Microbiology Lab Exercises 9-12 Review

Exercise 9 Review: The Genus Staphylococcus

• Characteristics of Staphylococcus Species

  • Staphylococcus epidermidis

    • Gram stain/Morphology: Gram-positive, cocci in clusters.

    • Biochemical properties: Catalase positive, coagulase negative.

    • Movement: Non-motile.

    • Pathogenicity: Rarely pathogenic.

  • Staphylococcus aureus

    • Gram stain/Morphology: Gram-positive, cocci in clusters.

    • Biochemical properties: Catalase positive, coagulase positive.

    • Movement: Non-motile.

    • Significance: Considered the most important pathogen within this genus.

• Artificial Culture Media Fundamentals

  • Purpose: Most medically important bacteria can be grown in a lab using artificial culture media.

  • Composition of Base Media: Made of digests from animal or plant proteins.

  • Supplements: Media are often supplemented with compounds such as glucose, yeast extract, serum, or blood.

  • Additives for Isolation: Specific components may be added to allow for rapid isolation of certain bacteria, including salt, alcohol sugars, bile, pH indicators, and dyes.

• Categorization of Artificial Culture Media

  • Nutrient Media

    • Description: Contain ingredients designed to satisfy the growth requirements for most bacteria.

    • General Ingredients: Water, salt, agar, and amino acids.

    • Examples: Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB).

  • Selective Media

    • Description: Encourages the growth of a desired microbe while inhibiting the growth of most other microbes.

    • Mechanism: Incorporates ingredients such as dyes, antibiotics, or other chemical additives.

    • Examples: Columbia Nalidixic Acid Agar (CAN), Mannitol Salt Agar (MSA), Eosin Methylene Blue Agar (EMB), and MacConkney Agar (MAC).

  • Differential Media

    • Description: Used when differentiating a desired bacterium from another, closely related bacterium found in the specimen.

    • Examples: Blood Agar Plate (BAP), Mannitol Salt Agar (MSA), Eosin Methylene blue Agar (EMB), MacConkney Agar (MAC), and Salmonella Shigella Agar (SSA).

• Media Specifics: MAC and MSA

  • MacConkey Agar (MAC)

    • Inhibition/Promotion: Inhibits the growth of gram-positive bacteria and promotes the growth of gram-negative bacteria.

    • Biochemical components: Contains lactose and a pH indicator.

    • Differentiation: Separates bacteria that can make acid from fermenting lactose from those that do not.

    • Lactose-fermenting bacteria Example: E. coli.

    • Non-lactose-fermenting bacteria Example: Salmonella.

  • Mannitol Salt Agar (MSA)

    • Selective Property: $7.5\%$ sodium chloride ($NaCl$). Staphylococci are facultative halophiles and can grow here.

    • Differential Property: Mannitol (a sugar alcohol).

    • Staph epidermidis reactions: Does not ferment mannitol; the medium color remains unchanged.

    • Staph aureus reactions: Ferments mannitol and produces an acid that turns the agar yellow.

    • Primary Use: Isolation and differentiation of bacteria in the genus Staphylococcus.

• Media Specifics: BAP and DNase

  • Blood Agar Plate (BAP)

    • Nutrient property: Trypticase soy agar enriched with $5\%$ sheep blood. Grows most bacteria.

    • Differential property: Identifies bacteria that produce hemolysins (enzymes that break down red blood cells).

    • Alpha-hemolytic colonies ($\alpha$): Partial breakdown of red blood cells; results in a greenish-brown zone around colonies.

    • Beta-hemolytic colonies ($\beta$): Complete breakdown of red blood cells; results in a clear zone around colonies.

    • Gamma-hemolytic colonies ($\gamma$): No breakdown of red blood cells; no zone exists around colonies.

  • DNase Agar

    • Purpose: Used to identify bacteria producing the enzyme DNase.

    • Components: Contains DNA and the indicator methyl green.

    • Negative Result: If the agar color stays green, DNase is not present.

    • Positive Result: If the agar turns blear, DNase is present.

• Biochemical Testing for Staphylococci

  • Coagulase Test

    • Purpose: Used to identify bacteria producing the enzyme coagulase.

    • Mechanism: Coagulase causes blood plasma to clot or clump. Rabbit plasma is used for this test.

    • Coagulase negative: No clotting observed.

    • Coagulase positive: Clots are present.

  • Catalase Test

    • Purpose: Checks for the presence of the enzyme catalase.

    • Context: Hydrogen peroxide ($H_2O_2$) is produced as a toxic byproduct of oxygen used in cellular respiration.

    • Reaction: Catalase breaks down hydrogen peroxide to produce water and oxygen ($2H_2O_2 \rightarrow 2H_2O + O_2$).

    • Utility: Useful for differentiating Staphylococcus (positive) from Streptococcus (negative).

Exercise 10 Review: The Genus Streptococcus and Related Gram-Positive Cocci

• Characteristics of Streptococcus and Enterococcus

  • Enterococcus faecalis

    • Morphology: Gram-positive, cocci in chains.

    • Biochemistry: Catalase negative, coagulase negative.

    • Movement: Nonmotile.

  • Streptococcus pyogenes

    • Morphology: Gram-positive, cocci in chains.

    • Biochemistry: Catalase negative, coagulase negative.

    • Movement: Nonmotile.

• Antibiotic Sensitivity: Bacitracin Test

  • Bacitracin Overview: Main ingredient in Neosporin.

  • Mechanisms of Action:

    • Inhibits bacterial cell wall synthesis.

    • Alters bacterial cell membrane function.

  • Test Purpose: To detect Group A beta-hemolytic strep.

  • Group A Context: Streptococcus pyogenes is the most common species in Group A.

  • Sensitivity Profile: Group A strep are beta-hemolytic and sensitive to bacitracin.

  • Interpretation: Bacteria with a zone of inhibition around the bacitracin disk are presumed to be Group A beta-hemolytic strep.

• Bile Esculin Test

  • Selective Property: Sodium azide inhibits any gram-negative bacteria in the sample.

  • Differential Property: While gram-positive bacteria grow, Enterococci are differentiated by their ability to hydrolyze esculin in the presence of bile.

  • Reaction: Esculetin produced in a positive test reacts with ferric citrate.

  • Positive Result: Dark brown or black color change in the medium.

  • Species Result: Enterococcus species are all positive, including Enterococcus faecalis.

Exercise 11 Review: Family Enterobacteriaceae and IMViC Series

• Overview of Enterobacteriaceae

  • General Characteristics: Gram-negative, rod-shaped, non-endospore forming bacteria.

  • Member Example: E. coli.

  • Classification by Fermentation:

    • Lactose-fermenters (Coliforms): Generally only cause disease if spread outside the GI tract (e.g., urinary tract). Examples: Enterobacter aerogenes, Serratia marcescens, E. coli.

    • Non-lactose fermenters: Proteus vulgaris (normal flora), Salmonella typhimurium (pathogen), Shigella sonnei (pathogen).

• The IMViC Series

  • Components: Indole, Methyl red, Voges-Proskauer, Citrate.

  • Voges-Proskauer (VP) test: First test performed; take $15-30$ minutes using O’Meara’s reagent.

• EMB and MAC Differentiation

  • Eosin Methylene Blue Agar (EMB)

    • Selective Property: Methylene blue dye and bile salts permit growth of gram-negative enteric bacteria while inhibiting gram-positives.

    • Differential Property: Lactose differentiates between Lactose Fermenters (LF) and Non-lactose Fermenters (NLF).

    • Organism Results:

      • E. coli: Positive growth, green colonial morphology, lactose fermentation positive.

      • Enterobacter aerogenes: Positive growth, red "fish-eye" colonial morphology, lactose fermentation positive.

  • MacConkey Agar (MAC)

    • Organism Results:

      • E. coli: Positive growth, small round raised colonies, red/hot pink agar with halo, lactose fermentation positive.

      • Enterobacter aerogenes: Positive growth, pink/light purple colonies, lactose fermentation positive.

• Specific IMViC Tests

  • Indole Test

    • Description: Determines if bacteria can break down the amino acid tryptophan into indole.

    • Reagent: Ehrlich’s Reagent.

    • E. coli: Positive growth, red ring, test result positive (+).

    • Enterobacter aerogenes: Positive growth, no red ring, test result negative (-).

  • Methyl Red Test

    • Description: Determines if bacteria can break down glucose into acidic end products.

    • E. coli: Positive growth, red broth color, test result positive (+).

    • Enterobacter aerogenes: Positive growth, yellow broth color, test result negative (-).

  • Voges-Proskauer Test

    • Description: Determines if bacteria can break down glucose into acetoin.

    • E. coli: Positive growth, yellow broth color, test result negative (-).

    • Enterobacter aerogenes: Positive growth, red band, test result positive (+).

  • Citrate Test

    • Description: Determines if bacteria can use citrate as the only source of carbon in the medium.

    • E. coli: Positive growth, green broth color, test result negative (-).

    • Enterobacter aerogenes: Positive growth, blue broth color, test result positive (+).

Exercise 12 Review: Enteric Pathogens and Biochemical Profiles

• MacConkey (MAC) Results for Pathogens

  • Salmonella: Positive growth, small round raised colonies, lactose fermentation negative (-).

  • Shigella: Positive growth, small round raised colonies, lactose fermentation negative (-).

• Additional Biochemical Tests

  • DNase Test

    • E. coli: Positive growth, DNase negative (-).

    • Serratia marcescens: Positive growth, DNase positive (+).

  • Urease Test

    • Mechanism: Determines if bacteria can break down urea into ammonia ($NH_3$) and carbon dioxide ($CO_2$) using urease.

    • Proteus vulgaris: Positive growth, urease positive (+).

    • Serratia marcescens: Positive growth, urease negative (-).

  • Phenylalanine Deaminase (PAD) Test

    • Mechanism: Determines if bacteria can break down the amino acid phenylalanine into phenylpyruvic acid.

    • Proteus vulgaris: Positive growth, PAD positive (+).

    • Serratia marcescens: Positive growth, PAD negative (-).

• Triple Sugar Iron (TSI) Agar

  • Contents: Glucose, lactose, and sucrose.

  • Mechanism: Media changes color when bacteria ferment one or more sugars.

  • Differentiation Zones:

    • Lactose and Sucrose: Ferment at the slant. Slant is yellow (A for acid) if positive, red (K for alkaline) if negative.

    • Glucose: Ferments at the bottom (butt) of the tube. Butt is yellow or black (A for acid) if positive, red (K for alkaline) if negative.

  • Interpreting Results:

    • K/K: Red/red; Alkaline/alkaline (No fermentation of any sugars).

    • K/A: Red/yellow (or black); Alkaline/acid (Glucose fermentation only).

    • A/A: Yellow/yellow (or black); Acid/acid (Glucose and lactose/sucrose fermentation).

  • Special Indicators:

    • Black color: Presence of hydrogen sulfide ($H_2S$).

    • Gas Bubbles: Splits, cracks, or popping up of the medium indicates gas production.

  • Organism Profiles on TSI:

    • E. coli: A/A fermentation, hydrogen sulfide negative (-), gas positive (+).

    • Salmonella: K/A fermentation, hydrogen sulfide positive (+), gas positive (+).

    • Shigella: K/A fermentation, hydrogen sulfide negative (-), gas negative (-).

• SIM Media (Sulfide, Indole, Motility)

  • Functions: Detects $H_2S$ production, indole production, and the ability to move (motility).

  • Motility Interpretation:

    • Positive: Cloudy, hazy appearance throughout.

    • Negative: Growth only at the cloudy stab line.

    • Special Case (Black agar): If black, cloudy at top = motility; clear stab line = no motility.

  • Indole Interpretation:

    • Positive: Red ring at the top of the agar.

    • Negative: Colorless or slightly yellow ring at the top.

  • Organism Profiles in SIM:

    • E. coli: Motility (+), Hydrogen Sulfide (-), Indole (+).

    • Salmonella: Motility (+), Hydrogen Sulfide (+), Indole (-).

    • Shigella: Motility (-), Hydrogen Sulfide (-), Indole (-).

Summary Comparison Tables

• Gram-Positive Species Summary

  • Staph aureus

    • Colony: Small, white, round.

    • MSA: Growth-yellow.

    • BAP: Beta-hemolytic ($\beta$).

    • Catalase: (+).

    • Coagulase: (+).

    • DNase: (+).

    • Bacitracin: Sensitive (S).

    • Bile Esculin: (-).

  • Staph epidermidis

    • Colony: Small, white, round.

    • MSA: Growth-pink.

    • BAP: Alpha/Gamma-hemolytic ($\alpha / \gamma$).

    • Catalase: (+).

    • Coagulase: (-).

    • DNase: (-).

    • Bacitracin: Resistant (R).

    • Bile Esculin: (-).

  • Strep pyogenes

    • Colony: Small, opaque, cream-colored.

    • MSA: No growth-pink.

    • BAP: Beta-hemolytic ($\beta$).

    • Catalase: (-).

  • Enterococcus faecalis

    • Colony: Translucent, grey.

    • MSA: Pseudo growth-pink.

    • BAP: Alpha/Gamma-hemolytic ($\alpha / \gamma$).

    • Catalase: (-).

    • Bile Esculin: (+).