Announcements

1
Announcements

• REMEMBER THERE IS NO EXTRA CREDIT IN THIS CLASS.
• ALTHOUGH YOU ARE WORKING IN PAIRS FOR YOUR
  ENVIRONMENTAL ISOLATE, YOU NEED TO DO ALL OF THE
  STAINING AND IDENFICATION ON YOUR OWN AND THEN
  COMPARE.
• Does anyone have a gram-negative cocci?

2
What we are doing today

• Discuss Hamburger Report
• -Make an appointment to go over fine details
• Bacterial Growth
• Environmental Isolate

3
Bacterial GrowthReview of Enumeration methods

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

4
Spectrophotometer

• Measure growth rates with a spectrophotometer
• Direct relationship between cell number and
  absorbance, Otherwise known as Optical Density
• More bacteria higher absorbance.
• less light reaches sensor
• Cells scatter light, not absorb light

• Measure living and dead cells
• Gives immediate assessment of the number of cells
  in a population.

5
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 bleach and rinse
  with distilled water

The cuvettes are glass and can get scratched.
Please treat them with care.
6
Notes about the Spectrophotometer

• 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

7
Bacterial Growth

• The spectrophotometer uses the turbidity of the
  culture to determine the amount of light that is
  deflected off the cells by measuring the optical
  density (OD) of the culture.
• The changes in absorbance are due to the light
  scattering off the cells (99 of which are
  viable)
• The greater the number of organisms, the more
  light they deflect from the photodetector.
• A cloudier culture, with more growth, will have a
  higher OD then a less cloudy culture, since more
  light is deflected by the denser culture.

8
Examples of Bacterial Growth

• Bacterial Cell Growth is the increase in the
  quantity of all cellular structures and
  components. This increases the cell's size until
  division occurs.
• A vegetative cell is a cell that is actively
  growing and dividing.
• Microbial Growth is measured by population growth
  as opposed to cell growth, measured by total
  number of cells and can be measured exponentially
  due to binary fission reproduction

9
Bacterial Growth

• Generation Time (or doubling time) is the time it
  takes an individual cell to divide or for a
  population of cells to double. Bacterial growth
  is logarithmic (exponential) - 2 cells divide
  into 4, 4 cells divide into 8, 8 cells divide
  into 16, etc.

10
Four Phases of Bacterial Growth

• 1) During the lag phase cells are metabolically
  active but not dividing. They are
  re-synthesizing enzymes, other proteins, and
  amino acids that are required for growth and
  division.
• 2) The log phase is the period of fastest growth
  as bacteria are dividing at an exponential rate.
  Generation time is maximal and constant as all
  nutrients and molecules needed for growth are in
  good supply.

11
Four Phases of Bacterial Growth

• 3) During the stationary phase the medium is
  becoming depleted in nutrients and toxic waste
  may be accumulating. The number of new cells is
  equal to the number of old cells dying, so the
  total viable cell count remains constant.
• 4) In the Death Phase the conditions are becoming
  less conducive to cell growth. The cells are
  dying more rapidly than they are growing. There
  is a logarithmic decrease in cell number.

12
Example Curve
13
Purpose of Bacterial Growth Experiment

• Determine Generation Times of bacteria in
  different growth media
• glucose minimal media
• YEP media

14
What is so remarkable about the organism we will
study (E. coli strain ML30)?

• It can manufacture
• Amino acids
• Proteins
• Carbohydrates
• Lipids
• Nucleic acids
• Vitamin
• All from a simple buffered solution containing
  glucose and a few salts.

15
Glucose Minimal Media

• Glucose will serve as both the
• sole carbon source
• sole energy source

16
Adding The Nutrition

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

17
Adding Nutrition

• Yeast-extract - digest of yeast, provides a good
  general base for culture media
• Components amino acids, small peptides, vitamins
• Peptone proteinaceous materials (meat, soy
  beans, etc.) digested by enzymes or acids

18
Shaker/Heat Bath

• 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 ANY OTHER SWITCHES, DIALS,
  ECT..(severe loss of Professional Points)

19
The Experiment

• Work in groups of three
• Find 50 mL E. coli culture in minimal medium in
  shaker/heat bath
• Add glucose, .5ml
• Immediately remove sample from culture (Time 0)
• Put 5ml sample in cuvette and measure turbidity
  with the spectrophotometer
• Record absorbance
• Remove samples from E. coli culture at 20 min
  intervals and record absorbance (record exact
  time if not 20 min.)
• Take at least 3 points, 4 would be better

20
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

21
Bacterial GrowthExample Graph

• Generation time Doubling time

22
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)
• Take in account for dropped 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)

23
Bacterial GrowthAddition of YE-P

• Add calculated amount of 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
• Again take at least 3 samples, 4 samples would be
  better.

24
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

25
The Experiment step by step
50 ml aliquots in 125 ml flask with group labels
First add 0.5ml 20 glucose to your flask
E. coli .0025 glucose in Fernbach Flask
Record initial OD 0.012-0.020
Shaker Bath (turn back on after sampling)
Groups stagger start times at 3 min intervals
Determine your doubling time and cleanup
Repeat 2-3 X 20min intervals and plot as you go
Then Immediately take your first time point Time
0 for glucose
Calculate the YE-P needed for 0,5, add YE-P to
flask and take YE-P Time 0
Store your cuvette upside down in the rack
Bleach Waste
Repeat 2-3 X 20min intervals (record your exact
times) and plot your data as you go
Dump the culture and the rinse into the waste
beaker
26
Your Hypothesis

• E. coli will have a faster growth rate in Glucose
  and Yeast extract-peptone (YE-P) than in Glucose
• This is your hypothesis. Include your hypothesis
  in the INTRODUCTION of Lab Report

27
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
  enriched media?

28
Environmental Isolate

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

29
Biochemical Tests

• Discard used tubes in proper places
• Redo any biochemical tests that were not done
  correctly.
• You will need these results to be able to
  identify your unknown.

30
Stains

• Remember to perform all of the stains (i.e.
  acid-fast, endospore, gram, capsule) on your
  unknown
• This will help you identify your unknown
  better!

31
Environmental Isolate

• Even though you dont have all of your
  information.
• Start working on
• environmental unknown reports now!!!