[BIO 120.12] Final Exam - Exercises 5 to 8

Sucrose

Example of an oligosaccharide

Glucose

Example of a monosaccharide

Starch, Glycogen

Examples of polysaccharides

Triple Sugar Iron Agar

Rich medium designed to differentiate bacteria based on glucose, sucrose, and lactose fermentation

Glucose, Sucrose, Lactose

What sugars are being tested in TSI agar?

Phenol red

pH indicator used in TSI agar

TRUE

T/F: Phenol red is a pH indicator in TSI agar

FALSE

T/F: Phenol red is an indicator of H2S production

Sulfur reduction

Aside from sugar fermentation, TSI agar is also used to test for?

Iron

What is used as H2S indicator in TSI agar?

Ferrous sulfate, Sodium thiosulfate

In TSI Agar, what are the sources of oxidized sulfur?

Ferments glucose and lactose/sucrose

TSI: Yellow slant and butt

Gas production from fermentation of any of the sugars

TSI: Fissures in the medium (lifting the agar off the bottom of the tube)

Ferments glucose but not lactose and/or sucrose

TSI: Red slant and yellow butt

H2S production; Ferments glucose and lactose/sucrose

TSI: Black butt, yellow slant

H2S production; Ferments glucose

TSI: Black butt, red slant

No fermentation; Peptone and amino acids as C and N sources under aerobic and anaerobic conditions

TSI: Red slant and butt

No fermentation; Peptone and amino acids as C and N sources under aerobic conditions only

TSI: Red Slant

Ability to use citrate as sole carbon source and perform citrate fermentation

What does the citrate utilization test for?

Enterobacteriaceae

Citrate utilization is best used for differentiating members of?

Simmons Citrate Agar

What agar is used for Citrate Utilization?

Citrate

Sole C source in SCA?

Ammonium phosphate

Sole N source in SCA?

Bromothymol blue

pH indicator in SCA?

Citrate permease

Citrate Utilization: Bacteria with ___ can transport citrate into the cell

Green at pH 6.9, Blue at pH 7.6

What are the results of SCA with bromothymol blue as pH indicator?

Citrate is hydrolyzed into oxaloacetate and acetate using citrate lyase

What happens to citrate after it has been transported into the cell?

Oxaloacetate is converted to pyruvate, Pyruvate can be converted to a variety of products depending on pH

In Citrate Utilization, after oxaloacetate and acetate has been produced from hydrolysis of citrate, what happens to these two?

Citrate is utilized; Ammonium phosphate is converted to ammonia and ammonium hydroxide (alkalinize the agar)

Citrate Utilization: Blue (even a small amount)

Citrate is utilized; Incomplete incubation

Citrate Utilization: No color change, with growth

Citrate is not utilized

Citrate Utilization: No color change, no growth

Enterobacter, E. coli

Give the most probable species that give the following results (L-R)

Starch Hydrolysis

Amylase Test is a test for?

0.1% glucose, 1% sucrose, 1% lactose

What are the %s of the sugars added in TSI?

a-D-glucose subunits bonded by 1,4-a-glycosidic (acetal) linkages

What is starch made up of in terms of subunits and linkages?

Amylose (linear), Amylopectin (branched)

What are the 2 forms of starch?

Amylopectin

What is the predominant form of starch?

α-amylase and Oligo-1,6,-glucosidase

2 extracellular enzymes that can hydrolyze starch

TRUE

T/F: α-amylase and Oligo-1,6,-glucosidase are extracellular enzymes

FALSE

T/F: α-amylase and Oligo-1,6,-glucosidase are intracellular enzymes

Break glycosidic linkages between sugar subunits

What is the mechanism of α-amylase and Oligo-1,6,-glucosidase in hydrolyzing starch?

Iodine

Detects the presence or absence of starch in the vicinity around the bacterial growth

Iodine

Substance that is later added in Starch Agar plate that reacts with starch

Blue or dark brown

What color is produced by Iodine in Starch Agar Hydrolysis?

Clear zone surrounding growth

What is the indication of starch hydrolysis in an SA plate?

B. subtilis

Bacteria used as a control for starch hydrolysis (+ starch hydrolysis)

S. aureus

Bacteria used as a control in SCA (+ for citrate utilization)

K

Symbol for alkaline in TSI

A

Symbol for acid in TSI

A/A with gas production

What is the expected result for E. coli in TSI?

FALSE

T/F: Gelatin is a carbohydrate derived from collagen

TRUE

T/F: Gelatin is a protein derived from collagen

Collagen

A component of vertebrate connective tissue from which gelatin is derived

Gelatinase

An extracellular enzyme and can hydrolyze gelatin

Gelatin → Polypeptides → Amino Acids

Explain how gelatinase works in hydrolyzing gelatin

Gelatin

Solidifying agent and substrate for enzymatic activity

Liquefied medium

What is an indication of a gelatinase positive bacteria?

S. aureus

Example of a gelatinase positive bacteria

S. epidermidis

Example of a gelatinase negative bacteria

Gelatinase Positive, Gelatinase Negative

Gelatin Liquefaction: Interpret the results (top - bottom)

Lipid

Water insoluble organic biomolecules that serve as good source of metabolic fuels when broken down to fatty acids

Lipase

Bacterial extracellular enzyme that hydrolyzes lipid

Tween 80

Non-ionic emulsifier that is added to cosmetics, pharmaceuticals, and foods as a source of lipid

Source of lipid

What is the function of Tween 80?

Polysorbate 80

Tween 80 is also known as?

Ca2+ and Na2+

These are the ions in ST (Sierra’s Medium with Tween 80) plate that react with fatty acids released due to lipolysis

Precipitates composed of crystals and calcium soap

The reaction between Ca2+ and Na2+ and fatty acids result to?

White or opaque zone of precipitation around line of streak

What is the expected result for lipase test for a bacteria that has lipolytic activity

Urease Test

What test is done to determine hydrolysis of urea?

Urease

Hydrolyzes urea into ammonia (usable form of nitrogen) and carbon dioxide

Hydrolyzes urea into ammonia (usable form of nitrogen) and carbon dioxide

What does urease do?

Proteus

Example of a bacteria with urease as a virulence factor?

Christensen’s medium

Differentiates rapid urease-positive bacteria from slow urease-positive and urease-negative bacteria

FALSE

T/F: Christensen’s medium differentiates rapid urease-positive bacteria from urease-negative bacteria

TRUE

T/F: Christensen’s media differentiates rapid urease-positive bacteria from slow urease-positive and urease-negative bacteria

Decarboxylation of certain amino acids

Urea is a product of?

Urine

Urea is the primary nitrogenous waste in ___.

Phenol red

pH indicator in Christensen’s medium

Yellow or orange (below pH 8.4), Red or pink (above pH 8.4)

What are the 2 expected results for Christensen’s medium with phenol red as a pH indicator?

Rapid urea hydrolysis; Strong urease production

Urease Test: Pink

No urea hydrolysis; No urease production

Urease Test: Orange or Yellow

Rapid urea hydrolysis; Strong urease production; No urea hydrolysis; No urease production

Interpret (L-R)

Indole production from tryptophan

What does the indole test test for?

Tryptone broth

Source of tryptophan in indole production test?

Tryptophanase

Hydrolyzes tryptophan to pyruvate, ammonia, and indole

Pyruvate, Indole, Ammonia

Tryptophanase can hydrolyze tryptophan to __

Tryptophan hydrolysis

What is detected by the addition of Kovac’s reagent in the indole test?

Kovac’s Reagent

Added to detect tryptophan hydrolysis

Para-dimethylaminobenzaldehyde (DMABA)

Component of Kovac’s reagent that reacts with indole and produces a quinoidal compound that turns the reagent layer red

Quinoidal compound

What compound is produced by DMABA that turns the reagent layer red?

Alcohol layer

In what layer of the Kovac’s reagent is the red color expected?

Tryptophanase production + Tryptophan hydrolysis; No tryptophanase production + No tryptophan hydrolysis

Interpret the results of the Indole production tests shown (L-R)

37 deg C for 24 h

Isolation temp and time for urea hydrolysis

37 deg C for 36-48 h

Isolation temp and time for indole production

37 deg C for 48 h

Incubation temp and time for H2S production

2 drops

How many drops of Kovac’s reagent is used in testing for indole production?

15 minutes

How many minutes are the incubated NG tubes placed in an ice bath to see if liquefaction occurs?

Lead Acetate Agar

Agar that is used to test for H2S production

Sodium thiosulfate

Component in LAA that serves as source of thiosulfate

Peptone (cysteine)

Component in LAA that serves as an organic sulfur-containing compound