Skin and Oral Microbiome

What is the main structural difference between Gram-positive and Gram-negative cell walls?

Peptidoglycan thickness

What percent peptidoglycan is in Gram-positive cells?

About 90%

What percent peptidoglycan is in Gram-negative cells?

About 20%

What extra structure do Gram-negative cells have?

Outer membrane with lipids

What does the alcohol decolorizer do to Gram-positive cells?

Dehydrates wall and closes pores

What does the alcohol decolorizer do to Gram-negative cells?

Dissolves outer membrane and opens pores

Why do Gram-positive cells retain stain?

Pores close and trap crystal violet-iodine complex

Why do Gram-negative cells lose stain?

Pores open and stain escapes

What is heat fixation?

Process that attaches bacteria to slide

What must be done before heat fixation?

Air dry smear

How is heat fixation performed?

Pass through flame or use slide warmer

What does heat fixation do to cells?

Kills cells and bonds proteins to glass

Why is heat fixation important?

Prevents cells from washing off

When is heat fixation required?

Before most staining procedures

Which stain should NOT use heat fixation?

Capsule stain

Why should capsule stain not be heat fixed?

It shrinks/distorts the capsule

What is the primary stain in Gram stain?

Crystal violet

What is the mordant in Gram stain?

Iodine

What is the function of iodine in Gram stain?

Forms complex with crystal violet to trap stain

What is the decolorizer in Gram stain?

Acetone-alcohol

Why is the decolorizer the most critical step?

Incorrect use causes false results

What happens to Gram-positive after decolorizer?

Stays purple

What happens to Gram-negative after decolorizer?

Becomes colorless

What is the counterstain in Gram stain?

Safranin

What color are Gram-positive cells at the end?

Purple

What color are Gram-negative cells at the end?

Pink

What happens if you over-decolorize?

Gram-positive appear Gram-negative

What happens if you under-decolorize?

Gram-negative appear Gram-positive

Why can Mycobacterium not be Gram stained well?

Waxy mycolic acid cell wall

What is the purpose of acid-fast stain?

Identify Mycobacterium

What is the primary stain in acid-fast stain?

Carbol fuchsin

What helps carbol fuchsin enter the cell?

Heat and detergent

What is the decolorizer in acid-fast stain?

Acid-alcohol

What does acid-alcohol remove?

Stain from non-acid-fast cells

What is the counterstain in acid-fast stain?

Methylene blue

What color are acid-fast bacteria?

Red/pink

What color are non-acid-fast bacteria?

Blue

Which genus is acid-fast?

Mycobacterium

What disease is caused by Mycobacterium tuberculosis?

Tuberculosis

What disease is caused by Mycobacterium leprae?

Leprosy

What is the purpose of endospore stain?

Detect endospores

What is the primary stain in endospore stain?

Malachite green

Why is heat used in endospore stain?

Drives stain into spore

What removes malachite green from non-spore cells?

Water

What is the counterstain in endospore stain?

Safranin

What color are endospores?

Green

What color are non-spore cells?

Pink/red

Which genus forms aerobic spores?

Bacillus

Where are Bacillus spores located?

Central

Which genus forms anaerobic spores?

Clostridium

Where are Clostridium spores located?

Terminal end

What disease is caused by Bacillus anthracis?

Anthrax

What disease is caused by Clostridium botulinum?

Botulism

What disease is caused by Clostridium tetani?

Tetanus

What is defined media?

Exact chemical composition known

What is complex media?

Contains digested material, composition not fully known

What is selective media?

Inhibits some organisms while allowing others to grow

What is differential media?

Shows visible differences between organisms

What is enriched media?

Contains extra nutrients for fastidious organisms

What type of media is PEA agar?

Selective only

What makes PEA selective?

Phenylethyl alcohol

What does phenylethyl alcohol do?

Inhibits Gram-negative bacteria

What does PEA select for?

Gram-positive bacteria

Which organism grows on PEA?

Staphylococcus aureus

Which organism is inhibited on PEA?

E. coli

What type of media is Mannitol Salt Agar?

Selective and differential

What makes MSA selective?

7.5% NaCl

What organisms grow on MSA?

Staphylococcus

What makes MSA differential?

Mannitol fermentation with phenol red indicator

What color indicates acid production on MSA?

Yellow

What color indicates no fermentation on MSA?

Red

What color indicates alkaline products on MSA?

Fuchsia/pink

Which organism ferments mannitol?

Staphylococcus aureus

Which organism does not ferment mannitol?

Staphylococcus epidermidis

What type of media is MacConkey agar?

Selective and differential

What makes MacConkey selective?

Crystal violet and bile salts

What does MacConkey select for?

Gram-negative bacteria

What makes MacConkey differential?

Lactose fermentation with neutral red

What color are strong lactose fermenters?

Deep pink/red

What color are non-fermenters?

Colorless

Which organism is strong fermenter?

E. coli

Which organism is moderate fermenter?

Enterobacter aerogenes

Which organism is non-fermenter?

Salmonella typhimurium

What type of media is EMB agar?

Selective and differential

What makes EMB selective?

Methylene blue dye inhibits Gram-positive

What makes EMB differential?

Lactose fermentation strength

What indicates strong fermentation on EMB?

Metallic green sheen

What indicates moderate fermentation on EMB?

Pink/mucoid colonies

What indicates no fermentation on EMB?

Colorless colonies

Which organism shows metallic green on EMB?

E. coli

Which organism shows pink colonies on EMB?

Enterobacter aerogenes

Which organism is colorless on EMB?

Salmonella typhimurium

What type of media is blood agar?

Enriched and differential

What makes blood agar enriched?

5% red blood cells

What does blood agar test for?

Hemolysis

What is beta hemolysis?

Complete destruction of RBCs (clear zone)

What is alpha hemolysis?

Partial breakdown (green/brown color)

What is gamma hemolysis?

No hemolysis (no change)

Which hemolysis is most clinically important?

Beta

Which organisms show beta hemolysis?

Streptococcus pyogenes and Enterococcus faecalis