188 DEPARTMENT OF THE NAVAL SERVICE 



8 GEORGE V, A. 1918 



same time. Absolute alcohol lias proved to be simple in application and quite satis- 

 factory. The cans were first cleaned with a weaker alcohol (70 per cent to 90 per cent) 

 then thoroughly treated with the absolute alcohol. Can openers, forceps, and dissect- 

 ing scissors were immersed in alcohol ^nd flamed immediately before use. When a 

 sufficiently large aperture had been made in the can, pieces of fish and a portion of 

 the oil or sauce were removed with forceps and pipettes and inoculated into tubes of 

 liquid medium. 



At the commencement, it was at once obvious that direct plating from the cans 

 would not be at all satisf,actory on account of the oily nature of the contents; liquid 

 media have therefore been used for the first inoculation from the cans, the procedure 

 having the additional advantage in that such media serve as enrichment fluids. I 

 first used peptone broth (Dunham), herring broth, and nutrient broth; later, the 

 addition to the series of glucose peptone broth proved to h^ve advantages. As a result 

 of the additional knowledge provided by a study of the strains of organisms already 

 worked out, it will be desirable in further work to use media having differential quali- 

 ties for the first inoculations; in addition to the broths already in use. 



The tubes were incubated at 37° C. except during the six weeks spent at St. 

 Andrews, when all cultures were kept at room temperature. The broths were examined 

 in 18-24 hours for growth; if no growth were apparent, further incubation was 

 resorted to; if growth could be noted, series of plates were made. The preliminary 

 incubation in broth tubes had the additional advantage to those already mentiouea, in 

 that the oil had risen to the surface leaving the sub-surface liquid comparatively free. 

 Finely drawn out pipettes with the finger over the end were passed throiigh the layer 

 of oil, and the culture fluid drawn up. After suitable dilutions had been made, plates 

 were poured using herring agar, clam agar, beef peptone agar, and glucose agar; in 

 the more recent work glucose agar being used almost solely. The plates were incu- 

 bated — temperatures as aforementioned — and when growth was sufficient, those 

 colonies most common were streaked on agar slopes; from these the necessary purifi- 

 cation by plates being made. 



Note. — The preliminary incubation in broth tubes was in some cases, but not 

 always, duplicated aerobically and anaerobically. 



The following apply to the main Class I: — 



Microscopic examinations. — The microscopic preparations were uniformly made from 

 beef peptone agar slopes incubated 18 to 24 hours at 37^ C. 



^Gram's Stain. — The gas-producing organisms. Class I pages 192-207, display an unu- 

 sual degree of resistance to decolorisation with alcohol in the Gram method of 

 staining. Wlien treated by the usual method, — decolorisation with alcohol un- 

 til no further colour can be washed out, — each of the eight strains recorded 

 would be classified as Gram positive. The shade of violet is not as deep as 

 that which is typical of the classic Gram positive reaction, but the result is 

 much nearer positive than negative. On prolonged soaking in absolute alcohol, 

 30 to 40 minutes, the reaction is definitely Gram negative. Films made from 

 a typical Gram positive lactic acid producing organism withstood the decolor- 

 isation with alcohol for 40 minutes 



The organisms herein discussed should therefore be described as Gram nega- 

 tive, displaying an unusual degree of resistance to the decolorisation with 

 alcohol. 



Molility. ^Hanging drops for these tests were made from the water of condensation, 



agar slopes; young cultures incubated at 37° C, never longer than twenty-four 

 hours. 



Inoculation of Media.— AW tubes of media used for the determination of cultural fea- 

 tures and biochemical reactions were inoculated from young peptone brotX 

 cultures of the particular organism. The use of peptone salt solution instead 



