Preparation of Minicolonies for Scanning electron microscopy:

1) SK1 minicolonies are grown as described in the basic protocol except that 400-600 ul (600-900 cells) is inoculated in each well of an 8-chamber Permanox slide (Lab-Tek, Catalog # 177445). 

2) After incubation, spent medium is removed from the well by touching a pipetman tip to the edge of the well, and the well washed 4X with 200 ml cacodylate buffer (0.2M sodium cacodylate [pH 7.2]).  After this, 400 ul of 2.5 % glutaraldehyde / sodium cacodylate buffer is added, and the sample incubated for 90 minutes at room temperature.

3) The fixative is then removed from the wells, and the wells washed two times with 200 ul sodium cacodylate buffer and two times with 1X O buffer (100 mM KH₂PO₄, 10 mM MgCl₂, pH 6.0).

4) In a fume hood, 200 ul of 1% OsO₄ / 1X O buffer is added, and allowed to incubate on ice in the hood for 1 hr. 

5) After the incubation, continuing to work in the hood, the OsO₄ solution is removed, and the wells washed 2X in O buffer and 3X in dH₂O (200 ul each wash). 

6) After removing the last wash, samples are removed from hood and cells progressively incubated for 5 minutes each in 200 ul 33%, 67%, 85%, 95% and 2X in 100% ethanol.  Immediately after the 100% ethanol incubations, ethanol was removed and 200 ul of hexamethyldisilazane (HMDS) added to the slide for five minutes.  After the HMDS was removed, the wells were removed from the slide, and the sample dried in a fume hood overnight.

7) The next day the samples are coated with gold/palladium.  Samples were visualized in a FEG ESEM XL30 scanning electron microscope (FEI/Philips, Hillsboro, OR).

Recipes:

VLN-YPA (liquid)

            10g Potassium Acetate

            5g  Peptone

            2.5g Yeast Extract

                        pH 5.8 with Acetic Acid

                        to 1L with ddH2O

                        Autoclave

 

2X O Buffer (200 ml)

            150 ml ddH₂0

            5.44g KH₂PO₄

            0.81 MgCl2*6H₂0

                        pH to 6.0 with KOH

                        ddH₂0 to 200 ml

                        Store at 4° C

 

1X O  Buffer

            Dilute 2X O Buffer to 1X in dH₂O

            Store at 4° C

 

1% OsO4 / 1X O Buffer

 

0.2M Na Cacodylate Buffer

            200 mM Na Cacodylate

                        pH to 7.2 with NaOH

Store at 4° C

           

2.5% glutaraldehyde/ sodium cacodylate

Dilute 50% glutaraldehyde to 2.5% in 0.2M Na Cacodylate Buffer (pH 7.2)

Store at -20° C

 

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