BIOLUMINESCENT BACTERIA MICROBIAL CULTURE INSTRUCTIONS

GROWING BIOLUMINESCENT BACTERIA

⌬ When making liquid bacteria cultures, it is best to use a nice half cm sized dab of fresh cultured bacteria to inoculate the liquid broth nutrients so we provide 2 x 5 ml vial of bioluminescent bacteria and enough nutrients (Agar) to grow multiple cultures! 

⌬ To grow additional bacteria, we have included 1 tube of powdered AMPICILLIN AGAR MEDIA (enough agar nutrients for 7 x plate cultures) and 3 tubes of powdered AMPICILLIN BROTH MEDIA (enough liquid nutrients for 3 x 250 ml liquid cultures) 

⌬ All this from just one tiny 5 ml vial of bacteria! 

WHAT'S INCLUDED:

⌬ 1 250 ml glass bottle

⌬ 1 AMP GEL AGAR tube

⌬ 3 AMP LIQUID BROTH AGAR tubes

⌬ 7 Petri plates

⌬ 10 Nitrile gloves

⌬ 5 Sterile inoculating loops

⌬ 1 Measuring conical 50 ml

⌬ 5 Disposable pipettes

⌬ 1 Vial sterile H2O 

⌬ 2 Vials non-pathogenic e.coli bacteria (1 extra!)

     - modified vibrio fischeri plasmid PJE202

⌬ STEP 1: MAKING AGAR PLATES

⌬ This step is crucial for growing more bacteria. You must prepare their nutrient dishes to grow and multiply on so you can collect more bacteria for further use in liquid cultures. 

• Pour the contents of the single tube labeled LB AMPICILLIN AGAR MEDIA in the 250 ml glass bottle. (This is Agar for pouring the plates)
• Add 150 ml of tap water to the glass bottle using the conical tube labeled MEASURE (3 tubes - 50 ml ea.)
• Shake well and place the glass bottle in the microwave with the lid unscrewed and lightly sitting on top. 
• DO NOT SCREW THE CAP ON TIGHT IN THE MICROWAVE.
• Heat the bottle in the microwave for a few seconds at a time (5-10 seconds) and be careful not to over boil the contents. 
• Open the microwave and let it sit for a few seconds to cool down. 
• Continue microwaving on short intervals without boiling for 2-5 minutes or when the liquid becomes a clear yellow with no visible particles.
• Remove the bottle from the microwave and let it cool to a lukewarm temperature (20-30 minutes).
• While the bottle remains warm, pour the Petri plates one at a time.
• Remove the lid off each plate and pour just enough Agar to cover the bottom half of the plate (1-2 mm deep).
• Gently put the lids back on and let the plates cool at room temperature for at least one hour. The Agar will solidify when cooled.

⌬ STEP 2: MAKING BACTERIA AGAR PLATES (7 Plates)

• Using a Pipette, add 4 drops of Sterile H2O to the vial labeled FREEZE DRIED BACTERIA. Screw on the cap and shake for a few seconds.
• Add all the bacteria onto ONE of the prepared Agar gel plates with the Pipette.
• Gently streak and spread the bacteria around with a Sterile inoculation loop.
• Be careful not to break the Agar. 
• Drag the inoculation loop around in different areas to grow large colonies.
• Place the lid back on the Agar Gel Plate and let the water dry / evaporate before storing. 
• Flip the plate upside down (Agar side up) so condensation doesn't drip off the lid onto the Agar.
• Let the plate incubate and grow at room temperature (18-20°C) for 12-18 hours. 
• You will see white colored bacteria colonies indicating growth.
• Check for bioluminescence after 12 hours by exposing the culture plate to darkness.
• Once the bacteria agar plate starts glowing it can be used to streak the other 6 Agar Gel Plates and make 3 Liquid Cultures.
• Avoid placing bacteria in areas where temperatures exceed 30°C or the protein responsible for bioluminescence will denature and stop glowing.
• You can store the plates in the fridge (4°C) for up to 2 weeks.

⌬ STEP 3: MAKING BACTERIA LIQUID CULTURES (3 Bottles)

• Pour the contents of ONE of the tubes labeled AMPICILLIN BROTH MEDIA in the 250 ml glass bottle.
• Add 200 ml of tap water to the glass bottle using the conical tube labeled MEASURE (4 tubes - 50 ml ea.)
• Screw on the bottle cap, shake well and place the glass bottle in a stable position at room temperature.
• Let the liquid settle for a couple minutes before adding bacteria.
• When all the powder is dissolved, use a STERILE INOCULATION LOOP to gently scrape a small amount of bacteria (0.1-0.5cm diameter bead) from one of the plates you made and gently drop or mix it into the liquid agar bottle.
• Let the bottle incubate and grow at room temperature (18-20°C) for 6-12 hours. 
• The liquid will become cloudy yellow after 6+ hours indicating growth.
• Do not shake the bottle during bacteria mixing or incubation.
• Check for bioluminescence after 10 hours by gently swirling the liquid culture to introduce oxygen.
• Once the liquid bacteria culture starts glowing, it is fully mature and should be stored in a cool dark place.
• To renew the glow, simply make a new bottle of AMP LIQUID AGAR and add a few drops of your old glowing liquid culture to the new bottle of nutrients and let it sit at room temperature for 6-12 hours to grow.
• You can store the liquid culture in the fridge (4°C) for up to 2 weeks.

ADDITIONAL NOTES

⌬ Bacteria vials provided are freeze dried and are ok to ship at room temperature but should be stored in the freezer upon arrival. 

⌬ Never store prepared agar media or inoculated bacteria cultures in the freezer. 

⌬ Avoid placing bacteria in areas where temperatures exceed 30°C or the protein responsible for bioluminescence will denature and stop glowing. 

⌬ Work in a sterile environment and wear a mask and gloves to avoid culture contamination. Agar is a nutrient rich medium and can grow many kinds of bacteria. Keep in mind you are trying to grow only ONE kind!