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Bacterial growth

An increase in the number of bacterial cells.

Binary fission

A process in which one bacterial cell divides into two cells.

Generation time / Doubling time

The time required for a bacterial population to double.

Growth curve

A graph showing changes in bacterial cell number or turbidity over time.

Log phase / Exponential phase

The phase in which cells multiply exponentially with a constant generation time under stable conditions.

Growth rate

The increase in cell number or a growth measurement per unit of time.

Culture

Microorganisms grown in a medium, or the process of growing them.

Culture medium / Media

Material that provides nutrients for microbial growth. Medium is singular; media is plural.

Broth

A liquid nutrient medium used to grow microorganisms.

TSB — Tryptic Soy Broth

The liquid nutrient medium used in the bacterial growth experiment.

Inoculation

Introducing microorganisms into a culture medium.

Incubation

Keeping an inoculated medium under specific conditions for a period of time.

Turbidity

The cloudiness of a liquid; an indirect indicator of bacterial concentration.

Spectrophotometer

An instrument that measures changes in light passing through a sample.

Absorbance — ABS

A measurement of light lost as it passes through a sample. Light scattering contributes to readings of bacterial suspensions.

Wavelength

The distance between corresponding points on successive waves. These experiments use light at 600 nm.

Cuvette

A small container that holds a sample inside a spectrophotometer.

Blank

A reference sample containing the medium without bacteria.

Blanking / Zeroing

Using a blank to set absorbance to 0.00 and correct for the medium’s contribution.

Enumeration

Counting or estimating the number of microorganisms in a sample.

Direct microscopic count

Counting cells directly under a microscope. Ordinary counts generally include both living and dead cells.

Counting chamber

A device with a defined volume used to count cells and calculate their concentration; an example is the Petroff-Hausser chamber.

Fluorescence microscopy

Using fluorescent stains to observe and count cells. Some staining methods distinguish living from dead cells.

Stock culture

The starting culture used to prepare dilutions.

Dilution

Reducing a sample’s concentration by adding a diluent, such as water or broth.

Serial dilution

A series of stepwise dilutions, each prepared from the previous dilution.

Twofold dilution

A dilution that reduces the concentration to one-half of the previous concentration.

Tenfold dilution

A dilution that reduces the concentration to one-tenth, or 10⁻¹, of the previous concentration.

Dilution factor

The reciprocal of the dilution fraction. A dilution fraction of 10⁻⁵ corresponds to a dilution factor of 10⁵.

Spread plate method

Spreading a measured sample across an agar surface so colonies can develop.

NA — Nutrient Agar

A solid nutrient medium used to grow bacteria.

Colony

A visible mass of microorganisms growing on a solid medium.

CFU — Colony-forming unit

A viable cell or group of cells capable of forming a colony under the culture conditions.

CFU/mL

The number of colony-forming units per milliliter of the original sample.

Plated volume

The volume of sample added to an agar plate. The slide calculation examples use 0.1 mL.

Countable plate

A plate containing a suitable number of colonies for counting. The course slides use 20–200 colonies.

Scientific notation

Expressing a number as a coefficient multiplied by a power of ten. Example: 50,000,000 = 5 × 10⁷.

UV-C

Ultraviolet radiation that can damage microbial DNA and reduce microbial survival.

Bacterial lawn

Dense, continuous bacterial growth covering an agar surface.

Control

A comparison condition used to evaluate a treatment’s effect. The UV experiment uses 0 minutes of UV exposure as its control.

Lag phase

The phase in which cells adjust to a new environment, with little or no increase in cell number.

Stationary phase

The phase in which cell production and cell death occur at approximately equal rates, so the population levels off.

Death phase / Decline phase

The phase in which viable cell numbers decrease because cells die faster than new cells are produced.

Order of bacterial growth phases

Lag phase → Log phase → Stationary phase → Death phase.

Why does bacterial growth slow down?

Nutrients become limited and waste products accumulate.

Which phase is used to determine generation time?

The log phase, when the population increases exponentially.

Axes of the growth experiment graph

X-axis: time. Y-axis: absorbance. Use a legend to distinguish the temperatures.

Advantages of direct microscopic counting

It is quick and relatively easy.

Limitations of direct microscopic counting

Only a small sample is counted, exposure may occur, and ordinary counts include both living and dead cells.

Limitations of fluorescence counting

Equipment cost, background fluorescence, and stain-dependent difficulty distinguishing living from dead cells.

Advantages of spectrophotometry

It is fast and convenient for repeated growth measurements.

Can absorbance distinguish living from dead cells?

No. Both living and dead cells can contribute to turbidity.

Does absorbance directly measure CFU/mL?

No. A calibration relating absorbance to viable plate counts is needed to estimate CFU/mL.

What does a viable plate count measure?

Cells or cell groups capable of forming colonies under the incubation conditions.

How does dilution affect colony number?

With the same plated volume, more diluted samples generally produce fewer colonies.

Dilution fraction of one step

Volume transferred ÷ total volume after mixing.

Total dilution fraction

Multiply the dilution fractions of all steps. Example: 10⁻¹ × 10⁻¹ = 10⁻².

Concentrations after successive twofold dilutions

Relative to the stock: 1/2, 1/4, 1/8, and 1/16.

CFU/mL formula

Colonies counted ÷ (dilution fraction × volume plated in mL).

Why must plated volume be included in CFU/mL calculations?

A count obtained from 0.1 mL must be converted to a concentration per 1 mL.

Calculate CFU/mL: 50 colonies, 10⁻⁵ dilution, 0.1 mL plated

50 ÷ (10⁻⁵ × 0.1) = 5 × 10⁷ CFU/mL.

Multiplying powers of ten

Add the exponents: 10ᵃ × 10ᵇ = 10ᵃ⁺ᵇ.

Dividing powers of ten

Subtract the exponents: 10ᵃ ÷ 10ᵇ = 10ᵃ⁻ᵇ.

Required unit for viable cell concentration

CFU/mL.

Temperatures compared in the growth experiment

4°C, 25°C, 37°C, 42°C, 55°C, and 65°C.

Spectrophotometer warm-up time

10 minutes, according to the slides.

Why should the cuvette sides be clean?

Fingerprints and residue can interfere with light measurements.

Reading order in the dilution experiment

Tube 4 → 3 → 2 → 1 → 0, from most dilute to most concentrated.

Organisms compared in the UV experiment

Escherichia coli and Bacillus cereus.

UV exposure times compared

0, 5, and 10 minutes.

How does UV damage bacterial DNA?

It can form pyrimidine dimers, such as thymine dimers, that interfere with DNA function.

Purpose of a bacterial lawn in the UV experiment

To provide continuous growth across the plate so the effect of UV exposure can be observed.