diagnostic microbiology diagnostic microbiology diagnostic microbiology is the study of microbial identification. since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. 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. new studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining. aerobic vs anaerobic anaerobic organisms require an oxygen-free environment. when culturing anaerobic microbes, broths are often flushed with nitrogen gas to extinguish oxygen present, and growth can also occur on media in a chamber without oxygen present. sodium resazurin can be added to indicate redox potential. cultures are to be incubated in an oxygen-free environment for 48 hours at 35 °c before growth is examined. gas-pak jar 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. incubation length incubation times vary based upon the microbe that requires culturing. traditional culturing techniques, for example, require less than 24 hours culture time for escherichia coli but 6–8 weeks for successful culturing of mycobacterium tuberculosis before definitive results are expressed. a benefit of non-culture tests is that physicians and microbiologists are not handicapped by waiting periods. incubation follows a growth curve variable for every microorganism. cultures follow a lag, log, stationary, and finally death phase. the lag phase is not well known in microbiology, but it is speculated that this phase consists of the microorganism adjusting to its environment by synthesizing proteins specific for the surrounding habitat. the log phase is the period where a culture experiences logarithmic growth until nutrients become scarce. the stationary phase is when culture concentration is the highest and cells stop reproducing. when nutrients in the environment are depleting, organisms enter the death phase where toxic metabolites become abundant and nutrients are depleted to the point where cell death exceeds reproduction. rapid identification after culture automated culturing systems 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. laboratories can also set incubation times to adjust for the lag period involved in bacterial growth. blood cultures blood cultures can allow for diagnostic results after culture. recent development of dna based pcr diagnostics have provided faster diagnostic results as opposed to overnight biochemical tests. dna diagnostic test can diagnose with near the same specificity as biochemical test, resulting in the same diagnostic result in 90% of cases. breath tests breath test for microbial diagnosis on patients has been used in a clinical setting for bacteria, including helicobacter pylori. diagnostic test using the breath of patients look for metabolites excreted that were manufactured by the infectious microorganism. h. pylori is tested by testing patients for co2 concentration, increased because of the organism’s ability to convert urea into other derivatives. conventional tests antibody detection a benefit of antibody detection (elisa) is that protein identification on a microorganism becomes faster than a western blot. antibody detection works by attaching an indicator to an antibody with a known specificity and observing whether the antibody attaches. elisa can also indicate viral presence and is highly specific, having a detection specificity of 10−9-10−12 moles per litre detection. by knowing the epitope sequence of the antibody, elisa can also be used for antigen detection in a sample. histological detection and culture histological methods used for microbiology are useful because of their ability to quickly identify a disease present in a tissue biopsy. rapid antigen tests immunofluorescence immunofluorescence immunofluorescence is performed by the production of anti-antibodies with a fluorescent molecule attached, making it a chemiluminescent molecule, which provides a glow when subject to ultraviolet light. antibodies are added to a bacterial solution, providing an antigen for the binding of fluorescent anti-antibody adherence. immunofluorescence mass spectrometry maldi-tof (matrix-assisted laser desorption/ionization - time of flight) is a specific type of mass spectrometry that is able to identify microorganisms. a pure culture is isolated and spread directly on a stainless steel or disposable target. the cells are lysed and overlaid with a matrix, which forms protein complexes with the bacterial proteins. 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. the resulting protein spectra is compared to a known database of previously catalogued organisms, resulting in rapid diagnosis of microorganisms. recent studies have suggested that these tests can become specific enough to diagnose down to the sub-species level by observing novel biomarkers. the maldi-tof identification method requires pure cultures that are less than 72 hours old. this places the organism in log phase with an abundance of ribosomal proteins, which are the most common proteins detected in the spectra. identifications with this technology can also be impacted if the culture is exposed to cold temperatures, as this would change the typical protein distribution. biochemical profile-based microbial identification systems phenotypic tests are used to identify microbes based on metabolic and biochemical pathways present in those microbes. there are many automated and semi-automated commercial systems available. these methods can be very informative but are not as accurate as maldi-tof or genotypic methods. 6.5% salt broth the 6.5% salt broth test is used to analyze the tolerance level of various bacteria under halophilic conditions. this test is used because most organisms cannot survive in high salt concentrations while staphylococci, enterococci, and aerococci are all expected to tolerate 6.5% nacl concentrations. acetate utilization the acetate utilization test is used primarily to differentiate between escherichia coli from members of the genus shigella. 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. since acetate utilization results in an increase in ph, an indicator is added that changes color under conditions of acetate utilization. ala an ala (delta-aminolevulinic acid) test is used to test for the presence of porphyrin and cytochrome compounds. finding hemin synthesis indicates that the organism is likely haemophilus. aminopeptidase the aminopeptidase test analyzes bacteria for the production of the enzyme l-alanine-aminopeptidase, an enzyme found in many gram-negative bacteria. 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. analytical profile index an analytical profile index is a fast identification system based on biochemical incubation tests. usually, this test is used to quickly diagnose clinically relevant bacteria by allowing physicians to run about 20 tests at one time. antibiotic disks antibiotic susceptible bacteria antibiotic disks are used to test the ability for an antibiotic to inhibit growth of a microorganism. this method, which is commonly used with mueller–hinton agar, is used by evenly seeding bacteria over a petri dish and applying an antibiotic treated disk to the top of the agar. by observing the ring formed around the disk formed due to the lack of bacterial growth, the zone of inhibition can be found, which is used to find the susceptibility of an organism to an antibiotic. bile esculin agar the bile esculin test is used to differentiate members of the genus enterococcus from streptococcus. bile solubility bile solubility is used to test for streptococcus pneumoniae due to their unique ability to be lysed by sodium deoxycholate. lysis indicates s. pneumoniae while no lysis does not. camp a positive camp test with the characteristic arrowhead shaped zone of hemolysis a camp test is used to differentiate between streptococcus agalactiae and other species of beta-hemolytic streptococcus. 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. catalase the catalase test tests whether a microbe produces the enzyme catalase, which catalyzes the breakdown of hydrogen peroxide. 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. bubbling is a positive test while nothing happening is a negative result. cetrimide agar cetrimide agar slants is a selective agar used to isolate pseudomonas aeruginosa. clo tests the clo test is used to diagnose h. pylori in patient biopsies. a sample of the biopsy is places in a medium containing urea, which h. pylori can use in some of its biochemical pathways. consumption of urea indicates a positive test result. coagulase the coagulase test determines whether an organism can produce the enzyme coagulase, which causes the fibrin to clot. inoculating a plasma test tube with the microbe indicates whether coagulase is produced. a clot indicates the presence of coagulase, while no clot indicates the lack of coagulase. dna hydrolysis dnase agar is used to test whether a microbe can produce the exoenzyme deoxyribonuclease (dnase), which hydrolyzes dna. methyl green is used as an indicator in the growth medium because it is a cation that provides an opaqueness to a medium with the presence of negatively charged dna strands. when dna is cleaved, the media becomes clear, showing the presence of dnase activity. dna hydrolysis is tested by growing an organism on a dnase test agar plate (providing nutrients and dna) and then checking the plate for hydrolysis. the agar plate has dna-methyl green complex, and if the organism on the agar does hydrolyze dna then the green color fades and the colony is surrounded by a colorless zone. gelatin the gelatin test is used to analyze whether a microbe can hydrolyze gelatin with the enzyme gelatinase. the gelatin makes the agar solid, so if an organism can produce gelatinase and consume gelatin as an energy and carbon source, the agar will become liquid during growth. gonocheck ii the gonochek ii test, a commercial biochemical test, is used to differentiate between neisseria lactamica, neisseria meningitidis, n. gonorrhoeae and moraxella catarrhalis. the principle behind this test is to use enzymes native to the organism to create a colored product in the presence of foreign molecules. the chemical hippurate the hippurate diagnostic test is used to differentiate between gardnerella vaginalis, campylobacter jejuni, listeria monocytogenes and group b streptococci using the chemical hippurate. the hippurate hydrolysis pathway, capable by organisms with the necessary enzymes, produces glycine as a byproduct. using the indicator ninhydrin, which changes color in the presence of glycine, will display either a colorless product, a negative result, of a dark blue color, a positive result. indole butyrate disk an indole butyrate disc is used to differentiate between neisseria gonorrhoeae (negative result) and moraxella catarrhalis (positive result). 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 blue color is the result of a positive test. lysine iron agar slant the lysine iron agar slant test is used to tell whether an organism can decarboxylate lysine and/or produce hydrogen sulfide. lysostaphin the lysostaphin test is used to differentiate between staphylococcus and micrococcus bacteria. lysostaphin can lyse staphylococcus, but micrococcus bacteria are resistant to the chemical. methyl red test color transition of methyl red solution under different acid-base conditions the methyl red test is used to analyze whether a bacterium produces acids through sugar fermentation. microdase microdase is a modified oxidase test used to differentiate micrococcus from staphylococcus by testing for the presence of cytochrome c. a positive result produces a dark color around the inoculant while negative result produces no color change. nitrite test the nitrite test is commonly used to diagnose urinary tract infections by measuring the concentrations of nitrite in solution, indicating the presence of a gram-negative organism. a simple nitrite test can be performed by adding 4 m sulfuric acid to the sample until acidic, and then adding 0.1 m iron (ii) sulfate to the solution. a positive