Skip to 0 minutes and 10 seconds There are a range of techniques which can be used to identify precisely which microbe is responsible for causing an infection. We’ve returned to the microbiology lab to show you some of the identification techniques our students use to identify pathogens in their practical classes. For this, I have set up pure cultures of three different human pathogens and have asked Harriet and Jordan to demonstrate the process of identification.
Skip to 0 minutes and 43 seconds The first plate has yellow-coloured, circular, shiny colonies. This has been examined under the microscope, and it is a species of bacteria, a gramme positive coccis. The plate was incubated in the standard incubator, so we know it can grow in the presence of oxygen. The next step is to perform a catalase test. To do this, a small amount of a single colony is transferred to a Petri dish, using a sterile plastic loop. A few drops of hydrogen peroxide are added. If it bubbles or effervesces, the reaction is positive. If no bubbles are produced, it is negative. This species of bacteria is catalase positive. The next step is to perform an oxidation fermentation or OF test.
Skip to 1 minute and 35 seconds Glucose is added to two tubes of molten OF agar. These are then inoculated with a pathogen. After they have cooled down and solidified, a small cap of molten agar is added to the top of one of the tubes. This restricts how much oxygen can reach the bacteria that are now growing in the green OF agar beneath it. After incubation for 24 hours, both tests have turned yellow. The bacteria have produced acid from the respiration of glucose in both tubes. This pathogen can ferment glucose, so we know it must be a species of staphylococcus. But which one is it? The Staphaurex test can be used to determine if it is staphylococcus aureus or not.
Skip to 2 minutes and 23 seconds A small amount of culture is transferred to two wells on the test card. Harriet adds a drop of the controlled reagent, which contains latex beads, to one well. She adds a drop of the test reagent to the other well. The test reagent contains latex beads that are bound with anti protein A antibodies.
Skip to 2 minutes and 46 seconds You can see that the test reagent has reacted with a culture to form visible clumps or aggregates of the latex beads. But this hasn’t happened in the control. The antibodies in the test reagent have bound to protein A in the bacterial cell wall, and this has caused the beads to clump together, which we call agglutination. This reaction is positive, so we can be certain this pathogen is staphylococcus aureus.
Skip to 3 minutes and 22 seconds Let’s take a look at the next plate. The second pathogen forms pale green flat colonies that look slightly gooey. This has been examined under the microscope, and it is a species of bacteria, but one that is a gramme negative rod. It grew in the presence of oxygen, so the next step is to do an oxidase test. To do this, Jordan puts a drop of reagent on a piece of filter paper. He then transfers a single colony using a sterile plastic loop onto the filter paper. If a purple colour develops, the reaction is positive. If no colour change is seen, the reaction is negative. The reaction has turned purple, so this species of bacteria is oxidase positive.
Skip to 4 minutes and 6 seconds The next step is to perform an oxidation fermentation test, in the same way Harriet showed us earlier in this video. In this case, acid has only been produced in the uncapped tube. The capped tube is still green. This pathogen can oxidise glucose, but is not able to ferment it. It therefore seems likely that the pathogen is a species of pseudomonas. As you can see, on the King’s B agar, it has a very green colour. If we place the plate under UV light, it is fluorescent. So we can be certain this pathogen is pseudomonas aeruginosa.
Skip to 4 minutes and 52 seconds The third plate has cream coloured, raised, circular colonies. This has already been examined under the microscope, and the cells stain purple, but they were much larger than what you would expect for a bacteria. The cells are oval shaped, and a few are budding. So this one looks like a type of fungi, yeast. To find out which type it is, we can streak it onto a yeast chromogenic agar. This will change colour based on the type of yeast. The colonies have turned green, which means this pathogen is candida albicans. The differential media made that one much easier. We started by examining gramme stains of the three cultures under the microscope.
Skip to 5 minutes and 37 seconds By following a logical series of tests, we have been able to identify all three pathogens to the species level, staphylococcus aureus, pseudomonas aeruginosa, and candida albicans.
How to identify the culprit of an infection
When someone has a serious infection, it is often necessary to work out exactly which microbe is responsible for causing the disease.
The process starts in the doctor’s surgery or hospital, where a patient’s symptoms can provide clues as to what the microbe is likely to be. Samples are taken (eg urine, blood, throat swab, skin scraping) and given to a pathology lab. Clinical microbiologists will then determine if the pathogen is a bacterium, fungus, protist or virus (no archaea have been found to cause disease) and produce a pure culture (if this is possible; as you explored in Week 2 - not all microbes can be cultured).
There are a range of techniques that can be used to identify precisely which microbe is responsible for causing an infection. This information can help doctors to select the best treatment, and health agencies to prevent the spread of the infectious disease to other people in the community.
In the video, Harriet and Jordan identify three pathogens using a variety of culture-based methods. In many hospitals, such traditional culture-based tests are being predominately replaced by automated machines. These include a technique called MALDI-TOF that identifies bacteria based on the proteins they contain (their proteome). You can watch a video of what’s involved in the MALDI-TOF process here. Other sensitive molecular techniques can be performed on clinical samples without needing to culture the pathogens in the laboratory. These include PCR and nucleic acid (DNA/RNA) sequencing. Continued development of molecular techniques ensures not only rapid, sensitive and accurate results, but can also substantially reduce costs.
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