Complete FTB Lab - lab PDF

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Microbiology Lab Notebook Report

Fluid Thioglycollate Broth - Exercise 2-6 March 3, 2020

Introduction Fluid thioglycollate broth is a liquid medium that can be modified to promote growth of a wide variety of fastidious to non-fastidious microorganisms. It can be used to illustrate microbial growth representing all levels of oxygen tolerance; however, its primary use is cultivation of anaerobic and microaerophilic bacteria.

Purpose The purpose of this lab is to support growth of multiple anaerobic and aerobic, fastidious or non-fastidious organisms. This allows practice of dilution technique and specific agar growth for unique microbes.

Materials • •

Four fluid thioglycollate broth (FTB) tubes Fresh cultures of these recommended organisms: - Clostridium sporogenes - Staphylococcus aureus - Pseudomonas auruginosa

ID #7330577

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Procedure Lab One: 1. Wear a lab coat, gloves, and chemical eye protection when performing this procedure. 2. Working with your group, obtain four fluid thioglycollate tubes and label them with your name, the date, and the medium. Label three of the tubes with the name of an organism, using a different organism on each tube; label the fourth tube “uninoculated.” 3. Using your loop, inoculate three broths with the organisms provided. (Note: when inoculating thioglycollate broth, it helps to dip the loop all the way to the bottom of the tube and gently mix the broth with the loop as you remove it. Do not shake the tube. Finish mixing by gently rolling the tube between your hands.) Do not inoculate the fourth tube. 4. Incubate the tubes at 35 ± 2 ℃ for 24 to 48 hours. 5. Save or dispose of the original cultures as directed by your instructor. Lab Two: 1. Check the uninoculated tube for growth to assure sterility of the medium. Note any changes that may have occurred as a result of incubation, especially in the colored region at the surface. 2. Using the control as a comparison, examine and note the location of the growth in all tubes. 3. Enter your observations and interpretations in the table provided on the data sheet. 4. Dispose of all tubes in the appropriate autoclave container when finished.

ID #7330577

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Results

Observations and Interpretations Organism

Location of Growth in Medium

Aerotolerance Category Not Applicable

Control Clostridium sporogenes

Grows at the bottom of FTB

Obligate anaerobe

Staphylococcus aureus

Grows best at surface, but spreads throughout FTB

Facultative anaerobe

Grows only at the top layer

Obligate aerobe

Pseudomonas auruginosa

Conclusion The pink at the surface of the media indicates that oxygen diffused at the top of the broth. This needs to be considered before inoculation to be sure that the environment is suitable for growth. I decided on the oxygen tolerance for each tube based on the amount of bacteria growth and where it grew the most.

ID #7330577

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Follow-Up Questions 1. Consider aerotolerance categories and their growth in FTB. a. Where would you expect to see growth of a strict aerobe? -Obligate aerobes grow at the top of the broth, where oxygen is abundant b. Anaerobe? -Anaerobe grows at the bottom of the broth, where oxygen is not present c. Microaerophile? -Microaerophile grows in the middle of the broth where oxygen is present, but not in large amounts d. Facultative anaerobe? -Facultative anaerobe is concentrated at the top (grows best when most oxygen is present), but spread throughout (can grow anaerobically). e. Aerotolerant anaerobe? - Aerotolerant anaerobe growth is relatively even throughout (anaerobic, but tolerates oxygen presence) 2. Consider resazurin dye. a. What purpose does the dye serve in FTB? -It indicates which parts of the tube that oxygen has diffused into and it shows you how far down the oxygen has gotten into your medium so you can use it as a reference point for what kind of metabolism is being utilized. b. Where in the broth column is resazurin dye located? -At the very top of the broth, forming a band. c. Why is the broth pink at its surface? -Resazurin turns pink in the presence of oxygen, and because oxygen has more chance to diffuse into the top portion (more oxygen in the top part of the broth). d. Considering the primary application of FTB, which is more desirable, a thick or a thin colored band at the surface? Explain your answer. -A thin colored band would be more desirable, as it would indicate that the medium is more fresh. The older the medium, the more oxidation occurs within the media, creating the indicator band to expand. Certain bacteria will not be able to grow or the results of their growth will become skewed.

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