Diagnostic microbiology
Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies.
[3] Anaerobic bacteria collection can come from a variety of sources in patient samples, including blood, bile, bone marrow, cerebrospinal fluid, direct lung aspirate, tissue biopsies from a normally sterile site, fluid from a normally sterile site (like a joint), dental, abscess, abdominal or pelvic abscess, knife, gunshot, or surgical wound, or severe burn.
[5] Automatic cell culturing systems are becoming popular because of their ability to maintain a sterile growth environment and remove strain on the laboratory staff involving repetitive experimentation.
MALDI-TOF (Matrix-assisted laser desorption/ionization - time of flight) is a specific type of mass spectrometry that is able to identify microorganisms.
The MALDI fires a laser and ionizes the protein complexes, which break off and travel up the vacuum where they are detected based on mass and charge.
Identifications with this technology can also be impacted if the culture is exposed to cold temperatures, as this would change the typical protein distribution.
Many of the Escherichia coli strains have the capability of the utilization of acetate for a sole carbon and energy source, while Shigella does not.
Adding L-Alanine-4-nitroanilide hydrochloride to a bacterial culture works as an indicator, changing to a yellow color in the presence of L-alanine-aminopeptidase.
[citation needed] Bile solubility is used to test for Streptococcus Pneumoniae due to their unique ability to be lysed by sodium deoxycholate.
This biochemical test uses the fact that Streptococcus agalactiae excretes a CAMP substance, making it slightly more hemolytic, which can be observed on blood agar media.
Smearing a colony sample onto a glass slide and adding a solution of hydrogen peroxide (3% H2O2) will indicate whether the enzyme is present or not.
The principle behind this test is to use enzymes native to the organism to create a colored product in the presence of foreign molecules.
Gamma-glutamyl-p-nitroanilide is added to the solution to indicate whether the bacteria is N. meningitides, which hydrolyzes the molecule with the enzyme gamma-glutamylaminopeptidase, producing a yellow end-product.
Prolyl-4-methoxynaphthylamide is in the solution to identify N. gonorrhoeae because of its ability to hydrolyze the molecule with the enzyme hydroxyprolylaminopeptidase, creating a red-pink derivative.
This test involves a butyrate disk, which when smeared with a culture, will change color for a positive result after 5 minutes of incubation.
A positive test for nitrite is indicated by a dark brown solution, arising from the iron-nitric oxide complex ion.
By using the chemical N,N,N,N-tetramethyl-1,4-phenylendiamin, an electron acceptor that changes color when oxidized by cytochrome c oxidase, one can deduce whether a microbe can perform aerobic respiration.
Because ammonia is alkaline, the media contains phenol red, an indicator that changes from orange to pink when a pH increases above 8.1.
When ammonia is increased to high enough concentrations, the media will change to a pink color, indicating the presence of urease production.
[38] Mycolic acid analysis has been an evolving field of study for gas-liquid chromatography, as it offers a solution to slow growth rates in Mycobacterium.
DNA will go to the density that reflects its own, and ethidium bromide is then added to enhance the visuals the nucleic acid band provides.
[40] New extraction techniques have been developed using magnetic beads for the purification of nucleic acids by taking advantage of the charged and polymeric nature of long strand of DNA.
Beads are both uncoated to increase surface are and yield, while others are more selective by being coated with functional groups that interact with the polymers present in microbes.
Phenol-Chloroform extraction is a liquid-liquid method used by biochemists to separate nucleic acids from proteins and lipids after cells have been lysed.
This is similar to magnetic beads, where the solid phase is fixed and selectively binds a cellular component, allowing for its isolation.
Gel electrophoresis is a technique to separate macromolecules by taking advantage of the charge on many of the molecules found in nucleic acids and protein.
Optical mapping is a technique using multiple restriction enzymes to create a genomic “barcode” which can be referenced back to diagnose an unknown microbe.
Pulsed-field gel electrophoresis is a technique used to separate large DNA in an electric field that periodically changes direction.
Ribotyping is a rapid automated method for microbial diagnostics, testing for rRNA in bacteria using restriction enzyme digestion and Southern blot technology.
[43] Multiple-locus VNTR analysis is a test used to detect variable number tandem repeats, which act as a DNA fingerprint in microbial diagnostics.
