Microbiology lab quiz #3, Dr. Collins

What are the steps for the pGLO plasmid experiment:

1) Cells are placed in transformation solution, which makes the cells competent.

2) Add pGLO plasmid, which will stick to the cells.

3) Heat shock, which allows DNA to get into transient pores.

4) Add Lb to help shrink the pores on the cell and repair them.

5) Plate cells on selective media, to see if transformation occurred.

What is the purpose of placing transformed bacteria onto selective media?

Any colonies that can grow on the selective media were successful in transforming their DNA with the selective marker.

Coagulase:

An enzyme produced by pathogenic species of Staphylococcus that causes clotting of blood plasma.

Fibrin clot:

Surrounds the bacteria and protects it from the host’s immune system.

What is a positive result for coagulase?

Clumping/clotting in the test tube

Catalase:

An enzyme that breaks down H2O2 (hydrogen peroxide) into O2 and H2O.

What is a positive result for catalase?

A positive result would be the release of oxygen and the appearance of bubbles on the plate.

What types of bacteria possess the catalase enzyme?

Obligate aerobes and facultative anaerobes

Oxidase:

This enzyme is directly involved in cellular respiration through the ETC. This enzyme oxidizes cytochrome C, which turns the substrate purple.

What color is a positive result for oxidase activity?

Purple is positive

What does the urease test determine?

It is used to determine whether enteric bacteria possess urease activity and can use the enzyme urease to degrade urea.

What does the degradation of urea produce?

NH3 (Ammonia), CO2, and H2O

Ammonia production makes the test media alkaline or acidic?

alkaline

What color is a positive result for urease activity?

pink

What color is a negative result for urease activity?

orange

What is bacterial transformation?

When a competent bacterial cell takes up naked DNA from the environment

What does a selective marker on DNA do?

It determines if the DNA has been transformed by the bacteria

What does the pGLO plasmid encode for?

the bla gene and GFP

bla gene:

Makes the bacterium resistant to the antibiotic ampicillin

What controls the expression of GFP in the pGLO plasmid?

The arabinose sugar

GFP is expressed in the _______ of arabinose

presence

What will colonies containing GFP look like?

They will fluoresce when illuminated by UV light

Bacteriophages:

Viruses that infect bacteria

Lytic viruses:

Lytic viruses use the bacterial cell to replicate

Lysogenic viruses:

Instead of immediately beginning the replication process leading to lysis, the virus enters into a stable state of existence with the host.

Lysis:

The release of new viral particles from the bacterial cell after replication

Plaques:

Clear zones on agar plates created by lysis

Antigens:

Components of the infectious agents that cause the immune response.

Antibodies:

The body produces millions of antibodies that bind to antigens and mark them for destruction by the cells of the immune system

What do ELISA assays do?

They detect for antibodies circulating in the bloodstream.

What does ELISA stand for?

Enzyme-linked immunosorbent assay

What does a positive ELISA assay look like?

It turns blue

Dental caries:

A bacterial infection that causes demineralization and destruction of the hard tissues

What do dental caries produce?

Dental caries produce acids via the fermentation of carbohydrates that adhere to the teeth’s surface

Snyder test:

Tests a person’s susceptibility to dental caries by testing the amount of acid produced by the action of lactobacilli on glucose

What does it mean when the snyder agar turns green?

It means that little to no fermentation by lactobacilli happened

What does it mean when the snyder agar turns yellow?

It means that glucose was fermented and the pH is 4.4 or lower

What are the components of snyder agar?

Glucose and bremocresol green

Coliforms:

Intestinal bacteria that ferment lactose and produce CO2

What does the presence of coliforms indicate?

The presence of coliforms in a water supply indicates fecal contamination

How can the number of coliforms in a water supply be quantified?

By the most probable number (MPN)

What 3 tests make up the MPN?

1) The presumptive.

2) The confirmed.

3) The completed.

The presumptive test:

Durham fermentation tubes containing lactose and an inverted durham tube are inoculated with water, then observed for lactose fermentation and CO2 production

What does a positive result for the presence of coliforms in the presumptive test look like?

Yellow broth and gas bubble in the durham tube

The completed test:

An EMB plate is inoculated with bacteria from the presumptive test.

What does a positive result for the presence of coliforms in the completed test look like?

Coliforms will form black or metallic green colonies on the EMB plates

Potable:

Means that a water source os safe to drink because it has less than one coliform per 100 mL.

The Colilert (Presence/Absence) test:

Is used to detect coliforms and E.coli in water

What are the main sources of carbon in Colilert?

The two nutrient indicators ONPG and MUG are the major carbon sources

ONPG

Can be metabolized by the coliform enzyme beta-galactosidase

MUG:

Can be metabolized by the E. coli enzyme beta-glucuronidase

Beta-galactosidase:

Breaks down lactose and ONPG. Coliforms use Beta-galactosidase to metabolize ONPG and turn the solution from colorless to yellow.

Beta-glucuronidase:

E. coli uses Beta-glucuronidase to metabolize MUG and create fluorescence.

What is a negative result for the presence of coliforms in water?

Solution is colorless

What is a positive result for the presence of coliforms in water?

Solution is yellow and will fluoresce under UV light.

What are some methods of treating water to make it fit for consumption?

• Chlorine

• Slow sand filtration

• Boiling

What is Heterotrophic Plate Count?

HPC is used to quantify the number of live, culturable heterotrophic organisms in a water sample.

What is the requirement for water to be considered potable?

Drinking water must have a concentration of coliforms less than or equal to 500 cfu/mL

What is the role of Horseradish Peroxidase antibody (HRP) in an ELISA?

If an individual possesses antibodies for a specific antigen, the HRP enzyme will oxidize the substrate and change it from colorless to blue.