Finally, an assessment of limits of the duration of storage of STGG medium prior to use, at various temperatures but especially frozen, would assist sites with limited ability to produce STGG themselves. An ideal culture ABT-263 ic50 medium should prevent growth of non-pneumococcal species without inhibiting growth of the pneumococci itself. To this end, defibrinated blood agar (from a non-human source such as sheep, horse or goat) supplemented with 5 μg/ml gentamicin has been the most widely used selective medium to culture pneumococci from NP samples [38], [39] and [40]. For culture of pediatric NP and

throat swabs, this medium has been shown to result in a similar yield of pneumococci to anaerobically incubated blood agar plates [41]. The concentration of gentamicin in agar has been shown to have a significant effect on isolation of pneumococci [42]. There are similar yields of pneumococci when culturing respiratory tract specimens on blood agar supplemented with 2.5–5 μg/ml gentamicin compared with culture on plain blood agar or by mouse inoculation [43], [44] and [45]. Alternative supplements used to improve the isolation of pneumococci by culture include

combinations of colistin and nalidixic acid (CNA) or colistin and oxolinic acid (COBA) [46]. Unlike blood agar-gentamicin and COBA, blood-CNA agar does not suppress the growth of staphylococci. Blood agar, either Columbia or trypticase soy agar base with

sheep, horse, or goat blood, supplemented with 5 μg/ml gentamicin is considered the core primary isolation media. Blood-CNA or COBA agars Depsipeptide chemical structure are acceptable alternatives, whereas human blood agar should never be used [45] and [47]. Thoroughly mix a fresh or fully-thawed NP swab-STGG specimen using a vortex and inoculate 10 μl onto a selective plate and streak into all four plate quadrants with sterile loops. Some investigators may choose to use larger volumes of STGG medium (e.g. 50 μl or 100 μl). As this will affect the sensitivity of detection, the volume used should be noted when reporting. Incubate the pneumococcal plate(s) overnight at 35–37 °C in medroxyprogesterone a CO2 enriched atmosphere, either by using a candle jar or 5–10% CO2 incubator. Plates with no growth should be re-incubated for another 24 h before being discarded as negative. If required, record the semi-quantitative growth of alpha-hemolytic colonies [1]. Single colonies are then picked and subcultured for analysis, including identification as described below. Culture of NP specimens, by scraping or drilling into the frozen STGG media using a sterile microbiological loop, might permit prolongation of specimen integrity. This technique has been used successfully in the sub-culture of pneumococcal isolates stored in STGG, but requires quantitative validation for use with NP samples.