Microbiology 2125 Occc Hovda Lab Final

Blood Agar Plates (BAP)

tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood cells

Oxidise test will show colonies on these plates. Which could be Neisseria species.

reagent: tetramethyl-p-phenylenediamine

Sulfur Indole Motility Media

H2S production (turns agar black)

tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile).

Mannitol Salt Agar

It's selective & differential & inhibits other MO's.

It's selective for Staphylococcus & differential for different species for Staphylococcus

Contains mannitol, 7.5% NaCl, and phenol red.

Staphylococcus aureus. A coagulase test is used to confirm this organism.

Coagulase test

This test is performed on Gram-positive, catalase positive species to identify the coagulase positive(clot) like Staphylococcus aureus.

This test differentiates Sa from other coagulase negative(clear) Sa (it's the SA in MRSA)

Simmon’s Citrate Agar

positive (blue agar)
negative. (remains green)

defined medium used to determine if an organism can use citrate as its sole carbon source

MacConkey agar

selective & differential. The selective ingredients are the bile salts and the dye, crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color

Escherichia coli

Enterobacteriaceae

* -NEG=cream= Lac- (Potentially pathogenic for salmonella Ex. Salmonella typhi causing typhoid fever)
* +POS=Pink= Lac+ (E. coli and other normal flora)

Urease test

This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease

pink is positive

* Streak aseptically w/ quadrant technique to isolate colonies

LAB MANUAL says determines the size of the smallest object that can be seen clearly under specified conditions.

The ability of a microscope to distinguish one point object from a nearby point object
RP= wavelength/ 2x Numerical Aperture

1. Primary stain: Stains cell
2. Mordant: Holds primary dye onto cell
3. Decolorizer: Removes some or all of the primary dye
4. Counter or Secondary stain: Recolors cells that lost stain through decolorization

determine cell wall structure & possible treatments

1. Transfer aseptically, dry, and heat fix

2. -crystal violet 60sec, rinse H20 (adheres to peptidoglycan)

3. Mordant-gram's iodine 60sec, rinse H20 (seals 1°)

4. Decolorizer- ETOH 15 sec, rinse H20 (Remove some/all of crystal violet dye)

5. 2°- Safranin 30 sec, rinse H20 (recolors) blot dry

Gram Positive=Purple
Gram Negative=Pink/red
Shapes: Cocci, rod
Arrangement: chains, clusters, pairs

* Transfer aseptically, dry, and heat fix
* Place over boiling H20 w/ filter paper
* 1°-Malachite green on paper for 3min
* Mordant- Steam during that 3min
* Decolorizer- H20 rinse
* 2°- Safranin 60 sec, rinse H20, blot dry

Heat (Based on growth in tube from None to thick+++)
-G- die off fastest due to thin cell wall Ex. E. coli
-G+ last longer due to thicker wall Ex.Staph
aureus
-Endospores thrive in heat Ex.Bac meg

UV light (Based on amount of growth on plate)
-G- die off fastest due to thin cell wall Ex. E. coli
-G+ last longer due to thicker wall Ex.Staph aureus
-Endospores help organism survive longer Ex.Bac meg
*Excessive UV kills all organisms

Based on how large the zone of growth/inhibition measured in mm

categorized as

S = susceptible, I = intermediate, R = resistant

uses a Kirby Bauer Plate

* Acid-fast= magenta (pinkish-purple)
* NOT Acid-fast=Blue

-Tuberculosis
-Myobacterium tuberculosis

S: allows only certain species of microorganisms grow, prevents growth of some organisms

D: causes diff. organisms to produce diff. results

-Differential based on its ability of hemolysis (lysis of RBC)

1. SHEEP BLOOD AGAR
2. α (alpha) hemolysis- "Partial" w/ green/brown color (Normal Flora)
3. β (beta) hemolysis- "Complete" Clear (Possibly pathogenic)
4. γ (gamma) hemolysis- No hemolysis (Normal flora or possibly Neisseria spp.)

-Strep pyogenes
-Strep throat

dimethyl-p-phenylenediamine hydrocloride

* -NEG=cream= Lac- (Potentially pathogenic for salmonella Ex. Salmonella typhi causing typhoid fever)
* +POS=Pink/purple/metallic green= Lac+ (E. coli and other normal flora)

* -NEG- Purple
* +POS- Yellow (acid) /Bubble (gas)

Neg-No bubbles
Pos-Bubbles

Sulfur (H2S)
-NEG- No Precipitate +POS- Black Precipitate
-NEG- Yellow Ring +POS- Red Ring Indole
-NEG- Visible stab line no growth (pretty clear)
+POS- Growth along stab line (cloudy) Motility
We use Kovac's as the reagent

* <30 colonies = TFTC "too few to count"
* >300 colonies = TMTC "too many to count"
* Always record in scientific notation

Nigrosin, Congo Red & India Ink

all carry a NEGATIVE charge

which will repell from Neg. charge bacteria

Flagella

major resource that covers all known prokaryotes based mostly on characteristics such as Gram stain and metabolic reactions. that is called phenetic.

2nd edition uses -phylogenetic:(evolutionary) history and relationships of the thousands of known species