PEHPS medium: an alternative for axenic cultivation of Entamoeba histolytica and E. invadens

Abstract

Knowledge of Entamoeba histolytica biology in the last 17 years has been acquired largely as a consequence of this parasite's axenic cultivation in TPS-1 or TYI-S-33 media. Unfortunately, there are often low yields in these media, due to variability of their main components, Panmede and yeast extract. We describe a medium, PEHPS, of which the main components are extracts of ox liver, and ox and swine pancreas (EHP). 5 strains of E. histolytica and 2 of E. invadens were quickly and easily adapted to PEHPS and serially cultivated for 3 years. Yields progressively rose initially, and then became stable. Depending on the strain, average yields in the last 6 months of this study were 1.3 to 3.1 x 10(5) amoebae/ml for E. histolytica and 5.5 to 5.7 x 10(5) for E. invadens. All the 18 EHP batches tested supported vigorous amoebal growth. PEHPS had 2 additional advantages: (a) it was stable at 4 degrees C or 25 degrees C for 9 months, and at -10 degrees C for at least 2 years, and (b) it supported amoebal growth with inocula as low as one trophozoite/ml. PEHPS avoids the variability shown by TPS-1 and TYI-S-33, and could therefore be a good alternative for axenic amoebal cultivation.

Methods for Cultivation of Luminal Parasitic Protists of Clinical Importance

“…Diamond et al later described YI-S as an alternative to TYI-S-33 (20); these two media are described below. A homemade axenic culture medium (PEHPS [55]) made from liver and pancreas extracts has been described as an alternative to the above media which rely on commercially produced products. A partially defined medium is also available (21) but is not further considered here.…”

Modified PEHPS Medium as an Alternative for theIn VitroCulture ofGiardia lamblia

“…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.…”

“…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  μ m filter (Millipore, Millipore Corporation, Billerica, MA, USA) and stored in 5.6 mL aliquots at −20°C.…”

“…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]. …”

Antiprotozoal Activity of a <i>Thymus vulgaris</i> Methanol Extract and Its Fractions

“…de Mexico, Mexico). Sterile bovine serum and PEHPS medium were prepared in our laboratory as described elsewhere [22]. Metronidazole (Sigma Chemical Co.), a nitroimidazole antibiotic and antiprotozoal agent was dissolved in DMSO to get 500 μg/mL concentration and further diluted adding 500 μL in 10 mL of dimethyl sulfoxide (DMSO), finally this solution was added to media 1:10 v:v to give 12.5 μg/mL final concentration.…”