1. Bacteria Cultures
Biotechnology II

2. 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

3. Culture medium Liquid growth culture
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

4. 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

5. 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

6. Bacteria Growth Curve

7. 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

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

9. Isolating Pure Culture of E. Coli
Use streak plate technique
Step 1: A loop full of bacteria cells are streaked in a Z pattern in one quadrant of plate
Step 2: Turn the plate 900
Step 3: Pass the innoculating loop after flaming through one corner of z pattern and create a new z pattern in next quadrant
Repeat steps 2 and 3 for quadrants 3 and 4;

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

11. 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

12. 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

13. BioLogical safety

14. Why important?
Biological safety is major issue in biotechnology facilities because they work with wide range of life forms

15. Strategies for Minimizing the Risks of Biohazards
All laboratories using microorganisms must follow standard microbiological practices.

16. Standard Microbiological Procedures
Wear the appropriate PPE
Washing hands after working with microorganisms and before they leave the laboratory
No eating or drinking
Decontaminating work surfaces after any spill or at end of work session
All biological materials must be properly decontaminated before disposal

17. Biosafety Level Determination
National Institute of Health classifies organism according the health risks to laboratory workers
The health risk is defined by
the pathogenicity of the organism
Infection rate after exposure
Availability of therapeutic agent if infected

18. Biosafety Level Determination
Biosafety levels determined by health risk
Lowest risk – biosafety level 1
Highest risk – biosafety level 4
BSL-1 –organisms not known to cause disease
BSL-3,4 – dealing with highly infectious agents where they may (BSL -3) or may not (BSL-4) be therapeutic agents to fight infection

19. Biosafety Level 1
Work with well- characterized strains of living microorganisms
not known to cause disease in humans
High school and college students commonly work with BSL1 organisms
Example of BSL 1 organisms -E. coli, yeasts, and most plants
E.Coli Bacteria
Yeast