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Antibacterial
response (hemolymph)
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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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