Trans–corneal inoculation of Acanthamoeba suspension: Diagrammatic illustration of intrastromal injection in rat’s cornea using 10μL Hamilton surgical micro–syringe with 33G needle (first phase of the experiment).

The intrastromal injection was precisely performed in four steps [40]; (i) A surgical forceps was used for proptosis of the eye by carefully retracting the lower and upper lids, (ii) Creation of a small intrastromal pocket with 33G needle in the mid–peripheral cornea, (iii) Inserting a smaller portion of needle and advancing it intrastromally toward the corneal centre, and (iv) Gentle inoculation of amoebal suspension (~2μl).