Chapter 9: Microbial Growth

Based on Microbiology Lecture Lesson on 9/23

What is microbial growth?

What is microbial growth?

• An increase in cell size

• An increase in population

Why is microbial growth important?

To control microbial growth

• Infectious diseases

• Food spoilage

• Biotechnology

• Etc.

How do bacteria normally reproduce?

Reproduce through binary fission

How does binary fission work for bacteria?

1. Replication of DNA as the cell elongates

2. Division septum forms in the center of the cell

3. Formation of two daughter cells, each receiving a copy of the original chromosome

Generation (doubling) Time

The time it takes for the population to double through one round of binary fission

• Time varies from minutes to days

Exponential Growth

Each new fission cycle increases the population by a factor of two

What is the equation for calculating population size over time?

Nn = (N0) 2^n

What does Nn stand for?

The number of cells at any generation “n”

What does No stand for?

The initial number of cells

What does n stand for?

The number of generations (# of divisions)

Lag Phase

There is no increase in the number of living bacterial cells

Log Phase

There is an exponential increase in the number of living bacterial cells

Stationary Phase

The number of living bacterial cells plateau; the rate of cell division roughly EQUALS rate of cell death

Death or Decline Phase

The exponential decrease in the number of living bacterial cells

What is a chemostat, and how does it maintain the culture’s logarithmic phase?

• A culture vessel fitted with an opening to add nutrients (feed) and an outlet to remove contents (effluent), effectively diluting toxic wastes and dead cells

• The addition and removal of fluids is adjusted to maintain the culture in the logarithmic phase

What is Direct (Total) Cell Count?

• Counting both dead and living bacterial cells

• Counting chambers

  • Fluorescence staining techniques (Use fluorescence dyes)

• Electronic Counters

What is Direct (Viable) Cell Count?

• Counting only the living bacterial cells

• Plating Methods:

  • Spread Plate

  • Pour Plate

• Membrane filtration method

Serial Dilution

Diluting a fixed volume of cells mixed with dilution solution using previous dilution as an inoculum.

• What is the density of microorganisms in this sample?

• The liquid gets lights in color

• The first sample has all colonies merged (TOO NUMEROUS)

Pour Plate Method

1. Bacterial sample mixed with warm agar

2. Sample poured onto a sterile plate

3. Sample swirled to mix, allowed to solidify

4. Plate inoculated until bacterial colonies grow

• The resulting colonies are counted and provided an estimate of the number of cells in the original volume sampled

Spread Plate Method

1. Sample (0.1) poured onto a solid medium

2. Spread the sample evenly over the surface

3. Plate incubated until bacterial colonies grow on the surface of the medium

• The sample is poured onto solid agar

• The resulting colonies are counted and provide an estimate of the number of cells in the original volume samples

What are other examples of Indirect Cell Counts?

• Turbidity/Cloudiness: Look at the cloudiness of the culture

• Metabolic Activity

• Dry Weight: Keep drying the sample until there is a constant weight

What are the alternative patterns of cell division?

• NOT COMMON

• Fragmentation: involves the breaking down of the parent organism into multiple parts, each part developing into a complete organism. This process does not require genetic recombination, making it faster than sexual reproduction

• Budding: a small bud forms at one end of the mother cell or on filaments called prosthecae. As growth proceeds, the size of the mother cell remains about constant, but the bud enlarges. When the bud is about the same size as the mother cell, it separates.

What are the environmental factors that influence microbes?

• Oxygen Requirements

• Temperature

• pH

• Osmotic Pressure

• Barometric Pressure

Aerobes

Microorganisms that utilize oxygen (and can detoxify toxic products)

• NEED oxygen to live

Anaerobes

Do not utilize oxygen

• Don’t have ENZYMES to detoxify OXYGEN

Obligate Aerobes

Microorganisms that cannot grow without oxygen

Obligate Anaerobes

Microorganisms that cannot survive in an oxygen-rich environment

Faculative Anaerobes

Microorganisms that uses oxygen but can grow in its absence

Aerotolerant Anaerobes

Microorganisms that do not use oxygen but can grow in its presence

Microaerophiles

Microorganisms that require only a small amount of oxygen

What toxic products could oxygen be transformed as?

• singlet oxygen

• superoxide ion

• hydrogen peroxide

• hydroxyl radicals

What enzymes in most cells neutralize toxic products?

• Superoxide dismutase

• Catalase: Made by peroxisomes

What is the bubbles on a microscope slides an indicator of?

• Sample is an aerobic microorganism

• Catalase enzymes is present

What if a microbe is not capable of dealing with toxic oxygen?

The microbe is forced to live in an oxygen-free environment!

How do you culture microbes that require oxygen?

• Use stab cultures: By using an inoculating needle to stab the nutrient agar on a test tube

• By reducing media: Combining the media with oxygen, then removing it from the medium

  Ex: Thioglycolate Broth

How do you culture microbes that require carbon dioxide?

• By increasing the amount of CO2

• Use Anaerobic Jar: Tool that is used to grow anaerobic microorganisms

• Products inside jar include H2, CO2, and water vapor

• Alternative to Anaerobic Jar: Candle Jar

Canophiles

Microorganisms that prefer high concentrations of CO2 (3-10% CO2)

Ex: Neisseria and Streptococcus pneumoniae

Acidophiles

Microorganisms that have optimal growth at pH values near three

Neutrophiles

Microorganisms that have optimal growth at pH values close to near-neutral pH (center curve)

Alkaliphiles

Microorganisms that have optimal growth at pH values above 9

Why does acidic food last longer?

Most microbes that cause food spoilage grow best at a near neutral pH and do not tolerate acidity well

What are the three distinct cardinal temperatures that organisms exhibit?

1. Minimum

2. Maximum

3. Optimum

Psychrophiles

Grows at 0 degrees Celsius; optimum temperature < 15 degrees Celsius

Mesopiles

Grows at the optimum temperature between 20-45 degrees Celsius (perfect for the human body!)

Thermophiles

Grows at the optimum temperature of 50 degrees Celsius to a maximum of 80 degrees Celsius

Hyperthermophiles

Growth temperature range from 80 degrees Celsius to a maximum of 110 degrees Celsius

What conditions do most microbes exist under?

hypotonic or isotonic conditions

Halophiles

Microorganisms that require a high concentration of salt

Osmotolerant

Microoganisms that do not require a high concentration of solute but can tolerate it when it occurs

Barophiles

Microorganisms that can survive under extreme pressure and will rupture if exposed to normal atmospheric pressure

Chemically Defined Media

The exact chemical composition of the medium is known

Complex Media

The precise chemical composition of the medium is not known

Selective Media

Suppress unwanted microbe and encourage desired microbes

• Ex: Blood agar, McConkey agar, Mannitol salt agar (MSA), Eosin methylene blue agar (EMB)

Differential Media

Make it easy to distinguish colonies of different microbes

• Ex: McConkey agar, MSA, Eosin methylene blue agar (EMB)

Enriched media

Encourages growth of desired microbes

• Ex: Blood agar and chocolate agar