keeping properties of such preparations have 

 been followed for periods exceeding 5 years. 

 For certain types of study, this method seems 

 to be superior to all others. Its advantages 

 make it a useful supplement for nearly any 

 examination and enumeration technique. 



MATERIALS AND METHODS 



We have employed 1-inch or 47-mm. di- 

 ameter molecular filters (pore size 0.45//) 

 together with the glass and/or stainless steel 

 filter holders. Additional top elements of dif- 

 ferent diameters have been fabricated for the 

 base of the stock filter assemblies so that the 

 filtering area of the molecular filter disk may 

 be reduced. 



The revised method of sample treatment 

 differs from the original in the following de- 

 tails: (1) a basic formalin fixative is substi- 

 tuted for Lugol's iodine, (2) alcoholic rather 

 than aqueous Fast Green stain is added at a 

 later stage in the dehydration process, and 

 (3) beechwood creosote or anisole have been 

 substituted for cedar oil as clearing agents. 



Sample Preparation 



Prior to sample preparation, the organisms 

 contained in 100 ml. of a freshly collected 

 sea-water sample are killed and fixed by 

 adding 3-5 ml. of 5 percent formalin made 

 basic (pH 11-12) by the addition of sodium 

 carbonate. If the sample does not contain 

 organisms of interest bearing calcium carbo- 

 nate skeletons (e.g. coccolithophorids, for- 

 aminifera), a more satisfactory fixative may 

 be substituted. This fixative (10 g. iodine, 

 20 g. potassium iodide, 20 g. glacial acetic 

 acid, and 200 g. water) suggested to me by 

 Wilhelm Rodhe has been found to preserve 

 the naked flagellates and dinoflagellates better 

 than the basic formalin in that flagellae are 

 not shed and cell deformation and shrinkage 

 are less severe (Lund, Kipling, and LaCren, 

 1958). Enough of the mixture is added to the 

 sample to give it a weak tea color; the sam- 

 ples are stored in the dark. In the concentra- 

 tions normally employed, this solution makes 

 the sea-water sample sufficiently acidic to 

 dissolve skeletal parts composed of calcium 

 carbonate. 



Following fixation, the sample is vigorously 

 agitated, and an aliquot is withdrawn with a 

 wide-bore pipette and passed through the 

 filter under suction. Before the organisms 

 have dried, the filter is washed with suc- 

 cessively more dilute sea-water (75, 50, 25, 

 and 10 percent) and finally with distilled water 

 made basic (pH ca. 8.0-8.5) by the addition 

 of ammonium hydroxide. The material on the 

 filter is next dehydrated by washing succes- 

 sively with 10, 30, 50, 75, and 95 percent 

 aqueous ethanol. The amount of reagent used 

 in each of the washings depends upon the 

 filter diameter — generally 10-15 ml. is ade- 

 quate. 



The filter disk is completely covered with 

 alcoholic Fast Green (0.1 percent in 95 per- 

 cent ethanol) and allowed to stand for about 

 20 minutes. The Fast Green solution is then 

 passed through the filter disk (with suction) 

 and the disk rinsed with 10-15 ml. of absolute 

 ethanol. Staining with Fast Green, although an 

 optional step, makes it easier to locate orga- 

 nisms on the filter disk and to distinguish 

 small detrital particles from small flagel- 

 lates. 



The filter disk is carefully removed from 

 the filter assembly, its edges are trimmed, 

 and it is placed, filtering surface up, on a 

 few drops of clearing agent on a glass micro- 

 scope slide. A variety of clearing agents 

 have been investigated. Beechwood creosote 

 clears well, and samples using this agent are 

 still in good condition after 5 to 6 years. Ani- 

 sole (methoxybenzene) is a better clearing 

 agent; its keeping properties have been fol- 

 lowed for 18 months with no signs of deterio- 

 ration. Although toluene and xylene also clear 

 the filter disk well, there is a tendency for 

 the filter disk to cloud with age unless the 

 filter is completely free of water prior to 

 mounting in balsam. 



After the filter disk is cleared (usually less 

 than 10 minutes) the excess clearing agent is 

 removed from the bottom of the filter disk by 

 dragging the filter across a clean microscope 

 slide. The filter is transferred with forceps 

 onto a few drops of toluene or xylene balsam 

 on a clean microscope slide. A few addi- 

 tional drops of balsam are added to the upper 



