Question 1

What are the four phases of a bacterial growth curve?

Accepted Answer

A typical bacterial growth curve progresses through four distinct phases. Lag phase is when bacteria adjust to their environment before rapid growth begins. Exponential phase follows, characterized by rapid cell division and doubling. Stationary phase occurs when growth plateaus due to nutrient depletion. Finally, death phase involves cell lysis from severe nutrient limitation.

Question 2

How do colony forming units differ from optical density measurements?

Accepted Answer

Colony forming units (CFUs) provide direct bacterial cell counts through serial dilution and plate counting, yielding precise CFU/mL values. Optical density measures light absorbance at 600 nanometers (OD600) using a spectrophotometer, offering instant results with fewer reagents. While OD600 is faster and more convenient, CFUs are more accurate, especially at higher cell densities.

Question 3

Why is the 30-300 colony range important when counting plates?

Accepted Answer

The 30-300 colony range minimizes counting error when calculating CFU/mL. Plates with fewer than 30 colonies show high stochastic variation, leading to unreliable estimates. Plates exceeding 300 colonies suffer from crowding and overlapping, causing underestimation. Using plates within this optimal range ensures accurate bacterial enumeration for each time point.

Question 4

What is doubling time and how is it calculated?

Accepted Answer

Doubling time is the fastest rate at which bacteria reproduce under specific growth conditions, measured during the exponential phase. To calculate it, identify two time points with the steepest slope, then compute the change in time and change in generations using the bacterial counts at each point. Dividing time change by generation change yields doubling time, allowing comparison of growth conditions.

Question 5

How does a standard curve relate optical density to colony forming units?

Accepted Answer

A standard curve plots CFU/mL against OD600 on a linear scale for readings at or below 1.0 OD600. A linear regression equation (Y = MX + B) is generated, where M is the slope and B is the y-intercept. This relationship allows researchers to estimate CFU/mL from OD600 measurements in future experiments, saving time and materials while maintaining accuracy.

Question 6

What preparation steps are necessary before measuring bacterial growth?

Accepted Answer

First, prepare LB broth and agar media in separate bottles, then sterilize in an autoclave at 121°C for 35 minutes. Cool agar to 50°C and pour into Petri dishes. Using pure cultures and streak plating isolation of single bacterial colonies, inoculate liquid media overnight at 37°C with shaking. Finally, inoculate a sterile flask with a 1:1000 volume of the overnight culture to begin growth curve measurements.

Question 7

Why does the relationship between OD600 and CFU/mL deteriorate at higher optical densities?

Accepted Answer

The OD600-to-CFU relationship becomes less accurate above 1.0 OD600 because bacterial cells begin changing shape and accumulating extracellular products in the media. These changes influence light absorbance readings independently of actual cell count, causing the spectrophotometer measurement to diverge from true bacterial enumeration. This error becomes pronounced during stationary and death phases.

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