Microscopy of blood cells

Question 1

How should a microscope be set up?

Answer 1

Step 1: Focus the image
The main focusing knob may consist of an outer and
inner part for coarse and fine focus, or there may be a single focusing knob which gives fine focus for the first quarter turn or so and then coarse focus engages

Step 2: Focus the condenser
Close the field iris and adjust the condenser focus knob until you get a sharp image of the edge of the disc of light coming through the iris diaphragm. This should be roughly in the centre of the field of view - if it is not, adjust it with the centering screws

Step 3: Adjust the field iris
Open the field iris again until the whole field of view is illuminated.

Step 4: Adjust the condenser iris
Start with the condenser iris fully open and then close it until it only just begins to darken the
image.

Question 2

What factors affect the resolution of the microscope?

Answer 2

• The wavelength of light
• The optical quality of lenses and all other components in the light path
• The refractive index of the medium through which the light passes (which is why the highest resolution lenses are designed to use oil instead of air between the objective lens and the slide)
• The physical properties of the objective lens
• The illuminating cone of light provided by the condenser lens (which depends in part on the design properties of the lens, but to a large degree on the way in which the condenser is focused and the iris diaphragms adjusted)

Question 3

What is numerical arpeture?

Answer 3

It is written on the lens next to its magnification, e.g. 10/0.25 means that lens has 10* magnification and a numerical aperture of 0.25. It follows that higher magnification does not necessarily mean higher resolution, though in fact it is normally the case that higher power lenses have better numerical apertures)

Question 4

Stains -H&E

Answer 4

• Very few cellular components absorb light at visible wavelengths so in order to see cells we stain them
• H&E is the most frequently used combination of stains.
• Haematoxylin is a purple-blue component that binds to acidic components of cells. In particular it binds DNA and shows up the nucleus.
• Eosin is a pinkish stain that binds protein components, particularly in the cytoplasm.
• H&E is a good general purpose stain, but others may be chosen for demonstrating particular chemical constituents of cells, such as carbohydrate.

Question 5

How to create a blood smear

Answer 5

• Obtain a drop of blood from your finger by using a sterile lancet to puncture the fleshy pad alongside the base of a fingernail.
• Immediately place the drop on a clean slide and slowly smear it out in a film using a second slide as a spreader, as indicated below.
• the direction of spreading is so that the blood drop remains behind the spreader, otherwise blood cells might become damaged between the two slides.
• Dry the blood smear by gently waving the slide
  around in the air, and check the blood film looks thin and even.

Question 6

What are some safety considerations that should be made when creating a blood smear?

Answer 6

• clean your skin with an alcohol swab before puncture
• do not share lancets, and dispose of them in the special “sharps” bin provided
• protect the wound with a plaster
• wear a lab coat, and put on protective gloves to handle any blood other than your own
• wash your hands immediately if they accidentally come into contact with blood
• never under any circumstance use a mouth pipette
• spills of blood must be decontaminated immediately
• at the end of the experiment dispose of glassware and anything which has come into contact with blood in
  the sharps bin

Question 7

What is the diameter of a RBC?

Answer 7

7 micrometers

Question 8

What is the diameter of a platelet?

Answer 8

2-4 micrometers

Question 9

What is the diameter of a lymphocyte?

Answer 9

8 micrometers

Question 10

What is the diameter of a eosinophil?

Answer 10

12 micrometers

Question 11

What is the diameter of a neutrophil?

Answer 11

10-12 micrometers

Question 12

What is the diameter of a basophil?

Answer 12

10-15 micrometers

Question 13

What is the diameter of a monocyte?

Answer 13

17-20 micrometers

Question 14

Leishman’s stain

Answer 14

Leishman’s contains a purple-blue dye which stains nuclei and a pink one staining components in the cytoplasm. Red cells look red, while white cells actually look blue due to the staining of the nucleus

Question 15

How should a blood smear be stained?

Answer 15

1. Put 8 drops of Leishman’s stain on the slide so that it completely covers the smear.
2. Add 8 drops of buffer pH 6.8 and rock the slide gently so this mixes in.
3. Pour off the stain and rinse well with buffer
4. Dry the slide by tapping the edge of the slide on filter paper and gently waving in the air.