Measurement of the red blood cell lysis by bacterial hemolysin

doi:10.1038/nprot.2007.483

Method Article

Measurement of the red blood cell lysis by bacterial hemolysin

https://doi.org/10.1038/nprot.2007.483

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Biochemistry

Computational biology and bioinformatics

red blood cells (RBC)

hemolysis

hemolysin

The lysis of the red blood cells can be monitored by the release of hemoglobin to the extracellular environment. After removing the intact RBC by centrifugation, the absorbance at 404 nm of the supernatant reflects the hemolysis. The RBC lysate obtained by total disruption by 1% Triton- X100, is used as a positive control.

The RBC lysis by bacterial hemolysin is measured as described₁ with modification.

Collect the RBCs into heparinized tubes.
Wash the RBC two times by gentle resuspension with 10 times volume of pyrogen free saline (0.9% NaCl), and centrifugation at 1000 x g for 10 min at room temperature.
Gently resuspend the RBC pellet with pyrogen free saline and dilute the RBC to 0.8% (v/v).
Culture the bacteria under test according to protocol #4, and adjust the population to 10⁷ cfu/ml.
Incubate the washed RBC with equal volume of bacteria at room temperature under steady rotation at 6 rpm.
Set up pyrogen-free saline (0.9%) as the negative control, and 1% Triton-X100 in saline as the positive control.
At time intervals, retrieve aliquots of the reaction mixture and pellet the RBC by centrifuging at 1000 x g for 10 min at room temperature.
Transfer the supernatant into 96 well microtitre plate. Monitor the absorbance at 404 nm with ELISA-reader as an indication of the hemoglobin released into the supernatant.

Hemolysin producing microbes, such as S. aureus, lyses the RBC and the typical A404= 1.2 – 1.6.

Shin, S. Y., Kang, J. H. & Hahm, K. S. Structure-antibacterial, antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides. J. Peptide Res. 53:82–90 (1999).

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Measurement of the red blood cell lysis by bacterial hemolysin

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Measurement of the red blood cell lysis by bacterial hemolysin

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