Medio de cultivo Pavlova Modificado

Medio Base:
Extracto de levadura 1.45 g
NaCl 7.27 g
Na2HPO4.12H2O 3.25 g
KH2PO4 0.42 g
H2O destilada o desionizada c.s.p. 1000 mL

Disolver todos los componentes en agua destilada o desionizada e enrrasar a 1 litro.

Autoclavar el medio base y dejar enfriar. Preparar el medio completo, adicionando 50
ml de suero humano inactivado (56 - 60°C por 45 minutos) o plasma humano fresco
proveniente de unidades de sangre y un tercio de una ampolla de amikacina (aprox.
700 uL). Mezclar bien y repartir 1.5 ml en microtubos tipo Eppendorf de 2mL de
capacidad. Guardar a 4 - 8 °C hasta por 6 meses.
En un tubo estéril, preparar almidón de arroz al 1% en agua destilada o desionizada y
guardar a 4 - 8 °C hasta por 6 meses.

Aislamiento y cultivo de Blastocystis sp.

 Llevar el medio de cultivo y el almidón de arroz por unos 15 minutos a temperatura

ambiente o por unos 5 minutos a 37°C.
 Adicionar una gota (aprox. 40 uL) de la solución de almidón de arroz a cada
microtubo conteniendo 1.5 ml del medio Pavlova.
 Coger una pequeña cantidad de la muestra fecal (aprox. 2 asadas de siembra) e
introducirla en el microtubo conteniendo el medio de cultivo completo y mezclar
bien. Incubar a 37°C por 48 horas.
 Con ayuda de una pipeta Pasteur o con una micropipeta de 1 ml, eliminar todo el
sobrenadante posible del microcultivo.
 Coger una pequeña gota del sedimento del cultivo y examinar las formas
evolutivas del parasito a través de montaje húmedo (lamina y laminilla), utilizando
objetivos de 10X y 40X.
 Opcional: Lavar el sedimento con solución salina fisiológica o buffer fosfato salino
centrifugando a 2,500 rpm por 5 minutos y eliminar el sobrenadante. Repetir el
proceso de lavado una vez más.
 Congelar el sedimento a -20°C hasta el momento de su uso.

En condiciones de cultivo, los trofozoitos de Blastocystis sp. puede crecer en

tamaño y tomar diversas formas diferentes a lo que usualmente se puede observar en
heces frescas, conforme descrito en la literatura: forma vacuolar clásica, forma
ameboide, forma granular y forma esquizogónica. También puede observarse formas
en división binaria o división múltiple.
Zerpa R y cols. A simplified culture method for Blastocystis hominis

A simplified culture method for

Blastocystis hominis
Key words: Blastocystis hominis culture. Rito Zerpa L,*,** Luis Huicho,*,** C Náquira,* I Espinoza *

Palabras clave: Blastocystis hominis * San Marcos University.

cultivo. ** Instituto de Salud del Niño, Lima Perú, Perú.

Recibido: 11/10/99 Corresponding author:

Aceptado: 25/11/99 Luis Huicho, MD
Laboratorio de Biofísica. Departamento de Ciencias Fisiológicas
Universidad Peruana Cayetano Heredia
Apartado 4314, Lima 100, Perú. Fax: 51-14897939

Abstract
A simplified method of culturing Blastocystis hominis is
described. Two hundred fresh stool samples were inoculated
in tubes containing a modified Pavlova’s medium. The
tightened tubes were incubated at 36º C without any
additional conventional system of anaerobiosis. Cultures were
examined after 24, 48 and 72 hours. Overall, 140 (70%)
cultures were positive for B. hominis, whereas wet mount
examination was positive only in 42 (21%) samples. The
different forms of parasite described with the standard
method were visualized. This method is proposed as an
alternative to the conventional one.
Resumen
Aquí es descrito un método simplificado de cultivo de
Blastocystis hominis. Se inocularon doscientas muestras fecales
frescas en tubos que contenían un medio de Pavlova modifica-
do. Los tubos herméticos fueron incubados a 36oC sin ningún
sistema adicional de anaerobiosis convencional. Los cultivos fue- 17
ron examinados después de 24, 48 y 72 horas. En conjunto,
140 (70%) de los cultivos fueron positivos para B. hominis,
mientras que el monto del examen microscópico en fresco fue
positivo sólo en 42 (21 %) muestras. Se observaron las diferen-
tes formas del parásito descritas con el método estándar. Este
método es propuesto como una alternativa al convencional.

lastocystis hominis is a strict anaerobic pro- described in a recent comprehensive review

B tozoan that occurs in the intestine of humans
and other mammals (Zierdt, 1991).
Recently, phylogenetic analyses of ribosomal
RNAs place B. hominis within the straminopiles
(Silberman et al., 1996). Its pathogenic role in
human beings is still disputed by some investigators
(Markell and Udkow, 1986; Miller, 1988). The
mechanism by which it can lead to diarrhea is not
understood. Diagnosis of clinical stool samples is
edigraphic.com
generally based on finding of typical forms
described in textbooks (Healy, 1991; Pessoa, 1988).
However, these references do not mention the
different forms of Blastocystis hominis such as those
(Zierdt, 1991) thus resulting most probably in
misdiagnosis of numerous cases.
The medium of choice for in vitro culture is a
modified whole-egg slant medium with Locke
solution overlay that requires addition of 30%
horse serum (Zierdt, 1991). Medium in screw-
capped tubes needs reduction by means of
incubation under anaerobic conditions for 3 days
or longer. The caps should be loosened to permit
gas exchange and tightened afterwards when re-
moved from the anaerobic atmosphere.
Inoculated tubes with stool samples should also
be anaerobically incubated. With this method

Revista Mexicana de Patología Clínica, Vol. 47, Núm. 1 • Enero - Marzo, 2000
 Zerpa R y cols. A simplified culture method for Blastocystis hominis

a e

b f

18
c g

d h

Figure 1. Different forms of Blastocystis hominis obtained from positive cultures are shown. Panel
a, spheric forms with different size. Panels b and c, forms with central body. Panel d, ameboid
forms. Panel e, binary fission. Panel f, budding forms. Panel g, endodyogeny.

Revista Mexicana de Patología Clínica, Vol. 47, Núm. 1 • Enero - Marzo, 2000
Zerpa R y cols. A simplified culture method for Blastocystis hominis
cultures become positive quickly and identification
after 24 hours is feasible. Stock cultures need to
be transferred every 3 to 4 days. A simplified
method of culturing B. hominis is described here.
A total of 200 fresh stool samples received for
parasitological examination were studied. The
specimens were inoculated in a slightly modified
Pavlova’s medium, using human plasma instead of
horse serum.
The medium used contains sodium acid phos-
phate 12 H20, 8.95 g; potassium phosphate, 1.15
g; chloride sodium, 20 g; yeast extract, 4 g; and
distiled water, csp 2,750 mL. Sodium hydroxide
(1 N) was used to adjust the pH to 7.2-7.4. In
addition, 5% human plasma was added.
Then 2.75 g of sterile rice starch was added,
potasic G penicillin (Squibb) 1,000 IU/mL and strep-
tomycin 50-100 µg/mL were added after steriliza-
tion with a Seitz filter. Then the medium was dis-
tributed in sterile glass tubes, 10 mL each (Barral
de Martínez, 1993). The amount of inoculum con-
sisted from stool fractions similar to those used
for microscopical examination, except that they
were first inoculated in the tubes containing the
culture medium. The tubes were tightened and
incubated without any additional conventional sys-
tem of anaerobiosis at 36ºC. Examinations were
performed after 24, 48 and 72 h. Similar inocula
were used for initial microscopic examination of
wet mount preparations to compare them with
culture results. The wet mounts of all specimens
were microscopically observed using saline and
parasitological lugol solutions.
Of a total of 200 stool samples studied, 140
(70%) were positive for B. hominis by the modified
culture. By contrast, wet mount examination was
positive only in 42 (21%) samples.
The different forms of B. hominis obtained from
edigraphic.com
positive cultures are shown in figure 1 (panels a to h).
They are similar to those described with the choice
medium (Zierdt, 1991).
Blastocystis hominis has been described as a fas-
tidious strict anaerobic organism for culture (Sten-
Revista Mexicana de Patología Clínica, Vol. 47, Núm. 1 • Enero - Marzo, 2000
zel and Boreham, 1996). Studies of this parasite
which include culture are rarely found in the liter-
ature (Healy, 1991; Pessoa, 1988).
The College of American Pathologists has
prompted reporting of B. hominis from clinical sam-
ples. Clinical diagnosis is facilitated through culture
when microscopic examination is uncertain.
An easily available culture method such as the
one described here may make feasible the study
of several aspects about B. hominis in the
laboratory, even in those with scarce resources.
The different forms of the organism such as those
that we show in the figure 1 can be observed, e.g.
central body forms, ameboid forms. Also, the
different division modes (binary fission, budding,
plasmotomy, and endodyogeny) can be noticed.
This method is cost-effective because it does
not require horse serum, reduction during its
preparation is not necessary, and it does not need
an additional anaerobiotic system. Stock cultures
need to be transferred every 3 to 4 days in order
to maintain the organisms viable. We propose the 19
use of this method as an alternative to the stan-
dard methods presently known.
Bibliography
1. Barral de Martínez AM. Perfil isoenzimatico de cepas de
Entamoeba histolytica (Schaudin 1903) provenientes das regiôes
amazônica e sudeste do Brasil e mantidas sob diferentes condicôes
de cultivo. Thesis. Universidade Federal de Minas Gerais. Belo
Horizonte 1993.
2. Healy GR, Smith JW. Intestinal and urogenital protozoa. In: Balows
A, Hausler WJ, Hermann KL, Isenberg HD and Shadomy HJ (eds).
Manual of Clinical Microbiology, Fifth Edition. American Society
for Microbiology 1991; 751-770.
3. Markell EK, Udkow MP. Blastocystis hominis: pathogen or fellow
travelor? Am J Trop Med Hyg 1986; 35: 1023-1026.
4. Miller RA, Minshew BH. Blastocystis hominis: an organism in
search of a disease. Rev Infect Dis 1988; 10: 930-938.
5. Pessoa SB, Vianna Martins A. Amebas nao patogenicas-amebas
de vida livre. In: Pessoa parasitología médica. 11º edition. Editora
Guanabra, 1988: 231-242.
6. Silberman JD, Sogin ML, Leipe DD, Clark CG. Human parasite
finds taxonomic home. Nature 1996; 380: 398.
7. Stenzel DJ, Boreham FL. Blastocystis hominis revisited. Clinical
Microbiology Reviews 1996; 9: 563-584.
8. Zierdt CH. Blastocistis hominis- Past and future. Clin Microbiol
Rev 1991; 4: 61-79.