GHH Lab (handout prelim)

General Histology and Histotechnique Laboratory (1st Semester; 2012-2013)

Activity #1

Microtechnique – it involves the instrument

microtome.

Microtomy – sectioning instrument

Microtome

- Indispensable in microtecnique

3 essential parts:

1. Block holder

2. Knife carrier/knife

3. Pawl, ratchet feed wheel and adjustment

screws

Pawl

- to line up the tissue block

- Adjust the thickness for successive

sectioning.

Sliding Rotary

-Slicing motion -Chopping motion

-The object to be cut is fixed and the knife is carried obliquely across it.

-Knife is fixed in a horizontal position intended to precisely cut equally thin sections of different materials.

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

B. Focusing parts:

1. Fine adjustment

2. Coarse adjustment

C. Mechanical parts:

1. Draw tube

2. Body tube

3. Adjustments screws

4. Revolving nosepiece

5. Dust shield

6. Stage and stage clips

7. Inclination joint

8. Pillar

9. arm

10. base

Other materials used in microtechnique

Staining bottle

Glass slides

Cover slips

Pipettes

Vacuum oven

Tissue stretcher

Staining rock

Slide box

Forceps

Dissecting pan

Reagent box


Activity #2:

Common Reagents used in Microtechnique

Reagents:

Fixatives

Ex: Ethanol, Xylene

Dehydrants

Clearers

Stains

Embedding matrix

Adhesives

Washing reagents

Mountant

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.

Physical Hazards

1. Combustible

- Substances that can ignite at certain

temperature.

2. Explosives

- Substances that can explode

3. Oxidizers

- May initiate combustion

Basophilic

Acidophilic

Metachromatic


Activity # 3:

Preparation of whole mounts

Whole mount purposes:

- Specimen is mounted whole; thin specimen

- To observe the gross anatomy

- To preserve chemical and morphological

features of the cell.

1. Temporary preparation

- Prepared in order to make possible the

observations in the live of normal state.

- Allows physiological observations to be

made directly such as mitosis, phagocytosis

etc.

- Allows true and natural colors to be

observed.

- Mountant used is water.

Advantages:

- Allows one to observe living characteristics

of specimen.

- Can be prepared quickly.

- Retain characteristics of the 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.

Disadvantages:

1. Processing kills and disintegrates many

tissue components preventing their

observations.

2. Improper processing may also leave behind

certain by-products called artifacts that may

interfere either the examination of slides.

Qualities of a good whole mount:

- Transparent

- Parts complete

- Non-overlapping

- Not distorted

Selection of whole mount specimen:

- Whole mount slide preparations is one

where the specimen is taken wholly without

part/s missing and carefully mounted as is is

on a slide.

- specimen on character must be:

a. Adequate size

b. Adequate bulk

c. All parts present and free of distortion

d. Fresh specimen.


Activity # 4: Separating Cells for Microscopic

study: Squashing, Smearing, and Teasing

Squashing

Qualities of a good squash preparation:

- Evenly stained cells

- Cells normal shape and contours are

retained.

- 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

4. Too much or too little stain is used

5. Staining time is not followed.

Teasing

Qualities of good slide:

Cells are distinct and clear

Cells are isolated

Cells exhibit correct form and shape.


Activity # 5: Preparation of specimen for

microtome sectioning

Features to consider in selecting specimen:

A specimen must be fresh, healthy, and in

live condition.

Specimen must be of sufficient numbers.

Specimen must be readily available when

needed

Reasons for cutting the specimen into 1x1 cm

dimensions

To facilitate process of microtomy

To obtain full penetration and satisfactory

fixation to avoid post mortem conditions to

occur.

For specimen to fit in congruence with the

paraffin block during embedding for easy

cutting.

Activity #6: Tissue Processing part I; Fixation,

Washing, Dehydration, Clearing.

Fixation

- To preserve cellular and structural

elements with least alteration possible

- Prevents post-mortem conditions

- Protects tissue by hardening naturally soft

tissues

- Increase visual differentiation of structures

upon application of stains.

Washing

- only necessary if the fixatives used contains

some reagents which may have undesirable

effects such as:

- Discoloration

- Precipitation

- 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


Activity #7: Tissue processing part II;

Infiltration and embedding

Infiltration

Embedding

- To encase the tissue in block of solid

paraffin.

- Block must be contiguous, clear and

homogenous, and free from crystallization.

- 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.

Activity #9: Staining microtome-sectioned

specimen

Hematoxylin

- Natural stain used for nuclear staining

- Stain the nuclei blue

Eosin

- Synthetic acid stain for cytoplasmic staining

- Counterstained for hematoxylin

GHH Lab (handout prelim)

Education

Transcript of GHH Lab (handout prelim)