Bacterial Growth Notes

Bacterial Growth

Introduction

Bacteria are prokaryotic organisms that replicate asexually through binary fission under favorable conditions.
Bacterial growth is graphically represented as the number of living cells in a population over time, known as a bacterial growth curve.
Bacterial growth is influenced by factors like oxygen, pH, temperature, light, osmotic pressure, atmospheric pressure, and moisture availability.

Mechanism of Bacterial Growth

There are four mechanisms of bacterial growth:

Binary fission
Budding
Fragmentation
Formation of sporangiospores & condiospores

Binary Fission

Bacteria reproduce asexually by transverse binary fission, where a cell divides into two identical cells after forming a transverse septum.
Bacteria increase in number through geometric progression or exponential growth, doubling with each generation (1, 2, 4, 8, etc., or $2^0, 2^1, 2^2, 2^3, …2^n$ where $n$ = number of generations).
Population size can be calculated using the formula: $Nt = (No)2^n$
- $N_t$ : final number of cells
- $N_o$ : original number of cells
- $n$ : number of divisions
Example: If $No = 10$ cells and $n = 12$ (4 hours, 20-minute generation time), then $Nt = 10 [times] 2^{12} = 10 [times] 4096 = 40960$

Budding

Some bacteria reproduce via budding, where a bud develops, enlarges, and separates into a new cell (e.g., Rhodopseudomonas acidophila).

Fragmentation

Bacteria with filamentous growth reproduce by fragmentation into small cells (e.g., Nocardia).

Formation of Sporangiospores and Condiospores

Some Streptomyces species produce spores by developing cross walls at hyphal tips, with each spore forming a new organism.

Kinetic of Bacterial Growth

Generation Time
Bacterial Growth Curve
1. Lag phase
2. Log (logarithmic or exponential) phase
3. Stationary phase
4. Decline (death) phase

Generation Time

Generation time is the time required for a cell to divide into two, varying by species and conditions.
Examples:
- E. coli doubles every 20 minutes.
- Mycobacterium tuberculosis doubles every 18 hours.
- Mycobacterium leprae doubles every 14 days.

The Bacterial Growth Curve

The bacterial growth curve is obtained by plotting the logarithm of cell number over time in a closed system.
It consists of four distinct phases:
1. Lag phase
2. Log (logarithmic or exponential) phase
3. Stationary phase
4. Decline (death) phase

The Lag Phase

The lag phase is a period of temporary inactivity immediately following inoculation.
Cells adapt physiologically to the new environment before resuming division.
Cells may grow in volume or mass, synthesizing enzymes, proteins, and RNA with high metabolic activity.
The length of the lag phase depends on inoculum size, recovery from physical damage, synthesis of essential coenzymes or division factors, and synthesis of new enzymes.
Duration: 1 hour to several days.

The Log Phase (Logarithmic or Exponential Phase)

Following the lag phase, binary fission occurs, and bacteria multiply at the fastest possible rate.
The rate of increase in cell number is geometric: 1, 2, 4, 8, etc., or $2^0, 2^1, 2^2, 2^3, …2^n$ (where $n$ = the number of generations).
Plotting log cell number versus time on a semi-log graph results in a straight line.
Bacterial division rate (generation time) is constant but varies with species, temperature, and media.
Microbes are most sensitive to adverse conditions and antibiotics during this phase.

The Stationary Phase

The stationary phase is a steady-state equilibrium where the rate of cell growth equals the rate of cell death (death rate = rate of reproduction).
Viable count remains the same; total cell count increases.
Cell death occurs due to:
- Exhaustion of nutrients and water
- Accumulation of catabolic end products
- Exhaustion of space
- Changes in oxygen concentration and pH
Bacteria produce secondary metabolites like antibiotics, and sporulation may begin.

The Decline Phase (Death Phase)

The stationary phase is followed by a die-off, where the death rate exceeds the reproduction rate (Death rate > rate of reproduction).
Cell death means cells cannot resume division when transferred to a new environment.
The number of viable cells decreases geometrically (exponentially), reversing the log phase.
Deaths are due to stationary phase factors and lytic enzymes released during lysis.

How Growth Is Detected

Qualitative Methods

On solid medium (agar), growth is visible as colonies (colonial morphology) after incubation.
In liquid medium (broth), growth is visible as turbidity (cloudiness) after incubation.
"Just barely visible" turbidity indicates a density of approximately $10^6 - 10^7$ (1 to 10 million) cells/mL.

Bacterial Growth on Solid Medium

Growth on solid medium is achieved by cultivating microorganisms on agar slopes, plates, or Roux flasks.
Large-scale growth is difficult on solid media; bacteria are typically grown in liquid culture.
Advantages of solid media:
- Easy to check for contamination (culture purity).
- Viable cell counts using spread plate or pour plate techniques.
Serial dilution is used, followed by inoculation on agar plates, assuming each cell forms a single colony.

Measurement of Cell Mass

Direct Methods

Direct physical measurement of dry weight, wet weight, or cell volume after centrifugation.
Direct chemical measurement of cell components like total nitrogen, protein, or DNA.

Indirect Methods

Indirect measurement of chemical activity (e.g., $O2$ production/consumption or $CO2$ production/consumption).
Turbidity measurement: Optical density is related to cell mass or number. This method is simple but limited to high cell concentrations (approximately $10^7$ cells per mL).
McFarland's Standards or Spectrophotometer.

Measurement of Cell Numbers

Total Count

A total count is a direct method that counts all cells, whether dead or alive, using microscopes.

Viable Count

A viable count is a direct method that counts only live cells.
Techniques include:
- Pour plating
- Spread plating
- Most probable number method

Total Versus Viable Count

How to Quantify Bacteria

Colony Forming Units (CFU)

The standard unit for CFU is the number of culturable microorganisms per 1 mL of culture (CFU/mL), determined by serial dilution and spread plating.

Total Plate Count Measurement

Pipette and spread 100 µL onto agar plates of the correct growth medium using a cell spreader.
Label plates with the Dilution Factor (DF) and incubate overnight (or as necessary). (DF = total volume of dilution/sample volume)
Choose a plate with a reasonable number of colonies (between 30 and 300 colonies).
Count how many colonies you can see on each plate.
Once you have the CFU for each plate, perform the following calculations:
Amount of colony x Volume plated (mL) x DF = Total CFU / mL
For example, if you counted 150 colonies on the plate with the dilution factor of 1:100. You plated 100 µL onto that plate:
Total DF = 0.1 mL x 1/100 (or 0.01) = 0.001
Total CFU = 150 / 0.001 = 150,000 CFU / mL

Optical Density (OD) Measurement

Optical density is determined by a spectrophotometer to estimate the number of cells present.
This technique is widely used to monitor cell growth during fermentation.
During batch culture, growth curves show four distinct phases: lag phase, exponential phase, stationary phase, and death phase.