Explorations in BioLuminescence

DIY bioluminescent mushroom :-)

From fireflies to stinky squids... the future is bright

drinking?

1 GENERAL INTRODUCTION
2 LUMINOUS BACTERIA
3 GLOW SHROOMS
4 BIOLUMINESCENT ALGAE
- 4.1 Art / Design / Products using dinoflagellates
- 4.2 Further links
5 HACKING FIREFLIES
- 5.1 Artists working with the topic of firefly
6 DIY BIOLUMINESCENT DORMICE
7 Glowing Mouse in HEL
8 Make your own GlowSticks
9 Glowing Wurst
10 Other experiments
11 GLOWING FOOD EXPERIMENTS
- 11.1 Further links
12 LUMINOUS WORKSHOPOLOGY
13 TOWARDS A MINI CITIZEN SCIENCE FESTIVAL IN ZURI
14 OTHER LINKS AND REFERENCES

GENERAL INTRODUCTION

Bioluminescence is visible light made by living creatures. Such creatures are rare on land but extremely common in the oceans. see more on Marine Bioluminescence by BioScience | Explained here: File:BiolumEN.pdf

Bioluminescence is not the same as phosphorescence

Even though Steinbeck, Hemmingway and even Darwin referred to the “phosphorescence of the sea” this is a literary rather than a scientific description. Phosphorescence is the delayed emission of light from a source that has been excited by light. Examples include glow-in-the-dark paints and toys.

Bioluminescence is not the same as fluorescence

As with phosphorescence light emission is stimulated by light not by a chemical reaction. With fluorescence the excitation wavelength is always shorter, that is, higher energy, than the emission wavelength and emission ceases as soon as the excitation source is turned off. Some of this confusion may have arisen because some, but not all, luciferins are fluorescent and a few pass their excitation energy along to fluorescent proteins like GFP.

LUMINOUS BACTERIA

from citizendium

Various bacteria on a luminous fish fillet (Magnification 1000x) Baggenstos/Rudolf

Luminous bacteria of a coalfish filet in a petri dish Baggenstos/Rudolf

At present, light-emitting bacteria are found in approximately 25 species of five genera in three families of Gammaproteobacteria: Shewanella (Shewanellaceae), Photorhabdus (Enterobacteriaceae), and Vibrionaceae (Aliivibrio, Photobacterium, and Vibrio) (Dunlap & Kita-Tsukamoto, 2006; Ast et al., 2009; Dunlap, 2009; Yoshizawa et al., 2009a, b).

Members of seven Photobacterium species produce luminescence: Photobacterium angustum (certain strains), Photobacterium aquimaris, Photobacterium damselae (certain strains), Photobacterium kishitanii, P.leiognathi, Photobacterium mandapamensis, and P.phosphoreum.

Vibrio fischeri (Beijerinck 1889) got a new reclassification from genus Vibrio to the newly created Aliivibrio in 2007. Source: Phylogeny, genomics, and symbiosis of Photobacterium Henryk Urbanczyk, Jennifer C.Ast and Paul V.Dunlap

Isolation of luminous bacteria from fish or squid

from bioScience | Explained, download File:BioSci expl PhotoLumen.pdf

P. phosphoreum is one of the commonest spoilage organisms of fish such as cod. It is not known to cause disease, but there are reports of people being startled by glowing fish fingers in the fridge!

To isolate glowing bacteria from fish, obtain a freshly-caught seafish or squid. It is very important that the fish has not been frozen or washed in fresh water. It is also better if the fish has not been stored on ice. (That‘s what you usually find everywhere on the Internet. But you can buy almost any saltwater fish fillet from the counter and you will be able to isolate glowing bacteria.) Place the fish in a container with 3% NaCl solution. The liquid should be deep enough to come half way up the fish.

Cover the container and store the fish for 24 hours at about 12–15 °C. Note: if this temperature is difficult to achieve, place the fish in a fridge at about 4 °C for 48–72 hours.

After incubation, take the container with the fish into a dark room. When your eyes have been adopted to dark, light spots will be visible on the skin of the fish. Use a sterile toothpick or disposable sterile loop or needle to aseptically transfer the brightest spots onto sterile fish enrichment agar plates. Tip: some people find it useful to use a dim red lamp (e.g., a photographic safety lamp) for this step. Turn the fish away from the lamp so that the glowing colonies are in the shade and therefore visible.

Transfer the cultures to new agar plates every second day if you are incubating them at temperatures around 12 °C or once a week if you are storing them in a fridge. P. phosphoreum will grow at 4 °C; V. fischeri will not. By selecting the brightest colonies when inoculating, it should be possible to isolate a pure culture.

Fish enrichment medium

Boil 250 g fish meat in 1 l of water.
Add 30 g NaCl and sieve to remove solids.
Add 10 g peptone, 10 ml glycerol and 1 g yeast extract.
Adjust the pH to 7.
Autoclave at 121 °C for 15–20 minutes.

For a solid medium add 15–20 g of agar to every litre of broth.

Safety note Several species of Vibrio are pathogenic. The chances of inadvertently isolating pathogens in this procedure can be reduced by using at least 3% salt solution and incubating fish and plates at no more than 15 °C. Human pathogens are unlikely to grow under such conditions.

Photorhabdus luminescens

Photorhabdus luminescens (sometimes called Xenorhabdus luminescens) is a bioluminescent microbe and lives in symbiotic relationship with soil entomopathogenic nematodes(family: Heterorhabditidae).

Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Photorhabdus
Species: luminescens

Draft Genome Sequence of Photorhabdus luminescens Strain BA1

Photorhabdus luminescens bacteria live in Nematodes and produce a toxin that is pathogenic for many insects. The nematodes infect insect larvae and kill them with the toxin. In the insect larvae, the bacteria excrete next to the fatal toxin also an antibiotic substance to prevent invasion of the insect by bacterial or fungal competitors, and a luminescent protein that causes a faint glow in the infected insect remains.

The life cycle of Photorhabdus luminescens. (Williamson et al. 2003, Nature, 21, 1294-1295)

For a detailed description see these two websites:

There have been some reported cases of human infection by Photorhabdus luminescens. Stories from the American Civil War and the First World War tell of soldiers with wounds that glowed in the dark. Their fluorescing tissues appeared to heal more cleanly and more quickly than the unilluminated wounds of their counterparts. This mysterious phenomenon became known as the „Angel’s Glow“. The afflicted soldiers had no idea that the glow was caused by a beneficial bacterial infection and instead interpreted it as the gift of survival from God, handed to them by angels, hence the name.

Nematodes are used as pesticides in organic farming; so for orchards, for young vines and ornamental plants, against the black vine weevil and the garden chafer. In Switzerland you can easily buy such nematodes at coop and migros.

Pictures by: Baggenstos/Rudolf

Further links P. Luminescence

Photorhabdus species: optimizing bioluminescence by Simon Park

A very good Investigation on the bacterium Photorhabdus luminescens from Jürg M. Grunder:

Art / Design / Products using luminous bacteria

A collaborative project between Anne Brodie and Simon Park 2009-2011
Bioluminescent Cities Simon Park
from Bioglyphs
Hunter Cole Living Drawings Created with Bioluminescent Bacteria
Rebecca Klee „Painting“ with bacteria
Kathy M. Takayama The symbiotic bacterial light project
Anne Brodie: Installation at The Old Operating Theatre Museum and Herb Garret, London
Tim Otto Roth Fisch, 2009 Luminogram
Teresa Van Dongen One Luminous Dot 2015 Installation
Marianne Engel Vibrio fischeri
Thomas Feuerstein Unmarked Space Oddity, 2007
Anna Dumitriu in collaboration with Dr Simon Park and Alex May Bacteria Light Lab, 2015

Further links

https://vimeo.com/115082758

GLOW SHROOMS

P.Stipticus in a agar plate

We found this wood near Zurzach AG Switzerland July 2014

Wood piece 23.8.2014

For various recipes on making agar growth plates check Agar is the Media

A full description of ideal growth conditions has been publisher here http://jresearchbiology.redolences.com/documents/RA0190.pdf

Influence of culture conditions on mycelial growth and luminescence of Panellus stipticus

IB Prasher, VC Chandel, AS Ahluwalia - Journal of research in Biology (2012) 3: 152-159

ABSTRACT:

Panellus stipticus (Bull.) P. Karst, a naturally bioluminescent tropical fungus, has been studied in vitro for the optimum culture conditions viz culture media, temperature, pH and days of incubation required for luminescence. Temperature and pH affect growth and bioluminescence to a great extent. Glucose-peptone medium has been found to be the best for optimum mycelial growth as well as luminescence. The fungus exhibits luminescence at 20-24°C. The maximum mycelia dry weight (mg/25ml of the basal media) and luminescence observed at pH 4.0. The fungus exhibits luminescence after eight days of incubation at 24°C and pH 4.0, whereas it intensified to maximum after 13 days of incubation (pH 4.0 and temperature 24°C).

tips for Shroom Cultures

bags: sterilizable bags
Recipes: Agar Medium

GLOWING WOOD

Glowing Wood is a bioluminescence phenomenon caused by a fungus. Some fungi have the ability for bioluminescence and one of them is Armillaria.

Armillaria --> Honey fungus, or Hallimasch (German) or оpenky (Ukrainian) is a genus of parasitic fungi that live on trees and woody shrubs. It includes about 10 species formerly lumped together as Armillaria mellea. It is known to be one of the largest living organisms, where scientists have estimated a single specimen (of the species Armillaria solidipes) found in Malheur National Forest in Oregon to have been growing for some 2,400 years, covering 3.4 square miles (8.4 km²). Some species of Armillaria are bioluminescent and may be responsible for the phenomena known as „foxfire“ and perhaps „will o‘ the wisp“. wikipedia.org

Die Gattung „Hallimasch“ (Armillaria) zählt zur grossen Klasse der Ständerpilze (Basidiomycetes). Innerhalb dieser Gattung gehört Armillaria zur Ordnung der Blätterpilze (Agaricales).

Actually Armillaria is known as a common tree pest, because it means certain death for the infected trees.

Das Licht, das Pilze zum Leuchten bringt entsteht durch eine chemische Reaktion in den Zellen, dabei reagiert Luciferin mit Sauerstoff und gibt ein Lichtteilchen ab, ein Photon. Das für diesen Prozess verantwortlichen Molekül ist bei Raumtemperatur instabil, und in Kontakt mit Licht zerfällt es sofort. —> Weshalb der Hallimasch im Dunkeln leuchtet - Von Atlant Bieri 2011

Schon lange versuchten Wissenschaftler die Struktur des Moleküls Luciferin, das Pilze zum Leuchten bringt, herauszufinden. Denn die „Leuchtsubstanz“ von Pilzen unterscheidet sich gänzlich von dem der Glühwürmchen. Im Sommer 2015 gelang es einem Forscherteam aus Krasnoyarsk, Moscow und Nagoya das Puzzle zu lösen.

A team of Russian and Japanese scientists has now unravelled the previously unknown chemistry responsible for the glowing fungi. The team was able to identify the luciferin precursor as hispidin, a fungal metabolite known to be a strong antioxidant. What makes a fungus able to luminesce isn't hispidin alone; it is the ability to produce the two enzymes necessary for bioluminescence. The researchers were able to extract these two enzymes from the biomass of luminescent fungi: The first is hispidin-3-hydroxylase, which converts hispidin to the actual luciferin, 3-hydroxylhispidin, by means of a hydroxylation reaction that also involves NADPH. -> Fungi get the green light: Chemical basis for bioluminescence in glowing fungi uncovered

The Chemical Basis of Fungal Bioluminescence Press-Information Abstract

Historic examples

Used as lighting for early submarines http://en.wikipedia.org/wiki/American_Turtle

"Six small pieces of thick glass in the top of the submarine provided natural light.[5] Illumination while submerged was provided by a piece of cork that gave off a fungus-powered bioluminescent foxfire. During trials in November 1775, Bushnell discovered that this illumination failed when the temperature dropped too low. Although repeated requests were made to Benjamin Franklin for possible alternatives, none were forthcoming, and the Turtle was sidelined for the winter."

Das erste Mal, dass wir von biolumineszierendem Holz hörten war durch die Erzählung eines Freundes der Andreas Brodbeck kannte und der ihm einmal leuchtendes Holz gezeigt hatte.

Andreas Brodbeck is a fotographer and bioluminescence geek. Here some Links to his pictures.

Art / Design / Products using GlowMashrooms

Marianne Engel Armillaria
Marianne Engel Panellus Stipticus
Natalie Andrew Growing fungi in crafted shapes...

Marianne Engel Hallimasch, 2010
Natalie Andrew Jar growing panellus
Baggenstos/Rudolf Desire 10, 2015
Tim Otto Roth Waldlicht > fungus luminogram, 2007

Further links

BIOLUMINESCENT ALGAE

DINOFLAGELLATES

What is a dinoflagellate? Latz Laboratory Scripps Institution of Oceanography

GEO Reportage: Die Leuchtkraft der Natur (About Dinoflagellates)

Bioluminescence in Dinoflagellates (Rüdiger Hardeland and Mona Hoppenrath)

Microorganisms 2013: Understanding Bioluminescence in Dinoflagellates—How Far Have We Come?

nature queer!!

Material for growing dinoflagellates:

from Microorganisms 2013

Dinoflagellates Pyrocystis Noctulica

A clear growing container (shallow containers with lots of surface area)Sanitation is necessary so your batch doesn‘t crash. REALLY wash out the grow container, make sure there is absolutely no residue left. Culture flasks -- Sterilized glassware if autoclave is available, otherwise use disposable tissue culture flasks. If you use sterile media and glassware, your cultures will continue forever; every month pour about 1/4 of the culture into some new medium. If you can‘t maintain sterile culture conditions, the cells will last for a few weeks to a month before bacteria overgrow the culture.

Water

Use purified water as tap water can contain chlorine or other things that might kill your batch.
No tap water, tap water may contain metal ions that are detrimental to algal growth.
Temperature: 22° C (Pyrocystis fusiformis 15°C - 25°C)

Sea Salt from pet or aquarium stores

Light and timer

Indirect natural light is good, but not direct sunlight.
Illumination with cool-white fluorescent lamps
Fluorescent light, either cool white or full spectrum, is also sufficient. Avoid incandescent lamps, as they can overheat the water and kill your algae.
A grow light or an aquarium/terriarium light
Recommendation of Sciento: We finde illumination under 30-40 watt Fluorescent tube or LED lighting ideal and avoid lighting like halogen bulbs or high wattage incandescent bulbs which tend to emit excessive heat.
12 hours of light and 12 hours of darkness every 24 hours

Nutrients (Growth medium)

Dinoflagellates require nitrate, phosphate, trace metals, and vitamins.
Growth medium:
Dino Food
Dino Refill
UCSB lab instructions "all the instructions you will need for growing temperate-tropical dinoflagellates in a lab"

A Starter Culture

A15 Bioluminescent Algae 250ml cultures from Sciento. Planktonic marine, bioluminescent dinoflagellate (Pyrocystis sp.)

Supplier of Dinoflagellates:

Sciento Manchaster UK http://www.sciento.co.uk/catalog/item/553/
Empco Holdings Menlo Park USA http://empco.org/edu/index.php/bioluminescent-algae.html
Sunnyside Sea Farms Goleta California USA http://seafarms.com/html/products.html
Carolina Burlington USA

Art / Design / Products using dinoflagellates

Dino Pet Powered by dinoflagellates We like to think of it as lighting in a bottle, only without the pesky thunder.
Dino Pet at Kickstarter
Sophia Sobers Heartbeats & Bioluminescence / Necklace, heartbeat monitor is a concept

Nicola Burggraf She is a product designer and works on the topic of bioluminescence from a design and research perspective.

Bioluminescent Field 2010

Andreas Greiner - Artist
Erika Blumenfeld - Artist

from biopop.com
from Sophia Sobers
from Nicola Burggraf Interference 2012
from Andreas Greiner toccata-pyrocystis-fusiformis
Bioluminescence Erika Blumenfeld

Further links

HACKING FIREFLIES

Gluehwuermchen.ch

LED-Falle

Artists working with the topic of firefly

from Marianne Engel Directly exposed film by glowworms
from Francesco Mariotti The Fireflies Factory
Living Lightning: Critical Making Workshop and Performance
from China Blue Firefly Trees 2011
from Robin Meier Synchronicity
from Silke Silkeborg Achenberg mit acht GW, 19 x 33 cm, Oel auf Holz, 2013

DIY BIOLUMINESCENT DORMICE

still working on it...

Glowing Mouse in HEL

Make your own GlowSticks

Glowing Wurst

see more about it on Randelab

Other experiments

Daphnia GlowStick experiments in Gaudilabs

http://instagram.com/p/sLMnA7Dm9F/?modal=true

GLOWING FOOD EXPERIMENTS

DIY glowing worms (edible)

Further links

http://www.funathomewithkids.com/2013/08/safe-and-edible-glow-water-for-baths.html

http://www.funathomewithkids.com/2013/08/edible-glowing-oobleck.html

http://www.funathomewithkids.com/2013/09/edible-glowing-spiderwebs-sensory-play.html

Jellyfish Ice Cream Glows When Licked

Glow in the Dark Ice Cream

Fox News - Glow in the Dark Ice Cream

LUMINOUS WORKSHOPOLOGY

--- the future is bright ---
Why the hell should everything glow?

Phototaxis is a kind of taxis, or locomotory movement, that occurs when a whole organism moves towards or away from stimulus of light.This is advantageous for phototrophic organisms as they can orient themselves most efficiently to receive light for photosynthesis. (wikipedia.org)

Phototropism is the movement or growth of organisms in response to lights or colors of light (e.g.- the sunflower) (wikipedia.org)

Heliotropism is the motion of plant parts (flowers or leaves) in response to the direction of the sun. A. P. de Candolle called this phenomenon in any plant heliotropism (1832). It was renamed phototropism in 1892, because it is a response to light rather than to the sun. (wikipedia.org)

TOWARDS A MINI CITIZEN SCIENCE FESTIVAL IN ZURI

starting with experimentations

Andreas shows the glowing wooden logs they found in the forest around Zurzach
Andreas chopping and preparing wood for the biolum-effect
Can we turn this artist's studio into a micro-GlowLab?
Let's go shopping... Coop in Letzipark will have all we need
... and the rest we get donated from some friends up in ETH Hönggerberg. Thanks to Edmondo for all the fine chemicals and containers
The Baggenstoss / Rudolf Atelier is slowly turning into a cozy neighborhood science lab
Let's make a nice clean surface and prepare some containers.
Sterilizing, cleaning, cooking...
Measuring, mixing, stirring, boiling and testing various agar agar powders from the supermarkets.
Making a potato extraxt stock solution.
Using a MAE agar plates the first Armillaria and P. Stipticus cultures are ready to grow. Aswell as some Grain-cultures of the P. Stipticus. Patience...
Culturing the dinoflagellates
The look ok, some seem to have survived the 9 day UK-mail trip...
Planning the Läb(e) am Egge
Agar plate nr.4 at 22.8.2014
Placing of some Armillaria Rhizomes on agar plate
Searching for Armillaria Rhizomes in the forest floor
Transferring of good parts on other agar plates
The remainings
The P. Stipticus is growing nicely and developed a beautiful glow
Inoculation of an beech tree piece
Beech tree pieces
Glowing Wurst experiment... It woooooorrkkks!
We found this wood near Zurzach AG Switzerland July 2014
Wood piece 23.8.2014
Wood search on 3 September above Triemli, Zurich
The first location, right next to the path
We were looking for ten locations
Armillaria paradise at Swiss Federal Institute for Forest, Snow and Landscape Research WSL
The professional kitchen
Renate transfers some mycelium into a new agar plate
Armillaria culture 19 Days old

Armillaria culture 45 Days old. A small part of the mycelium shows a faint glow

Armillaria culture 113 Days old (Once with back lighting)
Armillaria culture 150 Days old
Armillaria Rhizome after 13 days in a petri dish
Armillaria Rhizome after 13 days in a petri dish
Armillaria Rhizome after 15 days in a petri dish (A20)
Armillaria Rhizome after 15 days in a petri dish (A20 double exposure)
DNA sequence Elongation Factor 1α of the rhizome A20
We have turned our ticket counter from the performance „notion (Vorstellung 2010)“ into a sterile working area
We use UVC light with 254Nm for surface sterilization. For inside overpressure we want to use a converted vacuum cleaner and a HEPA filter. (Still at work)
Potato Agar on the Floor. What is living in our studio.
Potato Agar on the Floor. What is living in our studio.
Another toxic? whatsoever fungi species at our studio.