Last Updated: November 25, 2025
Estimated reading time: ~8 minutes
In scientific research, the methodology is just as critical as the results. Without robust limnological sampling techniques, data on biodiversity and water quality is meaningless. This post deconstructs the rigorous field and laboratory protocols employed in the Gujranwala freshwater study (2011–2012). It serves as a practical manual for zoology students and early-career researchers, detailing how to correctly collect water samples, preserve delicate specimens, and perform quantitative analysis using standard counting chambers. Whether you are sampling a flowing canal or a stagnant pond, these protocols form the backbone of credible aquatic science.
Search intent: This post satisfies the user intent to explain standard research methodologies, instruct on the use of specific equipment (nets, chambers), and apply formulas for population density calculations.
Key Takeaways
- Net Selection: A 70 µm mesh size is the standard for capturing copepods while allowing smaller phytoplankton to pass.
- Preservation: Immediate fixation with 4–5% formalin is crucial to prevent predation or decomposition in the bottle.
- Quantitative Counting: The Sedgwick-Rafter (S-R) cell is the preferred tool for estimating population density (Organisms/mL).
- Composite Sampling: Mixing water from multiple sub-sites ensures the chemical analysis represents the whole water body, not just one spot.
Field Collection: The Plankton Net and Towing
The primary tool for any zooplanktonologist is the plankton net. In this study, the choice of mesh size was a critical methodological decision. A net with a mesh size of 70 µm was selected. This specific aperture is small enough to catch adult copepods and their developmental stages (copepodids) but large enough to prevent clogging by heavy algal blooms.
“For collection of copepods sample 50 L water was passed through a planktonic net having mesh size 70 µm… Samples were collected just beneath the water surface by towing the net horizontally for 5 minutes” (Maqbool, 2012, p. 21).
The technique employed was horizontal towing. Unlike vertical hauls used in deep lakes, horizontal towing is ideal for the shallow ponds and canals of Gujranwala. By towing the net just beneath the surface for a fixed time (5 minutes) or filtering a fixed volume (50 Liters), the researcher ensures that the sample is quantitative—meaning the results can be expressed as “number of animals per liter” rather than just a random list of species.
Student Note: Mesh Size matters. If you use a 200 µm net, you will miss the nauplii and small Cyclopoids. If you use a 20 µm net, it will clog instantly with algae. 70–80 µm is the “Sweet Spot” for freshwater zooplankton.
Professor’s Insight: Standardization is key. You cannot compare Station A to Station B if you towed the net for 5 minutes at one and 10 minutes at the other. Consistency in speed and duration is the golden rule of field sampling.
Chemical Analysis: Composite Sampling and Preservation
Water chemistry drives biology, so collecting representative water samples is essential. The study utilized a composite sampling technique. Instead of taking one cup of water from the edge, samples were collected from three sub-sites (e.g., NS1, NS2, NS3 for Nandipur Canal) and mixed. This composite sample provides a true average of the water body’s condition, smoothing out patches of high or low concentration.
For the biological samples, time is the enemy. Once copepods are trapped in the collection bottle, larger predators will eat smaller ones, and bacteria will begin decomposition.
“The contents collected in the net were put in 50 ml plastic bottles and few drops of 4-5% formalin were added as preservative” (Maqbool, 2012, p. 22).
Formalin (a solution of formaldehyde gas) is the standard fixative. It stiffens the tissues, preserving the morphological features (legs, antennae) required for identification. Without this step, soft tissues would disintegrate, making taxonomy impossible.
Student Note: Safety First. Formalin is a carcinogen. Always add it in a well-ventilated area outdoors, or use a fume hood back in the lab.
| Step | Equipment / Chemical | Purpose |
|---|---|---|
| Collection | 70 µm Plankton Net | Filter zooplankton from water. |
| Concentration | 50 mL Plastic Bottle | Store the concentrated sample. |
| Fixation | 4–5% Formalin | Kill and preserve specimens instantly. |
| Labeling | Waterproof Marker | Record Station, Date, and Time. |
Fig: Standard workflow for field collection and preservation of zooplankton samples (Data source: Maqbool, 2012).
Professor’s Insight: Always rinse your sample bottles with 10% HCl and distilled water before the trip (as done in this study) to remove any contaminants that could skew chemical readings like pH or TDS.
Laboratory Analysis: The Sedgwick-Rafter Chamber
Back in the lab, the challenge is converting a bottle of “soup” into data. The study employed the Sedgwick-Rafter (S-R) counting chamber. This is a specialized glass slide with a raised rim that holds exactly 1.0 mL of liquid. It is grid-lined, allowing the researcher to scan “strips” of the sample under a microscope.
“Counting was started after the settlement of sample. Settlement time was about 5 min… Organisms present in strips were counted” (Maqbool, 2012, p. 22).
The calculation of population density is not a guess; it is derived from a specific mathematical formula utilized in the thesis:
$$ \text{Number/mL} = \frac{C \times 1000}{L \times D \times W \times S} $$
Where:
- C = Count (number of organisms seen)
- L = Length of strip (50 mm)
- D = Depth of chamber (1 mm)
- W = Width of strip
- S = Number of strips counted
This formula converts the raw count from the microscope view into a standardized density (Organisms per mL), allowing for statistical comparison between different months and stations.
Student Note: Settling Time is crucial. If you start counting immediately, the copepods will be floating and moving (if not fixed well) or at different focal planes. Waiting 5 minutes ensures they all sink to the bottom glass for easy counting.
Professor’s Insight: The S-R cell is preferred over the Haemocytometer for zooplankton because copepods (0.5–2.0 mm) are too large for blood-counting chambers.
Statistical Tools and Software
Modern limnological sampling techniques extend into the digital realm. Collecting data is only half the battle; analyzing it requires robust software. This study utilized a suite of statistical programs to find patterns in the chaos of raw numbers.
- Minitab: Used for Analysis of Variance (ANOVA) to check if the differences between stations were statistically significant, and for Pearson Correlation to link abiotic factors (like Temp/pH) to biotic factors (copepods).
- PAST (Paleontological Statistics): Used for Cluster Analysis. This software draws dendrograms (tree diagrams) that group species based on similarity.
- XLSTAT: Employed for Principal Component Analysis (PCA) to reduce the dimensionality of the data and find the primary drivers of variance (e.g., Seasonality).
“Software ‘the Minitab’ was used to find Pearson’s correlation and ANOVA… Dendrograms were prepared by using software ‘Past'” (Maqbool, 2012, p. 24).
Student Note: You don’t need to be a mathematician, but you must know which test to run. Use ANOVA to compare groups (e.g., Pond A vs. Pond B) and Correlation to check relationships (e.g., Temperature vs. Density).
Professor’s Insight: The use of multiple software packages demonstrates valid methodology. Cross-verifying results (e.g., checking if Cluster Analysis matches PCA groupings) strengthens the scientific conclusion.
The content above is a unique educational synthesis. This section was reviewed and refined by the Professor of Zoology editorial team. Direct citations from the thesis represent the only non-original text.
Real-Life Applications
Why do these specific techniques matter in the real world?
- Environmental Consulting: Consultants must use standard methods (like APHA protocols) when assessing water quality for construction projects. Using the wrong net or formula can lead to legal challenges over the data’s validity.
- Fisheries Management: Hatchery managers use Sedgwick-Rafter chambers to count the density of live food (rotifers/copepods) in culture tanks. Knowing the exact density (e.g., 5 copepods/mL) ensures fish larvae are neither underfed nor overfed.
- Public Health Surveillance: To detect vector mosquitoes or parasitic hosts (like Cyclops carrying Guinea worm), health officers use these exact concentration and identification techniques to screen local water sources.
Exam Relevance: You might be asked to “Describe the procedure for quantitative analysis of zooplankton.” You must mention the Plankton Net, Formalin, and the Sedgwick-Rafter formula to get full marks.
Key Takeaways
- Gear Standard: Use a 70–80 µm mesh net for freshwater copepods; finer nets clog, coarser nets lose data.
- Composite is Best: Always mix samples from multiple spots to represent the whole water body.
- Fixation: 4% Formalin is the industry standard for morphological preservation.
- The Math: Density is calculated, not estimated. Memorize the S-R cell formula variables.
- Software: Statistical validation (ANOVA, PCA) is mandatory for publishing modern ecological research.
MCQs
1. What is the standard mesh size recommended in the study for collecting freshwater copepods?
A. 20 µm
B. 70 µm
C. 250 µm
D. 1 mm
Correct: B
Difficulty: Easy
Explanation: A 70 µm mesh is efficient for catching adult copepods and their larval stages without retaining too much phytoplankton which causes clogging (Maqbool, 2012, p. 21).
2. Which specialized glass slide is used to count zooplankton in the laboratory?
A. Haemocytometer
B. Petri dish
C. Sedgwick-Rafter chamber
D. Neubauer chamber
Correct: C
Difficulty: Moderate
Explanation: The Sedgwick-Rafter chamber holds exactly 1 mL of sample and is grid-lined for the quantitative counting of organisms larger than 50 µm (Maqbool, 2012, p. 22).
3. In the counting formula $ \text{Number/mL} = \frac{C \times 1000}{L \times D \times W \times S} $, what does ‘C’ stand for?
A. Concentration of chemical
B. Count (number of organisms observed)
C. Capacity of the net
D. Celsius temperature
Correct: B
Difficulty: Moderate
Explanation: ‘C’ represents the actual number of individual organisms counted in the selected strips of the chamber (Maqbool, 2012, p. 22).
FAQs
Q: Why use Formalin instead of Alcohol?
A: Formalin is better at fixing tissues without causing shrinkage or brittleness, which is important for identifying the delicate bristles (setae) on copepod legs. Alcohol is better if you plan to do DNA analysis later.
Q: What is a “horizontal tow”?
A: It involves pulling the plankton net through the water parallel to the surface (usually behind a boat or by walking along a bank). It samples the surface layer where many zooplankton congregate.
Q: Can I use a microscope slide instead of an S-R cell?
A: No. A standard slide holds a tiny, unmeasured volume of water (drops). An S-R cell holds a calibrated volume (1.0 mL), which is required to calculate the density per Liter.
Lab / Practical Note
Maintenance: After every sampling trip, wash your plankton net thoroughly with fresh water and hang it to dry in the shade. Never use soap, as residues can kill organisms in the next sample. Sunlight (UV) degrades the nylon mesh, so avoid direct sun drying.
External Resources
- Standard Methods for the Examination of Water and Wastewater (The gold standard for water analysis protocols)
- Zooplankton Sampling Techniques (Springer Protocols)
Sources & Citations
Thesis Citation:
Studies on Abundance and Diversity of Copepods from Fresh waters, Asma Maqbool, Supervisor: Dr. Abdul Qayyum Khan Sulehria, GC University Lahore, Pakistan, Session 2009-2012 (Submitted ~2017).
Corrections:
If you are the author of this thesis and wish to submit corrections, please contact us at contact@professorofzoology.com.
Note: Placeholder tokens and formatting artifacts from the PDF conversion process were removed for clarity.
Author Box
Author: Asma Maqbool, Ph.D. Scholar, Department of Zoology, GC University Lahore.
Reviewer: Abubakar Siddiq
Note: This summary was assisted by AI and verified by a human editor.
Disclaimer: These methodologies reflect the specific protocols of the cited study; researchers should verify current best practices for their specific field conditions.
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