All Practicals

Question 1

How is HTLV1 transmitted

Answer 1

Breast feeding
Sexual contact
Blood produces

Question 2

How is HTLV 1 diagnosed

Answer 2

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

Question 3

How is HTLV 1 detected

Answer 3

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

Question 4

Electrophoresis

Answer 4

Analyzing molecules on basis of charge my measuring their migration in an electric field

Question 5

PCR

Answer 5

amplifies copies of a Specific dna fragment

Question 6

PCR steps

Answer 6

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

Question 7

What’s done after PCR

Answer 7

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)

Question 8

What’s the purpose of adding loading dye

Answer 8

It’s blue and allows mixture to sink due to increase in density

Question 9

Most common quantitative real time PCR

Answer 9

Fluorescent dye based
DNA probe based method

Question 10

Cycle threshold

Answer 10

Number of cycles needed for fluorescent signal to cross threshold. Lower cycle threshold indicates high load

Question 11

Which direction does dna move

Answer 11

DNA is negatively charged so moves towards possible electrode

Question 12

Do smaller or bigger dna fragments move faster

Answer 12

Smaller and it’s easier for them to move through agarose mesh

Question 13

How will you know if dna fragment has correct size

Answer 13

Based on dna ladder which runs parallel to

Question 14

Limits of PCR

Answer 14

Not sensitive enough so if low infection then this might not be detected

Question 15

Nested PCR

Answer 15

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.

Question 16

Advantage of PCR over culture based method

Answer 16

Results obtained more quickly thus less likely to get infected

Question 17

Mean cell haemoglobin

Answer 17

Hb/rbc pg

Question 18

Mean cell haemoglobin concentration

Answer 18

Hb/Hct (l/l)

Question 19

Mean cell volume

Answer 19

HCT/rbc

Question 20

Red blood cell count

Answer 20

Dilute blood and place on haemocytometer
Count red cellls
Count 5 medium squares then times by 10 to the power of 10

Question 21

Measuring haematocrit

Answer 21

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

Question 22

Haemoglobin measured

Answer 22

Using haemocue. Add blood into analyses and get results

Question 23

Gram staining

Answer 23

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

Question 24

Lactose fermenters

Answer 24

If plate is red or pink the bacteria are lactose fermenters
If yellow or colorless they are non lactose fermenters

Question 25

Blood agar plates

Answer 25

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

Question 26

Oxidase test

Answer 26

Checks if organism makes cytochrome c oxidase
Oxidases postive are aerobic bacteria

Question 27

Catalase test

Answer 27

End product of aerobic breakdown of sugar
Place bacteria with hydrogen peroxide
If you see bubbles then bacteria are catalase positive

Question 28

Coagulase

Answer 28

Checks for enzyme Coagulase which reacts with fibrinogen to form clumps
If clots seen the Coagulase pos

Question 29

Antibiotic sensitivity

Answer 29

Measured via inhibition zone
E test which measures minimum inhibitory concentration

Question 30

Lineweaver Burke plot

Answer 30

V max is the max enzyme velocity which is approached when substrate concentrating increases

Question 31

Michaelis constant

Answer 31

Is the substrate concentration at which the rate of reaction is exactly 1/2 v max

Question 32

Non competitive inhibitor

Answer 32

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

Question 33

Competitive inhibitor

Answer 33

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

Question 34

Turnover number

Answer 34

Kcat measueres the number of molecules that an enzyme can process in a given unit of time
Vmax/enzyme conc

Question 35

How to set up microscope

Answer 35

Focus image
Condenser
Field iris
Condenser iris
Measure using lowest magnification first

Question 36

Preparing blood film

Answer 36

Let blood drop run bending spreader
Add leishmans stain and then buffer
The purple die stains nucleus and pink stains cytoplasm

Question 37

Spectrophotometer

Answer 37

Add sodium dithionite to strip oxygen from haemoglobin. The measure Absorbance

Question 38

Electrophoresis

Answer 38

Place Hb onto tray. Add cellulose acetate strip budge and then using application to add Hb. Then place in electrophoresis tank

Question 39

Beer lambert

Answer 39

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

Question 40

Positive cooperation

Answer 40

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.

Question 41

Myoglobin

Answer 41

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.

Question 42

Hb absorption spectra

Answer 42

Oxy haemoglobin has two peaks at 540 and 580 wherr2 deoxygenation has one at 560

Question 43

Carboxyhaemoglobin

Answer 43

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.

Question 44

Methaemoglobin

Answer 44

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.