By popular demand, here is a procedure for the isolation of bioluminescent bacteria from a couple reliable source materials, namely shrimp and squid. The following procedure and media are from our old Bacteriology 320 lab manual (circa 1984), and isolation was generally successful when 10-20 enrichments were set up initially. The "Periods" shown below occur at one-day intervals except between Periods D and E when cultures can be refrigerated for one or more days.
For "official" methods and a general overview by experts in the field, one can consult the on-line edition of The Prokaryotes; type "luminous bacteria AND isolation" (without the quotation marks) in the Search box.
- Set up the "seafood enrichment" as follows, utilizing fresh (on ice is OK – but not frozen!) shrimp or squid from a local market: Place the shrimp or squid in a 500 ml flask and add just enough 3.0% NaCl solution such that approximately 10-20% of the seafood is above the level of the liquid.
- Incubate the flask in a cool dark room (18-22°C) and observe at intervals up to 24 hours. The room should be darkened totally such that the flask can be observed for luminous areas on the seafood. When such is seen, proceed to Period B.
- Streak one or more plates of Luminescent Agar (formula below) for isolated colonies from a luminous area of the seafood.
- Incubate the plates in the cool room (18-22°C) for 24 hours. (No more than 48 hours.) When isolated, luminous colonies are observed, proceed to Period C.
- Find two or more isolated, luminous colonies and streak each one on a new plate of Luminescent Agar. Incubate as above.
- Choose one or more of the more "brilliant" colonies and streak each onto a slant of Luminescent Agar. Incubate overnight or until luminescent growth is seen and then refrigerate until Period E.
- From the slant cultures, inoculate flasks of Luminescent Broth. You might try inoculating duplicate flasks – one to be shaken and one to remain still (for less chance of overgrowth) during incubation. You will see that exposure to oxygen enhances light production.
- Incubate the flasks in the cool room (18-22°C).
- Using a turbid, brightly luminescing culture, study the effects of inhibitors and uncouplers of oxidative phosphorylation such as cyanide, azide and dinitrophenol. (Caution: THESE ARE POISONOUS!) You can use a spectrophotometer with its own light source turned off, or you can utilize the "eyeball method." [If memory serves me correctly, I believe we used 5 ml samples of culture in test tubes and added one ml amounts of various dilutions of these materials to the tubes, vortexing the tubes for a short time and then observing for the cessation of light emission. One can always experiment!!]
- Identification to genus may be done. Check a reference such as Bergey's Manual or the on-line edition of The Prokaryotes (noted above). Various differential media used for other groups of bacteria (such as the enterics) can be utilized with the provision that 3% NaCl must be included.
One can also experiment to find optimal temperatures for growth and light production. A question to be answered experimentally: Will addition of nitrate cause the culture to glow more than it would otherwise under anaerobic conditions?
Especially good to have for demonstration purposes is a culture that can grow in a flask of broth – without shaking – that will suddenly glow when the flask is swirled vigourously a few times. Another great demo is to add a broth culture to a hollow glass tube (at least 2 feet long and an inch wide) such that there are a few inches of head space. As the tube is repeatedly tipped one way and then the other, the culture will glow where "activated" by the bubble as it passes through.
Peptone 10.0 g
NaCl 30.0 g
K2HPO4 2.0 g
MgSO4 0.25 g
Glycerol 2.0 g
Distilled H2O 1.0 L
Dehydrated Nutrient Broth 8.0 g
NaCl 30.0 g
Glycerol 10.0 g
CaCO3 5.0 g
Agar 15.0 g
Distilled H2O 1.0 L
Note: The CaCO3 should be the "fluffy" kind that does not settle out rapidly. Highly-refined CaCO3 settles out very rapidly in plates and tubes and tends to be a less effective buffer.