Lipopolysaccharide (LPS), also known as the pyrogen, resides on the outer membrane of the Gram-negative bacteria but not on the Gram-positive bacteria. Upon injection of the Gram-negative bacteria into the horseshoe crab, the LPS is necessary and sufficient to trigger the degranulation of the host hemocytes, which subsequently releases the intracellular factors to kill the invader effectively1. Previous study demonstrated that the intracellular serine proteases, such as Factor B, pro-clotting enzyme, and clotting enzyme can activate the prophenoloxidase activity of the hemocyanin (HMC/PPO) to phenoloxidase (PO)2,3. To verify that the microbial extracellular protease could activate HMC/PPO to PO and further mediate the antimicrobial response in the extracellular milieu, Gram-positive bacteria which lack LPS and therefore do not trigger hemocyte degranulation are used in the in vivo antimicrobial assay. Being ubiquitous and chemically very stable, LPS may contaminate the Gram-positive bacterial culture. In order to avoid the interference from the inadvertent introduction of exogenous LPS, every precaution must be taken to prepare the Gram-positive bacterial culture under pyrogen-free condition.
To verify the pyrogen-free status of the Gram-positive bacterial culture, we incubate the culture with the horseshoe crab hemocyte, and three examinations are conducted:
(1) morphological observations under light microscopy,
(2) Factor C activity assay in the cell free hemolymph. The principle of this test is that under physiological condition, Factor C resides in the large granules of the hemocytes in a zymogen form. It is only released into the cell free hemolymph upon hemocyte exocytosis/degranulation triggered by LPS1. To detect Factor C, LPS is applied to convert it from zymogen to active serine protease, which can be indicated by fluorescent substrates.
(3) Intact hemocytes sediment at the bottom of the test tube during incubation. The principle is as follows: the presence of LPS causes the hemocytes to degranulate and no intact cells would settle to the bottom of the glass tube, thereby presenting a translucent-to-clear ‘tube bottom’. In contrast, in the absence of LPS, Gram-positive bacteria alone will not cause hemocyte degranulation. After standing incubation, the hemocytes will aggregate into a dense button at the bottom of the borosilicate tube.