LAB 6
ASSAY OF LYSOZYME
The bacteriolytic enzyme lysozyme is widely distributed in nature, being found in
such diverse organisms as animals (e.g. tears), plants and bacteriophages. Lysozyme
is capable of breaking down the peptidoglycan (a.k.a. murein) in the cell walls of many
bacteria. The activity of the enzyme may be measured by the reduction in optical
density of a bacterial suspension.
The purpose of this exercise is to assess the susceptibility of a representative Grampositive (M. luteus) and Gram-negative (E. coli) bacterium to the lytic action of lysozyme
following exposure to the enzyme for zero, fifteen and thirty minutes.
Materials: (per pair)
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1 tube of lysozyme (8mL in Buffer #1, 0.8 mg/ml)
18 new tubes
1 tube of Buffer #1 (100mM Tris pH 6.8, 0.04% gelatin)
2 tubes of Buffer #2 (100mM Tris pH 6.8, 2mM EDTA, 0.04% gelatin)
2 tubes of Na2CO3 (0.5M)
Overnight culture of Escherichia coli suspended in Buffer #1
Overnight culture of Micrococcus luteus suspended in Buffer #1
OD520
M. luteus
0 min
15 min
E. coli
30 min
0 min
15 min
30 min
 A 
1.0mL buffer #1 + 0.5mL lysozyme
 B 
1.0mL buffer #2 + 0.5mL lysozyme
 C 
1.5mL buffer #2
1. Label your tubes with the bacteria (ML or EC), time (0, 15, and 30) and buffer (A,
B and C).
2. Add the buffers according to the chart above.
3. Add 100µL of M. luteus to the time=0 tubes (A, B and C). Immediately add 1.0 ml
of Na2CO3
4. Add 100µL of E. coli to the time=0 tubes (A, B, and C). Immediately add 1.0 ml of
Na2CO3.
5. Add 100µL of M. luteus to the time=15min tubes (A, B and C). Let sit at room
temperature for 15 minutes and then add 1.0 ml of Na2CO3.
6. Add 100µL of E. coli to the time=15min tubes (A, B, and C). Let sit at room
temperature for 15 minutes and then add 1.0 ml of Na2CO3.
7. Add 100µL of M. luteus to the time=30min tubes (A, B and C). Let sit at room
temperature for 30 minutes and then add 1.0 ml of Na2CO3.
8. Add 100µL of E. coli to the time=30min tubes (A, B, and C). Let sit at room
temperature for 30 minutes and then add 1.0 ml of Na2CO3.
9. Measure the OD520 of each tube, and record your readings in the chart above.
Remember to blank the spectrophotometer.
Student#_______________________
Questions
1.
Student # _______________________
Questions (Hand in)
1.
Graph the results of absorbance vs. time for the 3 buffer combinations, and both
bacteria.
2.
Based on your results, which type of bacteria (Gram-positive or Gram-negative)
was more susceptible to lysozyme killing? Why?
3.
What is the site of action of lysozyme? (Draw the structure of the target molecule
and indicate the point of attack.)
4.
Based on your results, did the addition of a chelating agents (i.e. EDTA) affect
the susceptibility of E. coli to lysozyme? Why?
5.
What was the effect of adding 0.5M Na2CO3? Explain your results.
6.
Why are archaea resistant to lysozyme?
7.
Why are antibacterial agents that target the bacterial cell wall effective in treating
bacterial infections in humans and also have a high therapeutic index (i.e. ratio of
toxic dose to therapeutic dose)?