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.