Aseptic technique + pure culture technique

what is the aseptic technique

set of specific procedures used to prevent the contamination of sterile media

Disinfectant

chemical used to kill pathogens and microorganisms on surfaces

Sterilizations

removal of all microorganisms + endospores

Inoculation + inoculating tools

introducing microorganisms into a growth medium (agar plate)

• tool used to transfer microorganisms

• tool used for stab cultures

Culture media

Broth - liquid growth medium in test tube

• bacteria grow throughout liquid

Agar slant - solid growth medium in a tube that is angled while cooling

Agar plate - Solid growth medium in a flat Petri dish

• nutrient agar plate with bacterial colonies

Why do we flame the inoculating loop or needle

we heat till red hot to sterilize the instrument preventing cross contamination

• then cool off instrument before collecting to prevent killing of microorganisms

how to collect microorganism from test tube

1. gently shake test tube to mix the culture

2. remove cap using pinky finger of hand holding loop (Do not place cap on table_

3. Flame mouth of tube using bunsen burner

4. insert loop into culture to collect microorganisms

5. insert loop and remove without touching sides of wall

   1. flame mouth of tube again, place cap, and flame loop again to sterilize

incubation factors for aseptic technique

• agar plates incubated upside down to precent condensation

• 37 C because many bacteria grow well at body temp promoting bacterial growth and reproduction

  • 24-48 hours for enough time for growth and reproduction of bacteria

• Agar slants

• zig zag pattern to spread bacteria across the agar plate and giving them space to grow

  • makes patterns easier to see and increases Surface coverage so bacterial growth is easier to observe

Different types of culture

Pure culture - only contains one kind of organism

Mixed culture - more than one kind of organism

Contaminated culture - desired organism plus unwanted organisms

Purpose of isolation technique (streaking)

isolating pure culture ( only one kind of culture)

• to separate bacteria from a mixed culture to obtain isolated colonies to be able to study and identify an individual bacteria without other bacteria interfering

Quadrant streak plate

1. Streak the first area with the original bacterial sample

2. Flame the loop and let it cool

3. open agar plate lid at angle to minimize contamination from microorganisms in the air

4. drag bacteria from previous area into second area

5. repeat for third and fourth area

6. flame loop to sterilize again

• Flame loop between quadrants to reduce the number of bacteria carried into the next area

• to separate bacteria to obtain isolated colonies

Subculturing

transferring an isolated colony to fresh sterile medium to establish a culture for further study

incubation temperature for pure culture method

22C (room temp) not 37 because the lab uses conditions appropriate for organisms being studied