METHODS OF INVESTIGATING BACTERIA. 793 



or 1 per cent, of agar-agar. The gelatine is dissolved by 



heat; the mixture is then neutralised, boiled for ten 



minutes in the steaming apparatus, and filtered through 



a warm filter. If the filtrate is not clear it must be 



again boiled, either with or without the addition of white 



of egg. The clear filtrate is introduced into the test 



tubes or flasks, and sterilised in the steaming apparatus 



for five minutes on three successive days. The agar Nutrient agar. 



mixture must be kept for a long time (ten to twelve 



hours) in a state of constant simmering over a small 



flame, the water being replaced as it evaporates ; it is then 



filtered through a warm funnel, or it is poured into a 



tall cylinder which is kept in warm water till the muddy 



portion is deposited ; it is then allowed to solidify, the 



upper clear portion is cut off, dissolved by boiling, and 



placed in the various vessels ; these are then again 



sterilised by exposure for half hour to the steam. 



If possible sheep's blood should be received with anti- Blood serum, 

 septic precautions in a sterilised vessel and covered with 

 a sterilised glass plate ; after forty-eight hours the clear 

 serum is removed by a pipette, transferred directly to 

 the vessels to be employed for cultivation, and kept at a 

 temperature of 68 to 70 C. till the serum is solidified. 

 The vessels are then placed in an incubator, and it is 

 generally found that the great majority are sterile. If 

 it is found impossible to obtain the blood with anti- 

 septic precautions, it must in the first place be sterilised 

 as above described at 55 C. on several successive days. 

 Instead of killing the germs we may sometimes try to 

 get rid of them by filtering the nutrient material through 

 plaster or porcelain (Chamberland's filter). 



All the nutrient substrata which are to be employed for 

 cultivation must be tested before use to see whether they 

 are sterile ; for this purpose they are kept for some time, 

 and generally at a high temperature (30 to 35 C.) ; where 

 the nutrient material is completely sterile no alterations 

 ought to occur. The substrata are protected against 

 drying by means of a caoutchouc cap placed over the 

 wool plug. Nutrient materials prepared with gelatine 

 must not be placed at a higher temperature than 20 to 



