Skip to 0 minutes and 10 secondsAs we've seen in this course, microbes are everywhere. I've gone out onto the university campus to collect a small sample of soil to bring back to the lab and study. In this small soil sample, there are trillions of microbes from all groups. Bacteria, archaea, fungi, protists, and viruses. I've asked my colleague, Harriet, to demonstrate how to isolate bacteria from this sample using common microbiology culture techniques.
Skip to 0 minutes and 49 secondsThe first step is to make a soil suspension. Harriet opens a vial of sterile water and adds this to the soil sample I collected earlier. The reason for this is that we can't plate dry soil-- this would be very messy. Harriet then uses a vortexer to release the microbes into the water. This is our soil suspension.
Skip to 1 minute and 16 secondsThe suspension we made probably contains a large number of bacteria so it is a good idea if we dilute the sample. Harriet makes a 1 in 10 dilution by using a pipette with a blue tip to transfer 1 millilitre of the soil solution into 9 millilitres of sterile water. Harriet uses aseptic technique to prevent her work from being contaminated by microbes that are in the environment around her. She works close to a Bunsen burner as this sterilises the air directly around the flame. She also flames the neck of the vial to sterilise it. She transfers 0.1 millilitre of the diluted sample onto the surface of an agar plate using a smaller pipette with a yellow tip.
Skip to 2 minutes and 6 secondsNotice, that she is very careful not to touch the glass vial with the pipette as this could contaminate her work. Harriet uses a sterile spreader to make sure the suspension covers the entire surface of the agar.
Skip to 2 minutes and 29 secondsThe platelets now needs to be incubated for 1 to 3 days to allow the bacteria to grow into visible colonies. During the incubation period, individual cells in the diluted sample will replicate if they can use the nutrients in the agar and the other conditions are right. Bacteria can replicate very quickly so within 24 to 48 hours, a single microscopic cell on the agar can produce so many cells that it forms a visible colony. Each species of bacteria forms characteristic colonies, which have a specific colour, shape, and texture. We refer to this as the colony morphology. There are clearly, some different coloured morphologies in this sample.
Skip to 3 minutes and 24 secondsIf we want to study a particular colony in more detail, we need to make a pure culture from that single colony. To do this, we use the streak plate method, Harriet sterilises an inoculation loop in the Bunsen flame. You can see, it is really hot. If she touched the colony with the hot loop, it would kill the cells and nothing would grow on the plate. Harriet makes sure the loop is cool before she carefully touches the colony, taking care not to touch any of the others. A small amount of the colony is transferred to an agar plate. This area will have a high concentration of cells and they will grow so close together, we won't see individual colonies.
Skip to 4 minutes and 12 secondsHarriet sterilises the loop by heating it in the Bunsen flame, waits for it to cool, and then, touches the area where she just placed the bacteria. She draws a few lines called streaks, to dilute the sample on the plate. This process is repeated several times. Once this is done, we incubate the streak plate.
Skip to 4 minutes and 39 secondsAfter the plate has been incubated, you can see that there is much more growth in the area that was streaked first. And the area streaked last has single isolated colonies. This is a pure culture. Only one type of colony morphology is present. But what does this microbe actually look like? We'll want to take a look at this under a microscope. To prepare the microscope slide, Harriet sterilises the inoculation loop and uses it to place sterile water onto the slide. Then, she sterilises the loop, again, over the Bunsen burner, waits for it to cool, and takes a small sample from a single isolated colony. She gently stirs this into the drop of sterile water to create a smear.
Skip to 5 minutes and 29 secondsHarriet waits for the smear to dry completely before she passes the slide through the flame to heat fix it. This firmly attaches the bacterial cells to the slide so that the stain doesn't wash them off. The sample is now ready to be stained.
Skip to 5 minutes and 50 secondsHarriet places the slide onto a staining tray. She then floods the smear with crystal violet and leaves it for one minute for the dye to absorb. And then, tips off the excess dye into the sink. Next, she floods the smear with Gram's iodine and leaves it for one minute.
Skip to 6 minutes and 12 secondsOnce she has tipped off the excess, Harriet keeps the slide tilted and the de-colourises the smear using ethanol until it runs almost clear. Harriet is careful not to overdo de-colourise with too much ethanol. Immediately, Harriet rinses the slide with tap water, before gently flooding with safranin to counter-stain the smear for 45 seconds, before rinsing with water.
Skip to 6 minutes and 41 secondsAfter this, slide is blotted dry with absorbent paper. The slide is now ready to view under the microscope.
Skip to 6 minutes and 55 secondsHarriet places a drop of immersion oil on the stained smear and uses a times 100 objective lens to view the bacteria. The cells are stained purple and they are rectangular in shape, which means it is a Gram positive rod. We know from our isolation method that this species of bacteria can grow in the presence of oxygen on nutrient agar. From these results, it is likely that this is a type of bacteria called bacillus, which is commonly found in soil. We would need to do more tests to identify exactly which species of bacillus this might be, but the process of identification has certainly begun. We will find out more about how we identify microbes later in the course.
How to isolate bacteria in a lab
As you saw in Week 1, there are many species of microbe and they differ in how they gain energy and building blocks (nutritional requirements) and what environmental conditions they are able to grow in (growth optima). This means there is no single technique that can be used to culture all the microbes present in a sample.
Recent advances in genomic sequencing of total DNA from environmental samples has revealed a much greater diversity of microbial species than we had ever imagined. Microbiologists working tirelessly over the last century have managed to find ways to culture thousands of different species of microbe in the lab, but it is becoming clear that these represent a very small percentage of microbes on Earth.
In this video, you’ll watch my colleague Harriet demonstrate how to isolate bacteria from a sample on petri dishes containing agar, a jelly-like substance derived from seaweed. Different combinations of nutrients are added to the agar in order to support the growth of particular species of bacteria and fungi. The type of agar Harriet uses in the video is called Tryptone Soya Agar (TSA).
It’s important that microbiologists use aseptic technique to protect themselves from any harmful microbes in the sample and also to prevent their work from contamination. You’ll see that Harriet works closely by a roaring flame of the Bunsen burner as this sterilises the air immediately above the flame, which helps to prevent contamination of her work from microbes, including fungal spores, floating about in the air.
After Harriet purifies a single colony of bacteria from the soil sample, she demonstrates how to make a heat-fixed smear and then stain the cells using the Gram stain technique. This enables us to see the cells under the light microscope and to distinguish two groups of bacteria (Gram positive and Gram negative) based on the structure of the cell wall (Figure 1).
Figure 1: Comparison of Gram positive (Left diagram) and Gram negative (Right diagram) bacterial cell wall structure © CNX OpenStax
Gram positive bacteria have a wall that has a thick layer of peptidoglycan, which retains the crystal violet-iodine dye after washing with ethanol. Gram positive cells therefore appear purple under the microscope. Gram negative bacteria have a much thinner layer of peptidoglycan and an outer membrane that contains channels called porins. When the cells are washed with ethanol the crystal violet-iodine dye is washed out and the cells decolourize. A pink dye (safranin or fuchsine) is used to counterstain Gram negative cells so that they are visible under the light microscope. The Gram positive cells also take up the counterstain but it is not noticeable because the crystal violet stain is much darker.
The Gram stain is often the starting point for identification. In the next Step, you can explore a 3D model of the structure of the Gram negative cell well in more detail.
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