General Histology and Histotechnique 2012-2013; 1st sem; Prelim Handout

1. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)
Activity #1 B. Focusing parts:
1. Fine adjustment
 Microtechnique – it involves the instrument 2. Coarse adjustment
microtome.
C. Mechanical parts:
 Microtomy – sectioning instrument 1. Draw tube
2. Body tube
 Microtome 3. Adjustments screws
- Indispensable in microtecnique 4. Revolving nosepiece
5. Dust shield
3 essential parts: 6. Stage and stage clips
1. Block holder 7. Inclination joint
2. Knife carrier/knife 8. Pillar
3. Pawl, ratchet feed wheel and adjustment 9. arm
screws 10. base
Pawl
- to line up the tissue block Other materials used in microtechnique
- Adjust the thickness for successive  Staining bottle
sectioning.  Glass slides
 Cover slips
Sliding Rotary  Pipettes
-Slicing motion -Chopping motion  Vacuum oven
-The object to be cut is -Knife is fixed in a
 Tissue stretcher
fixed and the knife is horizontal position
carried obliquely intended to precisely  Staining rock
across it. cut equally thin  Slide box
sections of different  Forceps
materials.  Dissecting pan
 Reagent box
Microscope
- Used to view micro-object
- Used to check if the specimen is stained
well or over-stain or not
- Used to view cytoplasmic part of the cell
and tissue.
A. Illuminating & magnifying parts:
1. Ocular/eyepiece
2. Iris diaphragm
3. Condenser
4. Bulb
5. Mirror

2. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)
Activity #2: Physical Hazards
Common Reagents used in Microtechnique 1. Combustible
- Substances that can ignite at certain
Reagents: temperature.
 Fixatives 2. Explosives
Ex: Ethanol, Xylene - Substances that can explode
 Dehydrants 3. Oxidizers
 Clearers - May initiate combustion
 Stains
 Embedding matrix Basophilic
 Adhesives
 Washing reagents Acidophilic
 Mountant
Metachromatic
Reminders / precautions to be considered to
properly utilize reagents:
 For nature specifically such as flammable
materials
 Toxicity
 For the safety precautions when handling
compounds
 Method of disposal of hazardous waste
 Read labels before using.
 Use only one dropper per reagent to avoid
mixing/contamination of reagent
Health hazards
1. Biohazards
- Can cause diseases in human
- Infectious agents, contaminated solutions.
2. Irritants
- It causes reversible inflammatory effects to
skin, eyes, & nasal passages.
3. Corrosive chemicals
- It destroys inanimate surface of living
tissues.
4. Carcinogens
- Substances that causes cancer and tumors.
5. Toxic Materials
- Harmful; it can cause death upon ingestion.

3. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)
Activity # 3: Disadvantages:
Preparation of whole mounts 1. Processing kills and disintegrates many
tissue components preventing their
Whole mount purposes: observations.
- Specimen is mounted whole; thin specimen 2. Improper processing may also leave behind
- To observe the gross anatomy certain by-products called artifacts that may
- To preserve chemical and morphological interfere either the examination of slides.
features of the cell.
Qualities of a good whole mount:
1. Temporary preparation - Transparent
- Prepared in order to make possible the - Parts complete
observations in the live of normal state. - Non-overlapping
- Allows physiological observations to be - Not distorted
made directly such as mitosis, phagocytosis
etc. Selection of whole mount specimen:
- Allows true and natural colors to be - Whole mount slide preparations is one
observed. where the specimen is taken wholly without
- Mountant used is water. part/s missing and carefully mounted as is is
on a slide.
Advantages: - specimen on character must be:
- Allows one to observe living characteristics a. Adequate size
of specimen. b. Adequate bulk
- Can be prepared quickly. c. All parts present and free of distortion
- Retain characteristics of the specimen d. Fresh specimen.
- Requires no reagent, if not minimal hence
cheap.
Disadvantages:
- Tissues prepared are thick and transparent
- Cannot be used repeatedly since it is prone
to dehydration.
2. Permanent preparation
- Have to undergo an elaborate processing of
specimen.
Advantages:
1. Specimens may be sectioned thinly.
2. Specimens are stained hence it enhances
the resolution of tissue components
3. Specimens may be stored for a long time.

4. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)
Activity # 4: Separating Cells for Microscopic 4. Too much or too little stain is used
study: Squashing, Smearing, and Teasing 5. Staining time is not followed.
Squashing Teasing
Qualities of a good squash preparation: Qualities of good slide:
- Evenly stained cells  Cells are distinct and clear
- Cells normal shape and contours are  Cells are isolated
retained.  Cells exhibit correct form and shape.
- Tissue components are well isolated
Note: Cells have the greater tendency to be
under-stained that to be over-stained.
Applying too much pressure when crushing the
stone cells may result to the:
 Distortion of the typical structure of the stone
cells
 Uneven staining since the distorted parts of
the cell may allow some stain to penetrate.
 Overlapping of stone cells
 Possible breakage of the glass slides.
Smearing
Qualities of good smear preparations:
 Cells must be isolated from each other
 Different cells must be observable
 Staining makes the cells distinct
 Shape of the cell and nucleus should be
normal and distinct
Bad qualities of Smearing:
a. Clumping of cells
b. Distortion
c. Homogeneity
d. Under or over staining resulting to:
1. Too large drop of blood
2. Poor or uneven spreading
3. Pusher used has nicks

5. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)
Activity # 5: Preparation of specimen for Activity #6: Tissue Processing part I; Fixation,
microtome sectioning Washing, Dehydration, Clearing.
Features to consider in selecting specimen: Fixation
 A specimen must be fresh, healthy, and in - To preserve cellular and structural
live condition. elements with least alteration possible
 Specimen must be of sufficient numbers. - Prevents post-mortem conditions
 Specimen must be readily available when - Protects tissue by hardening naturally soft
needed tissues
- Increase visual differentiation of structures
Reasons for cutting the specimen into 1x1 cm upon application of stains.
dimensions
 To facilitate process of microtomy Washing
 To obtain full penetration and satisfactory - only necessary if the fixatives used contains
fixation to avoid post mortem conditions to some reagents which may have undesirable
occur. effects such as:
 For specimen to fit in congruence with the - Discoloration
paraffin block during embedding for easy - Precipitation
cutting. - Corrosion
Note: washing must be done thoroughly and
gradually
Dehydration
- Where does water to be removed in
dehydration process come from?
- Extracellular – comes from humidity and
from water present around specimen
- Intracellular – comes from vacuoles and
cytoplasm of the cell
Clearing
- Renders the tissues translucent thus
increasing the tissue’s refractive index.
- Remove the opacity or darkening of the
specimen.
- Free specimen from opacity

6. General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)
Activity #7: Tissue processing part II; Activity #9: Staining microtome-sectioned
Infiltration and embedding specimen
Infiltration Hematoxylin
- Natural stain used for nuclear staining
Embedding - Stain the nuclei blue
- To encase the tissue in block of solid
paraffin. Eosin
- Block must be contiguous, clear and - Synthetic acid stain for cytoplasmic staining
homogenous, and free from crystallization. - Counterstained for hematoxylin
- If bubbles appear only at the sides can be
remedied by trimming off the block.
- If bubbles are found up to the center of the
block, re-embed the tissue.
Measures on blade and microtome setting
 Sharpen the blade
 Clean the knife edge
 Tighten the screw
 Adjust knife
 Re-adjust angulations’ of knife.
Activity #8: Microtome sectioning
Microtome sectioning
- Also referred to as Microtomy
- Involves 2 processes:
1. Microtome setting which involves proper
adjustment and alignment of microtome
parts in preparation of the cutting proper.
2. Sectioning which is the actual cutting of the
tissue block.