Introduction: Glowing Bacteria Ceparium
Hi again friends! This is my second Instructable and the first one in English! I thought that writing it in English could be good for more people so, here we go!
At first sorry for my bad English, I promise to improve it by practicing it on my tutorials.
Months ago I saw an instructable in which using a Peltier Cell someone build a refrigerator for drinks and other type of food. I´m student on the scientific area, using micro and macro-algae to do molecular studies about phosphorylated and non-phosphorylated proteins. We need to have a perfect refrigerated biomass to do this. I thought that having a portable refrigerated ceparium would be a great solution to combat against the heating in carrying this biomass from the sampling zone to the lab.
Now I need to keep in cold some colonies of Vibrio fischeri bacteria. This type of bacteria is used to detect toxic substances. The peculiarity of this organism is that GLOWS IN THE DARK!. This bioluminescence is used to estimate the toxicity. Toxic substances inhibit a respiration pathway that turns off the bioluminescence. Is amazing to see this microorganism glowing. I´m sure that the bioluminescence will have an important role in our everyday life.
Not much long ago, a day like any other, while I was walking on the street I found a piece of extruded polystyrene (a perfect insulating) so a bulb turn on in my head! I could build a system to keep my little bacteria in a cold but safety place. Great! Lets do something with this piece of "waste"!!
Step 1: Curiosities and Problem
Before build the device is good to take time on read information about systems, material specifications and in this case Vibrio fischeri conditions.
Here is some wiki information of this organism:
Aliivibrio fischeri is a gram-negative, rod-shaped bacterium found globally in marine environments. A. fischeri has bioluminescent properties, and is found predominantly in symbiosis with various marine animals, such as the bobtail squid. It is heterotrophic and moves by means of flagella. Free-living A. fischeri cells survive on decaying organic matter. The bacterium is a key research organism for examination of microbial bioluminescence, quorum sensing, and bacterial-animal symbiosis. It is named after Bernhard Fischer, a German microbiologist.
This organism survives In the deep ocean (high pressure and low temperatures)
Here in Spain the temperature in summer raises frequently 45ºC. In microbiology is important to keep in cold the bacteria cultures to control the growth of the microorganism but also in the case of Vibrios fischeri which optimus culture temperature is about (*) . My mom doesn't agree with the idea of putting my Petry´s culture dishes in the fridge... so a prototype that allows us to keep bacteria in cold but far from the fridge could be great to accelerate the subsequent analytic process.
*All the pictures of this section are not of my property, I only shows it as an explanation of the examples. :)
Step 2: Material
All you need to have is:
- 2 computer fans with it respective heat-sink system. Recycled!
- 1 Peltier Cell. 1,68 $
- 1 small syringe of thermal silicone. 2,5 $
- 1 external thermometer. 5 $
- Transformer 12V 3A. 3$
- Thermo-retractable tube. 0,6$
- 1 plastic jar. Recycled!
- 1 plug jack type 0,8 $
- Some biological agent you want to conserve out of your fridge and far from your food, in my case GLOWING BATERIA. You can choose another type of bacteria, microalgae, live fish food, marine plankton etc...
Step 3: Assambling
We want to build a cube to contain some Petry dishes and some algae samples so my design will be like the Autodesk draw shown on the pic! Simple, easy and low price!
We draw the pieces of the cube in the sheet of EP (Extruded Polyethylene) and we cut it using a hacksaw.
We put it together in order to compose a prism. Then I use silicone to fit and make it hermetic.
Our box must have a removable top but it must fix perfectly with the body of the box in order to be totally hermetic. To achieve it state of perfect hermeticity I use the EP sheet groove and I install some guides of plastic usually used to hide the wires, on the top that fix perfectly into the groove of the EP. With this we obtain a continuous circulation of the interior air which gets more and more cold each time.
The design of the cap is very important. It may have external contact to refrigerate the hot side of the Peltier Cell and internal contact to increase the cold inside the box. (See 3D images of cap)
- Electronic system:
In this case the electrical system is very simple.We are going to use three elements with similar electrical specifications;
- 2 Computer fans --> 12V 0,22A
- Peltier Cell --> 12V 3-5A. (A lower amperage may overheat the system and higher amperes may damage the Peltier Cell.)
The supply system is a laptop charger I buy in the scrap merchant for 2$. I weld some male and female plugs to give independence to the system and make it portable.
Is the time to install the Peltier Cell! We put thermic silicone and extend it all over the heat sink. *Make sure you put the cold side down inside the box and the hot side up outside the system. We install the inside down fan using thermal silicone to fix the union between the heat sink and the Cell
We make a hole to take out the inside fan and Peltier Cell wires and we weld it together with the outside fan . All weld in a female plug to connect and disconnect our refrigerator.
Then we install and weld the male plug to the supplier font. Perfect!! Now we can test our Ceparium!
Step 4: Culture of Vibrios Fischeri
If you are interested on this easy toxicity test you must to know how to culture Vibrios fischeri.
At first you need to buy a sample of the bacteria. You can do this on:
I found a perfect and detailed explanation to culture this bacteria by Courtney Chinn:
Before you add the bacteria you have to first make their food. This will serve as the the source of energy for the bacteria. The tryptic agar base is full of vitamins, minerals, amino acids, carbon, and salt.
- Mix agar medium (125 ml) and 40 grams of aquarium salt in a large saucepan.
- Cover with lid to create pressure cooker-like atmosphere (this will boil down the corse salt)
- Add 1/4 cup of distilled water. (like every other organism, Vibrio Fischeri need water too)
- Cover again and let sit for 45 minutes, stir mixture occasionally to remove lumps.
-Gather sterile petri dishes and open slightly
-Pour in agar mixture and replace lid back in place
-Since it's a gel it should set fairly quickly. now you're ready for the bacteria
- Turn off all lights and examine the Vibrio fischeri in the dish they came in. (give your eyes and the bacteria time to adjust to the darkness)
- Take a sterile Q tip and swap the bacteria and move it to the petri dish with the mixture you made (when doing this, make isolated colonies on all corners of the dish. This type of bacteria can be a bit selfish when it comes to sharing space)
- Cover back all lids and move the bacteria to a cool dark place. The bacteria need a dark area to thrive. Cover with newspaper to ensure no light gets in. (The bacteria should be left to culture 8-24 hrs)
If you are a good investigator you can choose the poor but better option:
Vibrios fischeri is a symbiotic bacteria who lives in the skin of some marine mollusks like sepia or squids. You can try to isolate it by trying to culture some samples of squid skin. When you get an isolated colony you´ll be the best Vibrios fischeri isolator in the world!!!
*All the pictures of this section are not of my property, I only shows it as an explanation of the examples.
Step 5: Testing Toxicity (Lab Aplications)
Preparation of test substances
- Hydrophilic: Weight the substance, disintegrate with a mortar or stomacher, and dilute in saline solution.
- Hydrophobic: Weight the substance, disintegrated with a mortar or stomacher, and dilute it in organic solvent (ethanol). Add 10 ul of sample in a paper disc. Dry for 24 h (or in an oven) to remove ethanol.
Inhibition of bioluminescence by inhibiting respiratory chain.
- Adjust the concentration of a culture of Vibrio fischeri at an approximate concentration of 108 cfu / mL (corresponding to approximately 0.5 McFarland turbidity).
- Planting Vibrio fischeri in a TSA saline plate (2% NaCl) using a swab or a spatula Digralsky.
- Incubate the plate at 22 ° C for 24 h.
- Once the bacteria grown, deposited on the plate 10 ul of the test substance to the desired concentration. Wait 15 minutes until the drop dries.
- Observe the decrease in bioluminescence in the area affected by the drop of the test substance under darkness conditions.
Step 6: Aplications and Conclusions (In Construction)
Ill write my test conclusion in a few days when I finish my experiments!
Videos and photos will be upload too!
Thanks for watching and reading :D