ABSTRACT

Objective

This study aimed to evaluate the efficiency of commonly available culture media in routine microbiology laboratories for the cultivation of Leishmaniatropica (L. tropica) promastigotes.

Methods

Various media including yeast extract agar, tryptic soy broth, sabouraud dextrose agar, brucella agar, and Columbia agar were tested. A total of sixteen media were prepared: eight blood-free (BY1-BY8) and eight supplemented with erythrocyte suspension blood-containing medium (KBY1-KBY8). Each medium was inoculated with L. tropica promastigotes at a concentration of 10⁵ promastigotes/mL and incubated for 12 days. Daily promastigote counts were performed to monitor growth.

Results

Among the tested media, BY7, BY8, KBY7, and KBY8 showed the most favorable growth patterns. In BY7 and BY8, the promastigote count increased from 10³/mL on day 1 to 10⁴/mL by day 5. BY7 supported continuous growth, reaching 10⁷ promastigotes/mL by day 8 and maintaining this level until day 12. BY8 peaked at 10⁵/mL on day 8 but declined to 10³/mL by day 12. KBY7 and KBY8 both demonstrated rapid growth, reaching 10⁷/mL by day 8 and sustaining this level through the end of incubation.

Conclusion

The presence of Columbia agar in BY7, BY8, KBY7, and KBY8 media significantly enhanced L. tropica promastigote proliferation. Due to its low cost, ease of preparation, and availability in routine laboratories, Columbia agar is proposed as a practical and effective alternative to the conventional Novy-McNeal-Nicolle medium for promastigote culture.

INTRODUCTION

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania, transmitted to humans through the bite of infected female phlebotomine sandflies. The World Health Organization estimates 700,000 to 1 million new cases annually, with 350 million people at risk (1). Classified as a neglected tropical disease (NTD), it disproportionately affects impoverished populations in developing countries and ranked second among all NTDs in 2017 in terms of disability-adjusted life years (DALYs), contributing to a global burden of 774,000 DALYs (2). The disease presents in three clinical forms: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis, with CL being the most frequently encountered form (3).

A wide range of diagnostic methods is employed for leishmaniasis. Microscopy remains the gold standard, owing to its high specificity. However, its sensitivity can be limited by factors such as insufficient sample volume, low parasite burden, smear preparation errors, and lack of technical expertise among laboratory personnel. In the case of VL, bone marrow biopsies are commonly used, since splenic biopsies—though highly sensitive—pose a significant risk of severe hemorrhage (4, 5). For CL, microscopic examination of parasites in skin lesions is a routine method, with reported sensitivity ranging between 42% and 70% (6).

Immunodiagnostic methods that detect Leishmania antigens or antibodies still play a fundamental role in diagnosis. Among antibody-based tests, immunochromatographic test (ICT) and enzyme-linked immunosorbent assay are widely used, employing antigens such as crude soluble antigen, recombinant K39 (rK39), and synthetic peptides in both commercial kits and in-house assays. The performance of these tests varies depending on the type and purity of the antigen, with rK39 ICT being a rapid and reliable method. However, the high cost of these tests and their limited applicability in all laboratories constitute a significant limitation in the diagnostic process (7, 8).

Molecular methods, such as direct DNA extraction from clinical samples, are also widely employed to detect Leishmania infection and determine strain types. However, PCR-based sequencing techniques can struggle to differentiate between closely related species, particularly when parasite DNA levels in the sample are low (9, 10). Recently, emerging technologies have started to complement traditional diagnostic methods. Promising innovations include nanoparticles, nanobiosensors, and novel point-of-care platforms such as breath analysis, portable PCR, nanotechnology-assisted rapid tests, next-generation sequencing, immunoinformatics, and artificial intelligence-based diagnostic systems (7).

Although clinical findings contribute to diagnosis, definitive identification of leishmaniasis relies on the direct detection of the parasite in clinical specimens and/or the cultivation of promastigotes using appropriate culture techniques. These methods are considered diagnostic gold standards, making culture media critical to the process. In 1904, Novy, McNeal, and Nicolle successfully cultured the promastigote form in a biphasic medium, which they named Novy-McNeal-Nicolle (NNN). This medium remains in widespread use today (11).

Culturing the parasite not only enhances diagnostic precision but also yields clinical isolates that facilitate advanced genetic studies-such as analyses of parasite evolution, lineage tracing, hybridization events, and identification of drug resistance genes. Expanding the collection of clinical isolates thus represents a valuable resource for Leishmania research.

This study aimed to achieve rapid and efficient cultivation of Leishmania tropica (L. tropica) promastigotes using culture media that are readily available in routine microbiology laboratories.

METHODS

Ethical Approval

No human or animal materials were used in this study. The experiments were conducted using parasite strains preserved in liquid nitrogen. Therefore, ethical approval is not required.

Parasite Isolate

For the evaluation of the modified media, the L. tropica strain MHOM/TR/2012/CBCL-LT, obtained from the Parasitology Bank of the Manisa Celal Bayar University Faculty of Medicine was used.

Preparation of NNN Medium

1.4 g of agar and 0.6 g of sodium chloride were dissolved in 90 mL of distilled water and sterilized in an autoclave (Hirayama HG-80, Japan) at 121 °C for 15 minutes. After cooling to 50-55 °C, 10 mL of defibrinated rabbit blood and 0.2 mL of penicillin/streptomycin solution were added to the medium. Then, 4 mL of the prepared medium was poured into sterile screw-cap tubes, which were placed at a 10° angle to allow solidification of the medium. The tubes were stored at +4 °C until use.

Preparation of Roswell Park Memorial Institute-1640 (RPMI-1640) Stock Liquid Medium

Commercially obtained RPMI-1640 was supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 1% gentamicin to prepare the stock liquid medium. The medium was stored at +4 °C until use.

Preparation of Modified Culture Medium

To formulate the solid phase of the modified media, culture media commonly available in routine microbiology laboratories were used, including yeast extract agar (113116, Merck), tryptic soy broth (TSB) (100525, Merck), sabouraud dextrose agar (SDA) with 2% glucose (110413, Merck), brucella agar (110490, Merck), and columbia agar (100214, Merck). Each medium was prepared in slant tubes following the manufacturer’s instructions.

A total of sixteen media formulations were created:

Eight blood-free modified media (BY1-BY8)

Eight blood-containing modified media (KBY1-KBY8)

Human erythrocyte suspension was added to the blood-containing media. For the liquid phase, either normal saline or RPMI-1640 was used. None of the media contained FCS. To prevent bacterial contamination, 1% penicillin/streptomycin and 1% gentamicin were included in all media formulations.

Inoculation into Modefied and NNN Medium

The strain retrieved from liquid nitrogen was thawed in a water bath at 37 °C (Memmert WBN-10, Germany) and inoculated into NNN medium for growth. The tubes were then incubated at 26 °C (Miprolab MSI-120, Türkiye). Upon observation of promastigote proliferation, the culture was transferred to RPMI-1640 liquid medium for further analysis. To obtain a sufficient number of promastigotes, fresh RPMI-1640 medium was added to the culture flasks every 2-3 days. L. tropica promastigotes were cultured until the logarithmic growth phase was reached with invert misroscope (Olympus CKX-41, Japan). Subsequently, the parasites were adjusted to a concentration of 10⁵ promastigotes/mL for inoculation into the test media. The modified culture media were incubated at 26 °C for 12 days.

Promastigotes in all tested media were counted daily using a Thoma hemocytometer under 40× magnification with a light microscope (Zeiss Primo Star, Germany). The results were compared with those obtained from the reference NNN medium. All experiments were repeated three times on independent days. All aseptic procedures included in the experimental design of the study were carried out in biosafety level II cabinets (Miprolab, Türkiye) while wearing personal protective equipment. The detailed compositions of the prepared media are provided in Table 1.

Statistical Analysis

All statistical analyses were performed using Python 3.10 with the pandas, numpy, scipy, and matplotlib libraries. The growth data of L. tropica promastigotes were collected over 12 consecutive days for each culture medium. The Shapiro-Wilk test was used to assess the normality of data distribution. As the data did not follow a normal distribution, non-parametric tests were employed. The Kruskal-Wallis test was initially used to assess overall differences among groups. For selected media (BY7, BY8, KBY7, and KBY8), pairwise comparisons with the standard NNN medium were conducted using the Wilcoxon signed-rank test. P-values less than 0.05 were considered statistically significant. Graphical representations of promastigote growth were plotted on a logarithmic scale, and the most successful media were visually emphasized using distinct colors and symbols.

RESULTS

No promastigote growth was observed in BY1, BY2, KBY1, and KBY2 media, and no viable promastigotes were detected in these media even as early as day 2. While no growth occurred in BY4 medium, in BY3 medium, 10² promastigotes/mL were counted on day 4, and 10³ promastigotes/mL on day 5. After day 5, the promastigote count gradually declined, and by day 12, no live promastigotes were observed. In KBY3 and KBY4 media, the initial promastigote concentration of 10²/mL on day 1 peaked at 10⁵/mL on day 7, then gradually declined to 10³/mL and 10¹/mL, respectively, by day 12. In BY5 medium, the initial count of 10² promastigotes/mL on day 1 increased to 10⁵/mL by day 6 and remained stable at this level until day 12. In BY6 medium, the promastigote count increased from 10²/mL on day 1 to 10³/mL on day 2, then progressively declined, with no promastigotes detected by day 9. In KBY5 and KBY6 media, the promastigote count started at 10³/mL on day 1, peaked at 10⁷/mL and 10⁵/mL, respectively, by day 8, and then declined to 10⁴/mL (KBY5) and 10²/mL (KBY6) by day 12.

In BY7 and BY8 media, the initial count of 10³ promastigotes/mL on day 1 increased to 10⁴/mL by day 5. In BY7, the count reached 10⁷/mL by day 8 and remained stable until day 12 (p=0.801). In BY8, 10⁵ promastigotes/mL were observed on day 8, decreasing to 10³/mL by day 12 (p=0.798). In both KBY7 and KBY8 media, the promastigote count started at 10³/mL on day 1, rose to 10⁷/mL by day 8, and remained stable through day 12 (p=0.987 for both KBY7 and KBY8). In the control NNN medium, the initial promastigote concentration of 10³/mL on day 1 increased to 10⁷/mL by day 7 and then declined to 10⁵/mL by day 12. Promastigote counts in blood-containing and blood-free media over time are presented in Table 2 and Table 3, and their graphical representations are shown in Figure 1 and Figure 2.

Statistical comparison was performed using the Wilcoxon paired-samples test. No statistically significant difference was observed in promastigote counts between the modified media and the NNN medium (BY7: p=0.139, BY8: p=0.110, KBY7: p=0.110, KBY8: p=0.110, false discovery rate-adjusted). These results suggest that both blood-free (BY7, BY8) and blood-containing (KBY7, KBY8) formulations exhibited a comparable level of promastigote growth to the standard NNN medium. Furthermore, BY7 and KBY7 demonstrated the highest yield, reaching 10⁷ promastigotes/mL by day 8 and maintaining this level through day 12. Based on these findings, the modified culture media BY7-BY8 and KBY7-KBY8 can be proposed as reliable alternatives to the traditional NNN medium for the in vitro cultivation of L. tropica promastigotes.

DISCUSSION

In the diagnosis of leishmaniasis, confirmation can be achieved either by demonstrating the parasite through various staining methods or by culturing promastigote forms. Culturing promastigotes not only supports clinical diagnosis but also enables the acquisition of a large number of parasites for scientific studies. These cultured parasites are essential for applications such as animal model development, vaccine research, elucidation of disease pathogenesis, evaluation of immune responses, development of diagnostic kits, screening of drug candidates, and assessment of therapeutic efficacy.

Over the years, various culture media have been developed for cultivating Leishmania species, primarily categorized as biphasic (semi-solid) and monophasic (liquid) types. Commonly used media, including RPMI-1640, Medium 199, brain heart infusion, NNN, and Schneider’s Drosophila medium, often require supplementation with fetal bovine serum (FBS) or blood to support long-term proliferation of Leishmania promastigotes (12, 13). FBS contains crucial components such as hormones, vitamins, growth factors, and carrier proteins, all of which are vital for the maintenance and propagation of cultured parasites (14).

Among these, the NNN medium remains the most widely used and accepted for both diagnostic and research purposes. While it is relatively easy to prepare, its reliance on rabbit blood and costly FBS limits its accessibility in routine microbiology laboratories. Therefore, recent years have seen a growing interest in developing cost-effective, serum-free, and autoclavable alternative media (15, 16).

The use of easily accessible and cost-efficient media in routine microbiology laboratories can significantly improve the detection of rare leishmaniasis cases, especially in non-endemic regions. In this context, various studies in Türkiye have investigated the potential of alternative biphasic and liquid culture media. In 1997, Limoncu et al. (17) compared a liquid P-Y medium supplemented with 10% FBS (containing peptone and yeast extract) with NNN and RPMI-1640+10% FBS media, reporting comparable results. Similarly, Ozbilgin et al. (18) observed promastigotes in nutrient broth medium containing 20% FBS as early as day 1, whereas detection in NNN medium began on day 2. Yereli et al. (19) also successfully cultivated L. infantum and L. tropica promastigotes in nutrient broth enriched with 10% FBS, reaching 3×10⁶ parasites/mL by day 5. However, the use of FBS in these media diminishes their cost advantage.

Internationally, several research groups have proposed alternative methods as well. Ali et al. (20) developed an egg-based biphasic medium free of FBS and blood, showing comparable proliferation to modified Tobie + FBS and Medium 199 + FBS. Kaddu and Nyamori (21) successfully cultured L. donovani, L. major, L. adleri, and a rodent Leishmania species in nutrient broth. Sadigursky and Brodskyn (22) reported similar promastigote proliferation in a serum-free liver infusion broth + tryptose (LIT) medium supplemented with 1% RPMI + Medium 199 (R9), compared to NNN, Warren medium, and LIT + 5% FBS. As also emphasized in a recent review, a major challenge in Leishmania culture remains the absence of a standardized, inexpensive, easy-to-prepare, and high-performance medium suitable for drug sensitivity testing (23).

In this study, the selected culture media are widely used in routine microbiology laboratories and serve different purposes in microbial cultivation. Yeast extract agar provides essential vitamins and nitrogen sources for the growth of various bacteria, yeasts, and molds. TSB supports the growth of both aerobic and anaerobic bacteria such as Staphylococcus aureus, Escherichia coli and Bacteroides fragilis. SDA, characterized by its high glucose content and slightly acidic pH, is ideal for the isolation of pathogenic fungi like Candida albicans. Brucella agar promotes the cultivation of fastidious and anaerobic microorganisms. Columbia agar, enriched with casein, heart, and meat peptones, provides a rich base for the growth of demanding bacteria such as Streptococcus and Haemophilus species (24).

In the our study, we evaluated the potential of routinely available microbiological media for the cultivation of L. tropica promastigotes. Compared to the NNN medium, the fastest and highest levels of proliferation were observed in Columbia agar-based BY7/BY8 and their blood-containing variants KBY7/KBY8. BY7 and BY8 media, which do not contain FBS or blood, achieved statistically comparable growth levels to NNN medium (p=0.139 for BY7, p=0.110 for BY8). This renders them advantageous in terms of cost and ease of preparation. Similarly, KBY7 and KBY8 media demonstrated comparable performance (p=0.110 for both). The use of human erythrocytes, which can be obtained from blood banks, instead of animal blood, adds practical utility. Additionally, promastigotes cultured in these media exhibited greater motility and typical fusiform morphology, suggesting preservation of their infective potential.

The enhanced growth observed in Columbia agar-based media (BY7, BY8, KBY7, KBY8) may be attributed to the balanced nutrient composition and favorable oxygen diffusion of the biphasic structure. Peptones, yeast extract, and maize starch provide essential amino acids, vitamins, and energy sources that support continuous promastigote metabolism, while the semi-solid matrix ensures optimal gas exchange similar to the sand fly midgut environment. In blood-containing variants, erythrocytes may further contribute to redox balance and iron availability, promoting sustained proliferation comparable to the NNN medium.

These culture media, which are easy and inexpensive to prepare and support high promastigote production, present a promising potential for anti-leishmanial drug screening when redesigned with viability-indicating colorimetric markers and supplemented with defined drug dilutions. Furthermore, the integration of such media into future automated analysis systems could facilitate the standardization of drug efficacy testing and enable the development of high-throughput screening platforms.

Study Limitations

The most notable limitation of this study is the lack of evaluation of parasite growth directly from clinical specimens in the high-performing media. Future studies are planned to assess the diagnostic potential of these media using both animal models and clinical samples.

CONCLUSION

Columbia agar (base) offers a highly supportive growth environment due to its rich composition of peptones derived from casein, meat, and heart tissue, along with yeast extract and maize starch. Yeast extract serves as a vitamin source, while maize starch contributes as an energy source. These nutritional components likely contributed to the enhanced proliferation of L. tropica promastigotes observed in the BY7, BY8, KBY7, and KBY8 media. Columbia agar stands out as a strong alternative for such applications, as it is inexpensive, easy to prepare, and readily available in routine microbiology laboratories. Therefore, it can be recommended as a practical and effective substitute for conventional NNN medium in promastigote cultivation.