Histology practical

Question 1

What is histology?

Answer 1

Histology is the study of tissues as viewed through a microscope.

Question 2

Why is histology important?

Answer 2

> It can reveal – through the use of various histological stains – whether a sample of
a tissue exhibits normal morphology and function, or whether abnormalities are visible within the tissue.
This is useful for diagnosing various diseases and pathologies, such as cancer. It is also useful for longer-term prognoses, and for confirming whether or not particular therapeutic strategies have been successful.

Question 3

What type of microscope is used?

Answer 3

Whilst using an electron microscope can reveal cellular details at the molecular level, routine work by Biomedical Scientists within a Cellular Pathology laboratory is carried out using a light microscope. As
such, understanding how a light microscope works, and how to use a light microscope correctly, is an essential part of a Biomedical Scientist’s training.

Question 4

What are artefacts

Answer 4

it is important to avoid
uneven illumination, as this can result in ‘artefacts’ within the image, such as shadowing and glare, which can make the
interpretation of the image difficult, and could therefore lead to incorrect diagnose

Question 5

steps to set up the light microscope with Köhler Illumination:

Answer 5

1. Switch on the microscope bulb.
2. Move the condenser until it is 2-3 mm below the stage.
3. Place a stained histological specimen slide on the stage.
4. Adjust the inter-pupillary distance (the distance between the eyepieces) until binocular vision is obtained; i.e. you should see a single image - not two overlapping images. Don’t rush this, it takes longer than you’d think to perfect
5. Select the low-magnification (10X) objective lens, and use the focus wheel to create a sharp image.
6. Using the brightness wheel on the side of the microscope, adjust the brightness to somewhere in the
   middle of the min/max range.
7. At least one of the eyepieces will have a movable focusing ring around its diameter. There is a scale around this focusing ring. Use this scale to set the eyepiece focus to 0.
8. Look at the image through the left eyepiece and rotate the eyepiece focus ring until the sharpest image is obtained.
9. Look at the image through the right eyepiece and rotate the eyepiece focus ring until the sharpest image is obtained

Question 6

What does a higher magnification mean?

Answer 6

Higher magnifications have smaller fields of view, meaning that if you move from a low- to a high magnification your field diaphragm will be illuminating excess specimen, producing glare… This indicates that the Field Diaphragm needs adjusting.

Question 7

What does a different objective lens mean?

Answer 7

Different objective lenses have different numerical apertures; therefore, the light angle provided for one will not be appropriate for another, producing sub-optimal contrast and resolution… This indicates that the Condenser Diaphragm needs adjusting

Question 8

Which stains are used?

Answer 8

Haematoxylin and eosin

Question 9

What does Haematoxylin dye and what colour?

Answer 9

It stains cell nuclei blue/purple

Question 10

What does Eosin dye and what colour?

Answer 10

stains most components of cytoplasm and connective tissues in shades of pink/red

Question 11

Dewaxing

Answer 11

1. Place your sections in the first container of XTF®. Ensure the sections are not touching each other. Leave the sections for ten minutes to dissolve the paraffin wax.
2. Transfer to the second container of XTF for two minutes.
3. Transfer to absolute alcohol for two minutes (this removes the XTF).
4. Transfer to 70% alcohol for two minutes
5. Wash gently in running tap water.
6. The slides are now ready to stain.
7. Leave slides submerged in water until you are ready to perform your stains

Question 12

Materials

Answer 12

• XTF
• Absolute ethanol
• 70% (v/v) ethanol
• Mounting medium
• Coverslips
• Yellow micropipette tips.
• Harris’s haematoxylin (a potassium alum haematoxylin)
• 0.5% (v/v) acid-alcohol (0.5ml concentrated hydrochloric acid, 100ml ethanol)
• 1% (w/v) eosin in 0.05% (v/v) acetic acid (1 g eosin Y, 50µl glacial acetic acid, 100ml distilled
  water)
• Scott’s tap water substitute (2g potassium bicarbonate, 20g magnesium sulphate, 1000ml distilled
  water)

Question 13

Step one

Answer 13

Stain in Harris’ haematoxylin for ten minutes

Question 14

Step two

Answer 14

Wash in tap water

Question 15

Step three

Answer 15

Remove excess stain from cytoplasm by differentiating in 0.5% v/v acid alcohol for 10 – 15 seconds (the blue staining of haematoxylin is changed to red by the action of the acid)

Question 16

Step four

Answer 16

Wash in tap water

Question 17

Step five

Answer 17

Regain the blue colour by treating with Scott’s tap water substitute for 2 minutes

Question 18

Step six

Answer 18

Wash in tap water

Question 19

Step seven

Answer 19

Stain with 1% eosin for two minutes

Question 20

Step eight

Answer 20

Rinse quickly in tap water

Question 21

Step nine

Answer 21

Dehydrate by washing in three changes of absolute alcohol for one minute each. Be sure to remove all the water by washing both sides of the slides

Question 22

Step ten

Answer 22

Clear in two changes of XTF for about 30 seconds each. Slides can now be left in XTF until you
are ready to mount them

Question 23

Step eleven

Answer 23

Mount a coverslip onto the slide using synthetic mounting medium

Question 24

Step twelve

Answer 24

Take the stained and cleared slide, and place it on the bench with the section facing upwards.

Question 25

Step thirteen

Answer 25

Carefully add 1-2 drops of mounting medium to form a blob on top of and directly in the centre of the section. Do not add too much, or too little (1-2 drops is normally enough). AVOID AIR BUBBLES

Question 26

Step fourteen

Answer 26

Hold a clean, dry coverslip with gloved hands between thumb and forefinger (hold the coverslip by the short edges) and place it gently on top of the blob.

Question 27

Step fifteen

Answer 27

Hold a yellow micropipette tip by the narrow end and use the wide end to press down gently on the top of the coverslip so that the mounting medium is spread evenly across the specimen, ensure that the coverslip aligns with the glass slide and that there are no air bubbles

Question 28

Step sixteen

Answer 28

Check your labels survived the protocol, and re-label if necessary.

Question 29

Step seventeen

Answer 29

Examine the slide with the microscope

Question 30

Step eighteen

Answer 30

Draw labelled diagrams in the space below.