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6-2 Protocol for making media

( 51625 Reads)

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Period 1

Materials

13 tubes of Nutrient Broth

2 250 ml flasks

1 graduated cylinder (100 ml)

magnetic stirrer and stir bar

scales and weighing paper

8 empty sterile Petri plates

1 tube of 50% glucose (2 ml)

1 bottle of 3% Agar + MgSO4 (50 ml, melted in 50 °C water bath)

Media ingredients (as listed below)

Minimal Medium - Part Ag/LNutrient Agarg/L
Ammonium Sulfate7.0Beef Extract5.0
Dipotassium Phosphate7.0Peptone3.0
Monopotassium Phosphate3.0Agar15.0
Minimal Medium -Part Bg/100 ml
Agar3.0
Magnesium Sulfate0.02
Minimal Medium Part Cg/100 ml
Glucose50

To become familiar with what goes into mixing up a batch of medium, 4 plates of a minimal medium and 4 plates of a complex medium will be prepared.

  1. Go to one of the designated measuring areas and weigh out each component required for the Minimal Medium - Part A. Use the recipe listed above. Note: The above recipe is in grams/liter and you are making 100 ml not 1 liter of each medium.
  2. Carry the measured amounts back to your lab bench. Use the graduated cylinder to measure 50 ml of distilled water (dH2O) and pour this into one 250 ml flask.
  3. Add the first ingredient on the list, gently swirl the flask, and let it dissolve completely. Then add the next ingredient. Continue until all ingredients have been added. When finished, place a foam plug in the flask.
  4. Fill the other 250 ml flask with 100 ml of dH2O. In this case we are using a dehydrated ready-made medium. Weigh out an appropriate amount of Nutrient Agar powder, add it to the flask, and swirl. That's it! Many types of media come in dehydrated, premixed form. They are convenient and cheap. Which brings up a rule to remember, scientists always try to make necessary chores, like media making, as quick and convenient as possible, thus freeing their time for more fruitful pursuits.
  5. Place the finished medium in the trays provided. When everyone is finished, attend the autoclaving demonstration. Autoclaving will take about 45 minutes total. During the waiting period, perform the other experiments scheduled for today.
  6. When the medium is sterile, place it in the 50 °C water bath to cool down. The minimal medium needs some additions (a carbon source and agar). Once the minimal medium is cool, add 0.4 ml of 50% glucose (Minimal Medium - Part C) to the medium and 50 ml of Minimal Medium - Part B (This is pre-made and is located in the 50 °C water bath). Swirl the flask and pour the now complete Minimal Medium into 4 plates. Also, swirl the Nutrient Agar flask and pour 4 plates.
  7. Let the plates cool and when completely solidified place them in your lab bench drawer, upside down, until next period.

Period 3

Materials

2 plates of Minimal Medium (MM)*

2 plates of Nutrient Agar (NA)*

Cultures for streaking

A picture of plates prepared plates

Figure 5.1. A picture of plates prepared plates. The appearance of plates after they are made. The minimal medium is colorless (left), while the nutrient agar is tan (right).

*Prepared last period by students

Media Making

  1. Bring out your plates of MM and NA. Examine them for contamination. If any colonies are present, do not use that plate. If you're unsure, have the instructors check them.
  2. To test the microbial acceptance of your media, streak the supplied culture for isolated colonies using a plate of minimal medium and rich medium.
  3. Incubate all tubes at 30 °C.

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