The diagnosis of tetanus is clinical and does not require a demonstration of C. tetani. Treatment should be started immediately based on clinical diagnosis. Laboratory diagnosis provides supportive evidence for confirmation.

Specimen

Excised tissue bits from the necrotic depths of wounds are more reliable than wound swabs.

Gram staining

Gram staining reveals gram-positive bacilli with terminal and round spores (drum stick appearance or tennis racket appearance). However, microscopy alone is unreliable as it cannot distinguish C. tetani from morphologically similar non-pathogenic clostridia like C. tetanomorphum and C. sphenoides.
Culture

Culture is more reliable than microscopy.

• Robertson cooked meat(RCM) broth – C. tetani being proteolytic turns the meat particles black and produces a foul odor.

• Blood agar with polymyxin B: C. tetani produce characteristic swarming growth when incubated at 37°C for 24-48 hours under anaerobic conditions.

Toxigenicity Test

As pathogenesis of tetanus is toxin mediated, the association of the isolated organism can only be established when its toxin production is demonstrated. Toxigenicity can be detected by both in vitro and in vivo methods.

• In vitro hemolysis inhibition test: C. tetani produces hemolysis on blood agar which is inhibited by adding antitoxin. This test indicates the production of tetanolysin only but not tetanospasmin.

• In vivo mouse inoculation test: RCM broth with black turbid growth is injected into the root of the tail of a test mouse. The test animal develops stiffness which begins with the tail and progresses to involve the hind limbs on the inoculated side- the other limb-trunk-forelimbs. Death occurs within two days. This test indicates the production of tetanospasmin.