Bacterial Growth, Enumeration, and Genetics

Bacterial Growth and Enumeration

Bacteria divide from one to two, then to four, then to eight, demonstrating exponential growth.
This growth can be represented as 2^n, where n is the generation number.
- 2^0 = 1 (Zero generation)
- 2^1 = 2
- 2^2 = 4
- 2^3 = 8
The number of bacteria increases exponentially, following a power law, not arithmetically, but logarithmically.
The exponent corresponds to the 'generation' of the bacteria, relating to the total number if starting with one bacterium.
In fresh media, bacteria divide as rapidly as possible to outcompete others, converting nutrients into copies of themselves.
Generation Time: The time it takes for a bacterium to replicate itself. This time is constant for a given species in fresh media, varying from species to species.

When graphed as time (or generations) versus the log number of cells, bacterial growth phases typically produce a characteristic curve.
A semi-log graph (log number of cells vs. time/generation) shows a straight line during exponential growth, indicating a constant generation time, allowing for interpolation and extrapolation.
Typical Phases of a Bacterial Growth Curve:
- Lag Phase (A): Initial period after introducing bacteria into fresh media. Bacteria must synthesize necessary molecules, especially if dormant, which can take time.
- Log Phase (B): Period of exponential growth. Represented as a straight line on a semi-log graph, indicating rapid division.
- Stationary Phase (C): Occurs when nutrients become limited, or waste products accumulate. The rate of replication equals the rate of death, resulting in a plateau in cell numbers.
- Death Phase (D): Number of deaths exceeds new replications. Represented as a straight line on a semi-log graph, indicating logarithmic death.
Phases B and D are logarithmic/exponential because they appear as straight lines on a semi-log plot.

A standard laboratory method to count bacteria by diluting a sample and plating to obtain countable colonies.
Not suitable for initial lecture discussion but a common lab technique.

Spread Plate: Bacteria are spread on the surface of an agar plate using a sterile spreader (e.g., bent glass rod). Used for non-pathogens.
Pour Plate: Liquid bacterial dilution is mixed with molten agar and poured onto a sterile plate. Bacteria grow within the agar.
- Advantage: Used for pathogens. Coating pathogens in agar prevents them from aerosolizing (becoming airborne) if the plate dries or is handled outside a biosafety cabinet.

Used for samples with very low bacterial counts (e.g., 30 bacteria per 10 gallons or 30 bacteria per mL but total of 30 in a large volume).
The water sample is poured through a filter (often paper).
Bacteria are retained on the filter.
The filter paper is placed directly on nutrient agar, and the bacteria grow into colonies.
Colonies are counted, and the total count per total volume can be calculated based on the known volume of water filtered.

Purpose: Primarily used to detect fecal contamination in water.
Principle: Fecal bacteria (e.g., E. coli) do not survive long in the environment (die within about 2 weeks). Their presence indicates recent fecal contamination.
Characteristics of Fecal Bacteria: Gram-negative and perform lactose fermentation.
- Few other environmental microorganisms exhibit this combination.
Procedure:
1. A series of tubes containing lactose broth and a pH indicator are prepared.
2. Decreasing amounts of a water sample are added to sets of these tubes (e.g., 5 tubes with 10 mL, 5 tubes with 1 mL, 5 tubes with 0.1 mL).
3. Positive tubes (indicating lactose fermentation and gas production) show a color change (e.g., from yellow to red, or production of gas bubbles).
4. The number of positive tubes for each dilution is recorded (e.g., 5, 3, 1).
5. This pattern (e.g., 531) is compared to a statistical table to estimate the MPN of bacteria in the sample (e.g., confident that the number is between 34 and 250 bacteria per 100 mL).
Real-world Relevance (Water Quality Standards):
- Drinking Water: Tolerates approximately 1 fecal contaminant per 100 mL.
- Swimming Water: Tolerates up to approximately 200 fecal contaminants per 100 mL. Exceeding this often leads to beach closures.

Method: Uses a Petroff-Hausser counting chamber (or hemocytometer), which is a special slide with a grid and a known volume.
Bacteria within a specific grid area are counted, then extrapolated to calculate the number of bacteria per mL.
Problems:
- Overcount: Counts both living and dead bacteria, leading to an overestimation.
- Difficulty: Distinguishing bacteria from debris under the microscope can lead to errors.
Advantage: Provides a rapid, maximum estimate of bacterial numbers, especially useful in milk samples.

Principle: When bacteria grow in a broth, they obstruct light passing through it (absorb light), a phenomenon called turbidity.
Instrument: Spectrophotometer quantifies the amount of light absorbed or transmitted.
Calibration: A standard curve is created by previously correlating absorbance/transmittance readings with known bacterial counts (obtained via serial dilution).
Application: Once calibrated, subsequent samples' bacterial numbers can be rapidly estimated by measuring their turbidity.

Mechanism:
1. A flask containing bacterial broth has two compartments separated by a small aperture (hole) and electrodes.
2. A siphon draws broth (containing salts for conductivity) through the aperture at a constant rate (e.g., 1 mL per 20 minutes).
3. A current passes between a positive electrode in one compartment and a negative electrode in the other.
4. As a bacterium (or other cell) passes through the aperture, it temporarily increases resistance to electron flow because it is non-conductive.
5. Each registered increase in resistance is a