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Tobacco Hornworms


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Antibacterial response (hemolymph)

Changes In Hemolymph During the Antibacterial Response of M. sexta

 

Protocol: Bacterial Treatment of M. sexta Larvae

 

Treatment with E. coli

An overnight culture of E. coli is prepared. Any common laboratory strain, one used in cloning for example, will work. The following morning 100 ul of the overnight culture is used to innoculate 10 ml of medium (any common medium will work, for example LB) in a 25 ml flask. This culture is grown until the OD600 is about 0.5. The bacteria in the culture are then pelleted by centrifugation (3,000 x g, 20 min). The pellet is resuspended in 0.37% formaldehyde diluted in 0.85% NaCl. This resuspension is left to sit at room temperature overnight. The following morning, the formalin-killed bacteria are washed three times in 0.85% NaCl to remove the formalin. After the final wash, the killed bacteria are resuspended in approximately 5 ml in 0.85% NaCl. This should give approximately 109 bacteria per ml final concentration. An injection of 10 µl &endash;50 µl is injected into each larva.

 

Treatment with Micrococcus lysodeikticus

Prepare a 1 mg/ml solution of M. lysodeikticus. These may be purchases from Sigma in lyophilized form. Ten minutes before performing the injection, heat the bacteria at 94C for at least 2 minutes. Inject first day larvae with 25 µl.

 

Control Treatment

Any one of a number of controls may be performed. The insects may not receive an injection. Alternatively, they may be injected with the same volumn of 0.85% NaCl. This solution should be filtered through a 0.2µm filter to remove bacterial particles that might be present and cause a response. Another option is to inject the insects with endotoxin free water (available from Sigma).

 

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On to protocol for injection of M. sexta larvae


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March 1999