Improvement of the observational method for Plasmodium berghei oocysts in the midgut of mosquitoes

BACKGROUND: There is a need for improving the method for counting oocysts of Plasmodium berghei in the midgut of Anopheles mosquitoes. The two methods currently used, the formalin fixation method and the mercurochrome staining method, have contradicting advantages and disadvantages. In the formalin fixation method, the specimen can be preserved but unstained oocysts were often indistinct from the insect tissue. While in the mercurochrome staining method, stained oocysts can be clearly distinguished from insect tissue but the specimen are not well preserved. These two methods were combined in this study to develop a new improved technique in counting the oocysts, in which the specimen can be both stained and preserved well. This technique was evaluated for its accuracy and suitability in observing the oocyst development. FINDINGS: In the improved technique, the parasite-infected midgut was first stained with mercurochrome, and then fixed with formalin. The specimens were finally observed using light microscopy. To evaluate the accuracy in the oocyst counting with the improved technique, mosquitoes were infected with the green fluorescent protein (GFP)-expressing parasite. Then, the midgut oocysts were counted using both the GFP marker and the improved technique. Results were then compared and showed that the improved technique retrieved 78%-123% (arithmetic mean = 97%) of the oocysts counted using the GFP marker. Furthermore, it was also possible to evaluate the oocyst development with a green filter using the light microscopy. CONCLUSIONS: The improved technique for oocyst counting will be a useful tool for evaluating midgut oocyst numbers and determining the developmental stage of oocysts in parasite-infected mosquitoes.