318 
from inshore areas of the Hawaiian Islands 
and adjacent pelagic environments. Seventeen 
sand samples were taken from the Phoenix and 
Line islands. The water samples were taken 
from four zones: the surf or spray zone; the 
inshore neritic zone, 2-200 m from shore; the 
offshore neritic zone, 300 m to 2 km from 
shore; and the oceanic zone, 2 km or more off 
shore. Of these water samples 56 were taken 
at the surface and 3 were taken at depths 
down to 600 m. The sand samples were 
taken from a depth of 2 inches in the supra- 
tidal, intertidal, and subtidal zones as delimited 
by Hedgpeth (1957). The sample sites were 
selected to include a variety of shore environ- 
ments: leeward, windward, or areas of special 
interest (e.g., South Point, Hawaii, the south- 
ernmost point in the Hawaiian Islands; Wai- 
kiki Beach, probably the most frequently used 
beach on Oahu; and Midway Island, the north- 
ernmost inhabited island of the Hawaiian island 
chain.) Other samples were obtained as oppor- 
tunity offered: those from Kure Island, the 
Phoenix Islands, the Line Islands, and the 
open ocean. Figure 1 gives the geographical 
location of the water and sand collection sites. 
The surface water samples were collected in 
sterile 600-ml glass bottles with plastic screw 
caps. The closed bottles were immersed in the 
water, then opened and allowed to fill with 
water, closed underwater and brought to the 
surface. The depth samples were taken by send- 
ing a plastic water sampler of the van Dorn 
(1956) type to the designated depths. After 
the sampler was brought to the deck of the 
ship, a sterile 600-ml bottle was filled with 
water from the sampler. 
All sand samples were collected with sterile 
implements and placed in sterile 100-ml jars 
with plastic screw caps or in sterile poly- 
ethylene bags (nasco Whirl-Pak, Hydro Prod- 
ucts Co., San Diego, California). 
Salinity measurements were obtained by use 
of Quantabs SO 51 (Linayer Corp., Detroit, 
Michigan). Temperature was determined by 
standard centigrade thermometer, and depth 
by standard oceanographic methods. 
Isolation 
Two principle means of isolation were em- 
ployed: the pour-plate method (Salle, 1954) 
PACIFIC SCIENCE, Vol. XXI, July 1967 
and the millipore filter method (Roth et al., 
1964). 
Standard materials and procedures were used 
in the pour-plate method. Amounts plated for 
the water samples ranged from 0.5 ml to 5.0 
ml. Dilutions of 1:10 to 1:100,000 were used 
for sand samples. Some samples were plated 
upon return to the laboratory; because of the 
distance between most sampling sites and the 
laboratory, however, most of the plating was 
done 24 to 48 hours after collection. All sam- 
ples were kept under refrigeration until plated. 
A control plate, uninoculated, was set up for 
each medium for every plating. Plates were 
also exposed to the air during plating opera- 
tions to determine the level of air contamina- 
tion in the laboratory. 
Materials for the millipore filter method 
included sterile cellulose-ester membranes with 
0.45 [x porosity (Millipore Filter Corp., Bed- 
ford, Massachusetts). The samples were run 
through the millipore filter apparatus using a 
vacuum pump. The membranes with retained 
fungal elements were cultured on selective me- 
dia in presterilized pastic petri dishes. The 
amount put through the filter ranged from 
100 ml to 600 ml per sample. Controls were 
included for testing agar sterility and air con- 
tamination. 
Several kinds of media were used: sodium 
caseinate agar (BBL 01-549, Fred and Waks- 
man, 1928, modified by Potter, 1957), Roth’s 
isolation medium (Roth et ah, 1964), Fell’s 
yeast agar (Fell et al., I960) and mycobiotic 
agar (difco 0689-02). All media except the 
mycobiotic agar were made with sea water col- 
lected at the sample sites. Bacterial growth was 
controlled by the incorporation of 0.05% chlor- 
amphenicol (Chloromycetin, Steri-Vial No. 65, 
Parke, Davis and Co.). Dilutions were made 
with Mcllvaine’s buffer solution, pH 7.0 
(Machlis and Torrey, 1956). The mycobiotic 
agar was made with 250 ml sea water and 
750 ml of distilled water in order to retain 
the selectivity of this medium in which Chloro- 
mycetin is already incorporated. 
The plates were placed in paper bags, sealed 
with adhesive tape, and incubated at 20°- 
24°C. Pour-plates were incubated for three 
weeks and the millipore filter plates for 10 
days at room temperature (20°-24°C). 
