Download Bacteria Killing Assay 1 Bacteria Killing Assay using agarose plates

Bacteria Killing Assay using agarose plates
Dan Ardia, Franklin & Marshall College: August 2008
Modified from protocols by Kirk Klasing and Anna Forsman
Original method: Millet, S., Bennet, J., Lee, K. A., Hau, M. & Klasing, K. C. 2007
Quantifying and comparing constitutive immunity across avian species. Developmental
And Comparative Immunology 31, 188-201.
Overview: All animals have an innate ability to recognize and kill microorganisms. This
is a coarse assay of innate immunity that assesses the ability of immune factors in the
blood to kill bacteria/yeast. With whole blood, the assay integrates phagocytosis by
macrophages, as well as complement and lysozyme activity. When just plasma is used,
phagocytosis is thus not included.
We mix whole blood or plasma with a known dilution of bacteria/yeast in a
growth medium. If the individual has the ability to kill off the pathogen, then the resultant
agar plates will show reduced bacterial growth relative to controls. The metric of interest
is the percent bacteria killed relative to control plates. Because of this, it is important to
pay close attention to the control plates. There should not be too many samples processed
without running control plates. We generally run our samples in batches of about 10-24
individuals with control plates done at the beginning and end of each run. Because the
bacteria/yeast are at a known dilution, it is important that they not be able to continue to
grow or degrade. Thus, whenever the bacteria/yeast are out of the fridge, they must be
kept on ice at all times.
This table shows common microorganisms used in this assay. The incubation times given
are for chickens and cockatiels from work done by Kirk Klasing (UC-Davis). Adjust
dilutions and incubation as required by your species.
Note: We’ve used E. coli (8739), S. aureus and Candida for tree swallows. E. coli gets an
incubation time of 45 minutes for nestlings, 30 minutes for adults. The others need 3
hours incubation for adults and nestlings. Only E. coli (#8739) is killed by plasma alone;
the others require whole blood.
Strain
Dilution
E. coli ATCC # 8739
Microbiologics #0483E7
1:10
E. coli ATCC #51813
Microbiologics #0791E8
C. albicans ATCC #10231
Microbiologics #0443E7
S. aureus ATCC # 6538
Microbiologics #0485E7
S. epidermidis ATCC
#12228
Microbiologics #0371E8
1:10
Bacteria Killing Assay
1:4
Incubation Comments
time
15-30 min You can use serum or plasma for
this strain – killing is complement
dependent
90 min
Mostly complement independent
and requires phagocytosis
4 hr
Killing mostly by phagocytosis
1:4
4 hr
1:4
4 hr
Mostly complement independent
and requires phagocytosis
Mostly complement independent
and requires phagocytosis
1
Materials:
-Petri dishes. I prefer Fisher Media Miser plates
-Tryptic soy agar (TSA)
-CO2 independent media (Invitrogen; Gibco media #18045)
-L-Glutamine (Sigma)
-Microorganisms (Microbiologics)
-Falcon tubes (50ml for stock solution, 15ml for working solution)
-Microcentrifuge tubes (1.5ml)
-Alcohol lamp or gas burner for sterilizing
-Dish of 70% EtOH for sterilizing
-Glass spreading rod
Lab equipment needed:
-Incubator (capable of 40ºC)
-Laminar flow (cell culture) hood
-Vortexer
Do beforehand:
-
Make plates ahead of time or the day of the assay.
Autoclave 1.5mL tubes, pipette tips, mix and autoclave 1x PBS.
Day of the assay:
-
Make bacteria stock and CO-independent media on the first day of the assay. It is
best to use the stock solution within 1 week.
Make fresh working solution of bacteria everyday.
Step 1: Pouring Plates (do up to one week beforehand)
1. Add 20g of tryptic soy agar to 500mL of tap water in a 1L bottle and mix by
shaking. Loosen the cap slightly, cover with aluminum foil and autoclave
for 20 min (no steam dry). This makes ~25-30 plates.
2. Remove from autoclave and tighten lid. Make sure the benchtop is sterile by
wiping down with alcohol (or bleach), and laying down bench paper.
3. Put on fresh gloves, and in a sterile, area, open a bag of Petri dishes (SAVE
BAG) and begin pouring the hot agar into each plate leaving the lids off
while they cool. Pour enough agar to cover the bottom of the plate. If agar
is too thick, reading the colonies is harder because the agar becomes cloudy.
Always make sure that the plates cool down completely (e.g. leave for a few
hours) before putting lid on – otherwise there will be condensation, which
results in spreading of colonies.
Bacteria Killing Assay
2
Swirl gently and begin pouring the agar immediately after autoclaving.
Cooling even for ~10min would make the agar clumpy and the plates at the
end uneven. Avoid bubbles. If bubbles are introduced, move them to the
edge of the plate with a sterile pipette tip. Put the lids back on ~40-60 min
after you start pouring (or until there is no more steam on the lids, plates
should be cool to the touch). Place the plates lid-side up in the plastic bag
they came in and store them in the refrigerator.
4. When the agar is hardened, place the lids back on and stack. Store the plates
upside down inside the bag the Petri dishes originally came in.
Step 2: Making media (30 mL/4 mM stock)
1) Pipet 29.4 mL of Co2 independent media into a sterile 50 mL falcon tube.
2) Add 0.6 mL (or 600 uL) of 200 mM L Glutamine. MIX WELL as it needs to
resuspend.
Step 2: Preparing Microorganisms (Epower microorganisms, Microbiologics)
If the stock solution never gets warm, it should last a couple of weeks. The main
concern is contamination which will show up as streaky or turbid solution. In
general, the bacteria decrease over time, so proper control plates are essential.
5. Remove a vial of lyophilized sample from the refrigerator and allow it to
equilibrate to room temp.
6. Warm hydrating solution (1X PBS) to 337 C. To make a stock solution, you
will need to dilute one pellet containing 107 bacteria in 40mL of sterile PBS.
If the pellet contains 108 bacteria, make an additional 10-fold dilution.
7. Transfer a pellet using sterile (generally flamed) forceps into the hydrating
fluid.
8. Place the hydrating microorganism suspension into a 35-37 C incubator for
30 min.
9. Following incubation, vortex or thoroughly mix the hydrated material. Store
this Stock suspension in the refrigerator. Keep on ice whenever it is
removed from the refrigerator.
10. To use the solution, vortex and then transfer the appropriate volume into a
working solution tube.
Bacteria Killing Assay
3
Step 3: Getting the proper dilution for the working solution
The goal is to add an appropriate amount of bacteria or yeast to get about 100 to 150
colonies per 50 µL of diluted blood (i.e. per plate). Over time the appropriate
concentration varies, but it normally runs from a low of 0.1ml microbe to 9.9 ml PBS to a
high 1ml microbe to 9.0ml PBS. The key is to get it right, which means to plate out a
range of dilutions the day before running the test. As a start I usually use duplicates of
0.1:9.9, 0.5:9.5, 1:9, 1.5:8.5. This allows me to titrate the right dilution.
Fresh working solution should be made daily and kept on ice at all times.
1. Fill and label tubes with the needed amount of PBS before removing the stock solution
from the fridge. You can use 15ml Falcon tubes with the exact amounts above or reduce
the volumes to use 1.5ml centrifuge tubes (i.e. 10ul microbe to 990ul PBS). The danger
of using smaller volumes is not getting a consistent sample of the microbes, thus you will
see more variation among your duplicates.
2. Remove stock solution from fridge and put on ice.
3. VORTEX STOCK SOLUTION BEFORE EACH USE!!
4. Add the appropriate volume of stock to the working solution tubes.
5. Return working stock tubes to ice or refrigerator
6. Prepare cell culture media (as noted below)
7. Follow ratios as listed below (I.e. Mix 20ul of each working volume of E. coli with
100ul of cell culture media in each centrifuge tube.)
8. Incubate at 40deg for the same time period as blood samples will be used (i.e. 45
minutes for tree swallow nestlings vs. E. coli).
9. Plate out as indicated in protocol below.
Step 4: Making cell culture media
Media = CO2 independent media (Invitrogen Inc; Gibco media #18045) + 4mM LGlutamine. Don’t make the entire bottle of media, use a 50ml Falcon tube to make about
40mls worth. It will become contaminated quickly (that’s why it is sold – L-glutamine)
For a 50ml Falcon tube: Pipet 29.4 mL of Co2 independent media into a sterile 50 mL
falcon tube, then add 0.6 mL (or 600 uL) of 200 mM L Glutamine. MIX WELL as
it needs to resuspend.
Bacteria Killing Assay
4
Step 5: Processing samples
Things to keep in mind:
- Assay up to 10 samples at a time with media control at the beginning and end.
- Keep sample tubes on ice at all times except during the 41C incubation.
- For stock and working bacteria solution as well as samples containing bacteria,
vortex thoroughly and aliquot immediately as bacteria settles very quickly.
- Keep the stock and working solution of bacteria on ice at all times.
- Use the offset method of placing the centrifuge tubes in a different row in the rack
after using to avoid using wrong tube twice.
Please note: volume and incubation times below here represent dilution used for testing
tree swallow blood against E. coli—changes dilutions as needed for 1:4 dilution vs. Staph
or Candida. To do duplicates, we would mix 5ul whole blood: 95ul media: 20ul microbe.
If you are running controls (or testing dilutions) increase media to 100ul to make up for
not having whole blood in mix.
Turn on incubator prior to starting!
How to calculate working bacteria solution:
1. Put 95ul of cell culture media in each centrifuge tube (20-200ul Pipettor will read 095).
2. Place 5ul of whole blood or plasma in each tube
3. Once plasma is in tubes, remove proper working stock dilution from fridge and place
on ice. VORTEX before using.
4. Add 20ul of bacteria from the working dilution into the proper tubes. E. coli into E
tubes (including controls), S. aureus into S tubes (including controls), Candida albicans in
C tubes.
5. Close tube tops tightly and VORTEX
6. Place tubes in incubator at 40deg for appropriate time (45 minutes E. coli, 3 hrs Staph
or Candida)
Step 6: Plating the samples To avoid contamination plate under a laminar flow (cell
culture) hood.
7. While tubes are incubating, remove and label Petri dishes on the underside (agar side).
When labeling, be sure to include sample number, microorganism and date. If doing
duplicates, then indicate this.. For example, a plate might say 94331-E-1 (bird 94331, vs.
E. coli, duplicate 1. Always do two plates for each control. To make things easier, we
generally label plates in reverse order of how they will be plated.
8. Once tubes are done in incubator, remove and VORTEX.
Bacteria Killing Assay
5
9. Plating bacteria
-Light burner and open alcohol dish. Be sure that the dish is not in the path of the
flaming spreader
-Flame sterilize spreader
-Remove 50 ul from the tube and place in the center of appropriate Petri dish.
DO A CONTROL PLATE FIRST AND THEN LAST
-Using the flame-sterilized spreader spread the liquid evenly over plate by turning the
plate.
-Place the plate under the hood to dry with the top next to it. Plates should stay in
hood for about 1-2 minutes.
-Flame sterilize spreader
-Repeat process (on other plates) and every couple of minutes, cover dried dishes and
stack to side AGAR SIDE UP.
10. When entire run is completed places plates AGAR SIDE UP in incubator. Incubate
the plates for min. 12 hours (overnight) at 40deg.
11. CLEAN WORK SURFACE AND HOOD SURFACE WITH BLEACH SOLUTION.
Bacteria Killing Assay
6