Aseptic Technique
- Aseptic = free of contamination
- Purpose:
- To keep our cultures from becoming contaminated
- To keep the lab environment from becoming contaminated
- To keep us from becoming infected by our cultures
Labeling cultures
- Always include:
- The name of the organism or specimen
- Your name or initials
- The date inoculated
- For broths/slants, label so you can still see growth
- For plates, label on the bottom, the side with the growth medium
- Label before you inoculate
Sources of Contamination
From our culture’s point of view:
- us
- the air
- objects & surfaces in the environment
From our point of view:
- the culture
Materials we’ll be using today
- Culture Media
- Broths (1 per student)
- Slants (1 per student)
- Plates (2 per student)
- Inoculating Loop
- Bunsen Burner
- Flint Striker
Demonstration of slant to broth transfer
(See Ex1-3, p. 32-37 in lab manual)
- Tube of broth & labeling
- Aseptic technique
- Inoculum
- Transfer to fresh broth
- Incubation at 37°C
Demonstration of slant to slant transfer
- Slant & labeling
- Aseptic technique
- Inoculum
- Transfer to fresh slant
- Incubation at 37°C
Streak Plate Method
- Purpose: To check or establish the purity of a culture
- Pure culture = contains a single species
- Mixed culture = contains more than 1 species
- Principle: Organisms are diluted out over the surface of a growth medium, so individual cells will grow to form visible colonies
- Colony = a visible mass of cells
- 1 cell = 1 colony-forming unit (CFU);
- The assumption is that 1 cell grows to form 1 colony
Streak Plate Method (see p. 43)
Quadrant Streak Method
* FLAME
Heat loop
Inoculate culture
Streak
1st quadrant
A good streak plate
- Is appropriately labeled
- Has appropriate pattern for streak lines
- Uses the entire plate
- Has well-isolated colonies
- Is free of contamination
- One colony type
Demonstration of Streak Plate Method
- Agar plate & labeling
- Aseptic technique
- Inoculum
- Streak pattern
- Incubation (inverted) at 37°C
For your review:
- Video of aseptic technique (2 min)
- Video of streak plate method (1½ min)
Expectations: Work as individuals
- Aseptically transfer from stock slant culture to a fresh broth and to a fresh slant – use “S. marcescens ” or “M. luteus” or “E. coli” or “S. sapro”
- Label appropriately with the organism, your name or initials, & date
- Aseptically Streak 1 plate using the quadrant method
- Use “culture used above”
- Label appropriately
- Incubate in plate in inverted position
- Aseptically Streak a 2nd plate using the quadrant method
- Use “mixed culture”
- Label appropriately
- Incubate in plate in inverted position
- Put all your cultures into the incubator (37°C), on the shelf for this section
Expectations: Work as individuals
- After inoculations are complete, Work on Simple Stain from last week
- Ex. 3.5, pg. 186-187 Making a Bacterial Smear and Simple Stain procedure.
Gather around for demonstration
(use real inoculum)
- Broth to broth transfer
- Plate to slant transfer
- Streak plate method
