SlideShare ist ein Scribd-Unternehmen logo
1 von 13
Downloaden Sie, um offline zu lesen
General Histology and Histotechnique (1st Semester; SY 2012-2013)


Preparation for whole mount
                                                            B. According to the reaction of the tissue stain.
Materials needed:                                           1. ) Basophilic
1. Glass slides                                             - stains the acid component
-    usually 25 x 75 mm                                     2. ) Acidophilic
- usually 1- 1.2 mm thick                                   - Stains the basic components
  Types:                                                    - Stains the cytoplasm of the cell
   a. flat slides
   b. concave slides                                        Some common stains:
                                                            1. ) Hematoxylin and Eosin (H & E)
2. Cover slips                                                 a. Hematoxylin – stain nuclei blue (basophilic)
Two types:                                                     b. Eosin – stain cytoplasm pink (acidophilic)
  Number 1 = 0.13-0.17 mm thick
  Number 2 = 0.17-0.25 mm thick                             2. ) Connective Tissue stains
  Functions:                                                   a. Masson’s trichome
   a. hold samples in place                                    b. Mallorys triple CT stain
   b. flattens out specimen                                 - both employ a nuclear, cytoplasmic and a third stain
   c. Protects the objective from immersion into the              specific for matrix
       water drop.
                                                            3. ) Silver impregnation
3. Stains                                                   - Used to trace nerves, stain Golgi, reticular fibers.
- Different stains have different affinities for the
    different organisms thus maybe used to                  4. )Wright stain – blood smear
    differentiate different types of organisms or to view
    specific parts of organisms.                            4. other chemicals
- Substance that give color to the specimen to                  Fixatives
    improve visibility.                                         Embedding materials
  Example:                                                      Mountants
  Counter stain – not all organisms can absorb.                 Clearers

Type of stains
A. According to the number of dyes used
1. ) Positive staining
   - Stains the specimen itself.                            Type of Slide Preparation
   a. Simple Stain
- Employs single dye (on color)                              Temporary Slide
- Primary dye                                               - water
        Ex. Crystal violet, Methylene blue                  - fresh specimen
   b. Differential Stain                                     Permanent slide
- Two or more stains                                        - reagents are used
- Primary dye – counter stain                               - dead tissues
- Gram-staining
        Ex.                                                  Fixatives – preserve specimen
        Primary dye = Crystal violet;                       - Function: to avoid post mortem conditions
        Counter stain = Safranin                            Ex: FAA, Bouin’s sol’n, Formalin (10%)
Ex. Acid fast stain (Mycobacterium), Ziel Neelsen
2. ) Negative staining                                       Dehydrants
- the background of the organism/specimen is the            - reagents that remove undesired fluid that may
      stain                                                     interfere with later processing
- Function: To emphasized the appendages                    Ex: Ethyl alcohol (ethanol/alcohol)
General Histology and Histotechnique (1st Semester; SY 2012-2013)


 Clearing agents                                           -  utilize when rapid diagnosis of tissues
- to clear                                                  -  specially recommended for lipids and nervous tissue
Ex: Xylene and Cloroform                                       elements
                                                            USES:
 Embedding medium                                            Rapid pathological diagnosis during surgery
- supports the specimen                                       Diagnostic and research enzyme histochemistry
- paraffin (wax time)                                         Diagnostic and research demonstration of soluble
                                                                substances such as lipids and carbohydrates
 Microtome                                                   Immunofluorescent and immunohistochemical
                                                                staining
 Stains                                                      Some specialized silver stains, particularly in
- Gives color to the specimen to differentiate the              neuropathology
    different parts of specimen.
Ex: Methylene blue, Hematoxylin, Wright stain, Crystal
violet, Eosin, Safranin, Fast green.                        Processing of Tissues

      Adhesive/Affixative                                   Fixation
- to stick or adhere the specimen to the slide              Purposes:
Ex: Mayer’s albumin                                           a. Confers chemical stability on the tissue
                                                              b. Hardens the tissue
       Mountant                                              c. Stops enzyme autolysis
- render permanency                                           d. many enhance later staining technique – act as
Ex: Balsam                                                       mordant

Fresh Tissue Examination                                    Five (5) major groups of Fixatives:
                                                            1. Aldehydes
1.  Teasing/Dissociation                                    - include formaldehyde or formalin and
-   use of needle to separate tissues in bulk                   glutaraldehyde
2.  Squashing                                               - non precipitating
-   cut a tiny piece on slide and with the use of another    10 % formalin
    slide to compress                                       - buffered to lessen acidity that causes precipitation
3. Smear preparation                                            and autolysis
- to spread specimen thinly onto the slide                   Glutaraldehyde
  a. Streaking                                              - 2-4 % buffered
- Applicator stick or the wine loop usage.                  - fixes very fast
- Spread specimen to the slide directly on a zigzag         - good for electron microscopy
    manner.                                                 - it penetrates poorly
- Uniform the distribution                                  - It gives best results on cytoplasmic and nuclear
  b. Spreading                                                  details.
- to spread thinly into the slide
- teasing is also needed                                    2.   Mercurials
- for fresh sputum mucoid secretion                         -    contain mercuric chloride and Zenker’s
  c. Full-apart                                             -    fixed the tissue by an unknown mechanism
- for blood smear, enzymes of the GIT                       -    penetrate very poorly
- it used pusher                                            -    causes tissue to harden
  d. Touch Preparation/impression smear                     -    fixed fast
- used of freshly cut fruit                                 -    give excellent nuclear detail
- touched on to the surface of the slide                    -    best in nematopoietic reticuloendothelial
- letting the cells stick on to the slide
4. Frozen section                                           3. Alcohols
General Histology and Histotechnique (1st Semester; SY 2012-2013)


-    include methanol and ethanol
-    protein denaturants                                      1. Buffering – pH 6-8
-    cause too much brittleness and hardness                  - phosphate, bicarbonates
-    good in cytological smears                               - Commercial formalin is buffered with phosphate at
-    give good nuclear detail                                    a pH of 7.
-    precipitating
                                                              2.   Penetration
4. Oxidizing agents                                           -    depends of diffusability of fixatives
- include Potassium permanganate, Potassium                   -    formalin and alcohol: best
   dichromate and Osmium tetroxide                            -    glutaraldehyde: worst
- none precipitating                                          -    diffusability – constant
- used in frequently                                          -    in between – mercurials
- causes an extensive denaturation                            -    in order to the organisms to be penetrated it should
                                                                   be cut in 2-3 mL
5.   Picrates
-    include fixatives and picric acid (e.g. Bouin’s sol’n)   3.   Volume
-    precipitating fixative                                   -    ratio 10:1
-    enhanced nuclear detail                                  -    10 – fixative
-    don’t cause hardens                                      -    1 – specimen / tissue
-    Dry form – explosive
-    in Solutions– stains everything it catches including     4. Temperature
     the skin; yellow color                                   - High temperature – Increases speed of fixation
                                                              Ex. Hot formalin will fix the tissue faster.
Fixative for General Usage
                                                              5.   Concentration of Fixation
a. 4 % / 10 % formalin                                        -    adjusted down to the lowest level possible
Advantages:                                                   -    10% formalin: best
1. easy to make                                               -    glutaraldehyde (3%)
2. Has good penetration power
3. Does not shrink the specimen                               6.   Time interval
4. Does not affect the material it kept for long time         -    FAA – up to 12 hrs.
5. Can be kept without deterioration for considerable         -    Carnoy’s – 1-3hrs
   time                                                       -    Zenker’s – 14-24hrs
6. Easy to handle
7. Less hazard                                                Organs for processing:
                                                              Kidney
b. FAA – Formalin-Acetic-Acid-Alcohol                         Liver
- Best for plant material (tissues)                           Stomach
- Composition: 50ml ethanol, 5ml glacial acetic acid,         Small Intestine
   10ml 37-40% formaldehyde, 35ml water                       Large intestine
                                                              Lungs
c. Zenker’s and Carnoys’s fluid                               Spleen
- best for animal tissues                                     Gonads
 Zenkers’s: 950ml distilled water, 25g potassium             Muscles
   dichromate, 50g mercuric chloride, 50g glacial
   acetic                                                     Specimen Class
 Carnoy’s: 60 ml ethanol, 30ml chloroform, 10ml              Class A – delicate tissues: lungs; membranes
   glacial acetic acid                                        Class B – moderately thick; spleen, live, kidney
                                                              Class C – bulky and thick; skin
Factors affecting fixation
General Histology and Histotechnique (1st Semester; SY 2012-2013)


Fixation process:                                       100% ethanol – 30 min
- submerge the specimen fully in fixative               100% ethanol– 30 min
- cover the container tightly                           ***To ensure complete removal of water during
                                                        dehydration, use two changes of 100% ethanol of at
Proper timing for each specimen if FAA is used:         least ½ hour each.
Class A – 6-9 hrs. (10hrs)
Class B – 12-18 hrs. (16-18 hrs)                        Timing of dehydration:
Class C – 24-36 hrs.                                    Class A – 30 min in each alcohol concentration
                                                        Class B – 1 hr
Dehydration                                             Class C – 3-4 hrs.
- removing undesired water inside the specimen
                                                        Clearing
Characteristics of an ideal dehydrating solution:
 It should dehydrate rapidly without producing              1. Alcohol –to provide rapid miscible in the tissue
   considerable shrinkage or distortion of tissues.          2. To facilitate impregnation or infiltration
 It should not evaporate very fast.
 It should be able to dehydrate even fatty tissues     Xylene
 It should not harden tissues exclusively.             - Commonly used; tends to harden tissues if left too
 It should not remove stain.                               long.
 It should not be toxic to the body
 It should not be a fire hazard.                           Advantages:
                                                        -    makes tissue transparent
Commonly used dehydrating agents:                       -    miscible with absolute alcohol and paraffin
- alcohol                                               -    evaporate quickly in paraffin oven and can
- acetone                                               -    cheap
- triethylphosphate
- tetrahydrofuran                                           Disadvantages:
- dioxane                                               -    highly in flammable
                                                        -    If used longer than 3 hrs. it makes tissues
   Alcohol                                                  excessively hard and brittle
-   a standard series of alcohols are used most         -    Causes considerable hardening and shrinkage of
-   Alcohol series: 70% - 80% - 95%                          tissues
-   Higher grades of alcohol: shrinkage and hardening   -    Becomes milky when an incompletely dehydrated
    of tissues                                               tissue is immersed in it.
-   concentrated alcohol tend to harden only the
    surface of tissue                                   Toluene
   Ethyl alcohol                                       - time recommended is 1-2 hours
-   best dehydrating agent
       fast acting                                     Advantages:
       mixes with water                                - it is miscible with both absolute alcohol and paraffin
       penetrates tissue easily                        - It acts fairly and rapidly and is recommended for
       not poisonous                                      routine purposes
       not very expensive                              - tissue do not become excessively hard and brittle
                                                           even if left in Toluene for 24 hrs
Dehydrating procedures:                                 - it is not carcinogenic
Water
50% ethanol                                             Disadvantages:
70% ethanol                                             -   it is relatively slower than Xylene and benzene
80% ethanol                                             - it tends to acidify in a partially filled vessel
90% ethanol
General Histology and Histotechnique (1st Semester; SY 2012-2013)


-   highly concentrated solutions will emit fumes that       -   complete clearing is difficult to evaluate
    are toxic upon prolonged exposure                        -   tissues tend to float in chloroform
-   it is more expensive                                     -   it evaporates quickly from a water bath

Benzene                                                      Impregnation, Embedding, Trimming
- clearing agent
- clear and penetrate tissues rapidly                        Impregnation
- carcinogenic                                               - Diffusion or accumulation in a cell or tissue
                                                                substances that are not normal.
Advantages:                                                  - To fill-out the tissues in each spaces or in-between
- It is rapidly acting, so recommended for urgent               spaces
   biopsies (15 to 60 min) and routine purposes.
- It volatilizes rapidly in paraffin oven and is therefore   Three (3) types of Tissue Impregnation:
   easily eliminated from the tissue.                        1. Paraffin wax impregnation
- It is miscible with absolute alcohol                       Precautions:
- it does not make tissues hard and brittle                  a. Prolong treatment in melted paraffin causes
- it causes minimum shrinkage                                    shrinkage and hardening of tissues making cutting
- it makes tissues transparent                                   difficult.
                                                             b. Infiltration in overheated paraffin (above 60°C) will
Disadvantages:                                                   produce shrinkage and hardening of tissues and
- it is highly in flammable                                      destroy lymphoid tissues completely
- if a section is left in benzene for a long time,           c. Paraffin wax must be pure
    considerable tissue shrinkage may be observed            d. Paraffin wax may be used only twice.
- Excessive exposure to benzene may be extremely             2. Celloidin impregnation
    toxic to man and may become carcinogenic or it           3. Gelatin impregnation
    may damage the bone marrow resulting in a plastic
    anemia.                                                  Embedding
                                                             - The process by which the impregnated tissue is
Chloroform                                                      placed into a precisely arranged position in a mold
- Used for routine clearing of tissues during the               containing a medium which is then allowed to
    embedding process.                                          solidify.
                                                             - Cooling allow hardening of tissue, giving them a
Advantages:                                                     firmer consistency and better support, thereby
- It is recommended for routine work (6-24 hrs.)                facilitating the cutting of sections.
- It is miscible with absolute alcohol                        Orientation
- It is recommended for tough tissues (ex. skin,             - A process by which a tissue is arranged in precise
   fibroid and decalcified tissues) for nervous tissues,        positions in the mold during embedding on the
   lymph nodes and embryos                                      microtome before cutting and on the slide before
- It is suitable for large tissue specimens                     staining.
- It is not inflammable.
                                                             Embedding Proper:
Disadvantages:                                                1. Prepare embedding boxes
- It is relatively toxic to the liver after prolonged         2. Pour melted paraffin into embedding box
    inhalation                                                3. Using a needle or forceps, position properly the
- wax impregnation after chloroform clearing is                  tissue inside the paraffin box according to the
    relatively slow                                              desired cut later. For cross section, position tissue
- it does not make tissues transparent                           vertically. Do not remove the needle or forceps ‘till
- it is not very volatile in paraffin oven                       paraffin has surrounded the tissue.
- it may even produce considerable deterioration of           4. Allow paraffin to solidify.
    the wax
General Histology and Histotechnique (1st Semester; SY 2012-2013)


    5. Label the box bearing the name and position of tissue contained.

 Trimming
-   Embedded tissue is trimmed and cut into thin uniformly slices.
-   The slides, top and bottom of the tissue block are trimmed until perfectly level and all slides are parallel, almost to
    the edge of the tissue.
- The block is placed in the microtome for final trimming and cutting.

Microtomy
- A process of cutting specimen to small or thin sizes.

Microtome
- An instrument used to cut the specimen.

Steel Blades
- Animal or plant tissue sections
- Light microscope

Glass Knives
- thin sections
- Light microscope and Electron Microscope

Industrial Grade
- Electron microscope

Diamond knives
- Hard materials

3 Essential parts of Microtome:
 1. Block holder – holds block in precision
 2. Knife carrier & knife – actual cutting of the tissue sections
 3. Pawl, Ratchet feed wheel and adjustment screws – line up the tissue flock in proper positioning with the knife
     adjusting the proper thickness of the tissue for successive section.

Kinds of Microtome’s:

1. Rocking (by Paldwell Trefall, 1881)
- Simplest
- Cut the paraffin embedded tissues

2. Rotary (by Minot, 1885-’86)
- Most common
- It involves staged rotary action

3. Sliding (by Adams, 1789)
- Cutting celloidin embedded tissue
   a. Base-sledge – a very hard tissue
   b. Standard sliding – with movable exposed knife. For cutting celloidin embedded tissue. This is the most
        dangerous style of microtome.
General Histology and Histotechnique (1st Semester; SY 2012-2013)


4.   Freezing (by Queckett, 1848)
-    Cutting embedded frozen sections
    Cryostat or cold microtome
-    Refrigerated apparatus used in fresh microtomy
-    For freezing tissue in the block holder to correct degree of hardness to facilitate easier and faster sectioning.
-    Provides a means of preparing a thin section of fresh frozen tissues especially for fluorescence antibody staining
     technique.

5.   Ultrathin
-    Cutting sections for electron microscope - celloidin
-    Specimen used should be small
-    Should be fixed in osmium tetroxide and embedded in plastics.

Microtome knives:

1.   Plane concave knife
-    25mm
-    one side flat, other concave
    Flat – recommended for cutting celloidin embedded tissue blocks using a sliding microtome.
    Concave – used for cutting paraffin embedded tissues. Using its Base Sledge microtome, Rotary, Rocking

2.   Biconcave knife
-    120mm
-    both sides are concave
    Concave – cutting paraffin embedded tissues using a rotary microtome.

3.   Plane wedge knife
-    100 mm
-    Both sides are straight
    Straight – recommended for frozen section or for cutting extremely hard/tough specimen embedded in a paraffin
     block.
-    Using a base sledge sliding microtome.


Sectioning
- Cutting thinly the specimen with the use of microtome
- Thin materials – sections

3 types of tissue sections:

1. Paraffin Section
- Leaded in a paraffin block
- Cut using the rotary microtome/Rocky microtome

2. Celloidin section
- Embedded in a celloidin
- Cut by sliding microtome

3. Frozen Section
- Fixed with carbon dioxide or cryostat
General Histology and Histotechnique (1st Semester; SY 2012-2013)


Faults occurring during tissue processing

              FAULTS                                  REASONS                                REMEDY
1. Brittle or hard tissue.            -     Prolong fixation, dehydration       -   Softened by soaking in a small
                                            clearing, and infiltration of           dish containing water with
                                            paraffin in overheated paraffin         detergent or phenol.
                                            oven.
                                      -     Dry out of tissue before actual
                                            fixation.
2. Clearing agent turns milky as      -     Water is not completely             -   Repeat dehydration with
   soon as tissue is placed in it.          removed due to incomplete               absolute alcohol then clear
                                            dehydration.                            again.
3. On trimming tissue smells of       -     Clearing agent not completely       -   Repeat paraffin impregnation.
   clearing agent.                          removed due to insufficient
                                            impregnation.
4. Tissue is opaque, section-         -     Insufficient clearing.              -   Repeat clearing and if the
   cutting is difficult due to                                                      object has been embedded,
   presence of alcohol                                                              prolong clearing up to 12 hrs.
                                                                                    And embed it again.
5. Tissue shrinks away from wax       -     Insufficient dehydration so         -   Repeat the whole process.
   when trimmed.                            there is incomplete clearing
                                            and impregnation.
6. Tissue is soft when block is       -     Incomplete fixation                 -   Repeat fixation procedure
   trimmed
7. Air holes found in tissue during   -     Incomplete infiltration or          -   Repeat impregnation.
   trimming.                                impregnation.
8. On trimming, wax appears           -     Contaminated wax                    -   Re-embed in a freshly filtered
   crystalline.                       -     The bluff Nat cold rapidly              wax.
9. Paraffin block after cooling is    -     there is insufficient paraffin      -   Repeat impregnation and re-
   moist and crumbles                       impregnation                            embed.

Faults observed during section-cutting:
              Faults                                    Reasons                               Remedy
1. Sections fail to form ribbons.    -      Surfaces and edges of the bluff     -   Re-trim the block
                                            are not parallel                    -   Re-adjust the block
                                      -     Horizontal surface of the block     -   Reduce the tilt of the knife
                                            is not parallel to the knife        -   Re-adjust the thickness of the
                                      -     Knife is tilted too much                section
                                      -     Sections are too thick              -   Re-sharpen the knife
                                      -     Knife is dull
2. Sections roll up on cutting so     -     Knife is blunt                      -   Re-sharpen the knife
   that they adhere and get           -     The tilt of the knife is great      -   Reduce the tilt
   broken against the knife edge.     -     The knife edge is dirty             -   Clean the knife edge
3. Ribbon is crooked, curved and      -     The knife is blunt or dull; there   -   Adjust the knife so that knife
   uneven instead of straight.              is a dull spot on the knife             edge will present a uniformly
                                            producing an irregular knife            sharp edge to the block.
                                            edge.
                                      -     The knife is not parallel to the    -   Re-adjust the knife and the
                                            block                                   block.
4. Sections are wrinkled,             -     The paraffin block is warm and      -   Cool the paraffin on ice water
General Histology and Histotechnique (1st Semester; SY 2012-2013)


    compressed or jammed.                 soft                                     until firmed.
                                      -   The knife edge is coated with        -   Clean the knife edge
                                          paraffin                             -   Re-adjust the thickness of
                                      -   The section are too thin                 section
                                      -   Microtome set screw is loose.        -   Tighten the screw
                                      -   The tilt of knife is too vertical.   -   Reduce the tilt
5. Sections are torn and crumble      -   Incomplete dehydration,              -   Repeat dehydration, clearing
   when cut.                              clearing and impregnation                and impregnation
                                      -   Paraffin is warmed and soft.         -   Cool and harden the paraffin in
                                                                                   ice water for ¼ to ½ hour.
6. A hole is formed in the section.   -   Bubble or dirt is formed in the      -   Re-embed in freshly filtered
                                          embedding medium                         wax.
7. Sections adhere to the knife or    -   There is a static electrically due   -   Breathe out or blow gently in
   other parts of the machine.            to low atmospheric humidity.             the block and knife to break-up
                                                                                   static electricity. Boil water
                                      -   Knife edge is dirty                      inside the laboratory.
                                      -   Knife edge is dull                   -   Clean the knife edge
                                      -   Knife tilt is great                  -   Re-sharpen the knife
                                                                               -   Reduce the tilt
8. Sections are lifted from the       -   Knife tilt is too great              -   Reduce tilt
   knife on upstrokes                 -   Knife is dull                        -   Re-sharpen the knife
                                      -   Paraffin is soft                     -   Cool the paraffin in ice water
9. Sections cut is sometimes thin,    -   Knife is blunt                       -   Re-sharpen the knife
   and sometimes thick                -   Knife is not completely clamp        -   Re-adjust the knife
                                      -   The tilt of knife is great           -   Reduce the tilt
                                      -   The knife or block holder is         -   Tighten adjusting and locking
                                          loose.                                   screws.

Paraffin Section
- Sections are usually cut between 4-6 micron in thickness.
- Micrometer gauge is set to the required thickness and the knife is positioned in such a way that the center of the
    blade is in line with the block and the knife has been securely clamped in place.
- Cutting is then started until complete sections come out of the block.
- A regular cutting rhythm is required
- The knife is usually tilted at 0-15 degrees angulations’ on a microtome to allow a clearance angle between the
    cutting facet and tissue block
- Sections are removed in ribbons of ten, to allow easy location of serial sections
- The sections are then floated out in water bath set at 45-50°C to flatten the sections


Staining
- To impart a color to the tissues
- Parts to be visible under microscope
Two Categories of dyes:

1. Natural Dyes
- Obtained from plants and animals
Hematoxilyn
- Derived by either extraction from the heartwood of Mexican tree known a Hematoxilyn campechianum.
- Most valuable stain in histology used by cytologist
General Histology and Histotechnique (1st Semester; SY 2012-2013)


- Powerful nuclear and chromatin staining capacity
- With striking polychrome qualities
Hematin – active coloring agents

2. Synthetic Dyes
- Known as Coal Tar Dyes
- Hydrocarbon benzene derivation
- Aniline dyes
Examples:
 Picric acid
- Ability to form salts with alkaline
- Fix and stain at the same time by itself
- Used as a fixative
- Decalcifying agen
- Tissue softener

   Eosin
-   a valuable stain in Histotechnique, important stain in connective tissue and cytoplasm.
-   Histopathology – counter-stain after hematoxilyn and before methylene blue
-   Background for contrasting stains because it gives a pleasing and colorful contrast to nuclear stain.

Red acid dye available in 2 shades:
-
a. Bluish
-   Deepen red color
   Ex. Eosin B
b. Yellowish
- Most usable
- Readily soluble in water and less in alcohol
- Available in both aqueous and alcoholic solutions
- Slows a green fluorescence especially in alcoholic medium.
   Ex. Eosin Y

3. Basic dyes
- coloring substance is found in the basic components
- Acid radical is taken from sulfuric, acetic and hydrochloric acid.
Ex. Methylene blue

*Note: On a chemical basis certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater
affinity for basic dyes, while basic constituents (e.g. cytoplasm) take more of the acid stains.


   Fixation of tissues with mercuric chloride and formaldehyde usually favors staining with basic dyes.
   Picric acid and Chromium – fixed tissues usually take an acidic dyes
   Ethyl alcohol or acetic acid – fixed tissues readily take in both basic and acidic dyes.

4. Neutral Dyes
- Combining aqueous solution of acid and basic dyes.
- Capable of staining cytoplasm and nucleus simultaneously and differentially
- Soluble in alcohol but not in water.
General Histology and Histotechnique (1st Semester; SY 2012-2013)




Ex. WBC differentiation
 Giemsa’s stain
 Leishman’s stain


3 main groups of tissue staining

1. Histological Staining
-   process by the tissue staining constituents are demonstrated in section by direct interaction with a dye or staining
   solution producing coloration of the active tissue components.
Example:
- Mircroanatomical stains
- Bacterial stains
- Specific tissues stains – muscle, connective tissue, neurological stains.
2. Histochemical staining (Histochemistry)
- A process whereby tissue constituents are studied through chemical reactions that will permit microscopy
   localization of a specific tissue substance.
3. Immunohistochemistry
- Combination of 1st and 2nd classification
- Allow phenotypic markers to be detected using fluorescent labeled or enzyme labeled.


Staining Technique

1. Direct Staining
- A process of giving color to the section by using aqueous or alcoholic dye solution.
Ex. Methylene blue, Eosin

2.   Indirect Staining
-    Whereby the action of the dye is intensified by adding another agent or mordant.
-    Mordant – serve as a bridge or link between the tissue and the dye.
-    Mordant + dye -> Lake + Tissue = “Tissue mordant dye complex

3.   Progressive Staining
-    The process whereby tissue elements are stained in a definite sequence.
-    Definite sequence? - to have a satisfactory differential coloration of tissues
-    Once the dye is taken by the tissue it is not wash anymore.

4.   Regressive Staining
-    The tissue is first over-stained then decolorized
-    Over-stained – to obliterate the cellular details decolorized until the desired color is achieved.
-    Differentiation – the selective removal of excess stain.

5. Metachromatic Staining
- Entails the use of specific dyes which differentiate particular substances by staining them with a color that is
   different from that of the stain itself.
Where is this used?
- Employ in staining cartilages, connective tissues, epithelial tissues
- Belong to basic dyes
General Histology and Histotechnique (1st Semester; SY 2012-2013)


Example: Methylene blue, Safranin.
6. Counterstaining
- Provide contrast and background
Example: Safranin

7. Microanatomical Staining
- General differentiation of nucleus and cytoplasm.

8.   Metallic Impregnation
-    Where specific tissue elements are demonstrated by colorless solutions of metallic salts
-    Metallic salts – reduced by the tissue ; reduced also by bacteria
-    It’s not absorbed by the tissues but it is held physically on the surface as precipitates
-    Valuable metals: Ag, Chloride, Silver nitrate

9. Vital Staining
- Selective staining of living cell constituents
- Demonstrates cytoplasmic structures by phagocytosis of the dye particle.

10. Intravital Staining
- By injecting the dye into any part of animal body.
- Purpose: To produce different coloration of cells
- Most specially in reticuloendothelial system
Examples: Lithium, Indian

11. Supravital Staining
- To stain living cells immediately after removal from the living body.
Ex. Neutral red

Staining of Paraffin Section

    Paraffin wax is poorly permeable to most staining solutions and should therefore remove from the section prior to
     staining.
    This is usually done by immersing the paraffin section in a solvent (xylene) two times, at 1-2 minutes duration.
    Xylene is not miscible with aqueous solutions and low graded alcohol and therefore be subsequently removed with
     absolute alcohol followed by descending grades of alcohol to prevent damage and detachment of sections due to
     possible production of diffusion currents.
    The alcohol is then finally replaced with water, before actual staining of section is performed
    After deparaffinization with xylene, the section is subjected to decreasing grades of alcohol.
    After staining, the section is again dehydrated with increasing grades of alcohol and cleared with two changes of
     xylene to prepare the section for mounting, since most mountants are miscible with xylene.
    The second change of xylene will raise the refractive index of the glass slide, thereby reducing light refraction during
     microscopic examination.

*Note: If section float off the slide during staining, fix section in a Bunsen flame
Slides must not be dirty of greasy.
General Histology and Histotechnique (1st Semester; SY 2012-2013)


Mounting
- A process of rendering slides permanency
- Used to keep the slide to its permanent position
- Seraphy Fluid – applied between the tissue and cover slip.

Mounting media importance:
 It protects the stained sections from getting scratched and from bleaching or deterioration due to oxidation, thereby
  preserving the slides for permanent keeping.
 Facilitate easy handling and storage
 Prevent the damage of sections which may lead to distortion of image during microscopic examination.

Characteristics of a good mounting medium:
 It should not dry quickly
 It should not dissolve out of fade tissue sections
 It should not cause shrinkage and distortion of tissues
 It should set hard, thereby producing permanent mounting of sections.

2 main groups of mounting media:

1. Aqueous media
- Design to mount water miscible preparations
Examples: Glycerin, water

2. Resinous media
- Used for preparations that has been dehydrated and cleared in xylene and are recommended for majority of
   staining method.
Examples: Balsan, Colorless nail polish

Types of Mounting

1. Dry Mount
- It requires no water
- Usually used in inanimate objects

2. Wet mount
- It requires water
- Used to prepare slides to cold living organisms whether they are motile or not.


Ringing
- A process of sealing the margins of cover slip.

Purpose:
1. To prevent the escape of fluid or semi-fluid mounts
2. To prevent evaporation of mounts
3. To immobilize the cover slip
4. To prevent sticking of the slides upon storage

Weitere ähnliche Inhalte

Was ist angesagt?

Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...
Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...
Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...Indian dental academy
 
Fixation & fixatives in histopathology, dr naveen reddy
Fixation & fixatives in histopathology, dr naveen reddyFixation & fixatives in histopathology, dr naveen reddy
Fixation & fixatives in histopathology, dr naveen reddyNaveen Parvathareddy
 
Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...
Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...
Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...akash mahadev
 
fixation and decalcification
fixation and decalcificationfixation and decalcification
fixation and decalcificationVasim ansari
 
Fixatives in Histopathology
Fixatives in HistopathologyFixatives in Histopathology
Fixatives in HistopathologyIshwar9
 
Histological techniques sections 1 2 3
Histological techniques sections 1 2 3Histological techniques sections 1 2 3
Histological techniques sections 1 2 3Mathew Joseph
 
Lecture (3) mechanisms of fixation
Lecture (3) mechanisms of fixationLecture (3) mechanisms of fixation
Lecture (3) mechanisms of fixationHafsa Hussein
 
Tissue preparation (group 4)
Tissue preparation (group 4)Tissue preparation (group 4)
Tissue preparation (group 4)sartika Amran
 
Tissue Fixation Histopathology
 Tissue Fixation Histopathology  Tissue Fixation Histopathology
Tissue Fixation Histopathology habibhasrat
 
Tissue processing incorporating Microarray
Tissue processing incorporating MicroarrayTissue processing incorporating Microarray
Tissue processing incorporating MicroarraySurbhi Mahajan
 
Histological Techaniques
Histological TechaniquesHistological Techaniques
Histological TechaniquesZahoor Ahmed
 
Histological Techniques: Section 2:Fixation of tissues
Histological Techniques: Section 2:Fixation of tissuesHistological Techniques: Section 2:Fixation of tissues
Histological Techniques: Section 2:Fixation of tissuesMathew Joseph
 

Was ist angesagt? (20)

15 histotechniques 1
15 histotechniques 115 histotechniques 1
15 histotechniques 1
 
Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...
Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...
Fixatives and fixation/certified fixed orthodontic courses by Indian dental a...
 
Fixation
Fixation Fixation
Fixation
 
Fixatives
FixativesFixatives
Fixatives
 
Fixation & fixatives in histopathology, dr naveen reddy
Fixation & fixatives in histopathology, dr naveen reddyFixation & fixatives in histopathology, dr naveen reddy
Fixation & fixatives in histopathology, dr naveen reddy
 
Lecture 1.introduction to histopathology ii
Lecture 1.introduction to histopathology iiLecture 1.introduction to histopathology ii
Lecture 1.introduction to histopathology ii
 
Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...
Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...
Histopathological techniques -sectioning, STAINING, EMBEDDING, fixaton, micro...
 
H & E STAIN
 H & E STAIN H & E STAIN
H & E STAIN
 
fixation and decalcification
fixation and decalcificationfixation and decalcification
fixation and decalcification
 
Fixatives in Histopathology
Fixatives in HistopathologyFixatives in Histopathology
Fixatives in Histopathology
 
6 histotechniques
6 histotechniques6 histotechniques
6 histotechniques
 
Histological techniques sections 1 2 3
Histological techniques sections 1 2 3Histological techniques sections 1 2 3
Histological techniques sections 1 2 3
 
Lecture (3) mechanisms of fixation
Lecture (3) mechanisms of fixationLecture (3) mechanisms of fixation
Lecture (3) mechanisms of fixation
 
Tissue processing
Tissue processingTissue processing
Tissue processing
 
Tissue preparation (group 4)
Tissue preparation (group 4)Tissue preparation (group 4)
Tissue preparation (group 4)
 
Tissue Fixation Histopathology
 Tissue Fixation Histopathology  Tissue Fixation Histopathology
Tissue Fixation Histopathology
 
Tissue processing incorporating Microarray
Tissue processing incorporating MicroarrayTissue processing incorporating Microarray
Tissue processing incorporating Microarray
 
Histological Techaniques
Histological TechaniquesHistological Techaniques
Histological Techaniques
 
Histological Techniques: Section 2:Fixation of tissues
Histological Techniques: Section 2:Fixation of tissuesHistological Techniques: Section 2:Fixation of tissues
Histological Techniques: Section 2:Fixation of tissues
 
Rahul singh
Rahul singhRahul singh
Rahul singh
 

Ähnlich wie GHH (prelim handout)

lecture3andlecture4techniquesincellbiology-210201105723.pdf
lecture3andlecture4techniquesincellbiology-210201105723.pdflecture3andlecture4techniquesincellbiology-210201105723.pdf
lecture3andlecture4techniquesincellbiology-210201105723.pdfZainabKhadhar
 
Chapter 3 observing microrganisms partial
Chapter 3 observing microrganisms partialChapter 3 observing microrganisms partial
Chapter 3 observing microrganisms partialBilalHoushaymi
 
Methods to-study-histology
Methods to-study-histologyMethods to-study-histology
Methods to-study-histologyHoolahoop13
 
Progressive staining/ oral surgery courses  
Progressive staining/ oral surgery courses  Progressive staining/ oral surgery courses  
Progressive staining/ oral surgery courses  Indian dental academy
 
Microtomy - Preparation of Histological Slides
Microtomy - Preparation of Histological SlidesMicrotomy - Preparation of Histological Slides
Microtomy - Preparation of Histological SlidesSyed Muhammad Khan
 
Lab 3-Staining 1.pptx
Lab 3-Staining 1.pptxLab 3-Staining 1.pptx
Lab 3-Staining 1.pptxFrtf1
 
Histological techniques
Histological techniquesHistological techniques
Histological techniquesRUPALIMUNDE
 
PREPARATION OF HISTOLOGICAL SPECIMENS.pptx
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxPREPARATION OF HISTOLOGICAL SPECIMENS.pptx
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxAnthonyMatu1
 
histopathology notes.docx
histopathology notes.docxhistopathology notes.docx
histopathology notes.docxrajesh kumar
 
Staining Technique in microbiology
Staining Technique in microbiologyStaining Technique in microbiology
Staining Technique in microbiologyPriyaDixit46
 
Commet Assay
Commet AssayCommet Assay
Commet AssayUlaa Iman
 

Ähnlich wie GHH (prelim handout) (20)

lecture3andlecture4techniquesincellbiology-210201105723.pdf
lecture3andlecture4techniquesincellbiology-210201105723.pdflecture3andlecture4techniquesincellbiology-210201105723.pdf
lecture3andlecture4techniquesincellbiology-210201105723.pdf
 
Techniques in Cell Biology
Techniques in Cell BiologyTechniques in Cell Biology
Techniques in Cell Biology
 
H and E staingin.ppt
H and E staingin.pptH and E staingin.ppt
H and E staingin.ppt
 
Histological Tools
Histological Tools Histological Tools
Histological Tools
 
Plant histology
Plant histologyPlant histology
Plant histology
 
Chapter 3 observing microrganisms partial
Chapter 3 observing microrganisms partialChapter 3 observing microrganisms partial
Chapter 3 observing microrganisms partial
 
Methods to-study-histology
Methods to-study-histologyMethods to-study-histology
Methods to-study-histology
 
Histopathology.pptx
Histopathology.pptxHistopathology.pptx
Histopathology.pptx
 
Progressive staining/ oral surgery courses  
Progressive staining/ oral surgery courses  Progressive staining/ oral surgery courses  
Progressive staining/ oral surgery courses  
 
Microtomy - Preparation of Histological Slides
Microtomy - Preparation of Histological SlidesMicrotomy - Preparation of Histological Slides
Microtomy - Preparation of Histological Slides
 
Lab 3-Staining 1.pptx
Lab 3-Staining 1.pptxLab 3-Staining 1.pptx
Lab 3-Staining 1.pptx
 
Histological techniques
Histological techniquesHistological techniques
Histological techniques
 
PREPARATION OF HISTOLOGICAL SPECIMENS.pptx
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxPREPARATION OF HISTOLOGICAL SPECIMENS.pptx
PREPARATION OF HISTOLOGICAL SPECIMENS.pptx
 
Ex1
Ex1Ex1
Ex1
 
HISTOPATHOLOGY ppt
HISTOPATHOLOGY ppt HISTOPATHOLOGY ppt
HISTOPATHOLOGY ppt
 
histopathology notes.docx
histopathology notes.docxhistopathology notes.docx
histopathology notes.docx
 
Staining.pptx
Staining.pptxStaining.pptx
Staining.pptx
 
Staining Technique in microbiology
Staining Technique in microbiologyStaining Technique in microbiology
Staining Technique in microbiology
 
Commet Assay
Commet AssayCommet Assay
Commet Assay
 
Principles of Staining
Principles of StainingPrinciples of Staining
Principles of Staining
 

Kürzlich hochgeladen

CAULIFLOWER BREEDING 1 Parmar pptx
CAULIFLOWER BREEDING 1 Parmar pptxCAULIFLOWER BREEDING 1 Parmar pptx
CAULIFLOWER BREEDING 1 Parmar pptxSaurabhParmar42
 
2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx
2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx
2024.03.23 What do successful readers do - Sandy Millin for PARK.pptxSandy Millin
 
Practical Research 1 Lesson 9 Scope and delimitation.pptx
Practical Research 1 Lesson 9 Scope and delimitation.pptxPractical Research 1 Lesson 9 Scope and delimitation.pptx
Practical Research 1 Lesson 9 Scope and delimitation.pptxKatherine Villaluna
 
3.21.24 The Origins of Black Power.pptx
3.21.24  The Origins of Black Power.pptx3.21.24  The Origins of Black Power.pptx
3.21.24 The Origins of Black Power.pptxmary850239
 
The Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George WellsThe Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George WellsEugene Lysak
 
How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17Celine George
 
5 charts on South Africa as a source country for international student recrui...
5 charts on South Africa as a source country for international student recrui...5 charts on South Africa as a source country for international student recrui...
5 charts on South Africa as a source country for international student recrui...CaraSkikne1
 
How to Solve Singleton Error in the Odoo 17
How to Solve Singleton Error in the  Odoo 17How to Solve Singleton Error in the  Odoo 17
How to Solve Singleton Error in the Odoo 17Celine George
 
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...Nguyen Thanh Tu Collection
 
Patterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptxPatterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptxMYDA ANGELICA SUAN
 
How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17Celine George
 
M-2- General Reactions of amino acids.pptx
M-2- General Reactions of amino acids.pptxM-2- General Reactions of amino acids.pptx
M-2- General Reactions of amino acids.pptxDr. Santhosh Kumar. N
 
DUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRA
DUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRADUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRA
DUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRATanmoy Mishra
 
The basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptxThe basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptxheathfieldcps1
 
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdfP4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdfYu Kanazawa / Osaka University
 
Practical Research 1: Lesson 8 Writing the Thesis Statement.pptx
Practical Research 1: Lesson 8 Writing the Thesis Statement.pptxPractical Research 1: Lesson 8 Writing the Thesis Statement.pptx
Practical Research 1: Lesson 8 Writing the Thesis Statement.pptxKatherine Villaluna
 
Diploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdfDiploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdfMohonDas
 

Kürzlich hochgeladen (20)

CAULIFLOWER BREEDING 1 Parmar pptx
CAULIFLOWER BREEDING 1 Parmar pptxCAULIFLOWER BREEDING 1 Parmar pptx
CAULIFLOWER BREEDING 1 Parmar pptx
 
Prelims of Kant get Marx 2.0: a general politics quiz
Prelims of Kant get Marx 2.0: a general politics quizPrelims of Kant get Marx 2.0: a general politics quiz
Prelims of Kant get Marx 2.0: a general politics quiz
 
2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx
2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx
2024.03.23 What do successful readers do - Sandy Millin for PARK.pptx
 
Practical Research 1 Lesson 9 Scope and delimitation.pptx
Practical Research 1 Lesson 9 Scope and delimitation.pptxPractical Research 1 Lesson 9 Scope and delimitation.pptx
Practical Research 1 Lesson 9 Scope and delimitation.pptx
 
3.21.24 The Origins of Black Power.pptx
3.21.24  The Origins of Black Power.pptx3.21.24  The Origins of Black Power.pptx
3.21.24 The Origins of Black Power.pptx
 
The Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George WellsThe Stolen Bacillus by Herbert George Wells
The Stolen Bacillus by Herbert George Wells
 
How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17How to Add a New Field in Existing Kanban View in Odoo 17
How to Add a New Field in Existing Kanban View in Odoo 17
 
5 charts on South Africa as a source country for international student recrui...
5 charts on South Africa as a source country for international student recrui...5 charts on South Africa as a source country for international student recrui...
5 charts on South Africa as a source country for international student recrui...
 
Personal Resilience in Project Management 2 - TV Edit 1a.pdf
Personal Resilience in Project Management 2 - TV Edit 1a.pdfPersonal Resilience in Project Management 2 - TV Edit 1a.pdf
Personal Resilience in Project Management 2 - TV Edit 1a.pdf
 
How to Solve Singleton Error in the Odoo 17
How to Solve Singleton Error in the  Odoo 17How to Solve Singleton Error in the  Odoo 17
How to Solve Singleton Error in the Odoo 17
 
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
CHUYÊN ĐỀ DẠY THÊM TIẾNG ANH LỚP 11 - GLOBAL SUCCESS - NĂM HỌC 2023-2024 - HK...
 
Patterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptxPatterns of Written Texts Across Disciplines.pptx
Patterns of Written Texts Across Disciplines.pptx
 
How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17How to Use api.constrains ( ) in Odoo 17
How to Use api.constrains ( ) in Odoo 17
 
M-2- General Reactions of amino acids.pptx
M-2- General Reactions of amino acids.pptxM-2- General Reactions of amino acids.pptx
M-2- General Reactions of amino acids.pptx
 
DUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRA
DUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRADUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRA
DUST OF SNOW_BY ROBERT FROST_EDITED BY_ TANMOY MISHRA
 
The basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptxThe basics of sentences session 10pptx.pptx
The basics of sentences session 10pptx.pptx
 
Finals of Kant get Marx 2.0 : a general politics quiz
Finals of Kant get Marx 2.0 : a general politics quizFinals of Kant get Marx 2.0 : a general politics quiz
Finals of Kant get Marx 2.0 : a general politics quiz
 
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdfP4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
P4C x ELT = P4ELT: Its Theoretical Background (Kanazawa, 2024 March).pdf
 
Practical Research 1: Lesson 8 Writing the Thesis Statement.pptx
Practical Research 1: Lesson 8 Writing the Thesis Statement.pptxPractical Research 1: Lesson 8 Writing the Thesis Statement.pptx
Practical Research 1: Lesson 8 Writing the Thesis Statement.pptx
 
Diploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdfDiploma in Nursing Admission Test Question Solution 2023.pdf
Diploma in Nursing Admission Test Question Solution 2023.pdf
 

GHH (prelim handout)

  • 1. General Histology and Histotechnique (1st Semester; SY 2012-2013) Preparation for whole mount B. According to the reaction of the tissue stain. Materials needed: 1. ) Basophilic 1. Glass slides - stains the acid component - usually 25 x 75 mm 2. ) Acidophilic - usually 1- 1.2 mm thick - Stains the basic components Types: - Stains the cytoplasm of the cell a. flat slides b. concave slides Some common stains: 1. ) Hematoxylin and Eosin (H & E) 2. Cover slips a. Hematoxylin – stain nuclei blue (basophilic) Two types: b. Eosin – stain cytoplasm pink (acidophilic) Number 1 = 0.13-0.17 mm thick Number 2 = 0.17-0.25 mm thick 2. ) Connective Tissue stains Functions: a. Masson’s trichome a. hold samples in place b. Mallorys triple CT stain b. flattens out specimen - both employ a nuclear, cytoplasmic and a third stain c. Protects the objective from immersion into the specific for matrix water drop. 3. ) Silver impregnation 3. Stains - Used to trace nerves, stain Golgi, reticular fibers. - Different stains have different affinities for the different organisms thus maybe used to 4. )Wright stain – blood smear differentiate different types of organisms or to view specific parts of organisms. 4. other chemicals - Substance that give color to the specimen to  Fixatives improve visibility.  Embedding materials Example:  Mountants Counter stain – not all organisms can absorb.  Clearers Type of stains A. According to the number of dyes used 1. ) Positive staining - Stains the specimen itself. Type of Slide Preparation a. Simple Stain - Employs single dye (on color)  Temporary Slide - Primary dye - water Ex. Crystal violet, Methylene blue - fresh specimen b. Differential Stain  Permanent slide - Two or more stains - reagents are used - Primary dye – counter stain - dead tissues - Gram-staining Ex.  Fixatives – preserve specimen Primary dye = Crystal violet; - Function: to avoid post mortem conditions Counter stain = Safranin Ex: FAA, Bouin’s sol’n, Formalin (10%) Ex. Acid fast stain (Mycobacterium), Ziel Neelsen 2. ) Negative staining  Dehydrants - the background of the organism/specimen is the - reagents that remove undesired fluid that may stain interfere with later processing - Function: To emphasized the appendages Ex: Ethyl alcohol (ethanol/alcohol)
  • 2. General Histology and Histotechnique (1st Semester; SY 2012-2013)  Clearing agents - utilize when rapid diagnosis of tissues - to clear - specially recommended for lipids and nervous tissue Ex: Xylene and Cloroform elements USES:  Embedding medium  Rapid pathological diagnosis during surgery - supports the specimen  Diagnostic and research enzyme histochemistry - paraffin (wax time)  Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates  Microtome  Immunofluorescent and immunohistochemical staining  Stains  Some specialized silver stains, particularly in - Gives color to the specimen to differentiate the neuropathology different parts of specimen. Ex: Methylene blue, Hematoxylin, Wright stain, Crystal violet, Eosin, Safranin, Fast green. Processing of Tissues  Adhesive/Affixative  Fixation - to stick or adhere the specimen to the slide Purposes: Ex: Mayer’s albumin a. Confers chemical stability on the tissue b. Hardens the tissue  Mountant c. Stops enzyme autolysis - render permanency d. many enhance later staining technique – act as Ex: Balsam mordant Fresh Tissue Examination Five (5) major groups of Fixatives: 1. Aldehydes 1. Teasing/Dissociation - include formaldehyde or formalin and - use of needle to separate tissues in bulk glutaraldehyde 2. Squashing - non precipitating - cut a tiny piece on slide and with the use of another  10 % formalin slide to compress - buffered to lessen acidity that causes precipitation 3. Smear preparation and autolysis - to spread specimen thinly onto the slide  Glutaraldehyde a. Streaking - 2-4 % buffered - Applicator stick or the wine loop usage. - fixes very fast - Spread specimen to the slide directly on a zigzag - good for electron microscopy manner. - it penetrates poorly - Uniform the distribution - It gives best results on cytoplasmic and nuclear b. Spreading details. - to spread thinly into the slide - teasing is also needed 2. Mercurials - for fresh sputum mucoid secretion - contain mercuric chloride and Zenker’s c. Full-apart - fixed the tissue by an unknown mechanism - for blood smear, enzymes of the GIT - penetrate very poorly - it used pusher - causes tissue to harden d. Touch Preparation/impression smear - fixed fast - used of freshly cut fruit - give excellent nuclear detail - touched on to the surface of the slide - best in nematopoietic reticuloendothelial - letting the cells stick on to the slide 4. Frozen section 3. Alcohols
  • 3. General Histology and Histotechnique (1st Semester; SY 2012-2013) - include methanol and ethanol - protein denaturants 1. Buffering – pH 6-8 - cause too much brittleness and hardness - phosphate, bicarbonates - good in cytological smears - Commercial formalin is buffered with phosphate at - give good nuclear detail a pH of 7. - precipitating 2. Penetration 4. Oxidizing agents - depends of diffusability of fixatives - include Potassium permanganate, Potassium - formalin and alcohol: best dichromate and Osmium tetroxide - glutaraldehyde: worst - none precipitating - diffusability – constant - used in frequently - in between – mercurials - causes an extensive denaturation - in order to the organisms to be penetrated it should be cut in 2-3 mL 5. Picrates - include fixatives and picric acid (e.g. Bouin’s sol’n) 3. Volume - precipitating fixative - ratio 10:1 - enhanced nuclear detail - 10 – fixative - don’t cause hardens - 1 – specimen / tissue - Dry form – explosive - in Solutions– stains everything it catches including 4. Temperature the skin; yellow color - High temperature – Increases speed of fixation Ex. Hot formalin will fix the tissue faster. Fixative for General Usage 5. Concentration of Fixation a. 4 % / 10 % formalin - adjusted down to the lowest level possible Advantages: - 10% formalin: best 1. easy to make - glutaraldehyde (3%) 2. Has good penetration power 3. Does not shrink the specimen 6. Time interval 4. Does not affect the material it kept for long time - FAA – up to 12 hrs. 5. Can be kept without deterioration for considerable - Carnoy’s – 1-3hrs time - Zenker’s – 14-24hrs 6. Easy to handle 7. Less hazard Organs for processing: Kidney b. FAA – Formalin-Acetic-Acid-Alcohol Liver - Best for plant material (tissues) Stomach - Composition: 50ml ethanol, 5ml glacial acetic acid, Small Intestine 10ml 37-40% formaldehyde, 35ml water Large intestine Lungs c. Zenker’s and Carnoys’s fluid Spleen - best for animal tissues Gonads  Zenkers’s: 950ml distilled water, 25g potassium Muscles dichromate, 50g mercuric chloride, 50g glacial acetic Specimen Class  Carnoy’s: 60 ml ethanol, 30ml chloroform, 10ml Class A – delicate tissues: lungs; membranes glacial acetic acid Class B – moderately thick; spleen, live, kidney Class C – bulky and thick; skin Factors affecting fixation
  • 4. General Histology and Histotechnique (1st Semester; SY 2012-2013) Fixation process: 100% ethanol – 30 min - submerge the specimen fully in fixative 100% ethanol– 30 min - cover the container tightly ***To ensure complete removal of water during dehydration, use two changes of 100% ethanol of at Proper timing for each specimen if FAA is used: least ½ hour each. Class A – 6-9 hrs. (10hrs) Class B – 12-18 hrs. (16-18 hrs) Timing of dehydration: Class C – 24-36 hrs. Class A – 30 min in each alcohol concentration Class B – 1 hr Dehydration Class C – 3-4 hrs. - removing undesired water inside the specimen Clearing Characteristics of an ideal dehydrating solution:  It should dehydrate rapidly without producing 1. Alcohol –to provide rapid miscible in the tissue considerable shrinkage or distortion of tissues. 2. To facilitate impregnation or infiltration  It should not evaporate very fast.  It should be able to dehydrate even fatty tissues Xylene  It should not harden tissues exclusively. - Commonly used; tends to harden tissues if left too  It should not remove stain. long.  It should not be toxic to the body  It should not be a fire hazard. Advantages: - makes tissue transparent Commonly used dehydrating agents: - miscible with absolute alcohol and paraffin - alcohol - evaporate quickly in paraffin oven and can - acetone - cheap - triethylphosphate - tetrahydrofuran Disadvantages: - dioxane - highly in flammable - If used longer than 3 hrs. it makes tissues  Alcohol excessively hard and brittle - a standard series of alcohols are used most - Causes considerable hardening and shrinkage of - Alcohol series: 70% - 80% - 95% tissues - Higher grades of alcohol: shrinkage and hardening - Becomes milky when an incompletely dehydrated of tissues tissue is immersed in it. - concentrated alcohol tend to harden only the surface of tissue Toluene  Ethyl alcohol - time recommended is 1-2 hours - best dehydrating agent  fast acting Advantages:  mixes with water - it is miscible with both absolute alcohol and paraffin  penetrates tissue easily - It acts fairly and rapidly and is recommended for  not poisonous routine purposes  not very expensive - tissue do not become excessively hard and brittle even if left in Toluene for 24 hrs Dehydrating procedures: - it is not carcinogenic Water 50% ethanol Disadvantages: 70% ethanol - it is relatively slower than Xylene and benzene 80% ethanol - it tends to acidify in a partially filled vessel 90% ethanol
  • 5. General Histology and Histotechnique (1st Semester; SY 2012-2013) - highly concentrated solutions will emit fumes that - complete clearing is difficult to evaluate are toxic upon prolonged exposure - tissues tend to float in chloroform - it is more expensive - it evaporates quickly from a water bath Benzene Impregnation, Embedding, Trimming - clearing agent - clear and penetrate tissues rapidly Impregnation - carcinogenic - Diffusion or accumulation in a cell or tissue substances that are not normal. Advantages: - To fill-out the tissues in each spaces or in-between - It is rapidly acting, so recommended for urgent spaces biopsies (15 to 60 min) and routine purposes. - It volatilizes rapidly in paraffin oven and is therefore Three (3) types of Tissue Impregnation: easily eliminated from the tissue. 1. Paraffin wax impregnation - It is miscible with absolute alcohol Precautions: - it does not make tissues hard and brittle a. Prolong treatment in melted paraffin causes - it causes minimum shrinkage shrinkage and hardening of tissues making cutting - it makes tissues transparent difficult. b. Infiltration in overheated paraffin (above 60°C) will Disadvantages: produce shrinkage and hardening of tissues and - it is highly in flammable destroy lymphoid tissues completely - if a section is left in benzene for a long time, c. Paraffin wax must be pure considerable tissue shrinkage may be observed d. Paraffin wax may be used only twice. - Excessive exposure to benzene may be extremely 2. Celloidin impregnation toxic to man and may become carcinogenic or it 3. Gelatin impregnation may damage the bone marrow resulting in a plastic anemia. Embedding - The process by which the impregnated tissue is Chloroform placed into a precisely arranged position in a mold - Used for routine clearing of tissues during the containing a medium which is then allowed to embedding process. solidify. - Cooling allow hardening of tissue, giving them a Advantages: firmer consistency and better support, thereby - It is recommended for routine work (6-24 hrs.) facilitating the cutting of sections. - It is miscible with absolute alcohol  Orientation - It is recommended for tough tissues (ex. skin, - A process by which a tissue is arranged in precise fibroid and decalcified tissues) for nervous tissues, positions in the mold during embedding on the lymph nodes and embryos microtome before cutting and on the slide before - It is suitable for large tissue specimens staining. - It is not inflammable. Embedding Proper: Disadvantages: 1. Prepare embedding boxes - It is relatively toxic to the liver after prolonged 2. Pour melted paraffin into embedding box inhalation 3. Using a needle or forceps, position properly the - wax impregnation after chloroform clearing is tissue inside the paraffin box according to the relatively slow desired cut later. For cross section, position tissue - it does not make tissues transparent vertically. Do not remove the needle or forceps ‘till - it is not very volatile in paraffin oven paraffin has surrounded the tissue. - it may even produce considerable deterioration of 4. Allow paraffin to solidify. the wax
  • 6. General Histology and Histotechnique (1st Semester; SY 2012-2013) 5. Label the box bearing the name and position of tissue contained. Trimming - Embedded tissue is trimmed and cut into thin uniformly slices. - The slides, top and bottom of the tissue block are trimmed until perfectly level and all slides are parallel, almost to the edge of the tissue. - The block is placed in the microtome for final trimming and cutting. Microtomy - A process of cutting specimen to small or thin sizes. Microtome - An instrument used to cut the specimen. Steel Blades - Animal or plant tissue sections - Light microscope Glass Knives - thin sections - Light microscope and Electron Microscope Industrial Grade - Electron microscope Diamond knives - Hard materials 3 Essential parts of Microtome: 1. Block holder – holds block in precision 2. Knife carrier & knife – actual cutting of the tissue sections 3. Pawl, Ratchet feed wheel and adjustment screws – line up the tissue flock in proper positioning with the knife adjusting the proper thickness of the tissue for successive section. Kinds of Microtome’s: 1. Rocking (by Paldwell Trefall, 1881) - Simplest - Cut the paraffin embedded tissues 2. Rotary (by Minot, 1885-’86) - Most common - It involves staged rotary action 3. Sliding (by Adams, 1789) - Cutting celloidin embedded tissue a. Base-sledge – a very hard tissue b. Standard sliding – with movable exposed knife. For cutting celloidin embedded tissue. This is the most dangerous style of microtome.
  • 7. General Histology and Histotechnique (1st Semester; SY 2012-2013) 4. Freezing (by Queckett, 1848) - Cutting embedded frozen sections  Cryostat or cold microtome - Refrigerated apparatus used in fresh microtomy - For freezing tissue in the block holder to correct degree of hardness to facilitate easier and faster sectioning. - Provides a means of preparing a thin section of fresh frozen tissues especially for fluorescence antibody staining technique. 5. Ultrathin - Cutting sections for electron microscope - celloidin - Specimen used should be small - Should be fixed in osmium tetroxide and embedded in plastics. Microtome knives: 1. Plane concave knife - 25mm - one side flat, other concave  Flat – recommended for cutting celloidin embedded tissue blocks using a sliding microtome.  Concave – used for cutting paraffin embedded tissues. Using its Base Sledge microtome, Rotary, Rocking 2. Biconcave knife - 120mm - both sides are concave  Concave – cutting paraffin embedded tissues using a rotary microtome. 3. Plane wedge knife - 100 mm - Both sides are straight  Straight – recommended for frozen section or for cutting extremely hard/tough specimen embedded in a paraffin block. - Using a base sledge sliding microtome. Sectioning - Cutting thinly the specimen with the use of microtome - Thin materials – sections 3 types of tissue sections: 1. Paraffin Section - Leaded in a paraffin block - Cut using the rotary microtome/Rocky microtome 2. Celloidin section - Embedded in a celloidin - Cut by sliding microtome 3. Frozen Section - Fixed with carbon dioxide or cryostat
  • 8. General Histology and Histotechnique (1st Semester; SY 2012-2013) Faults occurring during tissue processing FAULTS REASONS REMEDY 1. Brittle or hard tissue. - Prolong fixation, dehydration - Softened by soaking in a small clearing, and infiltration of dish containing water with paraffin in overheated paraffin detergent or phenol. oven. - Dry out of tissue before actual fixation. 2. Clearing agent turns milky as - Water is not completely - Repeat dehydration with soon as tissue is placed in it. removed due to incomplete absolute alcohol then clear dehydration. again. 3. On trimming tissue smells of - Clearing agent not completely - Repeat paraffin impregnation. clearing agent. removed due to insufficient impregnation. 4. Tissue is opaque, section- - Insufficient clearing. - Repeat clearing and if the cutting is difficult due to object has been embedded, presence of alcohol prolong clearing up to 12 hrs. And embed it again. 5. Tissue shrinks away from wax - Insufficient dehydration so - Repeat the whole process. when trimmed. there is incomplete clearing and impregnation. 6. Tissue is soft when block is - Incomplete fixation - Repeat fixation procedure trimmed 7. Air holes found in tissue during - Incomplete infiltration or - Repeat impregnation. trimming. impregnation. 8. On trimming, wax appears - Contaminated wax - Re-embed in a freshly filtered crystalline. - The bluff Nat cold rapidly wax. 9. Paraffin block after cooling is - there is insufficient paraffin - Repeat impregnation and re- moist and crumbles impregnation embed. Faults observed during section-cutting: Faults Reasons Remedy 1. Sections fail to form ribbons. - Surfaces and edges of the bluff - Re-trim the block are not parallel - Re-adjust the block - Horizontal surface of the block - Reduce the tilt of the knife is not parallel to the knife - Re-adjust the thickness of the - Knife is tilted too much section - Sections are too thick - Re-sharpen the knife - Knife is dull 2. Sections roll up on cutting so - Knife is blunt - Re-sharpen the knife that they adhere and get - The tilt of the knife is great - Reduce the tilt broken against the knife edge. - The knife edge is dirty - Clean the knife edge 3. Ribbon is crooked, curved and - The knife is blunt or dull; there - Adjust the knife so that knife uneven instead of straight. is a dull spot on the knife edge will present a uniformly producing an irregular knife sharp edge to the block. edge. - The knife is not parallel to the - Re-adjust the knife and the block block. 4. Sections are wrinkled, - The paraffin block is warm and - Cool the paraffin on ice water
  • 9. General Histology and Histotechnique (1st Semester; SY 2012-2013) compressed or jammed. soft until firmed. - The knife edge is coated with - Clean the knife edge paraffin - Re-adjust the thickness of - The section are too thin section - Microtome set screw is loose. - Tighten the screw - The tilt of knife is too vertical. - Reduce the tilt 5. Sections are torn and crumble - Incomplete dehydration, - Repeat dehydration, clearing when cut. clearing and impregnation and impregnation - Paraffin is warmed and soft. - Cool and harden the paraffin in ice water for ¼ to ½ hour. 6. A hole is formed in the section. - Bubble or dirt is formed in the - Re-embed in freshly filtered embedding medium wax. 7. Sections adhere to the knife or - There is a static electrically due - Breathe out or blow gently in other parts of the machine. to low atmospheric humidity. the block and knife to break-up static electricity. Boil water - Knife edge is dirty inside the laboratory. - Knife edge is dull - Clean the knife edge - Knife tilt is great - Re-sharpen the knife - Reduce the tilt 8. Sections are lifted from the - Knife tilt is too great - Reduce tilt knife on upstrokes - Knife is dull - Re-sharpen the knife - Paraffin is soft - Cool the paraffin in ice water 9. Sections cut is sometimes thin, - Knife is blunt - Re-sharpen the knife and sometimes thick - Knife is not completely clamp - Re-adjust the knife - The tilt of knife is great - Reduce the tilt - The knife or block holder is - Tighten adjusting and locking loose. screws. Paraffin Section - Sections are usually cut between 4-6 micron in thickness. - Micrometer gauge is set to the required thickness and the knife is positioned in such a way that the center of the blade is in line with the block and the knife has been securely clamped in place. - Cutting is then started until complete sections come out of the block. - A regular cutting rhythm is required - The knife is usually tilted at 0-15 degrees angulations’ on a microtome to allow a clearance angle between the cutting facet and tissue block - Sections are removed in ribbons of ten, to allow easy location of serial sections - The sections are then floated out in water bath set at 45-50°C to flatten the sections Staining - To impart a color to the tissues - Parts to be visible under microscope Two Categories of dyes: 1. Natural Dyes - Obtained from plants and animals Hematoxilyn - Derived by either extraction from the heartwood of Mexican tree known a Hematoxilyn campechianum. - Most valuable stain in histology used by cytologist
  • 10. General Histology and Histotechnique (1st Semester; SY 2012-2013) - Powerful nuclear and chromatin staining capacity - With striking polychrome qualities Hematin – active coloring agents 2. Synthetic Dyes - Known as Coal Tar Dyes - Hydrocarbon benzene derivation - Aniline dyes Examples:  Picric acid - Ability to form salts with alkaline - Fix and stain at the same time by itself - Used as a fixative - Decalcifying agen - Tissue softener  Eosin - a valuable stain in Histotechnique, important stain in connective tissue and cytoplasm. - Histopathology – counter-stain after hematoxilyn and before methylene blue - Background for contrasting stains because it gives a pleasing and colorful contrast to nuclear stain. Red acid dye available in 2 shades: - a. Bluish - Deepen red color Ex. Eosin B b. Yellowish - Most usable - Readily soluble in water and less in alcohol - Available in both aqueous and alcoholic solutions - Slows a green fluorescence especially in alcoholic medium. Ex. Eosin Y 3. Basic dyes - coloring substance is found in the basic components - Acid radical is taken from sulfuric, acetic and hydrochloric acid. Ex. Methylene blue *Note: On a chemical basis certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater affinity for basic dyes, while basic constituents (e.g. cytoplasm) take more of the acid stains.  Fixation of tissues with mercuric chloride and formaldehyde usually favors staining with basic dyes.  Picric acid and Chromium – fixed tissues usually take an acidic dyes  Ethyl alcohol or acetic acid – fixed tissues readily take in both basic and acidic dyes. 4. Neutral Dyes - Combining aqueous solution of acid and basic dyes. - Capable of staining cytoplasm and nucleus simultaneously and differentially - Soluble in alcohol but not in water.
  • 11. General Histology and Histotechnique (1st Semester; SY 2012-2013) Ex. WBC differentiation  Giemsa’s stain  Leishman’s stain 3 main groups of tissue staining 1. Histological Staining - process by the tissue staining constituents are demonstrated in section by direct interaction with a dye or staining solution producing coloration of the active tissue components. Example: - Mircroanatomical stains - Bacterial stains - Specific tissues stains – muscle, connective tissue, neurological stains. 2. Histochemical staining (Histochemistry) - A process whereby tissue constituents are studied through chemical reactions that will permit microscopy localization of a specific tissue substance. 3. Immunohistochemistry - Combination of 1st and 2nd classification - Allow phenotypic markers to be detected using fluorescent labeled or enzyme labeled. Staining Technique 1. Direct Staining - A process of giving color to the section by using aqueous or alcoholic dye solution. Ex. Methylene blue, Eosin 2. Indirect Staining - Whereby the action of the dye is intensified by adding another agent or mordant. - Mordant – serve as a bridge or link between the tissue and the dye. - Mordant + dye -> Lake + Tissue = “Tissue mordant dye complex 3. Progressive Staining - The process whereby tissue elements are stained in a definite sequence. - Definite sequence? - to have a satisfactory differential coloration of tissues - Once the dye is taken by the tissue it is not wash anymore. 4. Regressive Staining - The tissue is first over-stained then decolorized - Over-stained – to obliterate the cellular details decolorized until the desired color is achieved. - Differentiation – the selective removal of excess stain. 5. Metachromatic Staining - Entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself. Where is this used? - Employ in staining cartilages, connective tissues, epithelial tissues - Belong to basic dyes
  • 12. General Histology and Histotechnique (1st Semester; SY 2012-2013) Example: Methylene blue, Safranin. 6. Counterstaining - Provide contrast and background Example: Safranin 7. Microanatomical Staining - General differentiation of nucleus and cytoplasm. 8. Metallic Impregnation - Where specific tissue elements are demonstrated by colorless solutions of metallic salts - Metallic salts – reduced by the tissue ; reduced also by bacteria - It’s not absorbed by the tissues but it is held physically on the surface as precipitates - Valuable metals: Ag, Chloride, Silver nitrate 9. Vital Staining - Selective staining of living cell constituents - Demonstrates cytoplasmic structures by phagocytosis of the dye particle. 10. Intravital Staining - By injecting the dye into any part of animal body. - Purpose: To produce different coloration of cells - Most specially in reticuloendothelial system Examples: Lithium, Indian 11. Supravital Staining - To stain living cells immediately after removal from the living body. Ex. Neutral red Staining of Paraffin Section  Paraffin wax is poorly permeable to most staining solutions and should therefore remove from the section prior to staining.  This is usually done by immersing the paraffin section in a solvent (xylene) two times, at 1-2 minutes duration.  Xylene is not miscible with aqueous solutions and low graded alcohol and therefore be subsequently removed with absolute alcohol followed by descending grades of alcohol to prevent damage and detachment of sections due to possible production of diffusion currents.  The alcohol is then finally replaced with water, before actual staining of section is performed  After deparaffinization with xylene, the section is subjected to decreasing grades of alcohol.  After staining, the section is again dehydrated with increasing grades of alcohol and cleared with two changes of xylene to prepare the section for mounting, since most mountants are miscible with xylene.  The second change of xylene will raise the refractive index of the glass slide, thereby reducing light refraction during microscopic examination. *Note: If section float off the slide during staining, fix section in a Bunsen flame Slides must not be dirty of greasy.
  • 13. General Histology and Histotechnique (1st Semester; SY 2012-2013) Mounting - A process of rendering slides permanency - Used to keep the slide to its permanent position - Seraphy Fluid – applied between the tissue and cover slip. Mounting media importance:  It protects the stained sections from getting scratched and from bleaching or deterioration due to oxidation, thereby preserving the slides for permanent keeping.  Facilitate easy handling and storage  Prevent the damage of sections which may lead to distortion of image during microscopic examination. Characteristics of a good mounting medium:  It should not dry quickly  It should not dissolve out of fade tissue sections  It should not cause shrinkage and distortion of tissues  It should set hard, thereby producing permanent mounting of sections. 2 main groups of mounting media: 1. Aqueous media - Design to mount water miscible preparations Examples: Glycerin, water 2. Resinous media - Used for preparations that has been dehydrated and cleared in xylene and are recommended for majority of staining method. Examples: Balsan, Colorless nail polish Types of Mounting 1. Dry Mount - It requires no water - Usually used in inanimate objects 2. Wet mount - It requires water - Used to prepare slides to cold living organisms whether they are motile or not. Ringing - A process of sealing the margins of cover slip. Purpose: 1. To prevent the escape of fluid or semi-fluid mounts 2. To prevent evaporation of mounts 3. To immobilize the cover slip 4. To prevent sticking of the slides upon storage