PLATING OF JUVENILES FOR TOTAL CFUs PER LIGHT ORGAN
I. Things to have ready:
-
SWT plates (at least 3 per animal) that have been dried well (LBS plates will also
do in a pinch).
Sterile 1.5 ml microfuge tubes
Blue plastic pestles (make sure they fit the microfuge tubes that you'll be using)
Sterile Instant Ocean (IO) water
Forceps
Plating materials (either sterile plating beads, or spreader/spinner/ethanol)
Aspirator
Pasteur pipettes (drawn to a fine point)
II. Procedure:
1. Determine the level of light production by the juvenile squid using the photometer.
2. Rinse the juvenile 2 times with filter-sterilized or autoclaved IO water. Rinsing can
be done by sucking the water (but not the animal) out of a vial with the drawn point
of a pipette that is attached to a slight suction. Alternatively, the animal can be
transferred serially through 2 or 3 vials containing filter-sterilized IO water.
3. After the final rinse, leave the animal in a minimum amount of IO water in the
bottom of a vial.
4. Using forceps, grasp the animal by the tentacles (as he’s doing “pushups” along the
bottom) and transfer to a sterile 1.5 ml microfuge tube on ice; after5 minutes on ice,
add 700µl sterile IO water.
Optional: Instead of repeatedly rinsing the squid, moving them to directly from the
original vial to an empty microfuge tube and freezing at -80°C will effectively surfacesterilize the animal. When you're ready to plate (animals can be stored in the -80°C for
days to weeks), thaw the animals on ice, add 700 µl sterile IO water, and proceed to the
next step.
5. Dip the pestles in 95-100% ethanol and stand them (tip up) in a rack. Allow to dry.
6. Homogenize the juvenile well against the side of the tube using the ethanol-sterilized
pestle. When the water becomes gray and cloudy, the ink sac has ruptured, which is a
good indication that the light organ has been homogenized, too.
7. Make sure no animal tissue clings to the pestle, and close the tube.
8. Thoroughly vortex the tube for 15 seconds, and let the solids settle for 3 to 5 minutes.
9. Dilute the homogenate in sterile IO water in microfuge tubes (see step 10). When
removing sample from the original homogenate tube, take from the top of the tube
(i.e., do not suck up any of the tissue fragments on the bottom).
10. Plate the dilutions as follows onto SWT agar.
Original homogenate = “A” dilution
(plate 2 x 50 µl)
Dilute 50 µl into 1 ml and vortex = “B” dilution
(plate 2 x 50µl)
Dilute 50 µl into 1 ml and vortex = “C” dilution (plate 2x 50µl)
Note: Based on the light readings you get from the juveniles before
homogenization, you should be able to estimate whether the animals are colonized
fully, or to only a very little extent. If the former is true, the platings of “B” and
“C” will be most useful (“A” will be overgrown). If you expect few if any cells
in the light organ, plate “A” and “B”. If you see no V. fischeri CFUs on the “A”
plate, there are < 17 CFU/organ.
10. Incubate the plates for 24 to 48 hours at 25º to 28º C.
III. Calculations:
1. Count the total CFU (colony-forming-units) and calculate the CFU/organ. (Note: past
work has shown that, normally, all the CFUs are arising from the light organ. This is
an assumption you may need to confirm for yourself by dissecting out the organs and
plating the homogenized organ and the homogenized body separately)
2. To calculate the number of CFU/organ (based on the dilution scheme given above):
Mean CFU on “A” dilution plates
“B”
“C”
x 20 x 0.7
= CFU/organ
x 20 x 20 x 0.7
= CFU/organ
x 20 x 20 x 20 x 0.7 = CFU/organ
(The plates with the highest number of countable colonies give the most reliable
numbers)
3. After 48 hours observe the colonies for uniformity of appearance (color, texture, etc.).
Questionable colonies can be checked for luminescence (disclaimer: since V. fischeri
colonies on plates are sometimes non-luminous by 48 hours after plating, you may
need to inoculate the colony into liquid media to check for luminescence). The
colonies you counted yesterday should look like V. fischeri (golden-yellow colonies);
if they don’t, you'll need to adjust your counts accordingly.
Note: The “A” dilution plates often have a few non-V. fischeri colonies. However, if
there are many “spreader” colonies, that can be an indication that the juvenile was
“infected” with a pathogen (data from such animals are suspect at best). Contamination
of all plates by similar numbers of non-fischeri colonies usually indicates that the water
used for dilutions was not sterile.