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
- Glucose
- Lysine
- Ornithine
- H2S / Indole
- Adonitol
- Lactose
- Arabinose
- Sorbitol
- VP
- Dulcitol / PA
- Urea
- 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?
- Pick up a colony
- Pull wire through
- Reinsert wire
- Break wire
- Puncture air inlets
- 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?
- 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.
- Wash
- Serum is added, wait 5 min. to allow antibodies to bind to antigen coating.
- Wash
- Secondary antibody is added, wait 5 min. to allow it to bind to the serum's antibodies.
- Wash
- Add enzyme
- 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
Pbad - 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
- Fixation
- Crystal Violet, 1 min
- Iodine, 1 min
- Decolorization
- 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