The most frequent question I am asked by guests to the TIME room is “What is that shaking thing that looks like it has dumplings in flasks?” Many are fascinated by this strange tool and the microbes growing on it. Even Mr. Gibbs, Director of Human Resources at the Ed Center, stopped mid-presentation to say “I’ve got to ask: What is that thing jiggling in the back?” Now you too are wondering…what is this mysterious instrument?
THE SHAKING INCUBATOR was acquired by the TIME program last year for my group’s project. It has a rotating plate in which different flasks can be strapped in and spun for an infinite amount of time at any RPM. We use this to grow our microbes in broth cultures rather than petri dishes. The shaking helps to oxidize the culture and thus promotes respiration.
Currently growing on the shaker are some of my most beloved organisms. We have two positive controls. These are different species of Trichoderma, which is a known cellulase producer. One of these is growing great, and the other has no signs of growth. Next is my personal favorite, B4, which is a currently unknown organism we cultured from compost. B4 appears to be a fungus. Lastly is B5, another unknown organism from compost, who also appears to be producing the enzymes necessary to digest the cellulose. B5 resembles a bacteria.
If you are interested in observing this fascinating piece of equipment, feel free to take a visit to the TIME room at BHS during 4th period. We’d be happy to show you around.
Don’t worry; this is not some new illegal substance! Rather, it is an experiment that will be key to the BRELLA project. As my partners Bryce and Lauren already explained to you, our project took a little longer to get cranking than usual. Now, after several, SEVERAL attempts at project designs, we are ready to start our preliminary experiments.
DNS stands for dinitrosalicylic acid, a fairly common chemical which is also known to produce a color change when it bonds with glucose. (This is important because, if you recall from Bryce’s blog, when you are making ethanol, the enzymes break the substrate into glucose molecules. These are then fermented into ethanol.) We will be performing an assay using DNS to see if enzymes are being produced by both our consortium and the individual microbes.
Enzymes will be isolated from the broth in which the organisms are growing in. These isolates will be placed on a piece of filter paper and given time to degrade the paper. Then DNS will be added and will result in a color change when it bonds with the glucose molecules. This color change will indicate that the proteins have broken the bonds of the filter paper into straight glucose. This assay screens for “reducing sugars,” basically meaning that if glucose is present, enzymes had to have been present. Tomorrow, we will be using performing a positive control test in which there are no enzymes, but instead we just add the DNS to straight glucose. This will aid us in measuring the next tests. Stay tuned for data!
Over the past week, the Jolizen station has been quite colorful. Screening methods using dyes have been used to detect enzyme production. Originally, we were using dyed plates and looking for a change in color as the fungus grew on the plate over the period of one week or more. After unclear results with this type of method, a new idea for enzyme detection was adopted. After growing our fungus for a few days, the plates are then flooded with the dye and results are available in 10 minutes, rather than 1 week, and are much more definite. The first assay of this kind detected amylase, an enzyme produced in our saliva that breaks down starch. Our fungus was grown on potato dextrose and flooded with Gram’s iodine. The starch that had not been “digested” stained black, leaving a ring of clear fungal growth where our fungus had broken down the starch. Amylase is being produced! The second assay was stolen from Hannah and Ryan and was the answer to our prayers! Our fungus was grown on LDE plates. When we returned from the weekend, the fungus an the surrounding quarter inch were brown to orange in color while the rest of our plate was clear. This indicates the production of ligninolytic enzymes, or enzymes that break down lignin. Our last assay which detects for production of cellulases (they break down cellulose) will happen tomorrow. Our fungus has been growing on cellulose and will be flooded with Congo red dye. Let’s cross our fingers that it’s 3 for 3!
The pressure cooker has been running nonstop this past week in the TIME room, cooking every type of agar known to man. Our refrigerator is stocked full of potato dextrose, blue, red, and water agar, so come and get ’em! Yesterday and today, the infamous “Jolizen” team (made of members Eliza, Joe, and Lauren) has been transferring our fungus, Diaporthe sp., to plates filled with 3 different types of mediums. The red plates (making up the red stripes of our homeland’s flag) have cellulose and a special Congo Red dye (especially extracted and shipped to us on the back of African elephants straight from the heart of the Congo). If this dye shows discoloration by the start of next week, our fungus is producing some type of cellulase enzyme. We also are growing our fungus on blue plates (found in the blue star corner of the flag) which, if color change occurs, will indicate activity of an enzyme that breaks down lignin. Lastly, we cultured on potato dextrose agar (the white stars and stripes) for the purpose of maintaining a healthy base line fungus to take isolates from. We performed DNA extraction and PCR on these PDA cultures last week so that we can be sure they are still Diaporthe sp. Stay tuned for the results of our colorful tests!