Marine Biology (2+1)

Methods of Fixation and Preservation of Individual Groups of Zooplankton

Calcareous plankton

 These include planktonic foraminifers and certain gastropod molluscs such as Janthina and Creseis, larvae of pelecypods and gastropods which possess calcareous skeletons, namely tests or shells made of calcium carbonate. These are fixed and preserved in 2% formaldehyde (pH 8 - 8.5) as follows: 5 ml of 40% formaldehyde buffered with borax is added to 90 ml of sea-water containing living calcareous plankton . The buffered formaldehyde is prepared with 1000 ml of 40% formaldehyde to which are added 30 g of sodium tetraborate (NCI2S407 ' H20 - b or ax ) , 4 . 5 ml of propylene glycol and 0.1 ml of propylene phenoxetol . It is advisable to have the volume ratio of plankton biomass to preservative fluid at 1 : 9 and storage temperature at 15 ° C.

Coelenterates(Cninadarians)

The fixation of live cnidarians in a large glass jar may be done by gently adding 40% neutralised formaldehyde . Alternatively, the animals may be gently trans f e r r e d to a 4% formaldehyde fixative using a spoon .

These coelenterates are preserved in 2 % formal dehyde solution prepared with additives as follows: 10 ml of stock solu tio n (prepared using 500 ml of 40% formaldehyde to which the additives , viz. , 50 ml of propylene phenoxetol and 45 0 ml of propylene glycolare added) is added to 90 ml of sea - water or distilled water . Hydromedusae may be best preserved in 1 % phenoxetol in distilled water and i n the same fluid neutralised with 1 % hexamine.

 

Ctenophores(Acnidarians)  

The animals are fixed in 1 % trichloroacetic acid (1 g in 99 m l se a- water) for 30 minuts . They are then temporarily preserved in 100 m l of sea- water containing 1 ml of stock solution (50 ml of 4% formaldehyde containing 5 ml of propylene phenoxetol and 45 ml of propylene glycol). After a week, the anima l s are permanently transferred to 1 0 0 ml of sea -w ate r containing 5 ml of the above stock solution .

Chaetognaths

These are fixed with 5 % neutralised for maldehyde which is prepared as follows . A solution containing 1 I of tap-water and 5 g o f Ro c hel l e salt is prepared. Then 125 ml of 40 % commercial for maldehyde and 10 g or CaC03 are added to just 875 m l of the a fore saidsolution . Fixatives containing osmic acid or Souin's fluid can also be used for chaetognaths .

The chaetognaths can be preserve d in 2% form a ldehyde in sea-water at pH 6 - 7 . 1 % phenoxetol in distilled water and 5 0% e thylene glycol in distilled water are also added as preservative s .

Polychaetes

The fixation of adult planktonic polychaetes is done by slowly adding neutralised formaldehyde until a concentration of about 2% is reached. They are preserved in either 2% neutralised formaldehyde or 70 - 80% ethanol .

The f ixation of polychaete larvae i s done by pipetting Bouin's fluid heated to 60 ° C into s mall volume of sea-water containing the swimming larva e. The dead larvae are then transferred to cold Bouin's fluid f or 1 t o 3 hours and subsequently was hed and stored in ethanol .

C r u s t ace a ns

For either fixation or preservation of crustacean zooplankton the following solution is prepared and used. 5 - 10 ml of neutralised for maldehyde (prepared by adding 30 - 40 g of sodium tetraborate to 1000 ml of 4 0 % formal dehyde) is added to 90 - 95 ml of filtered sea-water . The pH of this solution should be between 7 and 8. The ratio of plankton biomass to the preservative fluid should be approximately 1 : 4 by volume .

Important type species may be preserved in a 70% ethanol-distilled water solution. Specimens from a formaldehyde-sea-water solution may be transferred to an ethanol-d i stilled water solution as follows: The specimens , after thorough rinsing in distilled water , are first immersed in 30% ethanol for 10 m i n . Subsequently, the animals are immersed i n 50% ethanol for 1 h and finally stored in 70% ethanol . A few drops of glycerol may be added to this solution , especially for the preservation of copepod and decapod plankton .

Planktonic mollusks These include bivalve and gastropod veligers, shelled pteropods and heteropods and naked pteropods. The shell of these organisms is composed of aragonite, a most suitable form of calcium carbonate. Hence the pH of preservatives suitable to these organisms should drop to 6. Other- wise severe corrosion of the shells will take place. Further, the shells, usually shiny, translucent, coloured and hard, may lose their sculpture and become opaque - white, then chalky-white, soft and brittle. Such shells may break easily when touched and eventually dissolve completely.

Veliger larvae: The veliger larvae of ' molluscs can be fixed with 90 - 95% aqueous ethanol which will prevent the dissolution of calcium carbonate. They may be preserved in a solution prepared as follows: 10 cc of 40% formaldehyde (buffered with sodium bicarbonate to pH 10) is added to 11 of filtered seawater containing 100 g of commercial sugar. The sugar besides reserving the colour of organisms, acts as a clearing agent and as a safety factor against evaporation.

The veliger larvae can also be fixed in a 40% 'formaldehyde solution buffered to pH 8 with sodium glycerophosphate. After washing in fresh- water, they can be stored in a solution of 1 % propylene phenoxetol and 5 - 10% glycerol in freshwater .

Pteropods and heteropods: These animals can be fixed using 70% ethanol or 4% formaldehyde sea - water buffered to pH 8 ' - While the shelled forms are preserved in 70% ethanol, the naked forms are best preserved in 3 - 4% formaldehyde buffered to pH 8.

Thaliaceans : The salps, doliolids and pyrosomas may be fixed in 4% formaldehyde . Subsequently, they are best preserved in 70% aqueous ethanol .

Appendicularians : The appendicularians , for example Oikopleura and Fritillaria , are fixed using 4% formaldehyde in sea-water. While fixing, equal volumes of plankton and 4% formaldehyde are selected . Subsequently, they are preserved in 2% formaldehyde in sea-wate r with a pH o f 6,