Comprehensive Tools for Culturing Blastocystis: A Standardized Resource for Research and Diagnostics

Abstract

Blastocystis spp. is a widely prevalent anaerobic protozoan of uncertain pathogenicity found in the gastrointestinal tracts of over 1 billion people worldwide. Despite its potential significance in health and disease, Blastocystis spp. remains challenging to culture axenically due to its anaerobic nature and the diversity of its genetic subtypes. This manuscript presents a standardized toolkit for culturing Blastocystis spp. in xenic and axenic conditions, detailing protocols for the preparation of appropriate liquid and solid media, cryopreservation, and inoculation. By providing a comprehensive set of tools and methodologies, this work aims to streamline research on Blastocystis spp., enabling reproducibility, subtype characterization, and advancements in understanding its role in the gut microbiome and host health. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Establishment of xenic Blastocystis culture from stool samples in liquid medium

Basic Protocol 2: Subculturing from existing Blastocystis liquid culture

Support Protocol 1: Preparation of modified Jones’ medium for Blastocystis culturing

Support Protocol 2: Preparation of trypticase‐yeast extract‐serum‐gastric mucin‐9 (TSGYM‐9) medium for Blastocystis culturing

Support Protocol 3: Preparation of liver extract‐yeast extract‐serum‐gastric mucin (LYSGM) medium for Blastocystis culturing

Basic Protocol 3: Subculturing xenic Blastocystis cultures in diphasic medium

Support Protocol 4: Preparation of Robinson's medium for Blastocystis culturing

Support Protocol 5: Preparation of Ringer's agar slants for Blastocystis culturing

Basic Protocol 4: Axenization of Blastocystis cultures

Basic Protocol 5: Subculturing axenic Blastocystis cultures in diphasic medium

Support Protocol 6: Preparation of Boeck and Drbohlav's Locke‐egg serum (LES) medium for Blastocystis culturing

Support Protocol 7: Preparation of Boeck and Drbohlav's diphasic modified medium (BDMM) for Blastocystis culturing

Basic Protocol 6: Establishment of axenic Blastocystis cultures in Iscove's modified Dulbecco's medium (IMDM)

Basic Protocol 7: Establishment of axenic Blastocystis cultures in soft IMDM agar

Basic Protocol 8: Establishment of axenic Blastocystis cultures on solid IMDM agar

Basic Protocol 9: Optimized method for establishing axenic Blastocystis cultures on solid IMDM agar

Basic Protocol 10: Establishment of axenic Blastocystis cultures in semi‐solid Locke's agar

Basic Protocol 11: Cryopreservation of xenic Blastocystis cultures

Basic Protocol 12: Cryopreservation of axenic Blastocystis cultures

Basic Protocol 13: Inoculation of liquid medium with xenic Blastocystis cultures from frozen stocks

Basic Protocol 14: Inoculation of liquid medium with axenic Blastocystis cultures from frozen stocks

Introduction

Blastocystis spp. is an anaerobic gastrointestinal protozoan of considerable interest due to its widespread prevalence and controversial role in health and disease. It is estimated to inhabit the guts of over 1 billion individuals globally (Scanlan & Stensvold, 2013), spanning both asymptomatic carriers and individuals with gastrointestinal disturbances, such as diarrhea and abdominal pain. The duality of its presence in healthy and symptomatic individuals continues to fuel the debate about whether Blastocystis spp. acts as a commensal organism or a pathogen.

The diversity of its subtypes (STs) further underscores the complexity of Blastocystis spp. To date, at least 44 STs have been identified in mammalian and avian hosts (Šejnohová et al., 2024), based on gene polymorphisms in the small subunit ribosomal RNA gene (SSU rDNA; Scicluna et al., 2006), with ST1‐ST4 being most commonly found in humans. This heterogeneity highlights the importance of ST‐specific studies, particularly in understanding transmission pathways, ecological niches, and host‐specific interactions.

Culturing Blastocystis spp. for research purposes has historically been challenging because of its anaerobic growth requirements and dependence on coexisting microorganisms for xenic cultivation. Although a limited number of STs have been axenized successfully, maintaining viable cultures often necessitates xenic conditions, in which other microbes play a critical role in oxygen consumption and nutrient provision. Overcoming these barriers with standardized and reproducible culturing techniques is essential for advancing research into Blastocystis spp. biology, its interactions with the gut microbiome, and its potential impact on host health.

This article consolidates the current knowledge of and methods for culturing Blastocystis spp., offering a comprehensive guide to both xenic and axenic cultivation techniques (definitions of some key terms used in this article are provided in Table 1). By detailing protocols for the preparation of liquid and solid media, growth monitoring, and cryopreservation (see Fig. 1), we aim to equip the scientific community with the tools necessary for consistent and successful initiation and maintenance of Blastocystis spp. cultures. The article consists of eight main parts: medium preparation and methods for xenic cultures in liquid media (Basic Protocols 1 and 2 and Support Protocols 1, 2, and 3); medium preparation and methods for xenic cultures in diphasic media (Basic Protocol 3 and Support Protocols 4 and 5); axenization (Basic Protocol 4); medium preparation and methods for axenic cultures in diphasic media (Basic Protocol 5 and Support Protocols 6 and 7); medium preparation and culturing methods for axenic cultures in liquid media (Basic Protocol 6); medium preparation and methods for axenic cultures in semi‐solid and solid media (Basic Protocols 7‐9); cryopreservation (Basic Protocols 10 and 11); and establishment of xenic and axenic cultures from frozen stocks (Basic Protocols 12 and 13). This work addresses the current bottlenecks in Blastocystis research and facilitates further exploration into its enigmatic role in the gut microbiome.

Table 1.

Key Terms

Anaerobic	An environment that is completely free of oxygen
Axenic	A culture of Blastocystis devoid of other microbial contaminants (bacteria, fungi, etc.)
Cryopreservation	The process of preserving biological samples at extremely low temperatures to maintain viability over time
Diphasic	A type of culturing system that includes both a solid and liquid phase
Inspissation	A technique involving the coagulation of medium via heating
Microaerophilic	Conditions with very low levels of oxygen, but not fully anaerobic
Monophasic	A type of culturing system consisting of a single phase (solid, semi‐solid, or liquid)
Pre‐reducing	The process of removing oxygen from culture medium before inoculation, typically by storing in an anaerobic chamber
Pre‐warming	Bringing culture medium to incubation temperature before inoculation, preventing thermal shock
Xenic	A culture of Blastocystis that contains other microorganisms (bacteria, fungi, etc.) obtained from the original sample

Figure 1.

Schematic of the topics covered in this paper.

The methods described herein are recommended as a guide. It is important to be aware that different STs might have distinct culture requirements; thus, although these protocols should work for most STs, there may be STs, or strains within STs, that require a certain level of trial and error to obtain an optimized protocol. This is especially true for axenic cultures. This protocol overview does not include discussion of how to interpret results because it is a review of the methods and protocols used within the field; for specific results related to each protocol, please refer to the studies cited therein. This article is designed to provide a comprehensive resource for Blastocystis culturing methods that can be implemented throughout laboratories within the field.

MEDIA AND CULTURING METHODS FOR XENIC CULTURES IN LIQUID MEDIA

Basic Protocol 1. Establishment of Xenic Blastocystis Cultures from Stool Samples in Liquid Medium

The following procedure should preferably be carried out in a class II biological safety cabinet. See Figure 2 for an overview of the protocol.

Figure 2.

Inoculation of liquid medium from stool samples. Created in BioRender (D. Shaw, 2025; https://BioRender.com/h58m072).

Materials

• Liquid medium

• Fresh human stool samples (within 2 hr of collection, or within 24 hr if refrigerated)

• Class II biosafety cabinet

• Incubator, 37°C

• 1Aliquot 12 ml of your chosen liquid medium into 15‐ml centrifuge tubes and place in a 37°C incubator to pre‐warm.
• 2Working in a class II biosafety cabinet, transfer 50‐500 mg (a pea‐sized amount) of fresh stool sample from the collection tube to the pre‐warmed liquid medium.
• 3Incubate at 37°C and subculture every 3‐4 days.

  Growth can be variable depending on ST, so frequent observation of the culture under a light microscope should be carried out.

  Results and validation from this protocol can be found in Clark & Stensvold (2016), as well as in Figure 3.

Figure 3.

Subculturing of liquid medium from existing liquid cultures. Created in BioRender (D. Shaw, 2025; https://BioRender.com/y69j358).

Basic Protocol 2. Subculturing from Existing Blastocystis Liquid Culture

The following procedure should preferably be carried out in a class II biological safety cabinet.

Materials

• Liquid medium

• Blastocystis liquid culture

• Class II biosafety cabinet

• 15‐ml centrifuge

• Incubator, 37°C

• 1Aliquot 12 ml of your chosen liquid medium into 15‐ml centrifuge tubes and place in a 37°C incubator to pre‐warm.

  In the existing culture, Blastocystis spp. cells will fall to the bottom of the tube

• 2Working in a class II biosafety cabinet, use a sterile transfer pipet to take up Blastocystis spp. and bacterial cells from the bottom of the tube.

  The amount transferred can be adjusted depending on cell density.

• 3Transfer to the fresh, pre‐warmed medium, avoiding introducing air into the culture.
• 4Incubate xenic cultures at 37°C and repeat the subculturing process every 3‐4 days.

  Growth can be variable depending on ST, so frequent observation of the culture under a light microscope should be carried out.

  Results and validation from this protocol can be found in Jones (1946).

Support Protocol 1. Preparation of Modified Jones’ Medium for Blastocystis Culturing

Xenic cultures of Blastocystis spp. can be maintained in a range of different media. The most common monophasic liquid medium is modified Jones’ medium (see Fig. 4), which is based on the study published by W. R. Jones for the cultivation of Entamoeba histolytica in 1946 (Jones, 1946). In fact, most of the xenic culturing media used for Entamoeba spp. are appropriate for the cultivation of Blastocystis spp. (Clark & Diamond, 2002; Leelayoova et al., 2002).

Figure 4.

Preparation of modified Jones’ medium for liquid culture. Created in BioRender (D. Shaw, 2025; https://BioRender.com/f80s670).

Modified Jones’ medium consists of a buffered saline solution of disodium phosphate, monopotassium phosphate, and sodium chloride mixed with yeast extract and heat‐inactivated horse serum. It is used to cultivate and detect Blastocystis spp. from human and animal stool samples, with the organisms’ presence being confirmed by conventional PCR (Lhotská et al., 2020; Šloufová et al., 2022) and qPCR (Šloufová et al., 2022).

Pros and cons

Modified Jones’ medium is the standard medium of choice for the maintenance of xenic cultures and is selective for Blastocystis over bacteria. It was developed to be used with human samples and is not always optimal for the establishment of cultures from animal samples.

Materials

• Milli‐Q‐purified water

• Disodium phosphate (Na₂HPO₄)

• Monopotassium phosphate (KH₂PO₄)

• Sodium chloride (NaCl)

• Yeast extract

• Horse serum, heat inactivated

• 1
  To prepare modified Jones’ medium, first make the following base solution:

  • 900 ml Milli‐Q water
  • 1.27 g Na₂HPO₄
  • 419.5 mg KH₂PO₄
  • 7.36 g NaCl
  • 1 g yeast extract
• 2Autoclave 30 min at 121°C at 15 psi, allow to cool, and add 100 ml heat‐inactivated horse serum to 1 L final volume.

  OPTIONAL: Base medium can be frozen at –20°C indefinitely, with 10% (v/v) heat‐inactivated horse serum being added before use.

  Many laboratories buy horse serum in non‐heat‐inactivated form; therefore, horse serum should be heat inactivated before being added to any culture medium. Heat inactivation is achieved by heating horse serum for 30 min at 56°C. The serum can then be stored for up to 3 weeks at 4°C or several months at –20°C.

  OPTIONAL: Working in a sterile environment, filter the medium using a 0.22‐µm‐pore‐size filter.

• 3Store medium at 4°C and pre‐warm to 37°C before inoculation with Blastocystis spp.

  Results and validation from this protocol can be found in Clark & Diamond (2002), Jones (1946), Leelayoova et al. (2002), Lhotská et al. (2020), and Šloufová et al. (2022).

Support Protocol 2. Preparation of Trypticase‐Yeast Extract‐Serum‐Gastric Mucin‐9 Medium for Blastocystis Culturing

Trypticase‐yeast extract‐serum gastric mucin‐9 (TYSGM‐9) medium (Clark & Diamond, 2002) was derived from trypticase‐yeast extract‐iron‐serum (TYI‐S‐33) medium from Diamond et al. (Diamond, 1982; Gillin & Diamond, 1978), originally developed for axenic culture of Entamoeba histolytica, but it is now used for the growth of xenic cultures of Blastocystis spp. TYSGM‐9 consists of dipotassium phosphate, monopotassium phosphate, sodium chloride, yeast extract, gastric mucin, heat‐inactivated adult bovine serum, and Tween‐80 (see Fig. 5).

Figure 5.

Preparation of TYSM‐9 for liquid culture. Created in BioRender (D. Shaw, 2025; https://BioRender.com/y59e408).

Pros and cons

TYSGM‐9 can support both human and animal xenic isolates of Blastocystis spp. well, but may not support axenic growth.

Materials

• Milli‐Q‐purified water

• Monopotassium phosphate (K₂HPO₄)

• Dipotassium phosphate (KH₂PO₄)

• Sodium chloride (NaCl)

• Yeast extract

• Gastric mucin

• Adult bovine serum, heat inactivated

• Tween‐80

• Absolute (100%) ethanol

• 1
  To prepare 1 L of TYSGM‐9, combine the following reagents:

  • 950 ml Milli‐Q water
  • 2.8 g K₂HPO₄
  • 0.4 g KH₂PO₄
  • 7.5 g NaCl
  • 1 g yeast extract
  • 2 g gastric mucin
• 2Autoclave 30 min at 121°C, 15 psi, allow to cool, and add 50 ml heat‐inactivated adult bovine serum and 1 ml of 5% (v/v) Tween‐80 in 100% ethanol

  OPTIONAL: Base medium can be frozen indefinitely at –20°C, with 5% (v/v) heat‐inactivated adult bovine serum being added prior to use.

  OPTIONAL: Filter the medium in a sterile environment using a 0.22‐µm‐pore‐size filter.

• 3Store medium at 4°C and pre‐warm to 37°C before inoculation with Blastocystis spp.

  Results and validation from this protocol can be found in Clark & Diamond (2002), Diamond (1982), and Gillin & Diamond (1978).

Support Protocol 3. Preparation of Liver Extract‐Yeast Extract‐Serum‐Gastric Mucin Medium for Blastocystis Culturing

Liver extract‐yeast extract‐serum gastric mucin (LYSGM) medium was derived from trypticase‐yeast extract‐serum‐gastric mucin (TYSGM‐9) medium, which was developed by Diamond et al. for the xenic cultivation of Entamoeba histolytica (Diamond, 1982); however, LYSGM medium can support the growth of high densities of Blastocystis spp. (Clark & Stensvold, 2016). LYSGM consists of dipotassium phosphate, monopotassium phosphate, sodium chloride, yeast extract, liver extract, gastric mucin, and heat‐inactivated horse serum (see Fig. 6).

Figure 6.

Preparation of LYSGM for liquid culture. Created in BioRender (D. Shaw, 2025; https://BioRender.com/n43a396).

Pros and cons

LYSGM is rich in nutrients and can support xenic cultures, being especially good for initial isolation from fecal samples; however, its inclusion of complex components such as liver extract, yeast, and serum can lead to batch‐to‐batch variability, so it is recommended that the same lot number be used.

Materials

• Milli‐Q‐purified water

• Monopotassium phosphate (K₂HPO₄)

• Dipotassium phosphate (KH₂PO₄)

• Sodium chloride (NaCl)

• Yeast extract

• Liver extract

• Gastric mucin

• Adult bovine serum, heat inactivated

• 1
  To prepare 1 L LYSGM, combine the following reagents:

  • 950 ml Milli‐Q water
  • 2.8 g K₂HPO₄
  • 0.4 g KH₂PO₄
  • 7.5 g NaCl
  • 2.5 g yeast extract
  • 0.5 g liver extract
  • 1 g gastric mucin
• 2Autoclave 30 min at 121°C, 15 psi, allow to cool, and add 50 ml heat‐inactivated adult bovine serum for a final volume of 1 L.

  OPTIONAL: Base medium can be frozen indefinitely at –20°C, with 5% (v/v) heat‐inactivated adult bovine serum being added before use.

  OPTIONAL: Filter the medium in a sterile environment using a 0.22‐µm‐pore‐size filter.

• 3Store medium at 4°C and pre‐warm to 37°C before inoculation with Blastocystis spp.

  Results and validation from this protocol can be found in Clark & Stensvold (2016) and Diamond (1982).

MEDIA AND CULTURING METHODS FOR XENIC CULTURES IN DIPHASIC MEDIA

Xenic cultures of Blastocystis spp. can also be maintained in diphasic media, which consist of two media with distinct physical states: i.e., a solid component topped with a liquid component.

Examples of diphasic medium used for Blastocystis spp. include Robinson's medium (Support Protocol 4; Clark & Stensvold, 2016; Robinson, 1968) and Ringer's agar slants (Support Protocol 5; Stenzel & Boreham, 1996).

Basic Protocol 3. Subculturing Xenic Blastocystis Cultures in Diphasic Media

This procedure is based on that of Robinson (1968). It should preferably be carried out in a class II biological safety cabinet (Fig. 7).

Figure 7.

Culturing of xenic STs in diphasic medium. Created in BioRender (D. Shaw, 2025; https://BioRender.com/x93j637).

Materials

• Blastocystis spp. culture in diphasic medium

• Fresh diphasic medium in fresh culture tubes

• Class II biosafety cabinet

• Incubator, 37°C

• 1Pre‐warm diphasic medium to 37°C before inoculation.
• 2Working in a class II biosafety cabinet, use a sterile transfer pipet to remove Blastocystis spp. and bacteria from the liquid phase and solid surface of the diphasic medium.
• 3Transfer cells to the liquid phase of the fresh, pre‐warmed diphasic medium tubes.
• 4Incubate at 37°C and subculture every 3‐4 days.

  Growth can be variable depending on ST, so frequent observation of the culture under a light microscope should be carried out.

  Results and validation from this protocol can be found in Robinson (1968).

Support Protocol 4. Preparation of Robinson's Medium for Blastocystis Culturing

Robinson's medium (Robinson, 1968) can also be used for Blastocystis spp., which grows well in it (Clark & Stensvold, 2016), but its use is not that widespread, likely because of its complex preparation (see Fig. 8). This medium is another whose primary usage is for Entamoeba histolytica culturing. It is composed of Bacto Peptone, phthalate, and BRS medium, which is inoculated with Escherichia coli.

Figure 8.

Preparation of Robinson's medium. Created in BioRender (D. Shaw, 2025; https://BioRender.com/a32z897).

Pros and cons

Supports robust growth of xenic Blastocystis, but can be labor intensive to prepare, and components such as egg can lead to batch‐to‐batch variability. The following steps make 1 L of Robinson's medium.

Materials

• Milli‐Q‐purified water

• Erythromycin

• Bacto Peptone

• Potassium hydrogen phthalate (C₈H₅KO₄)

• Sodium hydroxide (NaOH)

• Citric acid

• Dipotassium phosphate (KH₂PO₄)

• Ammonium sulfate [(NH₄)₂SO₄]

• Magnesium sulfate heptahydrate (MgSO₄·7H₂O)

• Lactic acid

• Escherichia coli stock

• Erythromycin

• 1Prepare 0.5% (w/v) erythromycin in Milli‐Q water and filter sterilize using a 0.22‐µm‐pore‐size filter.
• 2Prepare 20% (w/v) Bacto Peptone in Milli‐Q water and autoclave 30 min at 121°C, 15 psi.
• 3Prepare a 10× phthalate stock solution by combining 102 g of potassium hydrogen phthalate (C₈H₅KO₄), 50 ml of 40% (w/v) NaOH, and 950 ml Milli‐Q water, and then autoclave. Use at 1× concentration.
• 4Prepare a phthalate/Bacto Peptone stock by combining 1.25 ml of 20% (w/v) Bacto Peptone and 100 ml of 1× phthalate.
• 5Prepare a 10× R medium stock by combining 1 L Milli‐Q water, 50 g NaCl, 20 g citric acid, 50 g KH₂PO₄, 10 g (NH₄)₂SO₄, 0.5 g MgSO₄·7H₂O, and 40 ml of 85% (w/v) lactic acid solution. Autoclave and use at 1× concentration.
• 6Prepare BR medium by inoculating 1× R medium with E. coli and incubating for 48 hr at 37°C.
• 7Prepare BRS medium by combining equal volumes of BR medium and heat‐inactivated bovine serum and incubating for 24 hr at 37°C.
• 8Prepare 1.5% (w/v) Noble agar in 0.7% (w/v) NaCl in Milli‐Q water and autoclave.
• 9Aliquot 5 ml of agar solution into tubes or bottles and leave to set at an angle.
• 10Once ready to inoculate with Blastocystis spp., add 3 ml of 1× phthalate/Bacto Peptone, 1 ml BRS medium, and 50 µl erythromycin to the 5‐ml agar slants
• 11Incubate at 37°C before use.

  Results and validation from this protocol can be found in Clark & Stensvold (2016) and Robinson (1968).

Support Protocol 5. Preparation of Ringer's Agar Slants for Blastocytis Culturing

Ringer's agar slants are composed of a solid layer of sodium chloride, calcium chloride, potassium chloride, Bacto Agar, and l‐asparagine, topped with a solution of sodium chloride, calcium chloride, and potassium chloride (see Fig. 9).

Figure 9.

Preparation of Ringer's agar slants. Created in BioRender (D. Shaw, 2025; https://BioRender.com/u98p989).

Pros and cons

Ringer's agar slants are simple and quick to prepare, but are not the most nutrient rich medium and cannot support axenic cultures.

Materials

• Milli‐Q‐purified water

• Sodium chloride (NaCl)

• Calcium chloride (CaCl₂)

• Potassium chloride (KCl)

• l‐Asparagine

• 1 M sodium hydroxide (NaOH)

• Bacto Agar

• Horse serum, heat inactivated

• 15‐ml Falcon tubes

• 1
  To make Ringer's agar slants (Stenzel & Boreham, 1996), first combine the following reagents to make the solid phase:

  • 1 L Milli‐Q water
  • 8.6 g NaCl
  • 0.3 g CaCl₂
  • 0.3 g KCl
  • 1 g l‐asparagine
  • Adjust pH to 7.4 with 1 M NaOH, and then add:
  • 15 g Bacto Agar
    The solution is acidic and must be adjusted to pH 7.4 in order for Blastocystis spp. to grow.
• 2Autoclave 30 min at 121°C, 15 psi.
• 3Allow to cool to ∼60°C and add 10% (v/v) heat‐inactivated horse serum. Transfer 5‐ml aliquots into 15‐ml Falcon tubes.

  Make sure to dispense the agar into the Falcon tubes while it is still warm, to keep it from setting beforehand.

• 4Leave the tubes to set at room temperature at a 30° angle.
• 5
  For the liquid phase, combine the following reagents:

  • 1 L Milli‐Q water
  • 8.6 g NaCl
  • 0.3 g CaCl₂
  • 0.3 g KCl
• 6Autoclave 30 min at 121°C, 15 psi.
• 7Once cool to touch, add 10% (v/v) heat‐inactivated horse serum.
• 8Add 5 ml of the liquid phase to each tube containing the slanted solid phases.
• 9Store up to 2 months at 4°C.
• 10Incubate at 37°C prior before.

  Results and validation from this protocol can be found in Stenzel & Boreham (1996).

AXENIZATION

Basic Protocol 4. Axenization of Blastocystis Cultures

Axenization of Blastocystis (see Fig. 10), that is, its culturing in the absence of any other microorganisms, remains one of the main challenges in this field. Currently, only a handful of STs have been successfully axenized and subsequently maintained in culture. These are ST1 (Zierdt et al., 1988), ST4 (Chen et al., 1997; Deng & Tan, 2022), and ST7 (Ho et al., 1993). The most commonly used method for axenization includes the elimination of bacterial populations by antibiotics (Zierdt & Williams, 1974), but it is possible that this is not always successful in removing all bacteria (Ng & Tan, 1999). It is therefore recommended to follow this with clonal growth on soft (Tan, Singh, Thong, et al., 1996) or solid (Tan et al., 2000) agar, where individual colonies of Blastocystis can be isolated.

Figure 10.

Axenization of Blastocystis cultures. Created in BioRender (D. Shaw, 2025; https://BioRender.com/y38z642).

NOTE: The following procedure should preferably be carried out in a class II biological safety cabinet.

Materials

• Xenic Blastocystis spp. culture

• Fresh medium

• Ampicillin

• Streptomycin

• Amphotericin B

• IMDM soft (0.36%) agar (prepared as in Basic Protocol 7)

• Pre‐reduced liquid IMDM (see Basic Protocol 6, steps 1‐5), containing 10% (v/v) heat‐inactivated horse serum

• Class II biosafety cabinet

• Incubator, 37°C

• Anaerobic chamber

• 1Pre‐warm and pre‐reduce fresh medium to 37°C before inoculation.

  To pre‐reduce medium, place in anaerobic conditions for 24 hr before use.

• 2Working in a class II biosafety cabinet, inoculate fresh medium with a xenic culture of Blastocystis (Basic Protocol 2).
• 3Add an antimicrobial mixture of 4000 µg/ml ampicillin, 1000 µg/ml streptomycin, and 500 µg/ml amphotericin B.
• 4Incubate at 37°C under anaerobic conditions.

  For anaerobic culturing, we recommend using an anaerobic jar system, such as the Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets

• 5After 3 days, add another 4000 µg/ml ampicillin, 1000 µg/ml streptomycin, and 500 µg/ml amphotericin B.
• 6Incubate at 37°C for 7 days.
• 7After 7 days, subculture to fresh medium (see Basic Protocol 2) with 4000 µg/ml ampicillin, 1000 µg/ml streptomycin, and 500 µg/ml amphotericin B.
• 8Repeat subculturing until microscopic observation shows that Blastocystis cells outnumber bacterial cells.
• 9Transfer Blastocystis cells to IMDM soft (0.36%) agar (see Basic Protocol 7).
• 10Once colonies are visible (after ∼7 days), isolate colonies using a Pasteur pipet and transfer to pre‐reduced liquid IMDM containing 10% (v/v) heat‐inactivated horse serum.

  Results and validation from this protocol can be found in Chen et al. (1997), Deng & Tan (2022), Ho et al. (1993), Ng & Tan (1999), Tan et al. (2000), Tan, Singh, Thong, et al. (1996), Zierdt et al. (1988), and Zierdt & Williams (1974).

MEDIA AND CULTURING METHODS FOR AXENIC CULTURES IN DIPHASIC MEDIA

Basic Protocol 5. Establishment of Axenic Blastocystis Culture in Diphasic Medium

The following procedure should preferably be carried out in a class II biological safety cabinet (Fig. 11).

Figure 11.

Culturing of axenic Blastocystis subtypes in diphasic medium. Created in BioRender (D. Shaw, 2025; https://BioRender.com/x44u072).

Materials

• Fresh diphasic medium

• Ampicillin

• Streptomycin

• Amphotericin B

• Class II biosafety cabinet

• Incubator suitable for anaerobic culture, 37°C

• 1Pre‐reduce diphasic medium and pre‐warm to 37°C before inoculation.

  To pre‐reduce medium, place in anaerobic conditions for 24 hr before use.

• 2Working in a class II biosafety cabinet, use a sterile transfer pipet to remove Blastocystis spp. from the liquid phase and solid surface of the diphasic medium.
• 3Transfer cells to the liquid phase of the fresh, pre‐warmed diphasic medium tubes.
• 4Incubate at 37°C anaerobically and subculture every 3‐4 days.

  To culture anaerobically, it is recommended to use an anaerobic jar system, such as the Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets

  Growth can be variable depending on ST, so frequent observation of the culture under a light microscope should be carried out.

  Results and validation from this protocol can be found in Lanuza et al. (1996).

Support Protocol 6. Preparation of Boeck and Drbohlav's Locke‐Egg Serum (LES) Medium for Blastocystis Culturing

Boeck and Drbohlav's Locke‐egg serum (LES) medium (see Fig. 12) was also first used to establish cultures of Entamoeba histolytica (Boeck & Drbohlav, 1925). This medium consists of a solution of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium phosphate (dibasic), sodium bicarbonate, and potassium phosphate (monobasic) combined with whole eggs. This egg mixture undergoes inspissation to increase its viscosity and form the solid phase. It is then topped with Locke's solution, supplemented with heat‐inactivated horse serum.

Figure 12.

Preparation of Boeck and Drbohlav's Locke‐egg serum (LES) medium. Created in BioRender (D. Shaw, 2025; https://BioRender.com/u13l909).

Pros and cons

Boeck and Drbohlav's LES medium is rich in nutrients and effective for isolation of axenic cultures from fecal samples, but is labor intensive to prepare, requiring inspissation, and contains components such as eggs, which can lead to batch‐to‐batch variability.

Materials

• Milli‐Q‐purified water

• Sodium chloride (NaCl)

• Calcium chloride (CaCl₂)

• Potassium chloride (KCl)

• Magnesium chloride (MgCl₂)

• Disodium phosphate (Na₂HPO₄)

• Sodium bicarbonate (NaHCO₃)

• Monopotassium phosphate (KH₂PO₄)

• Horse serum, heat inactivated

• 70% (v/v) ethanol

• Whatman filter paper, grade 1

• Gauze

• 1
  To prepare Locke's solution, combine the following reagents and autoclave 30 min at 121°C at 15 psi:

  • 1 L Milli‐Q water
  • 8 g NaCl
  • 0.2 g CaCl₂
  • 0.2 g KCl
  • 0.01 g MgCl₂
  • 2 g Na₂HPO₄
  • 0.4 g NaHCO₃
  • 0.3 g KH₂PO₄
• 2Place on ice to allow precipitation, filter out insolubilized salts using grade 1 Whatman paper, and then re‐autoclave.
• 3
  Prepare the egg slants (Clark & Diamond, 2002; CDC, 1948):

  • a. Sterilize eggs by spraying each one with 70% ethanol and running it through a flame.
  • b. Break whole eggs into a flask, add 12.5 ml Locke's solution (from step 2) for every whole egg (∼45 ml) and shake to emulsify.
  • c. Filter the mixture through gauze to remove all unmixed egg white.
  • d. Transfer 5 ml to a sterile culture tube and apply a vacuum for 1 hr to remove air bubbles.
• 4Let tube rest at a 30° angle to form a slant and cook at 120°C for 30 min.
• 5Wait until cool, add 5 ml sterile Locke's solution (from step 2) on top of the solid phase, and then inspissate by autoclaving.
• 6Store at 4°C and add 25% (v/v) heat‐inactivated horse serum before use.

  Results and validation from this protocol can be found in Boeck & Drbohlav (1925), Clark & Diamond (2002), and CDC (1948).

Support Protocol 7. Preparation of Boeck and Drbohlav's Diphasic Modified Medium (BDMM) for Blastocystis Culturing

In 1996, Lanuza et al. published their use of a modified recipe of Boeck and Drbohlav's LES medium (BDMM; see Fig. 13), which contained the same ingredients as the standard recipe at twice the original concentration, as well as glucose (Lanuza et al., 1996).

Figure 13.

Preparation of Boeck and Drbohlav's diphasic modified medium (BDMM). Created in BioRender (D. Shaw, 2025; https://BioRender.com/w55n055).

Pros and cons

BDMM is rich in nutrients and effective for growth of axenic cultures, but is labor intensive to prepare, requiring inspissation, and contains components such as eggs that can lead to batch‐to‐batch variability.

Materials

• Milli‐Q‐purified water

• Glucose

• Sodium chloride (NaCl)

• Calcium chloride (CaCl₂)

• Potassium chloride (KCl)

• Magnesium chloride (MgCl₂)

• Disodium phosphate (Na₂HPO₄)

• Sodium bicarbonate (NaHCO₃)

• Monopotassium phosphate (KH₂PO₄)

• Phenol red

• Locke's solution (see Support Protocol 6)

• 70% (v/v) ethanol

• Whatman paper, grade 1

• Anaerobic chamber

• 1
  To make BDMM solution, combine the following reagents:

  • 1 L Milli‐Q water
  • 5 g glucose
  • 16 g NaCl
  • 0.4 g CaCl₂
  • 0.4 g KCl
  • 0.02 g MgCl₂
  • 4 g Na₂HPO₄
  • 0.8 g NaHCO₃
  • 0.6 g KH₂PO₄
• 2Autoclave 30 min at 121°C, 15 psi.
• 3Filter through grade 1 Whatman paper to remove insolubilized salts. Add 0.02% (w/v) phenol red.
• 4Autoclave again and store at 4°C.
• 5For the solid phase of BDMM, first sterilize eggs by spraying each with 70% ethanol and running it through a flame.
• 6Combine the whole eggs with Locke's solution (see Support Protocol 6) at 4:1 (v/v) and homogenize.
• 7Transfer 5 ml of this mixture to sterile culture tubes and inspissate at a 30° angle for 30 min at 80°C.
• 8Autoclave 30 min at 121°C, 15 psi.
• 9Top with 5 ml of the BDMM liquid medium from step 4.
• 10Store at 4°C until ready for use.
• 11Pre‐reduce in an anaerobic chamber before use.

  Results and validation from this protocol can be found in Lanuza et al. (1996).

MEDIUM AND CULTURING METHODS FOR AXENIC CULTURES IN LIQUID MEDIUM

Very few STs of Blastocystis spp. have been successfully axenized. In 1993, Ho et al. optimized the axenic culturing of Blastocystis spp. in Iscove's modified Dulbecco's medium (IMDM), supplemented with 10% (v/v) heat‐inactivated horse serum (Ho et al., 1993). IMDM medium (see Fig. 14) used for cultivation of Blastocystis spp. must contain l‐glutamine, sodium bicarbonate, HEPES buffer, phenol red, and sodium pyruvate. Medium should be pre‐reduced prior to inoculation with Blastocystis spp. and then incubated inside anaerobic jars to maintain an anaerobic environment.

Figure 14.

Preparation of Iscove's modified Dulbecco's medium (IMDM). Created in BioRender (D. Shaw, 2025; https://BioRender.com/x27r491).

Basic Protocol 6. Establishment of Axenic Blastocystis Cultures in Iscove's Modified Dulbecco's Medium (IMDM)

Pros and cons

The following procedure (Fig. 15) should preferably be carried out in a class II biosafety cabinet. Note that because it contains HEPES, IMDM is a buffered medium and can easily be supplemented for growth of axenic cultures; however, it can be more expensive that other culture media.

Figure 15.

Culturing of axenic Blastocystis subtypes in liquid medium. Created in BioRender (D. Shaw, 2025; https://BioRender.com/a49r646).

Materials

• Milli‐Q‐purified water

• Horse serum, heat inactivated

• IMDM powdered medium

• If not included in IMDM powder:

• l‐Glutamine

• Sodium bicarbonate (NaHCO₃)

• 4‐(2‐Hydroxyethyl)‐1‐piperazine ethanesulfonic acid (HEPES)

• Phenol red

• Sodium pyruvate

• 10,000 U/ml penicillin/10 mg/ml streptomycin (optional)

• NaOH and/or HCl, for pH adjustment

• 0.22‐µm‐pore‐size filter

• 14‐ml round‐bottom culture tubes with vented caps

• Anaerobic jar with anaerobic‐gas‐generating sachets (e.g., Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets)

Medium preparation

• 1
  To make 1 L of complete IMDM medium, add the following reagents and filter sterilize using a 0.22‐µm‐pore‐size vacuum filter (do not autoclave IMDM):

  • 900 ml Milli‐Q water
  • 100 ml heat‐inactivated horse serum
  • 17.67 g IMDM powder
• 2
  OPTIONAL: If your manufacturer's IMDM powder does not contain any of the following, add these to the solution:

  • 3.024 g/L NaHCO₃
  • 15.93 mg/L phenol red
  • 582 mg/L l‐glutamine
  • 5.96 g/L HEPES
  • 110 mg/L sodium pyruvate
    There are also many pre‐prepared IMDM powders pre‐supplemented with some or all of these components, so this step may not be necessary.
  • OPTIONAL: Add 1% (v/v) 10,000 U/ml penicillin/10 mg/ml streptomycin.
• 3The pH of the final solution may vary depending on the source of horse serum. Adjust pH using NaOH and HCl to achieve a pH of 7.6‐7.7.
• 4Filter using a 0.22‐µm‐pore‐size filter and store at 4°C until use.

Inoculation and culturing

Before inoculation with Blastocystis spp., the IMDM medium must be pre‐reduced (by being placed in anaerobic conditions) and pre‐warmed at 37°C.

• 5Transfer 8 ml complete IMDM medium to 14‐ml round‐bottom culture tubes, closing the cap to the vented position.
• 6Place the tubes in an anaerobic jar alongside an anaerobic‐gas‐generating sachet to produce a hypoxic environment.
• 7Incubate anaerobically at 37°C for 24 hr.

  For anaerobic culturing, we recommend using an anaerobic jar system, such as the Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets.

• 8Inoculate each tube with the visual sediment from the previous culture of Blastocystis spp. and culture anaerobically at 37°C.
• 9Subculture into fresh medium every 4‐5 days.

  Growth can be variable depending on ST, so frequent observation of the culture should be carried out.

  Results and validation from this protocol can be found in Ho et al. (1993).

MEDIA AND CULTURING METHODS FOR AXENIC CULTURES IN SEMI‐SOLID AND SOLID MEDIA

Monophasic solid or semi‐solid media are less commonly used than liquid media, although it is possible to culture colonies of Blastocystis spp. in this way.

Basic Protocol 7. Establishment of Axenic Blastocystis Cultures in Soft IMDM Agar

In 1996, Tan et al. published the first description of axenic Blastocystis spp. colony formation on soft IMDM with 0.36% (w/v) agar (Tan, Singh, Yap, et al., 1996). In the same year, they updated the protocol (see Fig. 16) as they found that adding the reducing agent sodium thioglycolate to the soft agar improved colony yield of biconvex disc‐shaped colonies, which could have previously been hindered by time needed to reach anaerobiosis after plating (Tan, Singh, Thong, et al., 1996).

Figure 16.

Culturing of axenic Blastocystis subtypes in IMDM 0.36% soft agar. Created in BioRender (D. Shaw, 2025; https://BioRender.com/o40p601).

Pros and cons

IMDM soft (0.36%) agar is optimal for anaerobic culturing due to the addition of sodium thioglycolate, supporting axenic colony formation; however, growth on this medium is slow.

NOTE: The following procedure should preferably be carried out in a class II biological safety cabinet.

Materials

• Milli‐Q‐purified water

• Bacto Agar

• Sodium thioglycolate

• 2× liquid IMDM medium

• Horse serum, heat inactivated

• Blastocystis culture in liquid medium

• Class II biosafety cabinet

• Anaerobic jar with anaerobic‐gas‐generating sachets (e.g., Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets)

• 1
  To prepare IMDM soft agar, first make the agar by combining the following reagents:

  • 1 L Milli‐Q water
  • 7.2 g Bacto Agar
  • 1 g sodium thioglycolate
  • Autoclave 30 min at 121°C, 15 psi.
• 2Working in a class II biosafety cabinet, add 1 part agar to 1 part 2× IMDM medium (adapted from Basic Protocol 6), along with 10% (w/v) heat‐inactivated horse serum, and inoculate with all visual sediment of Blastocystis spp. from culture.
• 3Pour ~20 ml/dish into 90‐mm petri dishes and allow to set for 10 min at –20°C.
• 4Incubate at 37°C in an anaerobic jar for 8‐10 days, until colonies form.

  For anaerobic culture, we recommend using an anaerobic jar system, such as the Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets.

  Growth can be variable depending on ST, so frequent observation of the culture under a light microscope should be carried out.

  Results and validation from this protocol can be found in Tan, Singh, Yap, et al. (1996).

Basic Protocol 8. Establishment of Axenic Blastocystis Cultures on Solid IMDM Agar

Tan et al. later successfully grew Blastocystis spp. colonies on solid IMDM agar (Tan et al., 2000). This method does not require the addition of sodium thioglycolate, as the plates are pre‐reduced before inoculation with Blastocystis spp. (see Fig. 17).

Figure 17.

Culturing of axenic Blastocystis subtypes in IMDM solid agar. Created in BioRender (D. Shaw, 2025; https://BioRender.com/w71 × 738).

NOTE: The following procedure should preferably be carried out in a class II biological safety cabinet.

Pros and cons

IMDM solid agar (2%) does not require chemical reducers, instead relying on being pre‐reduced in an anaerobic chamber; however, this medium requires an even longer incubation time for colony formation than soft agar.

Materials

• Milli‐Q‐purified water

• Bacto Agar

• 2× liquid IMDM medium, pre‐warmed to 40°C

• Horse serum, heat inactivated, pre‐warmed to 40°C

• Blastocystis culture in liquid medium

• Petri dishes

• Class II biosafety cabinet

• Anaerobic jar with anaerobic‐gas‐generating sachets (e.g., Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets)

Medium preparation

• 1
  To prepare solid IMDM agar, first make a 2% (w/v) agar stock by combining the following reagents:

  • 1 L Milli‐Q water
  • 20 g Bacto Agar
• 2Autoclave 30 min at 121°C, 15 psi.
• 3Once cooled, add equal parts of 2% agar to 2× IMDM supplemented with 10% (v/v) heat‐inactivated horse serum, both at 40°C.
• 4Pour ~20 ml/dish into 90‐mm petri dishes and leave to solidify at room temperature.

Inoculation and culturing

• 5Transfer to an anaerobic chamber to pre‐reduce for 24 hr before inoculation with Blastocystis spp.

  To pre‐reduce medium, place in anaerobic conditions for 24 hr before use.

• 6Working in a class II biosafety cabinet, pipet an inoculation‐loop‐sized amount of Blastocystis spp. onto the solid agar and gently rock to distribute the protozoa around the plate.

  Alternatively, plating using the classical streaking technique employed in bacteriology may also be used to obtain single colonies from undefined initial inoculum concentrations.

• 7Incubate in an anaerobic chamber for 24 hr at 37°C, face up to allow the protozoa to colonize the agar surface.
• 8After 24 h, invert the plates and continue to incubate in an anaerobic chamber at 37°C for 7‐21 days.

  For anaerobic culture, we recommend using an anaerobic jar system, such as the Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets.

  Growth can be variable depending on ST, so frequent observation of the culture under a light microscope should be carried out.

  Results and validation from this protocol can be found in Tan et al. (2000).

Basic Protocol 9. Optimized Method for Establishing Axenic Blastocystis Cultures on Solid IMDM Agar

Some groups have optimized the standard protocol for use with specific subtypes. For example, the IMDM solid agar recipe has been optimized for culturing ST8 (see Fig. 18) using the following protocol, which was established for an axenic culture of ST8 (E. Viscogliosi lab, personal communication).

Figure 18.

Culturing of axenic Blastocystis subtypes in IMDM solid agar (specifically optimized for ST8). Created in BioRender (D. Shaw, 2025; https://BioRender.com/z01w572).

NOTE: This method is not widely used in the literature, and any deviations from the standard protocol should be optimized for the lab that will be using it.

NOTE: The following procedure should preferably be carried out in a class II biological safety cabinet.

Pros and cons

IMDM solid agar (2.5%) has a higher percentage of agar than other solid media, making it easier to inoculate; however, is specifically optimized for ST8 and so may not produce reliable results for other STs.

Materials

• Bacto Agar

• Milli‐Q‐purified water

• IMDM powdered medium

• Horse serum, heat inactivated

• Blastocystis culture in liquid medium

• 10,000 U/ml penicillin/10 mg/ml streptomycin

• Water bath, 50°C

• 0.22‐µm‐pore‐size vacuum filter

• Class II biosafety cabinet

• Petri dishes

• Anaerobic jar with anaerobic‐gas‐generating sachets (e.g., Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets)

Medium preparation

• 1
  First, prepare a 2.5% (w/v) agar stock by combining:

  • 25 g Bacto Agar
  • 1 L Milli‐Q water
• 2Autoclave 30 min at 121°C, 15 psi.
• 3Keep mixture warm in a water bath at 50°C to prevent it from solidifying.
• 4
  Prepare the IMDM portion of the medium by combining:

  • 7.1 g IMDM powder
  • 200 ml Milli‐Q water
  • 100 ml heat‐inactivated horse serum
  • 5 ml of 10,000 U/ml penicillin/10 mg/ml streptomycin
• 5Filter sterilize using a 0.22‐µm‐pore‐size vacuum filter.
• 6Remove agar preparation from the water bath and allow to cool for 5 min.
• 7Preferably working in a class II biosafety cabinet, add the IMDM solution to the agar preparation.

  Always mix the solutions in the agar bottle to avoid having the agar stick to the sides.

• 8Pour ~20 ml/dish of the solution into 90‐mm petri dishes and allow to set inside the category II biosafety cabinet.
• 9Store at 4°C until use.

Inoculation and culturing

• 10The night before use, place the petri dish at room temperature in the biosafety cabinet.
• 11The next day, using an inoculation loop, transfer Blastocystis spp. culture to the agar, making small circular motions.
• 12Incubate at 37°C in an anaerobic chamber with an anaerobic sachet, replating monthly.

  Results from this protocol are presented below in Figure 19.

Figure 19.

ST8 culture grown on IMDM agar. Photograph courtesy of Constance Denoyelle.

Basic Protocol 10. Establishment of Axenic Blastocystis Cultures in Semi‐Solid Locke's Agar

Later, in 2007, Valido and Rivera published an adapted recipe for soft Locke's agar (see Fig. 19), with a final concentration of 0.7% (w/v) agar, which resulted in biconvex disc‐shaped colonies as seen previously (Valido & Rivera, 2007).

NOTE: The following procedure should preferably be carried out in a class II biological safety cabinet.

Pros and cons

Semi‐solid Locke's agar (0.7%) supports the formation of distinct biconvex disc‐shaped colonies; however, because of its consistency, it can be difficult to handle.

Materials

• Milli‐Q‐purified water

• Bacto Agar

• Sodium thioglycolate

• Locke's solution (see Support Protocol 6, step 1), sterilized

• Horse serum, heat inactivated

• Blastocystis culture in liquid medium

• Class II biosafety cabinet

• Petri dishes

• Anaerobic jar with anaerobic‐gas‐generating sachets (e.g., Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets)

• 1
  To prepare semi‐solid Locke's agar, first make a 0.7% (w/v) agar stock by combining the following reagents (Fig. 20):

  • 1 L Milli‐Q water
  • 14 g Bacto Agar
  • 1 g sodium thioglycolate

Figure 20.

Culturing of axenic Blastocystis subtypes in semi‐solid Locke's agar. Created in BioRender (D. Shaw, 2025; https://BioRender.com/g82t511).

• 2Autoclave at 121°C for 30 min at 15 psi.
• 3Add in equal parts to sterilized Locke's solution.
• 4Allow to cool to 40°C and add 10% (v/v) heat‐inactivated horse serum.
• 5Working in a class II biosafety cabinet, inoculate with Blastocystis spp. and pour ~20 ml/dish into 90‐mm petri dishes.
• 6Cool for 10 min at –20°C to allow the agar to set.
• 7Transfer the plates to an anaerobic chamber and incubate at 37°C for 7‐10 days.

  Growth can be variable depending on the ST, so frequent observation of the culture under a light microscope is recommended.

  Results and validation from this protocol can be found in Valido & Rivera (2007).

CRYOPRESERVATION

Basic Protocol 11. Cryopreservation of Xenic Blastocystis Cultures

This procedure is based on that of Clark & Stensvold (2016). The procedure should preferably be carried out in a class II biological safety cabinet.

Materials

• Culture medium base

• Appropriate serum (e.g., horse serum or adult bovine serum), heat inactivated

• Dimethyl sulfoxide (DMSO)

• Xenic Blastocystis culture to be cryopreserved

• Class II biosafety cabinet

• Centrifuge

• Cryovials

• Isopropyl alcohol freezing container

• –80°C freezer or liquid nitrogen (N₂) freezing container

• 1
  Prepare two tubes:

  • a. In tube 1, combine 5 ml culture medium base (without serum) and 5 ml of the appropriate serum.
  • b. In tube 2, transfer 4.25 ml from tube 1, add 0.75 ml DMSO, and invert to combine.
• 2Working in a class II biosafety cabinet, transfer the culture to be cryopreserved to a centrifuge tube and centrifuge 4 min at 275 × g, room temperature

  This pellets the Blastocystis cells while leaving most bacterial cells in suspension. As the bacterial cells will survive the cryopreservation process better than Blastocystis, this will improve the growth rate of Blastocystis upon revival.

• 3Remove and discard the supernatant.
• 4Resuspend the Blastocystis pellet in medium from tube 1.
• 5Aliquot the suspension equally between an appropriate number of cryovials.

  The number of cryovials needed will be dependent on the density of cell growth from the original sample.

• 6Add an equal volume of medium from tube 2 and gently mix with a pipet.
• 7Incubate at 37°C for 15 min.

  DMSO is used to prevent the formation of ice crystals, which can rupture cells. This incubation allows the DMSO to equilibrate and diffuse into cells.

• 8Transfer cryovials to an isopropyl alcohol freezing container and place in a –80°C freezer overnight.

  The isopropanol container reduces the rate of freezing to –1°C/min to reduce cell damage.

• 9Once frozen, cultures can be stored at –80°C, but storage in liquid nitrogen (N₂) is recommended for long‐term storage

  Results and validation from this protocol can be found in Clark & Stensvold (2016); see also Figures 21 and 22.

Figure 21.

Cryopreservation of xenic cultures. Created in BioRender (D. Shaw, 2025; https://BioRender.com/q61 × 071).

Figure 22.

Cryopreservation of axenic cultures. Created in BioRender (D. Shaw, 2025; https://BioRender.com/x16f847).

Basic Protocol 12. Cryopreservation of Axenic Blastocystis Cultures

The following procedure should preferably be carried out in a class II biological safety cabinet.

Materials

• Axenic Blastocystis cultures to be cryopreserved

• Liquid IMDM base medium

• Appropriate serum (e.g., horse serum or adult bovine serum), heat inactivated

• Dimethyl sulfoxide (DMSO)

• Class II biosafety cabinet

• Centrifuge

• 0.22‐µm‐pore‐size syringe filter

• Cryovials

• Isopropyl alcohol freezing container

• –80°C freezer or liquid nitrogen (N₂) freezing container

• 1Obtain axenic culture to cryopreserve and, working in a class II biosafety cabinet, transfer to a centrifuge tube.
• 2Centrifuge the tube for 10 min at 1000 × g, room temperature, to gently pellet the Blastocystis cells.
• 3Remove as much supernatant as possible without disturbing the pellet.
• 4Resuspend in 1 ml IMDM.
• 5Prepare 1 ml of cryoprotective agent containing 700 µl IMDM base medium, 200 µl heat‐inactivated horse serum, and 100 µl DMSO.
• 6Filter through a 0.22‐µm‐pore‐size syringe filter.
• 7Transfer 800 µl of resuspended pellet to a cryovial tube.
• 8Add 800 µl cryoprotective agent (from step 5) and mix gently with a pipet.
• 9Transfer cryovials to an isopropyl alcohol freezing container and place in a –80°C freezer overnight.

  The isopropanol container reduces the rate of freezing to –1°C/min to reduce cell damage.

• 10Once frozen, cultures can be stored at –80°C, but storage in liquid nitrogen (N₂) is recommended for long‐term storage.

CULTURING BLASTOCYSTIS FROM FROZEN STOCKS

Basic Protocol 13. Inoculation of Liquid Medium with Xenic Blastocystis Cultures from Frozen Stocks

This procedure is based on that of Clark & Stensvold (2016). It should preferably be carried out in a class II biological safety cabinet.

Materials

• Cryopreserved xenic Blastocystis cultures (Basic Protocol 11)

• Appropriate culture medium

• Class II biosafety cabinet

• 1Preheat a water bath to 37°C.
• 2Pre‐warm aliquots of culture medium to 37°C.
• 3Remove a cryovial of culture from liquid nitrogen storage and, working in a class II biosafety cabinet, place it into the water bath, ensuring all contents of the cryovial are immersed.
• 4Once thawed, transfer culture to the pre‐warmed medium, incubate for 2 hr at 37°C, and then gently mix by inverting the cryovials a few times by hand.

  DMSO is viscous, so it will settle to the bottom of the culture tube. It is best for the cells to incubate for a while without DMSO being dispersed in the culture medium, before gently mixing it.

• 5Incubate at 37°C.
• 6Monitor growth over subsequent days and subculture when appropriate.

  Results and validation from this protocol can be found in Clark & Stensvold (2016), as well as Figures 23 and 24.

Figure 23.

Inoculation of liquid medium with xenic cultures from frozen stocks. Created in BioRender (D. Shaw, 2025); https://BioRender.com/d38v281).

Figure 24.

Inoculation of liquid medium with axenic cultures from frozen stocks. Created in BioRender (D. Shaw, 2025; https://BioRender.com/a43y606).

Basic Protocol 14. Inoculation of Liquid Medium with Axenic Blastocystis Cultures from Frozen Stocks

The following procedure should preferably be carried out in a class II biological safety cabinet.

Materials

• Cryopreserved axenic Blastocystis cultures (Basic Protocol 12)

• Appropriate culture medium containing 10% (v/v) heat‐inactivated horse serum

• 15‐ml centrifuge tube

• Class II biosafety cabinet

• Centrifuge

• Anaerobic jar with anaerobic‐gas‐generating sachets (e.g., Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets)

• 1Preheat a water bath to 37°C.
• 2Pre‐reduce and pre‐warm aliquots of an appropriate culture medium at 37°C.

  Medium for initial inoculation of axenic cultures should contain 20% heat‐inactivated horse serum instead of the 10% usually used for subculturing, because the additional growth factors will help to revive the frozen culture (G.C. Ng, personal communication).

  To pre‐reduce medium, place in anaerobic conditions for 24 hr before use (see below).

• 3Remove a cryovial from liquid nitrogen storage and, working in a class II biosafety cabinet, place in the water bath, ensuring that all contents of the cryovial are immersed.
• 4Once thawed, transfer the contents of the cryovial to a 15‐ml centrifuge tube and slowly add 10 ml of medium with 10% heat‐inactivated horse serum.
• 5Centrifuge the tube for 10 min at 1000 × g, room temperature, to gently pellet the Blastocystis cells.
• 6Remove as much supernatant as possible without disturbing the pellet.
• 7Resuspend the pellet in 1 ml of the pre‐reduced, pre‐warmed medium, and then transfer the suspension to the pre‐reduced, pre‐warmed culture medium.
• 8Incubate at 37°C anaerobically and subculture every 3‐4 days.

  For anaerobic culture, we recommend using an anaerobic jar system, such as the Anoxomat III Anaerobic Jar System or the Oxoid AnaeroJar with BD GasPak EZ sachets.

COMMENTARY

Background Information

The protocols presented in this article represent a significant step toward gathering together all Blastocystis spp. culturing techniques and, thus, addressing one of the most challenging aspects of studying this enigmatic protozoan. By providing detailed methodologies for xenic and axenic cultivation, cryopreservation, and the preparation of various media, these tools aim to facilitate reproducibility and enhance the accessibility of Blastocystis spp. research across laboratories globally. These protocols also pave the way for large‐scale studies of Blastocystis spp. prevalence, ST distribution, and host‐microbe interactions, which are crucial for elucidating its role in health and disease.

Impact of current culturing protocols

The ability to culture Blastocystis spp. effectively has direct implications for multiple areas of research (Figueiredo et al., 2025). For microbiome studies, xenic cultures provide an opportunity to investigate interactions between Blastocystis spp. and other gut microorganisms, shedding light on its ecological role, which is particularly advantageous for epidemiological studies, where obtaining current circulating STs is important. The establishment of culturing media has allowed the extraction of numerous STs from biological STs, which can be used for potential future use in culturing (Lhotská et al., 2020). Although more technically demanding, axenic cultures are invaluable for characterizing this microbe at the molecular and cellular levels without interference from other microbial species. This is particularly critical for genomic, transcriptomic, proteomic, and metabolomic studies, which rely on pure cultures to generate high‐quality, ST‐specific data (Cao et al., 2024). By streamlining xenic cultivation and offering guidance for transitioning to axenic conditions, these protocols contribute to a deeper understanding of Blastocystis spp. biology and its impact on the host. In addition, it is essential to highlight the importance of long‐term preservation of cultures, for example in liquid nitrogen, to allow subsequent culturing at future times.

Need for more robust axenic culturing protocols

Despite these advances, the field faces significant gaps in the availability of protocols for axenic cultivation, particularly for STs beyond those already successfully agenized (Tsaousis et al., 2024). Multi‐omics approaches, including single‐cell transcriptomics and metabolomics, are increasingly recognized as essential tools for deciphering the complex biology of Blastocystis spp. and its interactions with the host and microbiome (Aykur et al., 2024). However, these techniques require consistent access to axenic cultures of diverse STs, which remains a significant bottleneck. Developing more robust, scalable, and ST‐inclusive axenization protocols is therefore a priority for future research.

Future directions in blastocystis culturing

Integrating state‐of‐the‐art technologies into Blastocystis spp. culturing as the field progresses represents an exciting frontier. Microfluidic platforms, such as gut‐on‐a‐chip technology, offer the potential to mimic the gut environment more precisely, providing controlled conditions for observing Blastocystis spp. behavior and growth dynamics in real‐time. Such systems can facilitate high‐throughput culturing and enable studies of environmental factors, such as oxygen gradients and nutrient availability, that influence Blastocystis spp. viability. Microfluidic devices may also aid in maximization efforts by isolating individual Blastocystis spp. cells and allowing for their growth in defined, sterile microenvironments.

Other innovative methods, such as organoid cultures or co‐culture systems with human intestinal epithelial cells, may provide further insights into Blastocystis‐host interactions, including identifying potential immune responses (Deng et al., 2021). These advanced approaches can simulate the host environment, enabling the study of host‐specific responses to Blastocystis spp. colonization and elucidating its potential pathogenicity.

Although the protocols presented here provide a strong foundation for Blastocystis spp. culturing, further advancements are needed to unlock the potential of this research field fully (Tsaousis et al., 2024). Developing more reliable axenization methods and adopting cutting‐edge technologies will be critical for addressing the current limitations and expanding the scope of Blastocystis spp. research. By continuing to refine and innovate these techniques, we can move closer to understanding the multifaceted roles of Blastocystis spp. in human and animal health, with implications for diagnostics, therapeutics, and public health strategies.

Critical Parameters

Although important for the study of Blastocystis, its culturing in vitro remains a challenge. The main obstacles involve the difficulty of keeping axenic cultures alive, and it is important to remember that different STs have different culturing requirements. Cautions to be aware of include the need to consciously limit the introduction of oxygen into samples to promote optimal growth and survival of the cultures. It is also important to be aware that the protocols detailed here are primarily focused on Blastocystis derived from human sources; when working with animal‐derived samples and STs of Blastocystis, adjustments to requirements, such as incubation temperature, may be necessary to reflect the host animal's physiology. It is recommended that all culturing of Blastocystis be undertaken in a category II biological safety cabinet to prevent contamination, and to prevent thermal shock, medium should always be pre‐warmed to the incubation temperature prior to culturing or subculturing, and pre‐reduced in the case of medium for axenic cultures.

Troubleshooting

A list of common problems, along with their likely causes and solutions, is provided in Table 2.

Table 2.

Common Problems That Can Occur When Culturing Blastocystis

Problem	Reason	Possible solution
IMDM cultures turning yellow	Most likely bacterial contamination, but may also be due to Blastocystis overgrowth	For bacterial contamination, discard the contaminated culture if there are spare stocks or cultures available. If not, treat with 1% (v/v) 10,000 U/ml penicillin/10 mg/ml streptomycin for at least two subcultures.
Axenic cultures not growing well in IMDM	Horse serum can differ depending on origin and supplier	Adjust the pH of complete IMDM to ∼7.6 before filtering can help.
Axenic cultures not growing well in IMDM	IMDM composition can differ depending on supplier	Supplement any missing components of IMDM powder as described in Basic Protocol 6.
Axenic cultures not growing well in IMDM	Possible Mycoplasma contamination	Eliminate the Mycoplasma infection using an elimination kit, or discard the contaminated culture if there are spare stocks or cultures available.
Cultures in glass tubes are not growing well	Remnants of soap inhibit the cultures	Glass tubes should not be washed with soap; instead, wash with 0.5% HCl, rinse with water, and autoclave.
Cultures in egg media are not growing well	Egg media are porous and may retain gas	Degas the egg solution thoroughly before aliquoting using gentle vacuum or an anaerobic chamber, and dispense the solution slowly to minimize bubble formation.
TYSGM‐9 not filtering	Gastric mucin can clog vacuum filters	First use a filter with a larger pore size to remove any particles not in solution from the mixture of dipotassium phosphate, monopotassium phosphate, sodium chloride, yeast extract, and gastric mucin. Then, add the serum and Tween‐80, and filter using a 0.22‐µm‐pore‐size filter.
Liquid xenic cultures of Blastocystis are not growing	Too much oxygen is getting into the culture	Fill culture tubes up to the top with liquid medium to reduce air and create a more microaerophilic environment.

Author Contributions

Daisy Shaw: Conceptualization; visualization; writing—original draft; writing—review and editing. Constance Denoyelle: Methodology; writing—review and editing. Kevin Tan: Methodology; writing—review and editing. C. Clark: Methodology; writing—review and editing. Hisao Yoshikawa: Methodology; writing—review and editing. Eric Viscogliosi: Methodology; writing—review and editing. Eleni Gentekaki: Methodology; supervision; writing—review and editing. Kateřina Jirků: Methodology; writing—review and editing. Anastasios Tsaousis: Conceptualization; funding acquisition; supervision; writing—original draft; writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Shaw, D. , Denoyelle, C. , Tan, K. S. W. , Clark, C. G. , Yoshikawa, H. , Viscogliosi, E. , Gentekaki, E. , Jirků, K. , & Tsaousis, A. D. (2025). Comprehensive tools for culturing blastocystis: A standardized resource for research and diagnostics. Current Protocols, 5, e70175. doi: 10.1002/cpz1.70175

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.