Gram Staining and Microbial Testing Review

Flashcards to review key concepts related to Gram staining, microbial testing, and biochemical responses.

What is the purpose of Crystal Violet in the Gram Stain? 

A primary stain used to color microbes the color purple. Stains gram positive bacteria purple after complete Gram’s stain has been performed.

What is the purpose of Gram's Iodine in the Gram Stain?

Gram’s iodine acts a mordant locking in crystal violet

What is the purpose of decolorizer/ethanol in the Gram Stain?

The decolorizer removes the primary stain from gram negative bacteria(thin peptidoglycan)

What is the purpose of safranin in the Gram Stain? 

Safranin counter stains the Gram negative bacteria; stains cells pink 

What color are Gram positive cells after the Gram stain?

Purple

What color are Gram negative cells after the Gram stain?

Pink

What is a mordant?

A substance that fixes/sets a dye onto a specimen. In Gram stains: Gram’s Iodine binds to primary stain setting it into the peptidoglycan layer of gram positive bacteria

What color are Spores after the endospore stain? 

Green

What color are vegetative cells after the endospore stain? 

Pink

What is the purpose of Malachite Green in the Endospore stain? 

Used as a primary stain for staining endospores

Why did you need to steam the cells for the endospore stain? 

Steam and heat acts as a mordant for malachite green, forcing it into the endospore.

What color are Acid Fast (+) bacteria after Acid Fast staining? 

Pink

What color are Acid Fast (-) bacteria after Acid Fast staining? 

Blue

What is peptidoglycan made of?

NAG + NAM sugars cross linked by peptides. 

What is the oxidase test testing for? What indicates a positive result? What indicates a negative result? 

Useful for identifying bacteria, including providing clues about oxygen requirements. Positive result is purple, and no color change is negative.

What is the catalase test testing for? What indicates a positive result? What indicates a negative result? 

Tests for the presence of enzyme catalase

Bubbles - positive

No bubbles - negative

What is the MR-VP test testing for? 

The methyl red – Voges Proskauer (MR-VP) test detects the ability of an organism to perform fermentation via two different pathways

What does a positive result for MR look like? What does it indicate?

Positive is pink/red. Indicates mixed acid fermentation of glucose.

What does a positive result for VP look like? What does it indicate?

Positive is pink/red. Ferments glucose 

What does growth on Mannitol Salt Agar indicate? 

Halophilic bacteria; tolerates salt.

What changes color on Mannitol Salt if the bacteria can ferment Mannitol?

Phenol red and the plate change color

What changes color on Eosin Methylene Blue agar if the bacteria can ferment lactose? What is the color?

The bacteria colonies changes color, purpleish pink, black, metallic green 

How is Eosin Methylene Blue selective? What does it select for?

Selective because it inhibits the growth of Gram positive bacteria. Selects for Gram negative bacteria.

What is a Selective Plate?

Allows certain types of microbes to grow while inhibiting others

What is a Differential Plate?

Stain different types of cells or structures differently from one another

What does TSI stand for? 

Triple sugar Iron 

What does a yellow base in the TSI indicate?

Indicates glucose was fermented

What does a red base in the TSI indicate?

The base of TSI starts out red, if the base is still red this indicates that the bacteria did not ferment glucose

What does a yellow slant in the TSI indicate?

Indicates lactose and/or sucrose was fermented

What does a red slant in the TSI indicate?

Indicates bacteria did not ferment lactose and/or sucrose

What does the thioglycolate test show?

Shows bacteria’s ability to grow or not in the presence of oxygen.

Results will show either: anaerobe/aerobe/microaerophilic/facultative anaerobe, aerotolerant aerobe

What is Phenol Red?

pH indicator dye that shows alkaline or acid

What are the three colors of Phenol Red and what does each indicate?

Yellow - acidic

Orange/Red - neutral

Pink - alkaline

What is the citrate test testing for? What indicates a positive result? What indicates a negative result?

Using the pH indicator dye bromothymol blue, Tests if bacteria can use citrate as a sole carbon source. Blue (alkaline) indicates a positive result, Green (neutral) and yellow (acidic) indicates a negative result

What is the Urease test testing for? What indicates a positive result? What indicates a negative result?

Using the pH indicator dye phenol red, tests for the presence of the enzyme urease which breaks down Urea. Pink (alkaline) indicates a positive result for urease and orange (acidic) indicates a negative result

What does the Sulfur, Indole, Motility (SIM) test show?

Sulfur tests for the breakdown of AA cystine or thiosulfate to hydrogen sulfide (H2S). Indole tests for the breakdown of tryptophan into indole/pyruvate. If Indole is present, it will react with oxalic acid on the paper placed inside and turn the paper pink. SIM test has semisolid agar consistency which allows motile bacteria to migrate away from the stab line while nonmotile bacteria remain confined to the inoculation site. The agar solution will be either clear or fuzzy/cloudy.

What does the Nitrate test show?

Tests for presence of nitrate reductase enzyme to reduce nitrate. If bacteria can break down nitrate to nitrite, or nitrate to nitrogen gas

What are the three colors of Bromothymol Blue and what does each indicate?

Yellow - Acidic

Green - neutral

Blue - alkaline

What is Bromothymol Blue?

pH indicator dye

Draw and label a Gram (+)cell wall.

Draw and label a Gram (-) cell wall.