TRANSACTIONS OFTHEROYALSOCIETY OFTROPICAL MEDICINE ANDHYGIENE (1992) 86,

Axenic cultivation of Entamoeba histolytica in suspension Salvador Said-FernlndezlJ* and Benito David 1Divisidn de Biologia Celular, Unidad Mata-CQrdenasl de Investigacidn Biom6dica de1Noreste, Institute Mexican0 de1 Seguro Social, Aptdo Postal 020-E, 64720 Montemy, N. L., Mkxico; Tentro International de Biologia Celulary Molecular, Edificio Kales, Despacho 324, Monterrey, N. L., Mkico

Mass axenic culture of Entamoeba histolytica trophozoites is required for both molecular and biochemical studies of this protozoon (MARTINEZ-PALOMO, 1989) and in the production of antigenic material for use in the serodiagnosis of amoebiasis (THOMPSONet al., 1968). Large scale suspension culture of E. histolytaca strain HM-1 trophozoites has previously beendescribedusing a vibratory microfermentor containing 2 litres of TYI-S-33 medium with yields of 1.05x 105amoebae/ml(GOMEZREYESet al., 1986). Here we describe an alternative method for the mass axenic cultivation of E. histolytica trophozoites in suspension using a magnetic stirrer in 0.41 to 15 litres of PEHPS medium (SAID-FERNANDEZ et al., 1988), giving yields of 2.36~ 105to 3.08~ 105amoebae per ml.

HOURS

24 OF INCUBATION

&

72

AT 37 “C

Figure. Growth of amoeba1 cultures in PEHPS medium at 36°C over 72 h: without agitation, in 16x 125 mm screw-capped borosilicate tubes (A); in spinner flasks containing 390 ml of medium, without agitation (A); in spinner flasks with agitation for the last 24 h only (0); and with agitation throughout (0). Averages from 3 different experiments in triplicate.

Table.

173

Trophozoites from the axenic HK9 strain of E. histolytica were used. The reference culture amoebaewere ser-

1Short Report1

001.

173-174

Yields of axenic E. histolyfica

cultivated

Culture Cultivation volume (ml) mode 390 Stationarya’b Stationary followed by suspensiona’c 390 780 1560

ially subcultivated, every fourth day, at 36X, in 16x 125 mm borosilicate screw-cappedtubes, contaming 11 ml of PEHPS medium. The tubes were inoculated, in triplicate, with 1x 103amoebaeper ml, which were in logarithmic growth. Twenty-one spinner culture flasks of 620 ml capacity, (catalogueno. 1969-00250,Bellco Glass Inc., New Jersey, USA), filled with PEHPS medium to their full utilizable capacity (390 ml, to the base of the side arm), were inoculated with 5 x 103 amoebae/ml. The trophozoite concentrationsin 3 separateflasks were determined by haemocytometer counts every 24 h of incubation at 36°C. The flasks were chilled in ice-water for 15 min before counting. Trophozoite growth in the following culture conditions was recorded: (a) in stationary tubes, (b) in spinner flasks without agitation, (c) in spinner flasks, stationary for 48 h followed by 24 h in suspensionwith magnetic stirring at 50 rpm, and (d) in suspension culture throughout, with magnetic stirring at 50 rpm. With conditions (a) and (b) the logarithmic growth phaselasted for 3 d (Figure) and the deceleratedgrowth phasefor another 2 d. Longer periods of cultivation resulted in trophozoite lysis or in morphological changes & SAIDsuch as those described by MATA-URDENAS FERNANDEZ(1986). Analysis of variance revealed that the growth rate in the logarithmic phasein conditions (b) and (c) was not significantly different from that in condition (a) (P

Axenic cultivation of Entamoeba histolytica in suspension.

TRANSACTIONS OFTHEROYALSOCIETY OFTROPICAL MEDICINE ANDHYGIENE (1992) 86, Axenic cultivation of Entamoeba histolytica in suspension Salvador Said-Fern...
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