Ready to get your hands dirty? This is where theory becomes practice. Below you will find everything you need to start cultivating Janthinobacterium lividum and dyeing textiles with violacein, from preparing LB broth and agar plates to inoculation, incubation, and finally applying the pigment to fabric.
I have documented my own process in detail, including the failures and the small victories. All protocols are shared as open resources. You can adapt them to your own workspace, whether it is a professional lab or a modest makerspace, as long as you have a certification for a Bio-Safety Level 1 Laboratory.
Here is every piece of equipment and ingredient I used for my project.
1. Lab Consumables (Sterile, Single-Use)
Petri Dishes (Sterile, Plastic): Dishes for creating agar plates.
Inoculation Loops (Sterile, Plastic): Tools for transferring bacteria without contamination.
Autoclave Bags: Heavy-duty plastic bags to sterilize fabric and safely dispose of contaminated waste.
Nitrile Gloves (Powder-Free): For personal protection and to prevent contamination. A box of 100 pairs is a good start.
Bunsen Burner or Gas Burner: Used to create a sterile airflow zone while inoculating plates. (Open flame is a common lab technique)
Autoclave or Pressure Cooker: The most critical piece of equipment for sterilization. An industrial autoclave is ideal, but a standard pressure cooker can be used for small-scale sterilization.
2. Lab Equipment
Incubator: A machine to keep a constant temperature of 25°C. A small, desktop microbiology incubator is perfect for this.
Refrigerator (4°C): To store unused agar plates, the bacterial culture, and extracted pigment.
Freezer (-80°C or -20°C): For long-term storage of the bacterial strain. A standard -20°C freezer is a common alternative.
Digital Scale (0.1g precision): For weighing LB broth powder and agar.
Glass Bottles / Erlenmeyer Flasks: For preparing LB broth and agar.
Magnetic Stirrer & Stir Bar: To dissolve the LB broth and agar into the water effectively. (Optional, but highly recommended).
Automatic Pipettes (P1000, P200): For measuring and transferring liquids accurately. (Recommended for smaller volumes, but graduated cylinders work for larger ones).
3. Bacterial Cultivation and Maintenance
LB Broth (powder or pre-mixed): The food for the bacteria. An absolute must-have for growth.
Agar Powder (Bacteriological Grade): Added to LB broth to create solid agar plates.
Distilled or Deionized Water: For making all media.
Janthinobacterium lividum Strain:
Best Option: Order a lyophilized (freeze-dried) strain from a certified culture collection, such as DSMZ (German Collection). Search for strain number DSM 1522 (or DSM 16524).
Alternative: If you have a connection to a university or open biolab, they might provide an already activated culture.
4. Dyeing and Application Materials
Fabric & Fiber Samples: Test on natural fibers (cotton, silk, wool) and synthetics (polyester, nylon). Remember, the final color varies depending on the fiber.
5. Optional / Stress Induction Supplies (For Troubleshooting)
Glycerol: Adding glycerol can help trigger violacein production.
Ampicillin: A mild antibiotic stressor that has been shown to increase violacein production in research labs.
This list is comprehensive but not overly complicated. It contains everything you need to set up a functional workspace for bacterial dyeing.
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:
Mix water, LB broth, and agar in the bottle. Add a magnetic stir bar if available.
Heat and stir until completely dissolved.
Close the bottle loosely (do not tighten the cap – it may burst). Cover with aluminium foil.
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”.)
After sterilisation, let the medium cool to about 50 °C (warm but not burning your hand).
Pour the liquid into sterile Petri dishes inside a clean area (near a Bunsen burner or in a laminar flow hood).
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:
Dissolve all ingredients in the water. Use a magnetic stirrer or heat gently.
Pour into a glass bottle (blue cap). Close loosely, cover with aluminium foil.
Sterilise in an autoclave at 121 °C for 30 minutes.
For liquid medium (broth), let it cool. For solid plates, cool to about 50 °C, then pour into sterile Petri dishes.
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.
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:
Light the Bunsen burner. Work close to the flame; the heat creates a cone of sterile air.
Take a frozen or refrigerated culture. If it is freeze‑dried, rehydrate it following the supplier’s instructions.
Sterilise the loop by holding it in the flame until red hot. Let it cool for 10 seconds.
Open the Petri dish slightly. Touch the loop to a single colony or to the frozen pellet.
Gently streak the loop across the surface of the fresh agar plate in a zig‑zag pattern. Do not dig into the agar.
Close the plate, seal it with breathable tape (not airtight), and write the date and strain.
Place the plate upside down in the incubator at 25 °C.
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.
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:
Pour the purple liquid culture into a sterile container (e.g., an autoclave bag or glass jar).
Submerge the fabric completely.
Incubate at 25 °C for 3–7 days. The pigment will bind to the fabric.
After dyeing, autoclave the bag again to kill all bacteria (121 °C, 30 min).
Remove the fabric, rinse with water, and let it dry.
Shibori/folding:
Fold, twist, or bind your fabric using rubber bands or string (create your own pattern).
Place the folded fabric in the liquid culture.
Incubate and autoclave as above.
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.
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.