Bacterial Diagnosis

This post will likely be fairly long (even by MICR3350 standards), as it will essentially cover 2 hours worth of content. As the title states, this post will cover bacterial diagnosis: mainly the detection of pathogens and their identification. Without further ado, let's get started!

Microscopy

Microscopy involves, well, looking at stuff under a microscope. There are different types of microscopy that can be used for different purposes. Note: the stuff about microscopy here is going to be massively oversimplified as I'm not a physicist.

Bright field microscopy

Bright field microscopes are the type that you're probably more familiar with: the object appears dark compared to the background. Usually, some kind of stain, such as Gram's stain, is used to make bacteria more visible.

Dark field microscopy

In dark field microscopy, the light hits the specimen at an angle, resulting in a bright subject against a dark background. This is typically used for detecting spirochaetes, such as T. pallidum.

Phase contrast microscopy

Phase contrast microscopes use two beams of light, which are out of phase by a quarter of a wavelength. This setup increases visible contrast, making it easy to see your specimens without using a stain. Phase contrast microscopy tends to be used more for looking at fungi, rather than looking at bacteria.

Fluorescence microscopy

Fluorescence microscopy uses fluorescent dyes which, as their name suggests, fluoresce. Common dyes include auramine-rhodamine, which stains mycobacteria, and acridine orange, which stains nucleic acids. There are two main types of stains that can be used: fluorochroming, which stains everything, and immunofluorescence, which stains only target molecules via the use of an antibody-labelled dye.

Electron microscopy

Electron microscopy uses electrons which are focused via electromagnets. Electron microscopy can be used to view very small pathogens, such as viruses. It can also be used to visualise bacterial organelles.

Culture

Culturing pathogens can allow us to determine if pathogens are present, and purify pathogens so that we can test and identify them. Bacterial culture can also help us to perform antibiotic susceptibility testing. Most pathogens will form a colony after overnight incubation, though some are very slow-growing. The original bacterium that the colony arose from is called the "colony forming unit" (CFU).

Culture requirements

In order to culture bacteria, you need to have some idea of what is likely to be in the sample, which can be determined by the type of specimen (sputum, urine, etc.) and from symptoms of the illness. Next, optimal conditions need to be provided. Things to take into consideration include nutrients, temperature, atmosphere, pH, and osmolality:

• Nutrients: Pretty much all bacteria need sources of carbon, nitrogen, phosphorous, water and certain trace elements. Fastidious bacteria may need more than this.
• Temperature: Some organisms, known as psychrophiles, prefer cooler temperatures (15-20°C), while mesophiles prefer 30-37°C and thermophiles prefer 50-60°C.
• Aerobic: Strict aerobes only grow when oxygen is present, strict anaerobes only grow when oxygen is absent, facultative aerobes/anaerobes can grow in either condition, microaerophilic microbes like only small amounts of oxygen (2-3%), and capnophilic microbes grow better when CO₂ is present.
• pH: Most organisms prefer neutral or near-neutral pH.
• Osmolality: Some organisms (not many) require salt.

Culture phases 

There are four main phases of growth:

1. Lag phase: Little to no growth occurs as the organism is still adjusting to the medium.
2. Log or exponential phase: Exponential growth occurs.
3. Stationary phase: Cell growth equals cell death, so the overall number of cells remains constant.
4. Death/decline phase: Cells die off faster than they grow. This may be because all of the nutrients have been consumed.

Media types

There are many different types of culture media, and there are different ways in which they can be classified. Many can be prepared in solid (agar plate) or liquid (broth) form. A solid plate can detect a range of organisms and single colonies can be separated out, but it is not as sensitive as broth culture, and some specimens might be difficult to plate. Liquid broth is more sensitive (i.e. can detect very low numbers of organisms) but cannot produce pure cultures, and does not allow for quantification of how many organisms there are.

Media can also be separated according to function:

• Enriched media: Media that contains one or more compounds that stimulate growth, such as yeast or meat. May also include special compounds for culturing more fastidious organisms.
• Selective media: Media that only allow a specific organism or group of organisms to grow. For example, colistin and/or salt stop growth of Gram-negative bacteria. Salt can also be used to select for Staphylococci.
• Differential media: Media that allows multiple organisms to grow, but provides some means of separating between them. For example, mannitol salt agar not only selects for Staphylococci, but can be used to distinguish between Staphylococcus species. S. aureus ferments mannitol, turning the plate yellow, whereas S. epidermidis, which does not ferment mannitol, leaves the plate pink.

Now for some more examples of media, because why not?

Blood agar is made up of Columbia agar and 5% blood, which usually comes from a horse or sheep. Most bacteria (except for some Haemophilus species) can grow on blood agar. Blood agar can also be used to test for alpha- or beta-haemolysis, which can be used to identify species. Chocolate agar, so called because of the chocolate colour (not actual chocolate, sorry) used to be made up of heated blood agar, but is now made up of Columbia agar, haemin and isovitalex. Virtually everything grows on chocolate agar, even Haemophilus.

Chromagenic agar, or Chromagar, can turn many different colours depending on species. There are different Chromagar types for different specimens: for example, Chromagar Candida can distinguish between different Candida species, whereas Chromagar Orientation can distinguish between E. coli, Enterococcus, Klebsiella and more.

New blood culture systems have been developed in order to detect organisms in the blood. A patient's blood is taken and put into a bottle. There are different bottles for different specimens, according to culture requirements. Bottles are put into an automated machine, which signals positive when growth is detected. When growth is detected, a Gram stain and traditional culture may then be performed.

Mycobacteria Growth Indicator Tubes (MGIT) are used for detecting mycobacteria. They contain Middlebrook 7H9 broth and are put into a special machine that scans the tubes for increased fluorescence. Mycobacteria grow much faster in these tubes than they do on plate culture.

Biochemical tests

Following culture, a variety of tests may be done to identify the bacteria. Some commonly conducted tests include the catalase, coagulase and oxidase tests.

The catalase test is used to distinguish between Gram-positive cocci. The presence of catalase is detected by adding hydrogen peroxide (as mentioned here, catalase breaks hydrogen peroxide down into water and oxygen). If the test is positive, bubbles of oxygen will appear, whereas if the reaction is negative, no bubbles will appear. Gram-positive cocci that test positive include Staphylococcus species, whereas Gram-positive cocci that test negative include Streptococcus and Enterococcus. Note that there are many other pathogens that are catalase-positive (but of different shapes etc.).

The coagulase test is used to distinguish between S. aureus from most other Gram-positive, catalase-positive cocci. Coagulase is an enzyme that converts fibrinogen into fibrin (a protein involved in clots). Coagulase-positive bacteria will form clumps, whereas coagulase-negative bacteria will not form clumps. S. aureus is the main coagulase-positive bacteria- it's not the only one, but it's probably by far the most important in human specimens. S. aureus actually produces two forms of coagulase: the bound form can be detected in a slide coagulase test, whereas free coagulase can be detected with a tube coagulase test.

The oxidase test is used to differentiate Pseudomonas (oxidase-positive) from Enterobacteriaceae (an oxidase-negative group that includes Escherichia, Klebsiella, Salmonella and Shigella). It contains a reagent that turns dark-blue when oxidised, and colourless when reduced.

Of course, this is only the tip of the iceberg. As mentioned earlier, selective and differential media can be used to test for certain organisms. There are also biochemical tests in kits, such as the API system and the miniature Vitek card, which can run a lot of tests at once and have their results evaluated by machine.

Recently, even newer bacterial identification techniques, which don't rely on biochemical reactions, have been developed. One of these is MALDI-TOF, or Matrix Assisted Laser Desorption Ionisation - Time of Flight Mass Spectrometry. Once again, I'm not going to go into the physics of it, but it involves vaporisation and ionisation of the specimen, movement of ions through a flight tube, and detection by a detector at the other end of the flight tube. MALDI-TOF produces a spectra, which can be compared with other samples in a database. MALDI-TOF is reliable, relatively inexpensive and very fast, but it may be expensive at the start (due to having to buy a shiny new machine etc.).

16S RNA, which comprises part of the 30S subunit of bacterial ribosomes, can also be used for bacterial identification. 16S RNA has some highly conserved regions, making it easy to design primers, and highly variable regions, which can be used to distinguish between different species. 16S RNA identification is particularly useful for difficult-to-identify organisms.

Yet another bacterial identification technique is the use of the Fatty Acid Methyl Ester (FAME) profile, though that wasn't really covered much in the lecture (bottom right corner of one slide... whoop-de-doop).

Detection of Microbial Components

Not all microbes are easily cultured or viewed under the microscope, so being able to detect their components in other ways is crucial. Let's see how it's done!

Antigen detection 

Antigens can be detected by using monoclonal antibodies. And... that's pretty much all I have to say.

Toxin detection

Toxins can also be detected by antibodies, or measured by mass spectrometry. They can also be measured more indirectly by looking at the functional properties of the toxins.

Nucleic acid amplification

The main technique here is a nucleic acid test (NAT)/nucleic acid amplification test (NAAT), which I also mentioned here. NAAT is pretty much just PCR (see here if you don't know what PCR is). PCR can be single or multiplex (i.e. search for multiple genes at once). PCR products can be analysed by electrophoresis (as described here). Alternatively, real-time PCR, which analyses products of PCR while the reaction is in progress, can be used. Real-time PCR uses fluorescent dyes that react with the product. The amount of fluorescence can be detected and quantified.

Serology

There weren't actually any slides on serology, but it did keep coming up on the "Types of laboratory tests" slide, so I might as well give a quick note here. Apparently serology is the detection of specific antibodies to a microorganism in the serum (so I guess this would be something like looking for anti-HIV antibodies or whatever).