500 Marine Microbiology 



however, that if this method is to be successful one must be able 

 to identify accurately and quickly the microorganisms concerned. 

 Our first problem, then, was differentiation at the generic 

 level, and our early results ( 45 ) suggested some broad categories 

 corresponding roughly to the Pseudomonas, Achromobacfer, Vi- 

 brio and Bacteraceae of Bergey (6) and from the experience 

 gained since then, we have developed the scheme now to be 

 described (46). 



THE DETERMINATIVE SCHEME 



The medium used for the isolation and cultivation of our 

 bacteria was "Lab-Lemco" nutrient agar ("Oxoid") made up 

 either in tap water or with 75 per cent aged sea water (61) as re- 

 quired. We are, therefore, purposely excluding from our discus- 

 sion any of the Gram negative bacteria, requiring specialised 

 media, such as the Azotobacter, Acefobacter, CeUuIomonas sp. 

 etc. 



The original isolate is plated out to obtain a pure culture 

 and then put through the series of operations outlined in Table 

 1. Colony appearance. Gram's stain, morphology and the Kovacs' 

 (37) oxidase test are carried out on the agar plate culture; 

 motility and morphology (under phase contrast) are done on 

 the "Lab-Lemco" broth culture. This latter culture is also used 

 to surface seed on agar plate for the sensitivity tests of Shewan 

 et al. ( 45 ) ; and to inoculate the two tubes required for the Hugh 

 and Leifson (30) test. The production of diffusible fluorescent 

 pigment is determined by using the media of King et al. (35) or 

 that of Paton (43). When growth occurs in the latter medium 

 the production of 2 keto-gluconic acid from the glucose present, 

 an important diagnostic feature for many Pseudomonas sp. (6), 

 is recorded, using an aniline oxalate or aniline phthalate reagent. 

 A sheet of chromatographic paper ( Whatman No. 1 ) is impreg- 

 nated with a saturated solution of recrystallised aniline oxalate 

 or phthalate (29) and the culture is heavily spotted on the paper 

 which is then heated in an oven at 105C for two to three minutes. 

 A red spot indicates the presence of the 2 keto-gluconic acid. 

 Occasionally, the gluconate (or the glucose) appears to be dis- 

 similated beyond the 2 keto-gluconic acid stage and gives rise to 

 yellow colors with the aniline reagents (Floodgate, private com- 



