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14 Mar 2019

Starch hydrolysis ( amylase test)

a)What are the names of the 2 enzymes that degrade starch (linear and branched) and what do they degrade the starch into? ( Starch hydrolysis test )

b) What is the reagent used to visualize the degradation of starch on the plate? In what order procedure have you already use this reagent? ( Starch hydrolysis test)

c) During other labs, you grow the organism on 1) the test agar and 2) om nutrient agar as a control. Why is this type of control not necessary for this test? ( Starch hydrolysis test)

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15 Mar 2019

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nothing is missing now.you just have to tell what are expected observations.AS WE HAVE TO DO EXPERIMENT AFTER BUT FIRST WRITE THAT WHAT WILL BE EXPECTED FROM THE RESULTS?WE WILL GET PHOTOS WHEN WE PERFORM BUT THEN WE WILL COMPARE WHETHER WHAT WE EXPECTED IS WHAT WE GOT OR IS THE ACTUAL RESULT DIFFERENT.
INTRODUCTION
For acquiring food material, bacteria excrete extracellular enzymes that split larger molecules into smaller units.For example, amylase and cellulase degrade polysaccharides such as starch and cellulose into simple sugars, which are then transported into the cell for metabolic purposes. A variety of proteases degrade protein molecules such as gelatin and casein into amino acids, and lipases split fats into fatty
acids and glycerol. These physiological or biochemical characteristics are used for isolation and identification of bacteria in conjunction with other morphological and cultural methods. In this exercise, you will perform tests related to hydrolytic reactions.
Starch hydrolysisâSince many bacteria are capable of hydrolyzing starch, this test has a fairly wide application. Bacteria that hydrolyze starch produce amylases that yield molecules of maltose, glucose, and dextrins.
Casein hydrolysisâCasein is the predominant protein in milk and its presence causes milk to have its characteristic white color. Many bacteria produce proteases that degrade proteins into peptides and individual amino acids in a process called proteolysis.

MATERIALS
ï·
Nutrient broth cultures of Bacillus subtilis, Escherichia coli andStaphylococcus epidermidis
ï·
Starch agar culture plate
ï·
Casein agar culture plate
ï·
Gramâs iodin

1.Label and streak in a straight line with all the 3 cultures on the starch agar plate ANDon the casein agar plate.

2.Incubate the plates at 37oC for 24-48 hours.

Week 2 âStarch Hydrolysis

1.Add several drops of Gramâs iodine over the growth on the medium. Rotate and tilt the plate gently to spread the iodine. Iodine is a starch indicator and when it comes in contact with a medium of starch, it turns blue.

2.If starch is hydrolyzed and no longer present, the medium will have a clear zone next to the growth.

3. Take a photograph of your plate against a white background. (If you do not have a phone equipped with a camera, ask a lab mate to take a picture for you and have the picture electronically sent to you.)

Week 2 âCasein Hydrolysis

1.Examine the streaks on the casein agar plates. A clear zone adjacent to the growth is evidence of casein (protein) hydrolysis. Discussion Amongst other points, you should address in the discussion whether the observations you made were expected. (You will need to do research to answer address this issue. Make sure you include proper citations?

What is expected observation for it with reference if possible

I need a good introduction and conlcusion of this experiment below is the expermeint and my results.

In this experiment you will observe the action of the enzyme amylase on starch. Amylase changes starch into a simpler form: the sugar maltose, which is soluble in water. Amylase is present in our saliva, and begins to act on the starch in our food while still in the mouth.

Exposure to heat or extreme pH (acid or base) will denature proteins. Enzymes, including amylase, are proteins. If denatured, an enzyme can no longer act as a catalyst for the reaction.

Benedict's solution is a test reagent that reacts positively with simple reducing sugars like maltose, but will not react with starch. A positive test is observed as the formation of a brownish-red cuprous oxide precipitate. A weaker positive test will be yellow to orange

PRE-LAB:

Add 1g of cornstarch to a beaker containing 100ml of cold distilled water. While stirring frequently, heat the mixture just until it begins to boil. Allow to cool.

PROCEDURE:

1. Fill the 250-mL beaker about ³/₄ full of water and place on the hot plate for a boiling water bath. Keep the water JUST AT BOILING.

2. Mark 3 test tubes A, B and C. "Spit" between 1 and 2 mL of saliva into each test tube.

3. Into tube A, add 2 mL of vinegar. Into tubes B and C, add 2 mL of distilled water. Thump the tubes to mix.

4. Place tube B into the boiling water bath for 5 minutes. After the five minutes, remove from the bath, and place back into the test tube rack.

5. Add 5 mL of the starch solution to each tube and thump to mix. Allow the tubes to sit for 10 minutes, occasionally thumping the tubes to mix.

6. Add 5 mL of Benedict's solution to each tube and thump to mix. Place the tubes in the hot water bath. The reaction takes several minutes to begin.

OBSERVATIONS:

Tube A: Starch + saliva treated with vinegar (acid)

Was the test positive or negative? Negative

What does this indicate? The vinegar stops the action of amylase from catalyzing the starch therefore the solution becomes cloudier. This indicates that vinegar (acids) distort the shape of proteins in the salivary amylase.

Results: The solution turns cloudy.

Conclusion: Acids denature the shape of proteins in the salivary

Tube B: Starch + saliva and water, treated in a boiling water bath

Was the test positive or negative? Negative

What does this indicate? It indicates that when enzymes are exposed to extreme temperature for some time they are denatured and therefore become inactive.

Results: The benedictâs solution retained its color as the reaction did not occur. Extreme temperatures denatures enzymes, therefore when the salivary amylase was introduced to a hot water bath for five minutes the amylase was denture.

Conclusion: Salivary amylase/enzymes when introduced to hot temperatures it is a denatured therefore does not react on the substrate.

Tube C: Starch + saliva

Was the test positive or negative? Positive

What does this indicate? This indicates that the salivary amylase successfully worked on the starch.

Results: The benedictâs solution turned brown as an indication of the presence of sugar.

Conclusions: Salivary amylase acts on starch in favorable conditions, ie is optimum te

Please provide a lab report that explains your procedure, results and conclusions. Remember to include pictures of the lab set-up and results. Also, I want you to expand this initial experiment by proposing and testing (in the form of an additional experiment) a testable hypothesis that involves the above general concept. You will be graded on your use of the scientific method and the creativity/complexity of your experiment. Both the above experimentation and your original experiment may be included together into one experiment

CATALASE TEST

1. Would a false positive from the reaction between the inoculating loop and hydrogen peroxide be caused by poor specificity or poor sensitivity of the test system? Explain.

OXIDASE TEST

2.Think about theadvice given in the procedure to test a known oxidase-positive organism along with an unknown. a.) Is this a positive or a negative control? b.) What information is provided by the results?

3. Provide a possible explanation as to why the phenylenediamine reagent reduces cytochrome c (aa3) oxidase and not other oxidases (such as cytochrome d of E. coli shown in Fig. 5.18).

STARCH HYDROLYSIS (AMYLASE TEST)

2. How would you expect both positive and negative results to be affected if you were to add glucose to the medium?

I WOULD REALLY APPRECIATE SOME HELP WITH THESE THREE QUESTIONS REGARDING "MICROBIOLOGY LAB". I WILL RATE ANSWERS. THANKS A LOT.

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