PAG 1.2 Flashcards
1
Q
- Could you use a sample of your own blood for this practical? What precautions would you need to take if you did?
A
- Could you use a sample of your own blood for this practical? What precautions would you need to take if you did? Yes, students could use their own blood. Their proposed precautions should include measures to ensure that the puncture wound from which they extract their own blood does not become infected (pre- and post-swabbing with ethanol, covering with a sterile dressing) and measures to ensure that nobody else is contaminated with their blood (students work only with their own blood, all materials disposed of in bleach, good general hygiene (handwashing).
2
Q
- Make an estimate of the concentration of erythrocytes in this sample of blood. What facts do you need to produce this estimate? How accurately can you gauge each of these things? What do you judge to be the uncertainty in this estimate? How would you go about making a more accurate measurement and what additional apparatus would you need?
A
- Make an estimate of the concentration of erythrocytes in this sample of blood. What facts do you need to produce this estimate? How accurately can you gauge each of these things? What do you judge to be the uncertainty in this estimate? How would you go about making a more accurate measurement and what additional apparatus would you need? The way students deal with the uncertainty here is as interesting as the actual ‘right’ answer. They are being asked to pile estimates on top of estimates to get to a number – what is the volume of the drop they added to the slide?, how can they work out what fraction of that volume they are seeing in any one field of view?, what is the nature of the ‘wedge’ of blood they have created by smearing (how much thicker is the start than the end)? 5 billion (5 x 109) erythrocytes per cm3 is probably close to the truth. A haemocytometer would of course be the ideal way to get an accurate measurement and this in turn brings in the concept of serial dilution (students should realise from this practical that the concentration is going to be too high to easily count the cells in ‘neat’ blood – so dilution or smearing is required).
3
Q
- Using your calculation of the diameter of a typical erythrocyte, and assuming these cells are spherical, what is the volume of a typical erythrocyte? Given what you know about the shape of erythrocytes do you think this is likely to be an over-estimate or an under-estimate?
A
- Using your calculation of the diameter of a typical erythrocyte, and assuming these cells are spherical, what is the volume of a typical erythrocyte? Given what you know about the shape of erythrocytes do you think this is likely to be an over-estimate or an under-estimate? Check the student’s calculation of diameter to allow ‘error carried forward’. If the calculated diameter is 7 µm the volume of a spherical cell would be 180 µm3 or 1.8 x 10-16 m3.
Given their biconcave shape the true volume of erythrocytes will be considerably lower.
4
Q
- What are the functions of the various different cell types you have identified in the sample?
A
- What are the functions of the various different cell types you have identified in the sample? Erythrocytes – carry and deliver oxygen (from lungs to tissues) and carbon dioxide (from tissues to lungs)
Thrombocytes – blood clotting
Lymphocytes – specific immunity
Neutrophils – non-specific immunity (phagocytosis of pathogens)
Eosinophils – inflammation and response to parasites
Basophils – inflammatory response.
5
Q
equipment
A
Equipment
- Light microscope with x10 and x40 objective lenses and an eyepiece graticule
- Stage micrometer
- Microscope slides
- 70% ethanol
- Buffered water
- Leishman’s Stain
- Mammalian blood
- Disposal pot containing 1% sodium hypochlorite (bleach)
- Glass rod
6
Q
procedure
A
- Gently shake your sample of blood to re-suspend the cells.
- Dip the glass rod into the blood and place a drop of blood about a quarter of the way along a microscope slide. Put the rod into the disposal pot so the contaminated end is immersed in the hypochlorite.
- Hold a second slide at an angle of about 45˚. Push it backwards until it just touches the drop of blood and then pull it forwards towards the far end of the slide, dragging a smear of blood behind it. It is important that the second slide makes the briefest possible contact with the drop of blood, otherwise too much will be collected and far too many cells will spread on the slide. Most of the drop should remain at the origin.
- Put the slide used to smear the blood into the disposal pot.
- Leave the smear to air-dry on the bench. This will stick the cells to the slide but only if it is fully dried.
- While the smear is drying, use the stage micrometer to calibrate the eyepiece graticule with the x40 objective. Make a note of this calibration – you will refer to it later when viewing cells at high power.
- Pour ethanol onto the dry smear and leave for 2 minutes before pouring off. (This fixes and dehydrates the cells.)
- Put 5 drops of Leishman’s stain on the smear and leave for 1 minute.
- Add 5 drops of the buffered water and leave for 5 minutes.
- Wash the slide in buffered water until it looks pale pink, gently blot dry with filter paper and place a cover slip over the smear (at the end where it is thinnest).
- Examine your preparation under the microscope. Use the x10 objective to focus the microscope and identify the right part of the smear.
- Next use the x40 objective to make high power drawings to show the appearance of the different cells. Add labels, annotations and a scale bar to each drawing.
- Calculate the diameter of a typical erythrocyte from your slide.
- When you have finished dispose of the slide in the disposal pot and wash your hands.
7
Q
list of things to things to look out for, including the appearance of cells when stained with Leishman’s stain:
A
- Erythrocytes (red blood cells) – red
- Thrombocytes (platelets) – purple grains (so small they will be difficult to see even at high power)
- Lymphocytes – very dark nuclei (purple or dark blue) and light blue cytoplasm
- Neutrophils – dark purple nuclei, pale pink cytoplasm
- Eosinophils – blue nuclei, pale pink cytoplasm, red granules within the cytoplasm (these cells are rare and you will probably not see any examples in your sample)
- Basophils – dark blue nuclei, very dark granules in the cytoplasm (these cells are rare and you will probably not see any examples in your sample)
8
Q
why are you provided with new, clean microscope slides.
A
This is important for good results.
9
Q
why handle the slides by the edges
A
to avoid contaminating the surface.
