Media Sterilization Lab

Media

The nutrient preparations used to culture (grow) bacteria in the lab

Liquid broth culture

Example: nutrient broth

Used to obtain actively growing populations of bacteria to carry out biochemical tests or observe movement

Solid medium

Example: nutrient agar

Used to observe colony appearance (morphology), isolate pure cultures, and to carry out certain biochemical tests

May be prepared in a Petri plate (agar plate) or in a tube (agar deep or agar slant)

Semi-solid medium

Usually prepared in tubes for specific biochemical tests

Agar

Complex polysaccharide

Solidifying agent in culture media

Derived from marine algae

Most microbes don’t eat/degrade

Liquefies at 100* C

Great for growing all kinds of bacteria

Nutrient agar

Nutrient broth + agar

Is broth culture or solid medium better for starting a pure culture?

Solid medium

Is it easier to distinguish different microbes/colony morphologies in broth culture or solid medium?

Solid medium

Complex media

Rich media containing yeast extract, beef extract, or a similar complex material containing proteins, complex carbohydrates, and fats

Used for routine lab work

Supports a wide variety of heterotrophic bacteria

Exact chemical composition is not known

Chemically defined media

Exact chemical composition is known

Can be used to grow organisms w/ special growth requirements

Can be used to exclude certain organisms

Must contain a source of energy and sources of C, H+, O2, N, S, P and any other growth factor the organism cannot manufacture itself

Selective media

Suppress the growth of unwanted bacteria or other microorganisms

Differential media

Allows to distinguish visually between 2 bacterial species growing on the same plate b/c they appear different colors

Usually reflects different by-products of metabolism

Enriched media

Provides added nutrients to favor growth of target bacteria

Sterilization

The complete removal or destruction of all forms of microbial life

Why do we need to sterilize culture media and equipment in a health care setting?

Prevention

Confidence in determining organism

Why do we need to sterilize culture media and equipment in a research setting?

Decrease contamination

Autoclaving

Method of sterilization

Most effective on glassware and media in lab

Exposes items to steam at high temperature and pressure in a sealed chamber

Extreme heat denatures proteins at 121* C and 15 lbs. of pressure

Kills all vegetative cells and bacterial endospores and viruses in 15 minutes

Autoclave tape

Often lead carbonate

Before autoclaving → faint strips on tape

After autoclaving → black stripes on tape

Incineration

Uses dry heat to destroy contaminated material

Flame sterilization works by incinerating material on the loop or needle

Filtration

Used to sterilize media that are inactivated by high heat such as many antibiotic solutions

These media can be passed through a filter or screen w/ pores small enough to exclude microorganisms

Usually carried out in a sealed disposable unit using a vacuum to pull the solution through the filter

Ultraviolet irradiation

Lethal to many microorganisms b/c it damages DNA

Mutates DNA

UV lamps used to sterilize surfaces or the air in a room

UV light used to sterilize treated wastewater before release into the environment

Chemical sterilization

Uses ethylene oxide gas

Method of sterilizing heat sensitive items such as plastic Petri plates

Gas rapidly penetrates the plastic wrappers of packaged materials and denatures the proteins of microorganisms killing both vegetative cells and endospores in 4-18 hours

Disinfectants

Part of aseptic technique

Do not achieve complete sterilization

Reduces microbial numbers on surfaces

How to prep nutrient agar

1. Mix ingredients

   1. Nutrient agar (powder) + ultra pure H2O

2. Heat nutrient agar mix

   1. Mix occasionally

3. Autoclave nutrient agar

Suppose after prepping nutrient agar broth when going to pour the plates the nutrient agar is cloudy. What mistake was made?

Aseptic technique was not done correctly

Pouring plates steps

1. Have Petri dish poring into ready and near the flame

2. Light Bunsen burner and work close to it

3. Hold media bottle in dominant hand

4. Open media bottle

5. Flame bottle

6. Pour into Petri dish just enough to cover the bottom of the plate leaving about the size of a nickel

7. Flame bottle

8. Repeat

Recipe for nutrient agar

• Peptone (partially digested protein) = 5.0 g

• Beef extract = 3.0 g

• Sodium chloride = 8.0 g

• Agar = 15.0 g

• Water = 1 L

Recipe for chemically defined medium

• Glucose = 5.0 g

• Ammonium phosphate, monobasic (NH4H2PO4) = 1.0 g

• Sodium chloride = 5.0 g

• Magnesium sulfate (MgSO47H2O) = 0.2 g

• Potassium phosphate, dibasic (K2HPO4) = 1.0 g

• Water = 1 L

6 basic chemical elements required to build the structure of cells

1. Carbon

2. Hydrogen

3. Oxygen

4. Nitrogen

5. Sulfur

6. Phosphorus

Which ingredients in nutrient agar contain the 6 basic chemical elements required to build the structure of cells

• Carbon = peptone and beef extract

• Hydrogen = water

• Nitrogen = peptone

• Oxygen = water

• Phosphorus = beef extract

• Sulfur = peptone

Why was the boiling water bath used to melt the agar not sufficient to sterilize the medium?

Bacterial endospores are resistant, dormant structures that are designed to survive extreme heat and may not be reliable sterilization

Reliable sterilization requires heat above boiling temperatures achieved by steam and pressure in the autoclave

How would you sterilize a heat sensitive solution?

Filtration

How would you sterilize a plastic syringe?

Chemical sterilization

How would you sterilize a package of plastic Petri plates?

Chemical sterilization

How would you sterilize the bench top?

Chemical sterilization - microban

How would you sterilize glass test tubes containing nutrient broth?

Autoclave

How would you sterilize a reusable metal tool such as a loop?

incineration - flame sterilize