Diagnostic Clinical Microbiology Quiz

Answer:

In other medical science disciplines such as biochemistry and haematology, automation was easy to achieve (as you are nearly always counting something). It is the logical choice for maximum productivity. In microbiology, you are checking to see if there is a microbe in your sample or not. In other words, you need to prove microbial growth, which is time-dependent.
The backbone of 20th-century microbiology is M/C/S: Microscopy (quick); culture (days) and sensitivity testing (an additional 24 hours).

20th Century Innovations

The simplest request for M/C/S is for urine specimens. Urine is normally sterile. When you have an infection (UTI), there are bacteria, leukocytes, and sometimes erythrocytes. All are visible in a specimen with results available within minutes. Two pieces of equipment are needed: A counting chamber and a polarising microscope.

Counting chambers for hematology have been used since the 1880s. These were glass slides with a grid etched into them to quantify cells. These counting chambers were unsuitable for microbiology samples. The Petroff-Hausser counting chamber or variations (patented in the early 1920s by Carl Adolph Hausser and Sara Petroff) has been used routinely in microbiology since the 1930s.

A phase contrast microscope is useful because it allows imaging a specimen without the need to stain it first. It works. by definition, by converting small phase shifts in light passing through transparent specimens into visible differences in image contrast. Frits Zernike invented the phase-contrast microscope in the early 1930s and, collaborating with Zeiss, a leading microscope manufacturer, these microscopes started to be used routinely in 1941 (Zernike was awarded the Nobel Prize in Physics in 1953).

Danish pathologist Hans Christian Gram invented the Gram stain in 1884 to make pneumonia-causing bacteria stand out against lung tissue under a microscope. However, it instantly differentiates Gram-positive bacteria from Gram-negative bacteria, as well as sorting bacteria into one of two forms: spheres (cocci) or rods (bacilli). Over 140 years later, it remained a cornerstone of clinical microbiology because it was fast, inexpensive, simple, and provided immediate critical guidance for patient care, including guiding empirical antibiotic therapy.

In order to grow bacteria to find a pathogen, an inert material on which the bacteria can grow is essential. It must be a solid and inert material (bugs can't eat it). Agar, a gelatin-like substance, is ideal. It is a solid at room temperature but gel-setting temperature at 37C / 100°F, so nutrients can be added without altering their original properties. All-purpose media such as Horse Blood Agar will grow most common bacteria, but selective and differential media are also used. Selective media is needed where the area sampled (e.g., throat swab) will be full of common, harmless bacteria that grow there normally and may overshadow any pathogen. Ingredients are added to suppress the growth of normal flora. Differential media is where something is added to the medium to help differentiate normal flora from pathogens. MacConkey Agar is a good example. It is a very common medium used with most bacteriological specimens. It contains bile salts and crystal violet, which inhibit most Gram-positive bacteria. It is also a differential medium, as it contains lactose and a pH indicator. If the bacterium can utilise lactose, the colony turns pink; otherwise, it stays colourless. As E.coli is the most commonly isolated pathogen in urine, and it ferments lactose, MacConkey agar is a highly rated medium in the laboratory.

When you get bacteria into your bloodstream, which is normally sterile, you have a disease called septicaemia (often mistakenly called sepsis). This is one of the most serious of all bacterial infections and needs immediate treatment. Diagnosis traditionally involved injecting the patient's blood into bottles of highly nutritious broth and watching for visible signs of bacterial growth. This often took days. In the 1980s, this process was able to be automated. Patient blood was injected into broth bottles with a pH indicator on the base. The blood culture bottles were loaded into an instrument that detected colour change at the base of the bottle. When bacteria were present in the specimen, they grew quickly in the bottle and blew off CO2. This changed the pH of the medium, which changed the colour of the base, which was flagged by the instrument. This made results available in hours, not days. It revolutionised diagnostic microbiology.

Disc sensitivity testing is a semi-quantitative method used to evaluate the in vitro susceptibility of bacteria to antibiotics. These methods were first used in the 1940s. Paper discs, 6mm in diameter, soaked in antibiotics are placed on agar plates that have been covered with a solution of the bacterial pathogen. The resulting zone of bacterial growth inhibition is a measure of the antibiotic's effectiveness against the specific organism. If the organism grows up to the disc, then the pathogen is resistant to that antibiotic. When there is a zone of growth inhibition, results are then compared to standardised breakpoints to categorise the pathogen as susceptible, intermediate, or resistant to specific antibiotics.


A cell line is a population of cells originating from one cell or tissue source and cultured so they grow and divide continuously in the lab. These cell culture lines are passaged in sealed tubes. As viruses are too small to be seen, damage to inoculated cell lines (called cytopathic effect) is visible under a microscope. If the inoculated sample contains a virus known to damage a particular cell line, this is diagnostic of a specific viral infection. This test, though, takes from several days to several weeks. These methods were first undertaken in the 1910s but became popular in the diagnostic lab in the 1960s.

ELISA (Enzyme-Linked Immunosorbent Assay) is a sensitive diagnostic test used for detection of microbiological antigens or antibodies to microbiological pathogens. With this test, the sample or target molecule attaches to the solid surface of a microplate (about 12.8 cm x 9 cm) containing up to 96 wells. Then, an enzyme-linked antibody is added. It binds to the target molecule only if it is present. After binding, if and when it occurs, a substrate is added, which results in a colour change in the enzyme (and, by association, the target molecule it was bound to). Invented in 1971, the technique became routine in the lab by 1975. It was widely used (among other diagnostic purposes) as a screening test when HIV/AIDS was first identified in the early 1980s

The Western blot method was originally a biochemical test developed in 1981 to identify specific proteins in biological specimens in a three-stage procedure. In 1985, it was adapted to identify anti-HIV antibodies in human serum samples. It is costly and difficult to perform but yields accurate results. As HIV ELISA tests occasionally gave false positives, Western blot techniques were used as HIV confirmatory tests.

21st Century Innovations

MALDI-TOF Mass Spectrometry rapidly identifies microorganisms by analysing their unique protein mass spectra. Results can be obtained within two hours. First used in 2009.

Multiplex PCR facilitates the simultaneous amplification of multiple DNA/RNA targets in a single reaction, allowing rapid and efficient detection of microbial pathogens and resistance genes in a single specimen. Introduced in the clinical microbiology lab in 2008-10.

Total Laboratory Automation (TLA) is the use of integrated robotics, instruments, and software to automate the entire laboratory workflow with minimal or no human intervention. In addition to automating individual tests, the scope of TLA reaches all pre-analytical, analytical, and post-analytical stages in a single coordinated system. TLA for microbiology labs commenced in 2014.

Next-Generation Sequencing (NGS) facilitates sequencing of microbial DNA or RNA at a massive scale. It is particularly useful for outbreak investigations, detection of antimicrobial resistance genes, and identification of unusual or novel pathogens. This method was introduced into hospital labs in the early 2020s, but its cost limits its use to large public health and clinical reference labs.

Loop-mediated isothermal amplification (LAMP) is a rapid, highly specific, and cost-effective nucleic acid amplification technique that does not require an expensive thermal cycler, thereby making it accessible to smaller microbiology laboratories. While it was first used as a research tool, in 1998, it was not used in diagnostic microbiology labs until 2011-12.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a revolutionary microbiology tool that allows scientists to selectively and precisely edit, delete, or replace DNA in living organisms. While this technology is available in research settings, its implementation and instrumentation costs exclude it from becoming a routine microbiological testing platform.