Layered plates

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General Information

The cells being plated are sandwiched between layers of cell-free agar. The submerged cells grow into smaller colonies and many more colonies can be counted per plate (I've been able to distinguish over 1000 yeast colonies per plate using this method--Jgritton 14:46, 22 Aug 2005 (EDT)). In addition by avoiding the use of a spreader you can eliminate the error due to cells sticking to your spreader. The result is more accurate estimates of cell numbers in a sample.

Procedure

1. Prepare petri plates with 0.4 cm of base agar medium.
   • LB (or other nutrient) with 1.2% agar for bacteria
   • YPD (or other nutrient) with 2% agar for yeast
2. Prepare 0.7% agar medium (soft agar medium)
3. Warm small test tubes to 45 C and add 3 mL of soft agar medium.
4. Pipette sample to be plated onto inside rim of test tube.
5. Immediately pour soft agar onto the base agar, pouring over the pipetted sample.
6. Swirl plate to spread sample/agar mix and allow to set
7. Pipette or pour 2 mL of soft agar onto plate and swirl to spread. Allow to set.
8. Incubate plates

Materials

YPD Soft Agar

1. In a 2L flask add:
   • 10g Yeast Extract
   • 20g Peptone
   • 7g Agar
2. Add water to 950 mL
3. Autoclave
4. Add 50 mL 40% glucose
5. Dispense into bottles (~330 mL into each of three 500 mL bottles) and allow to cool
6. Microwave to melt agar before use

Reference

Koch, A.L. Growth Measurement in Methods for General and Molecular Biology 1994 pg 249-277

• MIT Library Call #: QR65.M26 1994 (Hayden Library)