TITLE: Preparation of agarose pads suitable for viewing filamentous cyanobacterial microbial communities using time-lapse imaging
AUTHORS: Jerko Rosko, Mary  Coates, Kelsey Cremin, Christian Zerfass, Orkun  Soyer

[DESCRIPTION]
This protocol is for viewing cultures on top of agar pads, e.g filamentous cyanobacterial microbial community (from a freshwater environment) that form biofilms and granular structures, using time-lapse imaging. 

[STEPS]
SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
1. 
 


Into a 100 mL Duran bottle, add 1 g agarose and make up to 47.35 mL with DW.

SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
2. Autoclave at  120 °C for 20 – 30 minutes. Remove at 80 °C. 

 

SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
3. After autoclaving, gentle swirl the bottle to ensure the agarose is fully dissolved.
SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
4. Once removed from the autoclave, keep in a hot water bath (55 °C) to prevent agarose from setting.

SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
5. Working in a sterile environment e.g Microbiological safety cabinet (MSC), add the following sterile BG11+ components:

 



SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
6. At this point, there are two optional routes to take:
SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7. 
SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7. 
Optional route 2



SECTION: Pipetting agarose pads (Makes 10 Pads)
8. Sterilise 20 22-mm2 cover glass slides with 70% Ethanol inside a MSC, allowing time for drying (~60 min).

SECTION: Pipetting agarose pads (Makes 10 Pads)
8. Sterilise 20 22-mm2 cover glass slides with 70% Ethanol inside a MSC, allowing time for drying (~60 min).
SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7.1. Add 50 mL vitamin B12 or vitamin mix (0.02 g/L) to the solution in the Duran bottle.
 


SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7. Optional route 1
SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7.1. For agarose pads (Step 8, below), only 10 mL medium is required per session, thus, it is best to aliquot the media at this stage to avoid repeated solidification/melting cycles of the medium.

SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7.3. Before using any aliquot, add 5 µL vitamin B12 (or your preferred vitamin[s]) and swirl to mix.
 


SECTION: STEP 1: Agarose pad medium (makes approx. 50 pads)
7.2. Using sterile glass bijou bottles with screw cap lids, aliquot 9.995 mL medium per bijou.
 


SECTION: Pipetting agarose pads (Makes 10 Pads)
9. Keep the agarose solution at 55 °C using a water bath before moving into the MSC.

SECTION: Pipetting agarose pads (Makes 10 Pads)
9. Keep the agarose solution at 55 °C using a water bath before moving into the MSC.
SECTION: Pipetting agarose pads (Makes 10 Pads)
10. Place five glass cover slips in each if 2 x 90 mm petri dish.
SECTION: Pipetting agarose pads (Makes 10 Pads)
10. Place five glass cover slips in each if 2 x 90 mm petri dish.
SECTION: Pipetting agarose pads (Makes 10 Pads)
11.  Working on one glass cover slip at a time, pipette 800 mL of cooling agarose solution evenly across the cover slip (as the agarose does not spread well when sandwiched, try to reach as far into the corners without unevenly distributing the gel). Quickly remove any air bubbles using the suction of the pipette. 
 


SECTION: Pipetting agarose pads (Makes 10 Pads)
11.  Working on one glass cover slip at a time, pipette 800 mL of cooling agarose solution evenly across the cover slip (as the agarose does not spread well when sandwiched, try to reach as far into the corners without unevenly distributing the gel). Quickly remove any air bubbles using the suction of the pipette. 
 


SECTION: Pipetting agarose pads (Makes 10 Pads)
12. Gently lower another cover slip on top of the agarose solution, to sandwich it  between the two cover slips. Quickly adjust the cover slip, if required, to ensure the agarose appears level.
 


SECTION: Pipetting agarose pads (Makes 10 Pads)
12. Gently lower another cover slip on top of the agarose solution, to sandwich it  between the two cover slips. Quickly adjust the cover slip, if required, to ensure the agarose appears level.
 


SECTION: Pipetting agarose pads (Makes 10 Pads)
13. Using a new pipette tip each time, repeat steps 11-12 for each pad. 

SECTION: Pipetting agarose pads (Makes 10 Pads)
14. Allow the pads to solidify, then seal the lid with parafilm and store in the cupboard until required. 
 

SECTION: Pipetting agarose pads (Makes 10 Pads)
15. The  BG11+ pads are best used the day after making, but we found them also store well for 1-2 weeks (and even longer). 

  

SECTION: Pipetting agarose pads (Makes 10 Pads)
13. Using a new pipette tip each time, repeat steps 11-12 for each pad. 

SECTION: Pipetting agarose pads (Makes 10 Pads)
14. Allow the pads to solidify, then seal the lid with parafilm and store in the cupboard until required. 
 

SECTION: Pipetting agarose pads (Makes 10 Pads)
15. The  BG11+ pads are best used the day after making, but we found them also store well for 1-2 weeks (and even longer). 

  

SECTION: Agarose ‘pads’ in 60mm dish (makes 15) (Alternative Step 2)
16. 
 

Pipette 7 mL medium per dish, leave to dry with lids on in the MSC for 60 min, then a further 60 min with lids off.
SECTION: Agarose ‘pads’ in 60mm dish (makes 15) (Alternative Step 2)
16. 
 

Pipette 7 mL medium per dish, leave to dry with lids on in the MSC for 60 min, then a further 60 min with lids off.

SECTION: Agarose ‘pads’ in 60mm dish (makes 15) (Alternative Step 2)
17. Parafilm close the dishes and store in the cupboard for use the next day. 
 

SECTION: Agarose ‘pads’ in 60mm dish (makes 15) (Alternative Step 2)
17. Parafilm close the dishes and store in the cupboard for use the next day. 
 

SECTION: STEP 3: Microscopy on the agarose pads
18. When required for use with microbial cultures, the top cover slip on an agarose pad should be removed from the selected pad. The pad can then be cut into smaller pieces (if required) or the whole pad placed into a 60mm dish.
 

SECTION: STEP 3: Microscopy on the agarose pads
19. Add bacteria culture and start imaging.
SECTION: STEP 3: Microscopy on the agarose pads
18. When required for use with microbial cultures, the top cover slip on an agarose pad should be removed from the selected pad. The pad can then be cut into smaller pieces (if required) or the whole pad placed into a 60mm dish.
 

SECTION: STEP 3: Microscopy on the agarose pads
19. Add bacteria culture and start imaging.