Aquatic ecology and Biodiversity

Unit 12- Biodiversity and conservation

Practical 5- Preparation of permanent slides
Experiment No. 5
Staining and mounting of plankton:
Staining and mounting techniques are primarily used for microscopic examination, and anatomical studies of plankton. This technique is best suited for animals which are transparent-invertebrate larvae (doliolids, salps, oikopleura, sagitta, medusae, etc.). Staining and mounting techniques differ, however according to the species of plankton.
Methods
• Fixing
• Staining
• Mounting
Fixing:
Keep the animal in warm Bouin's solution for 30min to 2hrs. Dehydrate the specimens by passing through different grades of alcohol - 30%, 50% and 70% with time duration of 30min in 30% and 50% and several days in 70% alcohol.
Staining:
Important stains used for plankton are methylene blue, methyl green, alum carmine, borax carmine, erythrosin, chlorazol balck E, fast green, acid fuchsin, etc.
Preparation:
Erythrosin solution (1%):Dissolve 1g of erythrosin powder in 99ml of 5% phenol.Alum carmine: Add 3-5g of alum and 2g of carmine to 100ml of d.w in a beaker. Boil the mixture for an hour, cool and filter. To this add 1ml of 40% formaldehyde solution.
Procedure
Wash the specimens with distilled water. Dehydrate the specimens by passing through different grade alcohol (30%, 50%&70%). Immerse the specimens in the respective stains, Immersion time may vary with the nature as well as thickness of the integument. Staining period varied between 30sec. to 1.0min. For example, copepods take 30-60 sec. after immersion. Specimens should be washed in distilled water after staining. Excess stain may be removed by immersing the specimens in a mixture of acid-alcohol (70% ethanol and a few drops of concentration HC1). The stained zooplankters are then washed in D.W. and passed through a reverse series of dehydrating baths - 70% 50% and 30% ethanol. Stained specimens are kept in clove oil for overnight, which yields transparency and reduces the effect of alcohol treatment.
Mounting
Common mountants used for zooplankton are
1. Canada balsam 2. Gum chloral 3.Glycerine-jelly
4. Hoyer's medium 5. DPX mountant 6. Turtox CMC-10 7. CMC-S
Procedure
Wash the slide and coverslip with 95% ethanol. Place a drop of mountant on the slide using a slender glass rod. Transfer the stained specimen to the drop. Place the coverslip at one edge of the drop and lower gently to avoid trapping air bubble in the mountant. Keep the slide horizontal and do not use until dry. Drying time may be reduced by heating in an oven at about 35°C.