Title: Announcements
1Announcements
- 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?
2What we are doing today
- Discuss Hamburger Report
- -Make an appointment to go over fine details
- Bacterial Growth
- Environmental Isolate
3Bacterial 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
4Spectrophotometer
- 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.
5Bacterial 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.
6Notes 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
7Bacterial 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.
8Examples 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 -
9Bacterial 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.
10Four 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.
11Four 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.
12Example Curve
13Purpose of Bacterial Growth Experiment
- Determine Generation Times of bacteria in
different growth media - glucose minimal media
- YEP media
14What 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.
15Glucose Minimal Media
- Glucose will serve as both the
- sole carbon source
- sole energy source
16Adding 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?
17Adding 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
18Shaker/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)
19The 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
20Bacterial 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
21Bacterial GrowthExample Graph
- Generation time Doubling time
22Bacterial 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)
23Bacterial 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.
24Bacterial 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
25The 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
26Your 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
27Questions 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?
28Environmental Isolate
- After TSA slant tubes have incubated (one room
temp, one 4ºC), inspect - Best storage conditions?
- Fridge?
- Room Temperature?
- Record Results
29Biochemical 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.
30Stains
- 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!
31Environmental Isolate
- Even though you dont have all of your
information. - Start working on
- environmental unknown reports now!!!