Module 2 - Preparing and Staining Slides

Question 1

Preparing a Slide:

Answer 1

1. ) Fixation
2. ) Dehydration
3. ) Embedding
4. ) Sectioning
5. ) Mounting
6. ) Staining
7. ) Observation

Question 2

Why preserve specimens?

Answer 2

• To enable them to be cut into sections for observing.
• To enable them to be treated with a variety of stains. (Permanent or temporary stains).
• To enable different structures to be revealed as a result of staining.

Question 3

Temporary slides:

Answer 3

Fixation - use of 70% alcohol.
Mounting - cover with a coverslip to exclude dust and air, and to protect the objective lens.
Staining - add few drops of stain to the specimen on the slide.

Question 4

Permanent slides:

Answer 4

Fixation - immerse in glutaraldehyde.
Dehydration - place specimen in increasing concentrations of ethanol or propanone.
Clearing - use xylol to replace the ethanol or propanone.
Embedding - immerse in epoxy resin or plastic.
Sectioning - use microtome to cut into very thin sections.
Staining - add temporary or permanent stain.
Mounting - place on slide (LM), or copper grid (EM).

Question 5

Define Staining

Answer 5

• Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image.
• Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes.

Question 6

Simple Staining:

Answer 6

-This method uses only one stain.
-It imparts only one colour to all bacterial cells.
-It reveals the size, shape and arrangement of bacterial cells.
Example: Methylene blue staining method.

Question 7

Differential Staining:

Answer 7

-This method uses more than one stain.
-It imparts two or more different colours to bacterial cells.
-It reveals size, shape and arrangement. In addition, it differentiates two groups of bacteria.
Example: Gram’s staining method, Acid Fast staining method.

Question 8

Preparing for Differential Staining:

Answer 8

1. ) Smear loopful of microbes onto slide.
2. ) Let air dry.
3. ) Drip methanol onto specimen to fix.
4. ) Flood slide with stain.
5. ) Rinse with water. Blot dry.
6. ) Examine with x100 objective (oil immersion).

Question 9

Stains in Plants:

Answer 9

Eosin = Permanent; Cellulose - RED
Toluidine = Permanent; Lignin - BLUE
Cellulose - PURPLE
Iodine (KI) = Temporary; Starch - BLUE-BLACK

Question 10

Blood Smear Staining:

Answer 10

• Involves use of differential staining to identify different red blood cells (erythrocytes) and white blood cells (leucocytes).
• Stains highlight the different size and nuclei shapes of different leucocytes.
• Romanowsky stains are a group used by pathologists which use Leishman’s and Wright’s stain.

Question 11

Cryostat Sectioning:

Answer 11

• Tissue is rapidly frozen to -20 or -30 *C.
• Specimen is embedded in a gel medium.
• The embedded specimen is then cut using a cryostats (microtome at very low temperatures).
• Sections of 5-10 μm are cut & stained.
• Rapid process - 10mins appox.

Question 12

Disadvantages of Cryostat Sectioning:

Answer 12

• Lower quality is achieved.
• Distorts the cell.
• Ruptures cell surface membrane.
• Introduces ice crystals.
• Introduces artefacts.

Question 13

Uses of Cryo-sectioning:

Answer 13

• Pathology.
• Oncology.
• During operations - biopsy, determining benign or malignant tissue.

Question 14

Different types of Sectioning:

Answer 14

Longitudinal Sectioning (LS) = vertical section.
Transverse Sectioning (TS) = horizontal section.
Oblique Sectioning (OS) = angular section.

Question 15

Areas/Volumes of Sections:

Answer 15

Transverse Area Section = π x radius^2
Oblique Area Section = π x radius A x radius B
Volume of Sphere = 4/3 x π x radius^3

Question 16

Calculating Cross-Sectional Areas:

Answer 16

1. ) Calculate Magnification using A = I/M
2. ) Calculate the ACTUAL radius of section.
3. ) Put value into formula (π x r^2) to find ACTUAL area.
4. ) Put correct units. e.g. μm^2.