MICROSCOPY RESEARCH AND TECHNIQUE 35:357-358 (1996)

Brief Communication
Processing Trichomonas vaqinalis for Scanning Electron Microscopy

S.R. COSTAMAGNA*,V. SORRIVAS** and M. PRADO FIGUEROA*

*Departamento de Biologia y Bioquimica, Universidad Nocional del Sur, San Juan 670, 8000 Bahia Blanca,
Argentina. e-mail:rcostama@criba. edu.ar
**Centre Regional de Investigaciones Basicos y Aplicadas de Bahia Blanca (CRIBABB-CONICET)

INTRODUCTION
A rapid, simple and economical method useful for processing Trichomonas vaainalis samples for subsequent
observation by scanning electron microscopy (SEM) is described.
Processing protozoa samples for SEM presents some difficulties. Sample manipulation during successive
reagent changes renders the preservation of mobile structures difficult. Based on previous studies published by
Marchant (1973), Newel and Roath (1975) who used small containers for critical point drier of delicate samples, and
Forrest (1986) who used a syringe to contain small nematodes during SEM processing, we have designed a container
for the complete processing of the flagellate.

MATERIALS AND METHODS

A Trichomonas vaeinalis sample from a 48 hour culture (in Merck code 5447 medium) of human vaginal
secretions was processed. The viability of the sample was verified by optical microscopy. It was centrihged at 120g
for 3 minutes. The sediment was fixed with 2% glutaraldehyde in 0.05M sodium phosphate buffer, pH 7.2 and stored
at 4C for 8 days before processing. The fixed material was centrifuged at lOOg for 3 minutes. The supernatant was
discarded, and the sedimented material carefidly resuspended in the same buffer.
The sample container is a cylinder made from a 1.5 ml polypropylene Eppendorf tube (Sigma, St. Louis,
USA) sectioned at its conical end. Filters (Millipore: FHLP 01300, pore size 0.5 pm) were placed at both ends of the
tube and were secured with caps from two tubes. The caps which held the filters in each place had a 4mm hole (Fig.
1).
A steel needle 0.5 x 1.5 mm (25G) was inserted through the capsule wall and connected to a 5 ml
hypodermic syringe. Reagents were introduced by gently pressing the syringe plunger (Fig. 1). The syringe was filled
with 5 ml washing liquid (0.05 M phosphate buffer, pH 7.2) and a soft current was generated, ejecting the liquid
through the filters at both ends of the cylinder. This process took approximately 10 minutes. Then the gradual
dehydration used 25%, 50% and 75% acetone, 5 ml each, 10 minutes for each concentration. The procedure was
carried out at 4 O C . For 100% acetone, three 15 nil changes were used for a total of 15 minutes at room temperature.
The filters were then dried by critical point (Polaron, Watford, England) using as intermediate fluids liquid CO2 and
acetone. Filters containing samples were mounted on a stub with double sided tape.
Samples were subsequently coated with gold in a sputter coater (Ted Pella Inc., Redding, CA, USA).
Observation was carried out with a JEOL 35 CF at 6 KV to decrease charging. The working distance was 15 mm.
Photographs were taken using Kodak VP 120 film.

RESULTS AND CONCLUSION

The method described in this paper permits the passage of washing media and dehydrating agents over the
sample without exerting high pressures, and all the processing steps are accomplished without removing the sample
from the container. In view of all this and according to the results obtained (Figs. 2 and 3), it is concluded that this
method is recommended for the processing of protozoa samples for SEM.

REFERENCES
Forrest, J.M.S., (1986) A niodified syringe for processing second stage juveniles of G. pallida for SEM.
Nematologica 32 ISS 1:123-124.
Marchant, H.J., (1973) Processing small delicate biological specimens for scanning electron microscopy. 1. Microsc.
97: 369-371.
Newel, D.G. and Roath, S . , (1975) A container for processing small volumes of cell suspensions for critical point
drying. J. Microsc. 104:321-323.
Q 1996 WILEY-LISS. INC kivd 18.1995;ampted in revised form August 3,1996.
358 S.R. COSTAMAGNA ET AL

L. Fig. 1

Fig. 1 . The syringe connection to the sample containing cylinder.

Fig. 2. The parasite under division (binary fission). Flagellum and its undulating membrane have already appeared.
Axostyles (Ax)emerging from the bottom can be observed. Bar: pm.
Fig. 3. At low magnification, many well-preserved Trichomonas vaginalis (T.vag.) can be observed with their
typical ovoid or pyriform shape, as well as vaginal epithelial cells (Ep. C.) 48h. culture. Bar: pn.