Lab 4e: Making Media for Bacteria Cell Culture (Media Prep) (smelly lab)

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Why are we growing bacteria?

To be used as a source of DNA

Where is bacteria grown in?

In or on their food source (MEDIA)

media

• sterilization is done prior to inoculation with organism

Medium is solid

agar

what type of mixture is agar

suspension

medium is a liquid

broth

what type of mixture is broth

solution

How do we decide what medium to use

depends on the bacteria grown

If single, isolated colonies are needed, you must grow the cells in

agar (normally petri plates)

• A single cell on the surface will divide to create a colony of identical cells (no genetic variation) (clones).

A plate colony is ideal

for separating bacteria into individual colonies as isolated, pure, uncontaminated cultures

Why would we transfer a single colony to broth after isolated colonies are grown

maximizing cell growth and reproduction because there is better access to nutrients. Culture clone bacteria from single colony

What are broth cultures used for

manufacutrng to obtain maximum number of cells to make max protein product

LB agar

• Luria Bertani

• All purpose

• supports growth of many types of fungi and bacteria

• other types of nutirents (ampicillin-will select for the growth of bacteria transformed with plasmids containing an ampicillin resistance gene) for different tests

LB agar ingredients

• 10 g SELECT Peptone 140

  • growth promoting

• 5 g SELECT Yeast Extract

  • provides a bunch of organic compounds needed for growth

• 5 g Sodium Chloride

  • electrolytes for transport and osmosis balance

• 12 g SELECT Agar

  • gel in which bacteria can grow

LB broth base

hygroscopic

media base

dry mixture of media components

agar texture vs broth

agar: thick jello

broth: liquid

both smell like a petsmart and give you a headache for days

autoclave

sterilize using high temperature and preassure

A 250ml Media bottle can hold a max of

125ml of Agar

why do we only fill media bottles half way

so it doesnt boil over

autoclave tape

special tape that shows black lines when exposed to preassure