Aseptic Technique:
Media and Equipment
Growth Medium
• A growth medium or culture medium is
a liquid or gel designed to support the
growth of microorganisms or cells
• The most common growth media for
microorganisms are nutrient broths
and agar plates
Defined Media
• An important distinction between
growth media types is that of defined
versus undefined media
• A defined medium will have known
quantities of all ingredients
Undefined Media
• An undefined medium has some
complex ingredients that consist of a
mixture of many chemical species in
unknown proportions
• Undefined media are sometimes
chosen based on price and sometimes
by necessity - some microorganisms
have never been cultured on defined
media.
Undefined Media
• We will use this in the lab
• Nutrient Agar contains:
– Pancreatic digest of gelatin
– Beef Extract
– Agar
Forms of Culture
• The most common growth media for
microorganisms are nutrient broths
(liquid in a test tube)
• Liquid media are often mixed with agar
and poured into petri dishes to solidify
• These agar plates provide a solid
medium on which microbes may be
cultured. They remain solid, as very few
bacteria are able to decompose agar.
Slants
• There are several reasons a slant is
best for culture storage:
• 1) Surface area – slant increases it
• 2) Size – test tubes smaller to store
• 3) Moisture – won’t dry out
• 4) Ease – once the microorganism is
transferred, you can store it for up to 6
months in a refrigerator
Aseptic Technique
• Aseptic technique refers to a procedure
that is performed under sterile
conditions
• This includes medical and laboratory
techniques (such as with
microbiological cultures)
• It includes techniques like
flame sterilization
(Bunsen burner)
Aseptic Technique
• Sterile surfaces must be protected from
microbes in the air or on non-sterile
surfaces
• In sterile technique, only sterile
surfaces touch other sterile surfaces
and air exposure is kept to a minimum
Aseptic Technique
• It is important in microbiology to work
with pure cultures
• This is difficult because the world
around us is covered with MO’s (even
on dust particles in the air)
• In order to protect broth, plates, slants
and pure cultures from MO’s around
us, we practice aseptic techniques
Aseptic Technique
• In this class you will need to practice
sterile technique when we inoculate a
pure culture into fresh medium (tube of
sterile broth or an agar plate)
Aseptic Technique
• “A” = Negative prefix
• “Septic” = Infection
• All techniques and procedures which
prevent contamination.
Safe Patterns
• Safe patterns include:
– Standard Lab Rules
– Inoculating Techniques
– Use and Disposal of Equipment
– Washing Hands
Safe Patterns
• Sterilize all materials before beginning
Safe Patterns
• Spray the lab top down with a
commercial disinfectant or a 10%
bleach solution and allow this to stand
for a minute. You may then wipe down
the bench with the paper towel.
Safe Patterns
• Wash hands before and after lab.
Safe Patterns
• You should have only the materials
you'll need and the written lab
procedures on your bench top or desk.
Safe Patterns
• Keep petri dishes and test tubes
covered as much as possible.
• If top must be removed completely do
not LAY IT on the lab top. This lowers
the probability of contamination and
prevents “false positive” results.
Safe Patterns
• Hold bottles and tubes at an angle to
minimize the amount of airborne microbes
that can fall into them (blue circle). Remove
the caps as shown above and do not set the
caps down. Keep the mouth of the facing cap
down (red circle).
Safe Patterns
• When using metal inoculating loops, HEAT
the entire piece of metal of the inoculation
instrument in the flame: it should be RED
HOT. Be sure to COOL your inoculation
instrument before picking the inoculum
(broth or agar).
Safe Patterns
• To inoculate a Petri plate: Lift one edge
of the Petri plate cover to gain access
to the culture medium. Keep the cover
over the plate bottom to prevent dust
and microbes from falling onto the
agar.
Safe Patterns
• Report spills to me immediately
• Cover the spill with paper towels and
squirt disinfectant onto the towels. Wait
20 minutes then clean up the spill.
Safe Patterns
• Label all test tubes and petri plates with
your name (initials), date, and name of
organism BEFORE you add any
solutions, bacteria, etc.
Safe Patterns
• Do not dump ANY microbial
suspension down the drain or in the
trash can. I will collect them for proper
disposal.
Safe Patterns
• Place test tubes in racks
when working at your table:
never lay the tubes down—they leak.
• Keep test tube caps and petri dish
covers on media to reduce
contamination (matters not whether it
is sterile media or already cultured).
Safe Patterns
• All agar plates are
incubated UPSIDE DOWN
to reduce bacterial
contamination and to
reduce the possibility of water
condensation that may be on the lid
dropping onto the agar, causing fluid to
run across the agar medium.
Media
• Sterilize after preparation, in
storage, or working containers.
Handle aseptically.
• If no goofs – Sterile indefinitely
E. Autoclave
• Pressurized steam
• Water boils at 100ºC, but if pressure is
increased the boiling point rises
– At 15 PSI the Boiling Point = 121ºC
– At 20 PSI the Boiling Point = 126ºC
• Minimum = 15 minutes @ 15 PSI
• A pressure cooker is the same as an
autoclave
Boiling
• 100ºC kills most MO, fungi, and viruses
but a few are resistant.
– Example: Inf. Hepatitis, Endospores
• But very good
nonprofessional use
Equipment
Technique depends on article:
Does it burn or melt?
Do you want it back?
Equipment
1. Autoclave
•
If it fits into machine and won’t melt
•
15 minutes @ 121ºC (15 PSI) minimum
Equipment
2. Boiling
•
Never in a lab
Equipment
Oven
•
Dry heat used for metal and glass
•
90 minutes @ 170ºC minimum
Equipment
Incineration – Burning
•
1000ºC Plus
•
Loops, test tubes
Equipment
5. Presterilized Equipment
•
Usually plastic and paper materials
•
Gas: Ethylene Oxide
•
Irradiation