Sam:LabNotes/Microbiome-new/2011-4-5: Difference between revisions

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>Sam Chiang
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='''Single E.coli cell micromanipulation new test on TrypenBlue stained E.coil'''=


==Background==
*From previous test on 03-31-11, we learned how to set up some basic conditions for targeting E.colis
**Use cover slide to keep sample from evaporation.
**Able to target E.coli cell without any staining.
**Still can see many debris >= the size of E.coli. Hard to define
==Objective==
*Test Trrypen BLue stained E.coli
*Improve glass slide washing using isopropanol
*Improve E.coli washing using 0.2um filtered 1XPBS.
*Make new micro-pipette tips with narrower mouth-open between 10~20um (by Andy)
==TrypanBlue(TB) staining==
*Use the same E.coli culture from 03-31-11 test
*E.coli washing at low-speed (4000 rpm, 5min at RT)with 0.2um filtered clean PBS.
**Use low speed to avoid the precipitation of cell debris.
*Resuspend the E.coli cells in 1.5mL PBS -> low-speed pelleting -> Discard supernatant
*Repeat the last step again.
*Resuspend in 1.5mL PBS (estimated conc. as 10^9/mL).
*Prepare two 1.7mL tube labeled as TB(E.coli) and TB(Neg)
*Transfer 10uL E.coli (10^9/mL) culture into the 1.7mL tube. Transfer 10uL PBS in the TB(Neg.) tube.
*Add 10uL undiluted TrypenBlue into 1.7mL tubes. Mix by repeat pieptting and incubate at RT for about 10min.
*Add 980uL 1XPBS (estimated conc. became 10^7/mL)
*Perform anther 1/100 dilution by adding 2uL stained sample in 198uL PBS (estimated conc. became 10^5/mL)
**since I use low-speed washing in the first step, I lost more E.coli during washing so I start with 10^5/mL for micromanipulation.
==Results==
*E.coli cells look like black spots no matter is a TrypenBlue stained or not. In a diluted sample. since we are not using eye piece to observe and manipulate. The camera is monochrome so the TB stained E.coli look the same as non-stained E.coli cell.
**There is no significant improvement using TrypenBlue staining (Or I didn' stain E.coli cell in the right way?)

Latest revision as of 20:27, 12 April 2011