Aseptic Technique:Media and Equipment

Growth Medium

• A growth medium or culture medium is a liquid or gel designed to _______ ____ _______ of microorganisms or cells

• The most common growth media for microorganisms are ________ _______ and _______ plates

Defined Media

• An important distinction between growth media types is that of ________ versus __________ media

• A defined medium will have _________ quantities of all ingredients

Undefined Media

• An undefined medium has some complex ingredients that consist of a mixture of many chemical species in _________ _____________

• Undefined media are sometimes chosen based on _______ and sometimes by ____________ - some microorganisms have never been cultured on defined media.

Undefined Media

• We will use this in the lab

• Nutrient Agar contains:– ________ _______ ___ ________– ________ ________– ________

Forms of Culture• The most common growth media for

microorganisms are ________ _______(liquid in a test tube)

• Liquid media are often mixed with ______ and poured into ______ _______ to solidify

• These agar plates provide a _______ 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) ________ ______ – slant increases it

• 2) ______ – test tubes smaller to store

• 3) ________ – won’t dry out

• 4) _______ – 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 ________ ____________

• This includes ________ and _________ techniques (such as with microbiological cultures)

• It includes techniques like

_________ ______________

(Bunsen burner)

Aseptic Technique• Sterile surfaces must be protected from

________ ___ ____ ____ or on non-sterile surfaces

• In sterile technique, only ________ surfaces touch other ________ surfaces and ____ exposure is kept to a minimum

Aseptic Technique

• It is important in microbiology to work with ________ __________

• This is difficult because the world around us is covered with ________ (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 Technique

• In this class you will need to practice sterile technique when we _________ a pure culture into ______ __________ (tube of sterile broth or an agar plate)

Aseptic Technique

• “A” = Negative prefix

• “Septic” = Infection

• All techniques and procedures which

__________ _______________

Safe Patterns

• Safe patterns include:

– Standard _______ _________

– ______________ Techniques

– ______ and ________ of Equipment

– _________ Hands

Safe Patterns

• ________ all materials before beginning

Safe Patterns

• Spray the lab top down with a _________ _________ or a ______ _______ __________ and allow this to stand for a _______. You may then wipe down the bench with the paper towel.

Safe Patterns

• Wash hands _______ and _______ lab.

Safe Patterns

• You should have only the _________ ______ ________ and the written _____ __________ on your bench top or desk.

Safe Patterns

• Keep petri dishes and test tubes ___________ as much as possible.

• If top must be removed completely do ______ _____ _____on the lab top. This lowers the probability of contamination and prevents “false positive” results.

Safe Patterns• Hold bottles and tubes ____ ___ _______ to

minimize the amount of airborne microbes that can fall into them (blue circle). Remove the caps as shown above and ___ _____set the caps down. Keep the mouth of the cap facing _________ (red circle).

Safe Patterns• When using metal ________ ________, HEAT

the _______ piece of metal of the inoculation instrument in the flame: it should be RED HOT. Be sure to ________ your inoculation instrument ________ picking the inoculum (broth or agar).

Safe Patterns

• To __________ 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 ______ and __________ from falling onto the agar.

Safe Patterns

• Report _________ to me immediately

• Cover the spill with ______ ________ and squirt _________ onto the towels. Wait _____ _________ then clean up the spill.

Safe Patterns

• ________ all test tubes and petri plates with your name (initials), date, and name of organism ________ you add any solutions, bacteria, etc. 

Safe Patterns

• Do _______ dump ANY microbial suspension down the ______ or in the _______ ______. I will collect them for proper disposal.

Safe Patterns

• Place _____ ________in racks

when working at your table:

never lay the tubes down—____ _____

• Keep test tube ______ and petri dish _______ on media to reduce _______________ (matters not whether it is sterile media or already cultured).

Safe Patterns

• All agar plates are

incubated _______ ______

to reduce bacterial

_______________ and to

reduce the possibility of water

_______________ that may be on the lid

dropping onto the agar, causing fluid to

run across the agar medium.  

Media• __________ after preparation, in

storage, or working containers.

Handle _____________.

• If no goofs – Sterile ___________

E. Autoclave

• _____________ ________

• Water boils at 100ºC, but if pressure is

__________ the boiling point _______

– At 15 PSI the Boiling Point = 121ºC

– At 20 PSI the Boiling Point = 126ºC

• Minimum = ____ minutes @ ____ PSI

• A pressure cooker is the same as an

____________

Boiling

• _________ kills _______ MO, fungi, and

viruses but a few are ____________.

– Example: Inf. Hepatitis, Endospores

• But very good

nonprofessional use

Equipment

Technique depends on article:

Does it _____ or _____?

Do you want it back?

Equipment

1. Autoclave

• If it _____ into machine and ____ ____

• ___ minutes @ 121ºC (15 PSI) minimum

Equipment

2. Boiling

• ________ in a lab

Equipment

Oven

• _____ ______ used for metal and glass

• ____ minutes @ _________ minimum

Equipment

___________ – Burning

• ________ Plus

• Loops, test tubes

Equipment

5. _____________ Equipment

• Usually _______ and ______ materials

• Gas: _______ _______

• ____________