Graber, July 2005
COLONIZATION ASSAY PROTOCOLS
I. Preparing the bacterial inoculum
Determine the relationship between optical density (OD600) and the number of colony-formingunits (CFU) of the bacterial strain you will be using.
Example: ES114
(shaken in SWT)
2 x 108 CFU/ml per OD unit
(unshaken in SWT) 5 x 108 CFU/ml per OD unit
(unshaken in HM) 1.4 x 109 CFU/ml per OD unit
One Day Pre-Hatching:
1. Streak out from a frozen stock your strain(s) onto plates of appropriate media. If the
strain is carrying a plasmid, you will need to maintain antibiotic pressure on as it grows
up and perhaps while it is colonizing the animal (experiments focusing on the first 12
hours of colonization may not require antibiotics).
On Hatching Day:
1. In the morning, inoculate liquid culture tubes (3-4 ml) of appropriate media with the
strains that you streaked out last night, and shake at 28°C. These are your "preinoculation cultures".
2. When the pre-inoculation cultures reach an OD of ~0.4 - 0.6, inoculate a fresh set of
culture tubes (same media) with each strain.
Helpful Hint: It's easiest to get accurate inoculation numbers when you use cultures
that are all very close to the same OD (usually between 0.2 to 0.3). For this reason,
it's often useful to make a series of tubes for each strain with 50% reductions in
amount of the pre-inoculation culture serving as the inoculum (i.e. inoculate one
tube with 100 µl, a second with 50 µl, a third with 25 µl, etc). This way, you can
pick the set of tubes that is closest to the desired OD when your animals are ready to
inoculate.
3. Allow these cultures to grow up while you're hatching squid (see below).
II. Hatching Squid
1. Several days prior to your experiment, sign up for animals on the white board outside
of the squid room. Nights available to either the Ruby or McFall lab are indicated by
an "R" or "M". It's best to talk to one of senior grad students or postdocs in your lab to
find out what the current etiquette is regarding signing up as primary vs. secondary
user, taking squid more than once a week, etc.
Graber, July 2005
2. On the morning of hatching, check the clutch tracking sheet on the door of the squid
room to find out which clutches have been actively hatching (or are likely to start).
3. Check all the clutches to see if any animals are present. These are referred to as
"earlies", and may or may not already be colonized by V. fischeri (which is present at
low levels in the tank water). Remove them from the tank, set them aside in an "E"
bowl (see note), and label them as earlies with the date (check the board to see if
anyone wants to use these animals).
4. Use a vacuum filtration apparatus to filter-sterilize several hundred mls of Instant
Ocean (IO) water.
5. Check for hatching squid at 15 to 30 minute intervals (letting animals sit in the clutch
for longer periods can result in colonization by V. fischeri present in the tanks).
Remove animals from the clutch cages and place them in E bowls containing filtersterilized IO water (keep a separate bowl for each clutch). In general, animals will
trickle out throughout the morning, but the most animals will hatch just after the lights
go out at noon (you are responsible for collecting animals for the entire period though).
6. Keep track of the numbers of animals hatching from each clutch. When you have the
number that you'll need for your experiment, inform the person that's signed up as
secondary that they can take over hatching. If no one is signed up as secondary, place
any extra animals in a bowl of filter-sterilized IO water and label them as "extras" with
the date.
7. As primary hatcher, it is your responsibility to record the final number of animals that
have hatched from each clutch and record it on the clutch tracking sheet (if necessary,
check with the person who was secondary to get their numbers and add them to the
total).
III. Inoculating Squid
1. Use plastic squid pipets to transfer the squid that you'll be using to a fresh E bowl
containing 50 ml sterile IO water. If you're using squid hatched from multiple clutches,
be sure to thoroughly mix the animals between your experimental treatments (squid
from different clutches can vary considerably in colonization characteristics). Also, set
up a separate bowl of squid (usually 3-5) to use as an aposymbiotic control (this insures
that the squid weren't infected with unknown strains of V. fischeri).
2. For each experimental treatment (i.e. bowl of squid) add 50 ml filter-sterilized water to
a sterile 50 ml plastic screw cap tube.
Graber, July 2005
3. Measure your culture OD's, and pick the strain (or strains) that are around 0.2 to 0.3.
Based on OD, calculate the amount of culture that you'll need to give the desired final
inoculum in the sea water bowls (which will contain 100 ml final volume). Add this
amount to the 50 ml screw cap tube, and mix thoroughly.
A note on inoculation levels: In general, about 1000 CFUs per ml are used for
overnight inoculations, and about 3000 CFUs are used for 3 hour inoculations. For
example, for an overnight competition experiment, you would add 500 CFU's per ml
of strain A and 500 CFUs per ml of strain B. However, the inoculation level that
you pick will be highly dependent on the goals of your experiment. Using greater
than 10,000 CFUs tends to depress the final population in the light organ and should
thus be avoided.
4. Plate 50 µl of water from the 50 ml tube onto 2-3 SWT plates and incubate overnight.
You'll count these the next day to determine if your inoculation level was on target.
5. Add the 50 ml of inoculated IO water from the screw cap tubes to the bowls containing
your squid treatments. Add 50 ml sterile IO water to the aposymbiotic control bowls.
Lightly cover the bowls with saran wrap (make sure not to seal them, as this will
suffocate your squid)) and label appropriately. From this point on, you will need to use
separate pipets for each treatment to make sure that you don't cross contaminate the
animals.
For overnight inoculations: Place the bowls (labeled with tape) on the shelves in
squid room. The next morning, the squid will need to be individually transferred
from the bowls to E vials (flats of scintillation vials dedicated for squid work)
containing 4 ml filter-sterilized IO water. Transfer as little water as possible. The
squid should be kept in the squid room for the duration of the experiment (to
maintain their artificial day/night cycle) and are transferred to vials containing fresh
filtered IO water every morning.
For three hour inoculations: Do not put the bowls in the squid room (three hours of
incubation in the dark will cause the squid to vent when you bring them into the light
to transfer the animals, altering the normal progression of colonization). Instead, put
the bowls in a protected area of your bench. After three hours, transfer the animals
the animals to vials of filter-sterilized IO water (as described above), and place the
flat in the squid room. From this point, proceed as described for the overnight
inoculations.
6. The next morning, count the inoculum control plates from the previous day to make
sure that your levels were correct (remember, these counts will be 2X the actual final
inoculation level). If you're doing competitions, you'll also need to patch 100 colonies
from these plates to determine whether your strain ratios were correct.
Graber, July 2005
7. After transferring squid to fresh vials, measure the luminescence levels of both
experimental and aposymbiotic control animals using the Turner luminometer.
Compare measurements of vials containing squid with one containing sterile IO water
(which does have a small amount of background luminescence). Apos should not have
detectable luminescence higher than background.
This covers the inoculation process. For more information on monitoring colonization kinetics by
luminescence and/or plating squid to determine colonization levels/competition ratios, see the relevant
protocol.
A Note On Chemical Contamination and "E-Glass":
All glassware used for squid is dedicated for that task and should not be used for any other purpose.
E-bowls (small- and medium-sized custard cups and larger mixing bowls) are marked with paint.
Flats of scintillation vials used for squid should also be marked with an E (along with the user's name).
E-glassware should never, for any reason, come into contact with detergent or other chemicals. Squid
are extremely sensitive to chemical toxicity, and even trace contamination will kill them. For this
reason, E-glassware should only be rinsed with RO water, and this should only be done in the sink in
the squid room. You should do this immediately after use (i.e., don't let sea water begin to dry down
in the bowls or vials). Whenever this glassware is brought out of the squid room, it should be kept on
a plastic tray lined with clean paper towels. Do not set it on an unprotected bench top or other surface
that may have traces of chemical contamination. This also goes for flats containing E-vials.
In the same spirit, nothing that may have been contaminated with chemicals should ever be brought
into the squid room (this basically means anything that's been within ten feet of your bench). Also,
you should always thoroughly wash and rinse your hands before coming into contact with the squid
tanks or E-glass (in the past, squid have been killed by exposure to insect repellent, sun block, and
moisturizing lotion).
Strict adherence to these rules prevents catastrophes for everyone. If you are working with a rotation
student, undergraduate, or otherwise new lab member, it is your responsibility to make sure they
understand these rules and why they are in place.