Preparing Agar Makes 500 mL

1
Preparing Agar(Makes 500 mL)

• By Laurie Brier

2
Materials Needed

• Digital gram scale
• Weigh boats
• Glass bottle
• Spatula
• p1000 micro pipette
• With appropriate tips
• Thumbwheel pipetter
• With sterile 30 mL pipette tube
• A large graduated cylender
• NaCl
• Bactopeptone
• Difco agar
• Distilled water
• 1 M CaCl3
• 1 M MgSO4
• 1 M KH2PO4 _at_ pH 6
• Cholesterol
• Access to an autoclave
• Plenty of petri dishes

3
Step 1

• Place weigh boat on the scale and set the weight
  to zero grams by pressing the TARE button.
• Remember to make sure that the scale is set to
  grams

4
Step 2

• Use the spatula to measure out 1.50 g of NaCl
  into the weight boat

5
Step 3

• Empty the contents of the weigh boat into the
  bottle

6
Step 4

• Repeat steps one through three with
• 1.25 g bactopeptone
• and
• 8.5 g difco agar

7
Step 5

• Use the graduated cylinder to measure 487.5 mL of
  distilled water
• Add the water to the bottled mixture

8
Step 6

• Gently shake the bottle to mix contents

9
Step 7

• Use the p1000 micro pipette to measure out 250 µL
  or 1 M CaCl2 and add it to the mixture.

Photographed by Kathy Szeniawski
10
Step 8

• Repeat step seven with 500 µL of 1M MgSO4.

11
Step 9

• Using the thumbwheel pipette, add 12.5 mL of 1M
  KH2PO4 (pH 6) to the bottal.

12
Step 10

• Autoclave the bottled mixture.
• REMEMBER
• To use autoclave tape
• To make sure that the lid of the bottle is on
  LOOSELY

13
Step 11

• Remove agar from autoclave and let cool few a few
  minutes.
• USE GLOVES!

14
Step 12

• Using the p1000 pipette add 500 µL of cholesterol
  to the agar.

15
Step 13

• Gently swirl the agar to mix.
• Be careful when mixing. You do not want any air
  bubbles in your agar.

16
Last Step!

• Use the thumbwheel pipette to distribute the agar
  into petri dishes.
• Small dishes get 6-10 mL each
• Large dishes get 20-25 mL each