Microbial Growth

The Requirements for Growth

Learning Objectives

6-1: Classify microbes into five groups based on preferred temperature range.
6-2: Identify how and why the pH of culture media is controlled.
6-3: Explain the importance of osmotic pressure to microbial growth.

Physical Requirements

Temperature
- Microbes have minimum, optimum, and maximum growth temperatures.
- Minimum growth temperature: The lowest temperature at which the species will grow.
- Optimum growth temperature: The temperature at which the species grows best.
- Maximum growth temperature: The highest temperature at which growth is possible.
pH
- The acidity or alkalinity of a solution significantly affects microbial growth.
Osmotic pressure
- Microbes require water for growth, and their environment's osmotic pressure affects water availability.

Chemical Requirements

Carbon
Nitrogen, sulfur, and phosphorus
Trace elements
Oxygen
Organic growth factors

Temperature

Psychrophiles:
- Cold-loving microbes that can grow at 0°C; optimum growth is around 15°C.
- They thrive in consistently cold environments such as deep oceans and polar regions.
Psychrotrophs:
- Can grow at 0°C, with an optimum growth temperature between 20°C and 30°C.
- These microbes are often responsible for food spoilage in refrigerated conditions.
Mesophiles:
- Optimum growth occurs at temperatures between 25°C and 40°C.
- Most normal microbiota and pathogens of animals, including humans, fall into this category.
Thermophiles:
- Optimum growth temperature ranges from 50°C to 60°C.
- Commonly found in hot springs and organic compost piles where temperatures are elevated.
Hyperthermophiles (extreme thermophiles):
- Optimum growth temperature is greater than 80°C.

pH

Most bacteria prefer a neutral pH range, growing best between pH 6.5 and 7.5.
Molds and yeasts tend to favor more acidic conditions, growing optimally between pH 5 and 6.
Food preservation techniques often utilize bacterial fermentation, producing acids that inhibit the growth of other microorganisms (e.g., sauerkraut, pickles, some cheeses).
In laboratory settings, growth media may include buffers - substances that help maintain a stable pH by neutralizing acids or bases.
Acidophiles are microorganisms that thrive in acidic environments, often with a pH much lower than neutral.

Osmotic Pressure

Hypertonic environments, where the solute concentration is higher outside the cell than inside, can lead to plasmolysis.
- Plasmolysis involves the shrinkage of the cell’s cytoplasm as water moves out of the cell via osmosis.
Extreme or obligate halophiles have adapted to require high salt concentrations for growth, sometimes as high as 30% NaCl.
Facultative halophiles are more versatile and can tolerate high salt concentrations, typically ranging from 2% to 10% NaCl, but do not require it.

Chemical Requirements

Learning Objectives

6-4: Name a use for each of the four elements (carbon, nitrogen, sulfur, and phosphorus) needed in large amounts for microbial growth.
6-5: Explain how microbes are classified on the basis of oxygen requirements.
6-6: Identify ways in which aerobes avoid damage by toxic forms of oxygen.

Carbon

Carbon is the structural backbone of organic molecules, essential for all life forms.
Chemoheterotrophs rely on organic molecules not only as carbon sources but also as energy sources.
Autotrophs, in contrast, utilize $CO_2$ as their primary carbon source, converting it into organic compounds.

Nitrogen

Nitrogen is a crucial component of proteins, DNA, RNA, and ATP, playing a vital role in cellular functions.
Most bacteria obtain nitrogen by decomposing protein-containing materials.
Some bacteria can directly use $NH_4^+$ from organic material as a nitrogen source.
A few specialized bacteria are capable of nitrogen fixation, converting atmospheric $N_2$ into usable forms.

Sulfur

Sulfur is a key element in certain amino acids, as well as vitamins like thiamine and biotin.
Many bacteria acquire sulfur through the decomposition of proteins.
Some bacteria can utilize $SO_4^{2-}$ as a sulfur source.

Phosphorus

Phosphorus is essential for the synthesis of DNA, RNA, and ATP, all critical for genetic information and energy transfer.
It is also found in cellular membranes in the form of phospholipids.
$PO_4^{3-}$ serves as a common source of phosphorus for microbial growth.

Trace Elements

Trace elements are inorganic elements required in very small amounts.
These elements often act as enzyme cofactors, assisting in various enzymatic reactions within the cell.
Common trace elements include iron, copper, molybdenum, and zinc.
Often, tap water used to prepare laboratory media provides these trace elements.

Oxygen

Obligate aerobes: Require oxygen for aerobic respiration.
Facultative anaerobes: Can grow in the presence or absence of oxygen; when oxygen is absent, they switch to fermentation or anaerobic respiration.
Anaerobes: Cannot use oxygen and are often harmed by its presence.
Aerotolerant anaerobes: Tolerate the presence of oxygen but cannot use it for growth.
Microaerophiles: Require oxygen but at concentrations lower than those found in the atmosphere.

Toxic Forms of Oxygen

Singlet oxygen ( $^1O_2^*$ ): Oxygen boosted to a higher energy state and is extremely reactive.
Superoxide radicals ( $O2^-$ ): Highly reactive and toxic; superoxide dismutase (SOD) neutralizes them in organisms that use oxygen. $2O2^- + 2H^+ \rightarrow H2O2 + O_2$
Peroxide anion ( $O2^{2-}$ ): A reactive form of oxygen found in hydrogen peroxide; catalase and peroxidase enzymes can neutralize it. $2H2O2 \rightarrow 2H2O + O2$ (catalase) and $H2O2 + 2H^+ \rightarrow 2H2O$ (peroxidase)
Hydroxyl radical ( $OH•$ ): The most reactive of the toxic oxygen species, damaging nearly all macromolecules.

Strategies to Detoxify Harmful Forms of Oxygen

Superoxide dismutase (SOD) catalyzes the reaction: $2O2^- + 2H^+ \rightarrow H2O2 + O2$
Catalase converts hydrogen peroxide to water and oxygen: $2H2O2 \rightarrow 2H2O + O2$
Peroxidase reduces hydrogen peroxide: $H2O2 + 2H^+ \rightarrow 2H_2O$

Organic Growth Factors

Organic compounds that an organism cannot synthesize on its own and must obtain from the environment.
Include vitamins, amino acids, purines, and pyrimidines.

Biofilms

Learning Objective

6-7: Describe the formation of biofilms and their potential for causing infection.

Biofilm Characteristics

Microbial communities consisting of one or more species of bacteria.
Formation involves the secretion of a matrix that forms a slime or hydrogel, allowing the bacteria to adhere to surfaces.
Bacteria within the biofilm communicate via quorum sensing.
- Bacteria produce and secrete an inducer (signaling chemical) that attracts other bacterial cells to the site.
Nutrients are shared among the community members.
The biofilm structure provides shelter, protecting the bacteria from harmful environmental factors, such as antibiotics and disinfectants.

Biofilm Relevance

Biofilms are prevalent in various environments, including the digestive system, sewage treatment systems, and indwelling medical devices.
- They can cause persistent clogging of pipes and contribute to equipment malfunction.
Bacteria in biofilms can be up to 1000 times more resistant to microbicides compared to their planktonic (free-living) counterparts.
Biofilms are implicated in approximately 70% of human infections.
- Common sites of biofilm-related infections include catheters, heart valves, contact lenses, and dental surfaces (causing caries).

Culture Media

Learning Objectives

6-8: Distinguish chemically defined and complex media.
6-9: Identify one use of each of the following: selective, differential, and enrichment media.
6-10: Justify the use of each of the following: anaerobic techniques, living host cells, and candle jars.
6-11: Differentiate biosafety levels 1, 2, 3, and 4.

Basics

Culture medium: A nutrient-rich preparation designed for microbial growth.
Sterile: The state of being free from living microbes and their spores.
Inoculum: The introduction of microorganisms into a culture medium to initiate growth.
Culture: The population of microbes growing in or on a culture medium.

Agar

Complex polysaccharide derived from marine algae.
Serves as a solidifying agent for culture media in Petri plates, slants, and deeps.
Microbes typically cannot metabolize agar, making it an ideal support structure.
It liquefies at 100°C.
Solidifies at approximately 40°C.

Types of Media

Chemically defined media:
- A growth medium in which the exact chemical composition is known.
- Often used for culturing fastidious organisms, which require specific growth factors.
- Fastidious organisms require many growth factors provided in their growth media.
Complex media:
- Growth media that contain extracts and digests of yeasts, meat, or plants, resulting in a variable chemical composition from batch to batch.
- Examples include nutrient broth and nutrient agar.

Anaerobic Growth Media and Methods

Reducing media:
- Specifically designed for the cultivation of anaerobic bacteria.
- Contain chemicals such as sodium thioglycolate that combine with oxygen, reducing its concentration.
- Often heated to further drive off oxygen before use.

Special Culture Techniques

Some bacteria cannot be grown on artificial laboratory media and require special culture techniques.
- Mycobacterium leprae is typically grown in armadillos.
- Rickettsia and Chlamydia are obligate intracellular parasites and are grown in tissue culture.
Capnophiles: Microorganisms that thrive in environments with higher CO2 concentrations.
- To create such environments in the laboratory:
 - Utilize $CO_2$ generating packets in enclosed containers.
 - Incubate cultures in a candle jar (achieves approximately 3% $CO2$ and about 17% $O2$ ).
 - Use a specialized $CO_2$ incubator to precisely control gas concentrations.

Selective Media

Selective media are designed to suppress the growth of unwanted microbes while encouraging the growth of desired ones.
These media contain inhibitors that prevent certain microorganisms from growing.
Examples:
- Bismuth sulfite agar is used to isolate Salmonella Typhi by inhibiting gram-positive and most gram-negative bacteria.
- Sabouraud’s dextrose agar is used to selectively grow fungi by inhibiting bacterial growth.

Differential Media

Differential media enable the differentiation of colonies of different microbes on the same plate.
These media often contain indicators that react differently based on the metabolic activities of the microorganisms.
Some media possess both selective and differential characteristics, allowing for both isolation and differentiation of specific microbes.

Enrichment Culture

Enrichment culture is used to enhance the growth of a desired microbe when it is present in very small numbers, increasing its population to detectable levels.
This technique is often applied to soil or fecal samples to identify rare organisms.
Typically, enrichment culture involves the use of a liquid medium.
The medium provides specific nutrients and environmental conditions that favor the growth of the target microbe while inhibiting the growth of others.

Biosafety Levels

BSL-1: Requires no special precautions; standard microbiology teaching labs fall into this category.
BSL-2: Requires lab coats, gloves, and eye protection.
BSL-3: Designed for working with highly infectious airborne pathogens.
- Requires biosafety cabinets to prevent airborne transmission.
- Facilities are negatively pressurized and equipped with HEPA (High-Efficiency Particulate Air) filters.
BSL-4: The most stringent level, used for working with extremely dangerous and exotic microbes.
- Requires sealed, negative pressure environments referred to as “hot zones.”
- Exhaust air is filtered twice through HEPA filters.
- Workers wear specialized “space suits” with a dedicated air supply.
- There are only a few BSL-4 labs in the United States and worldwide.

Obtaining Pure Cultures

Learning Objectives

6-12: Define pure culture.
6-13: Describe how pure cultures can be isolated by using the streak plate method.

Basics of Pure Cultures

A pure culture contains only one species or strain of microorganism.
A colony is a visible population of cells arising from a single cell, spore, or cluster of attached cells.
A colony is often referred to as a colony-forming unit (CFU).
The streak plate method is a common technique used to isolate pure cultures by diluting the sample across the surface of an agar plate.

Preserving Bacterial Cultures

Learning Objectives

6-14: Explain how microorganisms are preserved by deep-freezing and lyophilization (freeze-drying).

Preservation Methods

Deep-freezing: Cultures are rapidly cooled to temperatures between -50°C and -95°C to preserve them.
Lyophilization (freeze-drying): Cultures are frozen and then dehydrated under vacuum to remove water, allowing for long-term storage.

The Growth of Bacterial Cultures

Learning Objectives

6-15: Define bacterial growth, including binary fission.
6-16: Compare the phases of microbial growth, and describe their relation to generation time.
6-17: Explain four direct methods of measuring cell growth.
6-18: Differentiate direct and indirect methods of measuring cell growth.
6-19: Explain three indirect methods of measuring cell growth.

Bacterial Division

Bacterial growth is defined as an increase in the number of cells in a population, rather than an increase in cell size.
Binary fission: The primary method of cell division in bacteria, where one cell divides into two identical daughter cells.
Budding: An asexual reproductive process used by a few bacterial species, where a new cell grows from a small bud on the parent cell.
Conidiospores (actinomycetes): Chains of spores formed at the tips of filaments in actinomycetes, allowing for dispersal and reproduction.
Fragmentation of filaments: A process where filamentous bacteria break into smaller fragments, each capable of forming a new individual.

Generation Time

Generation time: The time required for a cell to divide, varying from about 20 minutes to 24 hours depending on the species and conditions.
Binary fission results in a doubling of the number of cells each generation, leading to exponential growth.
The total number of cells after $n$ generations can be calculated using the formula: Total number of cells = $2^n$
Growth curves are typically represented logarithmically to better visualize the exponential increase in population size.

Phases of Growth

Bacterial growth curve: A graphical representation of the change in population size over time in a closed culture.
- A small number of bacteria is inoculated into a liquid growth medium.
- The population of bacteria is counted at regular intervals.
- The logarithm of the number of bacteria (y-axis) is plotted against time (x-axis) to create the growth curve.
Phases of growth:
- Lag phase
- Log phase
- Stationary phase:
  - Bacteria approach the carrying capacity of the environment.
- Death phase

Lag Phase

During the lag phase, there is little or no immediate increase in cell number.
Cells are metabolically active, synthesizing enzymes and other molecules needed for growth; they are “tooling up” for rapid reproduction.

Log Phase (Exponential Growth Phase)

The log phase is characterized by a period of rapid reproduction and exponential increase in cell numbers.
Cells divide at a constant rate, with a minimum and constant generation time.

Stationary Phase

As resources become limited and waste products accumulate, growth slows, and the population reaches the carrying capacity.
During the stationary phase, the number of new cells produced is balanced by the number of cells that die.
Diminished nutrients and accumulating wastes inhibit further growth.

Death Phase

In the death phase, the number of deaths exceeds the production of new cells.
The population decreases logarithmically as cells die off.

Direct Measurement of Microbial Growth

Direct measurements involve physically counting microbial cells to determine population size.
Common methods:
- Plate count
- Filtration
- Most probable number (MPN) method
- Direct microscopic count

Plate Counts

Plate counts involve counting colonies on agar plates to estimate the number of viable cells in a sample.
Only plates with 30 to 300 colonies (colony-forming units, or CFUs) are considered statistically valid.
To obtain the appropriate colony count, the original inoculum must be serially diluted to reduce the number of cells plated.
Counts can be performed using either the pour plate method (bacteria mixed into molten agar) or the spread plate method (bacteria spread on the surface of solidified agar).

Filtration

Filtration is used to concentrate bacteria from dilute solutions.
A solution is passed through a filter with pores small enough to trap bacteria.
The filter is then transferred to a Petri dish containing nutrient medium, and colonies grow on the filter surface.

The Most Probable Number (MPN) Method

The most probable number (MPN) method is a statistical estimation technique used to estimate the concentration of viable microorganisms in a sample.
It involves a multiple tube test where a series of dilutions are made and inoculated into tubes of broth.
The number of positive tubes (showing growth) is counted, and the results are compared with a statistical table to determine the MPN.

Direct Microscopic Count

A known volume of a bacterial suspension is placed on a specially designed slide (e.g., Petroff-Hausser cell counter).
The average number of bacteria per viewing field is calculated by counting cells under a microscope.
The Petroff-Hausser cell counter has a defined volume, allowing for direct estimation of cell concentration.

Estimating Bacterial Numbers by Indirect Methods

Indirect methods of measuring microbial growth do not involve direct cell counts but instead rely on measurable parameters that correlate with cell density.
Common techniques include:
- Turbidity: Measurement of cloudiness (turbidity) of a liquid culture using a spectrophotometer.
- Metabolic activity: Assessing the rate of production of certain metabolic products (e.g., $CO_2$ , acid) to estimate bacterial numbers.
- Dry weight: Bacteria are filtered, dried, and weighed to determine the biomass; this method is particularly useful for filamentous organisms.