Isolation of Pure Cultures Of Bacteria

Vibrio phosphoreum from Squid or Ocean Fish

Bioluminescent bacteria are known in four genera and are discussed below.

Vibrio (Photobacterium) phosphoreum is one of my favorite bacteria. A log-phase culture of this bacterium in a clear plastic bottle such as a pop bottle makes an interesting, low-power, light weight flashlight for camping trips. My son and I have done that. This bacterium has long been known as Photobacterium but it has been reclassified as Vibrio.

Vibrio phosphoreum requires oxygen in order to produce light. Here is a nice demonstration that I use in my classes. I use a section of glass or plastic tubing or a pipet about 0.5 to 1.0 centimeter in diameter and 20 to 50 cm long. I stopper each end. I fill the tube about 1/3 full with a growing liquid culture of Vibrio phosphoreum and stopper. As I pour the bacteria into the tube and the kids are impressed by the bluish-green glow. In less than a minute, the glow is almost totally gone. The kids are not too surprised that the show lasted for such a short time, kids are used to disappointments. I mention the light production requires oxygen. Then I invert the tube; bright light re-appears as the liquid culture flows past the rising air bubble.

Not all related species produce light and some strains from other genera produce light. Bacteria which produce light have been found in these genera: Photobacterium, Vibrio, ---- more. At least one instance of a light producing strain from a wound has been reported.

A Safety Comment

Recall that some serious pathogens occur in the genus Vibrio. Especially Vibrio cholerae which produces cholera a serious, sometimes fatal, intestinal disease. Vibrio cholerae sometimes emits a faint glow. While pioneer towns have been wiped out by Vibrio cholerae, it is said a quart of water with a little table salt added would have replaced the lost liquids and saved the lives of most of these patients. For safety, teachers might use only bioluminescent species which require 30 grams of NaCl per liter of medium. Such bacteria should not be able to grow in any animal. Another safety precaution is to use bacteria which can't grow at human body temperature. Many marine and soil bacteria can't grow at such temperatures.

Description of the Obsolete Genus Photobacterium

Isolation Protocol for Vibrio phosphoreum

Many bacteria grow on dead seafish. Since Vibrio phosphoreum grows at low tempature and may glow on complex media, we use these to enrich for V. phosphoreum.

Incubate fresh fish, squid, or octopus at 10-15 deg Celcius, half submerged in natural or synthetic seawater. Examine at half-day or daily intervals for glowing colonies of bacteria (in total darkness after your eyes are dark adapted). Streak these colonies on LM (Luminescence Medium) or SSW. LM is synthetic or natural seawater containing per liter: 3 ml glycerol (glycerine), 5 gram yeast extract, 5 g tryptone, 1 g CaCO3 (powdered limestone or chalk) and 10 g of agar. Since P. phosphoreum also grows slowly at 4C (usual refrigerator temperature), you could conduct this incubation in a covered dish in a refrigerator and then examine at intervals of one or two days as the colonies will take longer to develop at the colder temperature.

Since streaking plates in total darkness is difficult, scientists use a red light. Light has no effect on biolumiescence of photobacteria, but the glowing colonies are too small and too faint to be seen in white light. A tube of sterile toothpicks is a convenient way to trasfer the colonies. Do your streaking, and place your supplies nearer the red light, but place the dead fish away from the light or in the shadow of your body so that you can see the faint glowing colonies on the fish. Marine labs use a rheostat (variable resistor) in series with one wire of the red lamp and turn the light down low.

If you lack petri plates, use LM agar in flat glass bottles such as small flat whiskey or medicine bottles. Lay these flat bottles on their side before the sterile medium gels.

LM or SSW without agar is the liquid medium used for the bubble in tube glow experiments. Test tube agar slants of P. phosphoreum glow nicely. A test tube slant emits enough light to light up the face of a person or read a newspaper which the tub is touching.

Squid or fish purchased in markets on seacoasts almost always produce luminescent colonies, but fresh seafood purchased inland is usually not successful. Water from a salt water aquarium gives varying, but usually poor results. If you live inland and happen to be on a seacoast, place a fresh squid in a plastic bag with trapped air and place it in crushed ice for storage for the trip home. For better results place fish or part of a fish in a jar that is pushed into crushed ice in a cooler. The purpose of the jar is to protect any growing colonies from smearing. By the time you get home glowing colonies may be present. After one or two days at 4 to 18C, luminous colonies may be present. Examine the squid or fish daily. Incubation at 15 to 20C may be the best temperature. Or use your home refrigerator which is about 4 C or above.

Glowing bacteria may be isolated from the light organs of deepsea fish, from shrimp and other seafoods, or from the intestinal contents of marine fish.

Luminous Bacteria are Found in Three Genera

Bioluminescense is an unusual trait and early bacteriologists tended to group luminous bacteria separate from similar bacteria. I had to relabel one of my cultures three times as bacteriologists tried to find better classifications. [This page is based on the classifications of Bergey's Manual of Systematic Bacteriology, Volume 1 (1984)]. Later, most (all?) members of the old Photobacterium genus were moved to the Genus Vibrio.

Under optimum conditions, at least one strain of the following bacteria emit at least a feeble light

Today, Xenorhabdus luminescens is classified in the Enterobacteriaceae family and all the other luminous bacteria are placed in the closely related family Vibrionaceae. Photobactium phosphoreum and P. leiognathi have been isolated from the light producing organs of fish but are common in seawater and on the bodies of sea animals. Fish are able flash the luminous organs at will.

Except for V. cholera and X. luminescens, all these luminous bacteria are found only in marine environments and require seawater medium (30 g NaCl per liter). V. cholerae is found in fresh and marine waters and sewage infected by cholera patients and carriers. X. luminescens was isolated in 1975 from an Australian nematode of the Genus Heterorhabdiits and also from insect larvae which this nematode had parasitized.

Xenorhabdus luminescens grows best at ~25C, and poorly or not at all at 36C. It produces orange pigment on Loeffler's blood serum. It produces no growth or negative reaction on the media commonly use for classification of the Enterobacteriaceae. It grows slowly and most assays require 3 or 4 days. While it has the common antigen of the Enterobacteriaceae, it is very distantly related to Escherichia coli ( 4% DNA/DNA hybridization) which is the type species of the family. See Int. J. Syst. Bacteriology 29: 352-360.

Web Sites of Interest

"Complete " list of bioluminescence web sites - Bacteria and other organisms.

The official bioluminescence Web Page - Don't miss this page

Luminescent Marine Bacteria Site - The species, literature, use in teaching - don't miss this site

If you have any questions send me an email    Let me know how this experiment worked for you. Since I live inland and seldom get to the sea, I will welcome cultures of any glowing or other marine bacteria and I am always interested in exchanging cultures.

First installed January 1998      Revision #7 1999 Sep 9   H2SO4     1022m
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Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164