Measuring the intracellular proliferation of Salmonella

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that is a major cause of food-acquired gastroenteritis. One key characteristic of S. Typhimurium is its ability to survive and replicate within macrophages, white blood cells that normally function to destroy invasive pathogens. This practical is designed to quantify this intracellular proliferative capacity and test how it is modified in a mutant strain.

Salmonella Survival Assay: 24 well plate

Day before (done for you)

Set up 24 well tissue culture plate with 2×105 macrophages per well in each of six wells.
Set up o/n cultures of the bacterial strains in 3ml LB

24-well plate layout:

Strain 1
T0

Strain 2
T0

Strain 1
T16

Strain 2
T16

Strain 1
Reserve well

Strain 2
Reserve well

Day 1 (Wednesday 4th Feb)

Bacterial preparation

You are provided with a PBS suspension of 2 different Salmonella (Keep these for your INPUT dilutions later)
dilute each Salmonella suspension 1/100 into DMEM+g (3ml for each strain) = infection medium
wash macrophages 1x with 1ml PBS
Add your 1ml infection medium to each well. Infect THREE wells with each strain (i.e. six wells in total).
incubate 20min at 37°C
INPUT dilution

Using the PBS Salmonella suspensions, perform a serial dilution.
You have been provided with 6 tubes for each strain (12 total) containing 1.8 ml sterile saline.
Starting from the PBS suspension, transfer 200 μl into tube 1. This is a 1/10 dilution (10-1). Continue this process until you have a 10-6 dilution for each strain.
Using the sterile plastic spreaders, plate 100 μl of your 10-5 and 10-6 dilutions for each strain onto LB agar plates (4 plates total).

End of infection

After the 20 min infection is finished, remove infection medium from all wells and replace with 1ml DMEM+g+100 μg/ml gentamycin.
incubate further 30min, to kill external bacteria = Time point 0 (T0)

T0 dilution

after the 30 min gentamycin incubation is over, remove antibacterial media
Add DMEM+g+FBS+10 ug/ml gentamicin to your T16 wells
For ONE well for each mutant (the “T0 well”):
wash macrophages 1x in warm PBS
add Triton-X 0.02% 1ml
incubate 15min
Serial dilute the lysate from T0 wells down to 10-2
Using the sterile plastic spreaders, plate 100 μl of the 10-1and 10-2 dilution
Return tissue culture plate to the incubator (continue infection of T24 wells).

Day2 (Thursday 5th Feb)

T16 dilution

after 16 hours (from T0)
wash macrophages 1x in warm PBS
add Triton-X 0.02% 1ml
incubate 15min
Serial dilute the lysate from T24 wells down to 10-3
Using the sterile plastic spreaders, plate 100 μl of the 10-2 and 10-3 dilution

The following week

Count the colonies on the most appropriate dilution plate for each sample. Using these data, calculate:

what percentage of your initial inoculum successfully invaded a host cell
whether that intracellular population then grew or died over 16hrs and by what percentage

The Write-Up

The write-up of this practical forms part of the continual assessment mark for this project. Your write up should consist of three parts:

Part A (<500 words)

Briefly outline the biology and pathogenesis of Salmonella Typhimurium and describe its intracellular lifestyle.

Part B (<1000 words)

In the form of a brief scientific paper, describe and interpret the practical work and the data you obtained. Briefly introduce the question to be addressed, describe the methods used and use appropriate figures to display your results. Include a short discussion to describe your findings and their implications.

Part C (<500 words)

The Salmonella mutant strain you were provided with carried a deletion in the tolC gene. Briefly describe the function of tolC and how this function may relate to intracellular pathogenesis in this organism