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 blood 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 culture 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 blood cultures 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 blood is comparatively lower. A 1:10 to 1:5 ratio of blood to culture medium is suggested to optimize microbial growth. For routine blood cultures 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 blood drawn in each set. In children, the amount of blood 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. Culturing After the blood 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 bloodstream pathogens are detected within 48 hours. The incubation time may be extended further if manual blood culture 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 blood, which is called