Microbiology Lecture Flashcards

Selective and Differential Media of Gram-Positive Bacteria

Blood Agar & Hemolysis

  • Hemolysis: Breakdown (lysis) of red blood cells (RBCs).
  • Caused by enzymes called hemolysins.
  • Blood Agar:
    • TSA-like medium that contains 5% sheep blood, giving it a red color.
    • Used to determine if bacteria can lyse RBCs (perform hemolysis).

Types of Hemolysis

  1. Beta (β) Hemolysis:
    • Complete hemolysis.
    • RBCs are fully lysed.
    • Transparent/clear zone observed around the colony.
  2. Alpha (α) Hemolysis:
    • Partial hemolysis.
    • RBCs are partially degraded.
    • Greenish or dark halo around colonies.
    • Medium appears darker, not fully cleared; bacterial cells damage RBCs converting hemoglobin to methemoglobin.
    • Color changes from red to greenish/brown due to methemoglobin.
  3. Gamma (γ) Hemolysis:
    • No hemolysis.
    • No RBC breakdown.
    • Medium remains unchanged red around colonies.
    • Considered a negative result.

Interpretation Flow

  1. Step 1: Is there any clearing around colonies?
    • No: Gamma (no hemolysis).
    • Yes: Proceed to step 2.
  2. Step 2: Can you see through the plate clearly?
    • Yes: Beta (complete hemolysis).
    • No: Alpha (partial hemolysis).

Blood Agar Plate Results Examples

  • Staphylococcus aureus: Beta Hemolytic
  • Staphylococcus epidermis: Alpha Hemolytic
  • Staphylococcus saprophyticus: Weakly Alpha Hemolytic
  • Micrococcus luteus: Beta Hemolytic
  • Streptococcus pyogenes: Beta Hemolytic
  • Streptococcus agalactiae: Beta Hemolytic
  • Streptococcus salivarius: Alpha Hemolytic
  • Enterococcus faecalis: Alpha Hemolytic
  • Branhamella catarrhalis: Gamma Hemolytic
  • Corynebacterium xerosis: Gamma Hemolytic

Mannitol Salt Agar (MSA)

  • Selective and Differential Media
  • Contains:
    • Mannitol (fermentable carbohydrate)
    • 5% Salt (NaCl)
    • Phenol red (pH indicator)
  • Selective: Some organisms can grow with high salt concentrations.
  • Differential: If growth occurs, assess mannitol fermentation:
    • Fermentation of mannitol produces acid end products.
    • Acid lowers the pH, causing the plate to change color.
      • Red = Neutral pH
      • Yellow = Acidic pH
  • If no growth occurs, no conclusions can be made about mannitol utilization.

Mannitol Salt Agar Results Examples

  • Staphylococcus aureus: Thick growth (+), yellow (mannitol fermented)
  • Staphylococcus epidermis: Small dots growth (+), Red (no fermentation)
  • Staphylococcus saprophyticus: Weakly Alpha Hemolytic
  • Micrococcus luteus: Thick growth (+), yellow (mannitol fermented)

DNAse Agar

  • Differential Media
  • Tests for the ability of an organism to degrade deoxyribonucleic acid (DNA) with the enzyme DNAse.
  • DNA \rightarrow Nucleotides + Phosphate + Deoxyribose
  • Contains methyl green bound to DNA.
  • If DNA is degraded, the dye has nothing to bind to and volatilizes.
    • (+) Result: Clear zone around the growth appears.
    • (-) Result: No clearing or no growth.
  • Few nutrients are provided; some organisms may not grow.
  • Inoculate with enough cells for a sufficient starting cell population (double inoculation).

DNAse Agar Results Examples

  • Staphylococcus aureus: Positive (clearing around the growth)
  • Staphylococcus epidermis: Negative (no clearing)
  • Staphylococcus saprophyticus: Negative (no clearing)
  • Micrococcus luteus: Negative (no clearing)

Salt Broth

  • Selective Media
  • Nutrient broth with 6.5% NaCl.
  • Some organisms will be able to grow with this much salt; others will not.
    • (+) Result: Turbidity (cloudiness) in the tube after suspending cells.
    • (-) Result: Broth remains clear even after flicking the tube.

Salt Broth Results Examples

  • Streptococcus pyogenes: positive for growth
  • Streptococcus agalactiae: positive for growth
  • Streptococcus salivarius: no growth
  • Enterococcus faecalis: positive for growth
  • Neisseria flava: positive for growth
  • Branhamella catarrhalis: no growth
  • Corynebacterium xerosis: no growth

Bile Esculin Slant

  • Selective and Differential Media
  • Contains:
    • Bile: Inhibits the growth of some organisms.
    • Ferric Citrate: Source of iron.
    • Esculin: A sugar alcohol that can be broken down by bacteria releasing sulfur.
      • Esculetin, specifically sulfur groups, binds to iron in the media to form FeS (Iron sulfide).
      • (+) Result: Black color found in the slant due to FeS.
      • (-) Result: Color of the slant remains yellowish-green.

Bile Esculin Slant Results Examples

  • Streptococcus pyogenes: No blackening (-)
  • Streptococcus agalactiae: No blackening (-)
  • Streptococcus salivarius: No blackening (-)
  • Enterococcus faecalis: Blackening (+)
  • Neisseria flava: Blackening (+)
  • Branhamella catarrhalis: No blackening (-)
  • Corynebacterium xerosis: No blackening (-)

Micrococcaceae

  • Incubate all tests at 37 ℃.
  • Includes the genera Staphylococcus and Micrococcus.
  • Gram-positive cocci in clusters or tetrads, always catalase-positive.
  • Catalase degrades toxic end products of using oxygen into something non-toxic.
  • H2O2 \rightarrow H2O + \frac{1}{2} O2 (g)
  • Organisms included in exercise:
    • Staphylococcus aureus
    • Staphylococcus epidermidis
    • Staphylococcus saprophyticus
    • Micrococcus luteus

Streptococcaceae

  • Incubate all tests at 37 ℃.
  • Includes the genera Streptococcus, Enterococcus, and Lactococcus.
  • Gram-positive cocci in chains or pairs, always catalase-negative.
  • Obligate fermenters; do not use oxygen or need to deal with toxic oxygen products.
  • Organisms included in this exercise:
    • Streptococcus pyogenes
    • Streptococcus agalactiae
    • Streptococcus salivarius
    • Enterococcus faecalis

Neisseriaceae and Corynebacterium

  • Incubate all tests at 37 ℃.
  • Neisseriaceae includes the genera Neisseria, Branhamella, and Moraxella.
    • Gram-negative diplococci, always catalase-positive.
    • H2O2 \rightarrow H2O + \frac{1}{2} O2 (g)
    • Catalase degrades toxic end products of using oxygen into something non-toxic.
    • Capnophiles
  • Corynebacterium are gram-positive short rods.
    • Catalase-positive.
    • Irregular branching of cells (Palisades structures, ‘Chinese letters’).
  • Organisms included in this exercise:
    • Neisseria flava
    • Branhamella catarrhalis
    • Corynebacterium xerosis

Metabolic and Biochemical Activities

Fermentation of Carbohydrates

  • Tests for utilization of a carbohydrate as a carbon and energy source.
  • Test Glucose, Lactose, Mannitol, or any other carbohydrates.
  • Media contains phenol red (pH indicator).
    • If a carbohydrate is fermented, the organism will release acid end products into the media.
    • As the media becomes acidic, the color of the pH indicator will change from red (neutral) to yellow (acidic)
  • Tubes contain a Durham tube (small, inverted tube for collection of non-water-soluble gases).
    • Only non-water-soluble gases collect here (O2, N2, H2). Water-soluble gases (CO2) will dissipate into the air.
  • Conducting the Fermentation of Carbohydrates Test
    • Inoculate by adding a single loop of culture to each tube
      • Pseudomonas aeruginosa
      • Escherichia coli
      • Enterobacter aerogenes
      • Serratia marcescens
    • Incubate 24-48 hours at 37℃
    • Observe for fermentation of the carbohydrate
      • Red = Negative for fermentation
      • Yellow = Positive for fermentation
    • Observe for production of a non-water-soluble gas (N2, H2, O2)

Carbohydrates Test Results examples

  • Lactose Fermentation
    • Pseudomonas aeruginosa: Negative
    • Escherichia coli: Positive
    • Enterobacter aerogenes: Positive
    • Serratia marcescens: Negative
  • Mannitol Fermentation
    • Pseudomonas aeruginosa: Negative
    • Escherichia coli: Positive
    • Enterobacter aerogenes: Positive
    • Serratia marcescens: Positive
  • Sucrose Fermentation
    • Pseudomonas aeruginosa: Negative
    • Escherichia coli: Negative
    • Enterobacter aerogenes: Positive
    • Serratia marcescens: Positive
  • Glucose Fermentation
    • Pseudomonas aeruginosa: Negative
    • Escherichia coli: Positive
    • Enterobacter aerogenes: Positive
    • Serratia marcescens: Positive

O/F Glucose Test (Two Tubes)

  • O: Tests to determine if the organism can undergo aerobic respiration (with energy gain from using O2).
  • F: Tests to determine if the organism can undergo fermentation in the absence of O2.
  • Tube must be sealed with mineral oil.
    • Oil prevents O2 from diffusing into the media.
  • When either form of metabolism takes place, the color of the tube changes from green to yellow.
  • Conducting an O/F Glucose Test (Two Tubes)
    • Using the inoculating needle, inoculate both tubes with culture
      • Pseudomonas aeruginosa
      • Escherichia coli
      • Enterobacter aerogenes
      • Serratia marcescens
    • Seal one tube with sterile mineral oil (to prevent new O2 from entering)
    • Incubate 24-48 hours at 37℃
    • If tubes are green, there was NO metabolism in the tube
    • if tubes are yellow, some form of metabolism DID occur in the tube

O/F Glucose Test Results

  • O/F Glucose Test with Mineral Oil
    • Pseudomonas aeruginosa: Green, Negative (no metabolism occurred)
    • Escherichia coli: Blue at top (non-saccharolytic) (no metabolism occurred)
    • Enterobacter aerogenes: Oxidizer (glucose metabolized)
    • Serratia marcescens: Oxidizer (glucose metabolized)
  • O/F Glucose Test without Mineral Oil
    • Pseudomonas aeruginosa: (non-saccharolytic) (no metabolism occurred)
    • Escherichia coli: Blue at top (non-saccharolytic) (no metabolism occurred)
    • Enterobacter aerogenes: Blue at top (non-saccharolytic) (no metabolism occurred) green blue and yellow
    • Serratia marcescens: Oxidizer (glucose metabolized)

Nitrate Test

  • Tests for utilization of nitrate (NO3-) as a terminal electron acceptor in anaerobic respiration.
  • Metabolic capabilities of microbes:
    • Incapable of using nitrate
    • Capable of only a single-step reduction of nitrate to nitrite (NO3- ->NO2-)
    • Capable of multi-step reaction of nitrate to nitrite to nitric oxide to nitrous oxide to nitrogen gas.
      • NO3- \rightarrow NO2- \rightarrow NO \rightarrow N2O \rightarrow N2 (g)
  • To inoculate: Add one loopful of culture to the nitrate broth, incubate at 37 for 24-48 hrs
  • To perform the test: Add 8 drops of each reagent and mix thoroughly
    • Sulfanilic Acid (Nitrate I)
    • - naphtholamine (Nitrate II)

Interpreting the Nitrate Test (The test with two positives)

  • When combined, the reagents will react with any nitrite in the media
    • This would indicate a POSITIVE result for a single-step reduction
    • NO3- \rightarrow NO2-
    • Color of the broth will change to RED
  • If no color change occurs, this indicates that there is no nitrite in the broth
    • This could be due to either:
      • Inability to use nitrate OR
      • All nitrate was reduced completely to N2(g)
  • Add a few grains of Zinc dust
    • Zinc dust will chemically reduce any nitrate that is in the tube (if the organism could not reduce the nitrate to nitrite or beyond)
      • Remember that reagents react with nitrite
    • If color changes to RED, this indicates that nitrate was reduced chemically by the zinc and NOT by the bacteria
      • Interpret as a NEGATIVE result
    • If broth remains COLORLESS, this indicates that nitrate was reduced completely by the bacteria to N2 gas
      • Interpret as a POSITIVE result

Nitrate Test Results

  • Pseudomonas aeruginosa: Colorless (+)
  • Escherichia coli: Red (+)
  • Enterobacter aerogenes: Red (+)
  • Serratia marcescens: Red (+)

Oxidase Test

  • Tests for cytochrome oxidase activity.
  • Unique test because it tests for something happening right now – NOT testing an organism’s capabilities
  • Conducting an Oxidase Test
    • Grow culture on a complex media such as NA, TSA, BHI
      • Pseudomonas aeruginosa
      • Escherichia coli
      • Enterobacter aerogenes
      • Serratia marcescens
    • Incubate until visible growth is seen
    • Aseptically collect some cell mass on the loop
    • Add the cell mass to the dry slide
    • Watch for the color change to purple in a positive result within 15 seconds

Oxidase Test Results

  • Pseudomonas aeruginosa: Positive (+)
  • Escherichia coli: Negative (-)
  • Enterobacter aerogenes: Negative (-)
  • Serratia marcescens: Negative (-)

Citrate Test

  • Tests for the ability of organisms to utilize citric acid as the sole carbon and energy source.
  • Inoculate slant surface with a LIGHT inoculum
    • Pseudomonas aeruginosa
    • Escherichia coli
    • Enterobacter aerogenes
    • Serratia marcescens
  • Incubate at 37 for 24-48 hrs
  • Observe for color change
    • Positive = Electric Blue
    • Negative = Media remains green

Citrate Test – Basis for Color Change

  • If the organism uses citric acid as the sole carbon and energy source, it will remove citric acid from the media.
  • The pH indicator in the media will sense the ALKALINE pH created with less acid remaining in the media.
  • pH indicator changes color of media due to the alkaline pH, and the color of the media shifts from green to blue.

Citrate Test Results

  • Pseudomonas aeruginosa: Positive (+)
  • Escherichia coli: Negative (-)
  • Enterobacter aerogenes: Positive (+)
  • Serratia marcescens: Positive (+)

MR-VP Broth

  • Two different tests
    • Methyl Red: Tests for Mixed Acid Fermentation
    • Voges-Proskauer: Tests for 2,3-Butanediol Fermentation
  • Mixed Acid Fermentation
    • In a positive result, the organism ferments glucose and produces a variety of acid end products (lactic, acetic, succinic, and formic acids).
    • pH decreases as acid end products are produced.
  • 2,3-Butanediol Fermentation
    • Unique in some organisms
    • Begin with mixed acid fermentation, but if pH gets dangerously low, these organisms can switch to making 2,3-Butanediol

Methyl Red Test

  • Inoculate one tube of MR-VP broth
    • Pseudomonas aeruginosa
    • Escherichia coli
    • Enterobacter aerogenes
    • Serratia marcescens
  • Incubate 24-48 hours at 37 ℃
  • Add 5-10 drops Methyl Red reagent, Mix
  • Positive result:
    • pH will be below 4.4 when the reagent is added
    • Broth will turn red
  • Negative result:
    • pH will be above 6 when the reagent is added
    • Broth will turn yellow

Methyl Red Test Results

  • Pseudomonas aeruginosa: Yellow (-)
  • Escherichia coli: Red (+)
  • Enterobacter aerogenes: Red (+)
  • Serratia marcescens: Red (+)

Voges-Proskauer Test

  • Inoculate a second tube of MR-VP broth
    • Pseudomonas aeruginosa
    • Escherichia coli
    • Enterobacter aerogenes
    • Serratia marcescens
  • Incubate 24-48 hours at 37 ℃
  • Add 8 drops of -naphthol, and 8 drops of KOH, mix vigorously, leave for 60 minutes
  • Positive result:
    • Acetoin (a precursor to 2,3-butanediol) reacts with reagents
    • Broth will turn brownish-red
  • Negative result:
    • No acetoin is present
    • Broth turns brownish-yellow

Voges-Proskauer Test Results

  • Pseudomonas aeruginosa: Negative (-)
  • Escherichia coli: Negative (-)
  • Enterobacter aerogenes: Red top yellow bottom (+)
  • Serratia marcescens: Red top yellow bottom (+)

Biochemical Testing – Part 2

Starch Hydrolysis

  • Starch is an extremely large molecule
    • Composed of many repeating units of glucose (3 or more, but usually in the hundreds or thousands)
    • This molecule is difficult to degrade because it is too large to be transported across the membrane.
    • Requires one or more exoenzymes
      • Amylase
  • Starch Agar
    • Tests for presence of amylase
      • Amylase removes one glucose ring at a time from the starch molecule.
      • Amylase reaction:
        • (Glucose)n \rightarrow (Glucose)n-1+Glucose
        • (Glucose)3 \rightarrow Maltose+Glucose
    • Media contains potato starch; we add bacteria to test whether they have amylase
      • Conducting a Starch Test
        • Inoculate one line of culture on a starch agar plate
          • Escherichia coli
          • Proteus mirabilis
          • Staphylococcus aureus
          • Bacillus subtilis
        • Incubate 24-48 hours at 37℃
        • Pour several drops of iodine on the plate
          • (+) result will have a clearing around the growth
          • (-) result will have no clearing
          • iodine forms a brown to blue/black complex with starch but does not react with glucose

Starch Test Results

  • Escherichia coli: Black positive (+)
  • Proteus mirabilis: No clearing negative (-)
  • Staphylococcus aureus: No clearing negative (-)
  • Bacillus subtilis: No clearing negative (-)

Casein Hydrolysis

  • Tests for caseinase (example of a protease enzyme)
  • Milk is composed of milk protein (casein), lactose, and water
  • Caseinase degrades casein (milk protein) into individual amino acids
  • Casein is a white, opaque protein and causes the plate to appear white and opaque
    • Conducting a Casein Hydrolysis Test
      • Inoculate one line of culture on one half of a milk agar plate
        • Escherichia coli
        • Proteus mirabilis
        • Staphylococcus aureus
        • Bacillus subtilis
      • Incubate 24-48 hours at 37℃
        • (+) result will have a clearing around the growth
        • (-) result will have no clearing

Casein Hydrolysis Test Results

  • Escherichia coli: No clearing around growth (-)
  • Proteus mirabilis: Clearing around growth (+)
  • Staphylococcus aureus: No clearing around growth (-)
  • Bacillus subtilis: No clearing around growth (-)

Lipid Hydrolysis

  • Tests for presence of the exoenzyme lipase (an enzyme that degrades fats)
  • Triglycerides (a possible bacterial carbon and energy source) are too large to enter the bacterial cell
  • Bacteria that produce and secrete lipase hydrolyze triglycerides into glycerol and 3 fatty acid chains
  • Spirit Blue Agar
    • Media begins blue
    • Spirit Blue dye binds to lipids in the agar
    • if bacteria can use the lipid as a carbon and energy source, they secrete lipase.
      • Lipase breaks down the fat into fatty acids and glycerol
      • Fatty acids are transported into the cell
      • Glycerol backbone is left behind with the spirit blue dye attached
      • Positive results will show a dark blue line of dye around the growth
        • Conducting a Lipase Test
          • Inoculate one line of culture on a spirit blue agar plate
            • Escherichia coli
            • Proteus mirabilis
            • Staphylococcus aureus
            • Bacillus subtilis
          • Incubate only 24 hours at 37℃.
            • Positive results will have a dark blue precipitate around the growth

Lipase Test Results

  • Escherichia coli: No precipitate negative (-)
  • Proteus mirabilis: Blue precipitate positive (+)
  • Staphylococcus aureus: No precipitate negative (-)
  • Bacillus subtilis: No precipitate negative (-)

Urea Broth

  • Tests for presence of Urease
  • Urease breaks down urea into 2 molecules of ammonia and one molecule of CO2
  • Reaction is useful for detoxifying a cell’s environment
    • Inoculate with one of the following
      • Escherichia coli
      • Proteus mirabilis
      • Staphylococcus aureus
      • Bacillus subtilis
    • Incubate at 37 for 24-48 hr
    • Urease test Broth contains a pH indicator that is peach/orange at neutral pH and fuschia at alkaline pH
      • Negative result: No increase in pH, the color remains peach/orange
      • Positive result: pH becomes alkaline due to release of NH4+, the color changes to fuchsia

Urease test Results

  • Escherichia coli: Negative (-)
  • Proteus mirabilis: Positive (+) fuchsia
  • Staphylococcus aureus: Negative (-)
  • Bacillus subtilis: Negative (-)

Triple Sugar Iron (TSI)

  • Three sugars
    • Slant: Sucrose and Lactose at 1% each
    • Butt: Glucose at 0.1%
    • Phenol Red (pH indicator)
      • If one or more of the sugars are fermented, that portion of the tube turns yellow
    • Some bacteria may reduce sodium thiosulfate
      • If thiosulfate is reduced, the organism will release H2S
      • Sulfide will react with iron in media to form FeS (black ppt)
      • Sulfurases are only active at acidic pH
      • Whenever black is visible in the slant, we assume positive for fermentation underneath
        • Inoculated by stabbing the butt of the tube with a needle and then streaking the surface of the slant
          • Using a needle, inoculate with one of the following
            • Escherichia coli
            • Proteus mirabilis
            • Staphylococcus aureus
            • Bacillus subtilis
          • Incubate at 37 for 24-48 hrs
          • Read Slant/Butt
            • Alkaline (K)/Acid (A)
            • Acid (A)/Acid (A)

Triple Sugar Iron (TSI) Results

  • Escherichia coli: A/A+gas
  • Proteus mirabilis:K/A+H2S
  • Staphylococcus aureus:K/K
  • Bacillus subtilis:K/A

Sulfide Indole Motility Test

  • Three tests in one!
    • Sulfide production from thiosulfate
    • Indole production from tryptophan
    • Motility
      • Using a needle, inoculate with one of the following
        • Escherichia coli
        • Proteus mirabilis
        • Staphylococcus aureus
        • Bacillus subtilis
        • Incubate at 37 for 24-48 hrs

Sulfide Indole Motility (Sulfide)

  • Sodium Thiosulfate included as a sulfur source
    • If thiosulfate is reduced, the organism will release H2S
    • H2S reacts with iron in the media to form FeS, a black precipitate

Sulfide Indole Motility (Indole)

  • Indole is a waste product of tryptophan utilization.
  • Tests for tryptophanase
  • Uses Kovac’s reagent

Sulfide Indole Motility (Motility)

  • Tests for motility away from the stab line
  • Assume the organism is positive for motility if the tube turns black

Sulfide Indole Motility Test Results

  • Escherichia coli: Negative for sulfide, Positive for Indol and Motility
  • Proteus mirabilis: Positive for sulfide, Negative for Indol, and Positive for Motility
  • Staphylococcus aureus: Negative for Sulfide, Indol, and Motility
  • Bacillus subtilis: Negative for Sulfide, Indol, and Motility

Phenylalanine Slant

  • Tests for phenylalanine deaminase
    • Inoculate with one of the following:
      • Escherichia coli
      • Proteus mirabilis
      • Staphylococcus aureus
      • Bacillus subtilis
      • Incubate at 37 for 24-48 hrs

Phenylalanine Deaminase Test

  • Tests for phenylalanine deaminase
    • Phenylalanine -> Phenylpyruvic Acid + NH3
    • Reagent: 10% FeCl3

Phenylalanine Deaminase Test Results

  • Escherichia coli: Negative (-)
  • Proteus mirabilis: Positive (+)
  • Staphylococcus aureus: Negative (-)
  • Bacillus subtilis: Negative (-)

Selective & Differential Plate Media of the Enterobacteriaceae and One Last Biochemical Test

Eosin Methylene Blue (EMB)

  • Dark cherry-red color
  • Fermentable Carbohydrate(s): Lactose and Sucrose
  • Selective Agents: Eosin Y and Methylene Blue
  • Lactose Fermentors: Pink colonies
    • Acid pumps are antiporter systems – when acid end products of carbohydrate fermentation are pumped out of the cell, another material is pumped in; Eosin Y is brought into the cell
  • Vigorous Lactose Fermentors: Metallic Green
    • As more and more lactose is fermented, more Eosin Y is brought into the cell.
    • Eventually, the internal concentration of Eosin Y is great enough to cause a metallic green sheen
  • Non-Lactose Fermentors: any color other than Pink or Metallic Green (colorless, purple, etc)

Eosin Methylene Blue (EMB) Results

  • Escherichia coli: Green (+)
  • Enterobacter aerogenes: Pink (+)
  • Serratia marcescens: Purple (-)
  • Klebsiella pneumoniae: Pink (+)
  • Citrobacter freundii: Green (+)
  • Proteus mirabilis: Blue (-)
  • Salmonella typhimurium (Frank pathogen): Purple (-)
  • Shigella flexneri (Frank pathogen): n/a (-)

MacConkey Agar (MAC)

  • Pink plate with a slight purple coloration (purple is especially visible when viewing the plate from the side)
  • Fermentable Carbohydrate(s): Lactose
  • Selective Agents: Bile Salts and Crystal Violet
  • Lactose Fermentors: Pink colonies
  • Vigorous Lactose Fermentors: Pink colonies with a bile precipitate
    • So much acid waste product gets dumped into the media that the bile added to the media falls out of solution (a bile precipitate)
    • Bile precipitate is observed as an opaque pink cloud IN the media
  • Non-Lactose Fermentors: any color other than pink

MacConkey Agar (MAC) Results

  • Escherichia coli: Vigorous lactose fermenter (+)
  • Enterobacter aerogenes: Pink (+)
  • Serratia marcescens: (-)
  • Klebsiella pneumoniae: Vigorous lactose fermenter (+)
  • Citrobacter freundii: Vigorous lactose fermenter (+)
  • Proteus mirabilis: (-)
  • Salmonella typhimurium (Frank pathogen): (-)
  • Shigella flexneri (Frank pathogen): (-)

Salmonella-Shigella Agar (SS)

  • Pink plate with an orange cast, especially as the plates age
  • Fermentable Carbohydrate(s): Lactose
  • Selective Agents: Bile salts, Brilliant Green, Sodium Citrate
  • Sodium thiosulfate (Source of sulfur)
  • Lactose Fermentors: Pink colonies
  • Vigorous Lactose Fermentors: Pink colonies with a bile precipitate
    • Same reaction as seen in MacConkey plates
    • Bile precipitate is observed as an opaque pink cloud IN the media
  • Non-Lactose Fermentors: any color other than Pink (colorless, purple, etc)
  • Black colonies: Indicate that sulfurases are present in the organism
    • Thiosulfate used as a sulfur source for anaerobic respiration
    • H2S released
    • H2S reacts with iron in the media to form FeS, a black precipitate IN the colonies (not the media)

Salmonella-Shigella Agar (SS) Test Results

  • Escherichia coli: Vigorous lactose fermenter (+)
  • Enterobacter aerogenes: (+)
  • Serratia marcescens: (-)
  • Klebsiella pneumoniae: (+)
  • Citrobacter freundii: Vigorous lactose fermentor (+)
  • Proteus mirabilis: Black colonies (+)
  • Salmonella typhimurium (Frank pathogen): Black colonies (+)
  • Shigella flexneri (Frank pathogen): (-)

Decarboxylase Test

  • Contains:
    • Growth Factors
    • Glucose (Fermentable Carbohydrate)
    • Bromcresol Purple (pH Indicator)
      • Purple at neutral pH
      • Yellow at acidic pH
  • Tests for an enzyme to decarboxylate a particular amino acid (ex: ornithine decarboxylase, lysine decarboxylase, arginine decarboxylase)
  • All decarboxylase enzymes are only active at acidic pH
    • i.e., The pH must become acidic from glucose fermentation for the enzymes to be turned on
  • Must be sealed with mineral oil to FORCE fermentation (otherwise organisms go through aerobic respiration)

Decarboxylase Test Results

  • Decarb Base
    • Escherichia coli: (+)
    • Enterobacter aerogenes: (+)
    • Serratia marcescens: (+)
    • Klebsiella pneumoniae: (+)
    • Citrobacter freundii: (+)
      *