Microbial Growth: Temperature & Chemical Needs

• Microbial growth refers to an increase in cell number, not cell size
• Studying microbial growth is essential in various fields, including medicine, food science, and biotechnology
• Binary fission is the primary mode of reproduction in bacteria

Requirements for Growth

• Microbes require both physical and chemical elements for growth
• Physical requirements include temperature, pH, and osmotic pressure
• Chemical requirements include carbon, nitrogen, sulfur, phosphorus, trace elements, oxygen, and organic growth factors

Temperature

• Microbes are classified into three primary groups based on their optimal growth temperatures: psychrophiles (cold-loving), mesophiles (moderate-temperature-loving), and thermophiles (heat-loving)
• Psychrophiles grow best at low temperatures (0-15°C)
• Mesophiles grow best at moderate temperatures (25-45°C); most human pathogens fall into this category
• Thermophiles grow best at high temperatures (45-80°C)
• Hyperthermophiles grow at very high temperatures (above 80°C)

pH

• Most bacteria grow best at a neutral pH (6.5-7.5)
• Acidophiles grow best in acidic environments
• Alkaliphiles grow best in alkaline environments

Osmotic Pressure

• Microbes require water for growth, and osmotic pressure affects water availability
• Hypertonic solutions (high solute concentrations) can inhibit microbial growth by causing plasmolysis (water loss from the cell)
• Halophiles can tolerate high salt concentrations
• Obligate halophiles require high salt concentrations for growth

Chemical Requirements: Carbon

• Carbon is the structural backbone of organic molecules
• Chemoheterotrophs obtain carbon from organic materials
• Autotrophs obtain carbon from carbon dioxide

Chemical Requirements: Nitrogen, Sulfur, and Phosphorus

• Nitrogen is a component of amino acids, proteins, DNA, and RNA
• Most bacteria obtain nitrogen from the decomposition of proteins; some can use ammonium ions or nitrates
• Nitrogen fixation is the process of converting nitrogen gas into ammonia
• Sulfur is used to synthesize sulfur-containing amino acids and vitamins
• Phosphorus is used in DNA, RNA, and ATP synthesis

Chemical Requirements: Trace Elements

• Trace elements, such as iron, copper, and zinc, are required in small amounts
• They are typically used as enzyme cofactors

Chemical Requirements: Oxygen

• Oxygen can be both beneficial and toxic to microbes
• Obligate aerobes require oxygen for growth
• Facultative anaerobes can grow with or without oxygen
• Obligate anaerobes cannot grow in the presence of oxygen
• Aerotolerant anaerobes can tolerate oxygen but do not use it for growth
• Microaerophiles require low concentrations of oxygen

Toxic Forms of Oxygen

• Singlet oxygen (¹O₂⁻) is highly reactive
• Superoxide free radicals (O₂⁻) are neutralized by superoxide dismutase
• Peroxide anion (O₂²⁻) is neutralized by catalase or peroxidase
• Hydroxyl radical (OH•) is highly reactive

Organic Growth Factors

• Organic growth factors are essential organic compounds that an organism cannot synthesize
• Examples include vitamins, amino acids, purines, and pyrimidines

Culture Media

• Culture media are used to grow microorganisms in the laboratory
• Media must provide all the nutrients required for growth

Agar

• Agar is a complex polysaccharide derived from marine algae
• It is used as a solidifying agent in culture media
• Agar is not metabolized by most bacteria

Chemically Defined Media

• Chemically defined media have a precisely known chemical composition

Complex Media

• Complex media contain extracts and digests of yeasts, meat, or plants
• The exact chemical composition is not known
• Nutrient broth and tryptic soy agar (TSA) are examples

Anaerobic Growth Media

• Anaerobic growth media are used to culture obligate anaerobes
• Reducing media contain chemicals that combine with oxygen, depleting it
• Petri plates can be incubated in anaerobic jars or chambers

Special Culture Techniques

• Some bacteria, such as Mycobacterium leprae, have never been grown on artificial media
• Tissue cultures or animal hosts may be required

Selective Media

• Selective media suppress the growth of unwanted bacteria and encourage the growth of desired microbes
• Example: Mannitol Salt Agar (MSA) allows for the growth of Staphylococci spp. while inhibiting other bacteria

Differential Media

• Differential media distinguish between different groups of microorganisms based on their metabolic activities
• Blood agar differentiates bacteria based on their ability to hemolyze red blood cells
• MacConkey agar differentiates between lactose fermenters and non-fermenters

Enrichment Culture

• Enrichment culture is used to encourage the growth of a desired microbe that is present in small numbers
• The medium and incubation conditions are designed to favor the growth of the desired microbe

Obtaining Pure Cultures

• A pure culture contains only one species or strain
• The streak plate method is commonly used to isolate pure cultures

Preserving Bacterial Cultures

• Refrigeration can be used for short-term storage
• Deep-freezing involves storing cultures at -50°C to -95°C
• Lyophilization (freeze-drying) involves removing water from the culture and sealing it under vacuum

Bacterial Growth

• Bacteria reproduce by binary fission, a process of cell division that results in two identical daughter cells
• Some bacteria reproduce by budding, fragmentation, or spore formation

Binary Fission

• The bacterial cell elongates and duplicates its chromosome
• The cell wall and plasma membrane invaginate
• A cross-wall forms, completely separating the two DNA copies
• The cells separate

Generation Time

• Generation time is the time required for a cell to divide (or the population to double)
• The generation time varies depending on the species and environmental conditions

Bacterial Growth Curve

• The bacterial growth curve illustrates the growth of a bacterial population over time
• It consists of four phases: lag phase, log phase, stationary phase, and death phase

Lag Phase

• During the lag phase, cells are adjusting to their environment and preparing for growth
• There is little or no cell division

Log Phase

• During the log phase (exponential growth phase), cells divide rapidly and exponentially
• The population doubles with each generation time
• Bacteria are most susceptible to antibiotics during this phase

Stationary Phase

• During the stationary phase, the rate of cell division equals the rate of cell death
• Nutrient depletion and accumulation of toxic wastes contribute to this phase

Death Phase

• During the death phase (decline phase), the rate of cell death exceeds the rate of cell division
• The population declines exponentially

Direct Measurement of Microbial Growth

• Direct methods involve counting cells
• Plate counts, filtration, microscopic counts, and electronic counters are examples

Plate Counts

• Plate counts involve diluting a sample and plating it on agar
• After incubation, the number of colonies is counted, and the original cell density is calculated
• Plate counts assume that each colony arises from a single cell (or CFU)

Serial Dilution

• Serial dilution is used to reduce the number of cells to a countable range
• The sample is diluted in a series of steps, and aliquots are plated

Filtration

• Filtration is used to count bacteria in dilute solutions
• The sample is passed through a filter, which traps the bacteria
• The filter is then placed on agar, and the bacteria grow into colonies

Microscopic Counts

• Microscopic counts involve counting cells directly under a microscope
• A Petroff-Hausser cell counter is used
• Microscopic counts do not distinguish between live and dead cells

Electronic Counters

• Electronic counters, such as Coulter counters, are used to count cells
• Cells pass through a narrow channel, and each cell is counted electronically
• Electronic counters do not distinguish between live and dead cells

Indirect Methods of Measuring Microbial Growth

• Indirect methods estimate cell density without directly counting cells
• Turbidity, metabolic activity, and dry weight are examples

Turbidity

• Turbidity measures the cloudiness of a liquid culture
• A spectrophotometer is used to measure the amount of light that passes through the culture
• Higher turbidity indicates higher cell density

Metabolic Activity

• Metabolic activity measures the rate of metabolic product formation or substrate utilization
• This can be used to estimate cell density

Dry Weight

• Dry weight involves weighing the dry mass of a culture after removing the water
• This method is useful for filamentous organisms

Sepsis

• Sepsis refers to microbial contamination

Asepsis

• Asepsis is the absence of significant contamination

Sterilization

• Sterilization is the removal or destruction of all microorganisms
• Autoclaving and filtration are examples of sterilization methods

Commercial Sterilization

• Commercial sterilization is aimed at killing Clostridium botulinum endospores from canned goods

Disinfection

• Disinfection is the destruction of vegetative pathogens on inanimate objects

Antisepsis

• Antisepsis is the destruction of vegetative pathogens on living tissue

Degerming

• Degerming is the mechanical removal of microbes from a limited area

Sanitization

• Sanitization is the treatment to lower microbial counts on eating and drinking utensils to safe levels
• High-temperature washing or chemical disinfectant use are examples

Biocide or Germicide

• Biocide or germicide refers to a substance that kills microorganisms

Bacteriostasis

• Bacteriostasis refers to a substance that inhibits bacterial growth

Rate of Microbial Death

• Microbial death occurs at a constant rate when exposed to a lethal agent
• Factors influencing the effectiveness of antimicrobial treatment include the number of microbes, environmental influences, time of exposure, and microbial characteristics

Actions of Microbial Control Agents

• Microbial control agents can damage the cell wall, alter membrane permeability, or damage proteins and nucleic acids

Physical Methods of Microbial Control: Heat

• Heat is a common method of microbial control
• Moist heat is more effective than dry heat
• Thermal death point (TDP) is the lowest temperature at which all cells in a culture are killed in 10 minutes
• Thermal death time (TDT) is the minimal time needed to kill all cells in a culture at a given temperature
• Decimal reduction time (DRT or D value) is the time required to kill 90% of the microbial population at a given temperature

Moist Heat Sterilization

• Boiling kills vegetative cells and some viruses within 10 minutes
• Autoclaving uses steam under pressure to sterilize materials

Pasteurization

• Pasteurization is the use of heat to reduce spoilage organisms and pathogens in food and beverages
• High-temperature short-time (HTST) pasteurization uses 72°C for 15 seconds
• Ultra-high-temperature (UHT) pasteurization uses 140°C for a few seconds

Dry Heat Sterilization

• Dry heat kills by oxidation
• Examples include flaming, incineration, and hot-air sterilization

Filtration

• Filtration is used to remove microbes from liquids or air
• High-efficiency particulate air (HEPA) filters are used in ventilation systems
• Membrane filters are used to filter liquids

Low Temperature

• Low temperatures inhibit microbial growth
• Refrigeration slows the growth of most microbes
• Freezing can preserve cultures but does not sterilize

High Pressure

• High pressure denatures proteins

Desiccation

• Desiccation (drying) inhibits microbial growth

Osmotic Pressure

• High concentrations of salts or sugars create a hypertonic environment, inhibiting microbial growth

Radiation

• Radiation can be used to control microbial growth
• Ionizing radiation (gamma rays, X-rays) damages DNA
• Nonionizing radiation (UV light) damages DNA

Principles of Effective Disinfection

• Concentration of the disinfectant
• Organic matter
• pH
• Time

Types of Disinfectants

• Phenol and Phenolics: Disrupt plasma membranes, denature proteins
• Bisphenols: Disrupt plasma membranes
• Biguanides: Disrupt plasma membranes
• Halogens: Oxidizing agents
• Alcohols: Denature proteins, dissolve lipids
• Heavy Metals: Denature proteins
• Surface-Active Agents (Surfactants): Decrease surface tension
• Chemical Food Preservatives: Inhibit metabolism
• Aldehydes: Denature proteins
• Gaseous Sterilants: Denature proteins
• Peroxygens: Oxidizing agents

Evaluation of Disinfectants

• Use-dilution test determines the effectiveness of a disinfectant against specific microorganisms
• Disk-diffusion method evaluates the effectiveness of chemical agents

Explore microbial growth essentials, focusing on physical factors like temperature, pH, and osmotic pressure, and chemical needs, including carbon, nitrogen, and oxygen. Learn how these conditions influence microbial classification and growth patterns. Understand the role of binary fission in bacterial reproduction.