Quiz 3 Flashcards
What is extracellular degradation? How does it work? What classes of enzymes are
involved?
- Extracellular degradation - The breakdown of large molecules for necessary nutrients to enter the cell
- Extracellular enzymes make and release enzymes into the surrounding medium.
- Hydrolytic enzymes break up large molecules (polymers)
- Classes of enzymes - Esterases (Break fats and lipids), Glycosidases (Break up polysaccharides) , and Proteinases (Break apart proteins)
What is translocation ?
Movement of molecules across the membrane
What is a polysaccharide? How is it degraded?
- Carbohydrates: Compounds of carbon, hydrogen, and oxygen.
- hydrolytic enzymes are a way polysaccharides are degraded
Types of carbohydrates
- Monosaccharides, Oligosaccharides, and Polysaccharides
Structural Classification VS Nutrient Classification
- Structural- Cellulose, pectin, hyaluronic acids
- Nutrient - Glycogen and starch
What is starch made from?
A chain of glucose molecules. Formed through larger chains called polysaccharides
Types of starch
Amylose and Amylopectin
Amylase
Enzymes that break down starch into simple sugars such as maltose and glucose
What molecule is formed as starch is degraded?
Maltose and smaller amounts of glucose
How can we evaluate if degradation occurs (technique employed)? What is the purpose
of iodine in this experiment? (Degradation of starch)
- Use starch agar. Zone of hydrolysis results from the enzymatic breakdown of the insoluble starch
- Iodine reacts with starch to create a dark color on the plate. Hydrolysis of starch can’t be seen and iodine makes the starch visible
Amylose (gives a deep deep-blue color when it reacts with iodine).
Amylopectins (Gives a red to brown color when they react with iodine)
What do positive/negative reactions look like? (When looking at the degradation of polysaccharides starch)
Positive result: clear, colorless zone surrounds growth of colony or streak.
Negative result: Entire plate stains deep blue or red-brown with no clear, colorless zones.
What is a protein? How is it degraded? Which protein is in milk?
Protein - Long complex folded chains of amino acids that are linked through peptide bonds
- Proteins are degraded through Proteinases. Proteinases break down proteins through hydrolytic cleavage of the protein molecule outside the cell.
- Casein proteins are found in milk
Peptidases
Enzymes that degrade peptides
Rancidity
Food spoilage in which unpleasant odors and flavors develop due to the release of free fatty acids from triglycerides
Casein
- When casein binds to calcium it forms calcium caseinate (Water-insoluble)
- Casein being insoluble makes it a good indicator of a reaction
Gelatin
- Formed by boiling collagen in water or acid solution.
- It liquefies at temperatures above 28 Celsius
How can we evaluate if degradation occurs? How do the casein and gelatin tests differ?
Casein - To study the degradation of casein we use a milk plate. If bacteria can digest casein, clearing will appear around the growth
Gelatin - To study the degradation of gelatin we are using gelatin deeps. If bacteria is able to digest gelatin, the deep will no longer be a solid, it will liquify
- The difference between casein and gelatin tests is that casein is not temperature dependent like the gelatin is
What do positive/negative reactions look like? (Degradation of proteins Casein and Gelatin)
Casin
- Negative will look like nothing and have no clear zone (Doesn’t produce casein)
- Positive will have a clear area around the site (Produce proteases to degrade casein)
Gelatin
- Positive gelatin will liquify at the top. Gelatin was degraded
- Negative gelatin will remain solid at the top. Gelatin wasn’t graded
What is a lipid? How is it degraded?
- Lipids are organic compounds that are mostly insoluble in water. Composed of fats and oils
- Lipases are used to degrade lipids
Triglycerides
Glycerol with three fatty acid chains
Lipases
Enzymes that hydrolyze glycerol by separating the ester linkages between glycerol and the fatty acid molecules
Phospholipids
hydrophilic (polar head) and Hydrophobic (Non-polar) tail
How can we evaluate if degradation occurs? How do the lipid and phospholipid tests
differ?
Lipids - To study lipases we are using spirit blue agar. Spirit blue agar shows the release of fatty acids as the medium contains a pH indicator and the fatty acid release will change the pH
Phospholipases - Egg yolk agar is used. The agar will become opalescent when the fatty acids are broken down due to the accumulation of water-insoluble fatty acids
What do positive/negative reactions look like? (Phospolipids and Lipids)
For the lipid test, positive results will turn the agar a pale lavender or deep blue color. For the phospholipids test, positive results will be an oily surface film that appears opalescent.
How is citrate used in bacterial metabolism? How do we test if it is used?
- Citrate is used as a carbon source within metabolism.
- Simmons citrate agar is used through slants for differentiating gram-negative bacteria based on citrate utilization.
Aerogenes vs Coli
Aerogenes - Can use citric acid as a sole carbon source
Coli - Cannot use citric acid as a sole carbon source
What is the role of bromothymol blue in this media?
Bromothymol blue is used as a pH indicator. Green under acidic conditions and blue under alkaline (basic) conditions.
What does a positive/negative result look like? What does this tell us about the bacterial
metabolism? (Citrate experiment)
Positive - Blue
Negative - Green
- The aerogenes group can use citric acid as a sole carbon source but the coli group cannot. The aerogenes group can remove the citric acid and excrete alkaline waste products
What are enteric bacteria?
- Large group of gram-negative bacteria found within the intestines within animals and humans
What is fermentation?
the catabolic process in which
sugars are partially degraded to
produce ATP (energy)
How can we observe fermentation ?
For a positive reaction - Turn the phenol red indicator yellow (pH at or below 6.9). Gas bubbles occur
For negative reaction - Growth occurs, but media remains the same color or yellow only in the Durham tube and/or in the bottom of the culture tube. Growth occurs but no bubbles on top of the test tubes
What is respiration ?
complete oxidation of organic
compounds to produce ATP
(energy)
Anaerobic
occur in the absence of O2
Electron deficient
Lacks the electron transport chain
Inefficient
Less energetically favorable than respiration
Excretion
Releases fermentation end products
Homolactic Acid bacteria
Primarily excrete lactic acid (Lactobacillus and Streptococcus)
Heterolactic Acid Bacteria
Excrete acetic acid, ethanol, glycerol, along with lactic acid (Lactobacillus and Leuconostoc)
What is a pH indicator and how does it work?
- Used to detect and study acid production
- A pH indicator is a chemical compound added in small amounts to a solution so the pH of the solution can be determined visually by gradually changing color over a range of pH levels.
- pH indicators detect the presence of H+ and OH-.
How do we evaluate growth, acid production, and gas production?
Positive Reaction: Acid: turns phenol red indicator yellow in the entire tube (pH at or below 6.9). Gas bubbles.
Negative Reaction: Growth occurs, but media remains the same color or yellow only at the bottom of the culture tube. no bubbles on the top of the tubes.
What types of bacteria do these tests differentiate? (The Methyl Red and Voges-Proskauer Tests )
- Differentiation two major types of anaerobic enteric bacteria
What is the difference between mixed acid and butylene glycol fermentation?
- In the Mixed acid type equal amounts of CO2 and H2 are created.Large amounts of lactate are formed
- In butylene glycol fermentation it produces more than twice the amount of CO2. A little amount of lactate produced
What do negative/positive results look like and what do they tell you about bacterial
metabolism? (The Methyl Red and Voges-Proskauer Tests)
- In the MR test, Red indicates lots of acid via mixed acid fermentation which drives down the pH. Yellow indicates that there is no acid production
- In the VP test, Red indicates acetoin can be produced via butylene glycol fermentation. Copper indicates that the organism cannot produce acetoin
What is catabolite repression? How does it work?
- Catabolite repression is where sugar shuts down tryptophan use
- Glucose inhibits indole production. Cells get more energy from respiration with glucose than respiration with tryptophan
What do negative/positive results look like and what do they tell you about bacterial (indole Production from the Amino Acid Tryptophan and Catabolite Repression)
pink layer indicates the presence of indole, the absence of color means that it was not produced
What is tryptophan and how does it relate to indole?
- Tryptophan is an essential α-amino acid that is used in the biosynthesis of proteins and cannot be produced by the body.
- Tryptophan is translocated from outside of the cell inside and broken down into indole and pyruvate. Indole is excreted and pyruvate oxidized for energy (aerobic bacteria)
Durham Tubes
- Allows us to study gas production
- Inverted vials place broth filed test tube prior to sterilization
- Gas produced during fermentation is trapped and produced a bubble
How is sulfate used in bacterial metabolism? How can this be detected in the laboratory?
- Sulfate can serve as an electron acceptor in anaerobic respiration and can also be reduced for the formation of organic compounds.
Hydrogen Sulfide (H2S)
- Gas that is water soluble
- The product of sulfate used in bacteria. Smells like rotten eggs and when bonded to iron, turns to a black substance
What do negative/positive results look like and what do they tell you about bacterial
metabolism?
When H2S gas reacts with ferrous ions (iron, Fe+), it forms a ferrous sulfide, which is a black precipitate. The black precipitate is only present in iron-containing media in which growing microbes produce hydrogen sulfide.
What is the role of peptone in the agar?
It provides nutrients like carbon and nitrogen to bacteria
How does the litmus milk test work? What are the possible outcomes?
- Litmus is a substance obtained from lichens, as well as a pH indicator. In the litmus reduction, a change in color of litmus from light blue/gray to white (total lack of color), is caused by a lack of oxygen.
- Possible outcomes
- No change (Color remains light blue-purple).
- Acid ( Change in color of litmus).
- Litmus reduction ( Change of color from light blue-purple to white)
- Curd (Change in milk from fluid to solid)
- Proteolysis - Results in bluish brown to amber colored fluid
What do these outcomes of the litmus tell you about bacterial metabolism?
- Change in pH - Due to excretion of acid or basic fermentation products. Red color - Acidic conditions. Blue/Purple color - Basic conditions
- Change in O/R - Potential from oxygen depletion
- Curdling - Large quantities of excreted acid
- Proteolysis - Resulting from the action of excreted extracellular enzymes.
How does the oxidase test work? What does it tell you about bacterial metabolism?
- This test can differentiate enteric bacteria which are negative for this test.
- Test for Cytochrome C oxidase (Used in aerobic respiration for metabolizing compounds like sugars for energy)
How does the catalase test work? What does it tell you about bacterial metabolism?
- Identifies bacteria that produce the enzyme catalase
-Positive results show us that the bacteria produces catalase and can break down hydrogen peroxide; bubbles form. A negative result shows us that the bacteria doesn’t produce catalase and may not need to detoxify hydrogen peroxide ; no bubbles form
What do positive/ negative results look like for these tests? (est for Cytochrome C (Oxidase) and Catalase Activities)
- Oxidase
- Positive - Dark purple/Blue color
- Negative - No color change
- Catalase
-Positive - Rapid bubbling - Negative - No bubbling
What is the process for isolating an unknown organism? (you should know what primary
and secondary streaks are and why they are used)
Primary streaks - Used to separate individual bacteria from a mixed sample to form isolated colonies.
Secondary Streaks - Used to ensure the purity of the isolated colony by obtaining a pure culture.
What species are active and contribute to the different stages of yogurt production?
- The first stage favors the growth of heat-tolerant organisms. Streptococcus thermophilus. Milk contains lactose and the bacteria converts lactose to lactic acid through homolactic fermentation
The second stage of yogurt production is carried out at a moderate temperature. Lactobacillus Bulgaricus. The bacteria ferments the remaining lactose and gives yogurt its flavor and aroma.
What happens to lactose and casein during the fermentation process?
- Lactose gets hydrolyzed into glucose and galactose. Ferments into lactic acid lowers pH and contributes to sour flavor.
- Casein gets broken down into peptides and amino acids by proteolytic enzymes. Metabolized into compounds that contribute to flavor aroma, and texture.
How is this experiment linked to the litmus milk test ?
Both deal with lactose fermentation. In both yogurt production and the litmus milk test, lactose fermentation by lactic acid bacteria produces lactic acid.
