2.1 Culture media
Growing isolates from clinical samples in the laboratory is a useful part of diagnosing the cause of an infectious disease and is an essential step for determining the antibiotic sensitivities of bacteria.
All artificial media comprise a mineral base, a nitrogen source, a carbon source and an energy source. Supplements of organic compounds may be added to encourage or suppress the growth requirements of particular microorganisms. Media may be in liquid (broth) or solid form.
Most clinical samples are inoculated directly onto solid, agar-based culture media on a plate. Videos 2 and 3 demonstrate two common methods for preparing plates, streak plating and spread plating.
Transcript: Video 2 Streak plating
All bacteria in a single colony are usually identical. In a pure culture all the colonies will be alike in shape, size, colour, texture and smell. But in a mixed culture, like a clinical specimen, several different types of colony may be seen.
To check whether a culture is pure we use a technique called streak plating.
The first step is to label the plate with the date and the identity of the sample.
Labels are written on the part of the plate containing the agar so that if the lid is lost the culture can still be identified.
The Petri dish is kept upside down to minimise the chances of a contaminant falling onto the agar.
To make a streak plate we use aseptic technique and a bacteriologist’s loop.
A bottle containing the specimen is taken, flamed to remove any contaminating organisms, and the loop used to sample the microbes within. The loop full of microbes is touched to the surface of the agar and is spread across the plate in a series of streaks that act to successively dilute out the sample.
The plate is turned between each set of streaks. The final streak, a wiggly line, should allow individual microbes to be separated.
In order to more clearly see the streaking technique, we’ve drawn the pattern of streaks on the bottom of a Petri dish, so you can see the order in which they are made.
Bacteria that can grow in the human body are incubated at 37 °C.
After overnight incubation, at the more dilute end of the streak series, separate colonies, each resulting from the growth of a single microbe can be seen.
Transcript: Video 3 Spread plating
Using aseptic technique, we take out 0.1 ml of a particular dilution from its tube and place it onto the middle of the agar surface.
The inoculum is spread over the plate using a spreader. This one is pre-packed and sterilised. The spreader is moved over the surface of the agar, while the plate itself is rotated with the other hand to give an even spread of bacteria across the surface.
When grown at optimal temperatures in suitable conditions, this process enables the laboratory technologist to view individual colonies and select them for additional work according to their appearance (colonial morphology) and relative abundance. Table 1 provides details of the main media types used routinely in microbiology laboratories. Note that media may fall into more than one of the categories listed, for example it may be both selective and indicator.
| Media | Use |
|---|---|
| Basic | Standard nutritional content for non-fastidious organisms. Also used as a base for other media. |
| Selective | Contains substances (antibiotics, bile salts etc.) which suppress certain organisms. Used for specimens where a lot of competing |
| Enriched | Contains extra nutrients (blood, vitamins etc.) for |
| Indicator | Contains substances which change colour, for example when carbohydrate fermentation leads to acid production by certain organisms. |
| Chromogenic | Contains chromogenic substrates that change colour in response to the presence of bacterial enzymes. The colour change is species specific, therefore organisms can be identified by colony colour. Allows rapid, basic identification but is relatively expensive and requires well-controlled storage conditions. |
Activity 5: Culture media in your workplace
Use Table 2 to list the types of agar-based media you have in your laboratory and what you use them for. Note which category of media described in Table 1 each comes into. What considerations govern your laboratory’s choice of media?
| Name of medium | Use | Category |
|---|---|---|
Discussion
You may use many of the media in the example provided or use others not on the list. The media available to your laboratory will depend on factors such as local suppliers, transport and logistics, preferences in your country/region, and cost.
There may be more up-to-date media than the ones you are using as the technology continues to advance. These media save a lot of time, but may be too costly or complicated to use for many district laboratories or for those processing small numbers of specimens. Ready-made media save time in pouring, sterilising, and QC, but are not always available or practical outside of big cities.
| Name of agar medium | Use | Category |
|---|---|---|
| Nutrient | General purpose; supports growth of a wide range of non-fastidious organisms | Basic |
| Blood | For |
Enriched |
| Chocolate | Growing the most fastidious organisms | Enriched |
| CHROMagarTM Orientation urines | Identify common pathogens quickly | Chromogenic |
| Urine samples: only culture those samples with positive microscopy. This medium has characteristic colony appearance and colour for the common pathogens. It also stops Proteus from swarming over the plate. | Selective Indicator | |
| MacConkey | Isolate Gram-negatives | Selective Indicator |
| Modified New York City ( |
Isolate Neisseria gonorrhoeae | Selective Enriched |
| Mueller-Hinton ( |
Enriched | |
| Xylose Lysine Deoxycholate ( |
Isolate Salmonella and Shigella species | Selective Indicator |
2 Basic tests used to isolate and identify bacterial pathogens
