Practicals Flashcards
Romanowsky staining is used for staining blood smears. What compounds are used and what do they stain?
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
What compounds are used to stain lymphoid tissues?
haematoxylin: stains cell nuceli blue
eosin:
- stains cytoplasm and extracellular matrix pink
- acidic so binds to basic compounds like proteins
What is immuno-histological staining?
- primary antibody that recognises specific protein is added to tissue
- secondary antibody with fluorescent marker that is specific for primary antibody is added. Fluorescence can be observed with a fluorescent microscope.
How do you identify neutrophils in a stained blood smear?
- irregular horseshoe nucleus, which can be fragmented
- visible cytoplasm w faint granules
How do you identify eosinophils in a stained blood smear?
- lobed nuclei (like a teardrop)
- pink stained granules (pronounced)
How do you identify basophils in a stained blood smear?
- non-visible nucleus due to highly granular cytoplasm
- granules are dark (blue)
How do you identify lymphocytes (T + B cells) in a stained blood smear?
- round nucleus
- v little visible cytoplasm
- if activated, much larger cytoplasm to nucleus ratio
How do you identify monocytes in a stained blood smear?
- large cells
- irregular nucleus w variations in shape
What are some pathogens that can be observed in a stained blood smear?
malaria, small dots in an erythrocyte
trypanosomes, parasite looking ass parasite
fungi, idk not v clear
bacteria, look like bacteria
How do you identify erythrocytes in a stained blood smear?
no nucleus, just pink cytoplasm
What happens to carbon after it is injected intravenously (i.v)?
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
What are the two main areas of the spleen and what are their function?
white pulp: contains lymphocytes
red pulp : dead red blood cells taken up and recycled by macrophages
what is the proportion of different cell types in the blood?
erythrocytes: 94-96%
platelets: 4-6%
leukocytes: 0.1-0.2%
What is the proportion of leukocytes in the blood?
granulocytes: 20-80%
neutrophils: -19-75%
lymphocytes: 15-30%
monocytes: 2-12%
eosinophils: 0-7%
basophils: 0-2%
How is the total number of cells per ml calculated from a sample counted in a haemocytometer?
count per large square x dilution factor x 10^4
What properties of a cell do forward scatter and side scatter measure?
side scatter: relative granulation
forward scatter: relative size
What does the mean fluorescence intensity of staining indicate?
how many receptors on the cells
In the flow cytometry experiment, what is the isotype-matched antibody control for?
negative control, antibodies could not bind
probably controls for non-specific binding too
What does each quadrant in a flow cytometry experiment detecting for CD4+ and CD8+ represent?
LL: no T cells, other leukocytes
UL: CD4+
LR: CD8+
UR: ?
What condition might a patient with a lack of CD4+ T cells have?
HIV - attacks CD4+ T cells
What general property of proteins allows an ELISA plate to be coated w proteins?
proteins can easily bind non-specifically to plastics
Why is it important to block an ELISA plate?
Prevents competition
also prevent non-specific binding of Ab to elisa well (false positive)
Why are serum samples diluted? In what situation could a single dilution be used?
- to come below the saturation limit, allows comparison of resolutions as there are differences
- in big assays or when comparison is already available
What properties of antibodies contribute to the final ELISA colour?
- binding affinity
- competitive binding
- cross-reactivity: Ab binding to similar antigen
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?
- Ab remains after infection, so only diagnostic of exposure.
- Ab could be obtained without being exposed to pathogen eg through placenta or vaccination
- could have infection but Ab response not mounted yet
- confirm through PCR or plating and identification
What are mechanisms of protection against viruses by Ab?
- blocking of entry into cells eg neutralisation, agglutination
- NK binds Ab via FcgRIII and kills infected cells via perforins (ADCC)
If an antibody recognises an antigen in an ELISA, does this mean it is sufficient to make a sheep resistant to the associated pathogen?
Ab may be able to bind but does not necessarily have mechanism to eliminate that pathogen
How could an ELISA be used to screen for other potential vaccine Ag?
coat a plate w range of different viral Ag
take sera from individuals who have cleared virus and see what Ab recognise which Ag
How to calculate the OD titres?
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- Take mean of control values
- Take control values away from new mean
- Take mean of these values, square them
- Times by 3, add onto original mean
In the haemagglutination and haemolysis practical, what was the purpose of the row A and row H controls?
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…)
hmmm
Yes!
