MGY277H1 Study Guide - Final Guide: Dental Caries, Aerotolerant Anaerobe, Human Microbiota
8 Jun 2018
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Unit 5
Principles of Microbial Growth
Principles of Prokaryotic Growth
- Prokaryotic cells divide by binary fission
➢ Exponential growth: population doubles each division
➢ Microbial growth defined by increase in cell numbers, not cell size
➢ Generation time: time it takes to double population
➔ Varies among species
➔ Influenced by environmental conditions – temp, availability of nutrients, etc.
➢ Exponential growth has important consequences
➔ 10 cells of food-borne pathogen (e.g. salmonella) in potato salad at a picnic can become
40 000 cells in 4 hours
➔ A single E. coli cell, through binary fission, can grow into a population of cells with the
mass of the entire Earth within 43 hours
- Growth can be calculated
➢ Nt = N0 x 2n
➔ Nt = number of cells in population at time t
➔ N0 = original number of cells in population
➔ n = number of divisions
➢ Example: pathogen in potato salad at a picnic in the sun
➔ Assume 10 cells with 20 minute generation time
➔ N0 = 10 cells in original population
➔ n = 12 (3 divisions per hour for 4 hours)
➔ Nt = 10 x 212
➔ Nt = 40 960 cells of pathogen in 4 hours!
- The power of exponential growth
➢ Rapid generation time with optimal conditions can yield huge populations quickly
➢ Remember that generation time depends on species and growth conditions
- Fast goth is’t required for a microbe to become a pathogen
➔ Mycobacteria (cause TB), for example, have generation times of over 12 hours under
ideal conditions
Prokaryotic Growth in Laboratory Conditions
- Prokaryotes grown in culture medium:
➢ Tubes/flasks of broth (most simple)
➢ Agar plates (petri dishes)
- Need a sterile environment (aseptic technique)
- Addition of cells to culture media is called inoculation
- Shaking culture improves oxygenation
- Closed systems
➢ Nutrients not renewed; wastes not removed
➢ Termed batch cultures
➔ Batch media constantly changes throughout microbial growth
➔ Starts with lots of nutrients, but soon becomes depleted
➢ Yields characteristic growth curve
- Open system required to maintain continuous growth
➢ Termed continuous culture
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➢ Nutrients added, wastes removed continuously
➢ Involves use of pump – uses chemostat
The Growth Curve
- Characterized by 5 stages:
1. Lag phase
➔ Number of cells do not increase
➔ Begin synthesizing enzymes required for growth
➔ Delay depends on conditions
2. Log or exponential phase
➔ Cells divide at constant rate
➔ Generation time measured
➔ Medically important
• Most sensitive to antibiotics
➔ Important commercially
• Production of primary metabolites
➔ Later stages
• Nutrients gradually deplete, waste accumulates
➔ Cell activities alter – preparation for starvation
➔ Secondary metabolite
• Used for purposes other than growth
• Antibiotic protection (commercial application
• Can include toxins and antibiotics
3. Stationary phase
➔ Nutrient levels too low to sustain growth
➔ Total numbers remain constant – some die, release contents; others grow
➔ Viable cells maintain properties of late log phase
➔ Variable durations – depends on environmental conditions and species
4. Death phase
➔ Total number of viable cells decrease
➔ Cells die at constant rate
➔ Exponential, but usually much slower than cell growth
5. Phase of prolonged decline
➔ Some fraction my survive death phase
➔ Adapted to tolerate worsened conditions
➔ Multiply for short period of time
➔ Use nutrients from dead cells
➔ As conditions deteriorate, most die
➔ Survivors can persist
Continuous Growth
- Chemostats can maintain continuous growth
➢ Use of a pump
➢ Continually drips fresh medium into culture in chamber
➢ Equivalent volume of culture is removed at the same rate as fresh media is introduced
➔ Contains cells, wastes, spent medium
➢ Nutrient content and speed of addition can be controlled
➔ Achieve constant growth rate and cell density
➢ Produces relatively uniform population for study
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Obtaining a Pure Culture
- Pure culture defined as population of cells derived from a single cell
➢ Allows study of single species
➢ Difficulties with studying isolated organisms:
➔ Often behave differently than in natural environments
➔ Approximately 1% of prokaryotes in the environment can currently be grown in a lab
➢ Most medically relevant bacteria and human-associated microbes can be grown in pure
culture
- Pure cultures require the use of aseptic technique
➢ Minimizes potential contamination by other organisms
- Recent advances including genomics have enabled us to cultivate far more microbes that before.
About 90% of microbes assoiated ith huas the hua ioioe hae o ee
cultured in isolation
Growth on Solid Media
- Using liquid media makes it hard to isolate bacteria
- Requirements: culture medium, container, aseptic conditions, method to separate individual
cells
➢ With correct conditions, single cell will multiply
➢ Form visible colony (~1 million cells easily visible)
➢ Agar used to solidify culture media (polysaccharide extracted from seaweed)
➔ Few microbes can degrade
➔ Not destroyed by high temperature
➔ Liquefies above 95 degC
➔ Solidifies below 45 degC
➢ Culture media contained in Petri dish
- The use of solid media enables the easy and rapid separation of microbes from one another
- Colony morphology can tell you that there are a number of different microbes present
Colony Morphology
- How colonies look and behave on certain types of media can tell you something about what
tpe of ioe ou’e lookig at
- The pheoea of destoig ed lood ells is alled heolsis
Bacterial Lawn
- If you have too many bacteria/fungi on a petri dish the colonies will grow together to give an
alost ee distiutio of ateia oe the plate sufae. This is efeed to as a la of
bacteria
- Lawns are useful for many types of experiment, just not for isolating individual microbial
species/strains
Growth on Solid Media
- Streak plate method
➢ Simplest, most commonly used method for isolating prokaryotes
➢ Spread out cells to separate
➔ Obtain cells so that individual colonies can form
- Second method – take liquid culture, dilute
➢ When dilute media applied to petri dish, you get single colonies
Maintenance of Microbial Isolates
- Maintaining stock cultures
➢ Streak-plate method yields pure cultures
➢ Cultures can be frozen at -70 degC for long-term storage
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Document Summary
Microbial growth defined by increase in cell numbers, not cell size. Generation time: time it takes to double population. Influenced by environmental conditions temp, availability of nutrients, etc. 10 cells of food-borne pathogen (e. g. salmonella) in potato salad at a picnic can become. A single e. coli cell, through binary fission, can grow into a population of cells with the mass of the entire earth within 43 hours. Nt = number of cells in population at time t. N0 = original number of cells in population. Example: pathogen in potato salad at a picnic in the sun. Assume 10 cells with 20 minute generation time. N0 = 10 cells in original population. N = 12 (3 divisions per hour for 4 hours) Nt = 40 960 cells of pathogen in 4 hours! Rapid generation time with optimal conditions can yield huge populations quickly. Remember that generation time depends on species and growth conditions.
