Microbio Quiz 3

What is extracellular degradation? How does it work?

Extracellular degradation is the use of enzymes outside of the cell to break down materials for energy.

How classes of enzymes are involved in extracellular degradation?

glycosidase (polysaccharides), proteinase (proteins), and esterase (lipids).

What is translocation?

the movement of molecules across a membrane.

What is a polysaccharide?

a chain of multiple sugars

How is a polysaccharide degraded?

by amylase

What is starch made from?

amylose and amylopectin

What molecule is formed as starch is degraded?

Maltose (disaccharide)

How can we evaluate whether degradation of starch occurs?

There will be a zone of hydrolysis (clearing) surrounding growth.

What is the purpose of iodine in the polysaccharide starch degradation experiment?

Iodine reacts with the starch, changing the color if the agar, making the zone of hydrolysis more visible.

What does a positive/negative result look like for the starch experiment?

• Positive: zone of hydrolysis (clearing surrounding growth)

• Negative: maybe growth but no clearing surrounding

What is a protein?

A long chain of amino acids linked by peptide bonds.

How is protein degraded?

proteases hydrolytically cleave proteins and peptidases break down peptide chains into amino acids.

Which protein is in milk?

Casein

How can we evaluate whether degradation of protein occurs on a milk plate?

If degradation occurs, there will be a zone of clearing surrounding growth.

 How do the casein and gelatin tests differ?

In the casein test, we inoculate bacteria onto a milk plate; if there is a zone of clearing around the growth then casein was degraded in the media.

In the gelatin deep, we inoculate gelatin deeps. If the gelatin liquifies even under cold temperatures, it indicates a positive reaction (that the bacteria can degrade the gelatin).

What do positive/negative reactions look like for the casein degradation test?

• Positive for casein: zone of clearing around growth

• Negative for casein: maybe growth but no zone of clearing

What do positive/negative reactions look like for the gelatin degradation test?

• Positive for gelatin: liquefied gelatin deeps

• Negative for gelatin: gelatin remains solid and intact.

What is a lipid? What are the two types?

fatty acids

• Triglycerides: glycerol molecule with 3 fatty acid chains

• Phospholipids: similar to triglycerides except that one of the three fatty acid chains is replaced by a non-fatty acid molecule that is linked by a phosphate group

How are lipids degraded?

by lipases

How can we evaluate if degradation occurs? How do the lipid and phospholipid tests differ?

• Spirit blue agar is used for lipid test: shows the release of fatty acids; medium is pH indicator and fatty acid release will change the pH. Dark blue outline will appear around growth showing the degradation of lipids by lipases.

• Egg yolk agar is used for phospholipid test: the agar will become opalescent when the phospholipids are broken down due to the accumulation of water-insoluble fatty acids.

What do positive/negative reactions look like for the spirit blue agar lipid test?

• Positive: dark blue outline around growth

• Negative: no change to agar

What do positive/negative reactions look like for the egg yolk agar phospholipid test?

• Positive: opalescent sheen around growth

• Negative: no change to agar

How is citrate used in bacterial metabolism?

Citric acid can be used as a carbon source for fermentation (anaerobic respiration)

How do we test if a bacteria uses citrate as a carbon source?

Simmons Citrate Test

What is the role of bromothymol blue in this media for the Simmons Citrate test?

It is a pH indicator. The media will change colors based on whether there is acidic or alkaline conditions.

What does a positive/negative result look like for the Simmons Citrate test? What does this tell us about the bacterial metabolism?

• Positive: blue color of slant indicating basic conditions; this means the bacteria uses citrate as a carbon source

• Negative: green color of slant indicating acidic conditions; this means the bacteria did not use citrate as a carbon source

What are enteric bacteria?

Bacteria that are typically found in the intestines of animals.

• Escherichia coli & Enterobacter aerogenes

What is fermentation?

The catabolic process in which sugars are partially degraded to produce ATP.

How is fermentation observed?

Using tests that take place in anaerobic conditions or tests that indicate acid or gas production.

What is respiration?

The complete oxidation of organic compounds to produce ATP.

What is a pH indicator and how does it work?

A pH indicator is used to detect and study acid production. Different media that contain pH indicators will change colors based on whether there is acidic or basic conditions.

How do we evaluate growth, acid production, and gas production for sugar fermentation?

Using Durham tubes in a broth with a pH indicator. 

• Growth = cloudy media

• Acid production = change in color of broth

• Gas production = bubbles in the Durham tube
  

homolactic vs heterolactic acid bacteria?

Homolactic: primarily excrete lactic acid

Heterolactic: excrete lactic acid AND acetic acid, ethanol, and glycerol

What types of bacteria do the Methyl-Red and Voges-Proskauer tests differentiate?

Enteric Bacteria (E. coli vs Enterobacter aerogenes)

What is the difference between mixed acid and butylene glycol fermentation?

• Mixed acid = produces large amounts of mixed acids, causes a large decrease in pH

• Butylene glycol = produces less acid. Produces large amounts of butanediol and ethanol, neither of which alters the pH of the media.

What do negative/positive results look like for the Methyl-Red test and what do they tell you about bacterial metabolism?

• Positive: Red color of broth; tells you the bacteria ferments glucose via mixed acid pathway

• Negative: Yellow color of broth; tells you the bacteria doesn’t ferment glucose via mixed acid pathway (mixed acids were not produced)

What do negative/positive results look like for the Voges-Proskauer test and what do they tell you about bacterial metabolism?

• Positive: Red color of broth; tells you that a bacteria ferments glucose through the butanediol pathway, producing acetoin

• Negative: Yellow/brown color of broth; tells you the bacteria cannot ferment glucose via the butanediol pathway to produce acetoin

What is catabolite repression?

The process by which glucose shuts down tryptophan use.

How does catabolite repression work?

Glucose inhibits indole production (breakdown of tryptophan produces indole). The cells get more energy from respiration with glucose than with tryptophan.

What do negative/positive results look like for the indole test and what do they tell you about bacterial metabolism?

Positive: dark red/violet color at the top of the broth

Negative: any color other than red at the top of the broth

The test tells you if the bacteria can use tryptophan as an energy source, thus producing indole.

What is tryptophan and how does it relate to indole?

Tryptophan is an amino acid that can be used as an energy source when glucose is not present. When a bacteria uses tryptophan, it produces indole.

How is sulfate used in bacterial metabolism?

Bacteria can reduce sulfate which produces H2S.

How can sulfate reduction be detected in the laboratory?

Using iron peptone deeps. The iron in the media will react with the H2S byproduct from sulfate reduction to produce visible black precipitate.

What do negative/positive results look like for H2S production test and what do they tell you about bacterial metabolism?

• Positive: any black precipitate in deep

• Negative: no black precipitate

The results tell you whether that bacteria reduces sulfate.

What is the role of peptone in the agar?

Peptone contains sulfur. If the bacteria can use the sulfur in the peptone as an energy source/reduces it, it will produce H2S as a byproduct. The H2S will then react with the iron in the media making a visible black precipitate

How does the litmus milk test work?

 It grows bacteria in a milk medium containing the litmus indicator. As the bacteria use the milk's nutrients, they cause visible changes that reveal their metabolic activity.

What are the possible outcomes of the litmus milk test?

• Acid: pink/red color

• Slight acid: light pink/red color

• Litmus reduction: white color caused by lack of oxygen

• Curd: change from fluid to solid

• Alkaline: dark blue/purple color

• Proteolysis: watery translucent fluid

 How does the oxidase test work?

The oxidase (cytochrome c) test uses a reagent that reacts with the cytochrome c oxidase enzyme. If the bacteria produce this enzyme, the reagent is oxidized and changes color.

What does the oxidase test tell you about bacterial metabolism?

 It tells you whether the bacteria uses cytochrome c in aerobic respiration to metabolize compounds like sugars for energy.

What do positive/ negative result look like for an oxidase test?

• Positive: dark blue/violet color

• Negative: no color change

How does the catalase test work?

The catalase test adds hydrogen peroxide (H₂O₂) to bacteria. If the bacteria produce the catalase enzyme, it breaks down the hydrogen peroxide into water and oxygen, causing bubbles to form.

What does the catalase test tell you about bacterial metabolism?

It tells you whether the bacteria produce catalase to break down toxic hydrogen peroxide, a byproduct of aerobic respiration.

What do positive/ negative result look like for the catalase test?

• Positive: bubbling

• Negative: no bubbling

What is the process for isolating an unknown organism?