Microbio Lab Midterm / Final

USF - Dr Chowdhury

How do you calculate total magnification?

Ocular lens magnification × objective lens magnification

What is the ocular lens magnification?

10X

What objective lens magnifications are used?

4X, 10X, 40X, and 100X

How many focus controls are on a microscope?

2: coarse focus and fine focus

What does the objective lens do?

Looks at the sample and picks up resolution and magnification information

What does the ocular lens do?

Projects the image to the user

When is immersion oil used?

100X objective

What is sterilization?

Destroys all microorganisms and spores

What is a common way to sterilize media?

Autoclaving

How does an autoclave work?

Uses high heat, pressure, and steam to kill microorganisms including spores

What is the purpose of the streak plate technique?

Separates a mixed culture into individual colonies

What is the expected result of a proper aseptic TSB transfer?

Both TSB tubes should be clear with no outside contamination

Gram-positive bacteria

Thick peptidoglycan cell wall; stain purple

Gram-negative bacteria

Thin peptidoglycan cell wall; stain pink

What test determines Gram-positive vs. Gram-negative?

Gram stain

What is a capsule?

A protective coating around some pathogenic bacteria

What is the purpose of a capsule?

Helps protect the pathogen from phagocytic activity of the host

Direct stain

Stains the specimen, not the background

Negative stain

Stains the background, not the specimen

Simple stain

Uses a single dye to stain a bacterial smear

Differential stain

Uses at least 3 chemical reagents on a smear

Gram stain step 1

Crystal violet = primary stain; stains all cells purple

Gram stain step 2

Iodine = mordant; forms a complex with crystal violet

Gram stain step 3

95% ethanol = decolorizer; removes crystal violet from some cells

Gram stain step 4

Safranin = counterstain; stains decolorized cells red/pink

Gram stain order

Crystal violet → iodine → 95% ethanol → safranin

Staphylococcus aureus

Gram-positive cocci; grape-like arrangement

Staphylococcus epidermidis

Gram-positive cocci; grape-like arrangement

Streptococcus pyogenes

Gram-positive cocci; chains or pairs

Streptococcus pneumoniae

Gram-positive cocci; chains or pairs

Escherichia coli

Gram-negative bacillus

Klebsiella pneumoniae

Gram-negative bacillus

Salmonella typhimurium

Gram-negative bacillus

Yersinia enterocolitica

Gram-negative bacillus

What does catalase positive mean?

The organism produces the enzyme catalase

What does catalase break down?

Hydrogen peroxide

What does the catalase reaction release?

Oxygen

Positive catalase test

Bubbles

Negative catalase test

No bubbles

Staphylococcus catalase result

Positive

Streptococcus catalase result

Negative

What tests distinguish Staphylococcus from Streptococcus?

Catalase test and MSA

Why does MSA distinguish Staph from Strep?

Staph tolerates high salt; Strep does not

What tests distinguish S. aureus from S. epidermidis?

BAP and MSA

S. aureus hemolysis

Beta

S. epidermidis hemolysis

Gamma/non-hemolytic

S. pyogenes hemolysis

Beta

S. pneumoniae hemolysis

Alpha

Gamma hemolysis

No RBC lysis; media remains unchanged

Alpha hemolysis

Partial/incomplete RBC lysis; green halo

Beta hemolysis

Complete RBC lysis; clear zone around colony

What sugar is in MSA?

Mannitol

What is the inhibitor in MSA?

7.5% NaCl

S. aureus on MSA

Grows and ferments mannitol

S. epidermidis on MSA

Grows but does NOT ferment mannitol

S. pyogenes on MSA

Does not grow; not salt tolerant

S. pneumoniae on MSA

Does not grow; not salt tolerant

S. aureus catalase

Positive

S. epidermidis catalase

Positive

S. pyogenes catalase

Negative

S. pneumoniae catalase

Negative

S. aureus salt tolerant

Yes

S. epidermidis salt tolerant

Yes

S. pyogenes salt tolerant

No

S. pneumoniae salt tolerant

No

S. aureus mannitol fermentation

Yes

S. epidermidis mannitol fermentation

No

S. pyogenes mannitol fermentation

Negative / Does not ferment mannitol

S. pneumoniae mannitol fermentation

No

BAP media type

Differential

TSI media type

Differential

MSA media type

Selective and differential

MacConkey (MAC) media type

Selective and differential

EMB media type

Selective and differential

SS media type

Selective and differential

Citrate slant media type

Selective and differential

Tests/media used to distinguish Gram-positive species

BAP, MSA, and catalase test

Tests/media used to distinguish Gram-negative species

MAC, EMB, SS, TSI, and citrate

What is a coliform?

An organism that ferments lactose

Which organisms are coliforms?

E. coli and K. pneumoniae

What is a paracolon?

An organism that does NOT ferment lactose

Which organisms are paracolons?

S. typhimurium and Y. enterocolitica

What sugars are in TSI?

Glucose, lactose, and sucrose

What sugar is in MacConkey agar?

Lactose

What sugar is in EMB agar?

Lactose

What sugar is in SS agar?

Lactose

What is the carbon source in citrate slant?

Sodium citrate

MAC contents

Lactose + bile salts

EMB contents

Lactose

SS contents

Lactose + bile salts + sodium thiosulfate + Fe++

TSI contents

Glucose + lactose + sucrose + sodium thiosulfate + Fe++

Citrate carbon source

Sodium citrate

E. coli on MAC

Bright pink colonies

E. coli on EMB

Metallic green sheen

E. coli on SS

Inhibited

S. typhimurium on MAC

Colorless colonies

S. typhimurium on EMB

Colorless colonies

S. typhimurium on SS

Colorless colonies with black centers/precipitate

K. pneumoniae on MAC

Bright pink colonies

K. pneumoniae on EMB

Dark/deep purple colonies