Exercise 7

1
Exercise 7

• Bacteriophage One-Step Growth Curve and
  Determination of Burst Size
• Bacterial Growth Report Due Today
• Spec report due next week
• Continue Identification of Environmental Isolate

2
Phantoms

• You have had 2 exams in you lecture
• If you are thinking of dropping the lecture you
  can continue in the lab and retake the lecture
  and be a phantom in the lab.

3
Spectrophotometry report

• First draft due next week.
• Same format as first report
• Use previous comments about writing when putting
  this report together.

4
Report 2

• The doubling time or generation time is the time
  it takes the population of cells to double.
• Bacterial growth is logarithmic (exponential).

5
Report 2

• Calculating doubling time
• Where your graph begins to be logarithmic,
  multiply the OD550 by 2, extrapolate the
  corresponding time.
• Subtract the initial time from the extrapolated
  time. This is the time it takes for you
  population to double.

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Bacterial GrowthDefinitions

• Generation time Doubling time

7
Bacterial GrowthPhases of growth
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Report 2

• First submition is not a rough draft, if it
  appears to be it will not be graded.
• If a section is missing you will receive a zero
  on the report and will not be able to turn in a
  second submittion.

9
Report 2

• Your report will contain a table of the
  information and a graph with the growth curves.
• You need to turn in you hand drawn graphs and a
  computer generated graph.

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Purposes or objectives of this Experiment

• Measure bacterial growth in
• glucose minimal media
• YEP media (effect of amino acid and peptides)
• Determine Generation Times

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Hypothesis

• Will E. coli grow with a faster growth rate in
  Glucose or in Glucose and YEP?
• Include your hypothesis in the INTRODUCTION of
  Lab Report

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predictions

• E. coli will grow faster in YEP medium.
• E. coli will grow faster in glucose minimal
  media.
• There will be no difference in the growth of E.
  coli.

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Questions for Discussion

• How does glucose affect growth rate?
• How do amino acids and peptides affect growth
  rate?
• What is the generation time of a culture in
  minimal media?
• What is the generation time of a culture in
  supplemented media?

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What if your glucose minimal media had a shorter
doubling time than with your YEP medium?

• You left your culture out of the 37 degree water
  bath to long during the YEP step.
• For lab section J, your shaker speed was changed
  midway through the experiment.
• You did not add the appropriate amount of Glucose
  or YEP.
• Your spec was not set at zero with your blank
• You changed cuvet or did not clean it
  appropriately

15
Viruses

• Viruses are Ubiquitous.
• That is, they are capable of infecting animals,
  plants and bacteria and they are found
  everywhere.

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Viruses

• Usually consist of a protein coat that contains
  the DNA or RNA needed to make more viruses and
  some sort of structure that binds to receptors on
  the plasma membrane or cell wall of the host

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Virus history

• In 1946, the American microbiologist John Enders
  was the first to cultivate viruses in cell
  cultures
• Cultivating a virus that is causing a disease
  allows for identification of the causative agent
  of that disease

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Viruses

• Identification of the causative agent can allow
  for cultivation of a viral culture with the
  intent to create vaccines against viral disease.
• Currently, more than 40 genera of viruses have
  been implicated in human disease.
• The diseases caused by viruses can range from the
  common cold and gastroeneritis to fatal diseases
  such as AIDS, rabies and smallpox.

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bacteriophage

• Viruses that infect only bacteria are called
  bacteriophage or phage
• Bacteriophage replicates within a bacterial cell
• Some phage are species specific, and some have a
  broad host range and can replicate within various
  bacterial species

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Current Bacteriophage Research

• Studies have been completed looking at
  bacteriophage as antimicrobial agents in animal
  systems.
• They could be used to lyse pathogenic cells
  within the gut of animals.
• When the animal excretes, the phage goes with
  the excrement and continues to lyse pathogenic
  bacterial cells possessing the phage receptor
  protein.
• This could prevent future infection from the
  source of the pathogen.

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Bacteriophage

• Could they be used in place of antibiotics?
• Possibly in farm animals to reduce the use of
  antibiotics.
• This would help with our resistant bacteria
  problem.

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Bacteriophage

• How close is this to FDA approval?
• Studies are far from complete Bacteriophage can
  transfer genes from cell to cell.
• Transfer of genes from healthy biota to
  pathogenic biota and vice versa is of great
  concern in approval of this system for use.
• Genes being transferred could include functions
  such as antibiotic resistance. Which could cause
  a greater problem than we currently have with
  resistant bacteria.

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Bacteriophage

• T4 Phage
• Specifically infect Escherichia coli strain B
• The large body of the T4 phage makes it quite
  delicate
• SO, do not vortex a solution containing T4 phage
  because it could cause the tail to break off from
  the body making the former T4 phage nonfunctional

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T4 Phage
http//msnbc.msn.com/id/5870695
25
Phage Replication

• Adsorption
• Attachment phage binds to a receptor site on
  the surface of the host cell
• Release of DNA
• Replication
• Phage DNA directs the host cell to transcribe the
  phage DNA to make phage RNA
• Assembly
• The phage DNA and proteins are assembled inside
  the host cell to make mature phage particles.
• The time between initial infection of the
  bacterial host and the release of mature phage is
  called the latent period or the eclipse
  period
• Lysis
• Host cell lyses from the internal action of a
  phage-encoded lytic enzyme, releasing mature
  phage particles into the environment.
• Lysis of a population of infected cells does not
  occur spontaneously, and is represented as the
  rise period.

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Adsorption
http//www.cat.cc.md.us/courses/bio141/lecguide/un
it2/viruses/adlyt.html
27
Release of DNA into cell
http//www.cat.cc.md.us/courses/bio141/lecguide/un
it2/viruses/adlyt.html
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Phage Replication

• Replication
• Phage DNA directs the host cell to transcribe the
  phage DNA to make phage RNA

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Phage Replication

• Assembly
• The phage DNA and proteins are assembled inside
  the host cell to make mature phage particles.
• The time between initial infection of the
  bacterial host and the release of mature phage is
  called the latent period or the eclipse
  period

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Replication and Assembly
http//www.cat.cc.md.us/courses/bio141/lecguide/un
it2/viruses/adlyt.html
31
Phage Replication

• Lysis
• Host cell lyses from the internal action of a
  phage-encoded lytic enzyme, releasing mature
  phage particles into the environment.
• Lysis of a population of infected cells does not
  occur spontaneously, and is represented as the
  rise period.

32
Lysis
http//www.cat.cc.md.us/courses/bio141/lecguide/un
it2/viruses/adlyt.html
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Bacteriophage One-Step GrowthPurpose

• Phage Plaque Assay
• Determine the number of bacteriophage
• Plot the phage one-step growth curve
• Calculate burst size

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Bacteriophage One-Step GrowthProcedure

• Phage Plaque Assay
• Infect Escherichia coli strain B with T4 phage
• Remove samples from infected culture over time
• Make serial dilutions

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Procedure

• Plate diluted sample with E. coli lawn cells
• (a lawn is a confluent layer of bacterial cells
  contained in a thin layer of soft agar that has
  been poured on top of a nutrient agar plate)
• Count the number of plaque forming units (PFU)
  for each plate
• (a plaque is a cleared area in a lawn of
  bacterial growth that is casused by cell lysis
  due to phage infection) Each plaque is assumed to
  have originated from a single phage infection of
  one host bacterial cell)

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Bacteriophage One-Step Growth Procedure

• Review procedure
• Once you add E. coli to the phage, this
  experiment proceeds quickly!
• Work in groups of 3
• Decide what each partner will do
• Set up for serial dilutions and label plates in
  advance

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Bacteriophage One-Step Growth Procedure

• Add E. coli to T4 phage
• Multiplicity of infection (the ratio of phage to
  bacteria) is 150
• Gentle mixing!!
• Incubate in 37?C water bath
• ADS Adsorption
• Dilute 1100
• Prevent further adsorption
• All phage infections at the same time

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Bacteriophage One-Step Growth Procedure

• Incubate infected culture at 37?C
• Remove samples from infected culture at specific
  times
• Dilute sample (serial dilution)
• Add diluted sample and healthy E. coli lawn cells
  to melted agar
• Pour top agar plates
• Allow plates to cool and set

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Bacteriophage One-Step Growth Procedure

• Incubate plates at 37?C
• Next week
• Count plaque forming units (PFU)
• Complete and turn in the worksheet
• Plot one-step growth curve
• Calculate burst size

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Bacteriophage One-Step Growth Serial Dilution
50
Figure 7.1. Pattern of dilution tubes (Shand
2003).
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Environmental Isolate

• Continue identification of your unknown
• Have all your stains checked off by exercise 8
  (this is two weeks away)
• One submission only
• 80 points
• Paper due week of Nov 27
• Start working on methods and results