186 DEPARTMENT OF THE NAVAL SERVICE 



8 GEORGE V, A. 1918 

 MEDIA EMPLOYED. 



In this investigation I have used media prepared from fish concoctions, the ordinary 

 laboratory media, and certain special media. In the early part of the work when 

 experimenting with methods prior to the adoption of a definite procedure, difficulty 

 was experienced in growing some of the strains isolated. The colonies developing on 

 some of the plates at this time were too small to be subcultured. I therefore utilized 

 the marine resources at hand and prepared media from fresh herrings, from clams, and 

 from seaweed, using fresh sea water instead of tap or distilled water. It was found 

 later that the organisms which necessitated this media were those I have put in the 

 main Class II, the non-gas-producers. After successive subculturing in the laboratory 

 these same strains have grown moderately well on the usual standard media. 



The organisms of my main Class I, the gas-producing strains, have grown well in 

 the standard media. The growth of some strains has been more luxuriant on herring 

 media or clam media, but the use of such has gradually been eliminated for two 

 reasons : — 



(1) the satisfactory growth obtained on standard media, and the convenience 

 of its use; 



(2) the necessity of using the standard media in order to compare the 

 strains isolated with varieties already described in literature. 



Herring Broth. — Eresh herrings obtained direct from the weirs were washed in run- 

 ning water and ground up, no portions discarded, through a meat grinder, mixed 

 with sea water, 1 part ground herring to 1-2 parts sea-water, and heated for 

 several hours in the steamer or autoclav. The mixture was allowed to cool and 

 the fat skimmed off; again heated, and strained through cheese cloth. The 

 strained liquid served as the standard herring extract. Varying strengths of 

 broth were made up, good results being obtained with the following mixture: — 

 500 cc standard broth, 



1,000 cc. sea water, 



15 grams peptone. 



The ingredients were heated together in the steamer, neutralized with n/20 

 NaOH to + 10 (phenol phthalein indicator), cleared with white of egg, tubed 

 and sterilized in the usual way. 



Herring Agar. — To 500 cc. of the standard broth, mentioned above, were added 500 cc. 

 or 1,000 cc. sea-water, peptone at the rate of 1 per cent and agar at the rate of 

 1-2 per cent; the whole heated together until ingredients dissolved, neutralized 

 to +10, cleared with white of egg, filtered, tubed and sterilized in the usual way. 



Clam Agar. — Fresh clams were dug up on the bench, washed in running water, opened 

 and ground through meat grinder; to this was added sea water at the rate of 1 

 part clams to 2 parts sea water, and the whole heated for several hours in steamer 

 or autoclav. The stewed mixture was strained through cheese cloth; this filtrate 

 constituting the standard broth. To 500 cc. of the standard broth were added 

 1,000 cc. sea water, peptone at the rate of 1 per cent, and agar at the rate of 1-2 

 per cent; the whole heated together until ingredients dissolved, neutralized to 

 +10, cleared with white of egg, filtered, tubed and sterilized in the usual way. 



I have also steamed clams in the shell in sea water, approximately weight 

 for weight; retaining the juice which has a typical "sheen" ; then after open- 

 ing the clams using them as described above. 



In the earlier part of the work the medium was used successfully to some 

 considerable extent; and in comparison with standard beef peptone agar it 

 appeared to exercise a selective action towards certain strains of bacteria 



