330 Marine Microbiology 



examination of yeasts from waters and sediment as well as asso- 

 ciated plants and animals. 



METHODS 



Sampling stations in the Atlantic were located in Biscayne 

 Bay, an estuary along the SE coast of Florida, and selected lo- 

 cales in the Gulf Stream and the vicinity of Bimini, the Bahamas. 

 Pacific collection sites were coastal areas near La JoHa, California. 

 Water samples from open ocean areas were obtained with van 

 Doren samplers. Sediments were collected either with a shallow 

 water coring device (6), or a gravity corer in the deep sea. 



The most successful method for the isolation and enumera- 

 tion of yeasts from water samples was membrane filtration. In 

 this technique a known volume of water was filtered and the 

 membrane placed on an isolation medium (2% glucose, 1% pep- 

 tone, 0.5% yeast extract and 2% agar in filtered sea water ) . This 

 medium was made selective for yeasts by adjusting the pH to 

 4.5 with lactic acid or by addition of an antibiotic mixture ( final 

 concentration of 10 mg% chlortetracycline HCl, 2 mg % chloram- 

 phenicol and 2 mg % streptomycin sulfate. ) Solid materials (algae, 

 sediments and intestinal samples) were sui-veyed by incubation 

 in flasks containing sterile sea water, 0.5 per cent glucose, and 

 the antibiotic mixture. The samples were agitated for two to 

 four days and aliquots streaked on isolation medium. Quanti- 

 tative, as well as qualitative, results were obtained by the addi- 

 tion of a known quantity of the sample to 2 to 4 volumes of sterile 

 sea water and then agitated for approximately 50 minutes. This 

 procedure was followed either by direct plating or membrane 

 filtration of a known volume of the wasliings. Streaked plates and 

 filter membranes were incubated for two to three days at tempera- 

 tures of 18-20 C for Pacific samples and 25 C for those from the 

 Atlantic. Individual colonies were selected for further study on 

 the basis of their microscopic and macroscopic morphology. 

 Identification procedures were those advocated by Wickerham 

 (20), Lodder and Kreger van Rij (13), and van Uden and 

 Farinha ( 19 ) . The liquid assimilation tests were kept in continu- 

 ous agitation by means of a rotary shaker (2) or roller drum 

 apparatus. This technique assured optimal aeration of the culture. 



