as their arrangement. gram-positive cocci in clusters, for example, are typical of staphylococcus species. yeast and other fungi may also be identified from the gram stain. a gram stain identifying microbial growth from a blood culture is considered a critical result and must immediately be reported to the clinician. the gram stain provides information about the possible identity of the organism, which assists the clinician in the selection of a more appropriate antimicrobial treatment before the full culture and sensitivity results are complete. in traditional methods, the blood is then subcultured onto agar plates to isolate the organism for further testing. the gram stain results inform microbiologists about what types of agar plates should be used and what tests might be appropriate to identify the organism. in some cases, no organisms are seen on the gram stain despite the culture bottle showing indicators of growth or being reported as positive by automated instruments. this may represent a false positive result, but it is possible that organisms are present but cannot easily be visualized microscopically. positive bottles with negative gram stains are subcultured before being returned to the incubator, often using special culture media that promotes the growth of slow-growing organisms. it typically takes 24 to 48 hours for sufficient growth to occur on the subculture plates for definitive identification to be possible. at this point, the microbiologist will assess the appearance of the bacterial or fungal colonies and carry out tests that provide information about the metabolic and biochemical features of the organism, which permit identification to the genus or species level. for example, the catalase test can distinguish streptococci and staphylococci (two genera of gram-positive cocci) from each other, and the coagulase test can differentiate staphylococcus aureus, a common culprit of bloodstream infections, from the less pathogenic coagulase-negative staphylococci. loading a target plate containing microbial samples into a bruker biotyper, an instrument used for maldi-tof analysis in microbiology microorganisms may also be identified using automated systems, such as instruments that perform panels of biochemical tests, or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms), in which microbial proteins are ionized and characterized on the basis of their mass-to-charge ratios; each microbial species exhibits a characteristic pattern of proteins when analyzed through mass spectrometry. because bloodstream infections can be life-threatening, timely diagnosis and treatment is critical, and to this end several rapid identification methods have been developed. maldi-tof can be used to identify organisms directly from positive blood culture bottles after separation and concentration procedures, or from preliminary growth on the agar plate within a few hours of subculturing. genetic methods such as polymerase chain reaction (pcr) and microarrays can identify microorganisms by detection of dna sequences specific to certain species in blood culture samples. several systems designed for the identification of common blood culture pathogens are commercially available. some biochemical and immunologic tests can be performed directly on positive blood cultures, such as the tube coagulase test for identification of s. aureus or latex agglutination tests for streptococcus pneumoniae, and unlike pcr and maldi-tof, these methods may be practical for laboratories in low and middle income countries. it is also possible to directly inoculate microbial identification panels with blood from a positive culture bottle, although this is not as reliable as testing subcultured bacteria because additives from the growth media can interfere with the results. even faster diagnosis could be achieved through bypassing culture entirely and detecting pathogens directly from blood samples. a few direct testing systems are commercially available as of 2018, but the technology is still in its infancy. most panels detect only a limited number of pathogens, and the sensitivity can be poor compared to conventional blood culture methods. culturing remains necessary in order to carry out full antimicrobial sensitivity testing. antibiotic susceptibility testing further information: antibiotic susceptibility testing left: manual antibiotic sensitivity testing by the disk diffusion test. right: pre-formulated panels loaded into a bd phoenix m50, an instrument used for automated antibiotic sensitivity testing. antimicrobial treatment of bloodstream infections is initially empiric, meaning it is based on the clinician's suspicion about the causative agent of the disease and local patterns of antimicrobial resistance. carrying out antibiotic susceptibility testing (ast) on pathogens isolated from a blood culture allows clinicians to provide a more targeted treatment and to discontinue broad-spectrum antibiotics, which can have undesirable side effects. in traditional ast methods, such as the disk diffusion test, pure colonies of the organism are selected from the subculture plate and used to inoculate a secondary medium. these methods require overnight incubation before results can be obtained. there are automated systems which use pre-formulated antibiotic panels, measure microbial growth automatically, and determine the sensitivity results using algorithms; some of these can provide results in as little as five hours, but others require overnight incubation as well. rapid administration of effective antimicrobial drugs is crucial in the treatment of sepsis, so several methods have been developed to provide faster antibiotic sensitivity results. conventional ast methods can be carried out on young growth from the subculture plate, pellets of microorganisms obtained from concentration and purification of the positive blood culture, or directly from the culture bottle. because direct testing methods do not isolate the organisms, they do not provide accurate results if more than one microorganism is present, although this is an infrequent occurrence in blood cultures. another source of error is the difficulty in standardizing the amount of bacteria in the sample (the inoculum), which has a profound effect on the test results. genetic testing can be used for rapid detection of certain antimicrobial resistance markers. methods such as pcr and microarrays, which can be performed directly on positive blood culture samples, detect dna sequences associated with genes that confer resistance, such as the meca gene found in methicillin-resistant staphylococcus aureus or the vana and vanb genes of vancomycin-resistant enterococci. maldi-tof has been explored as a rapid antimicrobial sensitivity testing method; principles involve measuring microbial growth in the presence of antibiotics, identifying the breakdown of antibiotics by microbial enzymes, and detecting protein spectra associated with bacterial strains that exhibit antibiotic resistance. some of these methods can be performed on pellets from positive blood culture bottles. however, the lack of established methodologies for ast by maldi-tof limits its use in clinical practice, and direct ast by maldi-tof, unlike genetic testing methods, had not been approved by the food and drug administration as of 2018. limitations blood cultures are subject to both false positive and false negative errors. in automated culture systems, identification of positive bottles is based on the detection of gases produced by cellular metabolism, so samples with high numbers of white blood cells may be reported as positive when no bacteria are present. inspection of the growth curve produced by the instrument can help to distinguish between true and false positive cultures, but gram staining and subculturing are still necessary for any sample that is flagged as positive. blood cultures can become contaminated with microorganisms from the skin or the environment, which multiply inside the culture bottle, giving the false impression that those organisms are present in the blood. contamination of blood cultures can lead to unnecessary antibiotic treatment and longer hospital stays. the frequency of contamination can be reduced by following established protocols for blood culture collection, but it cannot be eliminated; for instance, bacteria can survive in deeper layers of the skin even after meticulous disinfection of the blood draw site. the clsi defines an acceptable contamination rate as no greater than 3% of all blood cultures. the frequency of contamination varies widely between institutions and between different departments in the same hospital; studies have found rates ranging from 0.8 to 12.5 percent. when faced with a positive blood culture result, clinicians must decide whether the finding represents contamination or genuine infection. some organisms, such as s. aureus or streptococcus pneumoniae, are usually considered to be pathogenic when detected in a blood culture, while others are more likely to represent contamination with skin flora; but even common skin organisms such as coagulase-negative staphylococci can cause bloodstream infections under certain conditions. when such organisms are present, interpretation of the culture result involves taking into account the person's clinical condition and whether or not multiple cultures are positive for the same organism. false negatives may be caused by drawing blood cultures after the person has received antibiotics or collecting an insufficient amount of blood. the volume of blood drawn is considered the most important variable in ensuring that pathogens are detected: the more blood that is collected, the more pathogens are recovered. however, if the amount of blood collected far exceeds the recommended volume, bacterial growth may be inhibited by natural inhibitors present in the blood and an inadequate amount of growth medium in the bottle. over-filling of blood culture bottles may also contribute to iatrogenic anemia. not all pathogens are easily detected by conventional blood culture methods. particularly fastidious organisms, such as brucella and mycobacterium species, may require prolonged incubation times or special culture media. some organisms are exceedingly difficult to culture or do not grow in culture at all, so serology testing or molecular methods such as pcr are preferred if infection with these organisms is suspected. history an early vacuum tube system for blood culture collection, described by c.e. simon & c.c.w. judd in 1915 early blood culture methods were labour-intensive. one of the first known procedures, published in 1869, recommended that leeches be used to collect blood from the patient. a microbiology textbook from 1911 noted that decontamination of the draw site and equipment could take over an hour, and that due to a lack of effective methods for preserving blood, the cultures would sometimes have to be prepared at the patient's bedside. in addition to subculturing the broth, some protocols specified that the blood be mixed with melted agar and the mixture poured into a petri dish. in 1915, a blood culture collection system consisting of glass vacuum tubes containing glucose broth and an anticoagulant was described. robert james valentine pulvertaft published a seminal work on blood cultures in 1930, specifying—among other insights—an optimal blood-to-broth ratio of 1:5, which is still accepted today. the use of sps as an anticoagulant and preservative was introduced in the 1930s and 40s and resolved some of the logistical issues with earlier methods. from the 1940s through the 1980s, a great deal of research was carried out on broth formulations and additives, with the goal of creating a growth medium that could accommodate all common bloodstream pathogens. in 1947, m.r. castañeda invented a "biphasic" culture bottle for the identification of brucella species, which contained both broth and an agar slant, allowing the agar to be easily subcultured from the broth; this was a precursor of some contemporary systems for manual blood cultures. e.g. scott in 1951 published a protocol described as "the advent of the modern blood culture set". scott's method involved inoculating blood into two rubber-sealed glass bottles; one for aerobes and one for anaerobes. the aerobic bottle contained trypticase soy broth and an agar slant, and the anaerobic bottle contained thioglycollate broth. the lysis-centrifugation method was introduced in 1917 by mildred clough, but it was rarely used in clinical practice until commercial systems were developed in the mid-1970s. automated blood culture systems first became available in the 1970s. the earliest of these—the bactec systems, produced by johnston laboratories (now becton dickinson)—used culture broths containing nutrients labelled with radioactive isotopes. microbes that fed on these substrates would produce radioactive carbon dioxide, and growth could be detected by monitoring its concentration. before this technique was applied to blood cultures, it had been proposed by nasa as a method for detecting life on mars. throughout the 1970s and 80s several manufacturers attempted to detect microbial growth by measuring changes in the electrical conductivity of the culture medium, but none of these methods were commercially successful. a major issue with the early bactec systems was that they produced radioactive waste, which required special disposal procedures, so in 1984 a new generation of bactec instruments was released that used spectrophotometry to detect co2. the bact/alert system, which indirectly detects production of co2 by measuring the decrease in the medium's ph, was approved for use in the us in 1991. unlike the bactec systems available at the time, the