2.Methods

B. Hypotheses

Azole antifungals are characterised by the ability to inhibit ergosterol synthesis in fungal membrane. The biosynthesis of ergosterol requires the enzyme lanosterol 14 a-demethylase.* This enzyme is needed to convert lanosterol to ergosterol. Targeting this enzyme prevents ergosterol production. Thus, the fungal membrane structure is depleted of ergosterol and the fungus dies. the various types of azole classified drugs include imidazole, triazole and thiazole antifungals. Abledrugs are broad-spectrum drugs and treat fungal infections of the skin or the mouth. An example of an azole drug is Clomtriazole, commonly used to treat athlete's foot, ringworm, vaginal yeast infections and oral thrush.

Allylamine antifungals are characterized by the ability to inhibit fungal squalene metabolism. The biosynthesis of ergosterol requires an enzyme called squalene peroxidase. Squalene peroxidase is responsible for catalyzing the first step in ergosterol biosynthesis; inhibition of this enzyme results in disruption of ergosterol synthesis. The inhibition of squalene metabolism is toxic to the fungi because of the buildup of squalene and the inhibition of ergosterol synthesis. An example of an allylamine drug is Terbinafine, which is commonly used to treat fungal skin infections.

Source: Boundless. “Antifungal Drugs.” Boundless Microbiology. Boundless, 09 Jan. 2015. Retrieved 22 Feb. 2015 from https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/antimicrobial-drugs-13/other-antimicrobial-drugs-160/antifungal-drugs-803-8338/

Using this knowledge that we retrieved from the website we make our hypothesis that miconzale and clotrimazole will be more effective than terbinafine.This is due to the fact that azoles are more effective than allyamines.Azoles have the ability to Inhibit ergosterol synthesis in fungal membranes and depleat the fungal membrane structure causing to die.Allyamines inhibit the fungal squalene metobolism which is toxic to fungus thus distrupting the ergosteral synthesis.In contrast to the Azoles Allyamines have only got the ability to distrupt the growth of the fungus.But Azoles have the ability to cause the fungus to die.Thus, we hypothesise Clotrimazole and Miconzale is more effective than Terbinafine.

*This enzyme belongs to the family of oxidoreductases(catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor.), specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with NADH or NADPH as one donor, and incorporation of one atom o oxygen into the other donor.
NADPH:is a cofactor used in anabolic reactions, such as lipid and nucleic acid synthesis
NADH: coenzyme found in all living cells. (Wikipedia,2015)


C.    Description in detail of method or procedures

Materials
  1. Sugar
  2. Yeast
  3. Miconazole (Antifungals)
  4. Terbinafine (Antifungals)
  5. Clotrimazole (Antifungals)
  6. Disposable gloves
  7. Spatula
  8. Measuring boats
  9. Gram balance
  10. 1 beaker
  11. 1 measuring bottle
  12. 2 Bottles
  13. 2 Carbon dioxide sensors
  14. 1 Data logger

Detailed Procedure



Part 1:


Setting up the control


Step 1: Measure about 100ml of water(tap water)
Step 2: Measure 2 grams of sugar
Step 3: Measure 2 grams of yeast
Step 4: Pour the 100ml of tap water ,2g of sugar and 2g of yeast into a bottle/beaker and stir well with a glass rod
Step 5 : Take another bottle.
Step 6: Pour 50ml of the solution into the bottle.
Step 7: Place a Carbon dioxide sensor in the bottle.
Step 8: Leave the experiment for 30 minutes for data to be collected.


Part 2:

Setting up the experiments with antifungals


Setting up experiment with Clotrimazole


Step 1: Measure about 100ml of water(tap water)
Step 2: Measure 2 grams of sugar
Step 3: Measure 2 grams of yeast
Step 4: Pour the 100ml of tap water ,2g of sugar and 2g of yeast into a bottle/beaker and stir well with a glass rod
Step 5: Take 2 bottle
Step 6: Pour 50ml of the solution into the bottle.
Step 7: Weigh 2 grams of Clotrimazole and pour it into ONLY 1 OF THE BOTTLES.(do not shake the bottle)
Step 8: Mix the solution well
Step 9: Place a Carbon Dioxide sensor which is connected to data logger into both of the    bottles.
Step 10: Leave the experiment for 30 minutes for data to be collected.


Setting up experiment with Terbinafine


Step 1: Measure about 100ml of water(tap water)
Step 2: Measure 2 grams of sugar
Step 3: Measure 2 grams of yeast
Step 4: Pour the 100ml of tap water ,2g of sugar and 2g of yeast into a bottle/beaker and stir well with a glass rod
Step 5: Take 2 bottle
Step 6: Pour 50ml of the solution into the bottle.
Step 7: Weigh 2 grams of Terbinafine and pour it into ONLY 1 OF THE BOTTLES.(do not shake the bottle)
Step 8: Mix the solution well
Step 9: Place a Carbon Dioxide sensor which is connected to data logger into both of the    bottles.
Step 10: Leave the experiment for 30 minutes for data to be collected.


Setting up experiment with Miconazole


Step 1: Measure about 100ml of water(tap water)
Step 2: Measure 2 grams of sugar
Step 3: Measure 2 grams of yeast
Step 4: Pour the 100ml of tap water ,2g of sugar and 2g of yeast into a bottle/beaker and stir well with a glass rod
Step 5: Take 2 bottle
Step 6: Pour 50ml of the solution into the bottle.
Step 7: Weigh 1 gram of Miconazole and pour it into ONLY 1 OF THE BOTTLES.(do not shake the bottle)
Step 8: Mix the solution well
Step 9: Place a Carbon Dioxide sensor which is connected to data logger into both of the    bottles.
Step 10: Leave the experiment for 30 minutes for data to be collected.

Risk and Safety:

1.    As this experiment involves handling hot water we have to be extra careful when handling the hot water.We should use mittens cloths when handling the water.

2.    As this experiment handles fungal medicine which is highly toxic it may hurt our hands if may damage our skin if they are not used properly.Therefore we should use synthetic gloves when handling the anti fungals



Procedure(summarised):



  1. Collect the data of the amount of carbon dioxide that is inside the bottle before and after the experiment has started so as to see the difference the anti fungal has brought about to the amount of carbon dioxide produced by the yeast.
  2. Record the results in tables(using the data collected on the amount of carbon dioxide collected)
  3. Plot graphs using the data in the table
  4. Conclude which anti fungal is best at stopping the growth of Fungus.

Data Analysis



Type of data
Source
Analysis
Carbon dioxide
Carbon dioxide sensor
Carbon dioxide would be given out by the yeast when it is activated, when but under favourable conditions
Eg.put in cold/tap watered sugar water.

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