Final Lab Practical

Question 1

What is metabolism?

Answer 1

all of the biochemical reactions that occur in a cell to sustain life

Question 2

What are anabolic and catabolic reactions?

Answer 2

anabolic (build up) and catabolic (break down) reactions.

Question 3

List some examples of fermentation

Answer 3

■ Alcohol
■ Bread
■ cheese

Question 4

Compare and contrast aerobic respiration and fermentation

Answer 4

Aerobic Respiration
● 50-60% effective
● 36 ATP per glucose
● Many microbes
● Unique enzymes

Fermentation (anaerobic)
●Less than 50% effective
●2 ATP per glucose
●Some microbes

Question 5

What are Durham tubes and why are they used?

Answer 5

Very small glass vials placed upside down within the test tubes containing
fermentation media. The inverted Durham tubes are able to trap carbon
dioxide gas produced by the microbes, as an indicator of fermentation.

Question 6

What does a positive fermentation results look like? What does a negative result look like?

Answer 6

■ Positive-yellow

■ negative-red/pink

Question 7

What is starch?

Answer 7

Starch is a complex polysaccharide made up of amylose and

amylopectin.

Question 8

Describe the starch hydrolysis experiment

Answer 8

Tests for the ability of the microorganisms to break down starch into glucose, which is then used for metabolism. Uses starch plates that contain starch for the carbon source. After incubation, the plate is flooded with iodine which will give a clear halo if no starch is present.

Question 9

Why do we use gram’s iodine in starch hydrolysis experiment?

Answer 9

Gram’s iodine (as we used in the Gram stain procedure) is used to indicate the presence of starch by forming a starch iodine complex that turns blue-brown or black. Once starch is hydrolyzed by alpha-amylase, the iodine will not change color, as the starch is no longer available to form this complex. A clear area (halo) visible around the bacteria after incubation on an agar plate containing starch, indicates that alpha-amylase has been produced and starch was hydrolyzed.

Question 10

What 3 things does a SIM test tell us about the bacteria?

Answer 10

■ S = Sulfur: indicates production of H2S

■ I = Indole: indicate hydrolysis of tryptophan

■ M = Mobility: the semi-solid medium allows for mobility of motile
bacteria

Question 11

What does each letter in SIM stand for? Describe positive and negative results for each.

Answer 11

■ H2S production-black precipitate formed/ no formed

■ Motility-growth down tube/ no growth

■ Indole-red line at top/ no red line

Question 12

What type of bacteria would we use a SIM test for? Why?

Answer 12

Useful for identifying enteric bacteria. You would use a gram-negative bacteria
for a SIM test.

Question 13

What does each letter stand for in IMViC? Describe positive and negative results
for each.

Answer 13

■ Indole-red band at top/ no band

■ Methyl Red-red/yellow

■ Voges-Proskauer-red brown/remains yellow

■ Citrate-blue/green

Question 14

Describe the biochemical reactions for each part of the IMViC test

Answer 14

Bacteria that contain the enzyme tryptophanase hydrolyze tryptophan into indole, pyruvic acid and ammonia.

Question 15

What is casein?

Answer 15

Casein is a milk protein that is too large to enter the microbial cell.

Question 16

Why can only certain bacteria use this as a nutrient source? How is it broken down?

Answer 16

Many bacteria are able to use casein as a source of carbon and energy because they secrete proteolytic enzymes which will degrade casein. Peptide bonds are broken through hydrolysis, which separates the protein into individual amino acids.

Question 17

Describe the test and what a positive/negative Casein Hydrolysis result looks like

Answer 17

If a microbe can degrade casein, a clear zone will form around the
colonies, indicating that it has secreted proteases that have broken down the casein

Question 18

What are hydrogen peroxide and superoxide radicals? Why are they bad for the
cell?

Answer 18

Oxygen can be harmful to aerobic organisms when it forms hydrogen peroxide and superoxide radicals. Hydrogen peroxide can then form a hydroxyl radical. Radicals degrade other molecules, contributing to cell damage and death.

Question 19

What two enzymes are necessary to protect against superoxide radicals?

Answer 19

This first enzyme is superoxide dismutase. Another enzyme, catalase, then changes the hydrogen peroxide into water and free oxygen.

Question 20

Describe the biochemical reaction formula for superoxide dismutase and catalase.

Answer 20

Question 21

What bacteria does the Catalase test differentiate between?

Answer 21

This is a definitive test among the gram-positive organisms in this lab
because it differentiates Enterococcus from Staphylococcus

Question 22

Would bacteria with DNase be pathogenic or non-pathogenic? Why?

Answer 22

It would be pathegnic. This is because DNase is an enzyme that degrades DNA for use as a carbon source. As a secreted enzyme, it can increase the virulence of a bacterial pathogen because of the damage it influcts on the host’s cells.

Question 23

Explain the DNase test and how the DNA is complexed with a green dye

Answer 23

The DNase test medium contains a dissolved DNA, agar and special dye that complexes with the DNA to form a green color. Free dye not complexed with DNA is colorless. When a bacterium that can secrete DNAse grows on the plate, the DNA is degraded which releases the bound dye. The dye is no longet visible, creating a zone of clearing around the bacterial colony.

Question 24

Why do you use a sterile wooden stick instead of metal?

Answer 24

because the metal could give a false positive result due to surface oxidation during flame sterilization.

Question 25

What does a positive Cytochrome Oxidase Test look like? Negative test?

Answer 25

Question 26

How do the byproducts of urease test affect the pH?

Answer 26

The ammonia increases pH and can be used by the microbes as a nitrogen source to synthesize amino acids or other nitrogen containing compounds.

Question 27

What kinds of bacteria are urease positive?

Answer 27

Common microbes that possess urease include Helicobacter, Klebsiella and Campylobacter. The urease test is frequently used to distinguish Klebsiella from certain other enterobacterial genera

Question 28

What is denitrification?

Answer 28

Denitrification is the process by which oxidized nitrogen is reduced to molecular nitrogen.

Question 29

How do you finish the nitrate test after incubation?

Answer 29

add 10 drops of Nitrate A and 10 drops of Nitrate B to each tube.

Question 30

Why could the test be a false negative in Nitrate reduction test?

Answer 30

If there is no color change (negative test result) this could mean that nitrate was be reduced to N2. This is why zinc is added to reduce it down to nitrogen gass. If it turns red, this means that nitrate is still present (-) if it remains clear this means that there has been nitrogen gas formed (+)

Question 31

What do you add to determine whether the nitrate reduction test is a false negative?

Answer 31

Zinc

Question 32

Understand how antibiotics and disinfectants affect microbial growth

Answer 32

Although both disinfectants and antibiotics achieve the same outcome in destroying bacteria, they work in wholly different settings. Disinfectants are used in the external environment while antibiotics work inside the body where the effects are hard to control.

Question 33

What is the difference between microbicidal and microbiostatic? Bactericidal and bacteriostatic? Cytocidal and cytostatic?

Answer 33

Microbicidal or bactericidal means that these agents kill the microorganism, while microbiostatic or bacteriostatic chemicals only inhibit the growth of the microorganism. Another set of related terms are cytocidal and cytostatic (cyto = cells)

Question 34

What is a disinfectant? List some examples

Answer 34

Disinfectants are synthetic antimicrobial agents that kill or prevent the growth of microorganisms and should only be applied to inanimate objects. Some examples of disinfectants are phenol, chlorine, and alcohol.

Question 35

What is an antiseptic? List some examples

Answer 35

Antiseptics are synthetic antimicrobial agents that can be safely applied to tissues such as skin. Examples of antiseptics are alcohol and iodine.

Question 36

What are antibiotics?

Answer 36

Antibiotics are chemicals made from microorganisms that inhibit growth or kill other microorganisms.

Question 37

List some ways to reduce the risk of antibiotic resistance

Answer 37

One way to reduce the risk of antibiotic resistance is to reduce the use of antibiotics for non-bacterial infections.

Question 38

What was the first antibiotic discovered?

Answer 38

Penicillin

Question 39

What test do we use to determine antibiotic sensitivity? Describe this test

Answer 39

Kirby-Bauer method. The spread plate method is used to inoculate the plate with an even layer of bacteria. Disks with known concentrations of antibiotic are placed on the surface of the plate and then the plate is incubated. Following incubation, the bacteria growth will form visible lawn on the plate. A zone of clearing around a disk where the bacteria have not grown indicates sensitivity of the bacteria to that antibiotic. The size of the zone can be measured to quantify the extent of the sensitivity.

Question 40

What is the difference between broad-spectrum and narrow-spectrum antibiotics?

Answer 40

Broad-spectrum antibiotics can kill a wide range of bacteria including both gram-positive and gram-negative species. Narrow spectrum antibiotics can only kill a few select species of bacteria.

Question 41

What is a nosocomial infection?

Answer 41

A nosocomial infection is one that is acquired in a hospital.

Question 42

What is normal flora? What is transient flora?

Answer 42

Most animals and humans have normal flora present in the gut such as colonies of Escherichia coli. Microorganisms that are present for a short time without causing disease are transient flora.

Question 43

What is a pathogen? What are opportunistic pathogens?

Answer 43

A pathogen is a microorganism that normally causes disease. Opportunistic pathogens are microorganisms that usually do not cause disease; however, if the situation becomes right, the microorganism causes a disease.

Question 44

What is a plasmid and how do we use it in the laboratory setting?

Answer 44

circular DNA molecule capable of replicating independently of chromosomal DNA

Question 45

What is genetic exchange?

Answer 45

Genetic exchange occurs when DNA from one cell is transferred to a second cell. Transformation, conjugation and transduction are the three mechanisms that can result in genetic exchange.

Question 46

What are competent cells?

Answer 46

In order for cells to take up or absorb DNA, they must be competent.

Question 47

What is electroporation?

Answer 47

Some bacteria that are not naturally competent can be made competent through the use of chemical treatment or exposure to strong electrical fields by a technique called electroporation. Chemically competent cells are treated with calcium and magnesium ions and then stored at -80°C.

Question 48

What is a plasmid?

Answer 48

Plasmids are typically circular pieces of DNA that can replicate inside cells separately from the chromosome.

Question 49

Define transformation

Answer 49

Exogenous pure DNA is taken up by cells through the cell wall

Question 50

Define conjugation

Answer 50

○ Transfer of genetic material through direct cell to cell contact • Equivalent to bacterial“mating”

Question 51

Know how pili are used to transfer plasmids from one cell to another

Answer 51

Question 52

Why are we using selective media with antibiotics for this experiment?

Answer 52

One strain has resistance to ampicillin (amp+r) on the plasmid and the other has resistance to streptomycin (str+r) on the chromosome. Determine donors

Question 53

Know the different types of genetic mutations.

Answer 53

Point substitution –> when a single nucleotide is changed Nonsense mutation –> when mutation introduces a stop codon, then translation will terminate early and a shortened protein will be produced Silent mutation –> other substitution mutations where the change does not alter the codon composition Missense mutation –> If a codon is changed and a different amino acid is inserted during the expression of the gene which alters the function of the protein Frameshift mutation –> occur when there is an insertion or deletion of nucleotides that is not divisible by 3. This affects the reading of the entire gene, incorporating incorrect anino acids into the protein, which produces an abnormal protein product.

Question 54

Understand the process of the Ames test and how to interpret the results

Answer 54

Ames test: determines if a chemical is a mutagen. Results –> if there is a large number of colonies on the test plate in comparison to the control then chemical is said to be a mutagen.

Question 55

What is mutagenesis?

Answer 55

Mutagenesis is the process of creating mutations in the DNA of an organism

Question 56

Why and when can mutations occur?

Answer 56

Mutations can occu spontaneously as a result of external forces (muragens) or because of errors during DNA replication. Mutagenic changes in the DNA can be inherited when the cell divides.

Question 57

List some common mutagens

Answer 57

Some common mutagens are radiation and chemicals, such as acridine orange, ethidium bromide, formaldehyde and ethyl methane sulfonate.

Question 58

Codon

Answer 58

sequence of 3 nucleotides that form a unit of genetic gone

Question 59

Genotype

Answer 59

DNA sequence

Question 60

Phenotype

Answer 60

physical expression

Question 61

Nonsense mutation

Answer 61

when mutation introduces a stop codon, then translation | will terminate early and a shortened protein will be produced

Question 62

Missense mutation

Answer 62

If a codon is changed and a different amino acid is inserted | during the expression of the gene which alters the function of the protein

Question 63

Frameshift mutation

Answer 63

occur when there is an insertion or deletion of nucleotides that is not divisible by 3. This affects the reading of the entire gene, incorporating incorrect amino acids into the protein, which produces an abnormal protein product.

Question 64

Insertion mutation

Answer 64

a type of mutation that involves the addition of one or more nucleotides into a segment of DNA

Question 65

Deletion mutation

Answer 65

the type of mutation that involves the loss of one or more nucleotides from a segment of DNA

Question 66

Reversion

Answer 66

the process of changing a mutation back to the unmutated form

Question 67

Suppressors

Answer 67

a second-site revertant that is created by a complementary | mutation that occurs at a different sitw than the original mutation

Question 68

Genetic selection

Answer 68

the most common way to identify desirable mutants is to use a genetic selection. A selection could consist of a medium with specific additions that will only allow the mutant to grow.

Question 69

What mutation can cause a frameshift mutation?

Answer 69

Insertion or deletion mutation that is not divisible by three

Question 70

What mutations are typically NOT reversible?

Answer 70

Deletion mutations are not revertible anf some insertion mutations

Question 71

How can a second-site mutation result in reversion?

Answer 71

Most second-site reversion mutations restore the original phenotype by compensating for the effect of the original mutation.

Question 72

What is replica plating? How is it done?

Answer 72

If the researcher is looking for a mutant unable to use a carbohydrate, then a process known as replica plating is frequently used. Cells are plated through a dilution series and allowed to grow. Once the colonies are large enough to see, the plate is pressed onto a piece of velvet and then a fresh plate without the carbohydrate is pressed onto the same piece of velvet making a plate that is a replica of the first.

Question 73

What colonies are tested in a genetic screen? Is this a selection experiment or are all colonies allowed to grow prior to testing?

Answer 73

A genetic screen allows all of the cells to grow and develop into colonies. However there is either something included in the media or sprayed onto the colonies after they are grown that will allow the researcher to differentiate between the mutants and the wild type.

Question 74

What is the Ames test used for?

Answer 74

The Ames Test is used to test for mutagentic chemicals

Question 75

What bacteria is used for the Ames test? Why?

Answer 75

Nonpathogenic strains of Salmonella typhimurium, which have been intentionally mutated in genes encoding enzymes necessary for the biosynthesis of the amino acid histidine. These strains require the addition of histidine for growth and are thus histidine auxotrophs.

Question 76

What is the purpose of histidine?

Answer 76

The histidine permits the inoculated test organism to undergo a limited number of divisions, but is insufficient to permit normal growth. If the strain undergoes a reverse mutation (spontaneous or induced by the mutagen or a positive control material), the organism no longer requires histidine to grow and can produce a visible colony (revertant). Only mutations to the test organism in the region of the histidine gene will cause the test organism to undergo a reverse mutation to an organism, which then no longer requires histidine. Note that a small concentration of histidine is added to the media to allow for limited growth of the histidine auxotrophs during which time mutations can arise.