Protocols
Making LB Plates

Making LB Plates

Bacterial Culturing

Procedure

Before you start: If you wish, you can cool your media in a 60° water bath. This is especially helpful if you are adding heat-sensitive components to the agar before pouring — especially antibiotics. Prepare a water bath at 60 °C with sufficient water to submerge ~75% of the flask containing your media. As it takes time for the water bath to come up to temperature, it is wise to set it up well before you put your media into the autoclave.

Step 1. Measure 25g of LB powder per L of media you’d like to make. The precise mass you measure out will be based on the number of plates you’d like to pour.

Example: The 100mm plates can hold a maximum of 25ml of media. If you want to pour a stack of 20 plates, you will need $$ \frac{25ml}{plate}\times 20 plates=500ml $$ of media total. To calculate the necessary amount of LB powder for 500ml of media total, you either need to use milliliters in your LB powder/volume ratio, or use 0.5L as the volume you are making. Remember that 1L contains 1000ml : $$ \frac{25g}{1000ml}\times500ml=12.5g $$ --OR-- $$ \frac{25g}{1L}\times0.5L=12.5g $$

Step 2. Transfer the LB powder you’ve measured out into an appropriately sized flask for autoclaving. Always use a flask that holds double the volume you intend to make. The extra empty volume is necessary to prevent your molten agar from boiling over in the autoclave.

Step 3. Measure 15g of agar powder per L of media you are making. As with the LB powder, amount will be based on the number of plates you are pouring pour.

Example: For 500ml of media needed for 20 plates: $$ \frac{15g}{1000ml}\times500ml=7.5g $$

Step 4. Transfer the agar powder to the flask.

Step 5. Fill the flask partially with distilled water and swirl vigorously to mix.

Step 6. Cover the opening of the flask with aluminum foil and put a piece of autoclave tape on the foil. The autoclave tape will darken during the autoclave process if your sample has spent at least 10 minutes at 123 °C.

Step 7. Place the covered flask on a tray and put the tray in the autoclave. Run on the slow-exhaust (liquid) cycle for 15 minutes. High pressure will prevent your media from boiling over at a temperature above water's boiling point, and the slow exhaust of steam pressure will make sure the solution doesn't boil over when the cycle is complete.

Step 8. Although 123 °C should kill most potential contaminants, certain types of spores will stubbornly survive even under the harsh conditions in the autoclave. If you have difficulties with contamination, especially with Bacillus species, autoclave twice. The first cycle will kill the majority of organisms. Let the media cool to room temperature — at which point surviving spores should 'hatch' — then autoclave again.

Step 9.

While your samples are sterilizing in the autoclave, you should prepare your plate pouring station.

Find an empty section of lab bench with a working bunsen burner.
Spray down the bench with a 70% ethanol solution and wipe down with a paper towel.
Count out the appropriate number of plates and stack them on your lab bench. Label the bottom of the plates with the date and the medium they will contain including any additives, like antibiotics, for instance. Use a sharpie.
You can batch label your plates with a sharpie — different number or color of stripes could correspond to different media types.
Position the flame just to the side of where you’ll be pouring your plates — be sure to leave room for your flask of molten media, a tube rack containing any additives, and a section for active pouring.

Step 10. Retrieve your molten agar mix from the autoclave. If you have set up a water bath, cool the media in the water bath prior to pouring. In a 60° water bath the agar agar will remain liquid at this temperature but most antibiotics will not break down at this temperature.

Step 11. Light the Bunsen burner at your plate pouring station

Step 12. Swirl the agar bottle to ensure even distribution of agar.

Step 13. Open one plate at a time next to the flame and begin pouring directly from the flask. fill the plates roughy half full. Cap each plate after pouring and stack as you pour.

Step 14. It takes roughly 30 minutes for plates to solidify at room temperature, but you might want to leave them out at room temperature overnight to allow them to dry.

Step 15. After they have set, if you are not going to use them right away, slip the plastic sleeve they came in over the stacked plates, tape the sleeve closed, and label the sleeve.

Required recipes

Target volume

Unit

Step 1. Prepare ingredients.

LB Broth (Lysogeny Broth)

25 g

Step 2. Prepare ingredients.

Agar

15 g

Step 3. Bring final volume to 1000 mL with water.

Used By

Culturing Bacteria from Soil Samples

View

Adding Antibiotics and other Additives to Agar Media

View