BioMed Research International

Volume 2014 (2014), Article ID 714173, 4 pages

http://dx.doi.org/10.1155/2014/714173

Research Article

Modified PEHPS Medium as an Alternative for the In Vitro Culture of Giardia lamblia

Javier Vargas-Villarreal,¹ Benito D. Mata-Cárdenas,² Magda E. Hernández-García,1,3 Jesús N. Garza-González,¹ Laura H. De La Garza-Salinas,⁴ and Francisco González-Salazar¹

¹Laboratorio de Bioquímica y Fisiología Celular, División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano Del Seguro Social, Administración de Correo No. 4, Apartado Postal 020-E Colonia Independencia, 64720 Monterrey, NL, Mexico

²Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, Avenida Universidad s/n, Ciudad Universitaria, 66451 San Nicolás de los Garza, NL, Mexico

³Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Avenida Pedro de Alba y Manuel L. Barragán s/n, Ciudad Universitaria, 66451 San Nicolás de los Garza, NL, Mexico

⁴Coordinador Auxiliar Medico, Instituto Mexicano Del Seguro Social, Delegación Nuevo León, Gregorio Torres de Quevedo No. 1950 Ote, 64000 Monterrey, NL, Mexico

Received 28 February 2014; Revised 28 April 2014; Accepted 13 May 2014; Published 21 May 2014

Academic Editor: Thean Hock Tang

Copyright © 2014 Javier Vargas-Villarreal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Commercial culture media present interlot variations in biological activity. We have previously designed a homemade and economic culture medium, PEHPS medium, for the axenic cultivation of Entamoeba histolytica and Trichomonas vaginalis. Trophozoites of amoebae and trichomonads grow well in this medium. Furthermore, the medium is stable for several months when stored frozen or refrigerated. The objective of this work was to modify PEHPS medium to support the in vitro growth of Giardia lamblia. Inocula of 5 × 10³ trophozoites/mL of G. lamblia were incubated at 36.5°C in modified PEHPS or TYI-S-33 medium. Then, the growths of the three Giardia strains in both media were compared. The logarithmic growth phase lasted 72 h; the mean yield of the strains ranged from 10.06 to 11.43 × 10⁵Giardia trophozoites/mL, and the range of duplication time in the three strains was from 5.67 to 6.06 in modified PEHPS medium. These growth characteristics were not significantly different from those obtained with TYI-S-33 medium. We conclude that modified PEHPS medium might be used for the axenic cultivation of G. lamblia.

1. Introduction

Several culture media have been developed to facilitate the in vitro study of microorganisms. Ideally, the growth and development of the parasites cultivated in these media should be similar to those in their natural habitat [1]. In order to introduce new culture media for the cultivation of protozoa, their applicability for diagnosis and their biochemical and immunological characteristics should be revised [2]. Protozoan flagellate Giardia lamblia has been recognized as an important cause of diarrhea and malnutrition in the world [3–6], and its study has been facilitated by the design of several growth media, including the manufactured TYI-S-33 [7]. On the other hand, PEHPS medium was designed in our laboratory more than 25 years ago [8]. This medium was designed by the need to create a homemade, economic medium without the interlot variability present in manufactured culture media, ranging from perfect growth in one lot to hardly any growth in another [1]. The PEHPS was developed from extracts of liver and pancreas obtained from beef and pork and can be produced in any laboratory. With appropriate storage, PEHPS medium lasts up to 3 years without losing its efficiency [9].

This culture medium has been used to grow Entamoeba histolytica and Trichomonas vaginalis with results equal to those observed with manufactured culture media [10, 11]. In addition, these microorganisms, when grown in PEHPS medium, retain the activity of virulence factors. PEHPS medium also supports the synthesis of cell duringaxenic encystment in E. histolytica [12].

The development of culture media is definitely not a diagnostic tool, since there are diagnostic methods that are more efficient and faster than cultivation. However, the development of new means of cultivation for intestinal protozoan allows learning about the biology of parasites, their growth rate, virulence factors, susceptibility to new drugs, and the development of resistant strains among others [1].

Modifications of the PEHPS medium have been realized to optimize culture conditions and eliminate possible inhibitory factors [13].

In this study we provide evidence that G. lamblia trophozoites grow and develop in modified PEPHS medium as they do in manufactured TYI-S-33.

2. Material and Methods

G. lamblia strains IMSS:0989, WB, and IMSS:3 were kindly donated by Dr. Roberto Cedillo Rivera from the Centro Médico Nacional Siglo XXI, México City [14]. The parasites were maintained in axenic conditions at 36.5°C by serial subcultivation. Briefly, trophozoites/mL were placed in  mm borosilicate screw-capped tubes containing 5.6 mL of TYI-S-33 described by Keister [7]; they were then harvested and reseeded every 72 h.

For assays, the parasites were maintained in axenic conditions during 30 days at 36.5°C by serial subcultivation in TYI S 33 [7] or in modified PEHPS medium. Modified PEHPS medium was prepared by supplementing standard PEHPS medium, described by Said-Fernandez et al. [9] (Table 1), with 0.6 g/L bovine bile (Sigma-Aldrich, St Louis, MO, USA) and 10% bovine serum (homemade, see below), and additionally by increasing the cysteine (Sigma-Aldrich, St Louis, MO, USA) and ascorbic acid (Sigma-Aldrich, St Louis, MO, USA) concentrations to 1.26 g/L and 1.00 g/L, respectively (Table 1). The medium was sterilized by filtration over a 0.22 μm filter (Millipore, Millipore Corporation, Billerica, MA, USA) and stored in 5.6 mL aliquots at −20°C. To prepare modified PEHPS medium, bovine bile was added from a 3% w/v bovine bile/phosphate buffered saline (pH 7.3) stock. Bovine serum was homemade. Briefly, fresh blood (about 15 L), from cows for human consumption, was collected in a sterile way at a local slaughterhouse. The serum was separated by blood coagulation and sedimentation. After complement inactivation by heat (56°C for 1 h), the serum was filtered (0.45 or 0.22 μm pores; Millipore, Millipore Corporation, Billerica, MA, USA). The serum was frozen at −70°C until use. For use, 0.5 mL of bovine serum was added in each culture tube containing 5 mL of basal medium. Lots of well-prepared bovine serum took 3 years to be consumed.

Table 1: Comparative ingredients of original PEHPS media [9] with modified PEHPS media.

Compound
Brand
PEHPS [9]
Modified PEHPS

EHP
Homemade [9]
250 mL
250 mL

Casein peptone
BD Bioxon
10 g/L
10 g/L

Ascorbic acid
Sigma Aldrich
0.2 g/L
1 g/L

Cysteine
Sigma Aldrich
1.0 g/L
1.26 g/L

Glucose
Tecnica Quimica
6 g/L
6 g/L

K₂HPO₄
J T Baker
1
1

KH₂PO₄
Monterrey
0.6
0.6

Bovine bile
Sigma Aldrich
0.0 g/L
0.6 g/L

Deionized water

Enough to fill a 1/L
Enough to fill a 1/L

EHP: pancreas-liver extract.

The growth of G. lamblia in TYI-S-33 or modified PEHPS medium was determined every 24 h for 4 days in triplicate. The number of trophozoites/mL was determined using a haemocytometer, and the doubling time was calculated by linear regression [11]. The yields in each culture were determined at the end of the logarithmic growth phase (72 h of incubation). Yield differences between experimental and reference cultures were assessed by comparing means of assays using the different media by Student’s -test. A value was considered significative.

3. Results

Trophozoites of G. lamblia from the 3 strains used in the present study grew immediately after the parasites were transferred from TYI-S-33 to PEHPS, but all strains grew significantly less in modified PEHPS medium as compared to reference cultures (Table 2). The diminishment in yield was 13% for the WB strain, 12.5% for the 0989: IMSS strain, and 7.5% for the IMSS:3 strain.

Table 2: Yield of Giardia lamblia grown in TYI-S-33 or PEHPS medium.

Strain
Medium
Final density

Absolute
Relative

WB
TYI-S-33
11.67 ± 0.33
1.00

PEHPS
10.06 ± 0.32
0.86†

0989:IMSS
TYI-S-33
12.52 ± 0.31
1.00

PEHPS
10.96 ± 0.62
0.88†

IMSS:3
TYI-S-33
12.35 ± 0.31
1.00

PEHPS
11.43 ± 0.43
0.92†

Trophozoites ×10⁵ per mL; means ± standard deviations of 8 triplicate experiments.

†.

The doubling times of the three strains grown in modified PEHPS medium were longer than those grown in TYI-S-33 as shown in Table 3.

Table 3: Doubling times (h) of three strains of Giardia lamblia in TYI-S-33 or PEHPS medium.

TYI-S-33
PEHPS

WB
5.21
6.00

0989:IMSS
4.94
5.67

IMSS:3
4.84
6.06

All strains adapted satisfactorily to allow for growth in modified PEHPS medium.

The addition of bovine bile improved growth substantially. Nevertheless, in our experience, satisfactory yields with TYI-S-33 medium appear to depend on the brand and batch of yeast extract.

4. Discussion

We previously demonstrated that trophozoites of E. histolytica and T. vaginalis grow well in standard PEHPS medium and that supplementation with bovine bile improved the yield of E. Histolytica [10, 13, 15]. The PEHPS medium described by Said-Fernandez et al. [9] does not allow for proper growth of G. lamblia. In this paper, we report the modifications to the standard PEHPS medium that allow the adequate growth of G. lamblia.

The modified PEHPS medium offers the advantage of being inexpensive and very stable during refrigeration and freezing, so that a large number of experiments can be performed with the same batch of medium for more than 6 months without affecting yields [9].

We provided evidence that G. lamblia trophozoites grow well in modified PEHPS medium. The major modifications to the original media were the increase of the concentration of cysteine, ascorbic acid, bile inclusion, and a filtration step over a 0.22 μm membrane. This filtration step not only sterilizes the medium but also may remove certain factors from the medium such as hormones, enzymes, and other proteins from the liver and pancreas extracts [9] that might have been parasite growth inhibitors. The addition of soluble bile factors may favor the synthesis of parasite membranes and virulence factors [16] and therefore may be essential for many biological assays [9].

Increasing the cysteine concentration was justified as an increased cysteine intake by G. lamblia  trophozoites that has been reported [17]. Ascorbic acid, present in standard PEHPS at 0.2 g/L [9], was increased to 1 g/L in the modified PEHPS medium in order to ensure the same ascorbic acid concentration that is present inTYI-S-33.

We have maintained an E. histolytica strain in continuous culture in PEHPS medium for more than 25 years and T. vaginalis for over 15 years. The successful culture of G. lamblia in modified PEHPS media allows for the execution of assays in an economical and stable environment, avoiding the risk of variability between assays due to interlot variability that occurs when using commercial culture media [18].

Although G. lamblia trophozoites, cultured in modified PEHPS medium, provide lower yields than the ones obtained in TYI-S-33, the yields in modified PEHPS are sufficient to maintain G. lamblia strains in a stable and reliable culture for an undefined time. A reliable cultivation of G. lamblia in modified PEHPS medium allows us to investigate the biology of this parasite, to study the virulence factors, to develop new better diagnostic method, and to test new drugs for better treatments.

Modified PEHPS medium is an alternative culture medium, which is economic and free of the interlot variability present in manufactured TYI-S-33.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

The authors thank Dr. Irene Meester for the critical reviewing of the paper.

References

C. G. Clark and L. S. Diamond, “Methods for cultivation of luminal parasitic protists of clinical importance,” Clinical Microbiology Reviews, vol. 15, no. 3, pp. 329–341, 2002.

M. Limoncu, A. Özbilgin, I. Balcioglu, and Y. Özbel, “Evaluation of three new culture media for the cultivation and isolation of Leishmania parasites,” Journal of Basic Microbiology, vol. 44, no. 3, pp. 197–202, 2004.

R. D. Adam, “Biology of Giardia lamblia,” Clinical Microbiology Reviews, vol. 14, no. 3, pp. 447–475, 2001.

D. E. Katz, D. Heisey-Grove, M. Beach, R. C. Dicker, and B. T. Matyas, “Prolonged outbreak of giardiasis with two modes of transmission,” Epidemiology and Infection, vol. 134, no. 5, pp. 935–941, 2006.

R. C. A. Thompson, “Giardiasis as a re-emerging infectious disease and its zoonotic potential,” International Journal for Parasitology, vol. 30, no. 12-13, pp. 1259–1267, 2000.

M. S. Wolfe, “Giardiasis,” Clinical Microbiology Reviews, vol. 5, no. 1, pp. 93–100, 1992.

D. B. Keister, “Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile,” Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 77, no. 4, pp. 487–488, 1983.

B. D. Mata-Cárdenas and S. Said-Fernández, “Wall synthesis in axenic cultures of Entamoeba histolytica grown in PEHPS medium,” Archivos de Investigacion Medica, vol. 17, supplement 1, pp. 25–30, 1986.

S. Said-Fernandez, J. Vargas-Villarreal, J. Castro-Garza et al., “PEHPS medium: an alternative for axenic cultivation of Entamoeba histolytica and E. invadens,” Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 82, no. 2, pp. 249–253, 1988.

D. B. Mata-Cárdenas and S. Said-Fernández, “Development of a simplified method for the axenic culture of Entamoeba histolytica,” Archivos de Investigacion Medica, vol. 21, pp. 247–251, 1990.

B. D. Mata-Cárdenas, J. Vargas-Villarreal, L. Navarro-Marmolejo, and S. Said-Fernández, “Axenic cultivation of Trichomonas vaginalis in a serum-free medium,” Journal of Parasitology, vol. 84, no. 3, pp. 638–639, 1998.

S. Said-Fernández, E. Campos-Góngora, F. González-Salazar, H. G. Martinez-Rodriguez, J. Vargas-Villarreal, and J. M. Viader-Salvadó, “Mg2+, Mn2+, and Co2+ stimulate Entamoeba histolytica to produce chitin-like material,” Journal of Parasitology, vol. 87, no. 4, pp. 919–923, 2001.

B. D. Mata-Cardenas, J. Vargas-Villarreal, H. G. Martinez-Rodriguez, and S. Said-Fernandez, “Entamoeba histolytica axenic growth improvement by ox bile,” Archives of Medical Research, vol. 26, no. 4, pp. 441–444, 1995.

R. Cedillo-Rivera, J. A. Enciso-Moreno, A. Martínez-Palomo, and G. Ortega Pierres, “Isolation and axenization of Giardia lamblia isolates from symptomatic and asymptomatic patients in Mexico,” Archivos de Investigacion Medica, vol. 22, no. 1, pp. 79–85, 1991.

J. Castro-Garza, F. Anaya-Velazquez, S. Said-Fernandez, and M. T. Gonzalez Garza, “Comparable growth of a Trichomonas vaginalis strain in PEHPS and TYI-S-33 media,” Archives of Medical Research, vol. 27, pp. 567–569, 1996.

D. Bansal, H. S. Bhatti, and R. Sehgal, “Role of cholesterol in parasitic infections,” Lipids in Health and Disease, vol. 4, article10, 2005.

H. D. Lujan and T. E. Nash, “The uptake and metabolism of cysteine by Giardia lamblia trophozoites,” Journal of Eukaryotic Microbiology, vol. 41, no. 2, pp. 169–175, 1994.

F. Li, N. Vijayasankaran, A. Shen, R. Kiss, and A. Amanullah, “Cell culture processes for monoclonal antibody production,” mAbs, vol. 2, no. 5, pp. 466–479, 2010.