The Enterotube II / EnteroPluri-Test is designed to identify ___ bacteria and other select oxidase-______, Gram-_____ bacteria.

1. Enteric

2. Negative

3. Negative

How many chambers are there? Name the mediums present in each one.

12 chambers

1. Glucose
2. Lysine
3. Ornithine
4. H₂S / Indole
5. Adonitol
6. Lactose
7. Arabinose
8. Sorbitol
9. VP
10. Dulcitol / PA
11. Urea
12. Citrate

E. coli Positives: Lys, Ind, Adon, Arab, Sorb

E. aerogenes Positives: Lys, Orn, Arab, Sorb, VP, Citrate

K. pneumoniae Positives: Lys, Arab, Sorb, VP, Urea, Citrate

What are the five digit codes?

E. coli: 04660

E. aerogenes: 06061

K. pneumoniae: 04063

For the Indole Test, what reagent is used? Positive result is what color?

Kovac's Reagent

Red

For the VP test, what reagents are used? Positive result is what color?

KOH Reagent & alpha-Naphthol Reagent

Red

Steps for performing the Enterotube Test?

1. Pick up a colony
2. Pull wire through
3. Reinsert wire
4. Break wire
5. Puncture air inlets
6. Add reagents AFTER incubation

ELISA is an acronym for

Enzyme Linked Immunosorbent Assay

It is used to ______

detect antigen or antibody in a sample

What are the steps to the ELISA test?

1. The purified antigen is added to the wells of the strip, wait 5 min. to allow it to absorb to the plastic wall of the wells.
2. Wash
3. Serum is added, wait 5 min. to allow antibodies to bind to antigen coating.
4. Wash
5. Secondary antibody is added, wait 5 min. to allow it to bind to the serum's antibodies.
6. Wash
7. Add enzyme
8. Observe result

Why is the secondary antibody needed?

It will be what gives us a positive or negative result.

What is the enzyme used and why is itneeded? What color is a positive result?

The enzyme is HRP and it will bind secondary antibody to indicate its presence. Blue indicates a positive result.

Where does the Kirby-Bauer method get its name?

Of the names who published it

What is the meaning of MIC?

Minimum inhibitory concentration

How is the antibiotic used in the Kirby-Bauer method?

Antibiotic-impregnated paper disks

What is the drug that kills bacteria?

Bactericidal

What is the drug that stops the bacteria from dividing?

Bacteriostatic

What do you read with McFarland turbitidy?

Cell Density

We diluted the cultures with sterile saline until ____ McFarland.

0.5

If a bacteria is susceptible to an antibiotic, the zone diameter would be ___.

What if it was resistant?

Large

Small

What bacteria did we use for the Kirby-Bauer lab?

E. coli and S. aureus

What antibiotics did we use for the Kirby-Bauer lab?

Chloramphenicol, Ciprofloxicin, Trimethoprim, Penicillin

What is bacterial transformation?

The process by which competent bacterial cells pick up DNA from the environment and make use of the genes it carries.

What are the structural and functional genetic units of prokaryotes?

Operons

Each operon minimally includes a ______ ______ and two or more ______ _______ coding for enzymes in the same metabolic pathway.

promoter site

structural genes

What is used as a vector in the pGLO lab?

a plasmid

Plasmids are _______

small, naturally occurring, circular DNA molecules that possess only a few genes.

What does the pGLO plasmid contain?

araC - allows RNA polymerase to bind to promoter

P_bad - arabinose promoter

GFP - green fluorescent protein, makes it glow

bla - antibiotic resistance gene, produces beta-lactamase which hydrolyzes ampicillin

ori - replication origin

Viruses that attack bacteria are called

bacteriophages

Once assembly is complete, the cell lyses and releases the phages, which then attack other bacterial cells. What is this process called?

Lytic Cycle

Lysis of bacterial cells growing in a lawn on an agar plate produce a clearing, what are these clearings called?

Plaques

Why are plaques important?

They allow us to calculate the phage concentration in a given sample.

What is the formula to calculate phage titer?

Phage titer = PFU / Original Sample Volume

Why is the soft agar, the tube that contains phage-host mix, important?

The consistency of it is sufficient enough to immobilize the bacteria while allowing the smaller bacteriophages to diffuse short distances and infect surrounding cells.

Which parasite causes an infection that involves a significant amount of surface area of the small intestine and can cause chronic diarrhea, dehydration, abdominal pain and other symptoms?

Giardia lamblia

What parasite causes malaria?

Plasmodium spp.

Review bacteria shapes & stains:

What does gram positive cocci look like?

What does gram positive bacilli look like?

What does gram negative spirochetes look like?

Be aware of difference between spirochetes, spirilla, and vibrio.

Purple circles

Purple rods / pill shaped

Pink coils

look up differences on google

Steps for Gram Staining

1. Fixation
2. Crystal Violet, 1 min
3. Iodine, 1 min
4. Decolorization
5. Counter Stain (Safranin), 1 min

Steps for a Wet Mount

1. Place a loopful of water on a clean glass slide.
2 Gently add bacteria to the drop by placing the loop over the drop for several seconds.
3 Gently lower a cover glass with your loop supporting one side over the drop of water. Avoid trapping air bubbles.
4 Observe under high-dry power

What are the chemicals that develop color as they oxidize?

Chromogenic reducing agents

What kind of culture do you have if you only have a single species?

pure culture

What kind of culture do you have if you have two or more species?

mixed culture

What is an important thing to do with the metal material when transferring microorganisms?

sterilize using bunsen burner

When do you use petroleum jelly?

Hanging Drop

What color should a positive and negative result for a McConkey agar look like?

Pink for Positive, Yellow for Negative

What enzyme transforms hydrogen peroxide into water and gaseous oxygen?

Catalase

In the Oxidase Reaction, what enzyme turns the color?

cytochrome c oxidase

What substance is designed to reduce the number of pathogens in living tissue?

Antiseptics