fibre typing (PRACTICAL)

Question 1

outline the staining procedure of the fibre-typing practical in 9 steps

Answer 1

1. look at muscle sections under microscope
2. draw around muscle cross sections with pap pen to create hydrophobic barrier to contain staining solution
3. incubate sections at 37°C in water bath for 15 mins in 10ml of solution B + 5mg ATP
4. rinse well in a beaker of distilled water
5. incubate sections in 2% cobalt chloride for 5 mins
6. rinse thoroughly in distilled water
7. incubate sections in ammonium sulphide for 30 seconds in fume cupboard and wear gloves
8. rinse well in distilled then running cold tap water in fume cupboard
9. look at sections under light microscope

Question 2

on a muscle slide, which fibres are slow fibres and which are fast fibres?

Answer 2

slow: darker spots

fast: lighter spots

Question 3

what difference might you expect to see between a weightlifter and a marathon runner on the muscle slides in the fibre typing staining practical?

Answer 3

on weightlifter slide: you would see more darker spots than lighter spots

on marathon runner slide: you would see more lighter spots than darker spots

Question 4

what is myosin ATPase?

Answer 4

the enzyme that catalyses the hydrolysis of ATP during cross bridge cycling

Question 5

what stain is used to distiguish between fibre types?

Answer 5

myosin-ATPase stain

Question 6

what pre-incubation pH inactivates the myosin-ATPase enzyme in fast mammalian type 2A/2X fibres?

Answer 6

acidic pre-incubations

Question 7

what pre-incubation pH inactivate the myosin-ATPase enzyme in slow, mammalian type 1 fibres?

Answer 7

basic pre-incubations

Question 8

in the fibre typing assay, what is the substrate, what is the reaction product and what is the enzyme?

Answer 8

substrate: ATP
reaction product: Pi (phosphate ion)
enzyme: myosin ATPase

Question 9

as Pi is invisible histochemically, what is required to do to Pi in the assay?

Answer 9

it is required for Pi to be chemcially reacted with calcium in order to form calcium phosphate precipitate (CaPO4)

Question 10

after calcium phosphate is formed, what is CaPO4 converted into using incubation iwth (NH4)2S), what colour is it and why is this done?

Answer 10

Converted to: cobalt sulfide
colour: brownish-black

• this is done so it is more easily viewable and less soluble, so as Pi is released by the myosin molecules consumption of ATP, a brownish black product is deposited on the muscle tissue section

Question 11

what is the pH that standard ATPase stain is done at and what fibre type does it stain darker as a result?

Answer 11

standard pH: 9.4

• thus it stains type 2 darker as a result as more type