Experiments to investigate the effects of O₂ on bacterial growth.

The Streptococci

Additional Readings:

Tortora, et. al.:

Read about the Streptococci and the Clostridia in your textbook, especially look up the diseases that these organisms can cause.

Read about oxygen requirements of various bacteria and how oxygen levels are manipulated in the laboratory

LeBoffe and Pierce:

Read about the following topics

• Aerotolerance and anaerobic culture methods
• Blood Agar
• Bile esculin agar
• Catalase test
• Bacitracin test
• Optochin test
• PYR test

Experiment 1 - The growth of organisms in thioglycollate broth

Day 1:

Work in groups of 2

Choose one organism from each of the following groups of organisms. Inoculate 4 thioglycollate broths, one with each of the organisms you chose.

A. Choose one from this list:

Escherichia coli

Serratia marcescens

Enterobacter aerogenes

Staphylococcus aureus

B. Choose one from this list:

Clostridium sporogenes

Clostridium perfringes

C. Choose one from this list:

Streptococcus pyogenes

Streptococcus bovis

Enterococcus faecalis

D. Choose one from this list:

Pseudomonas aeruginosa

Day 2:

• Observe and record the growth patterns in the thioglycollate broths. How do these correlate with the patterns you see on the plates which were incubated under aerobic and anaerobic conditions?

Experiment 2 - The growth of organisms under aerobic and anaerobic conditions

• Day 1:
  • Work in groups of 2.
  • Use 2 TSA plates per group.
    • Divide the plates like a pie into 4 sections by writing on the back of the plates with your marking pen.
    • Label the sections with the names of the same 4 organisms you used in experiment 1.
    • Inoculate each section with a simple streak of those organisms.
    • Incubate one of the plates in the anaerobe jar and incubate the other plate aerobically

  Day 2:

  • Check the color of the methylene blue indicator strip in the anaerobe jar before opening the jar.
  • Observe and record the growth patterns you see.
  • Perform a catalase test on each of the organisms by dripping on small drop of 3% H₂O₂ on the growth of each of the organisms. Do this on both of the plates -- the test should be performed on the anaerobic plate soon after removing it from the anaerobe jar!

Experiment 3 -The growth of the Streptococci

Day 1

• Work in groups of 2.
• Use 2 blood agar plates per group.
• Divide the plates like a pie into 4 sections by writing on the back of the plates with your Sharpie pen.
• Label the three sections A, B, C and D.
• Inoculate each section with a heavy streak of each of the following organisms
  • A = Streptococcus pyogenes
  • B = Enterococcus faecalis
  • C = Streptococcus pneumoniae
  • D = Streptococcus bovis
• Place a bacitracin disk onto the S. pyogenes
• Place an optochin disk onto the S. pneumoniae

Incubate one plate in the candle jar and the other aerobically.

Instead of placing a lit candle in the jar, one may also place a small beaker with a little bit of water and an alka-seltzer tablet in the jar.

(* Some groups may be asked to incubate their candle jar plates in a Campy jar instead.)

 

Day 2

• Blood agar is an important differential medium. Read the discussions in Leboffe and Pierce as well as in Tortora et. al. to familiarize yourself with what differential media do. What is alpha, beta and gamma hemolysis?
  • Determine and record the hemolysis reaction of each of the Streptococci after 48 hours of incubation.
  • Note any differences between the organisms grown under different oxygen tensions.
  • Note any zones of inhibition around the differentiation disks.
  • Perform the slide catalase test on these organisms:
    • Remove a small amount of growth with your loop and smear it on a microscope slide. (You can fit several specimens on the same slide. Look at the example in Leboffe.)
    • Drip a drop of 3% hydrogen peroxide on each of the smears and observe for bubbling.
  • Gram stain the Streptococcus pyogenes, Streptococcus pneumoniae, and Enterococcus faecalis.
• • Perform the PYR test on the Streptococcus pyogenes:
    • Moisten a PYR disk with a small amount of water. Use a loop to transfer a small amount of water onto the disk, do not saturate the disk, it should just be moist. (For these manipulations you can place the disk on the inner surface of a petri dish lid.)
    • Use a sterile loop to pick 2-3 colonies and rub them onto the moistened PYR disk.
    • Incubate for 2 minutes
    • Add one drop of p-dimethylaminocinnamaldehyde to the disk and look for the appearance of a pink or cherry-red color within one minute