Microbiology Lab Final Notes

Fermentation Labs

MacConkey Agar “Coliform Lab” (Lab 5-3)

• Purpose: To differentiate coliforms from the Enterobacteriaceae family.

• Media Name: MacConkey Agar

• Type of Media: Selective and Differential

  • Selective: Bile Salts & Crystal Violet

    • Eliminates Gram-positive bacteria (G+)

  • Differential: Lactose & Neutral Red Dye

    • Color Indicator: Neutral Red Dye

      • Positive result (+): Pink/acid

      • Negative result (-): Clear/no acid

BGLB Test (LAB 8-13): Liquid Green Lab

• Purpose: To identify the presence of Coliforms in contaminated samples.

• Media Name: Brilliant Green, Lactose, Bile Broth (BGLB)

• Type of Media: Selective and Differential

• Media Ingredient:

  • Selective:

    • Oxgall Bile (from Ox): Kills all Gram-positive bacteria (G+)

  • Differential:

    • Lactose: Since all Coliforms ferment Lactose

    • Durham Tube: Traps Gas (end-product of fermentation)

    • Brilliant Green Dye: Not a pH indicator; for visual turbidity as bacterial growth

EMB (Eosin Methylene Blue) Agar: Neon Lab (Lab 4-6)

• Purpose: To differentiate/distinguish possible vs probable fecal Coliforms using EMB Agar.

• Type of Media: Selective and Differential

• Media Ingredient:

  • Selective: Eliminates Gram-positive bacteria (G+)

    • Eosin Dye

    • Methylene Blue Dye

  • Differential:

    • Lactose: All coliforms ferment; “probable” Coliform

    • Sucrose: Some coliforms ferment; “Possible Coliform”

    • Eosin, and Methylene Blue Dye: pH Indicators

      • Probable Coliform (+): Very acidic (Dark Purple or green)

      • Possible Coliform (+/-): Light Acid (pink)

      • Not a Coliform (-): Neutral (no color); Basic (no color)

O/F Glucose Test (Lab 5-2)

• Purpose: Identify organisms capable of oxidation (aerobic) or fermentation (anaerobic) or both using glucose.

• Media Name: O/F media

• Type of Media: Selective/Differential

• Media Ingredient:

  • Selective: Mineral Oil (against aerobes)

  • Differential:

    • Bromthymol Blue (pH indicator)

    • Glucose (fermentation and oxidation)

    • Agar (motility)

• Color Indicator: Bromthymol Blue

  • Acid: Yellow

  • Neutral: Green

  • Base: Blue

• Results:

  • Oiled Tube

    • Yellow: (+) Fermentation

    • Green: (-) Fermentation

    • Blue: (-) Fermentation; (+) peptone usage

  • Unoiled Tube

    • Yellow: (+) Oxidation

    • Green: (-) Oxidation

    • Blue: (-) Oxidation; (+) peptone usage

Phenol Red “Sunset Lab” (Lab 5-3)

• Purpose: To observe if microbes ferment carbohydrates: glucose, lactose, sucrose. Detect fermentation.

• Media Name: Phenol Red

• Type of Media: Differential

• Color Indicator: Phenol Red (pH)

  • Positive (+): Yellow (acid)

  • Weak Positive (Weak +): Orange (slight acidic)

  • Negative (-): Light red (neutral)

  • Negative (-): Bright pink (basic)

• Color Indicator: Durham Tube (gas)

  • Positive (+): Bubble (fermentation) or no bubble

• Media Ingredient:

  • Carbohydrates (Glucose, Lactose, Sucrose)

  • Casein Digest (peptone)

  • Phenol Red

Phenylalanine Deaminase (Lab 5-12)

• Purpose: Detect if which organisms produce Phenylalanine Deaminase.

• Type of Media: Differential (Phenylalanine Slants)

• Media Ingredient: Phenylalanine, Yeast extract, Agar, Slight salt.

• Reactant: Phenylalanine

• Enzyme: Phenylalanine Deaminase (removal of amino group from Phenylalanine).

• End-product: Phenylpyruvic Acid

• Color Indicator: Ferric Chloride (FeCl_3) (coats Phenylpyruvic acid)

  • Positive (+): Green

  • Negative (-): Yellow

DNase or Deoxyribonuclease (Lab 5-14): “Beyonce Halo”

• Purpose: Detect which organism produce DNase.

• Type of Media: Differential (DNA Tall)

• Media Ingredient: DNA, salt, Tryptose (peptone), Methyl Green dye, Agar.

• Reactant: DNA (polymer)

• Enzyme: DNase

  • Extra-cellular enzyme

  • Catalyzes the depolymerization of DNA

• End-product: Nucleotide (monomer)

• Color Indicator: Methyl Green Dye (coats DNA, but not nucleotide)

  • Positive (+): Halo

  • Negative (-): No Halo

• Deoxyribonuclease breaks covalent bonds from each nucleotide using {H_2O}

Bile Esculin Experiment (Lab 4-3): “Black Lab”

• Purpose: To detect organisms which are capable of utilizing the molecule esculin in the presence of bile. Detect Enterococcus.

• Media Name: Bile Esculin

• Type of Media: Differential/Selective

  • Selective: Bile & Sodium Azide

    • Bile: eliminates Gram-positive bacteria (G+), except enterococcus (+)

    • Sodium Azide: eliminate Gram-negative bacteria (G-)

  • Differential: Esculin & Ferric Ammonium Citrate

• Color Indicator: Ferric Ammonium Citrate (coats esculetin, but not esculin)

  • Positive (+): Dark Brown

  • Negative (-): no color

Esculin + Esculinase = esculetin + glucose. Addition of ferric ammonium citrate to Esculin results in a dark brown color.

Lysine Iron Agar (LIA) (Lab 5-22): “Lakers Lab”

• Purpose: To detect organisms that produce:

  • lysine deaminase

  • lysine decarboxylase

  • glucose fermentation

  • hydrogen sulfide gas.

• Media Name: LIA Slants

• Type of Media: Differential

• Color Indicator: Ferric Ammonium Citrate (Slant; nothing to do with pH)

  • Positive (+): Red

  • Negative (-): Violet (ammonia waste)

• Color Indicator: Ferric Ammonium Citrate (Butt; Sodium Thiosulfate)

  • Positive (+): Black (H_2S) Gas

  • Negative (-): none

• Color Indicator: Bromcresol Purple (Butt; pH indicator)

  • Positive (+): Dark Purple (Basic, Cadaverine)

  • Negative (-): Light Purple (Basic, Ammonia Waste)

  • Negative (-): Yellow (Acidic, Fermentation)
    Lysine Deaminase: Addition of Ferric Ammonium Citrate results in red color.
    Lysine Decarboxylase: Addition of Bromcresol Purple results in Purple color.

Thiosulfate iniosulfate reductase H_2S(g). Addition of Ferric Ammonium Citrate results in Black.

Urease Test (Lab 5-18): “Slow-Rapid Lab”

• Purpose: To detect organisms which are capable of producing the enzyme urease. Detect H. Pylori

• Media Name: Urea Plate

• Type of Media: Differential

• Color Indicator: Phenol Red (pH indicator)

  • Acidic: Yellow (-)

  • Neutral: Light Red (-)

  • Basic: Bright pink (+)
    Urea + Urease = 2NH3 + CO2(g). Addition of Phenol Red results in Pink.

Decarboxylase Test (LAB 5-11): Purple lab

• Purpose: Detect presence of “inducible” Lysine Decarboxylase enzyme

• Media Name: Lysine Broth

• Type of Media:

• Media Ingredient:

  • Lysine: Reactant

  • Bromcresol Purple: pH Indicator

  • Pyroxidal Phosphate (Vitamin B): co-enzyme; helps activate Lysine Decarboxylase

  • Peptone: Nutrients

  • 0.1% Glucose for slight fermentation

  • Mineral Oil for anaerobic conditions

• Reactant: Lysine

• Enzyme: Lysine Decarboxylase

• End-product: Cadaverine

Starch Hydrolysis (Lab 5-13)

• Purpose: Identify organisms capable of breaking down polysaccharide: Starch using Amylase and Oligo 1,6 Glucosidase exo-enzymes.

• Media Name: Starch Tall

• Type of Media: Differential

• Media Ingredient:

  • Starch = substrate (Differential)

  • Peptone

  • Agar

  • After Incubation: Iodine (Color indicator)

• Reactant: Linear Starch (Polysaccharide: Amylose)

• Enzyme: Amylase

• End-product: Individual Glucose

• Reactant: Branched Starch (Polysaccharide: Amylopectin)

• Enzyme: Oligo 1,6 Glucosidase

• End-product: Individual Glucose

• Color Indicator: Iodine (coats starch, but not glucose)

  • Brown (No halo): (-) Individual Glucose; (-) Exo-enzymes

  • Clear/Halo: (+) Individual Glucose; (+) Exo-enzymes

Amylose and Amylase.
Amylopectin and Oligo 1,6 Glucosidase.

Citrate Experiment (LAB 5-9)

• Purpose: To detect organisms capable of utilizing citrate only as carbon source.

• Media Name: Citrate Permease Slants

• Type of Media: Differential Media/Utilization Media (since nutrient is very limited, organisms are forced to utilize it otherwise death)

• Media Ingredient:

  • Citrate = organic molecule, no nitrogen

  • Ammonium Phosphate = Nitrogen Source for growth along with Carbon Source

  • Bromthymol Blue = pH Indicator

• Reactant: Citrate

• Process: Fermentation

• End-product: Pyruvate

• Pyruvate + CO2 + {H2O} + Salts = Sodium Bicarbonate (Base)

• Reactant: Ammonium Phosphate

• End-product: Ammonia (Base)

• Color Indicator: Bromthymol Blue (pH Indicator)

  • Positive (+) Citrate Permease: Blue (Base, due to Sodium Bicarb and Ammonia); Green + Growth (neutral, slow bicarb producer as long as growth is observed)

  • Negative (-) Citrate Permease: Orange (Acidic)

Catalase Experiment (Lab 5-6)

• Purpose: Identify organisms that produce catalase to detoxify Hydrogen Peroxide

• Media Name: Nutrient Agar Slants

• Type of Media: Differential

• Media Ingredient:

  • Peptone

  • After incubation: Hydrogen Peroxide (substrate, visual)

• Reactant: Hydrogen Peroxide

• Enzyme: Catalase

• End-product: Oxygen and Water

• Reactant: Superoxide

• Enzyme: SOD

• End-product: Hydrogen Peroxide and Oxygen

• Results:

  • Bubbles: (+) catalase

  • No Bubbles: (-) catalase

SOD + Hydrogen Peroxide = {H2O2} + {O_2}.
*Catalase: *Hydrogen Peroxide = Oxygen + Water.*

Gelatinase Test (5-17)

• Gelatin (collagen) + gelatinase = amino acids.

Nitrate Reduction Experiment (Lab 5-8)

• Purpose: Identify organisms that use nitrate instead of or in addition to oxygen in anaerobic respiration

• Media Name:

• Type of Media: Differential

• Media Ingredient:

  • A and B (color indicator)

    • A reacts with Nitrite and B colors it red during SSNR

  • Zinc

    • Denitrifies Nitrate to Nitrite producing red color

  • Nitrate (substrate to be converted to Nitrite or all the way down)

• Reactant: Nitrate

• Enzyme: Nitrate Reductase

• End-product: Nitrite and/or Nitric Oxide and Nitrous Oxide and Nitrogen gas

• Color Indicator: A and B

• Results:

  • SSNR (First Test)

    • Red = (+) Nitrate Reductase/SSNR

    • Clear = (+) / (-) Nitrate Reductase/SSNR

  • Denitrification (add zinc; Second Test)

    • Red = (-) Nitrate Reductase/Denitrification

    • Clear = (+) Nitrate Reductase/Denitrification

Nitrate + Nitrate Reductase = Nitrite.
Nitrate + Zinc = Nitrite.

MR-VP (Lab 5-4): (part of IMViC)

• Purpose: Determine if microbe is capable of mixed acid fermentation (MR) OR 2,3 butanediol fermentation (VP)

• Summary: Vouges Proskauer + Acetoin

• Media Ingredient:

  • Glucose

  • Buffered Peptone

• Color Indicator: Methyl Red (MR / pH Indicator)

  • Acid: Red (<4.4 pH) (+)

  • Neutral: Orange (4.4 - 6.2) (Retest/negative)

  • Base: Yellow (>6.2 pH) (-)

• Summary: Methyl Red + Four Mixed Acids = Red

• Color Indicator: Vouges Proskauer (not pH indicator)

  • Positive (+): Red (more neutral)

  • Negative (-): Copper-like color

• Summary: Vouges Proskauer + Acetoin = Red

MR: Glucose goes through Glycolysis to produce Pyruvate which goes through Enzymatic Reactions to create Mixed Acids: Acetic Acid, Lactic Acid, Succinic Acid, and Formic Acid.
VP: Glucose goes through Glycolysis to produce Pyruvate which goes through Enzymatic Reactions to create Acetoin and Alcohol (2,3 Butanediol).
Vouges-Proskauer + Naphthol + KOH + Acetoin = Cherry Red.

SIM (Lab 5-20)

• Purpose: Test for

  • Hydrogen Sulfide production;

  • Indole Production;

  • Motility

• Media Name: SIM agar

• Type of Media: Differential

• Media Ingredient:

  • Animal Digest / Cystein Amino Acid (sulfur source)

  • Thiosulfate (sulfur source)

  • Tryptophan/Casein (amino acid / indole source)

  • Low-Agar (motility)

• Reactant: Animal Digest (has cysteine amino acid which has sulfur)

• Enzyme: Cysteine Desulfurase

• End-product: {H_2S} gas (black)

• Reactant: Thiosulfate

• Enzyme: Thiosulfate Reductase

• End-product: {H_2S} gas (black)

• Reactant: Tryptophan

• Enzyme: Tryptophanase

• End-product: Indole (red)

• Color Indicator: Iron (for sulfur)

  • Positive (+): black

  • Negative (-): none

• Color Indicator: Kovac (for Indole)

  • Positive (+): red

  • Negative (-): none

Thiosulfate + Thiosulfate Reductase = {H2S} gas, which reacts with Iron from media to produce a Black color.
Animal Digest (Cysteine Amino Acid) + Cysteine Desulfurase = {H2S} gas, which reacts with Iron from media to produce a Black color.
Tryptophan + Tryptophanase = Indole, which reacts with Kovac to produce a Red/Pink color.

Triple Sugar Iron (Lab 5-21)

• Purpose: Detect fermenters of Glucose only or Glucose/Lactose/Sucrose; Detect thiosulfate reductase; Detect gas production.

• Media Name: TSI slant

• Type of Media: Differential

• Media Ingredient:

  • 3 Sugar:

    • 0.1% Glucose

    • 1% Lactose, 1% Sucrose

  • Phenol Red = detect acid

  • Peptone

  • Thiosulfate and Animal Digest (which has cysteine which has sulfur) = either digest

  • Iron = detect {H_2S}

• Reactant: Sugar

• Enzyme: enzyme for fermentation

• End-product: Acid, Alcohol, Gas

• Reactant: Thiosulfate/Cysteine AA

• Enzyme: thiosulfate reductase/cysteine desulfurase

• End-product: {H_2S} gas

• Color Indicator: Phenol Red

  • Acid: Yellow / (+) Ferm

  • Neutral: Red

  • Base: Deep Pink/Dark Red

• Color Indicator: Iron

  • Positive (+) {H_2S} gas = black spots

  • Negative (-) {H_2S} = no black spots

Blood Agar Test (LAB 5-25)

• Purpose: To differentiate species within the Streptococcus genus using blood agar & presumptively identify the causative agent of strep throat from throat sample

• Media Name: Sheep Blood Agar Plate

• Type of Media: Enriched Media, hemolysinsal Media

• Media Ingredient:

  • Differential and Enriched: Sheep Blood

• Differentiation:

  • Beta Hemolysis = complete lysis of RBC resulting in Halo

  • Alpha Hemolysis = partial bruising of cell membrane causing Greenish pigment around the RBC

  • Gamma Hemolysis = no reaction

• Summary: Just to check Streptococcus Genus and categorize based on hemolysins.

• Application: Identify the Streptococcus Genus which is responsible for producing hemolysins.
  *Purpose: Pathogen-Specific Lab

Hektoen Enteric Agar (Lab 4-7)

• Purpose: Identify Shigella and Salmonella from coliforms under the same Enterobacteriaceae family

• Media Name: Hektoen Enteric Agar (HEA)

• Type of Media: Selective and Differential

• Media Ingredient:

  • Selective:

    • Bile = eliminates gram (+)

  • Differential:

    • Lactose, Sucrose, Salicin (both do not ferment sugar)

    • Bromthymol Blue (pH indicator)

    • Thiosulfate

    • Ferric Ammonium Citrate (colors Hydrogen sulfide black)

• pH Indicator: Bromthymol Blue

  • Acid: Yellow

  • Neutral: Green

  • Base: Blue

• Color Indicator: Ferric Ammonium Citrate

  • Black spots = (+) Hydrogen Sulfide/Thiosulfate Reductase

  • No Black spots = (-) Hydrogen Sulfide/Thiosulfate Reductase

Mannitol Salt Agar (Lab 4-4)

• Purpose: Differentiate pathogenic Staphylococcus aureus from non-pathogenic, differentiate halophiles vs non- halophiles.

• Media Name: Nutrient Agar, MSA

• Type of Media:

  • Nutrient agar = General Purpose Media

  • MSA = Selective & Differential Media

• Media Ingredient:

  • MSA Selective:

    • 7.5% NaCl = promote growth of facultative halophile, eliminate non

  • Differential

    • Mannitol = sugar that detects Staphylococcus aureus

    • Phenol Red = pH indicator detects acid from fermentation

• Reactant: Mannitol

• End-product: sugar, acid, alcohol (we only detected acid)

• Color Indicator: Phenol Red

  • Acid = Yellow / (+) Mannitol

  • Neutral = Pink / (-) Mannitol

  • Base = Deep Pink / (-) Mannitol / (+) Peptone

Kirby-Bauer Test (Lab 7-3)

Disinfectant Lab (16-1)

Gram Stain (3-7)

• alkaline stain

• positive auxochrome

• attracts

• Crystal Violet

• Safranin

Endospore Stain

• Malachite Green

• Safranin

Simple Stain

• only one stain used

• alkaline stain

• positive auxochrome

• attracts

• Methylene Blue

• Safranin

• Crystal Violet

• Safranin

Negative Stain

• negative auxochrome

• acidic

• repels

• Nigrosin

• Eosin

• Congo Red