Culturing Microorganisms and Measuring Their Growth

Culture Media

Culture media are nutrient solutions used to grow microbes in the laboratory. The choice of media is essential for supporting the growth of specific microorganisms and for differentiating between them. Media are typically sterilized in an autoclave to prevent contamination.

• Defined media: Exact chemical composition is known. Used for precise studies of microbial metabolism.

• Complex media: Composed of digests of microbial, animal, or plant products (e.g., yeast and meat extracts). Composition varies slightly from batch to batch.

• Enriched media: Complex media supplemented with highly nutritious substances (e.g., serum, blood) to support growth of fastidious microorganisms.

• Selective media: Contains compounds that selectively inhibit growth of some microbes but not others. Example: Mannitol Salt Agar (MSA) inhibits non-halotolerant bacteria.

• Differential media: Contains indicators (dyes or pH indicators) that detect particular metabolic reactions, resulting in color changes. Example: MSA uses phenol red to indicate mannitol fermentation.

Physical Forms of Media

Media can be solid, semi-solid, or liquid, each serving different purposes in microbiology.

• Solid media: Contains a solidifying agent (e.g., agar). Used in Petri plates and slants. Colonies form isolated masses called colony-forming units (CFUs).

• Semi-solid media: Contains less agar, used to study bacterial motility.

• Liquid media (broth): No solidifying agent, used in tubes, flasks, or bottles.

Colony Morphology and Culture Types

Colony morphology refers to visible characteristics of colonies (size, shape, color, texture, margin, elevation, opacity). These features are used to identify microorganisms and determine culture purity.

• Pure culture: Contains one species or strain.

• Mixed culture: Contains multiple species.

Quantifying Microbial Growth

Microbial growth can be measured by direct and indirect methods, each with specific advantages and limitations.

• Direct methods:

  • Wet weight (not accurate due to water content)

  • Dry weight (more accurate, 1 mg ≈ 109 cells)

  • Total nitrogen, DNA, RNA, protein quantification

• Indirect methods:

  • Consumption/production of metabolites (O2, CO2, acids)

  • Optical methods (turbidity): Light scattering is proportional to cell density. Optical density (OD) is measured spectrophotometrically and is proportional to cell number within limits.

Total Cell Counting

Total cell counting includes both viable (living) and dead cells. Methods include:

• Direct microscopic counts: Breed method (spreading sample on slide, staining, counting under microscope), Petroff–Hausser Counting Chamber (calibrated chamber).

• Electron microscopy: Used for viruses.

• Coulter Counter: Counts and sizes particles by measuring changes in electrical impedance.

Viable Cell Counting

Viable cell counting measures only living cells capable of forming colonies.

• Standard Plate Count: Assumes each bacterium grows into a single colony (CFU). Ideal range: 30–300 CFU. Serial dilution may be required.

  • Spread Plate Method: Bacteria spread on surface of plate.

  • Pour-Plate Method: Bacteria mixed into dish with agar.

• Most Probable Number (MPN): Used for liquid samples. Dilution series inoculated into triplicate tubes, growth evaluated, MPN determined from table.

• Membrane Filtration: Used for liquid samples with low cell numbers.

Culturing Anaerobic Microorganisms

Anaerobic microorganisms require environments devoid of oxygen. Reducing media chemically remove O2 using agents like sodium thioglycolate. Oxygen indicators (e.g., resazurin) are used to confirm anaerobic conditions. Anaerobic jars, chambers, or OxyPlate systems are used for incubation.

Control of Microbial Growth

General Principles

Controlling microbial growth is essential in clinical, laboratory, and industrial settings. The level of cleanliness required depends on the item's use and the infectious agent.

• Disinfection: Reduction of microbial load, including pathogens.

• Sanitization: Reduction of microbial load to safe public health levels.

• Sterilization: Destruction of all vegetative cells, endospores, and viruses.

• Antiseptic: Reduction of microbial load on living tissue using antimicrobial chemicals.

• Degerming: Reduction of microbial load with scrubbing and mild chemicals (e.g., soap, alcohol swab).

Microbial Death Curve

Bacteria die at a constant rate, which is best represented on a logarithmic scale. The decimal reduction time (D-value) is the time required for a 1-log (90%) reduction in the microbial population.

• Thermal death time: Time to kill all cells at a given temperature.

• Thermal death point (TDP): Lowest temperature at which all cells in a liquid culture are killed in 10 minutes.

Physical Agents for Controlling Microbial Growth

Temperature (Heat)

Heat is a common method for controlling microbial growth. It can be applied as dry or moist heat.

• Dry heat: Kills by oxidation (hot air, oven, incineration, flaming).

• Moist heat: Kills by denaturing proteins/enzymes (boiling, pasteurization, autoclaving).

• Pasteurization: Reduces microbial load but does not sterilize.

• Autoclave: Uses steam under pressure (121°C, 15 PSI, 10–15 min) to achieve sterilization.

Refrigeration and Freezing

Low temperatures are bacteriostatic, slowing or stopping microbial growth.

• Refrigeration: Slows microbial metabolism (0–7°C).

• Freezing: Stops growth, may kill some organisms (–2°C to –7°C).

Filtration

Filtration is used to sterilize thermolabile compounds and remove microbes from air or liquids.

• HEPA filter: Used for air filtration.

• Nitrocellulose membrane filter: Used for liquid filtration (pore size 0.2 μm).

Radiation

Radiation is used to control microbial growth by damaging DNA and cellular structures.

• UV radiation (270 nm): Non-penetrating, induces thymine dimers, bactericidal for surfaces.

• Ionizing radiation (X-rays, gamma rays): Penetrates tissues, produces reactive ions, destroys DNA.

• D10 value: Amount of radiation needed to reduce population/activity 10-fold.

Chemical Agents for Controlling Microbial Growth

Chemical agents act by various mechanisms to kill or inhibit microbes.

• Alkylating agents: Modify DNA, causing replication failure.

• Surfactants: Disrupt cytoplasmic membrane.

• Oxidizing agents: Remove electrons from organic matter.

• Protein inactivation: Denaturation, precipitation, iodination.

Effects of Antimicrobial Agents

Antimicrobial agents can be classified based on their effects:

• -cidal: Kills microbes (bactericidal, fungicidal, viricidal).

• -static: Inhibits growth; removal of agent allows growth to resume (bacteriostatic, fungistatic, viristatic).

• -lytic: Causes cell lysis (bacteriolytic).

• MBC: Minimum bactericidal concentration.

• MIC: Minimum inhibitory concentration.

Effectiveness of Antimicrobial Agents

Several factors influence the effectiveness of antimicrobial agents:

• Population size

• Microbial characteristics (vegetative cells vs spores, viral envelopes, Mycobacteria, Gram-positive vs Gram-negative)

• Time of exposure

• Environmental conditions (pH, temperature, presence of organic matter, biofilms)

• Mode of action and dosage

Assaying Antimicrobial Activity

Assays are used to determine the efficacy of antimicrobial agents.

• Phenol coefficient: Comparison against pure phenol (historical method).

• Minimum inhibitory concentration (MIC): Minimum amount required to inhibit growth.

• Dilution method: Serial dilution of agent, inoculation, and observation of growth.

• Diffusion method (Kirby-Bauer test): Discs with antimicrobial agents placed on inoculated agar; zone of inhibition measured.

Table: Resistance of Microbial Types to Antimicrobial Agents