1. Bacteria Cell Culture and Reproduction
Biotechnology I
Bacteria Cell Culture and Reproduction

2. Essential Question
What are the optimum conditions for growing E. coli?

3. Introductory Question
Describe the environmental conditions, that you find ideal to live in.

4. Growing Bacteria in the Laboratory
Must provide an environment that cells like including environmental factors such as
Temperature – grown in an incubator where temperature can be kept constant
pH (concentration of H+ ions)
Oxygen levels – liquid cultures are usually kept in a shaking incubator to aerate (provide O2) the culture

5. Culture medium
Definition: a medium that can support the nutritional needs of the bacterium
This medium can be in
A liquid form called a broth or
A solid form called agar.
Agar is a polymer of galactose isolated from red algae that cannot be degraded by bacteria.
Liquid growth culture
Solid growth medium of agar

6. Culture medium for growing E. Coli
The most common medium for growing E. coli is Luria broth.
This broth contains
Yeast extract –provides vitamins and trace elements
Tryptone- provides amino acids (building blocks of proteins)
NaCl – is an osmoticum the prevents bacteria cells from shrinking or swelling
NaOH to adjust pH between 7.5 and 8

7. Growth of E.Coli on Solid Medium
When E. Coli is grown on solid medium
First
Dissolve agar in LB Broth
Sterilize in an autoclave before pouring into sterile petri plates

8. PROPERTY OF PIMA COUNTY JTED, 2010
Think-Pair-Share
Why must the agar dissolved in LB broth be autoclaved before pouring into petri plates?
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class
PROPERTY OF PIMA COUNTY JTED, 2010 3

9. Reproduction of Prokaryotic Cells
Reproduce asexually by the cell dividing in half
Process of reproduction – binary fission

10. Binary Fission Cell gets larger DNA undergoes replication
Two molecules of DNA migrate to opposite ends of cell
Cell splits in half

11. Generation time of Prokaryotic Cell
Generation time = doubling time
Doubling time = time it takes a bacterium to do one binary fission
starts when cell has just divided and ends when the next division is just complete
Doubling time can range from 20 minutes for fast growing bacteria (Ex. E.coli) to hours or days for slow growing bacteria

12. Bacterial Growth Curve
Four phases
Lag phase
observed when cells are added to excess nutrient broth
cells increase in size but do not divide
the number of cells stays constant
Exponential phase
Back to back division cycles
Increase in cell number but not in cell size
Double in cell number each generation time
For example, if begin with 2 cells
First generation – 4 cells
Second generation -8 cells
Third generation – 16 cells

13. Bacterial Growth Curve
Stationary phase
rate of cell division = rate of cell death
cell number stays constant
usually occurs when the cell number becomes so great that something in the environment (i.e. nutritional needs) becomes limiting
Death phase-
rate of cell death > rate of cell division

14. Bacteria Growth Curve

15. Think-Pair-Share
Bacteria cells are growing in the exponential phase. You start with 6 cells, how many cells will you have after 3 generations? Be ready to share
3. Think-pair-share
teacher presents a question
teacher gives wait time for student to form answer
teacher instructs students to share their answer with a partner
teacher calls on non-volunteers to share with the class 3

16. Significance to Biotechnology
Because of E.coli short doubling time can:
Accumulate a large number of cells in short period of time
collect cells when they are close to the end of the exponential phase
Can isolate large amount of DNA and protein from those cells at that time

17. Isolating Pure Culture of E.coli
Definition: only E.coli and no other microorganisms are growing in the culture medium

18. Isolating Pure Culture of E. Coli
Use streak plate technique
Step 1: A loopful of bacteria cells are streaked in a Z pattern in one quadrant of the petri plate
Step 2: Turn the plate 900
Step 3: Pass the innoculating loop after flame sterilization through one corner of z pattern and create a new z pattern in next quadrant of petri plate
Repeat steps 2 and 3 for quadrants 3 and 4; Using the previous Z pattern as source of E.coli.

19. Streak Plate Technique
Purpose of this technique is to dilute out the cells so that individual bacteria colonies can be isolated

20. Bacteria colony Well-isolated colony arises from a single bacterium
Represents a clone of a pure culture
Samples of the isolated colony can be picked up with an inoculating loop and restreaked on fresh medium to maintain a pure culture

21. Confluent Growth
If the bacteria sample used to inoculate a plate is not diluted sufficiently, the bacteria cells are not separated and confluent growth will occur.
Confluent growth is where the bacteria colonies converge together

22. Bacteria Culture
Confluent Growth versus Individual Colonies

23. Think-Pair-Share
Explain the difference between isolated colonies and confluent growth.
Why would you want to collect bacteria from isolated colonies as opposed to confluent growth?

24. Measure Growth of bacteria Cultures
On solid medium – count the number of colonies over time
In liquid culture measure the turbidity of the culture over time at a wavelength of 600 nm using a spectrophotometer
At 600 nm, the spectrophotometer gives an optical density reading which measures how cloudy the culture is
The more cloudy the more bacteria cells are present

25. Answer the following questions
What are the key environmental factors that are controlled when growing E. coli?
List the components of LB broth and explain what nutrient molecules they provide for E. coli.
Explain how bacteria divide by binary fission
Draw a bacteria growth curve and label the curve with the appropriate phases.
Why would you want to isolate bacteria at the end of the exponential phase of the growth curve?
You start with 4 bacteria cells. How many cells will you have after 4 generations.
Why are isolating bacteria colonies important?
Explain how the spectrophotometer is used to measure bacteria growth?