Jan 22, 2025
Version 1
Accessible bacterial culture V.1
This protocol is a draft, published without a DOI.
- 1OBS
Protocol Citation: openbioscience Adrian 2025. Accessible bacterial culture. protocols.io https://protocols.io/view/accessible-bacterial-culture-dxx67pre
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We used this protocol and it's working with the particular set of reagents we used.
Created: January 15, 2025
Last Modified: January 22, 2025
Protocol Integer ID: 118494
Disclaimer
This protocol is for education purpose one. Please study observe the biosafety legislation applicable to your particular jurisdiction.
Abstract
Culturing bacteria in a Petri dish is pretty much a rite of passage in molecular biology.
This is a protocol created by the Ottawa Bio Science Group for students that do not have access to a well equipped lab.
Materials
Reagents
- Solid Broth(chicken or beef, e.g., Bouillon )
- Water (tap, distilled or deionized)
- Sodium Chloride (table salt)
- Agar-agar
- Salt
PPOP
- nitrile gloves
- oven gloves
- coat
- Alcohol wipes (for sterilization)
- Alcohol (for sterilization)
- Chlorine/Bleach
Devices
- Bunsen burner
- Microwave
- Pipette (for antibiotics, if required)
Labware/ Consumables
- 100 mL Beaker or alternative microwaveable container
- Aluminum Foil (thick for cap, turkey roasting pan foil preferred)
- Cotton balls or pads (for filtering)
- Syringe filter (0.2 micron or similar)
- Pipette (for antibiotics, if required)
- Alcohol wipes (for sterilization)
Alternatives
- For making an inoculating loop check out: https://specyal.com/diybio/loopproject.html or PipetteJockey's blog
- For an alternative to Benson burner one can use a flambee device. For environments where open flames are not permitted you can use https://specyal.com/diybio/flamerproject.html. Details to be updated.
- For a free tube rack : check up upcoming tofu rack.
- Great alternative microwaveable containers for flasks are small glass bottles like the ones for baby milk.
- Great alternatives for beakers are washing detergent measuring cups.
- Anything object marked pp-polypropylene or showing number 5 on bottom triangle mark for recycling is autoclavable and microwaveable so don't throw them in the trash.
- For incubator please check https://specyal.com/diybio/incubatorproject.html. That will be updated shortly with details. Also https://www.youtube.com/watch?v=-4bQF6Fxj0M
- For a pipette holder please check https://github.com/AdrianMolecule/pipetteholder
Alternatives
Alternatives
For students that do not have access to all the mentioned labware or devices check alternatives on the materials page. That contains tested references or designs from reliable sources.
Plate making
Plate making
Use either the classic LB-agar protocol with .37 grams LB-Agar for 10 ml water to make a classic 9 cm Petri dish.
OR use the following accessible protocol with the following alternative media
Take Bouillon (chicken or beef Bouillon, Bovril)
One example of commercial broth in solid form
In a 100 mL beaker or alternative microwaveable container add about 20 ml water + 1 gram Bouillon + 0.25 grams salt (sodium chloride)
Mix until becomes a paste.
Note
Can use a stirring rod or spatula or similar metal implement for that.
Slowly add more water while stirring until the volume is 50 ml.
Add an aluminum cap to the beaker.
Note
If not available, make an aluminum cap for the beaker by using thick aluminum foil manually formed around the top of the beaker. Cut excess foil and sharp edges. Can use thin foil but pie pan foil is better because is reusable and can be autoclaved. Warning: Beware of sharp edges!
Example Cap
This can be done in an autoclave, a pressure cooker or microwave. This is the microwave version and it's very fast.
Heat the microwave to dissolve the Bouillon and get a runny liquid. It should take anywhere from 20 to 40 seconds depending on the microwave.
Safety information
The content of the flask is hot and can boil over so heat it in 10 sec increments, always observing if a white foam raises high in the beaker. Stop and stir when the foam raises about half way in the flask.
Do not fill the flask more than 25% of capacity otherwise it's hard to control spills and boil overs.
Filter (optional but without it the plates will be dark and greasy )
Alternate media before filtering
Cotton option: Take a 100 ml syringe and some cotton ball or pad.
Aspirate the media from the flask in the syringe, wrap the tip of the syringe in cotton.
Push the syringe piston slowly and collect the clear, filtered broth in labware
Commercial Filter Option. Syringe filter -0.2 micron or similar membrane filters
See for membrane filters https://www.youtube.com/watch?v=esm6qMSoQ3Q
Aspirate the media from the flask in the syringe. You might need to transfer from the flask to a beaker first if the syringe head does not fit in the flask. Add the filter to the syringe. Push the piston of the syringe slowly to filter the broth out of the syringe and back into the flask.
Add more water to a total of 50 mL to the flask
Add 1 gram agar-agar
Sterilize for about 1 full minute. Use oven gloves microwave the content of the flask. Do it in 30 seconds increments with shaking in between until content is clear. Warning! Stop if the foam created by the microwave gets higher that 2 cm in the flask otherwise it will boil over. After stop, swirl the content of flask and continue microwaving.
Note the white foam level in the flask. That is normal.
Safety information
Stop if the foam created by the microwave gets higher that 2 cm in the flask otherwise it will boil over.
For large flasks the content can boil over when manually stirring even if it's out of the microwave.
If protocol requires, pipette in antibiotic in the flask when it cools to about 60 Celsius (can hold comfortably with a nitrile glove hand) before the pouring. Shake to mix the antibiotic with the media.
Mark plates on lid edge with content, author initials, date, and antibiotic. Ex fuGFP, AF, Jan 15, Kan
In a sterile place, use sterile technique and wipe the bench and instruments with alcohol.
Light the burner fire on the bench to create uplift air currents
Pour plates from minimal height when the flask gets cool enough that it can be handled comfortably. That is around 50-60 Celsius and it usually takes 10-30 minutes to cool down depending on the volume. Minimize the time when the lid is opened and don't breathe over the plate. If needed, gently swirl the media in the plate to cover all the bottom of the plate.
Gently, place the plate in opened zip-lock bag
Wait about 40 min for plates to solidify. Do not move the plates during that period
Store upside-down in a closed zip-lock bag in the fridge. Should be good for several weeks. It's important to be upside down. See video for pouring tips
Inoculate
Inoculate
Sterilize by fire an inoculating loop by burning sections of the wire until red. End at the loop tip
Cool the tip of the loop by stabbing the agar at the edge of the dish
Dip/stab the loop in container that is source of organism. For source you can use another plate, a stab, a liquid culture, glycerol stock etc. Please check the aliquoting technique is appropriate to the prelevation of the organism.
For stabs, try to observe where in the stab are the bacterial colonies and insert the loop in the stab by following the path left in the agar during inoculation
Streak 3 zig-zags by gently skating the loop tip on the agar in a large zig zag motion. Do not apply too much pressure as to not cut the agar
Rotate plate right one third of a circle
Re-flame the loop
Make sure you go over a previous line and streak again 3 zigzags by gently skating the loop tip on the agar
Re-flame the loop
Make sure you go over a previous line and streak again 3 zigzags by gently skating the loop tip on the agar
Bag the plate in the zip-lock, again upside down
Grow/Incubate
Grow/Incubate
Place/incubate plate upside down in an incubator or close to source of low heat (heated mattress/pad) or near a hot air vent or room temperature for 12 hours to 3 days
You should see colonies along the zig-zag shape as white dots If the plasmid expresses anything of color the color of the dots should match that. Place plate upside down in an incubator at 37 Celsius.
As an alternative build an incubator using info from materials section or place the place close to source of low heat (bed warmer) or near a hot air vent or room temperature for 12 hours to 3 days.
Note
Normally a culture is visible white dots in 10 hours but if the grow temperature (37 Celsius) for e-coli is not met it can take days. If the plasmid expresses anything of color the color of the dots should match that.
Observe the colonies. You should be able to see dozens of colonies as small 1 mm disks where your loop touched the plate. Like this:
Plate using alternative media incubated at 37 Celsius. No UV light.
For fuGFP you can see some fluorescence with the naked eye but it's more spectacular under low frequency UV light. See addGene fuGFP for wavelength.
A sample plate growing fuGFP on filtered media grown at 37 Celsius under UV light
Monitor Warning:
Dispose of plates without opening if you notice any large unusual growth as sometimes mold/unknown growth can develop. Treat it as you would treat black mold.
Disposal
Disposal
Please check mandatory local regulations
For disposal you need to check local regulation
As a guideline -10% bleach(chlorine) poured over the plate and left for 5 minutes should destroy the bacteria
Dispose of the bouillon in similar fashion
Protocol references
Based on an idea from https://practicalbiology.org/standard-techniques/making-up-nutrient-agars
Alternative devices/labware in Open Surce
For making an inoculating loop check out: https://specyal.com/diybio/loopproject.html or PipetteJockey blog
For tube rack if using stabs check up upcoming tofu rack.
Acknowledgements
Special acknowledgement to Nick Coleman for creating this amazing plasmid and having the courage to Open Source it. https://coleman-lab.org/