Inoculation of Culture Media

• Inoculation- adding a portion of the specimen to the medium.
• Involves the use of sterile inoculating loop to apply a portion of the specimen to the surface of the medium; a process commonly referred to as “streaking”.

Materials

1. petri dishes
2. test tubes
3. bunsen burners/alcohol lamps
4. wire inoculating loops
5. bottles of staining reagents
6. incubators

Importance of Using “Sterile Technique”

• Necessary to exclude all microorganisms from a particular area, so that area will be sterile.
• Media should remain sterile before inoculation.
• Contaminants- unwanted microorganisms
• Contaminated-if the sample contains contaminants

Streaking the Agar Plate: Simple Streak

• Flame sterilize the inoculating loop then let it cool for a few seconds, afterwards fish out a loopful of specimen from pure culture.
• Hold the sterile Petri dish with cover by your left hand, partially open the agar plate near the flame of the alcohol lamp.
• Place a loopful of specimen on one side of the agar medium away from you and streak the culture back and forth from edge to edge of the plate,
• When the entire medium has been streaked, flame sterilized the cover of the plate completely.
• Label the Petri dish then incubate for 24-48 hours at 37 C.

Inoculating the Agar Slant

• Fish out a loopful of bacterial culture using a flamed sterilized loop.
• Hold the agar tube with the left hand and screw cap should be held by the small finger. Never lay the screw cap of the test bacterial specimen anywhere.

• Pass the mouth of the test tube through the alcohol lamp’s flame. Flame immediately and inoculate the agar surface by moving the inoculating loop from the bottom to the top of the agar slant or in a snake-like motion.
• Withdraw the inoculating loop and immediately flame-sterilize it. Pass the mouth of the agar tube on the alcohol lamp’s flame.
• Sterilize the screw cap and cover the agar tube.
• Label the agar slant tube and place it in the test tube rack, then incubate.

Inoculating the NB

• Fish out a loopful of bacterial culture using a flamed sterilized loop.
• Hold the agar tube with the left hand and screw cap should be held by the small finger. Never lay the screw cap of the test bacterial specimen anywhere.
• Inoculate the medium from top to bottom. Assume equal distribution of inoculums by suspending the inoculating loop into the broth, then shake.
• Flame sterilize the mouth of the test tubes before and after inoculation.
• Sterilize the screw caps, cover, and label the broth culture, then incubate.