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Antibacterial
response (hemolymph)
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Changes In Hemolymph During the Antibacterial
Response of M. sexta
HINTS for Determination of Lysozyme Activity in
Manduca sexta Hemolymph
- M. lysodeikticus cells are available from
Sigma
Chemical Company (catalog number M 3770). We weigh
these out in the beakers, cover with parafilm, and store
in the freezer until ready to use.
- The NaN3 can be omitted from the assay
buffer. If this is done, the buffer is more likely to
become contaminated by bacteria or fungi when it is
stored.
- The volumes in this protocol are required for a 2 ml
sample in the cuvette for a spectrophotometer. Adjust the
volume of assay buffer and hemolymph accordingly for a
cuvette that holds more or less.
- If the beginning reading of the cell suspension is
too high to be read by the spectrophotomter, then dilute
the cell suspension with assay buffer. It is wise to
check this before lab begins.
- We have students work in groups of 4 but pairs would
also work.
- We typically complete this lab (along with the set up
for the cecropin assay) in a 3 hr lab period.
- Because of the variability in how quickly the
individuals mix and begin their assay, determining which
portion of the line to use for determining the reaction
rate is tricky. In general, we have selected the portion
over which most of the class has a simple linear
relationship.
- Generally, we have found lysozyme activity in the
hemolymph of non-treated and saline-treated insects. The
levels of activity are generally lower than for the
treated insects (by about 1/2).
Return to the Protocol for
Determination of Lysozyme Activity
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