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Histological and histochemical methods used in study of development of internal male sex organs
VÁVROVÁ, Martina
Presented thesis devotes to comparative study of usability of fixation agents and colouring methods for the study of histology and histochemistry of Culex pipiens s.l. mosquito males. The study was carried out in males of Culex pipiens quinquefasciatus Say, which is a significant transmitter of filariasis. Histological and histochemistry studies of the structure of the whole abdomens or prepared testicles were carried out on the material fixed by fixation agents Carnoy and Bouin of Dubosque-Brasil modification. Fixed tissue was cast in paraplast or resin. Histological sections were done by means of automatic microtome Leica and half-thin sections by means of ultra microtone Reichert. Section preparations were coloured by Mayer or Harris haematoxylin or by colouring method according to Mallory. Semi-thin sections from materials cast in resin were coloured by toluidine blue. Fixation agent Bouin, Dubosque-Brasil modification proved as more convenient than Carnoy, either for the preparation of paraffin sections or for fabrication of half-thin sections from tissues cast in resin. The next advantage of this fixation is that fixed tissue can be stored for unlimited period of time without over fixation of the tissue as it is in the case of fixation by Carnoy. For general histological preparations Mallory colouring is apparently the most convenient from the used methods; it provides better orientation in coloured tissue thanks to the whole spectrum of colours-pink to red colouring of cell nuclei, blue colouring of tissue and secretions. In case of colouring of male genital organs all used colourings (Mallory, Harris and Mayer haematoxylin) seem to be comparable. Casting in resin with connection to colouring by toluidine blue (also histochemistry proof for carbohydrates) has shown to be suitable for visualisation of finer structures, in male genital organs particular developmental phases of male sex cells were distinguished.
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Histopathological methods used in the study of development of female internal sex organs
DĚDOVÁ, Kristýna
Presented thesis is based on comparison and usability of histological methods for the study of body structure, especially inner female genital organs. The study was carried out in mosquitoes Culex pipiens quinquefasciatus Say. Studied tissue was fixed in a fixation agent Bouin, modified Dubosque-Brasil, for the period of minimum 6-8 hours or in Carnoy fixation for the priod of 5 and 10 minutes. Bouin proved as a more convenient fixation liquid because the tissue can be stored in it for non-limited period of time with no over fixation of the tissue. This can happen in case of the use of Carnoy fixation. In Carnoy fixation it is necessary to determine the period of tissue soaking because the used fixation agent penetrates particular tissues for a different period of time and degradation of material by over fixation threatens, which results in disintegration of tissues and the material is non-usable for histological studies. Fixed tissue was after that cast in paraplast. From the material fixed by alcoholic Bouin, modification Dusque-Brasil, the blocks cast in resin were made for the preparation of half-thin sections. Histological sections from paraplast blocks were made at automatic microtone Leica and half-thin sections from resin blocks at ultra microtone Reichert. Section preparations were coloured either by Meyer and Harris haematoxylins or by the mixture of colours according to Mallory colouring method. For general biological preparations Mallory colouring method is the most convenient; it distinguishes the structures of studied tissue better. Half-thin sections can be coloured by toluidine blue, which is simultaneously a histochemical reaction, proving the presence of carbohydrates. This method is convenient for watching particular fine structures. Its disadvantage is the complexity at the preparation of serial sections necessary for the study of particular phases of insect genital organs development. Half-thin sections have also a significant advantage, namely at processing of materials containing chitin structures-nor chipping nor laceration of studied tissue occurs, as it is in paraffin sections.
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