5 The role of quorum-sensing in the virulence of Pseudomonas ...

2.5.2 Organ retrieval & CFU counts
For acute infection experiments, mice were culled and samples were taken for
analysis 18 hours post-infection, blood was taken via cardiac puncture, 20µl was
immediately added to 180µl PBS, the nasopharynx and lungs were removed by
dissection and homogenised in 10ml of pre-weighted PBS.
20µl of the homogenate or blood suspension was serially diluted to 10 3 , in 180µl PBS.
Six 20µl drops of each dilution were plated onto LA plates, left to dry and incubated
at 37ºC overnight. The dilution with between 30 – 300 colonies was counted and the
mean of the six spots was counted, multiplied by 50 to calculate the number of
colonies per ml and then multiplied by the dilution factor. The result is the colony
forming unit per ml (CFU/ml).
2.5.3 Symptom scoring
At the end point of 18 hours post-infection and frequently during survival
experiments, the symptoms of each individual mouse were monitored. There were
three signs to monitor; hunched, starry coat and lethargic each monitored on a scale of
0 = no signs, 1 = starting to show symptom and 2 = obviously exhibiting the symptom
(Morton, Griffiths 1985). The three scores were added together to give a total
symptom score out of 6. Mice that had a total symptom score of 6 were culled
immediately as the experimental end point as defined by the home office project
licence.
2.5.4 Histopathology
After the mice were culled, the lungs were removed and placed within 5ml PBS.
Aluminium foil was made into a cylinder shape by firmly wrapping the foil around a
5ml Bijoux tube. A metal container was placed in dry-ice and iso-pentane was
carefully poured into the metal container. Tissue Tek OCT tissue embedding
compound (Miles) was poured into the cylinder aluminium mould to cover the
bottom. The lungs were then added to the cylinder aluminium mould and small
amount of OCT tissue embedding compound was added to just cover the lungs. The
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