Exercise 6 Part I

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Exercise 6Part I

• Go over Hamburger Report
• Bacterial Growth
• Environmental Isolate

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Hamburger reports 1st submittion

• Average 23.5 out of 40
• High grade 34.5 out of 40
• Most of you can improve your grade by simply
  including info you did not include in your first
  attempt and by making corrections.
• A few of you need to do a lot more work.
• I can not catch all mistakes so, make the
  corrections and then DO PEER REVIEWS before you
  submit your final copy!

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Introduction

• Hypothesis/Predictions what do you expect
• Objectives what is the purpose of the study
• Background on enumeration
• Info on food bacteria or burger bacteria
• Citations (author, year). You can not over
  cite!!!!

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Methods

• Start your paragraph with the purpose of the
  methods.
• Examples
• In order to estimate the number of bacteria in
  burger, _________ was done.
• To determine the number of bacteria in burger,
• CITE SHAND 2002!!!!! DO NOT RESTATE HIS METHODS
  WORD FOR WORD!!!!!!

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Results

• One paragraph for each graph
• One paragraph for each figure
• Dont restate what the figure shows, this is
  redundant.
• Do state trends highest, lowest, comparisons.
• Dont use the term significant when discussing
  these trends.
• Significant used this way can only be determined
  by statistics
• Significant is ok to use when referring to number
  of bacteria between 30 and 300.

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Results

• Start paragraphs with purpose.
• Estimating the number of bacteria on pour and
  spread plates, ________ was found.
• Not 20cfu/ml ? 20X106 cfu/ml
• Tables and graphs
• Titles are descriptive
• Headers are not simply titles.
• Headers go on top of tables and below figures.

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Discussion

• Restate your hypothesis from your intro.
• Was it proven?
• Restate predictions from your intro.
• Were they correct?
• Restate your objectives from your intro.
• Were they met?
• Analyze important data.
• Discuss any problems you encountered.
• Why is this study important to your reader?
• What other experiments would be useful in this
  study?

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Exercise 6 Part I

• Bacterial Growth
• Exercise
• Laboratory report due two weeks from today!
• Environmental Isolate
• Storage Conditions
• Biochemical Tests
• Endospore Stains
• Next week
• Exercise 6 Part II

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Spectrophotometric Determination of Bacterial
Growth

• Absorbance, or optical density, is a measure of
  the amount of light absorbed by a solution.
• There is a direct relationship between absorbance
  of the culture and cell number
• The more bacteria in the culture, the higher the
  absorbance.

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Spectrophotometric Determination of Bacterial
Growth

• Pros of spectrophotometry
• Immediate assessment of the number of cells in
  the population
• We do not have to wait for colonies to grow on a
  plate before we know how many cells are present

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Bacterial Growth Enumeration methods

• Hamburger (viable count) exercise
• Direct counting method
• Viable count assays (using plate counts)
• Bacterial population at one point in time
• Bacterial growth exercise
• Indirect counting method
• Measure turbidity (using a spectrophotometer)
• Bacterial population growth over time

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Spectrophotometer

• Measure growth rates with a spectrophotometer
• More cells ?
• less light reaches sensor ?
• higher absorbance
• Optical density
• Cells scatter light, not absorb light
• Measure living and dead cells

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How to UseSpectrophotometer

• Wavelength of light beam is 550 nm
• Optical density at 550 nm (OD550 )
• Set blank (or standard)
• Distilled H2O
• Absorbance reading set at 0.00
• Should not have to adjust blank cuvette after it
  is set

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Bacterial GrowthSpectrophotometer

• Cuvettes
• Place sample in cuvette
• Wipe clean with lens paper
• Align in spectrophotometer
• Use the same cuvette for all sample readings
• Dispose of sample in beaker with Osyl

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Bacterial GrowthDefinitions

• Vegetative growth
• Cells actively growing and dividing
• Microbial growth
• Population growth as opposed to cell growth
• Measured by total number of cells
• Exponential due to binary fission reproduction
• Generation time (or doubling time)
• The time for an individual or a population to
  double

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Bacterial GrowthDefinitions

• Generation time Doubling time

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Bacterial GrowthPhases of growth

• Four phases of growth in a culture
• Lag phase
• Log phase (or exponential phase)
• Stationary phase
• Death phase

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Bacterial GrowthPhases of growth
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Purposes 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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Glucose Minimal Media

• Measure E. coli ML30 growth rate in glucose
  minimal medium
• This strain of E. coli can manufacture all the
  amino acids, proteins, carbohydrates, lipids,
  nucleic acids and vitamins required for survival
  from glucose and a few salts!!!
• Glucose minimal medium contains
• Glucose
• Salts

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Minimal Media

• Glucose will serve as
• sole carbon source
• sole energy source
• Cells must make everything else they need (amino
  acids, proteins, carbohydrates, lipids, nucleic
  acids, and vitamins) for growth.

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Nutritional Shift-Up

• We will add 10 yeast-extract peptone (YEP) to
  the medium.
• How will the growth rate change after the media
  is supplemented?

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Nutrional Shift-Up

• Yeast-extract digest of yeast, provides a good
  general base for culture media, components are
  undefined
• Peptone proteinaceous materials (meat, soy
  beans, etc.) digested by enzymes or acids
• Agar complex polysaccharide that serves as a
  solidifying agent (seaweed)

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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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Bacterial Growth

• E. coli culture in shaker/heat bath
• Constant temperature and distribution of
  nutrients and oxygen
• Temperature set at 37?C
• Shaker set at 7
• When adding nutrients or removing samples
• Turn off shaker
• Be careful not to spill culture into water bath
• Remember to turn shaker on
• DO NOT TOUCH TOUCH ANY OTHER SWITCHES, DIALS,
  ECT..(severe loss of Pro. Pts.)

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Bacterial GrowthIncreased glucose concentration

• 50 mL E. coli culture in minimal medium
• Add glucose
• Immediately remove sample from culture (Time 0)
• Measure turbidity with the spectrophotometer
• Record absorbance
• Remove samples from E. coli culture at 20 min
  intervals and record absorbance

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Bacterial GrowthIncreased glucose concentration

• Plot absorbance readings on 2-cycle semi-log
  paper (in your lab manual)
• Record the exact time that you remove your sample
  from the culture
• Do not plot in 20 minute intervals unless your
  samples were taken at exactly 20 minute intervals
• Obtain 3 or 4 data points to get a straight line
• Calculate generation time (doubling time) from
  your graph
• Generate graph in Excel for lab report

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Bacterial GrowthAddition of YE-P

• Calculate remaining volume of E. coli culture
• 50 mL original volume
• plus 0.5 ml glucose
• minus (5 mL samples)(number of samples)
• Calculate amount of yeast extract-peptone (YE-P)
  to add for a final concentration of 0.5
• (C1)(V1) (C2)(V2)
• (10)(mL YE-P) (0.5)(mL of culture)

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Bacterial GrowthAddition of YE-P

• Add calculation amount YE-P
• Immediately remove sample from culture (Time 0)
• Measure turbidity with the spectrophotometer
• Record absorbance
• Remove samples from E. coli culture at 20 min
  intervals and record absorbance

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Bacterial GrowthAddition of YE-P

• Plot absorbance on 2-cycle semi-log paper
• Obtain 3 or 4 data points to get a straight line
• Calculate generation time from your graph
• Generate graph in Excel for your lab report

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Questions for Discussion

• How does glucose affect growth rate?
• How do aa 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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Environmental Isolate
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Environmental Isolate

• After TSA slant tubes have incubated (one room
  temp, one 4ºC), inspect
• Best storage conditions?
• Fridge?
• Room Temperature?
• Record Results

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Biochemical Tests

• Discard used tubes in proper places
• Redo any biochemical tests

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Endospore Stain

• Perform endospore stain
• Environmental Unknown on one side
• B. meg (endospore plate) on other side

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Environmental Isolate

• Start working on environmental unknown reports
  now!!!

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Once experiment finished

• Demonstrate plotting on log paper
• Demonstrate how to calculate doubling time
• Handout on Excel for Bacterial Growth
• Demonstrate how to make graph in Excel