Chapter 7: Microbial Growth and Decontamination

Chapter Learning Outcomes

This chapter introduces the fundamental concepts of microbial growth and laboratory methods for handling microbes. Students will learn to:

• Explain laboratory methods used in microbiology, including techniques for growing, isolating, and counting microbes.

• Describe and illustrate the processes of microbial growth and metabolism.

Microbial Growth Basics

Dynamic and Complex Growth in Nature

Microbial growth refers to the process of cell division that produces new (daughter) cells, increasing the total cell population. Most of our understanding of microbial growth comes from laboratory studies, although only about 1% of bacterial species can be cultured in the lab.

• Microbial growth: Cell division resulting in an increase in the number of cells.

• Laboratory studies often use pure, single-species cultures, while in nature, microbes coexist with other bacteria, archaea, and eukaryotes.

• Environmental factors such as temperature, pH, and nutrient availability significantly influence microbial life, metabolism, and structure.

• Example: Escherichia coli can change from a motile bacillus to a filamentous nonmotile form during urinary tract infections.

Bacterial Growth in Nature vs. Laboratory

• In the lab, bacteria are grown as pure cultures, but in nature, they form complex communities with other organisms.

• Biofilms are a realistic representation of microbial communities, where cells communicate and collaborate for survival.

• Biofilm formation begins when free-floating (planktonic) bacteria adhere to a surface and produce a sticky polysaccharide matrix, allowing other microbes to join.

• Biofilms are common on indwelling medical devices such as catheters and heart valves.

Generation Time

Generation time is the period required for a cell to divide. This time varies by species and environmental conditions.

• Generation time: The time it takes for a microbial cell to divide and produce two daughter cells.

• Ranges from about 15 minutes (e.g., E. coli: 20 minutes) to 24 hours or more (e.g., Mycobacterium tuberculosis: 15–20 hours).

Equation for Generation Time:

Where: N = final cell number N0 = initial cell number n = number of generations

Bacterial Growth Phases

Bacterial populations in batch culture typically progress through distinct growth phases:

• Lag phase: Adaptation period; little to no cell division.

• Log (exponential) phase: Rapid cell division and population growth.

• Stationary phase: Growth rate slows as nutrients deplete and waste accumulates; cell division equals cell death.

• Death phase: Cells die at an exponential rate due to lack of nutrients and accumulation of toxic products.

Chemostat: A device used in industry to maintain cells at a specific growth phase (usually log or stationary) by continuously adding fresh medium and removing waste and excess cells.

Prokaryotic Growth Requirements

Adaptation to Environmental Conditions

Prokaryotes adapt to a variety of growth conditions, finding ecological niches based on:

• Temperature

• pH

• Salinity

• Oxygen levels

• Available nutrients

Temperature Requirements

Microbes have specific temperature ranges for growth, defined by minimum, optimum, and maximum temperatures.

• Psychrophiles: Grow at 0–20°C; found in cold environments.

• Mesophiles: Grow best at 10–50°C; most human pathogens are mesophiles.

• Thermophiles: Grow at 40–75°C; found in compost piles and hot springs.

• Hyperthermophiles: Grow at 65–120°C; often found in extreme environments like thermal vents.

• Barophiles: Thrive under high pressure, such as deep-sea environments.

pH Requirements

• Acidophiles: Grow at pH 1–5; found in sulfur hot springs and volcanic vents.

• Neutrophiles: Grow best at pH 5–8; most bacteria, including pathogens, are neutrophiles.

• Alkaliphiles: Grow at pH 9–11; found in soda lakes.

Microbes often maintain a near-neutral cytoplasmic pH using proton pumps to export excess protons.

Salinity Requirements

• Halophiles: Thrive in high-salt environments (up to 35% NaCl); found in the Dead Sea and Great Salt Lake.

• Facultative halophiles: Tolerate higher salt concentrations (up to 15%); e.g., Staphylococcus aureus on human skin.

High-salt conditions can cause plasmolysis (loss of water from the cell), but halophiles avoid this by accumulating compatible solutes or ions.

Oxygen Requirements

Microbes vary in their oxygen requirements and tolerance:

• Obligate aerobes: Require oxygen for growth.

• Obligate anaerobes: Cannot tolerate oxygen.

• Facultative anaerobes: Can grow with or without oxygen.

• Microaerophiles: Require low levels of oxygen.

• Aerotolerant anaerobes: Tolerate oxygen but do not use it for growth.

Oxygen can be toxic due to the formation of reactive oxygen species (ROS) such as superoxide ions () and hydrogen peroxide (). Aerobic microbes use enzymes like superoxide dismutase and catalase to detoxify ROS:

Nutritional Requirements

• About 90% of a cell's dry weight is carbon, hydrogen, oxygen, and nitrogen.

• Other essential elements: sulfur, phosphorus, potassium, sodium, calcium, magnesium, chlorine, and trace metals (e.g., copper, zinc, iron).

• Macronutrients: Needed in large amounts (e.g., carbon).

• Micronutrients: Needed in small amounts (e.g., iron).

Carbon Sources

• Autotrophs: Use inorganic carbon (CO2) via carbon fixation.

• Heterotrophs: Require organic carbon sources (e.g., sugars, proteins).

Nitrogen and Phosphorus Sources

• Key for nucleic acids (DNA/RNA) and ATP.

• Nitrogen is a major component of amino acids.

• Some microbes fix atmospheric nitrogen (N2) into ammonia.

Growth Factors

• Some microbes cannot synthesize all required organic precursors (e.g., amino acids, vitamins) and must import them—these are called growth factors.

• Organisms requiring many growth factors are termed fastidious.

Energy Sources

• Phototrophs: Use light as an energy source.

• Chemotrophs: Obtain energy by breaking down chemical compounds.

Growing, Isolating, and Counting Microbes

Culture Media

Microbes are grown on various types of media, classified by physical state, chemical composition, and function.

• Liquid media: Ideal for growing large batches of microbes.

• Solid media: Useful for isolating colonies and observing culture characteristics.

• Semisolid media: Used for motility testing.

Complex vs. Defined Media

Selective and Differential Media

• Selective media: Inhibits growth of some microbes while allowing others to grow.

• Differential media: Contains indicators to distinguish between different types of microbes based on metabolic properties.

Isolation Techniques

• Streak plate technique: Most common method for isolating pure bacterial colonies. Involves spreading a diluted sample over the surface of an agar plate to separate individual cells, which then form colonies.

Counting Microbes

Several methods are used to quantify microbial populations:

• Manual cell counting: Using a microscope and counting chamber; does not distinguish live from dead cells.

• Flow cytometry: Automated method that can distinguish live and dead cells using fluorescent tags.

• Viable plate count: Only counts living cells capable of forming colonies.

• Turbidity measurement: Uses a spectrophotometer to measure cloudiness of a culture; provides an indirect estimate of cell density.

• Dry weight: Measures the mass of dried cells.

• Biochemical activity: Measures metabolic byproducts to estimate cell numbers.

Microbial Identification

• Physical analysis: Staining and microscopy to observe cell morphology.

• Biochemical analysis: Uses media to assess metabolic properties.

• Genetic methods: Includes PCR, DNA fingerprinting, and electrophoresis for rapid identification.

Basics of Microbial Growth Reduction and Decontamination

Control Strategies

• Decontamination: Reduces or removes microbial populations to make objects safe for handling.

• Sterilization: Eliminates all microbes, including endospores; required for medical and laboratory equipment.

• Disinfection: Reduces microbial numbers; used for surfaces, cosmetics, and external medical equipment.

Physical Control Methods

Temperature

• Refrigeration and freezing: Slow microbial growth; used for food preservation.

• Autoclaving: Uses steam and pressure to sterilize media and equipment.

• Boiling: Reduces microbial numbers but does not eliminate endospores.

• Pasteurization: Uses moderate heat to eliminate pathogens in liquids (e.g., milk); does not sterilize.

• Dry heat: Incineration or hot-air ovens for sterilization (e.g., heating loops, incinerating waste).

Radiation

• Ionizing radiation (gamma rays, X-rays): Damages nucleic acids; sterilizes medical supplies and food.

• Non-ionizing radiation (UV): Causes DNA mutations; used for surface disinfection.

Filtration

• Removes microbes from liquids or air using filters; HEPA filters remove microbes and allergens from air.

Chemical Control Methods

Germicides

• Disinfectants: Used on inanimate objects.

• Antiseptics: Used on living tissue.

• Microbiocidal: Kill microbes.

• Microbiostatic: Inhibit microbial growth.

Tiers of Medical Equipment Decontamination

• Critical equipment: Contacts sterile body sites; must be sterilized.

• Semi-critical equipment: Contacts mucous membranes; should be free of most microbes.

• Noncritical equipment: Contacts intact skin; requires less stringent disinfection.

Tiers of Germicides

• Low-level: Destroy some bacteria, fungi, and viruses; not endospores.

• Intermediate-level: Destroy all bacteria (including M. tuberculosis), fungi, and viruses; not endospores.

• High-level: Destroy all microbes and endospores.

Examples of Germicides

Selection of Germicides

• Consider use, reactivity, concentration, treatment time, type of microbe, presence of organic matter, residue impact, and toxicity.

Special Considerations for Different Microbes

• Mycobacterium: Waxy cell walls; require strong disinfectants and airborne control measures.

• Endospores: Highly resistant; best eliminated by autoclaving or sporicidal chemicals.

• Viruses: Enveloped viruses are sensitive to heat and detergents; naked viruses require chlorine-based agents.

• Protozoa: Some life stages are resistant; require filtration, UV, or ozone treatments.

• Prions: Infectious proteins; require combined chemical and high-pressure autoclaving for elimination.