SEA-PHAGES | Plaque Bottom Agar

Link to this post \| posted 08 Sep, 2015 17:31
GregFrederick@letu.edu	I may be missing this. But everywhere I have looked I just see "Agar Plate" listed. QUESTION: So from the Resource Guide I cannot really tell. The only agar listed in the Appendix is for Luria Agar and Top Agar (1x and 2x). Is the "Bottom Agar" when plating for plaques actually Luria agar or is it Middlebrook 7H9 Complete with CaCl2 and 1.5% Agar or what? I'm guessing it is the 7H9 complete agar. But I want to optimize our chances for success. It may be in the resource guide somewhere. But I'm not finding it. Thanks for being out there! Greg

Link to this post | posted 08 Sep, 2015 17:31

I may be missing this. But everywhere I have looked I just see "Agar Plate" listed.

QUESTION: So from the Resource Guide I cannot really tell. The only agar listed in the Appendix is for Luria Agar and Top Agar (1x and 2x).

Is the "Bottom Agar" when plating for plaques actually Luria agar or is it Middlebrook 7H9 Complete with CaCl2 and 1.5% Agar or what? I'm guessing it is the 7H9 complete agar. But I want to optimize our chances for success.

It may be in the resource guide somewhere. But I'm not finding it.

Thanks for being out there! Greg smile

Link to this post \| posted 09 Sep, 2015 16:15
bradleyk	Hi Greg, The bottom agar is L-Agar (Miller recipe) found on Part 1, Appendix 6, pg 56: Luria Agar (L-Agar) Plates (1L) Ingredient Amount Final Concentration L-Agar base 30.5 g ddH2O To 1L CB stock* 1.0 mL 50 μg/mL CHX stock* 1.0 mL 10 μg/mL To Prepare ** 1. Mix 30.5g of L-Agar base with 1L of ddH2O and mix well with a magnetic stir bar. 2. Heat while stirring and boil for up to 1 minute to completely dissolve the powder. 3. Autoclave at 121°C for 10 minutes. 4. Cool to 55°C in a 55°C water bath 5. Once the agar has cooled to 50-55°C, aseptically add the CB and CHX stock solutions. 6. Mix well by swirling, and avoid bubbles. 7. Using aseptic technique, pour agar into Petri dishes. To Sterilize Autoclave (see above) Store At 4°C. Luria Agar (L-Agar) Plates (1L) Notes 1. Media can be autoclaved and aliquoted into sterile bottles and allowed to cool. These bottles can be stored at room temperature and microwaved to melt the agar. The agar solutions are cooled to 55°C (as above) before addition of antimicrobial supplements and pouring of plates. The recipe for this ingredient is included in this appendix. * First, see manufacturer’s instructions, and use them if they differ from these. The bottom agar is what you use to pour all of your plates. The only additives are the anti-microbials (carbenicillin and cycloheximide). The bottom agar is used alone for streaking your bacteria for single colonies AND in combination with the 7H9 top agar overlay when plating phages. The 7H9 top agar, as described in the manual, is prepared and stored at 2X concentration and must be diluted to a working concentration of 1X with 7H9 “neat” and CaCl2 (to a final [CaCl2] of 1mM) - no anti-microbials are added to top agar. There is essentially no need to make the top agar at 2X in the classroom. That is how it is prepared in the SEA Lab, but you can make it directly at 1X if it’s more convenient to you. Kevin Edited 09 Sep, 2015 16:26

Link to this post | posted 09 Sep, 2015 16:15

bradleyk

Hi Greg,

The bottom agar is L-Agar (Miller recipe) found on Part 1, Appendix 6, pg 56:

Luria Agar (L-Agar) Plates (1L)

Ingredient Amount Final Concentration

L-Agar base 30.5 g
ddH2O To 1L
CB stock* 1.0 mL 50 μg/mL
CHX stock* 1.0 mL 10 μg/mL

To Prepare **

1. Mix 30.5g of L-Agar base with 1L of ddH2O and mix well with a magnetic stir bar.
2. Heat while stirring and boil for up to 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 10 minutes.
4. Cool to 55°C in a 55°C water bath
5. Once the agar has cooled to 50-55°C, aseptically add the CB and CHX stock solutions.
6. Mix well by swirling, and avoid bubbles.
7. Using aseptic technique, pour agar into Petri dishes.

To Sterilize Autoclave (see above)

Store At 4°C.

Luria Agar (L-Agar) Plates (1L)

Notes 1. Media can be autoclaved and aliquoted into sterile bottles and allowed
to cool. These bottles can be stored at room temperature and microwaved

to melt the agar. The agar solutions are cooled to 55°C (as above) before addition of antimicrobial supplements and pouring of plates.

*The recipe for this ingredient is included in this appendix.
** First, see manufacturer’s instructions, and use them if they differ from these.

The bottom agar is what you use to pour all of your plates. The only additives are the anti-microbials (carbenicillin and cycloheximide). The bottom agar is used alone for streaking your bacteria for single colonies AND in combination with the 7H9 top agar overlay when plating phages.

The 7H9 top agar, as described in the manual, is prepared and stored at 2X concentration and must be diluted to a working concentration of 1X with 7H9 “neat” and CaCl2 (to a final [CaCl2] of 1mM) - no anti-microbials are added to top agar. There is essentially no need to make the top agar at 2X in the classroom. That is how it is prepared in the SEA Lab, but you can make it directly at 1X if it’s more convenient to you.

Kevin

Edited 09 Sep, 2015 16:26

Link to this post \| posted 09 Sep, 2015 16:17
GregFrederick@letu.edu	Thanks Kevin. I appreciate the email too! I am so glad you all are there! gf

Link to this post \| posted 09 Sep, 2015 16:22
bradleyk	No worries, Greg - I'm sure you'll be replying to these posts pretty soon here and sharing your experience/expertise!

Recent Activity

Plaque Bottom Agar - Recipe???