Microbial Growth: Temperature

• Microbial growth requirements are categorized as physical and chemical

Physical Requirements

• Temperature, pH, and osmotic pressure are crucial physical factors
• Chemical components include carbon, nitrogen, sulfur, phosphorus, trace elements (Fe/Zn), and oxygen

Temperature

• Each microbe has a temperature range where it can grow/survive, spanning about 30°C
• The minimum temperature is the lowest that supports growth
• The optimum temperature is preferred and best supports growth
• The maximum temperature is the highest that supports growth

Bacteria Grouped by Temperature Range

• Psychrophiles are cold-loving microbes that grow between -5°C and +15°C; killed at 20°C; found in deep ocean/polar environments; no issues with food preservation
• Psychrotrophs have a very broad temperature range, from min(0) to max(~35°C) with optimal temperature ~15-30°C; cause food spoilage in fridge
• Mesophiles thrive in moderate temperatures ~10-45°C, with an optimal temperature of 30-37°C; most bacteria and pathogens grow in this range
• Thermophiles have a temperature range of 45-70°C, with an optimal temperature ~60°C; cannot cause disease in the human body
• Hyperthermophiles have a temperature range of 65-110°C; limited to very few places on earth with such high water temperatures (e.g., deep ocean vents)

Food Safety

• Hot and cold temperatures control bacterial populations
• Heat kills mesophiles and psychrotrophs on food products, like cooking
• Cold temperatures slow the growth rate of microorganisms
• Only psychrotrophs grow in a refrigerator, but very slowly, other microorganisms are not able to grow

pH

• pH measures the acidity or alkalinity of a substance, on a scale of 0-14
• Substances with pH below 7 are acidic, pH equal to 7 are neutral, and pH above 7 are alkaline
• Most bacteria prefer a neutral pH
• Acidophiles grow at a very low pH
• Alkalophiles grow at a very high pH
• Neutrophiles grow at pH levels between 5 and 8, with an optimum pH of ~7

Osmotic Pressure

• Osmosis: solvent molecules go across a membrane
• Water moves from low to high concentration
• The direction of water movement depends on the surrounding hypertonic, hypotonic, or isotonic conditions

Tonicity

• Hypertonic solutions have a high solute concentration; when a cell is placed in a hypertonic solution, water rushes out, cell shrinks or dries up
• Hypotonic solutions have a low solute concentration; when a cell is placed in a hypotonic solution, water rushes in and cell bursts
• Isotonic solutions are of equal concentration, there is no net movement of water

Osmotic Pressure in Food Preservation

• Osmotic pressure is important for food preservation (e.g., salted fish, honey)
• Some bacteria, called extreme halophiles, adapt to high salt concentrations of 30% NaCl
• Blood has a salt concentration of ~0.9%, which is too low to inhibit most microorganism growth

Chemical Requirements

• Carbon is the backbone of all organic molecules
• Heterotrophs obtain carbon from organic matter (sugars, proteins, lipids)
• Autotrophs obtain carbon from inorganic matter (carbon dioxide, CO2)
• Nitrogen, sulfur, and phosphorus are required in smaller amounts than carbon for synthesis of cellular material and enzyme function (e.g., protein, nucleic acid, ATP)
• Trace elements such as zinc (Zn) and copper (Cu) are needed in very small amounts and are critical for enzyme function

Oxygen

• Oxygen can be extremely toxic
• Microorganisms are classified into five groups by oxygen use/tolerance
• Obligate aerobes require oxygen for cellular respiration
• Facultative anaerobes can use oxygen for cellular respiration, but also grow well in anaerobic environments
• Obligate anaerobes cannot use oxygen for cellular respiration, they are killed by oxygen presence
• Microaerophiles require oxygen in very low amounts, becoming inhibited or killed by higher concentrations
• Aerotolerant anaerobes cannot use oxygen for cellular respiration but are not killed by its presence

Types of Culture

• Continuous growth occurs when microbes multiply continuously in a culture
• Inoculum is when microbes are introduced into a culture

Culture Types

• Batch cultures are liquid media where, once started, no nutrients are added and bacteria begin to die as nutrients are used up, so the amount of nutrients is the limiting component
• Continuous cultures are open systems where nutrients are continually added, and waste is continually removed which supports indefinite growth
• Solid media allows growth of colonies of densely packed cells, solid media contains all nutrients required, with agar as a solidifying agent, derived from marine algae to isolate pure cultures forming a huge population of cells

Culture Medium

• Nutrients are prepared for microbial growth
• Bacterial cultures can be pure grown in the lab
• Mediums that are chemically defined or undefined must be sterile prior to inoculation
• A chemically undefined medium contains unknown components, example is media containing yeast extract
• A chemically defined media has all known components, example is media containing known quantities of salts and sugars

Selective vs Differential Media

• Selective media suppress the growth of unwanted organisms and promote growth of desired organisms
• Bismuth Sulfite Agar is used to culture Salmonella typhi by inhibiting gram-positive and other gram-negative bacteria

Differential Media

• Used to distinguish between different types of bacteria, allows all types of bacteria to grow
• In this type of media colonies look different on the plate
• Blood Agar is used to culture Streptococcus pyogenes and other bacteria that lyse and eat red blood cells

Selective and Differential Media

• MacConkey Agar is both selective and differential
• Bile salts and dyes inhibit non-intestinal bacteria promoting the growth of intestinal bacteria
• Lactose fermentation happens as bacteria that ferment lactose sugar produce acid products which turn the pH indicator pink, like E.coli
• Lactose non-fermenters appear white, like most intestinal pathogens

Bacterial Growth

• Bacterial growth refers to increase in bacterial cell number
• The most common process of reproducing includes binary fission

1. A cell duplicates its chromosome
2. The cell forms a cross-wall
3. 2 cells separate with one parent and a genetically identical duplicate

Generation Time

• Calculating the growth of bacteria allows determining the generation time
• Generation time is the time for a bacterial population to double in size
• The generation time for E.coli in rich media, is 20 minutes
• The generation time for M.tuberculosis in rich media, is 24 hours

Exponential Growth

• Log of the cell number constructs the bacterial growth curve exponentially

Bacterial Growth Curve

• Lag phase includes cells that are adapting, adjusting to the new media, and preparing for growth
• Exponential (log) phase includes increasing cell numbers exponentially, maximum reproduction, and is used to calculate the growth rate
• Stationary phase includes cells that have reached maximum population density, nutrients that've been depleted, where death/growth rate is equal and no increasing cell number
• Death phase includes all nutrients are exhausted, toxic waste products accumulate, and death rate exceeds growth rate

Counting Bacteria

• Direct counts are completed using a light microscope, often in a counting chamber; inaccurate because it counts live and dead cells
• Viable counts accurately show the number of living cells; performed by diluting a liquid culture; plating the dilutions onto agar plates; plates are incubated until growth

Colonies

• Each cell from the original dilution develops into a single colony called colony forming units per mL (cfu/mL)
• The assumption is that 1 cfu=1 bacterial cell