such as "Introduction", "Conclusion"..etc
MATERIAL AND METHODS
The ciliates Tracheloraphis phenicoptera, Coleps sp. and Diophrys appendiculata were collected from Baía de Guanabara, Rio de Janeiro; Lacrymaria olor and Spirostomum minus were collected in bromeliad; Colpoda cucullus was collected, in form of cysts, from the filter in the air conditioner of the laboratory. Paramecium aurelia and Euplotes woodruffi were collected from Lagoa de Piratininga in Niterói. Fabrea salina was collected from Lagoa de Araruama in Rio de Janeiro. Trichonympha sp. was isolated from digestive apparatus of termites. The opalinates were isolated from the digestive apparatus of tadpoles Physalaemus signifer.
The ciliates were cultivated in petri dishes with rice or wheat grains, except the opalinates and flagellates. The fixatives used were: 2% Osmiun tetroxide (T. phenicoptera), alcoholic-Bouin (D. appendiculata and Coleps sp.), Stieves' solution(L. olor, S. minus, C. cucullus, P. aurelia, E. woodruffi and F. salina) and 2,5% glutaraldehyde (Trichonynpha sp. and the Opalinata).
Alcoholic-Bouin: 150 ml 80% alcohol + 60 ml formaldehyde PA + 1 g of picric acid + 15 ml glacial acetic acid. This is an excellent fixative, one of its advantage is facilitating attaching the ciliates in the bottom of the clock glass, facilitating the subsequent washes.
Solution of Stieves: (modified by Foissner, 1991); 3,8 ml of aqueous mercury chloride (dissolve 30 g HgCl2 in 500 ml of hot distilled water) + 2 ml of commercial formaldehyde PA + 0,3 ml of concentrated glacial acetic acid. freshly prepared; the components can be stored.
Glycerinated-albumin of Mayer: For the preparation of the Glycerinated-Albumin of Mayer is recommended the use of two chicken eggs. Remove the albumin of the egg and filter through a nylon filter and mix 30 ml of the filtered albumin with 20 ml of glycerin; add 3 drops of formaldehyde PA to avoid the contamination of the material.
1% hydroquinone: place 2,5 g of sodium sulfite in 50 ml of distilled water, dissolve, and later add 0,5 g of hidroquinone.
STEPS OF THE TECHNIQUE:
1) Isolation and fixation of the specimens.
1.1) Under a stereoscopic microscope with transmitted light, the ciliates, flagellates or opalinates should be isolated and taken out from a Petri dish and transfered to a small glass recipient or a glass slide with the aid of a micropipete.
1.2) The ciliates, separated with little water, can be fixed with alcoholic Bouin, solution of Stieves or with 2,5% glutaraldehyde and 2% osmium tetroxide, for 20 minutes. The kind of fixative can vary according to the species.
1.3) Immediately after fixing, wash the specimens three times in distilled water.
2) Clarification and cleaning of the cells.
2.1) To clarify and remove fragments of organic matter attached to the cells, add in the last two washes, two or three drops of 3% sodium hipochloride. Mix and incubate for about 30 seconds, but control the clarification under the magnifying glass, to avoid the bursting of the cells and then wash three times or more. If necessary, repeat the clarification, leaving the cells less time in the hipochloride. For some more sensitive protists 4% sodium lauryl sulfate can be used for the cleaning of the cells.
2.2) After the cleaning and clarification, the cells should be distributed in degreased slides. Add on the cells a small drop of glycerinated albumin of Mayer solution (albumin of egg + glycerin in the proportion of 1:1 or 2:1 ), diluted before use with distilled water in the proportion 1:10, to avoid albumin excess in the final preparation. The material should be spread to be well homogenized and left to dry in ambient temperature, for several hours, or in incubator at 40 to 50oC. For obtaining success it is important to use the minimum of albumin to attach the cells.
The albumin dipped in the distilled water can also be placed in a small watch glass recipient, on the clarified cells, before distributing them in the slides. 1% polylysine in PBS can also be used to attach the cells on the slides. Later proceed a wash in a staining jar with distilled water and then, without drying the preparation, cover the cells with silver proteinate, as in the stage 3.
3) Impregnation and development of the specimens.
3.1) Before covering the cells with silver proteinate, cover the preparation with a mixture of alcohol-formoldehyde in the proportion of 8:2. Let it act during one minute and wash it with distilled water for two minutes. This procedure avoids the detachment of the cells during the processing of the technique.
Use a rectangular glass tray, with a lid and cover the bottom with moisten filter paper. Distribute the dry slides with the ciliates attached to them on the bottom of the tray and using a Pasteur pipette cover the preparations with drops of 0,8% or 1% silver proteinate. Place the covered tray in the incubator at 45oC-50oC, for 30 to 60 minutes, depending on the size of the protozoan. It is advisable to avoid the total evaporation of the silver proteinate of the preparation.
3.2) Remove the tray from the incubator to process the revelation of the slides, one at time. Wash the slide quickly in distilled water and quickly place it in a staining jar with 0,4% hydroquinone to reveal the preparation for 10 to 20 seconds. After brief wash in distilled water, an observation is done under the microscope to control impregnation. If necessary, it can be left more time in the developer, which can vary in concentration.
3.3) After reaching the desired impregnation, place the slide for about 30 seconds in the 2,5% sodium thiosulfate, to fix the impregnation. If the impregnation of the cells decreases, return it to the developer.
3.4) After passing it in the sodium thiosulfate, wash the slide for one minute in distilled water and begin the dehydration with alcoholic series from 50%-100% and finally in three jars with xylene.
Set up the slides covering the preparation with two drops of Entellan Merck or in diluted balsam from Canada, before placing the coverslip.
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