BTC DSC 152P: Cell Biology, Biochemistry and Environmental Biotechnology

Experiment A.1: Preparation of solutions and buffers

(a) Preparation of Molar Solutions (e.g., 1M NaCl)

Objective

To prepare a solution of a specific molarity.

Principle

A Molar (M) solution contains one mole of a solute dissolved in a final volume of one liter (1L) of solvent. Molarity is a unit of concentration (mol/L).
Formula: Molarity (M) = (Weight of Solute in g) / (Molecular Weight of Solute × Volume of Solution in L)
To find the weight needed: Weight (g) = Molarity × Molecular Weight × Volume (L)

Procedure (Example: 100mL of 1M NaCl)

1. Calculate:
   • Molecular Weight (MW) of NaCl = 23 (Na) + 35.5 (Cl) = 58.5 g/mol
   • Weight needed = 1 mol/L × 58.5 g/mol × 0.1 L = 5.85 g
2. Weigh: Accurately weigh 5.85 g of dry NaCl using a weighing balance.
3. Dissolve: Add the NaCl to a 100mL beaker with ~70-80mL of deionized water. Stir until fully dissolved.
4. Make up Volume: Transfer the solution to a 100mL volumetric flask. Rinse the beaker with a small amount of water and add the rinsing to the flask. Carefully add water to the flask until the bottom of the meniscus touches the 100mL calibration mark.
5. Mix: Cap the flask and invert it 10-15 times to ensure a homogenous solution.

(b) Preparation of Buffers (e.g., Phosphate Buffer)

Objective

To prepare a buffer solution that resists changes in pH.

Principle

A buffer is a solution containing a weak acid and its conjugate base (or a weak base and its conjugate acid). It resists pH changes upon the addition of small amounts of acid or base. The Henderson-Hasselbalch equation is used to calculate the ratio of acid to base needed for a specific pH:
pH = pKa + log ( [Conjugate Base] / [Weak Acid] )

Procedure (Example: 0.1M Phosphate Buffer, pH 7.2)

1. Choose components: Use a weak acid/conjugate base pair with a pKa close to 7.2. For phosphate, the relevant pair is H₂PO₄⁻ (acid, pKa = 7.21) and HPO₄²⁻ (base). We use their salts, e.g., Monosodium Phosphate (NaH₂PO₄) and Disodium Phosphate (Na₂HPO₄).
2. Calculate Ratio:
   • 7.20 = 7.21 + log ( [Na₂HPO₄] / [NaH₂PO₄] )
   • -0.01 = log ( [Base] / [Acid] )
   • [Base] / [Acid] = 10^-0.01 = 0.977
3. Prepare Stock Solutions: Prepare 100mL each of 0.1M NaH₂PO₄ (Stock A) and 0.1M Na₂HPO₄ (Stock B).
4. Mix: Mix the two stock solutions in the calculated ratio (e.g., ~49.4 mL of Stock B and ~50.6 mL of Stock A).
5. Adjust pH: Place a pH meter electrode in the solution. Slowly add drops of Stock B (to raise pH) or Stock A (to lower pH) until the meter reads exactly 7.20.
6. Make up Volume: If necessary, add water to reach the final desired volume (e.g., 100mL).

Experiment A.2: Handling and working principle of simple and compound microscope

Objective

To understand the parts and working principle of a compound light microscope.

Principle

A compound microscope uses two sets of lenses to produce a magnified, two-dimensional image.

• The Objective Lens (near the specimen) produces an inverted, real, and magnified primary image.
• The Eyepiece Lens (Ocular) (what you look through) magnifies this primary image further to produce a final, virtual, and highly magnified image.

Total Magnification = Magnification of Objective × Magnification of Ocular (e.g., 40x Objective × 10x Ocular = 400x Total Magnification)

[Image of a labeled diagram of a compound light microscope]

Procedure (Handling and Use)

1. Handling: Always carry the microscope with two hands: one on the arm and one supporting the base.
2. Setup: Place a prepared slide on the stage and secure it with the stage clips.
3. Focusing (Low Power):
   • Start with the lowest power objective (e.g., 4x or 10x).
   • Look from the side and use the coarse adjustment knob to bring the stage as close to the objective as possible without touching.
   • Look through the eyepiece and turn the coarse adjustment knob slowly to move the stage *away* from the lens until the image comes into view.
   • Use the fine adjustment knob to get a sharp, clear image.
4. Focusing (High Power):
   • Once focused on low power, rotate the revolving nosepiece to the high power objective (e.g., 40x).
   • The microscope should be parfocal (remain mostly in focus). Only use the fine adjustment knob to sharpen the image. Never use the coarse knob with high power.
5. Oil Immersion (100x): Place a drop of immersion oil on the coverslip before rotating the 100x objective into place. This lens must make contact with the oil.

Experiment A.3: Study of mitosis in onion root tips

Objective

To prepare a slide of an onion root tip and identify the different stages of mitosis.

Principle

The meristematic tissue at the very tip of an onion root is a region of rapid cell division (mitosis). Mitosis is the process of cell division that results in two identical daughter cells. It is divided into four main stages: Prophase, Metaphase, Anaphase, and Telophase. (Interphase is the non-dividing stage).

Materials

Onion root tips (pre-fixed in Carnoy's fluid), 1N HCl, Acetocarmine or Feulgen stain, glass slides, coverslips, spirit lamp, microscope.

Procedure

1. Hydrolysis: Place one root tip in a drop of 1N HCl on a slide. Gently warm the slide over a spirit lamp (do not boil) for 30-60 seconds. This breaks down the middle lamella, softening the tissue.
2. Staining: Blot the excess HCl and add 1-2 drops of acetocarmine stain. Leave for 2-5 minutes. The stain binds to the chromatin (chromosomes).
3. Mounting: Place a coverslip over the tip.
4. Squashing: Place a piece of filter paper over the coverslip and firmly press down with your thumb. This squashes the tissue into a single cell layer. Avoid any lateral movement.
5. Observation: Observe the slide under low power (10x) to find a good region, then switch to high power (40x or 45x) to identify the stages.

Observations

• Interphase: Nucleus is intact, chromatin is not condensed (looks grainy).
• Prophase: Chromatin condenses into visible, thread-like chromosomes. Nuclear envelope begins to break down.
• Metaphase: Chromosomes (each with two sister chromatids) align at the center of the cell, forming the metaphase plate.
• Anaphase: Sister chromatids separate and are pulled to opposite poles of the cell. They are now individual chromosomes.
• Telophase: Chromosomes arrive at the poles, decondense, and nuclear envelopes re-form around the two new sets. A cell plate begins to form in the middle.

Experiment A.4: Study of structure of prokaryotic and eukaryotic cell

Objective

To observe prepared slides of prokaryotic (bacteria) and eukaryotic (plant/animal) cells and identify their key structural differences.

Principle

Cells are the fundamental units of life. They are broadly classified as prokaryotic (lacking a true nucleus) or eukaryotic (possessing a true nucleus and membrane-bound organelles).

Procedure

1. Prokaryotic Cell (e.g., Bacteria):
   • Observe a prepared slide of bacteria (e.g., Gram-stained E. coli or Bacillus) under the 100x oil immersion lens.
   • Identify the cell shape (coccus, bacillus, spirillum).
   • Note the absence of a visible nucleus and organelles. Only the cell wall, cell membrane, and cytoplasm are discernible.
2. Eukaryotic Cell (e.g., Human Cheek Cell):
   • Gently scrape the inside of your cheek with a clean toothpick.
   • Smear the cells in a drop of saline on a slide, add a drop of methylene blue stain, and cover with a coverslip.
   • Observe under 40x. Identify the prominent, dark-stained nucleus, the cytoplasm, and the cell membrane. Note the irregular shape.
3. Eukaryotic Cell (e.g., Onion Peel):
   • Mount a thin peel of onion epidermis in a drop of iodine solution.
   • Observe under 40x. Identify the rigid, rectangular cell wall, the cell membrane (pressed against the wall), a large central vacuole (unstained), and the nucleus (often pushed to the side).

Key Differences

Experiment A.5: To study the effect of pH and temperature on the activity of salivary amylase

Objective

To determine the optimal pH and optimal temperature for the enzymatic activity of salivary amylase.

Principle

Salivary amylase (Ptyalin) is an enzyme that breaks down starch (a polysaccharide) into smaller sugars (maltose). Enzyme activity is highly sensitive to pH and temperature. At optimal pH/temperature, the enzyme has the correct 3D shape (conformation) and maximum activity. Extreme pH or high temperatures cause the enzyme to denature (lose its shape) and its activity.
Assay: We measure activity by observing the disappearance of starch. Starch gives a blue-black color with iodine. As amylase breaks it down, the color fades, eventually reaching an achromic point (no color). Shorter time to reach this point = higher enzyme activity.

Procedure (Effect of pH)

1. Prepare Saliva Solution: Collect saliva and dilute it 10-fold with saline. This is the enzyme source.
2. Setup Test Tubes: Label 5 test tubes. In each, add:
   • Tube 1: 1mL pH 5.0 buffer
   • Tube 2: 1mL pH 6.0 buffer
   • Tube 3: 1mL pH 7.0 buffer (e.g., neutral saline)
   • Tube 4: 1mL pH 8.0 buffer
   • Tube 5: 1mL pH 9.0 buffer
3. Substrate: Add 1mL of 1% starch solution to each tube.
4. Incubate: Place all tubes in a 37°C water bath (optimal temp) for 5 minutes to equilibrate.
5. Start Reaction: Add 0.5mL of the saliva solution to *each* tube. Start a stopwatch.
6. Test for Activity: At 1-minute intervals, take a drop from each tube and add it to a drop of iodine solution on a spotting tile.
   • Blue-black color: Starch is still present.
   • No color (yellow-brown of iodine): Starch is fully digested (achromic point).
7. Record: Note the time taken for each pH tube to reach the achromic point.

Procedure (Effect of Temperature)

Repeat the same process, but use only the optimal pH buffer (e.g., pH 7.0) in all tubes. Place each tube in a different water bath (e.g., 0°C, 25°C, 37°C, 60°C, 90°C).

Results

• Effect of pH: The tube closest to pH 6.8-7.0 will show the fastest activity (shortest time). Activity will be very low at pH 5 and 9.
• Effect of Temperature: The tube at 37°C will be fastest. The 0°C tube will be very slow (enzyme inactive). The 90°C tube will show no activity (enzyme denatured).

Experiment A.6: Estimation of blood glucose by glucose oxidase method

Objective

To estimate the concentration of glucose in a given blood serum sample using the GOD-POD (Glucose Oxidase-Peroxidase) method.

Principle

This is a colorimetric estimation. The reaction occurs in two steps:

1. Glucose Oxidase (GOD):
   Glucose + O₂ + H₂O → Gluconic Acid + H₂O₂ (Hydrogen Peroxide)
2. Peroxidase (POD):
   H₂O₂ + 4-Aminoantipyrine + Phenol (colorless) → Quinoneimine (pink-colored dye) + H₂O

The intensity of the pink color is directly proportional to the amount of glucose in the sample. We measure this intensity using a colorimeter at ~530 nm and compare it to a known standard.

Procedure

1. Label Tubes: Prepare 3 cuvettes/test tubes:
   • Blank (B): 1.0mL of GOD-POD Reagent.
   • Standard (S): 1.0mL of GOD-POD Reagent + 0.01mL (10µL) of Standard Glucose (100 mg/dL).
   • Test (T): 1.0mL of GOD-POD Reagent + 0.01mL (10µL) of Blood Serum Sample.
2. Incubate: Mix all tubes and incubate at 37°C (or room temp) for 10-15 minutes for the color to develop.
3. Read Absorbance:
   • Set the colorimeter to 530 nm.
   • Use the Blank (B) to set the absorbance to zero.
   • Read and record the absorbance (Optical Density, OD) of the Standard (S) and the Test (T).

Calculations

(Absorbance of Test / Absorbance of Standard) × Concentration of Standard = Conc. of Glucose in Sample

Example:
If OD_Test = 0.25, OD_Standard = 0.20, and Conc._Standard = 100 mg/dL
Glucose Conc. = (0.25 / 0.20) × 100 mg/dL = 125 mg/dL

Experiment A.7: Estimation of protein by Lowry's method

Objective

To estimate the concentration of protein in a given sample using the Lowry method.

Principle

This is a highly sensitive colorimetric method that occurs in two steps:

1. Biuret Reaction: Cu²⁺ ions in an alkaline solution (Reagent C) complex with the peptide bonds of the protein, forming a blue-colored complex.
2. Folin-Ciocalteu (F-C) Reaction: The amino acids Tyrosine (Tyr) and Tryptophan (Trp) in the protein, along with the copper-protein complex, reduce the F-C reagent (phosphomolybdate/phosphotungstate). This reduction produces a deep blue-colored product.

The intensity of the blue color is proportional to the protein concentration, measured at 660-750 nm.

Procedure

1. Prepare a Standard Curve:
   • Take 6 test tubes. Add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of standard protein solution (e.g., Bovine Serum Albumin, BSA, at 200 µg/mL).
   • Make up the volume in all tubes to 1.0mL with water. (These tubes now contain 0, 40, 80, 120, 160, 200 µg of protein).
2. Prepare Test Sample: Take 1.0mL of the unknown (T) sample (diluted if necessary).
3. Step 1 (Biuret): Add 4.5mL of Alkaline Copper Reagent (Reagent C) to all 7 tubes. Mix and let stand at room temp for 10 minutes.
4. Step 2 (F-C): Add 0.5mL of F-C Reagent (Reagent D) to all tubes. Mix *immediately* (this is critical).
5. Incubate: Incubate in the dark at room temp for 30 minutes for color to develop.
6. Read Absorbance: Set colorimeter to 660 nm. Use the '0' tube as the Blank to set zero. Read the OD of all standards and the test sample.

Calculations

1. Plot Graph: Plot a standard curve (graph) with Protein Conc. (µg) on the X-axis and Absorbance (OD) on the Y-axis. It should be a straight line.
2. Find Unknown: Locate the OD of your Test sample (T) on the Y-axis. Draw a horizontal line to the standard curve, then a vertical line down to the X-axis.
3. The value on the X-axis is the concentration of protein in your 1.0mL test sample (e.g., 130 µg/mL).

Experiment A.8: Separation of amino acids by paper chromatography

Objective

To separate a mixture of amino acids using ascending paper chromatography.

Principle

Chromatography separates components of a mixture based on their differential distribution between two phases: a stationary phase and a mobile phase.

• Stationary Phase: The water molecules bound to the cellulose fibers of the Whatman No. 1 chromatography paper.
• Mobile Phase: A nonpolar organic solvent (e.g., Butanol:Acetic Acid:Water) that moves up the paper by capillary action.

Separation occurs based on partition coefficient (solubility). Amino acids that are *more soluble* in the mobile phase will travel *faster and further* up the paper. Amino acids that are *less soluble* (more polar, more affinity for the stationary phase) will travel *slower*.

Procedure

1. Prepare Paper: Cut a Whatman No. 1 paper strip. Draw a pencil line (origin) 2 cm from the bottom.
2. Spot: Using a capillary tube, apply a small, concentrated spot of the amino acid mixture on the origin. Let it dry. Repeat 2-3 times.
3. Prepare Chamber: Pour the mobile phase into a chromatography chamber/jar to a depth of 1 cm. Cover and let it saturate with solvent vapor.
4. Run: Hang or stand the paper in the chamber so the bottom edge (but not the spot) is submerged in the solvent. Cover the chamber.
5. Develop: Allow the solvent to run up the paper (ascending) for 1-2 hours or until the solvent front is ~2 cm from the top.
6. Dry: Remove the paper, mark the solvent front with a pencil, and let it dry completely (preferably in a fume hood).
7. Visualize: Spray the paper with Ninhydrin solution and heat it in a hot air oven (80-100°C) for 2-5 minutes. Amino acids will appear as purple spots (Proline gives a yellow spot).

Calculations (R_f Value)

The Retention Factor (R_f) is a ratio that helps identify the components. It is constant for a given amino acid under specific conditions.

R_f = (Distance traveled by the amino acid spot) / (Distance traveled by the solvent front)

Measure from the origin line. The R_f value is always less than 1. Compare the R_f values of the unknown spots to known standards to identify them.

Experiment B.1: Analysis of Soil Samples

(a) Determination of Moisture Content

Principle

Moisture content is the weight of water in a soil sample, expressed as a percentage of the soil's oven-dry weight.

Procedure

1. Weigh an empty, dry crucible (W1).
2. Add fresh soil sample to the crucible and weigh it (W2).
3. Place the crucible in a hot air oven at 105°C for 24 hours.
4. Cool the crucible in a desiccator and weigh it again (W3).

Calculation

Moisture % = [ (W2 - W3) / (W3 - W1) ] × 100
(W2 - W3) = Weight of water
(W3 - W1) = Weight of dry soil

(b) Determination of Soil pH

Procedure

1. Weigh 20 g of air-dried soil and add it to a 100mL beaker.
2. Add 40mL of deionized water (1:2 soil-to-water ratio).
3. Stir the suspension for 30 minutes and let it settle.
4. Calibrate a pH meter using standard buffers (pH 4.0, 7.0, 9.2).
5. Immerse the electrode in the supernatant (clear liquid) of the soil mixture and record the pH.

(c) Determination of Particle Size (Sieve Analysis)

Principle

Soil texture (sand, silt, clay) is determined by separating particles using a set of standard sieves of decreasing mesh size.

Procedure

1. Stack the sieves in order of decreasing mesh size (largest on top, e.g., 2mm, 1mm, 0.5mm, etc.), with a pan at the bottom.
2. Weigh 100g of dry soil and place it in the top sieve.
3. Shake the sieve stack (manually or with a mechanical shaker) for 10-15 minutes.
4. Weigh the soil retained on each sieve and in the bottom pan.
5. Calculate the percentage of soil retained in each fraction. (e.g., % Sand, % Silt, % Clay).

(d) Determination of Water Holding Capacity (WHC)

Procedure

1. Weigh a filter paper (W1).
2. Fold it and place it in a funnel. Saturate it with water and let it drain.
3. Weigh 10 g of dry soil (W2) and place it on the filter paper.
4. Slowly add water to the soil until it is fully saturated and begins to drip.
5. Let it drain until no more water drips (e.g., 30 min).
6. Weigh the funnel, wet filter paper, and wet soil (W3).

Calculation

WHC (%) = [ ( (W3 - W2) - W1 ) / W2 ] × 100
(W3 - W2) = Weight of wet paper + water in soil.
( (W3 - W2) - W1 ) = Weight of water in soil.

(e) Determination of Organic Matter Content

Principle

Organic matter is combustible. We can estimate it by burning it off at a high temperature (ignition) and measuring the weight loss.

Procedure

1. Weigh an empty, dry crucible (W1).
2. Add 10g of oven-dried (105°C) soil to the crucible and weigh (W2).
3. Place the crucible in a muffle furnace at 550°C for 4 hours. This burns off the organic matter.
4. Cool the crucible in a desiccator and weigh it (W3).

Calculation

Organic Matter % = [ (W2 - W3) / (W2 - W1) ] × 100
(W2 - W3) = Weight of organic matter lost
(W2 - W1) = Weight of dry soil

Experiment B.2: Analysis of Water Samples

(a) Determination of pH

Procedure

Calibrate a pH meter with standard buffers (4.0, 7.0, 9.2). Rinse the electrode with deionized water and dry. Immerse the electrode directly into the collected water sample and record the reading.

(b) Determination of Conductivity

Principle

Electrical Conductivity (EC) measures the water's ability to conduct electricity, which is directly related to the concentration of dissolved ions (salts). It is measured in µS/cm (microsiemens per cm).

Procedure

Use a calibrated conductivity meter. Rinse the probe with deionized water and then with the sample water. Immerse the probe in the sample and record the stable reading.

(c) Determination of Total Dissolved Solids (TDS)

Principle

TDS is the measure of all dissolved inorganic and organic substances in the water. It can be estimated from conductivity or measured gravimetrically.

Procedure (Gravimetric)

1. Weigh a clean, dry evaporating dish (W1).
2. Filter 100mL of the water sample to remove suspended solids.
3. Transfer the filtered 100mL to the evaporating dish.
4. Evaporate the water by heating in a water bath, then dry in an oven at 180°C for 1 hour.
5. Cool in a desiccator and weigh (W2).

Calculation

TDS (mg/L) = [ (W2 - W1) in mg ] × 1000 / (Volume of sample in mL)
e.g., (5 mg) * 1000 / 100 mL = 50 mg/L

Experiments B.3, B.4, B.5: Isolation of microorganisms from soil, air, and water

Objective

To isolate and culture microorganisms (bacteria and fungi) from different environmental samples.

Principle

Microorganisms are ubiquitous. We can grow them on a solid nutrient agar (NA) medium, which provides all necessary nutrients (carbon, nitrogen, etc.). Each viable cell will multiply to form a visible colony.

(a) Isolation from Soil (Serial Dilution-Plating)

1. Add 1 g of soil to 9 mL of sterile water (10⁻¹ dilution). Vortex.
2. Transfer 1 mL from this tube to another 9 mL tube (10⁻² dilution). Repeat to get 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶ dilutions.
3. Pipette 0.1 mL from the 10⁻⁴, 10⁻⁵, and 10⁻⁶ tubes onto separate, pre-poured nutrient agar plates.
4. Spread the liquid evenly over the surface using a sterile L-shaped glass rod (spread plating).
5. Incubate the plates (inverted) at 37°C for 24-48 hours.
6. Observe the plates for individual bacterial colonies.

(b) Isolation from Air (Settle Plate Method)

1. Take a sterile nutrient agar plate.
2. Open the lid and expose it to the air in a chosen location (e.g., classroom, lab) for 15-30 minutes.
3. Close the lid, seal, and incubate (inverted) at 37°C for 24-48 hours.
4. Observe the colonies (bacteria and fungi) that grow.

(c) Isolation from Water

1. Use the same serial dilution-plating method as for soil.
2. Start by taking 1 mL of the pond/lake water sample and adding it to 9 mL of sterile water (10⁻¹ dilution).
3. Plate 0.1mL from the 10⁻², 10⁻³, and 10⁻⁴ dilutions onto nutrient agar plates.
4. Incubate and observe.

Experiment B.6: Determination of total coliform bacteria in water sample

Objective

To test a water sample for the presence and quantity of coliform bacteria, which are indicators of fecal contamination.

Principle

The Most Probable Number (MPN) method is used. Coliforms are bacteria (like E. coli) that ferment lactose, producing acid and gas.
We inoculate the water sample into MacConkey Broth, which contains lactose and a pH indicator.

• Positive Result: Broth turns yellow (acid production) AND gas is trapped in the small, inverted Durham tube inside.
• Negative Result: Broth remains red/purple, and no gas is produced.

Procedure (Presumptive Test)

1. Set up tubes:
   • 3 tubes of double-strength MacConkey Broth (DSMB).
   • 6 tubes of single-strength MacConkey Broth (SSMB).
2. Inoculate:
   • Add 10 mL of the water sample to each of the 3 DSMB tubes.
   • Add 1.0 mL of the water sample to 3 of the SSMB tubes.
   • Add 0.1 mL of the water sample to the remaining 3 SSMB tubes.
3. Incubate: Incubate all 9 tubes at 37°C for 48 hours.
4. Record Results: After 48 hours, count the number of positive tubes (yellow color + gas) in each set.

Calculations

Record the result as a 3-digit combination (e.g., "3-2-1" means 3 positive in the 10mL set, 2 positive in the 1mL set, and 1 positive in the 0.1mL set).
Compare this combination to a standard MPN Statistical Table to find the "Most Probable Number" of coliforms per 100 mL of the original water sample.