Advances in the axenic isolation methods of Blastocystis sp. and their applications

Abstract Abstract Blastocystis sp. is a prevalent protistan parasite found globally in the gastrointestinal tract of humans and various animals. This review aims to elucidate the advancements in research on axenic isolation techniques for Blastocystis sp. and their diverse applications. Axenic isolation, involving the culture and isolation of Blastocystis sp. free from any other organisms, necessitates the application of specific media and a series of axenic treatment methods. These methods encompass antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined use of culture media. Critical factors influencing axenic isolation effectiveness include medium composition, culture temperature, medium characteristics, antibiotic type and dosage and the subtype (ST) of Blastocystis sp. Applications of axenic isolation encompass exploring pathogenicity, karyotype and ST analysis, immunoassay, characterization of surface chemical structure and lipid composition and understanding drug treatment effects. This review serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.


Introduction
Blastocystis sp. is a common intestinal protist found in humans and animals (Udonsom et al., 2018), and is distributed worldwide, encompassing both urban and rural settings across developed and developing countries (Scanlan et al., 2014;Udonsom et al., 2018).In Poland, infection rates range from 0.14 to 23.6% across various study groups from 1955 to 2022 (Rudzińska and Sikorska, 2023).This figure, meanwhile, often exceeds 50% in developing countries and even 100% among children in Senegal River Basin (Alfellani et al., 2013;El Safadi et al., 2014;Popruk et al., 2021).Blastocystis sp. is transmitted through the fecal-oral route, and people or animals are infected by ingesting contaminated food or water (Maloney et al., 2019).Although some cases are asymptomatic, Blastocystis sp.infection is closely linked to conditions such as irritable bowel syndrome, terminal ileitis and ulcerative colitis, manifesting mainly as abdominal pain, diarrhoea, flatulence and constipation (Tunalı et al., 2018;Basuony et al., 2022).
The morphological diversity of Blastocystis sp.includes vacuolar, granular, amoebic, cyst, avacuolar and multivacuolar forms (Stenzel and Boreham, 1996).Among these, the vacuolar form is the most prevalent and typical form of Blastocystis sp., which is often used as the classical morphological standard of Blastocystis sp.infection.However, the presence of abundant associated bacteria poses challenges for ultrastructural observation of Blastocystis sp.through an electron microscope (Zierdt and Williams, 1974).
Isolating Blastocystis sp. from fecal cultures often introduces bacterial contamination, rendering many animal experimental results unacceptable or leading to inconsistencies across studies.Axenic strains, free from contaminating organisms, facilitate more scientific and persuasive in-depth studies on various aspects of Blastocystis sp., such as karyotype modelling, antigen analysis, enzyme activity and surface biochemical components, thereby advancing our understanding of the biological aspects of Blastocystis sp.(Lanuza et al., 1996b).Since the successful axenic culture of Blastocystis sp. by Zierdt and Williams in 1974, numerous scholars have attempted axenic isolation methods.This paper builds upon previous studies, reflecting a deep understanding of Blastocystis sp. and the significance of its axenic culture.The review encompasses presently available axenic isolation techniques and factors influencing their applicability, emphasizing the importance of axenic isolation for studying Blastocystis pathogenicity, describing its surface biochemical components and elucidating therapeutic effects.The content serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.
The axenic cultivation of Blastocystis sp.
Axenic cultivation of Blastocystis sp.involves the culture of Blastocystis sp.free from contamination with other organisms.This necessitates the application of a suitable culture medium and a series of axenic treatment methods, with the selection of the optimal culture medium being crucial (Tan et al., 2000).An efficient and economical medium for isolating Blastocystis sp.serves as the foundation for subsequent studies on factors such as its life history, morphological classification and infection immunity (Tan, 2008).Axenic methods include antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined application of culture medium.Obtaining axenic Blastocystis sp.often requires the combined application of different methods, presenting a promising avenue for further research.
Commonly used media for the axenic isolation of Blastocystis sp.
The in vitro culture of Blastocystis sp.involves the utilization of monophasic and biphasic media.The biphasic medium comprises 2 media with distinct physical states, while the monophasic medium consists of only 1 physical medium.Based on physical state, the medium is categorized as solid, liquid and solid-liquid biphasic.The solid medium contains agar, and the liquid medium includes Jones medium, Iscove's modified Dulbecco's medium (IMDM) and Dulbecco's modified Eagle medium (DMEM).Typically, a single-phase medium is prepared by sequentially adding necessary raw materials, sterilizing the mixture under high pressure and then packaging it in suitable volumes according to experimental requirements.Biphasic medium includes Locke's egg serum (LES) medium and Boeck-Drbohlav biphasic modified medium (BDMM), composed of solid and liquid components.The preparation involves configuring the solid phase first, followed by the liquid phase and finally covering the solid medium's surface with the liquid medium.Table 1 provides details of specific culture media.
Methods for the axenic isolation of Blastocystis sp.

Antibiotic treatment
Antibiotic treatment has been a key approach for the axenic isolation of Blastocystis sp.since the seminal study by Zierdt and Williams (1974).The inclusion of antibiotics in the culture medium (Fig. 1) has become a widely employed method, although it presents challenges.While antibiotics contribute to asepsis, complete elimination of bacteria may not be achieved due to the potential development of antibiotic resistance among the microbial population.It is important to note that low bacterial counts might be mistakenly regarded as axenic (Ng and Tan, 1999).Ampicillin and streptomycin combination stands out as a straight forward and effective addition, proving capable of eliminating a significant portion of bacteria (Teow et al., 1991(Teow et al., , 1992)).Additionally, amphotericin is employed to target yeasts and moulds, common contaminants in the initial culture stages (Zierdt and Williams, 1974).Ceftizoxime and vancomycin can eliminate drug-resistant bacteria (Zierdt, 1991).Despite the benefits of antibiotic treatment in achieving asepsis, it comes with the potential drawback of inhibiting parasite growth, with alterations to the size and vacuole content of Blastocystis sp.(Teow et al., 1992).It is noteworthy that the introduction of antibiotics can impact the longevity of Blastocystis sp., with signs of decline observed after the third passage in a chloramphenicol-containing medium (Zierdt et al., 1983).The strategic addition of antibiotics poses a delicate balance, as the direct use of a mixture of multiple antibiotics may result in insufficient growth of Blastocystis sp., while the sequential addition of antibiotics, 1 at a time, has proven beneficial for promoting growth (Lanuza et al., 1996b).

Monoclonal culture
Agar cloning technology plays a pivotal role in achieving the physical separation of Blastocystis sp. from bacteria, ensuring the asepsis of Blastocystis sp.(Valido and Rivera, 2007) (Fig. 2).
Monoclonal culture, in the presence of certain bacteria, facilitates the distinct separation of pure Blastocystis sp.colonies from bacterial colonies.The pioneering work of Tan et al. marked the cultivation of axenic Blastocystis sp.colonies in Petri dishes containing soft agar, leading to the successful cultivation of a substantial number of amoebic Blastocystis sp.(Tan et al., 1996a).In the same year, Tan et al. introduced sodium thioglycolate to enhance the colony formation efficiency (%CFE = the number of growing colonies/inoculated cells × 100), resulting in the abundant production of Blastocystis sp. from a single clone (Tan et al., 1996b).The monoclonal culture was further applied by Ng and Tan to strains treated with antibiotics, leading to the successful isolation of pure Blastocystis isolates (Ng and Tan, 1999).
The advantage of monoclonal culture lies in the physical separation of Blastocystis sp.colonies and bacterial colonies, with bacterial colonies growing in 0.36% Bacto agar easily distinguishable from the diffuse Blastocystis sp.colonies visible under an inverted microscope.However, this method was associated with the following drawbacks: (1) counting colonies becomes challenging due to the semisolid nature of agar; (2) cloning and growth of Blastocystis sp. in agar necessitate various nutrients, requiring the preparation of fresh culture medium and (3) extracting Blastocystis sp.colonies proves difficult as they are generally embedded in agar.To address these challenges, Tan et al. introduced a simplified and efficient method for clonal growth on solid agar, increasing the agar content from 0.36 to 2%.This modification allowed the successful culture of Blastocystis sp.colonies on the agar surface, facilitating their isolation for further studies (Tan et al., 2000).It is crucial to note that abundant bacterial colonies can impede the identification and isolation of Blastocystis sp.colonies.Therefore, antibiotic treatment is deemed necessary in monoclonal culture to reduce the bacterial population and aid in the isolation of Blastocystis sp.colonies (Ng and Tan, 1999;Valido and Rivera, 2007).

Differential centrifugation technique
Teow et al. employed the differential centrifugation technique in conjunction with antibiotics to eliminate the associated bacteria from 8 Blastocystis sp.isolates (Teow et al., 1992) (Fig. 3).The procedure involved centrifuging Blastocystis sp. in the logarithmic growth phase at 3000 g for 15 min, followed by resuspension in ∼10 mL of phosphate-buffered saline (PBS, pH 7.0) containing 4000 μg mL −1 ampicillin and 1000 μg mL −1 streptomycin.Subsequently, the suspension underwent centrifugation at 1000 g for 15 min, the supernatant was discarded and 10 mL of PBS containing antibiotics was added.This entire process was repeated twice.However, limited studies have reported the axenic isolation of Blastocystis sp. using differential centrifugation, possibly attributed to the fragility of Blastocystis sp. cells (Zierdt and Williams, 1974;Teow et al., 1992).

Density gradient separation
To isolate Blastocystis sp. from bacteria, density gradient separation has proven effective (Fig. 4).Upcroft et al. utilized the Ficoll-Paque gradient method to purify Blastocystis sp. and eliminate more than 75% of the bacteria, although complete sterility was not attained (Upcroft et al., 1989).Lanuza et al. purified Blastocystis sp. using the Ficoll-metrizoic acid solution and subsequently inoculated them into a fresh medium containing active antibiotics against residual bacteria, thus achieving sterility (Lanuza et al., 1996b).Defaye et al. reported a method for purifying Blastocystis sp.cysts from feces, employing the Percoll gradient method and an antibiotic mixture to reduce the number of bacteria inoculated per animal to less than 250 colonies (Defaye et al., 2018).See Table 2 for details.

Parasitology
Other axenic isolation methods Hess et al. employed a micropipette, that is, micromanipulation, to isolate Blastocystis sp. from turkey caecal contents for cloning, offering a step towards asepsis (Hess et al., 2006).Shin et al. successfully established an axenic pure culture system for Blastocystis sp. using LES biphasic medium, mLES biphasic medium, liquid IMDM and solid IMDM (Shin et al., 2017).Furthermore, several reports have documented the axenic culture of Blastocystis sp. from diverse animal species, including rats, snakes, lizards and crocodiles (Teow et al., 1991(Teow et al., , 1992;;Chen et al., 1997;Ng and Tan, 1999).Table 3 provides a summary of axenic isolation methods specific to each animal.
Factors affecting the axenic isolation of Blastocystis sp.

Composition of the culture medium
The composition of the culture medium significantly influences the axenic isolation of Blastocystis sp., with components such as growth factors, glucose, mineral salt and sodium thioglycolate   4) isovitalex (1%) and ( 5) haemin (5 μg mL −1 ).
Given the strict anaerobic growth requirements of Blastocystis sp., the addition of sodium thioglycolate to the axenic culture medium enhances anaerobic conditions, improving colony growth and yield (Tan et al., 1996a).Furthermore, the combined application of various media was conducive to the axenic culture of Blastocystis sp.Optimization of the media composition, such as enhancing the traditional LES biphasic medium with agar replacement protein, simplifies preparation and reduces contamination risks (Shin et al., 2017).

Culture temperature
The optimal growth temperature for Blastocystis sp. in axenic culture is 37°C, and attempts to purify Blastocystis sp. in Diamond's TPS-1 medium (The components of TPS-1 medium contained 5 g Trypticase, 10 g Panmede, 2.5 g D-glucose, 0.5 g L-cystein HCl, 0.2 g Ascorbic acid, 2.5 g NaCl, 0.3 g K 2 HPO 4 , 0.5 g KH 2 PO 4 , 50 mL Bovine serum and 435 mL Distilled water) at 28°C have been unsuccessful (Molet et al., 1981).Notably, Blastocystis lapemi sp.nov.has an optimal growth temperature of 24°C, and attempts to adapt from 24 to 37°C resulted in the gradual demise of B. lapemi sp.nov.above 28°C (Teow et al., 1991).Blastocystis isolated from land turtles, iguanas, crocodiles and pythons were successfully axenized at 24°C (Teow et al., 1992), and rats were axenized at 37°C (Chen et al., 1997).It is highly conceivable that the variation in axenic culture temperature for Blastocystis sp. is linked to the different body temperatures of their respective animal hosts.

Culture medium character
Axenic culture of Blastocystis sp. can be achieved using both monophasic and biphasic media (Tan et al., 2000).Compared with the monophasic medium, biphasic medium preparation is complicated and prone to bacterial contamination.

Types and doses of antibiotics used
Due to Blastocystis sp.inhabiting the intestinal tract, the isolation and culture process often involves a mix of unknown species and drug-resistant bacteria.The combination of 1-2 antibiotics at conventional doses may prove ineffective against bacteria due to multiple drug resistance caused by antibiotic abuse (Li et al., 2022).In such instances, considering larger doses of drugs along with physical separation is suggested to achieve asepsis.

Other factors affecting axenic isolation
Axenic culture may be related to the frequency of inoculation, and some bacteria will decrease with each subsequent inoculation until   (Tan et al., 2000).The morphology of Blastocystis sp. may also influence axenic culture, with an increase in granular forms of Blastocystis being closely related to a significant increase in bacterial count (Zierdt and Williams, 1974).
Applications of the axenic isolation of Blastocystis sp.
Application of the axenic isolation techniques to study the pathogenicity of Blastocystis sp.
The application of axenic isolation techniques is crucial for studying the pathogenicity of Blastocystis sp., which is implicated in various gastrointestinal diseases and may play a role in irritable bowel syndrome (Ismail et al., 2022).Valsecchi et al. showed that the amoebic form of Blastocystis sp.causes urticaria by affecting intestinal immune homoeostasis (Valsecchi et al., 2004).In the study conducted by Moe et al., the injection of axenic Blastocystis sp.isolate B into mice resulted in the discovery of vacuolar and granular parasites in the caecum.Histological examination of the caecum and colon revealed significant inflammatory cell infiltration, lamina propria oedema and mucosal exfoliation (Moe et al., 1997).Intramuscular injection of the same strain led to extensive necrosis and interstitial oedema in the muscle, accompanied by polymorphonuclear leucocyte infiltration (Moe et al., 1998).Puthia et al. observed that the lysate of the pure cultured Blastocystis ST4 WRl isolate induced apoptosis of rat intestinal epithelial cell-6 (IEC-6), altered the distribution of F-actin, reduced epithelial resistance and increased epithelial permeability of the IEC-6 monolayer (Puthia et al., 2006).Furthermore, Ajjampur et al. demonstrated that axenic Blastocystis sp. could degrade mucin in various segments of the large intestine, consequently impacting the mucin barrier (Ajjampur et al., 2016).The observed pathological conditions in the host may be linked to the disruption of in vivo balance by specific isolates or rare subtypes (STs).Yason et al. reported that specific axenic isolates of Blastocystis ST7 could reduce the abundance of Bifidobacterium and Lactobacillus (Yason et al., 2019).Importantly, it is emphasized that studies on the pathogenicity of Blastocystis sp.ideally should be based on axenic isolation for accurate assessments.

Application of axenic cultures in karyotype and ST analysis of
Blastocystis sp.
To facilitate accurate karyotype and ST analysis of Blastocystis sp., Upcroft et al. recommended isolating Blastocystis sp. from the associated bacteria, as the presence of bacteria in non-axenic cultures could potentially mask the detection of chromosomes (Upcroft et al., 1989).Karyotype analysis of Blastocystis sp.isolated from various hosts, such as sea snakes, rats, tortoises, iguanas and pythons, revealed independent isolates, leading to the designation of distinct species, namely B. lapemi sp.nov., Blastocystis ratti sp.nov., Blastocystis geocheloni sp.nov., Blastocystis cycluri sp.nov.and Blastocystis pythoni sp.nov.respectively (Teow et al., 1991;Singh et al., 1996;Chen et al., 1997).However, Ho et al. found that the karyotypes of different axenic isolates of the same host were not identical, and there were marginal differences in chromosome bands (Ho et al., 1994).
The classification of the above-mentioned Blastocystis sp.species, isolated from non-human hosts, relied on electrophoretic karyotype as the criterion for defining new species.However, due to the karyotype heterogeneity of some protozoa, this standard may be insufficient to explain the species formation of Blastocystis sp.Host specificity and pathogenicity of different isolates have been correlated with sequence variations in small subunit-ribosomal ribonucleic acid (SSU-rRNA), leading to the division of the genus into 28 STs (Noël et al., 2003;Parija and Jeremiah, 2013;Martiny et al., 2014).Nineteen axenic isolates of Blastocystis sp. and 9 isolates containing bacteria were analysed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Martiny et al., 2014).The 19 axenic isolates produced protein maps with good resolution, suggesting that MALDI-TOF mass spectrometry could effectively distinguish the STs of axenic strains (Martiny et al., 2014).However, the growth hindrance of Blastocystis sp. in the presence of bacteria and fungi resulted in correct identification for only 3 out of the 9 non-axenic isolates (Martiny et al., 2014).
Application of axenic cultures for immunological studies of Blastocystis sp.
Current immunological studies on Blastocystis sp.infection primarily focus on the detection of antibodies and antigens (Sheela et al., 2020).The potential pathogenic mechanisms of Blastocystis sp. can be preliminarily elucidated through in vitro co-culturing of live cells, lysates, secretions and soluble antigens.Notably, since bacteria can act as antigens and potentially react with cultured cells, the success of these in vitro experiments relies on the use of pure cultures of Blastocystis sp.(Deng and Tan, 2022).
Kukoschke et al. reported at least 2 different peptide patterns and antigenic variants of axenic Blastocystis sp.(Kukoschke and Müller, 1991).Müller (1994) divided 61 axenic isolates of Blastocystis sp.into 4 groups using immunodiffusion, while Lanuza et al. (1999) divided 18 axenic strains into 3 related patterns using sodium dodecyl sulphate-polyacrylamide gel electrophoresis.The findings of the present study corroborated Kukoschke's findings.Serum obtained from mice injected with 20 μg mL −1 soluble antigen of symptomatic and asymptomatic axenic Blastocystis ST3 demonstrated specific cleavage of the corresponding live Blastocystis sp.(Sheela et al., 2020).Serum obtained from mice with soluble antigen of symptomatic Blastocystis ST3 added to asymptomatic Blastocystis ST3, and vice versa, exhibited only 17% cross-reactivity, indicating significant epitope differences between symptomatic and asymptomatic Blastocystis ST3.These results also substantiate Kukoschke's report.However, it is essential to note that there are currently no validated cultures of axenic Blastocystis ST3, and Sheela et al. did not provide relevant evidence of the successful axenization of Blastocystis ST3 isolate.
Blastocystis sp.exerts regulatory effects on cells by modulating cytokine expression.Axenic Blastocystis sp.antigen induces the expression of interleukin (IL)-1β, IL-6 and tumour necrosis factor-alpha (TNF-α) in intestinal explants, colons and macrophages of mice (Lim et al., 2014).The axenic soluble antigen of Blastocystis sp. was introduced into peripheral blood mononuclear cells (PBMCs) and human colon cancer cells (HCT116) in a study by Chandramathi et al. (2010).The study revealed that the gene expressions of interferon-gamma (IFN-γ) and TNF-α were downregulated, while those of IL-6, IL-8 and nuclear factor kappa B (NF-κB) were upregulated in PBMCs.In HCT116, the gene expression of IFN-γ was significantly downregulated, while the expressions of IL-6 and NF-κB were upregulated, suggesting that the Blastocystis sp.antigen (at a specific concentration) has the potential to promote the growth of existing tumours or cancer cells (Chandramathi et al., 2010).
Recently, Deng et al. (2022) conducted a study where axenic Blastocystis ST4 was orally injected into healthy mice and dextran sodium sulphate (DSS)-induced colitis mice.The study revealed Parasitology that Blastocystis ST4 colonization altered the composition of the intestinal bacterial community healthy mice (without adverse effects) (Deng et al., 2022).Moreover, the colonization of Blastocystis ST4 induced T helper 2 and regulatory T cell immune responses in DSS-induced colitis mice, promoting the recovery of experimentally induced colitis mice (Deng et al., 2022).Under similar conditions, orally injecting axenic Blastocystis ST7 into DSS-induced colitis mice exacerbated the severity of colitis by increasing the proportion of pathogenic bacteria and inducing pro-inflammatory IL-17A and TNF-α-producing CD4+ T cells (Deng et al., 2023).
In a nutshell, the axenic isolation of Blastocystis sp.plays a crucial role in establishing a Blastocystis sp.-cell co-culture system in vitro and an animal model of Blastocystis sp.infection in vivo.These models serve as the foundation for immunological research.
The application of axenic cultures to study the surface chemical structure and lipid biochemical analysis of Blastocystis sp.
Studies on the surface chemical structure of Blastocystis sp.aid in understanding its pathogenic potential and nutritional impact (Yason et al., 2018).Axenic cultures offer the advantage of avoiding bacterial interference in experiments.Keenan et al. conducted the pioneering analysis of lipids in 6 axenic strains of Blastocystis sp., revealing that the phospholipid content of all strains constituted ∼39% of the total lipids (Keenan et al., 1992).The primary neutral lipid components were sterol esters, and the predominant polar lipid component was phosphatidylcholine across all strains.However, the study noted the challenge of determining whether the lipids were synthesized by the organisms or derived from the culture media (Keenan et al., 1992).Subsequent research by Keenan and Zierdt further demonstrated that axenic Blastocystis sp. can synthesize lipids and accumulate cholesterol and intact cholesterol esters directly from the growth medium (Keenan and Zierdt, 1994).Lanuza et al. employed lectin probes to identify specific carbohydrates within the surface coating of axenic Blastocystis sp., revealing that the structure contained α-D-mannose, α-D-glucose, N-acetyl-α-D-glucosamine, α-L-fucose, chitin and sialic acid (Lanuza et al., 1996a).In addition, the axenic Blastocystis ST7 isolate B was not directly destroyed by the antimicrobial peptide LL-37, which may be attributed to the fact that the thicker surface-coated Blastocystis STs are more resistant to osmotic pressure, extreme pH and oxygen exposure (Yason et al., 2016;Yason and Tan, 2018).
The application of axenic cultures to study the chemotherapy of Blastocystis sp.
Given the individual variations in treating Blastocystis sp. and the absence of a specific therapeutic drug, combination drugs have been employed in clinical treatments to enhance efficacy, reduce toxicity, minimize adverse reactions and delay the onset of drug resistance.The use of sterile blastocysts can effectively evaluate the effect of drugs.Metronidazole is a commonly used drug for Blastocystis sp.infection, induced apoptosis-like features in axenic Blastocystis sp. as observed by Nasirudeen et al. (2004).These features included nuclear condensation, nuclear DNA fragmentation, reduced cytoplasmic volume, phosphatidylserine externalization and increased cell membrane permeability.Similarly, staurosporine, an apoptosis-inducing agent, prompted similar apoptosis-like features in Blastocystis sp.(Yin et al., 2010).In vitro testing of 10 antiprotozoal drugs against axenic Blastocystis sp.revealed the order of efficacy as emetine, metronidazole, furazolidone, trimethoprim sulphamethoxazole, 5-chloro-8-hydroxy-7-iodoquinoline (enteric violine) and pentamidine (Zierdt et al., 1983).However, certain Blastocystis sp.isolates demonstrated resistance to metronidazole, which has potential mutagenic and carcinogenic effects (Nagel et al., 2014;Eida et al., 2016).Studies on Nigella sativa aqueous extracts revealed their inhibitory effect on axenic Blastocystis sp.Eida et al. found that N. sativa aqueous extracts at concentrations of 100 and 500 μg mL −1 exerted potent lethal effects on axenic Blastocystis sp.isolates in vitro and prevented cytopathic changes in infected mice orally inoculated with Blastocystis sp. in vivo (Eida et al., 2016).Yason et al. used the axenic Blastocystis isolates NUH9, WR1 and B and found good efficacy of diphenyleneiodonium chloride, auranofin and BIX 01294 trihydrochloride hydrate in treating Blastocystis sp.infection, particularly in the axenic Blastocystis ST7 isolate B, which was insensitive to metronidazole (Yason et al., 2018).
Application of axenic cultures for molecular biology of Blastocystis sp.
The application of axenic cultures in the molecular biology of Blastocystis sp. has played a crucial role in addressing the controversy surrounding the association between Blastocystis STs and its symptomatology (Stensvold et al., 2007).Denoeud et al. took a step by reporting the complete genome sequence of axenic Blastocystis ST7 isolates from a patient in Singapore.They proposed candidate genes for the study of physiopathology, opening avenues for comparative genomics with other STs and contributing to the development of typing tools for characterizing pathogenic isolates (Denoeud et al., 2011).
In the post-genomic era, genetic tools have become instrumental in obtaining genetically modified cells, providing essential insights into the function of Blastocystis genes.Li et al. achieved a milestone by establishing a robust gene delivery protocol using axenic Blastocystis ST7 isolates.Their study involved testing ectopic protein expression with a highly sensitive nano-luciferase reporting system (Li et al., 2019).The study identified a promoter that includes the upstream region of the 5 ′ untranslated region (UTR) in legumain gene.A robust transient transfection technique was established in Blastocystis by combining this promoter with the legumain 3 ′ UTR.This breakthrough technique lays a strong foundation for further investigations into the functions of key molecules in Blastocystis sp.

Conclusion
In summary, the axenic isolation of Blastocystis sp.provides a robust foundation for morphological, immunological, drug and molecular biology studies.Currently, the common approach among scholars involves the use of antibiotic treatment in conjunction with another isolation technique to achieve axenic Blastocystis sp.cultures.However, limited reports have explored the combined application of 3 or more axenic isolation techniques.Through an analysis of the pros and cons of various methods, it is posited that employing a combination of several axenic separation techniques proves more advantageous in obtaining pure Blastocystis sp.cultures (Fig. 5).Finally, it is strongly recommended that sufficient data are needed to provide supporting evidence for the successful axenization of Blastocystis sp. in future.This may involve presenting sterile plates to showcase the absence of bacterial growth, utilizing 16S polymerase chain reaction for bacterial detection, and supplying SSU-rRNA sequences for the confirmation of Blastocystis STs.

Figure 1 .
Figure 1.Antibiotic treatment for the axenic isolation of Blastocystis sp.

Figure 2 .
Figure 2. Monoclonal culture for the axenic isolation of Blastocystis sp.

Figure 4 .
Figure 4. Density gradient centrifugation for the axenic isolation of Blastocystis sp.P, passage.

Figure 5 .
Figure 5. Combination of multiple methods for the axenic isolation of Blastocystis sp.

Table 1 .
Commonly used media for aseptic isolationAgarEqual amounts of 2× IMDM and 0.72% agar mixed in a 50 mL centrifuge tube, and different final volumes obtained according to the experimental requirements; an appropriate amount of serum and Blastocystis sp.inoculation then added and cultured in an anaerobic tank at 37°C.Na 2 HPO 4 -9.46 g L −1 of distilled water, 31.3 mL KH 2 HPO 4 -9.08 g L −1 of distilled water, 562.5 mL NaCl-9 g L −1 of distilled water, 0.1% yeast extract are mixed and packed in 12 mL Falcon tubes; an appropriate amount of serum and Blastocystis sp.added and cultured under anaerobic conditions at 37°C.Locke's solution added to 30°egg slope medium, sterilized under high pressure; 20% sterile serum (human, horse, chicken, monkey and calf), 2 mL mineral oil covering layer, an appropriate amount of antibiotics added; medium placed in a CO 2 incubator and cultured at 37°C.

Table 2 .
Introduction of different density gradient methods

Table 3 .
Summary of the aseptic isolation methods from humans, rats and some reptiles they disappear completely.Centrifugation is employed to separate sp.from agar, creating favourable conditions for its growth.Tan et al. found that emulsifying inoculum in a small amount of pre-reduced IMDM enhances the cloning process, improving colony isolation