In The Calcutta Chromosome, the unknown is both theme and symbol. It symbolizes the secret and elusive truths many of the book’s characters seek. The unknown, then, overlaps with silence. Silence is always about what remains untold and unspoken. Silence is the truth not seen. Phulboni talks about the secrets each city carries. Murugan speaks about unknown influences manipulating the research on malaria that Ross was undertaking in 1895. Laakhan is spoken of many times throughout the story, yet his character always remains elusive, ghost-like, and mainly silent. It's not what you know that counts, according to Murugan, but what you don't know , that's where the truth lies. Phulboni had written a story and later wondered if it was truly he who had created the story, for its story seem not to have been created by him but crafted out of the silence of mud and clay. 

The microscope is a symbol that is used periodically throughout the novel. On the one hand, the microscope symbolizes discovery; on the other, it is depicted as subservient to mystic powers of the demiurge. The microscope was a significant instrument that was used by Ross, Farley, and others to determine how malaria is transmitted.