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Methods of study



1. Study of the alive cells: Method “in alive”: it is made to organisms or alive cells in his natural state orgánulos even cellular. Colorantes called colorantes are used vital, these do not cause damage to the organism or cell during a short time, in the long term is toxic and mortal.
Method “in Vitro”: it studies to the alive but placed organism in artificial conditions. It is made in cells isolated and easy to separate. These cells are placed on a suitable substrate that is called culture means in which they stay with life while the known study lasts as cellular culture.
2. Study of dead cells
A) FIXATION: when the animal dies is necessary to stop its vital processes before a autolisis of its materials. This process is known like fixation. It conserves the cells and weaves in a state but the possible similarity in morphology and chemical composition to the alive state.
Way of performance:
1. The proteins are insolubilizan
2. It avoids autolisis of the components of the cells due to his own enzymes given off by lisosomas of the cells.
3. It protects to the cells of the bacterial attack.
4. the weave for later treatments like the tinción, the cut…
Physical fixation:
By heat: we applied heat so that the coagulation of proteins takes place. It is not good since the cells are destroyed.
By cold: the weave with carbon dioxide is congealed or N I eliminate. It is better than the previous one but crystals can be formed.

Chemical fixation: (by means of dissolved chemical substances)

1. A single one I eliminate locking device:
OPTICAL microscopy:
Formaldehyde: it produces the reticulación of proteins
Ac. Acetic: it changes the colloidal state of proteins.
Ac. Pírico and potassium dichromate: they act forming salts.
Alcohol etílico and methylic: they produce dehydration.
ELECTRONIC microscopy:
Glutaraldehido: reticulación of proteins
Tetraoxido de Osmio: reticulación of proteins.

2. Fijadoras mixtures:
OPTICAL microscopy: Bouin: formed by a mixture of formaldehyde, acetic acid and acid pírico.
ELECTRONIC microscopy: Paraformaldehido is used glutaraldehido mixed with.

Types of chemical fixation:
Perfusión: the locking device in a syringe is placed injecting it to it to the animal in the sanguineous torrent so that the locking device is distributed by all its organism. Once fixed we can open it to obtain our organ.
Immersion: the organ in a container with locking device puts. This technique as large as depends the sample the volume on the locking device and all this will influence in the time of fixation.
The fixation tb depends on the speed of penetration of the locking device (tetraóxido of slow osmium, formaldehyde fast). The weave must remove from the locking device since the locking device can spoil it. After removing the weave we will make the inclusion.
B) INCLUSION: it consists of placing the weave fixed to a substance that conserves it and him of consistency and volume to be able to cut it (in fine and thin sections).
Average of inclusion:
OPTICAL microscopy: We will use paraffin or paraplast that is means of nonhidrosoluble inclusion.
ELECTRONIC microscopy: We will use resins epoxi like epón, araldita and meth-acrylate or gelatins and agar that they are average hidrosolubles.
Stages of inclusion: (dehydration, explanation, and accomplishment of the block or inclusion)

Dehydration: it consists of clearing the water to him to the weave since the paraffin is nonhidrosoluble. For it we washed in water the locking device. It was made with alcohol of ascending purity until arriving at pure alcohol that is not soluble in paraffin.
Explanation: east alcohol by an intermediary liquid is replaced normally the benzene hydrocarbon (silol, benzene, tolueno).
Impregnating: the weave is placed in paraffin I eliminate, this paraffin is eliminates to 60 degrees (to tº atmosphere is solid). The weave is impregnated of paraffin replacing it by silol.
Inclusion: the weave removes from paraffin and we at heart put it of a metallic mold and we filled up it of paraffin eliminates placing it to tº atmosphere so that the paraffin is solidified being left the block made and prepared for the cut.

Dehydration: it is made with alcohol of increasing concentrations like with the M.O. Sometimes acetones can be used.
Explanation: a benzene hydrocarbon in this case is used I oxidize of propileno.
Impregnating: the weave removes from I oxidize of propileno and epoxi puts in a boat with the resin that to tº atmosphere is eliminates and the weave is impregnated.
Preparation of the block or inclusion: metallic molds are not used but the gelatin capsules of medicines. The weave removes from the resin and it puts at heart of the gelatin capsule. Next it is introduced to 60 degrees so that the resin epoxi is solidified.

Once we have the made block we come to make the cut.
OPTICAL microscopy: the block is necessary to cut it in small fine sections. These are cut in micrótomo (that has a metallic blade) where are made sections of a thickness of 3 to 5 microns. The obtained sections are placed upon an extended crystal call carries. Once in the porthole we can dye the sample. For liquid weaves we must make frotis or extension: a drop of blood is placed above upon the porthole and another porthole forming a perpendicular angle. This porthole moves dragging the drop and forming in the other porthole an extension of blood.
ELECTRONIC microscopy: the blocks for this microscopy are placed in extreme micrótomo (that has blades of diamond and glass) thus obtaining sections very fine ultrafine calls of a thickness between 200 and 500 pressure gauges. These sections take shelter in small round structures that are the grids. These grids (of metal, + frequent copper) with the pieces or ultrafine patches are resisted.


M. OPTICIAN (tinción): before beginning the tinción it is necessary to clear paraffin of the weave since this paraffin is not hidrosoluble. So is to introduce the xylene porthole clearing the paraffin, call to desparafinar. Next it is hydrated with alcohol of decreasing concentration from xylene happening through alcohol to arriving at water. This the prepared weave to be already colored with hematoxilina and eosin passing them to water to wash the colorante. We must seal the weave to clear the water to him and to leave conserved it (EUKIT, DPX). For this balsams are used that are not soluble in water to if that we must dehydrate the weave and soon to clarify in xylene that if he is soluble in the balsams. We threw balsam and we placed another crystal above but small call covers. With this tinción the biological structures are emphasized by means of colorantes able to pay attention selectively on them according to their affinity specifies, related to their chemical nature. Several types of tinciones exist:
Tinciones vital: to dye alive cells.
Of routine: the tinción of hematoxilina and eosin. By means of this tinción the relation between woven and orgánulos cells can be demonstrated.
Special: a structure in individual of the cell is dyed. (Ej. Technical histoquímicas).
Classification of colorantes:
According to its origin:
Natural: taken from natural means
Artificial: made
According to its composition:
Acids: eosin: they dye basic elements like cytoplasm of an orange pink color.

Basic: hematoxilina: they dye acid elements like the nucleus of violet color.
Eosin and hematoxilina are used meetings when making a tinción to see the whole cell.

Metacromáticos: tinción different from the one from the used colorante, this property is called metacromasia and the colorante changes its color when it acts on certain cellular components.
Blue of toluidina (more important) he is blue but when one puts in contact with substances or metacromáticos elements he returns violet. It negatively dyes loaded molecules, molecules of high molecular weight and you cover with mats of sulphate (glucosalinoglicanos).
Mechanisms of coloration:
1. Dissolution: liposolubles colorantes are used in addition the weave pays attention by freezing. It serves to dye lipids or fats. It has the property of which they are more soluble in the lipids that the solvent that contains them for that reason tend to leave the solution to get up itself to lipids of the weave.
They sweat III: it dyes lipids of red color
IV sweat: it dyes lipids of black color
Tetraóxido de Osmio: it dyes black lipids When these are not used and the lipids of the weave change by conventional techniques of tinción dissolve due to the alcohol so that white hollows by the dissolution of fats are observed called negative vision of fats.

2. Impregnating: metals like the silver, the used lead and osmium in form of oxides or salts are used which are reduced by the cellular components precipitating in insoluble metal form. The most used it is the silver, then we spoke of argénticas impregnatings:
Gomori (it specifies for reticulita fibers), Reticulina and Verhoeff (for elastic fibers) give precipitated black.
Classes of tinciones according to I number of colorantes:
Monocromicas: a colorante Policromicas: several colorantes (ticrómicos).

Ticrómico de Masson: it dyes of green color on all colágena conjuntivo weave fiber.
Ticrómico de Mallory: it dyes of blue color same colágenas fibers.

ELECTRONIC microscopy (it contrasts):
Colorantes are not used, are not observed white, gray and black colors single. Heavy metal salts are used to resist the weave.
- For Electronic M. of transmission:
Acetate of uranilo
Citrato of lead
- For Electronic M. of sweeping: The mixture with carbon and platinum is shaded.

METHODS OF STUDY OF the CHEMICAL COMPOSITION OF the CELL (they study the chemical components of the interior of the cell).

Histoquímicas techniques:
1. Aldehyde identification:
Plasmal reaction: in order to show free aldehydes of the cell.
Feulgen: in order to show the DNA.
PAS (acid newspaper of Schiff): it shows hydrates of C.
PAS +: with hydrates of C
PAS -: without them
Result: when there are aldehydes is observed pink color hard and in the three techniques the reagent of Schiff is used (colorless). This it is the fuchsina bleached with sulfuroso anhydride.
Plasmal reaction: simple, if in the studied weave there are free aldehydes hard reacted with the reagent of Schiff producing a pink color.
Feulgen: groups take place in the weave an acid hydrolysis with hydrochlorate acid (HCL) with which have left free aldehydes, these groups are dealed with with the reagent Schiff having seen itself the pink color hard.
PAS: a hydrolysis with periodic acid is made being left aldehydes free that are shown with the reagent of Schiff and that leaves the color again pink hard.

2. Enzyme identification (proteins that manipulated becomes denaturalized and are destroyed by them cannot be fixed):
The weave pays attention by freezing.
The 2 more important techniques: Acid Fosfatasa: it dyes enzymes brown dark. Alkaline Fosfatasa: it dyes them of black. A substrate is used on which the enzyme acts that there is in the cell whose presence we want to show.

Radiográficas techniques Car: radioactive substances are used (like radioactive isotopes) that have affinity to a certain cellular compound. Main Isotopes: Trimidinatrinitada: compatible with DNA, Uridinatrinitada: compatible with ARN
The cell in the radioactive substance is placed to frame some component. It is washed and it is placed in paraffin. It is cut and it is placed in a porthole in a photographic emulsion that has crystals of silver bromide. The porthole is revealed as if outside a photo. The crystal of silver bromide which it has affected transforms into a silver filament whereas the crystals which any isotope has not affected disappear. In the end the Optical Microscope we observed small points that are the silver filaments.
The Electronic M. we will see silver filaments perfectly.
Inmunocitoquímicas techniques: (base-antigen-antibody)
T. Direct fluorescence
T. Indirect PAP (peroxidasa-antiperoxidasa)

For Electronic M.:
1. Negative Tinción: suspensions of cells
2. Shaded: inorganic material and suspensions.
3. Criofractura: bacteriological agents.

1. Negative Tinción: cuts but suspensions are not used.
On the grid the drop of suspension of cells is placed the one that is placed a fosfotúnstico acid layer that puts by all the orifices of the cell. As result we see the bottom and the orifices of color since the heavy metals are had accumulated therefore the electrons hit disperse without crossing the sample. The material we will see brilliant it since the electrons can cross it when not depositing on him heavy metals. In the end we have left something to the negative of a photo similar.
2. Shaded: the very small particle surface is observed with detail. A bell to the emptiness is used. In a side of this we have a platina in whose edge the sample is deposited on a grid with carbon support. To the other side of the bell there are electrodes done of different metals (platinum, carbon). The electrodes are warmed up causing that metal affects to him a side of the sample. Next we dissolve the bacteriological agents having left a platinum mold or of the called metal she talks back. Sometimes it is needed to reinforce talks back it depositing in addition to the metal a continuous carbon layer and enzyme talks back it.
3. Criofractura: the sample is placed on a support and it is introduced in liquid nitrogen with which called process is congealed child fixation. Next the congealed sample puts in a bell with a blade that gives a dry blow him to the sample that always fractures doing it by the weakest site. Later on one of the fracture faces a continuous carbon layer is deposited, next shading is made platinum and finally the retort is made.
- Recorded Freezing: freezing and cut of blade separating by the membrane at level of the lipídica bilayer. Later the Tº is increased so that the ice of fracture surface is sublimated (solid to gas) with which the particles that are in the zone of fracture are noticeable much more.
A continuous carbon film is deposited and on this it is shaded with platinum to obtain talks back dissolving it the organic matter.

Cellular division: it serves to separate the different orgánulos from a cell, for it the cells are crushed and several times are centrifuged. In each centrifugalization orgánulo separates different from the cell (1º nucleus). It is observed the speed to which settles each orgánulo being able to know the sedimentation coefficient expressed in units S (Svedberg) (Ej. Ribosomas 70s).