Bacteria Cell Culture and Reproduction Biotechnology I Bacteria Cell Culture and Reproduction

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

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

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

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

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

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

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

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

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

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

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

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

Bacteria Growth Curve

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

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

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

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.

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

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

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

Bacteria Culture Confluent Growth versus Individual Colonies

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?

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

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?