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='''Bacteria labeling using SYBR green'''= ==Objective== * Using SYBR to stand E. coli and confirm using inverted flourescent microscope * Reference **[[Media:Bacteria staining protocol.pdf|Bacteria staining protocol]] **[[Media:Rapid Bacteria staining with laser cytometry.pdf|Rapid Bacteria staining with laser cytometry]] ==Samples & Materials== *Bacteria: E.coli suspension in PBS (OD600=0.913 -> around 10^9 CFU/mL = 10^6 CFU/uL) *SYBR 2X: Diluted from SYBR 50X stock solution ==Procedures== *Dilute SYBR 50X to 2X with RNAse-free H2O: 40 uL of 50X SYBR + 960 uL H2O -> 2X SYBR, 1000 uL. *Prepare four 1.5-mL tube, labeled and wrapped with foil. *Set up as following exp design: No-treat 100uL 200uL 400uL --------------------------- H2O 400 0 0 0 uL 2X SYBR 0 100 200 400 uL E. coli 1 1 1 1 uL --------------------------- *Mix by gently pipetting (p1000 pipettor). Incubate at dark place for 15 min. Agitate by vortexing for every 3~4 min. *After incubation, add H2O to 1 mL for each tube, the new concentration for E. coli -> 1000 CFU/uL) *Prepare two microscopy slide with labeling. *load 1 uL stained E. coli sample from each tube on the slide and place with cover slips. *Keep the slide in dark storage box for microscope visualizing. ==Results== [[Image:40X - B&W - 400 SYBG_(c1).jpg|500px]] [[Image:40X - B&W -N treatment.jpg|500px]] '''Left: 40X, 400 uL 2XSYBR treatment, B&W picture. Right: 40X, 400 uL No-treat, B&W picture''' ==Discussion== *No flourresecnce was observed using Grean light excitation. *The suspecious dot might be the E. coli, which is not too small to confirm by morphology. *A higher magnification such 100X~200X may be necessary for our application **E. coli size may be around 3~10 uM (according previous studies) **The common practice for visulizing microbes is based on the microscopy with 200X magnification and Epi-flourescent microscope type. For better flourescence capturing?)
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