Samples from the sewage treatment plant, from the Campbell 
factory, and from the Tenneco plant were iced in the field and 
stored in ice until they were used in the laboratory. Samples 
taken on board ship (Buoy Rock, Spaniard Bar, Baltimore Harbor, 
Tilghman Island) were used for microbiological analysis within 
15 min of sampling; the remainder of each sample was stored on 
ice until it was used in the laboratory. 
Microbiological Samples 
Water samples were used to prepare appropriate dilutions for 
plating. For sediment samples, 1.0 g (wet weight) was suspended 
in 9.0 ml of sterile estuarine salts; further dilutions were pre¬ 
pared from this suspension. 
Total viable counts of aerobic, heterotrophic bacteria were 
made using the spread plating technique, plating on Nelson's 
medium (Nelson et al. 1973). Nelson's medium contains casamino 
acids; 5.0 g; yeast extract, 1.0 g; glucose, 2.0 g; agar, 15.0 g; 
and salt solution, 1 liter. For sediment samples from estuarine 
sites, the estuarine salts solution contained NaCl, 10.0 g; 
MgCl2*6H20, 2.3 g; KC1, 0.3 g; and distilled water, 1 liter. 
For samples from freshwater sites, the salts solution was used 
at one-tenth strength. 
For viable counts of tin-resistant organisms, appropriate 
dilutions were spread on the surface of Nelson's medium prepared 
as above, supplemented with SnCl^ to yield 75 ppm tin suspended 
in the medium. Extensive preliminary testing (Table 19 ) indi¬ 
cated that this concentration of tin was appropriate to select 
for tin-resistant organisms. Addition of SnCl^ to the medium 
resulted in a fine precipitate of SnC >2 which was uniformly sus¬ 
pended in the medium by agitation. In one series of experiments 
the organisms resistant to organotin were estimated by plating 
on Nelson's medium containing 15 ppm tin as (CH 3 ) 2 SnCl 2 . 
All platings were prepared in triplicate. Plates were incu¬ 
bated at 25 + 2 C for 3 days prior to counting time. 
Two systems were employed to determine if the microbial flora 
in samples could transform tin to volatile organotin compounds: 
i) Bioflasks . 250-ml flasks containing 20 ml of Nelson's 
medium supplemented with 75 ppm tin as SnCl^, were inoculated 
with 1.0 ml of sediment suspended in estuarine salts. Each 
flask contained a 10-ml beaker embedded in the agar.^ The beaker 
held 5.0 ml of a solution of 8 percent (wt/vol) citric acid in 
10 percent HC1. The flask was sealed with a rubber stopper. 
After 14 days incubation at 27 + 2 C, material in the beaker was 
examined for tin. Thus, if organisms growing on the medium pro¬ 
duced volatile tin compounds, they would be detected in the acid 
solution. Sterile controls to check for nonbiological production 
RR 
