Giardia lamblia is responsible for causing diarrhoeal diseases in humans.
Infection occurs by fecal-oral route and is considered an important water pathogen, since many water surfaces are infected by
cysts. Most studies involve
cyst concentration procedures, followed by conventional microscopy, but are often tedious and influenced by
fatigue. Our main objective was to optimize a specific flow cytometric (FC) protocol for detection of G. lamblia, to establish its sensibility limit and also the
cyst viability. G. lamblia
cysts (Waterborne, Inc., USA) were used for protocol optimization. FC analysis was performed using
cyst suspensions stained with serial concentrations of a
fluorescein-labelled mouse
monoclonal antibody (Giardia-a-Glo, Waterborne). Serial concentrations (2 x 10(5), 1 x 10(5), 2 x 10(4), 1 x 10(4), 2 x 10(3), 1 x 10(3), 2 x 10(2) and 1 x 10(2)
cysts/ml) were stained with the optimized antibody concentration and analysed by FC. Specificity and sensibility limit of the method were established using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Dead
cysts were stained with 5.0 microg/ml of
propidium iodide (PI, Sigma), with and without the specific fluorescent antibody. As the antibody concentration decreased, a decline of peak intensity was registered; 1.5 microg/ml was considered as the optimal antibody concentration, yielding a histogram clearly separated. We established a threshold of detection of 2 x 10(2)
cysts/ml. Below threshold limit fluorescence was not enough to allow the discrimination of
cysts. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. When using both antibody and PI, we could distinguish the viable
cyst. With the use of specific
antibodies, a distinct cellular population corresponding to
cysts could be represented in the FC histogram.