Practicals

Question 1

Romanowsky staining is used for staining blood smears. What compounds are used and what do they stain?

Answer 1

eosin Y: acidic, binds to basic proteins and dyes them pink eg proteins in cytoplasm
polychromed methylene blue: contains azure B, stains nucleic acids purple (?)

this method was originally developed for staining pathogens in the blood

Question 2

What compounds are used to stain lymphoid tissues?

Answer 2

haematoxylin: stains cell nuceli blue

eosin:
- stains cytoplasm and extracellular matrix pink
- acidic so binds to basic compounds like proteins

Question 3

What is immuno-histological staining?

Answer 3

1. primary antibody that recognises specific protein is added to tissue
2. secondary antibody with fluorescent marker that is specific for primary antibody is added. Fluorescence can be observed with a fluorescent microscope.

Question 4

How do you identify neutrophils in a stained blood smear?

Answer 4

• irregular horseshoe nucleus, which can be fragmented
• visible cytoplasm w faint granules

Question 5

How do you identify eosinophils in a stained blood smear?

Answer 5

• lobed nuclei (like a teardrop)
• pink stained granules (pronounced)

Question 6

How do you identify basophils in a stained blood smear?

Answer 6

• non-visible nucleus due to highly granular cytoplasm
• granules are dark (blue)

Question 7

How do you identify lymphocytes (T + B cells) in a stained blood smear?

Answer 7

• round nucleus
• v little visible cytoplasm
• if activated, much larger cytoplasm to nucleus ratio

Question 8

How do you identify monocytes in a stained blood smear?

Answer 8

• large cells
• irregular nucleus w variations in shape

Question 9

What are some pathogens that can be observed in a stained blood smear?

Answer 9

malaria, small dots in an erythrocyte
trypanosomes, parasite looking ass parasite
fungi, idk not v clear
bacteria, look like bacteria

Question 10

How do you identify erythrocytes in a stained blood smear?

Answer 10

no nucleus, just pink cytoplasm

Question 11

What happens to carbon after it is injected intravenously (i.v)?

Answer 11

75% of particulate material is taken up by Kupffer cells in liver as part of its detoxification process
also taken up by spleen as part of its blood filtration process

99% of carbon injected this way is removed after 30 minutes

Question 12

What are the two main areas of the spleen and what are their function?

Answer 12

white pulp: contains lymphocytes
red pulp : dead red blood cells taken up and recycled by macrophages

Question 13

what is the proportion of different cell types in the blood?

Answer 13

erythrocytes: 94-96%
platelets: 4-6%
leukocytes: 0.1-0.2%

Question 14

What is the proportion of leukocytes in the blood?

Answer 14

granulocytes: 20-80%
neutrophils: -19-75%
lymphocytes: 15-30%
monocytes: 2-12%
eosinophils: 0-7%
basophils: 0-2%

Question 15

How is the total number of cells per ml calculated from a sample counted in a haemocytometer?

Answer 15

count per large square x dilution factor x 10^4

Question 16

What properties of a cell do forward scatter and side scatter measure?

Answer 16

side scatter: relative granulation
forward scatter: relative size

Question 17

What does the mean fluorescence intensity of staining indicate?

Answer 17

how many receptors on the cells

Question 18

In the flow cytometry experiment, what is the isotype-matched antibody control for?

Answer 18

negative control, antibodies could not bind

probably controls for non-specific binding too

Question 19

What does each quadrant in a flow cytometry experiment detecting for CD4+ and CD8+ represent?

Answer 19

LL: no T cells, other leukocytes
UL: CD4+
LR: CD8+
UR: ?

Question 20

What condition might a patient with a lack of CD4+ T cells have?

Answer 20

HIV - attacks CD4+ T cells

Question 21

What general property of proteins allows an ELISA plate to be coated w proteins?

Answer 21

proteins can easily bind non-specifically to plastics

Question 22

Why is it important to block an ELISA plate?

Answer 22

Prevents competition
also prevent non-specific binding of Ab to elisa well (false positive)

Question 23

Why are serum samples diluted? In what situation could a single dilution be used?

Answer 23

1. to come below the saturation limit, allows comparison of resolutions as there are differences
2. in big assays or when comparison is already available

Question 24

What properties of antibodies contribute to the final ELISA colour?

Answer 24

• binding affinity
• competitive binding
• cross-reactivity: Ab binding to similar antigen

Question 25

Is testing for Ab diagnostic of active infection or exposure history, and what could result in a false positive result? How could active infection be confirmed?

Answer 25

1. Ab remains after infection, so only diagnostic of exposure. 2. Ab could be obtained without being exposed to pathogen eg through placenta or vaccination 3. could have infection but Ab response not mounted yet 4. confirm through PCR or plating and identification

Question 26

What are mechanisms of protection against viruses by Ab?

Answer 26

- blocking of entry into cells eg neutralisation, agglutination - NK binds Ab via FcgRIII and kills infected cells via perforins (ADCC)

Question 27

If an antibody recognises an antigen in an ELISA, does this mean it is sufficient to make a sheep resistant to the associated pathogen?

Answer 27

Ab may be able to bind but does not necessarily have mechanism to eliminate that pathogen

Question 28

How could an ELISA be used to screen for other potential vaccine Ag?

Answer 28

coat a plate w range of different viral Ag take sera from individuals who have cleared virus and see what Ab recognise which Ag

Question 29

How to calculate the OD titres?

Answer 29

??💀???💀??💀💀?💀💀??????? 1. Take mean of control values 2. Take control values away from new mean 3. Take mean of these values, square them 4. Times by 3, add onto original mean

Question 30

In the haemagglutination and haemolysis practical, what was the purpose of the row A and row H controls?

Answer 30

row A: no antibodies in the serum that are specific for sheep blood cells so no agglutination occurs (showing that agglutination was in fact due to SRBC specific Ab) row H: negative control without serum. to observe any effect on RBCs without the presence of serum eg stability (other possible effects intrinsic to the cells/buffer: cell age, effect of buffer...)

Question 31

hmmm

Answer 31

Yes!