Microbiological Culture Media Preparation and Sterilization; Culture Transfer Instruments and
Techniques
Abstract
The growth of microorganisms depends mainly on available nutrients and a favorable growth
environment. The nutrient preparations that are used for culturing microorganisms are called
media. Liquid or broth media, semisolid media, and solid media are the three physical forms of
media.
The method of rendering a medium or material free of all forms of life is sterilization. Three
basic ways in which sterilization of media and supplies can be achieved are autoclaving, dry-heat
sterilization, and filtration. The most useful approach is autoclaving; items are sterilized by
exposure to steam at 121°C and 15lb of pressure for 15 minutes or longer, depending on the
nature of the item. Dry heat sterilization occurs when the glassware is placed in an electric oven
set to operate between 160°and 170°C. However, some media cannot withstand heat, so the
medium can be sterilized by passing it through a bacteriological filter, which physically removes
bacteria and larger microorganisms from the solution and thereby sterilizes them without heat.
Introduction
Culture media are sterilized before they are used to eliminate microorganisms, which are always
present. Media can come in liquid forms called broths. When a solidifying agent like agar is
added to the broth, is now a solid media. Solid media is prepared into Petri plates or in tubes as
slants or deeps. In this experiment, we will make a media called nutrient broth and nutrient agar.
Materials
Escherichia coli Petri plates Culture tubes
Test-tube rack Test-tube racks 10-ml pipettes with pipettor
Agar Water bath set at 48°C Boiling water bath.
Bunsen burner Latex gloves Autoclave
Inoculating needle Tryptic soy broth tubes Tryptic soy agar slants
Tryptic soy agar deeps Sharpie marker Beaker with disinfectant
Vortex mixer
Procedure
Part 1
1. Prepare your workspace (Laminar Air Flow Cabinet) or lab bench by wiping
down the area with disinfectant.
2. With a marking pen, label a tube of sterile nutrient broth with your initials, the
organism's name, and the date of inoculation. Place in the test tube rack.
3. Sterilize your wire inoculating loop by passing it at an angle through the flame of
a Bunsen burner until the entire length of the wire becomes glowing red/orange from
the heat.
4. Never lay the loop down once it is sterilized or it may become re-contaminated.
Allow the loop to cool a few seconds to avoid killing the inoculum.
5. Using your other hand, pick up the tube containing the culture (inoculum) and
gently shake it to disperse the culture.
6. Grasp the cap (cotton plug) of the inoculum tube with the little finger of your
hand that holds the inoculating loop and remove it from the tube. Flame the mouth of
the tube.
7. Keeping the culture tube at an angle, insert the loop into the broth and remove a
loopful of inoculum.
Techniques
Abstract
The growth of microorganisms depends mainly on available nutrients and a favorable growth
environment. The nutrient preparations that are used for culturing microorganisms are called
media. Liquid or broth media, semisolid media, and solid media are the three physical forms of
media.
The method of rendering a medium or material free of all forms of life is sterilization. Three
basic ways in which sterilization of media and supplies can be achieved are autoclaving, dry-heat
sterilization, and filtration. The most useful approach is autoclaving; items are sterilized by
exposure to steam at 121°C and 15lb of pressure for 15 minutes or longer, depending on the
nature of the item. Dry heat sterilization occurs when the glassware is placed in an electric oven
set to operate between 160°and 170°C. However, some media cannot withstand heat, so the
medium can be sterilized by passing it through a bacteriological filter, which physically removes
bacteria and larger microorganisms from the solution and thereby sterilizes them without heat.
Introduction
Culture media are sterilized before they are used to eliminate microorganisms, which are always
present. Media can come in liquid forms called broths. When a solidifying agent like agar is
added to the broth, is now a solid media. Solid media is prepared into Petri plates or in tubes as
slants or deeps. In this experiment, we will make a media called nutrient broth and nutrient agar.
Materials
Escherichia coli Petri plates Culture tubes
Test-tube rack Test-tube racks 10-ml pipettes with pipettor
Agar Water bath set at 48°C Boiling water bath.
Bunsen burner Latex gloves Autoclave
Inoculating needle Tryptic soy broth tubes Tryptic soy agar slants
Tryptic soy agar deeps Sharpie marker Beaker with disinfectant
Vortex mixer
Procedure
Part 1
1. Prepare your workspace (Laminar Air Flow Cabinet) or lab bench by wiping
down the area with disinfectant.
2. With a marking pen, label a tube of sterile nutrient broth with your initials, the
organism's name, and the date of inoculation. Place in the test tube rack.
3. Sterilize your wire inoculating loop by passing it at an angle through the flame of
a Bunsen burner until the entire length of the wire becomes glowing red/orange from
the heat.
4. Never lay the loop down once it is sterilized or it may become re-contaminated.
Allow the loop to cool a few seconds to avoid killing the inoculum.
5. Using your other hand, pick up the tube containing the culture (inoculum) and
gently shake it to disperse the culture.
6. Grasp the cap (cotton plug) of the inoculum tube with the little finger of your
hand that holds the inoculating loop and remove it from the tube. Flame the mouth of
the tube.
7. Keeping the culture tube at an angle, insert the loop into the broth and remove a
loopful of inoculum.
