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Bacterial Cell Division

• Focus on bacterial cell division, different stages of the bacterial growth curve, growth in nature and in the lab, bacterial growth requirements, and viable cell count

Robert Koch

• Robert Koch was a German physician from 1843 to 1910 who studied disease-causing bacteria and earned a Nobel Prize.
• Koch developed methods for cultivating bacteria, initially trying potatoes, but the nutrients were limiting.
• Solidifying liquid nutrient media with gelatin helped, but it had limitations due to its melting temperature and digestibility
• In 1882, Fannie Hess suggested agar to harden jelly

Growing Microbes

• Growing microbes is important for studying single species in pure culture, understanding medical significance, and exploring nutritional and industrial uses

Challenges of Microbial Growth

• Microorganisms are found in severe conditions, including ocean depths, volcanic vents, and polar regions
• Some scientists believe that life on other planets may resemble these organisms
• Each species grows under a limited set of environmental conditions with specific nutrient requirements

Principles of Microbial Growth

• Prokaryotic cells divide by binary fission, where one cell divides into two, then into four, and so on
• Exponential growth means the population doubles with each division
• Generation time is the time it takes for the population to double and varies among species and environmental conditions
• Microbial growth is defined as an increase in the number of cells in a population

Cell Growth Calculation Example

• Bacterial number can be calculated using the formula N₁ = N₀ × 2ⁿ
• N₁ is the number of cells at time t, N₀ is the initial cell number, and n is the number of generations.
• Example: Starting with 10 cells (N₀ = 10) of a pathogen with a 20-minute generation time (3 divisions per hour) in potato salad for 4 hours (n = 12)
• Number of cells after process = 40,960 cells

Microbial Growth in Nature

• Complex conditions in nature greatly differ from laboratory conditions, profoundly affecting microbial growth and behavior
• Cells adjust to changes by sensing chemicals and producing situation-appropriate materials
• Most microbes attach to surfaces and live in polymer-encased communities called biofilms

Biofilms

• Free cells adhere to a surface and multiply, releasing polymers that enable unrelated cells to attach and create a slimy appearance
• Nutrients and wastes pass through characteristic channels, and cells communicate via chemical signals
• Implications include dental plaque leading to tooth decay and gum disease
• Many infections involve biofilms, enhancing resistance to the immune system and antibiotics
• Accumulations in pipes and drains are caused by industrial concerns and biofilms can be hundreds of times more resistant to disinfectants
• Biofilms are helpful in bioremediation and wastewater treatment

Mixed Microbial Communities

• Microorganisms regularly grow in close association with many different species
• Interactions can be cooperative, fostering growth for species otherwise unable to survive; strict anaerobes can grow if others consume O₂
• Metabolic waste can serve as nutrient for another microbe
• Some species synthesize toxic compounds to inhibit competitors
• Some Gram-negative bacteria use a needle-like structure to inject toxic compounds into competing bacteria in a process called contact-dependent growth inhibition.

Microbial Growth in Laboratory Conditions

• Pure culture is a population of cells derived from a single cell, allowing the study of a single species where organisms may behave differently than in nature
• Only about 1% of microorganisms can be cultured
• Pure cultures are obtained using aseptic techniques to minimize accidental introduction of other organisms

Obtaining a Pure Culture

• Obtaining a pure culture requires a culture medium, a container, aseptic conditions, and a method to separate individual cells
• With correct conditions, a single cell will multiply to form a visible colony (approximately 1 million cells)
• Agar is used to solidify medium and is not destroyed by high temperatures making sterilization possible
• Agar liquefies above 95 degrees Celsius and solidifies below 45 degrees Celsius
• Growth in a Petri dish is with a two-part covered container, glass or plastic, that allows air to enter but excludes contaminants
• The agar plate is a Petri dish with agar nutrient medium

Upside Down Incubation of Petri Dishes

• Condensation droplets formed during incubation will not fall on the agar's surface, preventing contamination
• Agar containing media can be stored longer in an inverted position as the evaporation of water from media causes media dryness affecting the microbial growth.
• Inverted position decreases the rate of evaporation, resulting in proper microbial growth
• Lids may detach in handling if incubated normally, causing contamination from the air so petri dishes are labeled on the bottom for easy reading

Obtaining a Pure Culture

• The simplest and most commonly used method is a streak plate for isolating a single colony:
• The streak plate reduces the number of cells with each series of streaks allowing single cells to separate

Maintaining Stock Cultures

• A pure culture can be maintained as a stock culture, often stored in a refrigerator as an agar slant
• Cells can be frozen at -70 degrees Celsius for long-term storage after mixing with glycerol to prevent ice crystal formation and can also be freeze-dried

Phases of The Growth Curve

• Lag phase is when the number of cells doesn't increase, and the cells begin synthesizing enzymes required for growth
• Delay depends on conditions
• Log is the logarithmic/exponential phase where cells divide at a constant rate, generation time can be measured, cells are most sensitive to antibiotics, and cells produce primary metabolites like amino acids
• Stationary phase is when nutrient levels become too low to sustain growth, total numbers remain constant, some die, and others release nutrients
• Secondary metabolites (chemicals) continue to be produced but are not necessary for growth
• Endospore production begins for the bacteria that produce them
• Prolonged death phase is when the total number of viable cells decreases, cells die at a constant rate, and some fraction may survive by adapting to tolerate worsened conditions

Continuous Culture

• Open system: culture to which nutrients are continually added and waste products removed
• Chemostat provides an open system which maintains continuous growth
• Nutrient content and speed of addition can be controlled so that constant growth rate and cell density are achieved
• Produces a relatively uniform population so response to different conditions can be studied
• Cells in log phase of growth can be maintained to harvest commercially valuable products

Environmental Factors That Influence Microbial Growth

• All groups of microorganisms inhabit nearly all environments
• Some live in comfortable habitats humans favor
• Extremophiles live in harsh environments and are mostly archaea
• Major factors include temperature, atmosphere, pH, and water availability

Temperature Requirements

• Thermophiles proteins resist denaturing, stemming from amino acid sequence, number, & position of bonds
• Refrigeration slows spoilage by limiting growth; psychrophiles and psychrotrophs grow slowly
• Freezing preserves food but is not effective at killing microbes
• Different body parts differ in temperature
• M. leprae grows best in cooler regions, thus leprosy involves ears, hands, feet, fingers
• Each species has a well-defined temperature range
• Optimum growth is generally at the upper edge of tolerance
• Psychrophiles grow at -5 to 15 degrees Celsius and are found in Arctic regions
• Psychrotrophs grow at 15 to 30 degrees Celsius and cause spoilage in refrigerated foods. Mesophiles grow at 25 to 45 degrees Celsius with pathogens best at 35 to 40 degrees Celsius
• Thermophiles grow at 45 to 70 degrees Celsius

Oxygen Requirements

• Requirements are measured in a shake tube including boiling nutrient agar to drive off O₂ and solidified agar slows gas diffusion. top being aerobic and bottom anerobic
• Obligate aerobes require O₂
• Facultative aerobes use O₂, but don't require it
• Obligate anaerobes cannot use O₂
• Microaerophiles require small amounts of O₂ aerotolerant anaerobes are obligate fermenters although tolerant of O₂

Oxygen (O2) Requirements

• Reactive Oxygen Species (ROS) are produced from aerobic respiration
• Aerobic species include include superoxide (O2), hydrogen peroxide (H2O2).
• All cells must have protective mechanisms when with oxygen
• Most all organisms in oxygen produce superoxide dismutase which inactivates superoxide by converting it to O2 and H2O2
• All must produce catalase that converts (H2O2) to O2 and and H2Os, with exception of aerotolerant anaerobes - provides useful test

pH Requirements

• Bacteria survive a pH range but have an optimum
• Cells maintain a constant internal pH, typically near neutral with the pump of protons if necessary
• Most microbes are neutrophiles with a range of pH of 5 to 8 and optimal conditions near pH 7.
• Acidity is used to preserve foods
• Acidophiles grow optimally at pH below 5.5
• Acidophiles like Picrophilus oshimae thrive under pH of less than 1!

Water Availability

• All microorganisms require water for growth
• Dissolved salts, sugars make water unavailable to cell so if solute concentration is higher outside of cell, water diffuses out (osmosis)
• Salts and sugars are used to preserve food by inhibiting water availability
• Some microbes withstand or require high salt environments
• Halotolerant organisms withstand up to 10% salt environments, like Staphylococcus dry skin, Halophiles require high salt levels, like most marines at at ~ 3%

Nutritional Factors That Influence Microbial Growth

• Carbon source distinguishes different groups
• Heterotrophs - Use organic carbon
• Autotrophs - Use inorganic carbon
• Carbon fixation process converts inorganic carbon to organic form
• Nitrogen required for amino acids, nucleic acids
• Available at lowest concentration for microbial levels

Representive Functions of Major elements

Chemical & Function

• Carbon, oxygen, hydrogen - Component of amino acids, lipids, nucleic acids, and sugars
• Nitrogen - Component of amino acids and nucleic acids
• Slfur- Component of some amino acids
• Phosphorus- Component of nucleic acids, membrane lipids, and ATP
• Potssium, magnesium, calcium- Required for the functioning of certain enzymes; additional functions as well
• Iron - Part of certain enzymes

Growth Factors

• Growth factors - organic molecules that an organism cannot synthesize; must be present in environment
• Growth factor requirements reflect biosynthetic capabilities
• Most E. coli strains synthesize all cellular components from glucose and no added growth factors
• Neisseria species (fastidious) require numerous growth factors, including vitamins and amino acids
• Neisseria Species can be used to measure the quantity of added vitimins

Types Of Energy

• Two main types - Sunlight-Phototroph, Chemical compound- Chemotroph
• Sunlight- plants, algae, photosynthetic bacteria
• Mammalian cells, organic molecules- Fuingi, extract from prokaryotes
• Inorganic chemicls - Hydrogen gas, nitrates etc. in prokaryotes

Cultivating Microorganisms in the Laboratory

• Categorized in Complex VS Chemically defined medias
• Complex Medias - Variety of ingredients
• Chemically defined medias - composed of exact amounts of pure chemicals

Culture Medias

• Complex VS Chemically Defined type
• • Complex: can have highly variable ingredients
• -- Chemically defined: Pure chemicals
• Complex Example- complex E. coli is in complex media
• Chemically Defined Example -chemically defined media E. coli chemically define the medias

Selective Medias VS Other Type

• Selective- Certain Sample
• Selective media inhibit growth of certain species in a mixed sample, while allowing growth of species of interest Mannitol- salt agar (MSA) blocks growth of gram-negative bacteria and that select Staphylococcus aureus

Different Types In Culture Medias

• Allows to differentiate between groups of bacteria and identifable substances
• Alpha Clear and Beta Red Blood Cells

Providing Atmospheric Condtions

• Aerobis requires obligate and facultative
• aerobes cand requires incubation in air but the broth cultures is needed is shken to proivde maxiumation of bacteria
• many medically inportant requires Best increased carbon dioioxidee
• anaeroibs
• Can incubate in gas-tight container with chemical packet

Viable cell count

• Cell counts of growing and multipling
• Use for select and selective
• Sinc=gle cells ises to colonies
• Samples of cell diluted