Microbiology Lab

1
Microbiology Lab

• Oct 30 Oct 31

2
Overview

• Turn in first submission of hamburger report
• attach rubrics!
• Ex. 6
• Discussion/determination of Biochemical test
  results
• Environmental unknown
• Staining
• Storage conditions

3
Purpose of Exercise 6

• Identify bacteria
• How fast does it grow
• On what does it grow
• Find ideal grow medium
• Find ideal grow time

4
Exercise 6

• Add 0.5 ml of 20 glucose to the E. coli ML 30 to
  start growth
• This is time 0
• Monitor the growth over 20 min intervals
• Replace the blank between each reading
• get 4 points and plot the glucose line
• Make sure you are recording the of ml of E.
  coli you take out each time

5
Exercise 6

• You will need to graph your points as you record
  them
• Its ok if readings arent done exactly at 20
  minutes
• However, record the exact time
• Once you have 4 good points, you will perform the
  calculation for the nutritional shift-up

6
Exercise 6

• Use the following equation to determine the
  amount of YE-P you need to add to the E. coli ML
  30
• 10 (ml of YE-P required) (ml of culture
  remaining) x 0.5
• After you calculate this, you will add the YE-P
• Remember to use sterile technique and pipette
  accurately

7
The Mechanics

• You will take readings at 20 minute intervals
  again
• Plot your points on the same graph
• Then we will calculate the doubling time for
  each type of medium

8
Spectrophotometer Use

• What were measuring is optical density
• Optical density A/path length
• Optical density is not absorbance, it has a
  functional relationship to absorbance however
• Cuvettes
• These LOOK a lot like test/culture tubes but they
  arent
• Cuvettes are made of special materials and are
  designed not to interfere with the ability of the
  light to pass through the sample

9
Spectrophotometer Use

• To take readings
• Place sample in cuvette
• Wipe clean with lens paper
• Avoid getting finger prints on the cuvette
• Align in spectrophotometer
• Remember-align it the same way each time (use the
  ? as a guide)
• Use the same cuvette for all sample readings
• Dispose of sample in beaker with bleach

10
Time to start!
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Bacterial growth

• The growth of microorganisms is affected by a
  wide range of factors
• Temperature
• pH
• Nutrient availability
• Presence/absence of oxygen or other terminal
  electron acceptors

12
Bacterial Growth

• In this experiment we are focusing on the effect
  of nutrient availability in regards to the growth
  of E. coli ML 30
• What is E. coli ML 30?
• Why did we choose 20 minute intervals?
• Keep in mind this is not a nutrient concentration
  experiment
• What we are looking at is how this bacteria
  alters its growth rate (or doubling time) under
  two sets of conditions
• In a minimal medium
• In an enriched medium

13
Bacterial Growth

• Definitions
• Minimal medium
• Enriched medium
• Under which condition do you think this organism
  will grow best and why?
• What is your hypothesis for this experiment?

14
Bacterial Growth stages

• Remember- bacteria reproduce by binary fission so
  in each cycle the number of individual is doubled
  
• This is described as exponential growth
• There are four phases in the bacterial growth
  curve
• Lag
• Log
• Stationary
• Death

15
Time for the next reading

• Lecture will resume once everyone has done their
  second reading

16
Doubling Time

• Part of what we are doing today is calculating
  the doubling time of our culture
• We will do this for both conditions (the glucose
  minimal and the YE-P enriched)
• To do this we pick an initial concentration of
  cells, we double that and determine the amount of
  time that passed between those two points

17
Doubling Time

• To calculate doubling time

18
Graphs

• Plot absorbance on 2-cycle semi-log paper
• Obtain 4 data points for each condition to get a
  straight line
• Your Y-axis will be in OD550
• Calculate generation time (Doubling time) from
  your graph
• Generate graph in Excel for next week to turn in
• Also include a table of the optical densities we
  record today