[BIO 120.11] Module 3 Part 3: Batch and Continuous Culture

Batch Culture

Typical cultivation system for bacteria

Fixed volume

What can we say about the volume of liquid enclosed within a container for batch cultures?

Batch Culture

Describes the growth of microbes in a fixed volume of liquid enclosed within a container

Batch Culture

Culture wherein sources of nutrients are limited or not replenished

Constantly changing environment because of nutrient consumption and waste production

What can we say about the environment within a batch culture?

Waste accumulates

What can we say about the waste in a batch culture?

Finite

What can we say about the amount of nutrients in a batch culture

Growth cannot be supported indefinitely

In batch culture, what can we say about its effect or support to bacterial growth?

FALSE

T/F: The trend of a bacterial growth curve can be seen in both arithmetic and logarithmic scales or plots

TRUE

T/F: The trend of a bacterial growth curve can not be seen in an arithmetic scale.

Logarithmic scale

The trend of the bacterial growth curve can only be seen using what kind of scale?

y = Log of numbers in bacteria, x = time in hours

What are the variables in the y and x axis of a growth curve?

Lag, Log, Stationary, Death

What are the 4 phases of bacterial growth?

Lag Phase

Period between inoculation and the onset of growth

Lag Phase

Phase of bacterial growth wherein there is an initial pause in growth

TRUE

T/F: There is no general growth during the lag phase

Flat line

What kind of line is present in the lag phase?

Subculturing into fresh culture media

When does lag phase usually happen?

Increase in cell size, No increase in cell number

Describe the following for bacteria in lag phase: cell size, cell number

Cells may be old, Cells may be depleted (ATP, essential cofactors, enzymes), Cells may be injured

What are the 3 causes of the lag phase?

Previous history of the cells (old vs. young), Growth medium (rich vs. MM), Growth conditions (optimal vs. inhibitory)

What are 3 factors affecting duration of lag phase? His MeCo

Longer lag phase

What can be said about the lag phase of older inoculum?

Shorter lag phase

In terms of lag phase, what can be said about bacteria grown in rich growth media?

Log Phase

Exponential phase of the growth curve is also known as?

Growing and dividing at maximal rate

Describe the following for bacteria in log phase: growth and division

Balanced growth

Aside from growing at maximal rate, bacteria at exponential phase are also said to have what kind of growth?

When culture conditions can no longer sustain growth

When does the exponential phase of bacterial growth end?

Exponential Phase

Phase of bacterial growth wherein bacteria are said to be growing and dividing at the maximal rate

Double at regular intervals

The cell population is said to grow in what rate?

Culture media, Growth conditions, Genetics of organism

What are 3 factors affecting rates of exponential growth? CuCoGen

Cells are metabolically identical at this stage

Why is the exponential or log phase described to be a stage of balanced growth?

Measurement is accurate since cells are uniform

If we are to measure metabolite production at the log phase, what can be expected?

Continuous Culture

In what type of culture can we lengthen the log phase?

Bacteria have uniform chemical and physiological properties

Why are bacteria in log phase the best for research purpose?

Stationary Phase

Phase wherein there is no net increase or decrease in cell number

Zero population growth, Cell death = cell division

What can be said about the following factors for bacteria in the stationary phase: population growth, cell division

Nutrient limitation, Accumulation of toxic byproducts, Oxygen depletion (aerobic)

What are 3 causes for the stationary phase? TON

Remain metabolically active, Reduced metabolic and biosynthetic rate

What can be said about the following features of cells at stationary phase?

• Metabolic activity

• Metabolic rate

• Biosynthetic rate

Growth, Maintenance and survival

For cells at the stationary phase, cellular metabolism shifts away from ___ as the cell prepares for ___ and __

Stationary Phase

At what phase of bacterial growth can we expect the production of secondary metabolites

Secondary Metabolites

Substances produced by bacteria that are not essential for growth and development but have great potential for human and animal health

Antibiotics, Pigments

What are 2 examples of secondary metabolites?

Phenazine Derivatives, Acyl-Homoserine Lactone Derivatives

What metabolites does Pseudomonas produce that has relevance for biocontrol and plant-growth promotion?

Growth inhibition
Stimulation of sporulation
PGPR/ISR
Modulation of gene expression
Inducing of motility and biofilm formation
Signaling and diversification

The ecological roles of bacterial secondary metabolites?

Decline Phase

Death phase is also known as?

Cell death > Cell division

What can be said about the following factors for bacteria in the death phase: cell death, cell division

Detrimental Environmental Change: Nutrient Deprivation, Build up of toxic waste, Oxygen depletion

What is the main and 3 specific causes of the occurrence of the death phase?

Stationary, Death

What phases can cannibalism occur?

Reuse resources released from dying cells

Why are some subpopulation of cells adapted for cannibalism during the stationary and death phase?

Sporulating

Between sporulating and nonsporulating bacteria, which has immunity to killing factors?

Skf, Sdp

What are 2 examples of killing factors produced by sporulating bacteria?

AbrB represses SdpRI (immunity operon) causing cell lysis and release of nutrients

After killing factors have been released by bacteria with Spo0a, describe what happens to the nonsporulating cells receiving the killing factors

A known volume of sterile medium is added at a constant rate, while an equal volume of spent culture medium is removed at same rate

Why is continuous culture considered as an open system?

weeks or months

Continuous cultures can be maintained in the exponential growth phase for how long?

Exponential growth phase

Cultures can be maintained in what phase for long periods?

Sterility is challenging to maintain

What is the main problem with continuous cultures?

Culture volume, cell number, nutrient concentration, waste product concentration

In steady state, what factors are held constant? CuVol CeNu NuCo Wa

Chemostat

What is the most common type of continuous culture?

Tubings are prone to contamination

What is the problem with chemostats?

Inflow in inlet, Effluent in outlet

What are 2 liquid types involved in a chemostat?

Bacterial physiology, Isolation and enrichment, Industrial applications

What are 3 applications of a chemostat (continuous cultures)?

FALSE

T/F: Chemostats (continuous cultures) are widely used for industrial applications.

TRUE

T/F: Chemostats (continuous cultures) are not widely used for industrial applications

Single-cell protein production, Beer production, Municipal waste treatment processes

What are 3 industrial applications of a chemostat (continuous cultures)?

Semi-batch operation

Operation involved in fed-batch cultures?

Fed-batch culture

Semi-batch operation culture wherein nutrients are fed either intermittently or continuously via one or more feed streams

Obtain a pure or axenic culture

What is the goal of bacterial isolation?

Pure or axenic culture

A culture containing only a single kind of microorganism

Pure or axenic culture

Population of cells arising from a single cell

Streak Plating, Spread Plating, Pour Plating, Membrane Filter Technique, Agar Shake or Agar Dilution Tubes, Liquid Dilution Method, Roll Tube Method, SCI: Laser Tweezers, SCI: Flow Cytometry, High Throughput culture device, Microfluidic devices

What are isolation techniques?

Streak Plating

Isolation technique that depends on spatial separation of cells

Streak plating

What is the main method of isolation used?

Spread Plating, Pour Plating

Isolation techniques used when desired microorganism is present at higher levels than any other microorganism

Spread Plating, Pour Plating

Isolation techniques that depends on spatial separation of single cells

Low density mixed cells are spread on agar surface

What is the expected result for spread plating?

Prevent colonies from coalescing

What is the purpose of keeping spread plates dry?

we mix the bacteria in melted agar instead of using a tool to spread them

What makes pour plating distinct from spread plating?

FALSE

T/F: If you are dealing with heat-sensitive microorganisms, the best option would be pour plating

TRUE

T/F: If you are dealing with heat-sensitive microorganisms, the best option would be streak plating or spread plating

Circular colonies are likely obligate aerobes, Lenticular subsurface colonies are microaerophiles

What are 2 types of colonies formed in pour plating and what bacteria is the most likely composition?

45 to 50 deg C

What is the temperature of molten agar?

Single Cell Isolation Technique

What general technique is done for slow growing microorganisms or those present in low numbers in inculum?

Membrane Filter Technique

technique that is good if you have very few cells in the sample

0.22 micrometer

What should be the pore size of the membrane filter used in membrane filter technique for bacteria?

Agar Shake or Agar Dilution

If you have anaerobic microorganisms that are heat sensitive, what isolation technique can you recommend?

Paraffin-mineral oil seal

What is added in agar shake tubes to prevent transfer of oxygen?

Inverted light microscope with infrared laser and micromanipulation device

What equipment is used for SSCI: Laser Tweezers?

SSCI: Flow Cytometry

Isolation technique that also involves counting and examining microscopic particles

High-Throughput Culture Device

Isolation technique involving dilution/cell sorting of a sample to yield a single cell in each well of a microtiter plate

Microfluidic Devices

Isolation technique that uses a miniaturized platform of <10 cm?

Microfabrication

What technology is used for microfluidic devices?

3200 nanoliter sized wells

Microfluidic Devices holds how many of what size of wells?