Lab

1
Lab 10

• Evaluation of Media
• Effect of Osmotic Pressure on Growth
• Standard Plant Count

2
Evaluation of Media

• How oxygen, salt, and nutrients effect growth of
  bacteria
• Undefined Media- composed of Extracts for growth
  of the greatest variety of microbes
  fastidious
• -complex media
• Defined Media amount and identity of all
  ingredients known, grows a more narrower range
  of bacteria.
• -chemically defined media

3
Organisms for Evaluation of Media

• E. coli
• Streptococcus sanguinis

4
Medium with different nutritional components

• Yeast Extract Broth YEB (Undefined)
• Nutrient Broth NB (Undefined)
• Glucose Salt Broth GSB (Defined)
• Inorganic Synthetic Broth ISB (Defined)

5
Experiment set up

• Collect two tubes of NB, YEB, GSB, ISB
• Using the E. coli, and S. sanguinis, Inoculate as
  uniformly as possible, 0.1ml of each organism
  into each of their own tube of media.
• Mark each tube at the top with correct media and
  organism
• Place in incubator for 24-48 hours
• Measure growth with levels of turbidity 0,1,2,3

6
Osmotic Pressure on Bacterial growth

• Salts directly affect osmosis ( the movement of
  H2O across a membrane).
• A solution with a higher concentration of solutes
  is hypertonic.
• A solution with a lower concentration of solutes
  is hypotonic.

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Halophiles bacteria with the ability to grow
in high salt (hypertonic) environment. Ex.
Staphylococcus aureus Facultative halophile
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Osmotic Pressure on Bacterial growth

• A cell can find itself in one of three
  environments
• -isotonic, hypotonic, hypertonic
• Most bacteria are isosmotic at 0.85 NaCl
• A bacteria is hyperosmotic in greater than 11
  NaCl
• Some bacteria can withstand 3-15 NaCl
• -moderate halophile
• Some bacteria can withstand 15-25 NaCl
• -extreme halophile
• A bacteria is hypoosmotic in 0 NaCl
• Most bacteria live where NaCl concentrations are
  less than 3 NaCl

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Organisms for osmotic pressure experiment

• Staphylococcus aureus
• E. coli

10
Experiment

• Nutrient broths containing NaCl 1NaCl, 3NaCl,
  7NaCl, 11NaCl
• 1 drop of each bacteria into respective NaCl
  broth
• Incubate broths 24-48 hours
• Spectrophotometer to measure absorbance

11
Standard Plate Counts

• Indirect counting method
• - estimating microbial cell density using
  the number of colonies a portion of the sample
  produces when spread onto an agar plate.
• Serial dilutions are controlled transfers
  (diluted sample) until each sample is a fraction
  of the original sample or the previous sample .
• Systematically reduces cell densities and
  produces a plate of colonies that can be counted

12
Organism in the original sample

• E. coli

13
The Experiment

• 2, 9.9ml tubes sterile water
• 3, 9.0ml tubes sterile water
• 1 tube of E. Coli (original sample)
• 8 agar plates
• In controlled transfers, transfer 100µl (.1ml) of
  original sample into 1st tube of 9.9ml water. In
  second transfer, you transfer 100µl (.1ml) of the
  1st tube and transfer to 2nd tube of 9.9ml
  sterile water. The third, fourth,fifth,transfer
  is withdrawing 1.0ml of sample from the previous
  tube and inoculating the 3rd,4th,5th tube of
  9.0ml sterile water.
• The 2nd,3rd,4th,5th,6th samples are then plated
  out on nutrient agar, by transfering 100ul from
  each sample and spreading them out on the plate
  using a spreader dipped with alchohol and flamed.
• Incubate 24-48 hours

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Standard Plate Count using serial
dilutionsExample of a serial dilution technique
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plate counter