Schilke Lab

Spin Plate Method

Preparation of Petri Dishes

1. Label bottom of petri dishes.
2. Prepare agar:
   • Make appropriate amount of MRS broth. Dissolve using heat and stir bar — does not take much time.
   52.2g MRS boullion per 1L RO Water
   • Add appropriate amount of agar. Dissolve using copious amounts of heat and high stir bar setting — takes time to compeletely dissolve. The solution will become translucent when completely dissolved. Let agar cool for small amount of time: enough to not melt plate but not solidify.
   15 g agar powder per 1L MRS broth
3. Add between 12-20ml of liquid agar to plate — a sufficient amount will cover the bottom of the plate.
4. Let agar set in plates so that agar will be level. Store in cold room until further use.

Addition of sample to plate

1. Properly grow bacteria samples and prepare serial dilutions, if necessary. See Serial Dilutions.
2. Pipette 100µl of sample into corresponding plate (labelled correctly). Eject tip into autoclave bag.
3. Flame hockey stick. Let cool so as not to melt agar — test by touching the tip of the hockey stick to a portion of the plate without the sample (near edge).
4. Spread the sample over entirety of plate by moving hockey stick back and forth while rotating plate. Be careful to not break agar. Replace hockey stick in ethanol beaker.
5. Repeat steps 2-4, using a new pipette tip each time. Be vigilant about correspondingly labelled samples and plates.
6. After all plates are completed, store in incubator for set amount of time (approximately 2 days). Autoclave bag of used tips.

Pour Plate Method

It should be noted that this method, when initially performed, resulted in uniformly small and difficult to count colonies. Contamination of blank plates may also occur; take care to use appropriate microbiological procedure.

Preparation of Agar

1. Label bottom of petri dishes.
2. Prepare agar:
   • Make appropriate amount of MRS broth. Dissolve using heat and stir bar — does not take much time.
   52.2g MRS boullion per 1L RO Water
   • Add appropriate amount of agar. Dissolve using copious amounts of heat and high stir bar setting — takes time to compeletely dissolve. The solution will become translucent when completely dissolved. Let agar cool for small amount of time: enough to not melt plate but not solidify.
   15 g agar powder per 1L MRS broth
3. Aliquot approximately 15ml of liquid agar into screw-top test tubes. Should have equal number of agar-filled test tubes as desired number of plates. Store agar in cool room until further use.

Making Plates with Sample

1. Properly prepare bacterial samples and dilutions. (See Serial Dilutions.)
2. Melt the agar in the screw-top test tubes in a hot bath. Start this process early as it will take copious amounts of time (approximately 4-5 hours unless a better method is determined).
3. Pipette 100µl of bacterial sample into the center of the corresponding petri dish. Eject tip into autoclave bag. Repeat for all petri dishes, using a new tip each time. Lift lid only enough to insert the pipette.
4. Let aliquotted agar vials cool for small amount of time: enough to not melt plate but not solidify. Temperature should be fine if can hold test tube in hand.
5. Pour one vial of agar into one plate, lifting plate lid only enough to pour agar.
6. Thoroughly mix the contents of the petri dish by tilting and rotating the plate (or with a mechanical rotator). Take care to not splash mixture on lid or edges.¹
7. Repeat steps 8-9 for each plate.
8. Let agar solidify (no longer than 10 minutes¹).
9. After all plates are completed, store in incubator for set amount of time (approximately 2 days). Autoclave bag of used tips.

Colony Counting Techniques

• Pick a wedge fraction of the plate and mark this part off with a ruler – try to make it representative of the plate.
• Use a marker to dot over counted colonies.
• Limit distractions like Matt. Rat, however, is not a distraction – listen to him.
• Record the number of colonies in the counted wedge then multiply for colonies in the entire plate. Include ±error.

References

¹ Speck, Marvin L. Compendium of Methods for the Microbiological Examination of Foods. 2nd Edition. American Public Health Administration. 1984.

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