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

Usui M, Fukumoto S, Inoue N, Kawazu S - Parasit Vectors (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan.

ABSTRACT

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.

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Comparison of methods currently used for counting oocysts. (A) In the formalin fixation method, the midgut was opened as a single layered specimen that avoided the overlapping of oocysts. However, unstained oocysts were indistinct and difficult to distinguish from insect tissue. (B) On the other hand, the mercurochrome staining method made the oocysts distinct from the surrounding tissue, although the two layers of oocysts overlapped and they were difficult to distinguish from each other. (C) In the improved technique, the red-stained oocysts could be distinguished clearly from the insect tissue, and in the single layered specimen they were no longer overlapping each other. Arrows indicate oocysts in the midgut of A. stephensi at 12-14 days post-feeding of P. berghei ANKA strain infected mice.
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Figure 1: Comparison of methods currently used for counting oocysts. (A) In the formalin fixation method, the midgut was opened as a single layered specimen that avoided the overlapping of oocysts. However, unstained oocysts were indistinct and difficult to distinguish from insect tissue. (B) On the other hand, the mercurochrome staining method made the oocysts distinct from the surrounding tissue, although the two layers of oocysts overlapped and they were difficult to distinguish from each other. (C) In the improved technique, the red-stained oocysts could be distinguished clearly from the insect tissue, and in the single layered specimen they were no longer overlapping each other. Arrows indicate oocysts in the midgut of A. stephensi at 12-14 days post-feeding of P. berghei ANKA strain infected mice.

Mentions: Malaria is a disease caused by the infection with protozoan parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes. To study the life cycle of Plasmodium berghei in mosquitoes, qualitative and quantitative evaluation of midgut oocysts in experimental infections is needed [1]. There are two methods currently used for counting oocysts. One technique used is the formalin fixation method [2]. In this method, the midgut is fixed with 10% formalin and opened along the median line to prepare a single-layered specimen. The advantage of using this technique is that it can preserve the sample for storage and later observation. However, this technique leaves the oocysts unstained, making them indistinguishable from insect tissues (Figure 1A). Another technique is the mercurochrome staining method [3-5]. In this method, the midgut is stained with 0.5% mercurochrome in water which makes the oocysts be easily distinguished from insect tissues (Figure 1B). However, the resulting specimen from this technique should be observed at the same day since there was no included preservative. This may pose a problem when handling large numbers of mosquitoes for oocyst examination. Furthermore, the observation of bagged (unopened) midguts from a single perspective under the microscope often results in overlapping oocysts leading to inaccuracies in counting. In this study, a new technique for counting oocysts was done by combining the two methods available. This technique produces fixed and stained oocysts in opened midgut specimens which can be utilized for a more reliable counting (Figure 1C).


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

Usui M, Fukumoto S, Inoue N, Kawazu S - Parasit Vectors (2011)

Comparison of methods currently used for counting oocysts. (A) In the formalin fixation method, the midgut was opened as a single layered specimen that avoided the overlapping of oocysts. However, unstained oocysts were indistinct and difficult to distinguish from insect tissue. (B) On the other hand, the mercurochrome staining method made the oocysts distinct from the surrounding tissue, although the two layers of oocysts overlapped and they were difficult to distinguish from each other. (C) In the improved technique, the red-stained oocysts could be distinguished clearly from the insect tissue, and in the single layered specimen they were no longer overlapping each other. Arrows indicate oocysts in the midgut of A. stephensi at 12-14 days post-feeding of P. berghei ANKA strain infected mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141744&req=5

Figure 1: Comparison of methods currently used for counting oocysts. (A) In the formalin fixation method, the midgut was opened as a single layered specimen that avoided the overlapping of oocysts. However, unstained oocysts were indistinct and difficult to distinguish from insect tissue. (B) On the other hand, the mercurochrome staining method made the oocysts distinct from the surrounding tissue, although the two layers of oocysts overlapped and they were difficult to distinguish from each other. (C) In the improved technique, the red-stained oocysts could be distinguished clearly from the insect tissue, and in the single layered specimen they were no longer overlapping each other. Arrows indicate oocysts in the midgut of A. stephensi at 12-14 days post-feeding of P. berghei ANKA strain infected mice.
Mentions: Malaria is a disease caused by the infection with protozoan parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes. To study the life cycle of Plasmodium berghei in mosquitoes, qualitative and quantitative evaluation of midgut oocysts in experimental infections is needed [1]. There are two methods currently used for counting oocysts. One technique used is the formalin fixation method [2]. In this method, the midgut is fixed with 10% formalin and opened along the median line to prepare a single-layered specimen. The advantage of using this technique is that it can preserve the sample for storage and later observation. However, this technique leaves the oocysts unstained, making them indistinguishable from insect tissues (Figure 1A). Another technique is the mercurochrome staining method [3-5]. In this method, the midgut is stained with 0.5% mercurochrome in water which makes the oocysts be easily distinguished from insect tissues (Figure 1B). However, the resulting specimen from this technique should be observed at the same day since there was no included preservative. This may pose a problem when handling large numbers of mosquitoes for oocyst examination. Furthermore, the observation of bagged (unopened) midguts from a single perspective under the microscope often results in overlapping oocysts leading to inaccuracies in counting. In this study, a new technique for counting oocysts was done by combining the two methods available. This technique produces fixed and stained oocysts in opened midgut specimens which can be utilized for a more reliable counting (Figure 1C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus