Of the 30 or so mollicute species that have been isolated from small and large ruminants, only a handful have been shown to cause disease in their own right. This chapter will concentrate on methods for sampling, transporting, and isolating those pathogenic mycoplasmas, including Mycoplasma mycoides subsp. mycoides small colony (SC), Mycoplasma capricolum subsp. capripneumoniae, Mycoplasma bovis, Mycoplasma agalactiae, and M. m. mycoides large colony (LC), which cause economically important diseases, such as contagious bovine pleuropneumonia (CBPP), contagious caprine pleuropneumonia (CCPP), and contagious agalactia. Although DNA amplification techniques are being used with ever-increasing frequency for the detection and identification of mycoplasmas (see Chapters 18–20), the isolation of these organisms by conventional techniques is still required by most national and international authorities to confirm disease outbreaks.