histology

Question 1

what is histology

Answer 1

the study of tissues

Question 2

what is the main piece of equipment used in all histology practicals

Answer 2

a microscope

Question 3

why is histology important

Answer 3

it can reveal - through use of various histological stains - whether a sample of a tissue exhibits normal morphology and function or whether abnormalities are visible. This can be useful in diagnosis of disease

Question 4

what type of microscope is used for routine work by biomedical scientists within a cellular pathology lab

Answer 4

a light microscope

Question 5

which two stains did we use in the histology practical and what are they often described as

Answer 5

Haematoxylin and Eosin - general tissue stains

Question 6

what kind of tissue was used in the practical and why

Answer 6

porcine (pig) tissue as it closely resembles that of human tissue

Question 6

what PPE should be worn during a histology practical involving staining

Answer 6

nitrile gloves, safety glasses and lab coats

Question 7

going from top to bottom, name all the parts of a light microscope

Answer 7

• eye piece - oculars
• objectives (lenses)
• stage
• condenser; condenser diaphragm and condenser alignment screws (directly beneath the stage)
• stage adjustment knob (directly beneath condenser)
• field diaphragm (wheel beneath stage adjustment knob)
  on the side:
• course focus (big wheel)
• fine focus (little wheel)
• bulb voltage (brightness)

Question 8

why when visualising a stained section of tissue is it important to avoid uneven illumination

Answer 8

as this can result in ‘artefacts’ within an image, such as shadowing and glare

Question 9

what is the procedure called to avoid uneven illumination

Answer 9

Kohler illumination

Question 10

explain Kohler illumination

Answer 10

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.
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. Focus both eyepieces individually
8. Ensure the light source is in the centre of the sample and open the field diaphragm to where the entire field of vision is lit up

Question 11

why do we centre the light source and focus the condenser

Answer 11

to reduce stray light escaping from the sample, to minimise glare and increasing image quality

Question 12

what does the condenser diaphragm control and why is this important

Answer 12

the angle at which the light hits the objective - each objective is designed to gather light at a particular angle - this is called its numerical aperture

Question 13

when moving between different magnification lenses what should be adjusted and why

Answer 13

field diaphragm as the fields of view will change (decrease when magnification is increased)
condenser diaphragm as the numerical aperture of each lens is different therefore if not changed glare and shadows may appear

Question 14

what are the two tissue sections we stained

Answer 14

lung and liver tissue

Question 15

what does haematoxylin stain and what colour

Answer 15

cell nuclei - blue/ purple

Question 16

what does Eosin stain and what colour

Answer 16

most components of cytoplasm and connective tissues - shades of pink/ red

Question 17

what is the first stage of the practical called and why do we do it

Answer 17

dewaxing/ taking samples to water - paraffin wax does not mix with aqueous solutions (immiscible), therefore to allow stains to enter the tissue the wax must be removed

Question 18

how do we dewax samples

Answer 18

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 19

which stain is used first and for how long

Answer 19

haematoxylin - 10 minutes

Question 20

what do we use to remove excess stain from a slide after washing in tap water

Answer 20

0.5% v/v acid alcohol for 10-15 seconds

Question 21

why is Scott’s tap water substitute used after washing of Haematoxylin stain with acid alcohol

Answer 21

the acid alcohol will remove excess stain, but it will also change the colour of the stain from blue to red, so we use Scott’s water for 2 minutes to regain the blue colour

Question 22

how long do you stain with eosin for

Answer 22

2 minutes

Question 23

after staining and washing with both haematoxylin and eosin how do we dehydrate the slide

Answer 23

washing in 3 changes of absolute alcohol for one minute each

Question 24

after dehydrating what do we do to the slide

Answer 24

clear in two changes of XTF for about 30 seconds each

Question 25

how do you mount a coverslip onto a stained slide using synthetic mounting medium

Answer 25

1. Take the stained and cleared slide, and place it on the bench with the section facing upwards. 2. Carefully add 1-2 drops of mounting medium to form a blob on top of and directly in the centre of the section. 3. 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. 4. 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