Slides were loaded into a BioTek slide in both a coverslip down Optimal exposure settings were determined for each objective using a positive control smear and then applied to all images for both positive and negative controls. Our group used microscope BioTek to do gram staining protocol with sample was CF594 (red color/cell wall) and Dapi (Blue color/ Nuclei) With + DAPI 377/477 1225100 Rev J - Blue + GFP 469/526 1225101 Rev K- green + TEXAS 586/647 125102 Rev J - Red I very like this new technique, and I think it is more exactly than normal gram staining technique I have used before. I hope I will do it few more time in future so I can use to do this new method.
1. Materials - Crystal violet (primary stain) - Gram's iodine (mordant, to fix crystal violet in the cell wall) - Ethanol or Acetone (decolorizer) - Safranin (secondary stain or counterstain) - DI Water - Microscope slide 2. Procedure - Place a small drop of bacterial sample on a slide. Heat fix the bacteria to the slide by passing it through the flame of a Bunsen burner three times. Applying too much heat or for too long can melt the bacteria cell walls, distorting their shape and leading to an inaccurate result. If too little heat is applied, the bacteria will wash off the slide during staining. - Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1 minute. Gently rinse the slide with water no longer than 5 seconds to remove excess stain. Rinsing too long can remove too much color, while not rinsing long enough may allow too much stain to remain on gram-negative cells. ...
I did the Transformation of E. cloni 10G Chemically Competent cells: 1. Remove Recovery Medium from the freezer and bring to room temperature. 2. Remove E. cloni 10G cells from the -80°C freezer and thaw completely on wet ice (10-15 minutes). Tube (+) Tube (D) Tube (I) 3. Thaw the tube of pRham™ Vector DNA and microcentrifuge the tube briefly to collect the solution in the bottom of the tube. 4. Add 2 µL (25 ng) of the pRham Vector DNA and 1 to 3 µL (25 to 100 ng) of insert product to the cells. Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells. 5. Important: Transfer the mixture of cells and DNA to a pre-chilled disposable polypropylene 15- mL culture tube (17 x 100 mm). Performing the heat shock in the small tube in which the cells are provided will significantly reduce the transformation efficiency. Summary: Put 2 µL Prham ™ C-His + 3 µL transformation to tub...
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