Microbio Lab Quiz 5

Alexander Fleming

- Father of antibiotics

- Discovered penicillin on accident

Paul Ehlrich

- "Magic Bullet" Hypothesis

- Chemicals can be designed to bind to and kill specific microbes or tumor cells without harming the host itself

Selman Waksman

- Coined the term "antibiotic"

- He discovered and developed the antibiotic streptomycin, produced by Streptomyces griseus

- Streptomycin was the cure for tuberculosis

Alma Whiffen

- Mycologist

- Discovered the antifungal agent cycloheximide

Antagonism

an interaction between organisms where one organism benefits at the expense of another

How do you detect antagonism?

Zone of inhibition

How and why is antibiotic resistance a concern?

- Occurs spontaneous mutations, horizontal gene transfer via conjugation

- Concern: The effect of the over use of antibiotics and antibiotic resistance in the environment

Antibiotic Characteristics

1. They are therapeutic agents

2. They are selectively toxic towards microorganisms

3. They kill or inhibit the growth of bacteria

4. These are small secondary metabolites

5. The term "antibiotics" is used for either antibiotics produced by microbes or for synthetic antibiotic

Targets of antibiotics

- Narrow-spectrum antibiotics: target a few types of bacteria

- Broad-spectrum antibiotics: target many types of bacteria.

- Metabolic enzymes, Cell wall synthesis, Ribosomes, Cell membrane, DNA synthesis

What are the steps of PCR?

1. Denaturation

2. Annealing

3. Extension

What happens in the denaturation step of PCR?

The DNA strands are separate

What happens in the annealing step of PCR?

Primers adhere to the DNA strands

What happens in the extension step of PCR?

Taq polymerase builds complementary strands

What is PCR?

A laboratory technique for rapidly amplifying millions to billions of copies of a specific segment of DNA

What is the purpose of PCR?

to amplify a specific desired fragment of DNA

What is a thermal cycler?

Used to amplify DNA sequence by increasing and decreasing temperature

What is Thermus aquaticus?

the organism that is thermophile whose enzymes are stable at high temperature

Taq polymerase

DNA polymerase which is heat stable; taken from Thermus aquaticus

What are primers?

small nucleotide sequence

What are the PCR components?

- Taq Polymerase

- dNTP's

- Polymerase Buffer

- Primers

- di water

What is the function of Taq polymerase?

polymerization of dNTP into a DNA strand

What is the function of dNTP's?

mix of nucleotides building blocks of new DNA strand

What is the function of Polymerase Buffer?

creates optimum activity of Taq polymerase

What is the function of Primers?

locate target DNA fragments

Tris-HCl

maintain pH

What are the Buffer compositions?

- Tris-HCl

- EDTA

- MgCl2

- KCl

- Triton

EDTA

chelating agent, keep DNA intact

MgCl2

cofactor for Taq polymerase

KCl

neutralizes charges in DNA template

Triton

stabilizes DNA

What are the PCR applications?

Bacterial identification, DNA fingerprinting, bioengineering

What is the principle and purpose of Gel electrophoresis?

- Analysis of nucleic acids (DNA /RNA) and proteins

- A procedure that separatesmolecules on the basis of their rate of movementthrough a gel under the influence of an electricalfield, their charge, shape and size

How does DNA migrate?

From cathode (positive) to anode (negative)

What dictates how far a fragment will travel?

size, the smallest fragments will travel the greater distance

What is agarose?

a linear polymer extracted from seaweed

What is the use of TBE (Tris/Borate/EDTA) and/or TAE (Tris/Acetate/EDTA buffer?

used for analyzing DNA products from PCRs by using agarose gel electrophoresis or polyacrylamide gel electrophoresis.

What is the function of Gel Green Stain and Staining dye?

binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel

Loading and Tracking buffer

- Bromophenol Blue (for color)

- Glycerol (for weight)

What is the purpose of Loading and Tracking buffer?

this allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells

What is the DNA ladder? (1 Kb-1.5Kb ladder)

- DNAs of known sizes

- Makes it easy to determine the sizes of unknown DNAs.

How do we "read" the DNA migration?

DNA ladder

What are plaques?

clear zones that develop on lawns of host cells

What is coliphage?

virus that infects certain bacteria called coliforms

What is bacteriophage?

A virus that infects bacteria

What are obligate intracellular parasites?

viruses that depend on the host cell for replication

What is titer?

Amount of bacteriophage present in a sample

What is the Coliphage used in lab?

T4 phage

What is the bacteria used in lab?

E. coli

What is the Lytic cycle?

- clear plaques

- involves the reproduction of viruses using a host cell to manufacture more viruses; the viruses then burst out of the cell

What is the lysogenic cycle?

- The lysogenic cycle is a viral replication cycle

that does not kill the host immediately.

- Cloudy plaques

What is the purpose of Nutrient agar?

supports the growth of a wide range of non-fibrous organisms

What supplement is used in the bacteriophage experiment?

Na +supplement

What is the calculation of titer?

# of plaques times 1 over the dilution factor

What is the unit of titer?

PFU/mL

What does PFU stand for?

plaque forming units

What is the principle of the starch test?

to test the presence of carbohydrates

What is the developer of the starch test?

Iodine

How do you read the starch test? (positive and negative)

- Positive: produce brown-black color

- Negative: no brown-black color produced

Which organisms are positive and negative for the starch hydrolysis?

- Positive: B. megaterium

- Negative: E. coli

Which primers were used for the 16S rRNA?

27F and 1492R

How did we test for the production of antibiotics by our isolates?

Zone of inhibition