Hemolysis, Phenol Red Broth, and Catalase Test Lab

Hemolysis, Phenol Red Broth, and Catalase Test Lab Goals

  • Inoculate Sheep's Blood Agar (SBA) to test organisms for hemolysins production.

  • Classify bacterial hemolysis as beta, alpha, or gamma.

  • Inoculate phenol red broths with Durham tubes using aseptic technique.

  • Identify all products of bacterial fermentation using phenol red fermentation broths.

  • Classify bacteria as catalase positive or negative.

Activities for Today’s Lab

  • Inoculate SBA with:

    • Staphylococcus aureus

    • S. epidermidis

    • Alcaligenes faecalis

  • Inoculate Phenol Red Broth (PRB) with:

    • Escherichia coli

    • S. aureus

    • Pseudomonas aeruginosa

  • Perform a catalase test on:

    • S. aureus

    • S. epidermidis

    • A. faecalis

    • E. coli

    • Enterococci

    • P. aeruginosa

Follow Up: Hemolysis and Phenol Red Broth

Activity: Inoculate SBA with S. aureus, S. epidermidis, and A. faecalis

Introduction

  • Several species of gram-positive cocci excrete exoenzymes known as hemolysins.

  • Hemolysins are exotoxins that attack the cell membrane of red blood cells, leading to hemolysis, defined as the lysing or breaking of red blood cells (erythrocytes) and hemoglobin.

  • The ability of bacteria to cause hemolysis can be tested using SBA, a differential medium that changes appearance based on hemolysis type:

    • Beta Hemolysis: Complete clearing of red blood cells around the growth.

    • Alpha Hemolysis: Partial hemolysis causing a greenish or brownish discoloration surrounding the growth.

    • Gamma Hemolysis: No visible change around the bacterial growth.

Safety Reminders

  • Live bacteria are being used; lab coats should be buttoned, gloves worn, and goggles used.

  • A bactoincinerator will be in use; reduce clutter and avoid direct contact with it.

  • S. aureus is classified under BSL-2 due to its potential to cause human disease.

Special Precautions

  • Personal items must be stored in cubbies with remaining items placed on paper towels.

  • Thorough hand washing is mandatory before and after handling organisms.

Set Up for SBA Inoculation

  • Students will work in groups of four and receive three SBA plates.

  • Each group will inoculate one plate with:

    • S. aureus

    • S. epidermidis

    • A. faecalis

  • Equipment needed: microincinerator.

Procedure

  • Step 1: Obtain three SBA plates, label the bottom with initials, media, organism, and date.

  • Step 2: Aseptically inoculate each plate with a straight-line streak.

  • Step 3: Close the plates and store them upside down for next week.

Cleaning Up

  • Wipe down the bench and wash hands.

Activity: Inoculate PRB with E. coli, S. aureus, and P. aeruginosa

Introduction

  • PRB is a differential medium made with a single sugar that tests bacterial fermentation and identifies waste products.

  • In this lab, PRB will be made with:

    • Glucose (monosaccharide)

    • Sucrose (disaccharide)

    • Lactose (disaccharide)

  • PRB contains phenol red, a pH indicator:

    • Red: Neutral pH.

    • Yellow: Acidic (indicative of fermentation).

    • Fuchsia: Alkaline (no fermentation).

  • The Durham tube traps gas produced during fermentation; if filled, no gas is produced; if a bubble is present, gas was produced.

Safety Reminders

  • Same as above regarding LIVE organisms and using protective equipment.

  • S. aureus and P. aeruginosa are BSL-2 organisms.

Special Precautions

  • Adhere to same precautions previously stated regarding personal items and handwashing.

Set Up for PRB Inoculation

  • Students will work in groups of four, receiving nine PRB tubes (3 each of glucose, lactose, and sucrose).

  • The 3 bacteria to be inoculated: E. coli, S. aureus, P. aeruginosa.

  • Equipment needed: microincinerator.

Procedure

  • Step 1: Gather three green (glucose), three yellow (sucrose), and three red (lactose) PRB tubes.

  • Step 2: Label each tube with initials, media type, organism, and date.

  • Step 3: Inoculate each tube following the method outlined for each bacterial species.

  • Step 4: Place inoculated tubes on class tray.

Cleaning Up

  • Wipe down bench and wash hands.

Important Notes

  • The protocol and skills from this lab will be assessed in the Unknown Techniques Assessment later in the semester.

  • Ensure to document the protocol in your notebook and feel free to ask questions for clarification.

Activity: Perform a Catalase Test

Introduction

  • This test determines if bacterial species produce the enzyme catalase, which converts toxic hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2).

  • The presence of bubbles indicates a catalase positive organism; absence indicates catalase negative.

Safety Reminders

  • Same safety precautions apply, especially regarding live bacterial species.

Set Up for Catalase Test

  • Groups will gather slants of:

    • E. coli, S. aureus, S. epidermidis, A. faecalis, Enterococci, P. aeruginosa.

Procedure

  • Step 1: Collect slants of all six bacterial species.

  • Step 2: Record observations in provided table:

    • Organism | Were O2 bubbles observed? | Catalase positive/negative

    • E. coli | |

    • S. aureus | |

    • S. epidermidis | |

    • A. faecalis | |

    • Enterococci | |

    • P. aeruginosa | |

  • Step 3: Add 2-3 drops of hydrogen peroxide to each slant.

  • Step 4: Observe for vigorous bubbling and complete the table for each organism.

Cleaning Up

  • Wipe down your bench and wash hands after the activity.