test for nitrite is indicated by a dark brown solution, arising from the iron-nitric oxide complex ion. oxidase the oxidase test indicates whether a microbe is aerobic. 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. a color change to purple indicates oxidative respiration while no color change provides evidence that the organism does not have cytochrome c oxidase. phenylalanine deaminase the phenylalanine deaminase test is used to tell whether an organism can produce the enzyme deaminase. deaminase is the enzyme that can deaminate the amino acid phenylalanine into the products ammonia and phenylpyruvic acid. the test is performed by adding phenylalanine to the growth medium and allowing growth to occur. after incubation, 10% ferric chloride is added to the solution, which will react with phenylpyruvic acid in solution to make a dark green color, resulting in a positive test result. pyr the pyr test is used to check if an organism has enzymes to hydrolyze l-pyrrolidonyl- β-napthylamide. a positive result indicates that the organism is either group a streptococcus and/or group d enterococcus. reverse camp the reverse camp test utilizes the synergetic hemolytic abilities of the camp factor produced by streptococcus agalactiae with the α-toxin produced by clostridium perfringens. streaking these two organisms perpendicular to each other on a blood agar plate will yield a “bow-tie” clearing of the blood agar by the hemolytic capabilities of the two organisms’ toxins. incubation requires 24 hours at 37 °c. simmons' citrate agar simmons' citrate agar is used to test whether an organism can utilize citrate for its sole carbon source. spot indole tsi agar the spot indole test is used to determine if a microbe can deaminate tryptophan to produce indole. this test is performed by saturating a piece of filter paper with indole kovacs reagent and scraping a portion of microbe onto the paper. a color to a pink-red color indicates a positive result while no color change indicates the lack of tryptophanase. sulphide indole motility medium the sulfide indole motility medium is a three-part test for an organism’s ability to reduce sulfates, produce indoles, and motile ability. tsi slant the triple sugar iron (tsi) test is a differential media used to tell whether an organism can ferment glucose, sucrose, and/or lactose and whether an organism can produce hydrogen sulfide gas. urea agar slant raoultella planticola on urea agar the urease agar slant is used to measure an organism’s ability to produce urease, an enzyme capable to digesting urea in carbon dioxide and ammonia through hydrolysis. 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. voges–proskauer test the voges-proskauer test detects whether a bacterium is producing the product acetoin from the digestion of glucose. cellular fatty acid based identification mycolic acid analysis 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. mycolic acid is a fatty acid found in the disease tuberculosis, offering a chemical target for diagnosticians to look for. nucleic acid extraction techniques cesium chloride / ethidium bromide density gradient centrifugation tabletop centrifuge with high speed buoyant density ultracentrifugation, a density gradient is created with caesium chloride in water. 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. magnetic bead method 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. one common method is to use polyethylene glycol to drive dna binding to the magnetic beads. the molecular weight and concentration of the peg will control what molecular weight dna binds. phenol–chloroform extraction 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 method has fallen out of favor with scientists and microbiologists as there are easier methods available which require less hazardous chemicals. solid phase extraction solid phase extraction which separates long polymers like dna from other substances found in the cells. this is similar to magnetic beads, where the solid phase is fixed and selectively binds a cellular component, allowing for its isolation. methods with electrophoretic outputs 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. this is also the key method for sanger sequencing. fluorescent-labeled dna fragments move through a polymer and are separated with one base precision. a laser excites the fluorescent tag and is captured by a camera. the result is an electropherogram which reads the dna sequence. restriction enzyme based optical mapping 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 pulsed-field gel electrophoresis is a technique used to separate large dna in an electric field that periodically changes direction. by cutting segments of the dna with restriction enzymes, pulse-field can be used to separate out the segments of dna. restriction enzymes then gel electrophoresis restriction enzymes are first used to recognize and then cut specific nucleic acid sequences. these cut pieces of dna can be run through a gel electrophoresis to allow diagnostics of the organism by referencing back to previous gel electrophoresis results. ribotyping ribotyping is a rapid automated method for microbial diagnostics, testing for rrna in bacteria using restriction enzyme digestion and southern blot technology. pcr-based multiple loci vntr analysis multiple-locus vntr analysis is a test used to detect variable number tandem repeats, which act as a dna fingerprint in microbial diagnostics. dna sequence-based methods multi-locus sequence typing multilocus sequence typing (mlst) is the sequencing of numerous loci to diagnose an organism by comparing dna sequences to a database of known organisms. this method is often used to compare isolates or strains of the same species to see if they are indistinguishable or different from each other. this is common for tracking food-borne illnesses and public health outbreaks. most mlst assays are published in scientific journals so consistent methods are used worldwide. there are also public databases available for tracking and comparisons. single-locus sequence typing single-locus sequence typing (slst) is the sequencing of a single locus of an organism to produce data that can be used for strain-level comparisons between isolates of the same species. genotypic identifications for bacterial identifications, microbiologists sequence the 16s rrna gene and for fungal identifications, sequence the its regions. both regions are part of the ribosomal operon so they are well-conserved but provide enough variation to allow for speciation. accurate identifications require high quality sequence data, a robust data analysis, and a broad microbial database of known organisms. it is also useful to use a neighbor joining tree or some other phylogenetic approach to make the identification. whole genome sequencing (wgs) whole genome sequencing and genomics applications can be used for large-scale alignment and comparative analysis with both bacteria and fungi. wgs can be used to diagnose, identify, or characterize an organism down to the individual base pairs by sequencing the entire genome. wgs can also be used to compare the genomes or average nucleotide identity (ani) of the shared genes between two strains and can be a robust way to compare genetic relatedness and if often used for investigating organisms involved in foodborne illness and other outbreaks.