Pre-Lab Assignment #4

Question 1

What protein source, sugar (list 3 common sugars used in this assay) and pH indicator are present in liquid broth media used to assess carbohydrate fermentation?

Answer 1

a. Protein Source: peptone
b. Sugar: glucose, lactose and sucrose
c. pH indicator: phenol red

Question 2

What color change occurs when a bacterial species ferments a sugar? If a bacterial species produces gas, how is the presence of gas assessed?

Answer 2

a. Turns it from red to a yellow/clear color. The gas presence if assessed because of the Durham tube, it goes up the tube and produced a big gas bubble

Question 3

How is mannitol salt agar both selective and differential? What pH indicator and what sugar does this assay contain?

Answer 3

a. Mannitol salt agar is both selective and differential, it is selective for salt tolerant bacteria and differential for Mannitol fermentation. The pH indicator is agar and the sugar is mannitol.

Question 4

What is the name of the enzyme that catalyzes the breakdown of starch?

Answer 4

a. Amylase

Question 4

What does it mean if a bacterial species cannot grow on mannitol salt agar?

Answer 4

a. It means that the salt inhibited it and that it is not a salt tolerant bacteria

Question 5

What does it mean if a bacterial species can grow on mannitol salt agar, but the color of the medium does not change to yellow?

Answer 5

a. It means that it does not ferment mannitol

Question 6

What does a positive Simmon’s Citrate Assay reaction look like? What does a negative reaction look like?

Answer 6

a. A positive reaction looks blue fully or only blue on top green on bottom
b. A negative reaction looks green fully

Question 6

How do we detect a positive or negative reaction in a starch hydrolysis assay? What does a positive and negative reaction look like?

Answer 6

a. A positive reaction is detected because it is clearing while a negative reaction is purple. A negative reaction dyes the background purple while the positive has amylase so it clears the starch and clears it (no purple)

Question 7

Citrate can be metabolized by species containing the enzyme _______ which, in the presence of CO2, produces the organic compound_______ causing the pH of the medium to ______.

Answer 7

citrate lyase, OXALOACETATE, decrease

Question 8

How is EMB agar both selective and differential?

Answer 8

a. Selective- can they survive in the presence of the dyes or not
b. Differential-what they do with the presence of the dye

Question 9

What dye, pH indicator and sugar does EMB agar contain?

Answer 9

a. Methalyne blue and eosin are the dyes. The pH indicator is the dye that is sucked up.

Question 10

What does the growth of a Gram-negative fermenter look like vs. a Gram-negative non-fermenter on EMB agar?

Answer 10

a. Growth of gram negative fermenter looks like pink and is more spread and positive looks like purple and has more bacteria and are closer together

Question 11

What are gelatin and the enzyme gelatinase?

Answer 11

a. Gelatin is derived from collagen. Gelatinase digests proteins of gelatin.

Question 12

How do we determine if a bacterium can hydrolyze gelatin?

Answer 12

a. If it solidifies the gelatin has not been digested and the bacteria is gelatinase negative. If the gelatin fails to solidify the gelatin has been digested and the bacteria produces the enzyme gelatinous

Question 13

What enzyme breaks down urea, what product is produced and what does it do to the pH of the medium?

Answer 13

a. The enzyme that breaks down urea is urease
b. The products produced are carbon dioxide and two ammonias. It effects the pH because the ammonia elevates (increases) the pH.

Question 14

What color(s) does urea agar change when a bacterial species is capable of breaking down urea?

Answer 14

a. When it is capable of breaking down urea it turns red/pink

Question 15

In the SIM assay, where does indole come from? What enzyme catalyzes the formation of indole? What reagent is added to detect the presence of indole? What does a positive indole test look like?

Answer 15

a. Indole comes from the amino acid tryptophan and the enzyme that catalyzes is tryptophanase. Kovacs reagent is an indole indicator and a positive indole test looks red.

Question 15

In the SIM assay, how is motility assessed?

Answer 15

a. Motility is assessed by growth from the stab marking

Question 15

The SIM assay tests for what three properties?

Answer 15

a. Tests for hydrogen sulfide, indole and motility

Question 16

In the SIM assay, what is the black material that can form? What enzyme catalyzes the formation of this black material?

Answer 16

a. The black material is hydrogen sulfide being produced and reacting with irons makes ferric sulfide.

Question 17

What is hemolysis and what are hemolysins?

Answer 17

a. Hemolysis: Lysis of erythrocytes
b. Hemolysins: Catalyzes hemolysis

Question 18

Differentiate between alpha-, beta-, and gamma hemolysis and describe the difference in their appearance.

Answer 18

a. Alpha-partial hemolysis without transparency: has zone of change, no transparency
b. Beta- complete hemolysis with transparency has a zone of transparency, the brightest
c. Gamma-no hemolysis: no red blood cell destruction, only see the streak of bacteria

Question 19

What enzyme breaks down phenylalanine?

Answer 19

i. Phenylalanine Deaminase

Question 20

What reagent is added to detect the breakdown products of phenylalanine? What does a positive and a negative result look like?

Answer 20

i. The reagent that is added is Ferric Chloride. Negative is clear, with the yellow at the bottom. Positive is green up the slant