Embedding yeast colonies for light and electron microscopy
Incubate approximately 300 colonies on agar medium for the indicated time .
Remove an isolated colony ( 1 - 2 mm in diameter ) and a small amount of the underlying agar medium , and place on a microscope slide .
Place several drops of 2 % agar ( 42°C ) on a microscope slide , and immediately place the colony on the agar and then place several drops of agar on top of the colony and allow to solidify .
Trim the resulting agar block with a razor blade , and place in a 3.5 ml borosilicate screw - cap vial ( Fisher 03 - 339 - 21B ) containing 1.5ml , 2 % , paraformaldehyde / 2 % , glutaraldehyde .
Fix colonies by incubating for 7 days at 4°C .
Wash # 1 : Wash agar blocks on ice by incubating for 15 minutes with 1.5 ml of 0.15M sodium cacodylate ( pH 7.2 ) .
Wash # 2 : Wash agar blocks on ice by incubating for 15 minutes with 1.5 ml of 0.15M sodium cacodylate ( pH 7.2 ) .
Wash # 3 : Wash agar blocks on ice by incubating for 5 minutes with 1.5ml OS buffer ( 100 mM KH2PO4 , 10 mM MgCl2 , pH 6.0 ) .
Wash # 4 : Wash agar blocks on ice by incubating for 5 minutes with 1.5ml OS buffer ( 100 mM KH2PO4 , 10 mM MgCl2 , pH 6.0 ) .
If sections will be used for electron microscopy , add 1 % OsO4 in OS to vials to cover the agar blocks and incubate on ice in a chemical fume hood for 1 hr .
Otherwise skip to step 13 .
Dispose of 1 % OsO4 in hazardous waste .
WashA : Wash with 1.5ml OS buffer by incubating on ice for 10 minutes .
WashB : Wash with 1.5ml OS buffer by incubating on ice for 10 minutes .
Add 1.5 ml OS and incubate overnight at 4°C .
WashA : Wash blocks with 1.5ml cold water by incubating on ice for 10 minutes .
WashB : Wash blocks with 1.5ml cold water by incubating on ice for 10 minutes .
Wash # 1 : Add 1.5ml cold 25 % ethanol and incubate on ice for 10 minutes .
Wash # 2 : Add 1.5ml cold 50 % ethanol and incubate on ice for 10 minutes .
Wash # 3 : Add 1.5ml cold 75 % ethanol and incubate on ice for 10 minutes .
Wash # 4 : Add 1.5ml cold 95 % ethanol and incubate on ice for 10 minutes .
Wash # 5 : Add 1.5ml cold 100 % ethanol and incubate on ice for 10 minutes .
Wash # 6 : Add 1.5ml cold 100 % ethanol and incubate on ice for 10 minutes .
Remove ethanol and resuspend in 1.5ml cold 100 % ethanol .
Leave the blocks overnight at 4°C in 100 % ethanol .
Make Spurr's reagent by stirring slowly under chemical fume hood 5 grams ERL4221 , 4 grams DER736 and 13 grams NSA for 20 minutes ( Electron Microscopy Sciences ) .
Add 0.15 grams DMAE ( Electron Microscopy Sciences ) and stir for 20 minutes .
De - gas for 1 - 2 hrs .
Wash blocks with 1.5ml unopened room temp 100 % ethanol by incubating at room temp for 10 minutes .
Repeat 4 more times .
Treatment # 1 : Remove ethanol and add 1.5ml of a 2 : 1 ratio of 100 % ethanol : Spurr's reagent .
Treatment # 1 : Rotate vial for 15 minutes on wheel at room temperature .
Treatment # 1 : Allow to stand for 30 minutes at room temperature .
Treatment # 2 : Remove 100 % ethanol : Spurr's reagent and add 1.5ml of a 2 : 1 ratio of 100 % ethanol : Spurr's reagent .
Rotate vial for 15 minutes on wheel at room temperature .
Treatment # 2 : Allow to stand for 30 minutes at room temperature .
Treatment # 3 : Remove 100 % ethanol : Spurr's reagent and add 1.5ml of a 2 : 1 ratio of 100 % ethanol : Spurr's reagent .
Rotate vial for 15 minutes on wheel at room temperature .
Treatment # 3 : Allow to stand for 30 minutes at room temperature .
Treatment # 4 : Remove 100 % ethanol : Spurr's reagent and add 1.5ml of a 1 : 1 ratio of 100 % ethanol : Spurr's reagent .
Rotate vials for 15 minutes on wheel at room temperature .
Treatment # 4 : Allow to stand for 30 minutes at room temperature .
Treatment # 5 : Remove 100 % ethanol : Spurr's reagent and add 1.5ml of a 1 : 1 ratio of 100 % ethanol : Spurr's reagent .
Rotate vials for 15 minutes on wheel at room temperature .
Treatment # 5 : Allow to stand for 30 minutes at room temperature .
Remove 100 % ethanol : Spurr's reagent and add 1.5ml Spurr's reagent to vial .
Incubate for 4hrs at room temperature .
Replace with 1.5ml Spurr's reagent and rotate the vial overnight on the wheel at room temperature .
Replace with 1.5ml Spurr's reagent and rotate the vial until late afternoon on the wheel at room temperature .
Remove the Spurr's reagent and replace with 1.5ml freshly made Spurr's reagent .
Rotate the vial overnight on the wheel at room temperature .
The next day replace with 1.5ml Spurr's reagent and rotate until the following day on the wheel at room temperature .
The next day replace with 1.5ml Spurr's reagent and rotate until the following day on the wheel at room temperature .
Place each agar block in a mold with 0.2ml Spurr's reagent and incubate at 60°C for four hours .
Top off the molds with Spurr's reagent and incubate at 60°C for 3 days .
Collect sections ( 0.5u ) from the central region of the colony in a drop of dH20 on a glass slide .
Dry slide on a 52°C heat block .
Stain sections with 1 % toluidine blue , 1 % Sodium Borate for 5 - 15 seconds .
Wash slide under a stream of dH20 .
Dry on heat block .
Cover in Permount ( Fisher SP15 - 100 ) .
Examine by light microscopy .
