1. Preparing LB broth and agar plates. 

What you need:

• 200 ml distilled water (you can also use tap water or drinking water)

• 4 g LB broth powder

• 3.5 g bacteriological agar (only for plates)

• Glass bottle (blue cap) or flask

• Autoclave or pressure cooker

• Petri dishes (sterile, plastic or glass)

Steps:

1. Mix water, LB broth, and agar in the bottle. Add a magnetic stir bar if available.

2. Heat and stir until completely dissolved.

3. Close the bottle loosely (do not tighten the cap – it may burst). Cover with aluminium foil.

4. Sterilise in an autoclave at 121 °C for 30 minutes. (If you don’t have an autoclave, a pressure cooker can work, search for “sterilisation at 15 PSI”.)

5. After sterilisation, let the medium cool to about 50 °C (warm but not burning your hand).

6. Pour the liquid into sterile Petri dishes inside a clean area (near a Bunsen burner or in a laminar flow hood).

7. Let the plates solidify, then store them upside down in the fridge (4 °C) until use.

Safety notes:

• Always wear gloves and a lab coat.

• Work near a flame or in a sterile cabinet to avoid contamination.

• Label everything with date and contents.

Important note from my own experience: I initially used LB broth (Luria‑Bertani), which is a common all‑purpose medium. However, Janthinobacterium lividum produces much more violacein when grown in Nutrient Broth (NB). The reason is that NB contains glucose and meat peptone, which seem to trigger the pigment production pathway. Many online resources recommend LB, but my trials and advice from other researchers confirm that NB is significantly better. 

What you need (for 1 liter of NB):

• 2 g yeast extract

• 5 g meat peptone

• 10 g glucose

• 5 g NaCl (sodium chloride)

• (Optional: 15 g agar if you want solid plates)

• 1 liter distilled water

Steps:

1. Dissolve all ingredients in the water. Use a magnetic stirrer or heat gently.

2. Pour into a glass bottle (blue cap). Close loosely, cover with aluminium foil.

3. Sterilise in an autoclave at 121 °C for 30 minutes.

4. For liquid medium (broth), let it cool. For solid plates, cool to about 50 °C, then pour into sterile Petri dishes.

5. Store in the fridge (4 °C) until use.

Note: Pre‑made NB powder can sometimes give poor results. I recommend preparing your own from individual ingredients using the recipe above.

2. Inoculation and incubation guide. 

Goal: Transfer a small amount of Janthinobacterium lividum from a stored culture to a fresh agar plate.

What you need:

• Fresh agar plates (from step 1)

• Bacterial culture (stored at –80 °C or fridge)

• Sterile inoculation loop (plastic disposable or metal loop sterilised in a flame)

• Bunsen burner (to create a sterile zone)

• Incubator set to 25 °C

Steps:

1. Light the Bunsen burner. Work close to the flame; the heat creates a cone of sterile air.

2. Take a frozen or refrigerated culture. If it is freeze‑dried, rehydrate it following the supplier’s instructions.

3. Sterilise the loop by holding it in the flame until red hot. Let it cool for 10 seconds.

4. Open the Petri dish slightly. Touch the loop to a single colony or to the frozen pellet.

5. Gently streak the loop across the surface of the fresh agar plate in a zig‑zag pattern. Do not dig into the agar.

6. Close the plate, seal it with breathable tape (not airtight), and write the date and strain.

7. Place the plate upside down in the incubator at 25 °C.

8. Check after 3–7 days. You should see visible bacterial growth.

Tip: If you use a metal loop, re‑sterilise it after each use. Dispose of used plastic loops in an autoclave bag.

3. Dyeing techniques: immersion, shibori, folding. 

Once the bacteria have grown and produced violacein (the purple pigment), you can transfer the pigment to fabric. 

Preparation:

• Sterilise your fabric in an autoclave bag (121 °C, 30 min).

• Prepare a liquid culture: inoculate LB broth (without agar) with the bacteria and incubate for 5–7 days. The liquid will turn purple.

Immersion dyeing:

1. Pour the purple liquid culture into a sterile container (e.g., an autoclave bag or glass jar).

2. Submerge the fabric completely.

3. Incubate at 25 °C for 3–7 days. The pigment will bind to the fabric.

4. After dyeing, autoclave the bag again to kill all bacteria (121 °C, 30 min).

5. Remove the fabric, rinse with water, and let it dry.

Shibori/folding:

1. Fold, twist, or bind your fabric using rubber bands or string (create your own pattern).

2. Place the folded fabric in the liquid culture.

3. Incubate and autoclave as above.

4. Unfold after rinsing to reveal the pattern.

For the bacteria to produce violacein, they need oxygen. Therefore, the most effective dyeing happens when the fabric is placed on the surface of the liquid – slightly submerged but partially exposed to the air. Here, the bacteria grow at the air‑liquid interface, where they can access both the nutrients in the broth and the oxygen they need.

If the fabric is completely submerged, the bacteria may struggle to produce pigment due to a lack of oxygen, leading to weaker or no colour. I recommend experimenting with different positions: fully submerged, partially submerged, or even laid directly on wet agar plates. Each method will create unique patterns and intensities, revealing the collaborative nature of the process.

4. Troubleshooting: Why is my pigment yellow or white? 

I spent weeks getting only yellow colonies instead of purple. After consulting other researchers and testing different conditions, I identified several causes and solutions. Use this checklist. 

1. Wrong growth medium (most common issue)
LB broth does not work well for violacein production. Switch to Nutrient Broth (NB) with the recipe above (yeast extract, meat peptone, glucose, NaCl). Do not use pre‑made NB powder; it often fails. Prepare your own. 

2. Temperature too low
The ideal range is 25–28 °C. At 20 °C or below, growth is slow, and pigment may not form. Check your incubator’s calibration. 

3. Old or weak strain
Bacteria lose their ability to produce pigment after repeated subculturing or prolonged storage. If you have tried fresh medium and correct temperature for 7–10 days with no purple, order a new certified strain from DSMZ (strain DSM 1522).

4. No stress, pigment needs a trigger
Violacein is a secondary metabolite. The bacteria produce it when they feel mild stress. Try these methods:

• Cold shock: After 3 days of growth at 25 °C, move the plates to the fridge (4 °C) for 2 days, then back to 25 °C.

• Add a stressor: Add 0.2 mg/mL ampicillin or a very small amount of hydrogen peroxide (H₂O₂) to the medium.

• Starvation: Slightly reduce the nutrients (e.g., use half the glucose).

5. Lack of oxygen (for liquid cultures)
Remember: the bacteria need oxygen to produce pigment. In liquid broth, place the fabric on the surface of the liquid, not fully submerged. The best colour appears at the air‑liquid interface. 

6. Patience
Sometimes the bacteria simply need more time. Leave the plates or liquid culture for up to 14 days, checking every 2–3 days. Do not discard them too early. 

My own journey:
I tried all of the above. The biggest breakthrough was switching from LB to homemade NB. Unfortunately, by the time I learned this, my thesis deadline had passed. I hope this information saves you months of frustration.