Microbiology Lab Exam 2 Review Notes

E. Coli

• Durham tube: Used to detect gas production by fermenting a carbohydrate.
• Phenol red:
  • <7=yellow: Indicates an acidic pH level.

Salmonella

• Does not produce gas (no bubble).
• No fermentation occurs.
• No change in pH (pH remains at 7).

Alcaligenes

• Acid and gas production varies (XAMIX).
• pH remains at 7.

Carbohydrate Fermentation

• Glucose:
  • Acid and gas production varies (XAMIX).
• Sucrose: None.
• Lactose: None.

Bacterial Growth on Media

• Some bacteria can grow on media, while others cannot.
• All bacteria that grow exhibit different reactions.

MacConkey Agar

• Selective: Selects for gram-negative bacteria.
  • Crystal violet and bile salts inhibit gram-positive bacteria.
• Differential: Differentiates based on lactose fermentation.
  • Pink (Pos): Lactose fermenter.
  • Not pink (Neg): Non-lactose fermenter.
• Components:
  • Lactose
  • Neutral red

• S. epidermidis: Gram-positive, does not grow (dead).

MacConkey Agar Results

EMB (Eosin Methylene Blue) Agar

• Selective: Selects for gram-negative bacteria.
  • EMB inhibits gram-positive bacteria.
• Differential: Differentiates based on lactose fermentation.
  • Metallic green: Rapid lactose fermenter.
  • Pink: Lactose fermenter.
  • Not pink: Non-lactose fermenter.

EMB (Eosin Methylene Blue) Results

Nitrate Reduction

• Process: $NO<em>3 \rightarrow NO</em>2 \rightarrow NH_3$
• Detection: Add color reagents (naphthylamine & sulfanilic acid).
  • If not red, add zinc ($Zn^{++}$).
  • Red color after adding reagents: Nitrite ($NO_2$) present.
• Enzymes:
  • Nitrate reductase: $NO<em>3 \rightarrow NO</em>2$
  • Nitrite reductase: $NO<em>2 \rightarrow NH</em>3$
• Zinc mimics enzyme reaction.
• Redox:
  • Reduction: Accepting electrons.
  • Oxidation: Losing electrons.

Nitrate Reduction Explanation

• E. coli:
  • Has nitrate reductase.
  • Converts nitrate ($NO<em>3$) to nitrite ($NO</em>2$), turning red immediately.
• Alcaligenes:
  • Does nothing initially.
  • Zinc mimics nitrate reductase, converting nitrate to nitrite, turning red after zinc addition.
• Pseudomonas:
  • Has both nitrate and nitrite reductase.
  • Converts nitrate to ammonia ($NH_3$), never turning red.

TSI (Triple Sugar Iron) Agar

• Used for gram-negative enterics.
• Contains phenol red (pH indicator).
• Tests:
  • Slant (lactose fermentation).
  • Butt (glucose fermentation).
• Reactions:
  • Yellow slant/butt: Positive for fermentation.
  • Red slant/butt: Negative for fermentation.
  • Black precipitate: $H_2S$ production.

• $H_2S$ reacts with iron, forming FeS (black color).

Urea Hydrolysis

• Detects deamination of amino acids.
• Urea hydrolysis: Only Proteus can produce urease.
  • Positive: >7 Purple (presence of ammonia).
  • Negative: <7 Yellow.
• Reaction:
  • $Urea + H<em>2O \xrightarrow{urease} NH</em>3 + CO_2$
• Phenol red:
  • @7 is red.
  • <7 is yellow.

SIM (Sulfide-Indole-Motility)

• Tests for Sulfide production, Indole production, and Motility.
• Reagents:
  • Kovaks reagent: Detects indole.
  • Cherry red band: Positive for indole.
• Reactions:
  • Tryptophan + tryptophanase = indole.
  • $H<em>2S + Fe \rightarrow FeS (black precipitate) + H</em>2$

Methyl Red (MR) Test

• Detects glucose fermentation.
• Positive: Cherry red (acidic).
• Negative: Orange (alkaline).

IMViC

• Indole, Methyl Red, Voges-Proskauer, Citrate

Simmons Citrate Agar

• Detects the ability to use citrate as a carbon source.
• Bromothymol blue dye: Positive = royal blue.

IMViC Results

Blood Agar

• Differential test.

• Detects hemolysis (hemolysin enzymes).

• Types of Hemolysis:

  • Beta (\\beta\-hemolysis): Extreme activity, complete clearing of blood.
  • Alpha (\\alpha\-hemolysis): Partial activity (greenish look).
  • Gamma (\\gamma\-hemolysis): No activity.

• Clinical Relevance: Group A \\beta\-hemolytic Streptococcus (Streptococcus pyogenes, strep throat).

  • Susceptible to bacitracin.

• Enterococcus: Resistant to bacitracin.

• Lactococcus: Resistant to bacitracin.

Mannitol Salt Agar

• Selective: Salt tolerance (7.5% NaCl).
• Differential: Mannitol fermentation (yellow if acidic).

• S. aureus: Tolerant of 7.5% salt, ferments mannitol.
• S. epidermidis: Tolerant of 7.5% salt, no mannitol fermentation.
• E. coli: Not tolerant of 7.5% salt, cannot grow on plate.

DNase Test

• Detects the ability to break down DNA.
• DNase enzyme cuts DNA, resulting in clearing of green dye.
• Positive: S. aureus - Clearing zone indicates enzyme present.
• Negative: S. epidermidis - No clearing zone indicates enzyme absent.

Catalase Test

• Detects the enzyme catalase, which breaks down hydrogen peroxide.
• Reaction: $H<em>2O</em>2 \rightarrow H<em>2O + O</em>2$

Hydrolysis Tests

• Gelatin:
  • Liquification indicates gelatinase enzyme production.
  • Reaction: gelatin + gelatinase -> amino acids
• Casein:
  • Clearing zones indicate casein digestion/hydrolysis.
  • Reaction: casein + caseinase -> amino acids
• Starch:
  • Clearing zones after IKI application indicate starch digestion.
  • Reaction: starch + amylase -> maltose (oligosaccharide)

Fermentation Tests

• Phenol Red Media:
  • Contains pH indicator; <7 = yellow.
• Specimens:
  • Glucose, Sucrose, Lactose.
• Reactions:
  • Fermented sugar = Acid + Gas.
  • Non-fermented sugar = no color change.
• Durham tube: Traps gas produced during fermentation of a carbohydrate.

MacConkey Agar

• Used to differentiate gram negative enteric bacteria
• Selective: bile salts and Crystal Violet inhibit growth of gram positive cells
• Differential: Contains lactose and ph indicator - neutral red.
• Reactions: all grow but have different reactions.
  • No growth = gram positive organism (S. epidermids)
  • growth with strong lactose fermentation = Pink media & Pink Colonies (* E. Coli *)
  • colorless growth = gram-negative & non lactose fermenter(P. aeruginosa)
  • growth with lactose fermentation = pink colonies

Eosin Methylene Blue (EMB)

• used to differentiate gram- bacilli
• Selective: eosin and methylene blue inhibit gram-positive cells
• Differential: Contains lactose and ph indicator eosin
• Reactions: difference between MacConkey is rapid fast fermenter - Metallic Green
  • no growth = gram-positive
  • metallic green = gram-negative & strong lactose fermenter (E.Coli)
  • Colorless colonies = gram-negative & non-lactose fermenter (P. aeruginosa)
  • Pink / Purple colonies = gram-negative & lactose fermenter (Enterbacter)

Nitrate Reduction Broth Test

• Used to differentiate the ability to reduce Nitrate NO3
• Reactions: naphthylamine -> Not red… add zinc
• Sulfanilic Acid ->red
• NO3 + Nitrate reductase = NO2 Nitrite
• Not red ->red = Nitrite
• NO2 + Nitrite reductase = NH3 (ammonia) or N2 (Nitrogen gas)
• XEM Pseudomonas
• AZU
• CH
• E. Coli has nitrate reductase there Into NO 2(Nitrite) turns red in the prescence of nitrite immediately
• Alicaligenes does Nothing
• When zinc is added it mimicks Nitrate reductase Synthetically making NO2 (Nitrite) red after Zinc
• Psvedomonas has both Nitrate and Nitrite reductase there fore can make NO3 (Nitrate) into NH3(Ammonia)
• Does not turn red, NEVER

Triple Sugar Iron Agar (TSI)

• used to test ability to ferment Sugars & produce hydrogen sulfide fermentation of sugar = acid end products.
• Bacteria that produce H2S: H2S+ fe = FeS + H2 (iron sulfide) (black precipitate)
• Reactions:
  • Yellow = fermenter (E. coli)
  • red = non fermenter (Alicalgenes)
  • Neg lactose Pos glucose(Shigella & Salmonella)
• slant represents lactose
• Fes+H2
• butt represents glucose • Salmonella Fes +H2 producer

Urease Broth: Urea Hydrolysis

• Used to differentiate ability to rapidly reduce urea only organisms that can produce large amants of NH3 can change PH
• Reaction: Urea + urease = NH3 Calkaline)
  • Yellow = negative (E. coli)
  • "MBC" pink = positive (alkaline) (Proteus)
• Organisms capable of doing this cause UTI's

Sulfide - Indole Motility - (Sim)

• used to identify ability to produce indole, reduce sulfur and determine motility
• Reactions : Kovacs binds to indole to produce cherry red band at top of tube trypsin + tryptophanase= indole
• cherry red band = positive for indole (E .coli)
• bacteria that produce HzS= HzS+Fe= FeS (iron sulfide, black precipitate) + H2
• black precipitate = positive for HaS production (salmonella)
• cloudy outside stab = positive for motility (Klebsiella)
• Enterobacter Negative indole Methyl Red - (MR) used to differentiate based on usage of glucose Reactions : organisms that oxide glucose produce large amounts of acids that overcome buffers and result in a positive MR test
• red when acidic, positive result (E.Coli)
• yellow/orange when alkaline negative resultCenterobacter) VP Test Note Im V C E. Coli MR +VD-
• E. Coli + + - -
• Enterobacter MR-VP + Enterobacter - - + +
• Simmons Citrate Agar Slant Used to differentiate gram-negative enteric bacilli based On ability to utilize citrate as a carbon Source Reactions
• Positive results= changes from green to blue Centerobacter
• Negative results = no change (E.coli)

Blood Agar Plate with Bacitracin

• Only a differential test I used to identify Group A beta hemolytic strep (S. pyogenes) Results:
  • Beta B= complete RBC hemolysis (S. pyogenes)
  • Gammar = no hemolysis (Enteroco(CUS)
  • Alpha α = partial hemolysis (lactococcus)

Mannitol Salt Agar Plate

• used to differentiate salt tolerance and ability to ferment mannitol Often used to differentiate pathogenic Staph loccus epidermidis from non-pathogenic Selective: Staph loccus are tolerant to 7.5% NaCl Differential: for fermentation of mannitol Reactions:
  • Growth + Yellow media = salt tolerance & mannitol fermentation (S. aureus)
  • No growth = not salt tolerant (S. epidermids)
  • Growth + Pink media= salt tolerance & No mannitol fermentation (E. coli)

DNase Plate

• used to determine DNA hydrolysis Often used to differentiate S.aureus from S. epidermids Reaction: DNA + DNase = nucleotides

• clearing zones = positive DNA hydrolysis (S. aureus)

• lack of clearing zones = negative DNA hydrolysis (S. epidermids)

Catalase Test

• Used to differentiate gram-positive cocci If positive for enzyme can break down peroxide into water & no oxygen Reaction:
  • bubbles = catalase positive (S. aureus)
  • lack of bubbles = catalase negative (enterococcus)