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BACTERIAL GROWTH CURVE

Spectrophotometric Determination of Microbial Growth Kinetics

1 Aim

To determine the growth kinetics and distinct growth phases of a bacterial population by measuring the Optical Density (OD) of a liquid culture at regular time intervals utilizing a spectrophotometer.

2 Principle

Bacteria reproduce primarily by binary fission, leading to an exponential increase in cell numbers over time. When cultured in a closed system (batch culture) with a fixed amount of nutrients, the population follows a highly predictable, standardized pattern known as the Bacterial Growth Curve.

Turbidimetric Measurement (The Spectrophotometer)

As bacteria multiply, the broth becomes increasingly cloudy (turbid). A spectrophotometer passes a beam of light (typically at 600 nm) through the culture. The bacterial cells scatter this light. The instrument measures the unscattered light that successfully passes through, calculating the Optical Density (OD₆₀₀). Therefore, Optical Density is directly proportional to the cell concentration.

Fig 1: Spectrophotometer Principle. Bacterial cells scatter the incident light. Less light reaches the detector, resulting in a higher Optical Density (OD) reading.

3 The 4 Phases of Bacterial Growth

1. Lag Phase

Cells are acclimatizing to the new environment. They are synthesizing RNA, enzymes, and essential metabolites. Cell size increases, but cell number does not.

2. Log (Exponential) Phase

Cells undergo rapid binary fission. The population doubles at a constant, maximum rate. Cells are healthiest and most uniformly active here.

3. Stationary Phase

Nutrients are depleted and toxic waste products (like acids) accumulate. The rate of cell division exactly equals the rate of cell death. Total viable population plateaus.

4. Death (Decline) Phase

Severe nutrient exhaustion and extreme toxicity cause the death rate to exceed the division rate. The number of viable cells drops exponentially.

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Fig 2: The standard four phases of the bacterial growth curve mapped over time.

4 Materials Required

Equipment

• UV-Vis Spectrophotometer (set to 600 nm)
• Incubator Shaker (set to 37°C and 150 rpm)
• Glass or quartz cuvettes (1 cm path length)
• Sterile conical flasks (250 ml)
• Micropipettes and sterile tips

Reagents & Cultures

• Freshly autoclaved Nutrient Broth or LB Broth
• Active overnight culture of bacteria (e.g., Escherichia coli)
• Sterile uninoculated broth (used to zero/blank the machine)

5 Procedure Step-by-Step

1. Preparation: Prepare and sterilize 100 ml of nutrient broth in a 250 ml conical flask.
2. Inoculation: Aseptically inoculate the flask with 1 ml (or a loopful) of the active overnight bacterial culture.
3. Initial Reading (Time Zero): Immediately withdraw 1 ml of the culture into a cuvette. Blank the spectrophotometer using pure sterile broth, then read the OD₆₀₀ of your sample. Record this as t=0.
4. Incubation: Place the flask in the shaking incubator at 37°C. The shaking ensures constant aeration and mixing.
5. Sampling: Every 30 minutes, aseptically withdraw 1 ml of the culture, transfer to a cuvette, and measure the OD₆₀₀.
6. Continue this process for 6 to 8 hours until the OD values plateau (Stationary Phase) and slightly decrease (Death Phase).
7. Plotting: Plot a line graph with Time (hours) on the X-axis and OD₆₀₀ on the Y-axis.

6. Crucial Mathematical Formulas

Optical Density (OD)

OD = log₁₀ ( I₀ / I )

I₀ = Incident Light, I = Transmitted Light

Growth Rate Constant (k)

k = 2.303(log N₂ - log N₁) / (t₂ - t₁)

N = Cells at time t.

Generation Time (g)

g = t / n   or   g = 1 / k

Time required for population to double.

Number of Generations (n)

n = (log N - log N₀) / log 2

N = final cell number, N₀ = initial cell number.

7. Observation Data Table Example

Time (Hours)	Optical Density (OD₆₀₀)	Growth Phase Identified
0.0	0.05	Lag Phase
1.0	0.08	Lag Phase
2.0	0.25	Log Phase
4.0	0.85	Log Phase
6.0	1.20	Stationary Phase
8.0	1.10	Death Phase

Result: The bacterial growth curve was successfully plotted by evaluating the optical density. The graph clearly displayed the characteristic sigmoid curve featuring the lag, exponential, stationary, and decline phases.

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🧠 Interactive Viva Quiz

Test your knowledge! Click on the questions below to reveal the correct answers.

1. Why do we measure OD specifically at 600 nm wavelength?

✅ Answer: It prevents light absorption by the media.

At 600 nm, the yellow/amber color of the nutrient broth does not absorb the light, and the bacterial cells themselves do not absorb the light. The light is purely scattered by the physical mass of the cells. Measuring at this wavelength ensures you are measuring turbidity/scattering, not chemical absorption.

2. Does the Spectrophotometer measure living cells or dead cells?

✅ Answer: Both.

The spectrophotometer measures total biomass (turbidity). A dead, intact bacterial cell scatters light just as effectively as a living cell. This is why OD measurements might not immediately drop during the early Death Phase. To count only living cells, you must perform a viable plate count (CFU/ml).

3. Why is it important to use a "Blank" cuvette before taking readings?

✅ Answer: To cancel out background noise.

The pure, uninoculated broth and the glass/plastic of the cuvette itself will scatter a tiny amount of light. By using a blank (pure broth), you tell the machine to set this baseline to "Zero." This ensures that any OD reading you take afterward is strictly due to the bacteria, not the broth.

4. What happens if the OD reading goes above 1.0?

✅ Answer: The reading loses its linear accuracy.

Spectrophotometers are most accurate between OD 0.1 and 0.8. Above 1.0, the bacterial suspension is so dense that cells start blocking each other (shadowing effect), and some scattered light bounces back into the detector. If your reading is above 1.0, you must dilute the sample (e.g., 1:10), read it again, and multiply the result by 10.