1.2: Media Preparation

Learning Objectives

• Define medium/media.
• Tell that microbes require nutrients to grow in the laboratory.
• Differentiate between broth, slant, deep, and petri plate.
• Calculate the amount of medium powder required to make a specific volume of medium.
• Successfully prepare microbiological media.

 

Microbiological Media

Just like all other living things, microbes require nutrients (food) to grow and live. Microbiological media (singular is "medium") is a mixture of water and nutrients necessary to grow microbes. Different types of media also can be used to provide information about the different characteristics microbes have.

Many types of microbiological media can be ordered in powder form from a science supplier. Instructions for preparing the medium are presented on the medium container. Typically, this involves dissolving a certain number of grams of the media powder in one liter of distilled or deionized water ("grams per liter" which is written as g/L). This does not mean that one must always make one liter of medium. The amount can be modified by calculating the amount of microbiological media powder required for the concentration (g/L) listed on the bottle)

Figure 1: Media can be prepared as a broth (liquid), a slant (agar in a test tube that has been slanted when cooling to create a larger agar surface area), a deep (agar in a test tube typically inoculated using an inoculation needle by stabbing into the agar), and a petri plate (a larger surface area for growing microbes on the surface).

 

Calculating Amount of Medium

Use the following formula to determine the amount of powdered medium to weigh and dissolve into DI or distilled water:

amount of solution you want to make (mL) x [concentration (g/1000 mL)] = amount of media powder to weigh out (g)

Because 1 L ("one liter") is the same thing as 1000 mL ("one thousand milliliters"), when the instructions on the media powder bottle tell to make a concentration in g/L ("grams per liter"), this is the same thing as g/1000 mL ("grams per thousand milliliters").

 

Calculation Example

Calculation

• In this example, you want to make 500 mL of medium. Instructions on the bottle say to make a concentration of 40 g/L.
• 40 g/L = 40 g/1000 mL
• Set up the formula:
  • 500 mL x [40 g / 1000 mL]
• Calculate the amount to weigh out:
  • Do the parentheses first: 500 mL x [40 g / 1000 mL]
  • Multiply [note that mL units cancel out]: 500 mL x 0.04 g/mL
  • Answer is the amount to weigh out in g ("grams"): 20 g

How to Make the Medium

1. Measure 500 mL of distilled or deionized water (not tap water) with a graduated cylinder.
2. Weigh 20 g of media powder using a balance.
3. Put a magnetic stir bar into a beaker, flask or bottle (must be larger capacity than 500 mL in for this example - 800 mL or 1000 mL) and add about half of the water.
4. Put the beaker, flask, or bottle onto a stir plate and turn it on so the magnetic stir bar is swirling the water enough to stir it well, but not too much that it is jumping and/or creating lots of bubbles in the water.
5. Gradually add the 20 g of media powder to the stirring water.
6. Add the reminder of the water. By adding the remaining water after the media powder, it will help dissolve any powder floating at the top of the water.
7. Allow to stir until the powder is completely dissolved.
8. If the medium is agar, the solution will need to be heated just to boiling (but not boiling over) starting after step 3. Keep stirring and heating until the solution is clear. Be careful not to over-heat since it is easy for the solution to boil over.
9. If the medium will be distributed into test tubes, measure amount into each test tube and add caps.
10. If the medium is for flasks or bottles, measure the amount into each flask or bottle and add caps or other covers (make sure screw-caps are not screwed on tightly before they are autoclaved).
11. If the medium is for petri plates, make sure the medium is in a container with a cover (make sure screw-caps are not screwed on tightly before they are autoclaved).
12. Autoclave.
13. If test tubes contain agar that will be for slants, prop the rack of test tubes so the agar slants as it cools.
14. If the medium is for petri plates, disinfect a workspace and pour agar into sterile petri plates and allow to cool completely.

 

Practice Calculations

Exercise 2.1

In order to make 400 mL of medium with a concentration of 15 g/L, how much medium powder would you weigh in grams?

Answer

Exercise 2.2

In order to make 650 mL of medium with a concentration of 20 g/L, how much medium powder would you weigh in grams?

Answer

Exercise 2.3

In order to make 200 mL of medium with a concentration of 30 g/L, how much medium powder would you weigh in grams?

Answer

Exercise 2.4

In order to make 300 mL of medium with a concentration of 45 g/L, how much medium powder would you weigh in grams?

Answer

Exercise 2.5

In order to make 150 mL of medium with a concentration of 35 g/L, how much medium powder would you weigh in grams?

Answer

 

How to Make Petri Plates

 

Attributions

• Klamm’s Microbiology Laboratory Manual by Loretta Sanderson Klamm is licensed under CC BY-NC-SA 4.0
• LB luquid medium bottle-01.jpg by Lilly_M is licensed under CC BY-SA 3.0