Anaerobic, aerobic, and pediatric<unk>  bottles Collection<unk> s are typically drawn through venipuncture. Collecting the sample from an intravenous line is not recommended, as this is associated with higher contamination rates, although<unk> s may be collected from both venipuncture and an intravenous line to diagnose catheter-associated infections. Prior to the<unk>  draw, the top of each collection bottle is disinfected using an alcohol swab to prevent contamination. The skin around the puncture site is then cleaned and left to dry; some protocols recommend disinfection with an alcohol-based antiseptic followed by either chlorhexidine or an iodine-based preparation, while others consider using only an alcohol-containing antiseptic to be sufficient. If<unk>  must be drawn for other tests at the same time as a<unk> , the<unk>  bottles are drawn first to minimize the risk of contamination. Because antimicrobial therapy can cause false negative results by inhibiting the growth of microbes, it is recommended that<unk> s are drawn before antimicrobial drugs are given, although this may be impractical in people who are critically ill. A typical<unk>  collection involves drawing<unk>  into two bottles, which together form one<unk> " or "set". One bottle is designed to enhance the growth of aerobic organisms, and the other is designed to grow anaerobic organisms. In children, infection with anaerobic bacteria is uncommon, so a single aerobic bottle may be collected to minimize the amount of<unk>  required. It is recommended that at least two sets are collected from two separate venipuncture locations. This helps to distinguish infection from contamination, as contaminants are less likely to appear in more than one set than true pathogens. Additionally, the collection of larger volumes of<unk>  increases the likelihood that microorganisms will be detected if present.<unk>  bottles contain a growth medium, which encourages microorganisms to multiply, and an anticoagulant that prevents<unk>  from clotting. Sodium polyanethol sulfonate (SPS) is the most commonly used anticoagulant because it does not interfere with the growth of most organisms. The exact composition of the growth medium varies, but aerobic bottles use a broth that is enriched with nutrients, such as brain-heart infusion or trypticase soy broth, and anaerobic bottles typically contain a reducing agent such as thioglycollate. The empty space in an anaerobic bottle is filled with a gas mixture that does not contain oxygen. Many commercially manufactured bottles contain a resin that absorbs antibiotics to reduce their action on the microorganisms in the sample. Bottles intended for pediatric use are designed to accommodate lower<unk>  volumes and have additives that enhance the growth of pathogens more commonly found in children. Other specialized bottles may be used to detect fungi and mycobacteria. In low and middle income countries, pre-formulated<unk>  bottles can be prohibitively expensive, and it may be necessary to prepare the bottles manually. It can be difficult to access the proper supplies and facilities, and in some regions, it may not be possible to perform<unk> s at all. It is important that the bottles are neither underfilled nor overfilled: underfilling can lead to false negative results as fewer organisms are present in the sample, while overfilling can inhibit microbial growth because the ratio of growth medium to<unk>  is comparatively lower. A 1:10 to 1:5 ratio of<unk>  to<unk>  medium is suggested to optimize microbial growth. For routine<unk> s in adults, the Clinical and Laboratory Standards Institute (CLSI) recommends the collection of two sets of bottles from two different draws, with 20–30 mL of<unk>  drawn in each set. In children, the amount of<unk>  to be drawn is often based on the child's age or weight. If endocarditis is suspected, a total of six bottles may be collected.<unk> ng pellicle) on the surface; b) bubbles from gas production; c) turbidity from microbial growth (in right bottle); d) visible microbial colonies After the<unk>  is collected, the bottles are incubated at body temperature to encourage the growth of microorganisms. Bottles are usually incubated for up to five days in automated systems, although most common<unk> stream pathogens are detected within 48 hours. The incubation time may be extended further if manual<unk>  methods are used or if slower-growing organisms, such as certain bacteria that cause endocarditis, are suspected. In manual systems, the bottles are visually examined for indicators of microbial growth, which might include cloudiness, the production of gas, the presence of visible microbial colonies, or a change in colour from the digestion of<unk> , which is called hemolysis. Some manual<unk>  systems indicate growth using a compartment that fills with fluid when gases are produced, or a miniature agar plate which is periodically inoculated by tipping the bottle. To ensure that positive<unk> s are not missed, a sample from the bottle is often inoculated onto an agar plate (subcultured) at the end of the incubation period regardless of whether or not indicators of growth are observed. In developed countries, manual<unk>  methods have largely been replaced by automated systems that provide continuous computerized monitoring of the<unk>  bottles. These systems, such as the BACTEC, BacT/ALERT and VersaTrek, consist of an incubator in which the<unk>  bottles are continuously mixed. Growth is detected by sensors that measure the levels of gases inside the bottle—most commonly carbon dioxide—which serve as an indicator of microbial metabolism. An alarm or a visual indicator alerts the microbiologist to the presence of a positive<unk>  bottle. If the bottle remains negative at the end of the incubation period, it is generally discarded without being subcultured. A technique called the lysis-centrifugation method can be used for improved isolation of slow-growing or fastidious organisms, such as fungi, mycobacteria, and Legionella. Rather than incubating the<unk>  in a bottle filled with growth medium, this method involves collecting<unk>  into a tube containing an agent that destroys (lyses) red and white<unk>  cells, then spinning the sample in a centrifuge. This process concentrates the solid contents of the sample, including microorganisms if present, into a pellet, which is used to inoculate the subculture media. While lysis-centrifugation offers greater sensitivity than conventional<unk>  methods, it is prone to contamination because it requires extensive manipulation of the sample. Identification If growth is detected, a microbiologist will perform a Gram stain on a sample of<unk>  from the bottle for a rapid preliminary identification of the organism. The Gram stain classifies bacteria as Gram-positive or Gram-negative and provides information about their shape—whether they are rod-shaped (referred to as bacilli), spherical (referred to as cocci), or spiral-shaped (spirochetes)—as well 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<unk>  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<unk>  and sensitivity results are complete. In traditional methods, the<unk>  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<unk>  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<unk>  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<unk> stream 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<unk> stream 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<unk>  bottles after separation
Blood culture
Sputum culture
Microbiological culture
Chest radiograph
Antibiotic sensitivity testing
Phonocardiogram
Coagulation testing
High-resolution computed tomography