Bacteria Cultures Biotechnology II

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 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

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

Bacteria Growth Curve

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

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 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;

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

BioLogical safety

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

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

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

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

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

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