessment of phytoplankton used for validation of ballast water treatment systems

To limit the spreading of aquatic invasive species, regulations require ships’ ballast water to be treated before discharge. To validate ballast water treatment system (BWTS) performance, treated water is analyzed for living organisms in different size classes. Quantitative assessment of the size class 10–50 μm (mainly phytoplankton) is carried out using the vital stain method, which requires labor-intensive manual microscope counts of fluorescent (i.e., living) cells. The method is slow, demands specialized personnel, and is challenged by subjectivity and mobile organisms. Using a high-content screening platform (HCS-Platform) and image analysis, we developed an automated, objective and faster quantification method. The automated method neutralized subjectivity by using fixed cell recognition parameters for image analysis. The implementation of membrane filters gently manipulated the organisms into a 2D plane that reduced mobility. Quantifications were performed at different concentrations using monocultures of slow-moving Rhodomonas salina, highly mobile Tetraselmis suecica and natural algae. Results were compared to the standard manual counting procedure. Automated counts of monocultures were comparable to manual counts at low and medium concentration levels. Manual counts of T. suecica at high concentration levels were significantly lower compared to automated counts stressing the challenge to count mobile cells in 3D. Natural algal counts were similar for both counting approaches, but accuracy was challenged by colony forming species and high number of algal species ~ 10 μm. Automated counts were significantly faster than manual counts. In conclusion, the HCS-Platform showed promising results as an alternative quantitative phytoplankton assessment method for BWTS validation.

Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold-starvation using propidium monoazide real-time polymerase chain reaction.

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions.

Evaluation of Acrosomal Integrity and Viability in Bull Spermatozoa: Comparison of Cytochemical and Fluorescent Techniques

This experiment was aimed to compare two well known methods for assessment of acrosomal integrity and another two methods for assessment of viability in bull spermatozoa during cryopreservation. For this purpose, ejaculates were collected from four Hariana bulls using artificial vagina at biweekly interval. The semen samples which possesses more than 70% progressive motility and above 500 million/ml spermatozoa concentration was subsequently subjected to processing for liquid nitrogen (LN2) vapour freezing. Semen samples were extended in Tris-egg yolk-glycerol extender and further subjected to two staining methods, cytochemical by Giemsa staining and fluorescent method by fluorescein isothiocyanate - pisum sativum agglutinin (FITC-PSA) at two stages i.e. pre freeze and post thaw. In the same way to assess the viability eosin-nigrosin staining is compared with fluorescent method i.e. Annexin-V/PI method. Results showed that fluorescent staining were more sensitive as compare to cytochemical method for spermatozoa of Hariana bulls.

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro.

In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.