Skip to 0 minutes and 10 secondsWe can do practical work to investigate the bacteria in plants. These plants are called legumes and have lumps on their roots called nodules. These nodules are full of bacteria. The bacteria take nitrogen from the air and ‘fix’ it forming nitrogen compounds. They turn the nitrogen from the air into a form that the plant can use. The bacteria we are looking at are species from the group called Rhizobium. We are going to grow the bacteria from the root nodules on agar in a Petri dish. It’s essential to use a Mannitol yeast extract agar (MYEA) - containing nitrogen compounds that the bacteria can use.
Skip to 0 minutes and 52 secondsAlthough the bacteria in the root nodules usually take nitrogen in from the air they are unable to do it once they are removed from the plant. This nutrient agar provides them with the nitrogen they need. In microbiology it is standard practice to label the dishes, so that students can make sense of their results and other people know what the dishes contain. I’ve labelled the plates with the date, type of medium used to grow the bacteria, source of bacteria – in this case root nodules– and my initials. We are going to use aseptic technique to isolate and grow bacteria from the root nodules. Start by choosing a piece of root with plenty of nodules.
Skip to 1 minute and 41 secondsWash away any soil or dirt under a running tap. To get rid of any bacteria that might be living on the surface of the nodules we soak the nodules in 70% ethanol for about 1 minute.
Skip to 2 minutes and 4 secondsNow that I’ve got the root nodules sterile from here on I am going to work with the Bunsen burner on a roaring blue flame, to try to keep things sterile. This creates an updraft that carries warm air and any bacteria in the air away from the Petri dish, helping to keep things sterile. Make sure you keep the ethanol well away from the Bunsen burner flame. You need to work fairly close to the flame to make use of the updraft, within say 10 cm of the flame. Now we are going to rinse the root. Sterilise some forceps and transfer the root from the ethanol into a petri dish of sterile water. Rinse thoroughly.
Skip to 2 minutes and 47 secondsAdd a few drops of sterile water to a sterile watch glass and add your root nodules.
Skip to 3 minutes and 19 secondsNow macerate this with a glass rod so that you get a milky fluid.
Skip to 3 minutes and 30 secondsFlame a loop, let it cool and then transfer a loop full of the liquid.
Skip to 3 minutes and 39 secondsYou can streak this a zigzag pattern. The bacteria should grow in that same zigzag pattern so I can be more sure they came from the nodules and aren’t just contamination.
Skip to 3 minutes and 56 secondsYou’ll notice the tape is just holding the lid in place, but plenty of air can get in, making sure we are growing aerobic bacteria that need oxygen from the air, not anaerobic ones. You’ll need to incubate the plates for 2-3 days, at 20-25 degrees Celsius. Once the Rhizobium bacteria have grown they will be off white and follow the zigzag pattern.
In my first year of teaching I remember having to teach the nitrogen cycle for the first time and wondering how to make it interesting.
There are several diagrams of the nitrogen cycle available, but teaching this topic is often text-book based. The practical technique shown in the video below can be used to isolate the nitrogen fixing bacteria from root nodules in leguminous plants and help students to recognise the role of bacteria in the nitrogen cycle.
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This video demonstrates the aseptic technique. Note that to keep the sample sterile you should work within 10cm of the Bunsen burner. Ethanol should be kept well away from lit Bunsen burners.
See additional links below for further guidance.