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 will show reduced bacterial growth relative to controls. The metric of interest is the percent bacteria killed relative to the controls.

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.

To view a PDF of this protocol: Bacteria killing protocol w spec.pdf

There are also bacterial killing assay protocols using plates, and in vivo and in vitro protocols for insect hemolymph.

Materials:

- Tryptic soy broth (TSB)

- Microorganisms (Microbiologics; see below)

- PBS

- CO2-independent media plus 4mM L-glutamine

- ultrapure water

- sterile 1.5mL microcentrifuge tubes

- 15mL Falcon tubes

- Alcohol lamp or gas burner for sterilizing

Lab equipment needed:

- Incubator (capable of 40C)

- Laminar flow (cell culture) hood

- Vortexer

- Pipette

1.Make broth **Can be made 1+ week in advance

a.Make 500 ml Tryptic Soy Broth as per directions on package.

b.Autoclave and store in refrigerator.

c.Remove broth using sterile technique only (using sterile pipette tips in laminar flow hood, flame bottle mouth)!

2.Prepare Microbe (Epower microorganisms; Microbiologics)

**Night before experiment

a.Warm 10 ml sterile PBS to desired temperature overnight in incubator (37 for E. coli and Staph; 30 for Candida).

b.Remove 1 pellet of lyophilized microbe using sterile forceps, add to PBS.

c.Incubate suspension at growth temperature of microbe (37 for E. coli and Staph; 30 for Candida) for 30m.

d.Vortex until pellet is fully dissipated. This is your stock solution.

e.Make a working solution of 105 bacteria/ml using sterile PBS.

i.For example, if your lyophilized microbe pellet contained 4.4 x 107 organisms:

Math to make working solution:

(4.4 x 107 bacteria/10mL)(x mL)= (105 bacteria/mL)(10mL)

X = [(105 bacteria)(10mL)]/ 4.4 x 106 bacteria/mL= 0.23mL stock in 9.77mL sterile PBS

f.Store extra stock solution in refrigerator for up to 24 hours.

3.Killing Assay for plasma

a.Vortex plasma sample (at least 10sec).

b.Add 1.5uL plasma to 34.5uL sterile PBS (1:23 dilution) in a 1.5mL tube (each sample should be conducted in triplicate if possible).

c.Add 12.5uL of 105 bacteria/ml solution to each tube.

d.Vortex.

e.Incubate at desired temperature (37C for E. coli and Staph; 30C for Candida) for 30min.

f.Remove samples from incubator and vortex.

g.Add  250uL broth to each sample.

h.Make control sample by adding 250uL broth to 48.5 uL of 105 bacteria/ml solution (in triplicate).

i.Make blank by adding 250uL broth to 50uL sterile PBS (in duplicate).

j.Incubate all samples and controls.

i.E. coli- 12 hours, 37C

ii. Candida- 24 hours, 30C

4.Killing Assay for blood

a.Vortex blood sample (at least 10sec).

b.Add 1.5uL blood to 34.5uL (1:23 dilution) CO2-independent media plus 4mM L-glutamine.

c.Make blank tubes by adding 1.5uL blood to 34.5uL CO2-independent media plus 4mM L-glutamine.  This should be done for EACH blood sample being assayed, as each will have a slightly different read on the spectrophotometer.

d.Add 12.5uL of 105 bacteria/ml solution to each sample tube (there should be 3) and 12.5uL sterile PBS to each blank tube (only 1).

e.Vortex.

f.Incubate at desired temperature (37 for E. coli and Staph; 30 for Candida) for 1h.

g.Remove samples from incubator and vortex.

h.Add 250uL broth to each sample.

i.Make control samples by adding 250uL broth to 48.5uL 105 bacteria/ml solution (in triplicate); the blank for these controls is 250uL broth + 48.5uL sterile PBS.

j.Incubate all samples and controls.

i.E. coli- 12 hours, 37C

ii. Candida- 24 hours, 30C

5.Spectrophotometric read

a.After broth has incubated for appropriate amount of time, remove tubes from incubator and vortex.

b.Using cell culture setting on spectrophotometer.

c.Blank with 2uL blank solution.

d.Remove 2uL of sample solution, place on spectrophotometer.

e.Measure the absorbance at OD300.

f.Wipe off sample with KimWipe or similar product.

g.Repeat for remaining samples; when using blood samples, blank as needed (we blank after ever 10 samples or so).