104 MANUAL OF MICROBIOLOGICAL METHODS 



culture is the reverse of the filUng process. Enough nutrient broth to 

 hquefy the pellet is added by means of a pipet, then withdrawn and 

 transferred to a tube of broth or an agar slant. It is often desirable to 

 inoculate both a broth culture and a slant. The latter permits spreading 

 the inoculum out so that often discrete colonies develop from which sub- 

 sequent transfers can be made. The broth, on the other hand, permits 

 enfeebled cultures to start development when direct inoculation of a 

 slant might fail. 



Oil Sealing 



Cultures of many kinds of bacteria can be preserved for periods greatly 

 exceeding the viability of ordinary agar or broth cultures by sealing them 

 with sterile paraffin oil. This is essentially a process of preventing mois- 

 ture loss from the medium, although suppression of growth by excluding 

 oxygen may also contribute to longevity. Among the bacteria, as a rule 

 only species that produce a copious surface growth and can therefore be 

 easily transferred are well adapted to this technique. Some kinds of 

 Rhizobium (but not all species) have survived for 4 or 5 years by this 

 method. Other genera that survive well in oil-sealed cultures are 

 Achromohacter, Bacillus, most of the Enter ohacteriaceae, Flavohacterium, 

 Micrococcus (some species), Proteus, Pseudomonas, Sarcina, Serratia, 

 Streptococcus, Vibrio (saprophytic forms). On the other hand, cultures 

 of the following have not been successfully preserved under oil seals in 

 our experience: Azotohacter, Lactobacillus, Leuconostoc, Mycobacterium, 

 Rhodo spirillum. Salmonella. 



The procedure of oil sealing is simplicity itself. Screw-cap tubes are 

 preferable to cotton-plugged ones, as there is always some risk of getting 

 oil on cotton plugs. The preparation of the oil has been described. For 

 treating only a few cultures the oil is simply poured over the culture until 

 all the agar is covered. The use of a separatory funnel as a reservoir for 

 the oil and of a glass sleeve enclosing the tip of the funnel, into which the 

 culture tube is inserted as a shield against atmospheric contamination, 

 may be advisable when a considerable number of cultures are to be oiled. 

 It is important not to let the oil get too warm from repeated flaming of 

 the reservoir tube or from contact with the flamed lip of the culture tube. 

 No portion of the agar must remain uncovered; otherwise loss of water 

 will continue through any exposed surface. It is advisable to store oiled 

 cultures at the same temperature at which unsealed ones are kept for 

 preservation. 



Soil Cultures 



Soil is sometimes used directly as a growing medium (though more 

 often for molds and Streptomycetes than for bacteria), and it is also used 



