All Practicals Flashcards
How is HTLV1 transmitted
Breast feeding
Sexual contact
Blood produces
How is HTLV 1 diagnosed
Using ELISA assays where viral antigens are detected by viral antibodies and confirmed by western blot. Can be inconclusive so OCR used by detecting from peripheral blood mononuclear cells
How is HTLV 1 detected
Blood taken and peripheral blood mononuclear samples isolated from whole blood sampl , then dna isolated from them and sued as a template in a PCR reaction , primers amplify tax gene . Will see 300 Bp dna fragment is infected and nothing if not infected
Electrophoresis
Analyzing molecules on basis of charge my measuring their migration in an electric field
PCR
amplifies copies of a Specific dna fragment
PCR steps
Step 1 denaturation where you heat sample to 94 degrees
Step 2 annelaing where sample cooled to 54 degrees and forward and reverse primers anneal
Step 3 heat to 72 degrees
Repeat 30 to 40 times
What’s done after PCR
Set up agarose gel to separate PCR product. Add SYBR safe dna stain to agarose to allow visualization of the dna (will turn agarose pink)
What’s the purpose of adding loading dye
It’s blue and allows mixture to sink due to increase in density
Most common quantitative real time PCR
Fluorescent dye based
DNA probe based method
Cycle threshold
Number of cycles needed for fluorescent signal to cross threshold. Lower cycle threshold indicates high load
Which direction does dna move
DNA is negatively charged so moves towards possible electrode
Do smaller or bigger dna fragments move faster
Smaller and it’s easier for them to move through agarose mesh
How will you know if dna fragment has correct size
Based on dna ladder which runs parallel to
Limits of PCR
Not sensitive enough so if low infection then this might not be detected
Nested PCR
When two PCRs are performed one after the other and the product of the first PCR is used as a template for the second PCR.
Advantage of PCR over culture based method
Results obtained more quickly thus less likely to get infected
Mean cell haemoglobin
Hb/rbc pg
Mean cell haemoglobin concentration
Hb/Hct (l/l)
Mean cell volume
HCT/rbc
Red blood cell count
Dilute blood and place on haemocytometer
Count red cellls
Count 5 medium squares then times by 10 to the power of 10
Measuring haematocrit
Shake blood sample and transfer by capillary action. Seal end with wax and centrifuge. Ratio of length of red cell to total lefty of cells in Haematocrit
Haemoglobin measured
Using haemocue. Add blood into analyses and get results
Gram staining
Differentiated gram negative and positive bacteria
Smear base trait into slide and heat treat and add crystal violet to turn cells purple. Then add iodine which traps crystal violet in cell. Add and organic solvent eg alcohol and counter stain with safranin. Gram postive reamin purple as they have a thick cell wall that isn’t easily penetrated. Gram negative have a thinner cell wall allowing removal of purple dye, counter stain makes them pink
Lactose fermenters
If plate is red or pink the bacteria are lactose fermenters
If yellow or colorless they are non lactose fermenters