MicroBio Lab Mediums

For lab exam #3

MR-VP Methyl Red Test — This medium tests for the fermentation of what sugar?

Glucose

MR-VP Methyl Red Test — What is the product of this fermentation?

Mixed Acid

MR-VP Methyl Red Test — What reagent is added to this medium?

Methyl Red

MR-VP Methyl Red Test —What is the result for organism A?

Negative; Organism does not ferment mixed acids from glucose

MR-VP Methyl Red Test —What is the result for organism B?

Positive; Organism does ferment mixed acids from glucose

What test is this?

MR-VP; Methyl Red Test or Voges Proskaeur Test

Lysine Decarboxylase Medium — What reagent is added to this medium?

Mineral Oil

Lysine Decarboxylase Medium —When is the reagent for this medium added?

After inoculation, Before incubation

Lysine Decarboxylase Medium — A positive result results in a ___ of the pH

increase/raising (basic environment)

Lysine Decarboxylase Medium — What is the result for organism A?

Negative; Organism does not decarboxylate from lysine; pH was not raised

Lysine Decarboxylase Medium — What is the result for organism B?

Positive; Organism does decarboxylate from lysine; pH was raised

What test is this?

Lysine or Ornithine Decarboxylase Medium

Bile Esculin Agar — What characteristic is tested for in this medium?

Hydrolysis of esculin to esculetin by esculinase

Bile Esculin Agar — Is this medium differential? Selective? Both?

Selective against non-strep organisms

Differential for esculinase production

Bile Esculin Agar — With which genus is this medium used?

Streptococcus / Enterococcus

Bile Esculin Agar — What is the result for organism A?

Negative; Organism does not hydrolyze esculin to esculetin; Enzyme esculinase is not present

Bile Esculin Agar — What is the result for organism B?

Positive; Organism does hydrolyze esculin to esculetin; Enzyme esculinase is present

What test is this?

Bile Esculin Agar

Simmon’s Citrate — What enzyme is tested for in this medium?

Citrase (citrate permease)

Simmon’s Citrate — What reagent is in the medium?

Bromothymol Blue

Simmon’s Citrate — What is the result for organism A?

Negative; Organism does not use citrate; Citrate permease is not present in the organism

Simmon’s Citrate — What is the result for organism B?

Positive; Organism does use citrate; Citrate permease is present in the organism

Nitrate Broth: A & B added — What enzyme is tested for in this medium?

Nitrate Reductase

Nitrate Broth: A & B added — What is the result for organism A?

Positive; Organism reduces NO3 to NO2

Nitrate Broth: A & B added — What are the TWO possible results for organism B?

1) Negative for nitrate reductase and denitrification

2) Negative for nitrate reductase; Positive for denitrification (NO3 to N2)

Nitrate Broth: A & B added — What is added to differentiate the results for organism B?

Reagent C - Zinc Dust

Nitrate Broth — reagent A + reagent B = RED

Positive for NO3 reduced to NO2 (Nitrate Reductase present)

Denitrification not present

(+NO2, -N2)

Nitrate Broth — reagent A + reagent B = COLORLESS

Negative for NO3 reduced to NO2 (Nitrate Reductase not present)

Denitrification not present

(-NO2, -N2)

Nitrate Broth — reagent A + reagent B + reagent C = RED

Negative for NO3 reduced to NO2 (Nitrate Reductase not present; NO3 not reduced)

(-NO2)

Nitrate Broth — reagent A + reagent B + reagent C = COLORLESS

Positive for Denitrification of NO3 to N2

(NO3 not present)

(-NO2, +N2)

MSA — With which genus is this medium used?

Staphylococcus

MSA — What is the selective ingredient in this medium?

7.5% NaCl / Salt

MSA — What is the differential ingredient in this medium?

Phenol Red

MSA — What are the results for this organism?

• Staphylococcus organism

• Salt tolerant (halotolerant)

• Mannitol Fermenter

MSA — Which species of Staphylococcus could this be?

S. aureus or S. saprophyticus

What does phenol red do in MSA and CDT?

Causes a pH sensitive color change of red to yellow for differentiation of mannitol fermentation / acid production

Differential characteristics of S. aureus

• Mannitol Fermentation = Positive (+)

• Novobiocin Susceptibility = Sensitive (S)

• Coagulase Production = Positive (+)

• alpha-toxin Production = Positive (+)

• Pigment on Solid Media = Negative (-)

Differential characteristics of S. epidermis

• Mannitol Fermentation = Negative (-)

• Novobiocin Susceptibility = Sensitive (S)

• Coagulase Production = Negative (-)

• alpha-toxin Production = Weak Positive (+)

• Pigment on Solid Media = Negative (-)

Differential characteristics of S.saprophyticus

• Mannitol Fermentation = Variable (±)

• Novobiocin Susceptibility = Resistant (R)

• Coagulase Production = Negative (-)

• alpha-toxin Production = Negative (-)

• Pigment on Solid Media = Negative (-)

Differential characteristics of S.xylosus

• Mannitol Fermentation = Positive (+)

• Novobiocin Susceptibility = Resistant (R)

• Coagulase Production = Negative (-)

• alpha-toxin Production = Weak Positive (+)

• Pigment on Solid Media = Positive (+)

Rabbit Plasma — With which genus is this medium used?

Staphylococcus

Rabbit Plasma — For what enzyme does this medium test?

Coagulase

Rabbit Plasma — What are the results for this organism?

Positive for coagulation of blood plasma proteins; Coagulase is produced and present in organism

Rabbit Plasma — Which species of Staphylococcus could this be?

S. aureus

Blood Agar: Staphylococcus Identification — For which staphylococcal capability does this inoculation test?

Alpha toxin production

Blood Agar: Staphylococcus Identification — What type of hemolysis does this toxin produce?

Beta hemolysis of red blood cells

Blood Agar: Staphylococcus Identification — What are the results for this organism?

Negative; Organism does not produce alpha toxins to hemolyze beta red blood cells

Blood Agar: Staphylococcus Identification — Which species of Staphylococcus could this be?

S. epidermis, S. saprophyticus, or S. xylosus

What does a positive result on Blood Agar look like and indicate?

Look: Prominent yellow halo surrounding the cells

Indicates: Alpha toxin production was present to hemolyze beta red blood cells

What does a negative result on Blood Agar look like and indicate?

Look: No yellow halo surrounding cells

Indicates: Alpha toxin production is not present to hemolyze beta red blood cells

Blood Agar: Streptococcus Identification — For which capability does this inoculation test?

Hemolysis of red blood cells

Blood Agar: Streptococcus Identification — What is the differential ingredient in this medium?

Red Blood Cells (RBCs)

Blood Agar: Streptococcus Identification — Which hemolysis results from the production of hydrogen peroxide (H2O2)?

Alpha Hemolysis

Blood Agar: Streptococcus Identification — Which hemolysis results from teh production of streptolysin S or O?

Beta Hemolysis

Blood Agar: Streptococcus Identification — What result is pictured here?

Gamma Hemolysis

In Streptococcal Blood Agar testing, what does hemolysis refer to in the medium?

Hemolysis refers to the color of the red blood cells in the medium adjacent to the bacterial growth, not the colonies themselves

Is Streptococcal Blood Agar differential or selective?

Differential

How is Streptococcal Blood Agar Differential?

Differentiates bacteria based on their ability to damage RBCs

What does a dark halo on Streptococcal Blood Agar represent

Alpha — Oxidation of RBCs

What does a light/yellow halo on Streptococcal Blood Agar represent

Beta — Hemolysis of RBCs

What does no halo on Streptococcal Blood Agar represent?

Gamma — RBCs are unaffected

Blood Agar: CAMP Factor Production — For which genus is CAMP capability important?

Streptococcus/Enterococcus

Blood Agar: CAMP Factor Production — Which organisms are CAMP positive?

Weakly beta hemolytic streptococci

Blood Agar: CAMP Factor Production — Which organism is used to test for CAMP production?

Staphylococcus aureus

Blood Agar: CAMP Factor Production — What result is pictured here?

Positive; Organism produces CAMP factor by increased beta hemolysis of red blood cells

Stain used to determine cell shape

Gram Staining

Stain used to determine cell arrangement

Gram Staining

Stain used to determine Gram Reaction

Gram Staining

Medium used to determine motility

Motility Medium & SIM

Medium used to determine Oxygen Requirement

FTM - Fluid Thioglycollate Medium

Redox Indicator in FTM

Resazurin

Reducing Agent in FTM

Thioglycollate

O2 Stabilizer in FTM

Agar

What tests determine Respiration-Related Characteristics

• Cytochrome Oxidase

• Hydrogen Peroxide Test

• Nitrate Broth

Positive Result of Cytochrome Oxidase/Oxidase Test

Purple/blue color change of oxistrip

Negative Result of Cytochrome Oxidase/Oxidase Test

No color change of oxistrip

Cytochrome Oxidase test determines what?

Oxidase Production

• Production of membrane bound electron carrier of ETC cytochrome oxidase

Medium used in Oxidase Production

Agar

Reagent(s) used in Oxidase Production

Oxistrips

Enzyme used in Oxidase Production

Cytochrome Oxidase

Negative Result of Hydrogen Peroxide/Catalase Test

no bubbles

Positive Result of Hydrogen Peroxide/Catalase Test

bubbles

Hydrogen Peroxide Test determines what?

Catalase Production

• conversion of H2O2 to O2 & H2O by catalase

Medium used in Catalase Production

Agar

Reagent(s) used in Catalase Production

Hydrogen Peroxide

Enzyme used in Catalase Production

Catalase

Nitrate Broth Test determines what

Nitrate Reduction & Denitrification

• Conversion of NO3 to NO2 by nitrate reductase

What medium is used in Nitrate Reduction?

Nitrate Broth

What substrate is used in Nitrate Reduction

Nitrate

What Reagent(s) is used in Nitrate Reduction

reagent A and reagent B

What enzyme is used in Nitrate Reduction

Nitrate Reductase

What medium is used in Denitrification

Nitrate Broth

What substrate is used in Denitrification

Nitrate

What reagent is used in Denitrification

reagent C - Zinc Dust

What tests determine Protein Catabolism-Related Characteristics

• SIM

  • Hydrogen Peroxide

  • Indole Production

  • Motility

• Gelatin Deep

• Urea Broth

• Tryptone Broth

• Skim Milk Agar

• Phenylalanine Afar Slant

• Decarboxylase Media

  • Lysine

  • Ornithine

Positive Result of SIM-H2S Test

Black precipitate

Negative Result of SIM-H2S Test

No black precipitate

SIM— H2S Test determines what

H2S production

• Hydrogen Sulfide Production: Removal of sulfur group from amino acids (cysteine) by various amino acids desulfurases

What medium is used to determine H2S Production

SIM

What substrate is used to determine H2S Production

Sulfur-containing amino acids