pseudomonasexp3 page1procedures

EXPERIMENT 3: PSEUDOMONAS (NON-MUCOID FORM) INFECTION OF UPA KNOCKOUT MICE This is the first project I was involved with at the University of Michigan in July/August of 98. Below is a small description of the procedures and concepts involved.

Intra-Tracheally (IT) inject 30 uL of 1X10EE7 pseudomonas bacteria into 20 +/+ and 20 -/- mice. Record time after each mouse so that time points can be taken. Click here for IT PROCEDURE.

The following mice and time points were used:

8 +/+ 4 HR
8 -/- 4 HR
8 +/+ 24 HR
8 -/- 24 HR
4 +/+ Survival
4 -/- Survival

After four hours from initial IT, overdose mice with Pentabarbitol. Click here for overdose procedures.
The lungs were lavaged from 4 mice in each of the two groups (+/+, -/-). Click here for Bronchio-Alveolar Lavage (BAL) procedures and details.
1 ml of buffered Formaline was injected into the lungs through the same cathoder as described in BAL procedures above.
The following procedures detail the steps that were followed after the BALs.
Only four mice from each group have been used so far for each time plot (8 total). Four more mice from each group were obtained (16 total / time plot).
The remaining 8 mice were sacrificed and bled retro-orbitally for blood smears.
These mice were not all sacrificed at one time. Instead, one was sacrificed and then the following procedure was used.
While the heart is still beating, the lungs were expsosed and profused through the heart with3 ml PBS. The lungs were then removed. Click here for detailed procedures regarding these methods.
All the above procedures were repeated for the 24 hour time plot. The survival mice were observed to see if they cleared the induced bacterial infection.
The blood smear slide and the cyto spin slides were hema-stained. Click for procedure.
Neutrophils and Macrophages were counted in six fields on each slide. The hypothesis is that the UPA -/- should have less neutrophils.
The formaldehyde fixed samples were embedded in parafin using this procedure.
The samples were cut and stained.
The neutrophils and bacteriophages were counted in the cuts.
The lungs that were profused with PBS and then removed and snap frozen were minced in 1 ml sterile PBS.
A Colony forming units (CFU) assay was performed.
A Mylo-Peroxidase (MPO) assay is also performed using 100 uL of the lung mince solution. Mylo-Peroxidase is produced by neutrophils and indirectly measured here. Click here for MPO procedures.

That concludes this experiment. The results will be up as soon as possible.