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
Blood agar plates
Some pathogens produce haemolysins which lose or destroy rbc
Alpha haemolysis has a small zone of haemolysis and colonies look green.
Beta haemolysis has a large clear zone of haemolysis
Gamma has no haemolysis
Oxidase test
Checks if organism makes cytochrome c oxidase
Oxidases postive are aerobic bacteria
Catalase test
End product of aerobic breakdown of sugar
Place bacteria with hydrogen peroxide
If you see bubbles then bacteria are catalase positive
Coagulase
Checks for enzyme Coagulase which reacts with fibrinogen to form clumps
If clots seen the Coagulase pos
Antibiotic sensitivity
Measured via inhibition zone
E test which measures minimum inhibitory concentration
Lineweaver Burke plot
V max is the max enzyme velocity which is approached when substrate concentrating increases
Michaelis constant
Is the substrate concentration at which the rate of reaction is exactly 1/2 v max
Non competitive inhibitor
Bind outside enzyme active site causing conformational change
No effect on km but lowers v max
C axis remains unchanged and y axis decreases with no inhibitor
Competitive inhibitor
Adding more susbtrwfe will outcomepete antagonist
Inhibits km and has no effect on v max
Thus g axis the same and X axis is lower with no inhibition
Turnover number
Kcat measueres the number of molecules that an enzyme can process in a given unit of time
Vmax/enzyme conc
How to set up microscope
Focus image
Condenser
Field iris
Condenser iris
Measure using lowest magnification first
Preparing blood film
Let blood drop run bending spreader
Add leishmans stain and then buffer
The purple die stains nucleus and pink stains cytoplasm
Spectrophotometer
Add sodium dithionite to strip oxygen from haemoglobin. The measure Absorbance
Electrophoresis
Place Hb onto tray. Add cellulose acetate strip budge and then using application to add Hb. Then place in electrophoresis tank
Beer lambert
The absorbance of a solution is proportional to the concentration of the absorbing material within it and to the distance (or path length) travelled by the light through the sample
A=ecl
So,stiles may fail to obey law as at high conc proteins can form dimers
Positive cooperation
Put another way, the positive cooperativity displayed by the binding of oxygen to haemoglobin enables it to deliver almost twice as much oxygen than would be possible in the absence of cooperativity.
Myoglobin
Myoglobin, an oxygen binding protein found in muscle, has one haem group per molecule and displays a greater affinity for oxygen than haemoglobin, saturating at lower pO2 values (green curve). However, the lack of cooperativity means that it is poor at releasing oxygen under the same conditions.
Hb absorption spectra
Oxy haemoglobin has two peaks at 540 and 580 wherr2 deoxygenation has one at 560
Carboxyhaemoglobin
Carboxyhaemoglobin is generated by the binding of carbon monoxide to ferrous iron (Fe2+) in haemoglobin.
Since haemoglobin has a 200-fold greater affinity for carbon monoxide than oxygen, it can readily outcompete oxygen for binding to the four haem groups of haemoglobin.
Even relatively low levels of carbon monoxide can dangerous. Levels of 0.2% carbon monoxide can lead to death within an hour or two.
Methaemoglobin
Methaemoglobin is generated when the Fe2+ ion is oxidised to the Fe3+ (ferric) state which results in greatly impaired oxygen binding. This gives the blood a bluish/chocolate colour if present at high levels.
Relatively low levels of MetHb can cause the oxygen dissociation curves that we looked at earlier to shift leftwards, which can result in tissue anoxia, as oxygen is not readily released by MetHb.
The enzyme methemoglobin reductase reduces methaemoglobin back to haemoglobin. The disorder methaemaglobinaemia can be hereditary, for example as a deficiency in methemoglobin reductase or production of a mutant form of haemoglobin known as haemoglobin M, which is resistant to reduction.
Methaemaglobinaemia can also be acquired following exposure to chemicals including aniline dues such as p-chloroaniline, nitrates, and local anaesthetics such as benzocaine.
