9 Axenic Culture
9.1 Working with Microbes in Axenic (Pure) Culture
As microbiologists, we often work with microbes in axenic (pure) culture (Figure 9.1). Although microbes in nature almost always exist in complex, multi-species communities, it is generally easiest for microbiologists to work with, and identify, microbes when they are in axenic culture.
Being able to work with pure cultures is therefore an essential technique for any microbiologist, and is useful in many other disciplines as well (e.g., for any experimenter who wishes to clone or manipulate genes in their model organism; for the production and purification of proteins for structural or biochemical analysis; etc.)
To work with microbes in axenic culture, we normally would work with single colonies. The asumption is that a single colony is derived from a single cell, and that if you start a new culture from a single colony, it should contain a pure culture. (Note that this may not always be the case.)
Although microbiologists often work with pure cultures in the laboratory, and it can be simpler to study a single organism grown in pure culture, as a discipline, we are beginning to realise the importance of complex microbial communities and have begun to utilise co-cultures to study these communities in the lab. However, this is beyond the scope of your labs this semester.
You will want to read and discuss the discussion questions below - perhaps with your lab partners - to make sure you really understand these points.
Questions to consider/discuss with your lab partners:
Working as a clinical microbiologist, you perform a set of biochemical tests aimed at identifying an unknown pathogen (e.g., testing the ability of the microbe to ferment various sugars, testing whether particular enzymes are expressed…) However, you later discover that you were working with a mixed culture (not a pure one). How does this affect your interpretation of the biochemical test results?
Imagine that a colleague has given you a culture of a particular bacterial pathogen - say, for instance, a particular strain of Yersinia pestis that you are interested in working with. Of course you trust that your colleague is a good microbiologist and you have no reason to doubt their aseptic technique. However, just for the avoidance of doubt - how could you confirm that they have given you a pure culture that contains only your strain of interest?
As microbiologists, how can we work with microbes that do not grow in pure culture (e.g., obligate intracellular pathogens)?
- Consider the agar plate with a mixed culture of Corynebacterium species (Figure D1) - because the colonies are physically separated, you can see that some are cysteinase+ while others are cysteinase-. If these cells were mixed together in a liquid culture and you tested for cysteinase activity, what would you expect to observe?
Figure D1. A mixed culture of Corynebacterium on Tinsdale agar medium, with some colonies producing a halo due to cysteinase activity. Image credit: PHIL 15191.
- Consider the micrograph showing Gram-stained Y. pestis cells (Figure D2). What sort of tests could you perform to determine if these are all genetically identical cells?
Figure D2. A micrograph of Gram-stained Yersinia pestis cells (1100X magnification). Image credit: PHIL 20848
- What do you know about facultative intracellular pathogens such as Mycobacterium leprae, and how researchers typically work with them in the laboratory?
9.1.1 Streaking for Single Colonies
There are many different ways to streak a plate for single colonies; typically, each microbiologist develops their own habits and preferences. You will learn through experience the technique required to ensure that you are able to isolate single, well-separated colonies.
In this lab, you will be learning how to make a quadrant streak plate. You will divide the plate into (typically 4) sectors, and essentially perform a serial dilution - streaking fewer and fewer bacteria out across the agar in each sector.
You will initially inoculate the first quadrant (1 in Figure 9.2), usually with a single colony or a loopful of culture or sample.
You will then sterilise your loop by flaming it (Section 6.1.1.1). You will touch the sterilised, cooled inoculating loop to the first sector, and then drag it across the surface of the agar several times (2 in Figure 9.2).
You will repeat this process several times (3 and 4 in Figure 9.2). In each quadrant, you are inoculating the agar with fewer and fewer bacteria - the aim is to isolate single bacterial cells in the final quadrant.
Do not exert too much pressure when you drag your loop across the surface of the agar - you want to be gentle, so as not to gouge the agar.
Make sure you flame your loop between each quadrant, and only touch the previous quadrant once or twice - you don’t want to inoculate the new quadrant with too many bacteria.
Make sure you are working close to the Bunsen burner (with a roaring blue flame), and using good aseptic technique. Keep your Petri plate closed whenever you are not actually using it, as this prevents microbes from the environment falling onto the agar and starting to grow there.
9.1.2 Plating for Single Colonies
Streaking a plate is not the only way to isolate single colonies - you can also use a spreader, and plate bacteria out for single colonies. However, because bacterial cultures are typically very dense (containing many millions of bacterial cells), it is necessary to perform serial dilutions to isolate single colonies. You’ll learn about these in the next section.