Techniques for the Quantification Density of Microplankton Populations
279
The reagents used for fixation and staining, 10% solution of glutaraldehyde and primuline solution must be freshly filtered through 0.2-~m pore size
membrane filters. The solution of primuline is freshly prepared 20 mg of dry
stain is dissolved in 10 ml of 0.5 M Trisma-hydrochloride buffer at pH ~4; 2 ml
of this solution is diluted in lOml of distilled water and filtered as indicated
above. The solutIon ot acndme orange IS made by diluitmg 5 mg ot stain in 5U
ml 0.5% glutaraldehyde.
The most convenient funnel for filtration should have the diameter of its
filtering area not more than 15mm (optimum 12-14mm).1t is not a standard
diameter of Millipore funnels, but such a funnel (see Fig. 2.2) with an upper
tube 6 cm long can be easily made in any workshop from Plexiglas (as support
for filters in this case porous glass plate is used). The funnel is fixed at the stand
connected with a 1-1 collecting flask supplied with a sensitive vacuum meter
for O.1-0.2atm (Fig. 6.4). Just before the start of filtration, the microscopic
slides should be prepared to immediately make the oil preparations. To do this,
three small drops of nonfluorescent immersion oil are placed along the whole
length of the slide. The oil is then distributed in a very thin homogenous layer
over an area little more than the area of the filter, with the aid of a cover slide
sealed into a bent glass stick.
To start filtration, first the funnel is charged with the Nucleopore filters as
follows: the tap on the funnel is closed and the working surface of the funnel
is moistened with several drops of distilled water. The wet membrane filter is
immediately placed onto this surface and its surface is also moistened in the
same way. The Nucleopore filter is placed over the fine layer of water covering the membrane filter. The top tube of the funnel is fixed in its place and
Fig.6.4. Stand for filtration of water samples. a Funnel; b reserve of vacuum; c vacuum
meter
279
The reagents used for fixation and staining, 10% solution of glutaraldehyde and primuline solution must be freshly filtered through 0.2-~m pore size
membrane filters. The solution of primuline is freshly prepared 20 mg of dry
stain is dissolved in 10 ml of 0.5 M Trisma-hydrochloride buffer at pH ~4; 2 ml
of this solution is diluted in lOml of distilled water and filtered as indicated
above. The solutIon ot acndme orange IS made by diluitmg 5 mg ot stain in 5U
ml 0.5% glutaraldehyde.
The most convenient funnel for filtration should have the diameter of its
filtering area not more than 15mm (optimum 12-14mm).1t is not a standard
diameter of Millipore funnels, but such a funnel (see Fig. 2.2) with an upper
tube 6 cm long can be easily made in any workshop from Plexiglas (as support
for filters in this case porous glass plate is used). The funnel is fixed at the stand
connected with a 1-1 collecting flask supplied with a sensitive vacuum meter
for O.1-0.2atm (Fig. 6.4). Just before the start of filtration, the microscopic
slides should be prepared to immediately make the oil preparations. To do this,
three small drops of nonfluorescent immersion oil are placed along the whole
length of the slide. The oil is then distributed in a very thin homogenous layer
over an area little more than the area of the filter, with the aid of a cover slide
sealed into a bent glass stick.
To start filtration, first the funnel is charged with the Nucleopore filters as
follows: the tap on the funnel is closed and the working surface of the funnel
is moistened with several drops of distilled water. The wet membrane filter is
immediately placed onto this surface and its surface is also moistened in the
same way. The Nucleopore filter is placed over the fine layer of water covering the membrane filter. The top tube of the funnel is fixed in its place and
Fig.6.4. Stand for filtration of water samples. a Funnel; b reserve of vacuum; c vacuum
meter