TITLE: Monitoring of Citrobacter rodentium shed from infected mice using luminometry and viable counts
AUTHORS: Hannah Read, Priyali Patel, Siouxsie Wiles

[DESCRIPTION]
Citrobacter rodentium is a Gram-negative bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. As a gastrointestinal pathogen, C. rodentium is shed in the stools of infected animals. This allows in vivo infection dynamics to be monitored by plating out the bacteria present in shed stools. We routinely use a bioluminescent derivative of C. rodentium called ICC180 which contains the lux operon from Photorhabdus luminescens. This allows us to monitor infection dynamics using luminometry too.



[BEFORE_START]
Label and weigh individual microfuge tubes. Write the weight on the side of the tube.
Remove inserts from pipette tip boxes

[GUIDELINES]
Experiments involving animals and pathogenic bacteria require ethical and biological safety approval. When planning experiments involving animals, consult the PREPARE and ARRIVE guidelines.

Luminometry: Luminometers measure light emission using photomultiplier tubes, which convert photons into electrical pulses. Luminometers come in many formats, from a simple manual one-tube luminometer to a computer-controlled micro-titre plate instrument. Detected photons are often displayed as relative light units (RLU) or counts per second. Absolute values of light emission vary greatly from luminometer to luminometer for a given amount of luciferase protein. For this reason, samples from an experiment should be run on the same machine using the same protocol. Also note, when using micro-titre plate instruments, light can spill over from wells containing bright samples to the surrounding wells if using white or clear plates. We routinely use black plates to minimise such spill over.

[STEPS]
SECTION: Stool collection
1. Place each mouse in a clean pipette tip box which has had the insert removed.
SECTION: Stool collection
2. Once a mouse has produced 1-3 individual stools, transfer these to a sterile labelled microfuge tube of known weight using a pair of clean tweezers. 

Depending on the mouse strain, stools can become very loose and much more difficult to pick up using tweezers. In these instances, try using a small laboratory spatula instead. We've also observed mice placing their stools up on the walls of the tip box like they are moving them out of the way or making an offering!   
SECTION: Stool collection
3. Transfer the mouse back to its home cage.
SECTION: Stool collection
4. Clean the pipette tip box and tweezers using an approved disinfectant.
SECTION: Stool suspension and homogenisation
5. Weigh each tube again to find the weight of the stool. 

SECTION: Stool suspension and homogenisation
6. Add PBS to a final concentration of 0.1 g of stool per 1 mL  of PBS.
SECTION: Stool suspension and homogenisation
7. Homogenise using a vortex.
SECTION: Viable counts
8. Allow any large remaining stool particles to sink to the bottom of the tube. 
SECTION: Viable counts
9. Prepare a 10 fold serial dilution of the stool homogenate. This can be done by removing 100 µL of the stool homogenate and adding to 900 µL PBS in a microcentrifuge tube and repeating for as many dilutions as necessary. Alternatively, fill the wells of a 96 well plate with 90 µL of PBS and add 10 µL stool homogenate to the first well in each column. Then perform serial dilutions down the plate using a multichannel pipette. Make sure to use clean pipette tips for each dilution.

SECTION: Viable counts
10. Plate 3 aliquots of 25 µL of each dilution onto a dry LB Agar plate containing kanamycin. If the plate is wet, the droplets will roll into each other and you won't be able to count the individual colonies.

SECTION: Viable counts
11. Incubate plates   at 37 °C and then count the C. rodentium colonies present at each dilution. 

SECTION: Luminometry
12. Record the luminescence of the first 1-2 dilutions from the serial dilution of the stool homogenate using a luminometer according to the instrument's instructions. 

Depending on the sensitivity of the luminometer, very bright signals can saturate the machine. This is why it is useful to run the first two dilutions at the peak of infection when there are large amounts of bacteria present. If the luminescence reading from the second dilution is not 10 times lower than the first dilution then you will know the first sample was saturated and the reading won't be accurate. 

The light from bright samples can also spill over to adjoining wells if using a micro-titre plate luminometer which is why we prefer to use black microtitre plates which reduce such spill over.