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Authors: Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding 


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.

  1. Collect the RBCs into heparinized tubes.
  2. 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.
  3. Gently resuspend the RBC pellet with pyrogen free saline and dilute the RBC to 0.8% (v/v).
  4. Culture the bacteria under test according to protocol #4, and adjust the population to 10e7 cfu/ml.
  5. Incubate the washed RBC with equal volume of bacteria at room temperature under steady rotation at 6 rpm.
  6. Set up pyrogen-free saline (0.9%) as the negative control, and 1% Triton-X100 in saline as the positive control.
  7. 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.
  8. 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.

Anticipated Results

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


  1. 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).


Figure 1: Hemolysis of RBC by S. aureus laboratory strains PC1839 and AK3.

Fig 1

S. aureus strains, PC 1839 and AK3, at cell density of 107 cfu/ml, rupture the RBC within 5 min.

Associated Publications

Respiratory protein–generated reactive oxygen species as an antimicrobial strategy, Naxin Jiang, Nguan Soon Tan, Bow Ho, and Jeak Ling Ding, Nature Immunology 8 (10) 1114 - 1122 26/08/2007 doi:10.1038/ni1501

Author information

Naxin Jiang, Nguan Soon Tan, Bow Ho & Jeak Ling Ding, National University of Singapore

Source: Protocol Exchange (2007) doi:10.1038/nprot.2007.483. Originally published online 31 October 2007.

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