DETERMINATION OF THE PATHOGENICITY OF AEROBES VIL4s-5as peculiarities of the organi,the cultural conditions,the age of theculture and the nature of the filtering agent must be considered whentesting the toxic properties of bacterial filtrates.As a rule,exotoxinsare heat labile and deteriorate on standing.Scarlet fever is the mostheat stable of the exotoxins and approaches the endotoxins in thisrespect.Heat stability y assist in differentiating the two types,but the final criterion of a true exotoxin is its ability to stimulate theproduction of a specific antitoxin when injected into a suitable anil.The exotoxin in a filtrate y be neutralized by the addition of im-mune serum and any residual toxic action y then be assumed to bedue to other toxic principles.The different organs affected and thetype of tissue dage should be recorded.For a general discussion of the care and use of laboratory anilssee,e.g.,Meyer (1932),Farris et al (1945),Cumming (1947)andWadsworth (1947).Anils are necessary,not only for determiningthe etiology of specific infectious diseases and the pathogenicity ofparticular cultures of bacteria,but they are also utilized as a means ofisolation,to determine specific pathogenic properties,to intainorganis that grow only in vivo,to increase pathogenicity and toproduce antibos and other agents used in the growth and identi-fication of microorganis and in the diagnosis and therapy of disease.The choice of an experimental anil and the method of injectionand recovery of the organi depend upon the bacterial species andthe property to be stud.The hun anil would be mostsatisfactory in dealing with diseases of n but he is not availableexcept on rare occasions.This limits the application of Koch'spostulates in the case of n,but natural infections and accidentalinfection of laboratory workers are useful in supplying circumstantialevidence as to the pathogenicity of certain bacteria for n.Healthy,previously unused anils should be employed.Severaldays of observation prior to injection are necessary to insure that theanils are in good condition and to provide a period of accliti-zation.Following injection the anils should be observed dailyfor gross abnorlities and symptoms of disease and in certain casesit y be necessary to take daily temperature,pulse,respirationchanges,hetology,etc.Large anils y.be rked withmetal tags in the ears,and the ears of all anils y be tattooedor rked with an indelible pencil.Pathogenic bacteria produce different types of lesions in anilswhich y be specific and equally as important as immunological,serological and biochemical properties.To recognize them the stu-dent should be trained in pathologic technic and should be familiarwith the gross and microscopic appearance of norl and diseasedtissue.METHODS OF INJECTIONBacteria or their products which cause disease when injectedparenterally y fail to do so when placed on the skin or when intro-duced by insufflation or by mouth.Hence the importance of differ-ent routes of injection.VIl4s-6MANUAL OF METHODS FOR PURE CULTURE STUDYThe required amount of terial is drawn into a sterile syringe;with the needle held up,air and any excess terial is expelled ontocotton moistened with a suitable disinfectant,which should be keptaway from the tip of the needle.Any undesirable disinfectant ybe removed with cotton moistened with alcohol.The following typesof injection are used:Cutaneous.This is a rather loose term and includes rubbing into,or scratching the skin or placing the inoculum under an adhesivepatch.The precise method is determined by the object to be at-tained.If it is desired to determine whether an organi can pene-trate the norl skin,the terial should be spread over the skinIrritation from shaving or depilation should be avoided.The skinshould be cleansed and sterilized with an antiseptic that has briefaction.The inoculated area y be covered with sterile gauze pro-vided the adhesive does not affect the skin.Coating the skin withcollodion excludes air and y ke the conditions abnorl andaffect the skin-penetrating power of the organi.It is commonpractice in cutaneous inoculation to abrade the epidermis by scratch-ing or scraping with a sharp instrument.This aids penetration byremoving the outer defensive layer and is similar to intracutaneousinjection.Intracutaneous.By intracutaneous injection is meant the intro-duction of terial between the intraderl layers.The fortionof a bleb indicates successful injection.It is advisable to use anilswith unpigmented skin and rabbits should not be in moult.A 27-gage needle is'best.Shaving and the application of antiseptics,particularly those that penetrate the skin,y interfere with thetest and should be used judiciously.Subcutaneous.The skin y be shaved or the hair clipped with-out interfering with the test.The point of puncture before injectionand the puncture after inoculation should be disinfected with a non-irritating disinfectant such as tincture of zephiran chloride,alcohol,merthiolate or,best of all,green soap and water.The area y berked with an indelible pencil.Material should be injected intothe subcutaneous tissue,with care not to puncture the peritonealwall when done in the abdomen.If the terial'will not pass through the needle,the skin y besterilized,after removing hair,and a V-shaped opening cut in the skinwith sterile scissors.The flap is then lifted up and loosened until apocket is formed and the terial to be tested is inserted.The flapis replaced,sterilized and covered with collodion,or sutured asepti-cally.Intramuscular.The skin is treated as for subcutaneous injectionand the culture injected deep into the muscles.Intravenous.The choice of a vein is inly a tter of con-venience and varies with the experimental anil.Rabbits usuallyare injected in the rginal ear vein,mice and rats in the tail veins,guinea pigs in the ear vein or jugular vein,horses and cows in thejugular vein,swine in the ear,dogs and cats in the jugular or the veincrossing the inner suce of the thigh and fowl in the radial vein that