Marine Biology (2+1)

Fixation and Preservation of Zooplankton

Fixation is the application of a chemical (fixative) to kill an organism but retain its morphological characteristics as far as possible. Preservation is the maintenance of the fixed condition for extended periods of time. The chemical solutions for fixation and for preservation may be the same or a different one may be used for the latter .

Fixatives and preservatives and their preparation

Formaldehyde: Its characteristics and uses:

(i) The commercial formaldehyde available to biologists has the

following composition:

Formaldehyde : 37 - 41%* ( * For calculation , 40 % is considered . )

Methanol : 7-13%

Formic acid : 0.025% (by wt)

pH : 2.8 - 5

Ash content : 0.01 % (by wt)

Fe : 2 ppm

Cu : 5 ppm

Water : 46 - 56%

(i i) Formaldehyde is the cheapest among the known preservatives.

(iii) It is known to have preserved plankters in good condition for more than 50 yrs

(Anon., 1968 ).

(iv) 2 to 4% formaldehyde in sea-water is the best reagent both as a fixative and as ' a preservative for most marine zooplankters.

A 4% solution is normally used if the plankton-to-preservative ratio exceeds 1 : 9. and a 2% solution is used when the above ratio does not exceed 1 : 9. For example, for a 10 ml plankton concentrate , 90 ml of 2% formaldehyde solution may be added or 180 ml of a 4% solution .

(v) The preferable of pH of formaldehyde solution for preserving a mixture of collected zooplankton is 7.

In order to obtain such a favourable pH, sodium glycerophosphate is added at the rate of 5 g per 100 ml of concentrated formaldehyde.

(vi) Dilution of formaldehyde for preserving marine zooplankton mixtures is done with sea-water .

(vii) The additive propylene glycol is added at the rate of 5 ml per 1 00 ml diluted formaldehyde to increase the bactericidal and fungicidal properties of the formaldehyde solution .

Preparation of formaldehyde solutions : The concentrated formaldehyde ( i . e., 40%) is usually used for fixing the zooplankters after the catch . However , for preservation, 2 to 4% solution is commonly employed. In the preparation of a 4% solution , 10 ml of 40% formaldehyde is dissolved in 90 ml sea - water or distilled water . Similarly , 100 ml of 2% formaldehyde would contain 5 ml of 40% formaldehyde . The prepared solutions are normally kept in low temperatures , i . e. , below 20°C.

Neutralisation of formaldehyde : As the commercial formaldehyde has considerable acidity, it is always advisable to use the formaldehyde solution whether concentrated or diluted only after neutralising it . To neutralise the acidity of the fixing (40%) and preserving (2 - 4%) formaldehyde solutions , excess CaC03 may be added when the pH of these solutions rises to 7 . When formaldehyde solutions neutralised with CaC03 are stored in steel containers , the dissolved iron occasionally present may produce brown deposits. This can be prevented by using the following solutions:

(i) Neutralisation of 40% formaldehyde fixative : This is prepared by adding 10 g of Rochelle salt (potassium sodium tartrate - KNaC4H406 . 4 H20) and 10 g of CaC03 to 1 litre of 40% commercial formaldehyde.

(ii) Preparation of 2 - 4% neutralised formaldehyde solution : 5 g of Rochelle salt is first dissolved in 1 litre of tap-water . To 875 ml of this solution , 120 ml of 40% commercial formaldehyde and 10 g of CaC03 are added . '

Formaldehyde fixation of general zooplankton : Two methods are commonly employed.

To an empty container (say 500 ml capacity) neutralised 40% formaldehyde is added so as to make a final strength of 4% when the container is completely filled with sea-water and zooplankton . In this case, 50 ml of neutralised 40% formaldehyde is added to the 300 ml of sea - water earlier poured into the container . The living, drained or concentrated zooplankters are then added and the jar topped up with sea-water . The jar is temporarily screened down and gently inverted a few times to mix the contents .

(ii) In the second method , 4% neutralised formaldehyde in sea- water or in distilled water is first added to half the level of an empty jar . The freshly collected and concentrated zooplankters are then added with a minimum of sea - water . Subsequently , more of the 4% formaldehyde solution is added until the jar i s full .

Such fixed zooplankton . are left for a day or two , then filtered once again . The animals are finally preserved (stored) in small jars with plastic screw caps in the neutralised 5% formaldehyde solution or in 75% ethanol . In replacing the fixative with the preservative , the fixative is carefully decanted . The sample jar is then filled to about three-quarters with sea-water or distilled water to which the correct volume of concentrated preservative (2.5 ml of buffered 40% formaldehyde or 7 . 5 ml of preservative with additives per 100 ml container volume) is added. Subsequently, the jar is topped up with sea-water or distilled water and sealed with a secure lid. It is also better if the jar is inverted several times to scatter the animals through the preservative. Such bottles should be checked monthly or quarterly for evaporation or animal condition. The plankton bottles should preferably be stored in a cool dark room.

Plankton storage bottles and labeling: Generally glass containers with wide mouths and screw-on plastic lids or good quality polyethylene jars with polypropylene lids are suitable for plankton fixation and storage. It is also essential that apart from an external label, an internal label written on water - resistant paper be placed in every jar containing a zooplankton sample.

This internal label should contain the following information:

1. Station number

2. Date of sampling

3. Duration of sampling

4.Sampling depth

5.Type of net, mouth size and mesh size

6.Type of haul (horizontal , vertical, etc.)

7. Number of turns recorded by flowmeter

8. Collector's name

Details on external label

1 . Bottle no.

2. Station no .

3. Date of sampling

4. Day/Night

5. Sky

6. Time

7. Depth of sampling site

8. Type of net

9. Mesh aperture

10. Flowmeter reading

11 . Collector's name