The media was prepared on the surface and lowered to the habitat in sealed 

 containers the day before the air sampling. The samples were taken by 

 the group engineer for a pre-determined period of time, returned to the 

 surface soon after exposure, and subsequently to the base camp for in- 

 cubation and further processing. 



The exposed plates were incubated at 28C for 72 hrs . The number of bac- 

 terial and fungal colonies were recorded from the TSA and 2216 plates 

 using a Quebec colony counter. The number of mannitol fermenters was 

 recorded from the Mannitol Salt Agar and green pigmented or fluorescence 

 positive organisms were recorded on Pseudosel agar. Positive colonies 

 from the latter two media were isolated to TSA mini-slants (agar slants 

 in 1-dram vials) for conformation at NBRL. On selected days all organisms 

 growing on all six stages of the TSA and 2216 samples were isolated to 

 TSA or 2216 mini-slants and returned to NBRL for identification. 



Human microbiology. The divers were sampled at three body sites, forearm 

 (approximately 9 sq. in.) nose and ears, with the aid of saline-wetted, 

 cotton-tipped sterile swabs. The swab tips were broken off in 1-dram 

 vials containing 1 ml of Trypticase Soy Broth (BEL) . The samples were 

 taken by the divers, immediately returned to the surface and subsequently 

 to the base camp. Samples were collected from each site; the day before 

 the dive, the day of the dive, the middle of the mission (no ear samples 

 collected at this time) and the day decompression was to start. The swabs 

 and vials for sampling were transferred to the divers the day before 

 sampling so collection could be obtained prior to diving activity. 



The sample vials were shaken vigorously to suspend the bacteria in the 

 broth. A 0.1 ml aliquot of the sample was plated to the surface of media 

 according to the sample source; nose, Mannitol Salts Agar (MSA); ear, 

 blood agar (3% def ibrigenated rabbit blood) and Pseudosel; skin, blood 

 agar. The inoculated plates were incubated at 37C for 24 hr. The manni- 

 tol fermenters on MSA and pigmented organisms on Pseudosel were recorded 

 and isolated for confirmation. On the blood agar plates the numbers of 

 each colony type was recorded, described and three of each colony type 

 isolated to TSA mini-slants for identification at NBRL. No effort was 

 made to isolate the anaerobic or microaerophilic flora. 



Marine microbiology . Sea water samples were collected from depths greater 

 than 30 feet at four sites in the area of the habitat using the Cobet 

 water sampler (Hydro Products) . The sea water was analysed by the mem- 

 brane filter technique as outlined in the Standard Methods for Water 

 Analysis (1965) using m-Endo Medium (Difco) for coliform enumeration, 

 Pseudosel for 7s_. aeruginosa and Marine Agar for the total bacterial 

 count. 



Bacterial Identification . The samples were received at NBRL on mini- 

 slants and were stored at 4-6C until identification procedures were 

 started. Each isolate was streaked to blood agar to ensure purity and 

 identified by means of standard bacteriological procedures, biochemical 

 reactions and morphological characteristics. No attempt was made to 



IX-38 



