Transcript Sterile Technique & Pure Culture Concept

General Microbiology Laboratory
Sterile Technique & Pure Culture Concept
Why?
To protect your self from contact with bio
hazards
To protect your sample from become
contaminated
To protect others in the lab
In working with microorganisms, we must
have a method of transferring growing
organisms from a pure culture to a sterile
medium without introducing any unwanted
outside contaminants.
 This method of preventing unwanted
microorganisms from gaining access is
termed aseptic technique.
For the most part, bacterial physiology only can
be studied in pure cultures.
The best way to obtain a pure culture is to start
with a single bacterial cell.
This cell then divides quickly, and may produce
millions of cells within 24 hours.
A single unwanted contaminant cell can do the
same thing in an otherwise pure culture, making
the culture useless.
A. ASEPTIC TECHNIQUE
First
 The most commonly used device for
moving bacteria is the inoculating loop.
This is simply a piece of nichrome (an
alloy of nickel and chromium) or platinum
wire with a loop at one end and a handle
at the other.
A similar instrument is the inoculating
needle, essentially the same as the loop,
but with just a straight wire
Leave the Bunsen burner on
during your work
Sterilize both instruments by holding the wire
portions in a flame until they glow red. The
instruments should be allowed to cool in the air
for 10-20 seconds before using them to avoid
killing the inoculum.
 In this way all contaminants on the wire are
incinerated.
Do not blow on the instruments to cool them
Do not touch the instruments to agar to cool
them
Do not lay the loop down once it is sterilized
or it may again become contaminated.
The procedure for aseptically transfer
1. Flame the loop.
2. Without setting the loop down, open the first
culture tube and flame the mouth (why) . Do
not set the cap on the bench. The cap
should be held in the same hand as the
loop.
3. Insert the loop into the culture medium, then
withdraw it.
4. Flame the mouth of the first culture tube
again, and replace the cap.
5. Open the second culture tube and flame
the mouth. Do not set the cap on the
bench. The cap should be held in the
same hand as the loop.
6. Insert the loop into the second culture
tube and spread the culture suspension
(on the loop) inoculum into/onto the
second culture medium.
7. Flame the mouth of the second culture
tube, then replace the cap.
8. Flame the loop and set on the bench.
9. When in doubt about the sterility of an
instrument or container, sterilize it.
Remember
 Bacteria
1.
2.
3.
4.
5.
6.
7.
Are everywhere!
On every surface of the body
Including digestive tract
Harmless
Beneficial
Pathogenic
Absorb nutrients and release toxins that damage cells
and tissues.
8. Bacterial toxins can cause disease even when bacteria
are destroyed
9. Bacteria are Prokaryotes
Five Basic Techniques of Culturing
1. Inoculate
2. Incubate
3. Isolation
4. Examination
5. Identification
Pure Culture Concept
Attempts to identify bacteria in a clinical
sample cannot be done unless isolated colonies
are used.
To obtain well-isolated colonies, it is essential
to disperse the inoculum (sample) on the
surface of an enriched agar plate so that
individual bacteria are well separated from
each other.
 Contaminants = other microorganisms present in the sample
 Isolated colois= a population of millions of cells that are
identical and are descendent from a single founder cell
 Stock Culture = a culture that already contains cells.
 It is used a source of cells from which to inoculate new
cultures.
 culture medium: rich/selective
•
•
•
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growth inhibitors
liquid/solid
temperature
source of energy
sources of carbon, nitrogen, ...
 Aseptic technique:
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sterilization of medium and equipment
proper handling
Necessary equipment
Procedure
1.
2.
3.
4.
5.
6.
With the loop, spread the inoculum back and forth across the
upper 1/4 of the plate, keeping the lines of inoculation very
close together (area 1 in figure below).
Isolated colonies are not expected in this area. Do not use strong
pressure, which will break the surface of the agar. Use the end
of the loop, not its side when streaking. Dispose of the loop in
the biohazard bucket on the bench.
Turn plate approximately 90oC. Streak the plate as indicated in
the figure (area 2) across about 1/4 of the plate. Dispose of the
loop.
Repeat step 2 one or two times more.
In area 3 and/or 4 single colonies should appear.
Label plates on the bottom and incubate inverted at 37oC.
Note: Lids on test tubes are loose.
Always hold the glass test tube (not the lid)
when carrying them.
Streaking
FORMS OF CULTURE MEDIA
1. Broth tube: are tubes containing a liquid medium. A typical nutrient containing
broth medium such as Trypticase Soy broth , nutrient broth After incubation,
growth (development of many cells from a few cells) may be observed as one or
a combination of three forms:
a. Pellicle: A mass of organisms is floating on top of the broth.
b. Turbidity: The organisms appear as a general cloudiness throughout the broth .
c. Sediment: A mass of organisms appears as a deposit at the bottom of the tube.
2. Slant tubes: are tubes containing a nutrient medium
plus a solidifying agent, agar-agar. The medium has
been allowed to solidify at an angle in order to get a
flat inoculating surface .
3. Stab tubes (deeps): are tubes of hardened agar
medium which are inoculated by "stabbing" the
inoculum into the agar .
4. Agar plates: are sterile petri plates that are
aseptically filled with a melted sterile agar medium
and allowed to solidify. Plates are much less
confining than slants and stabs and are commonly
used in the culturing, separating, and counting of
microorganisms.
Agar Plate Culture of a Bacterium
An Agar Slant Tube
Agar Stab Culture of a Bacterium
Inoculation of agar slants and deeps.
Three common forms of agar media.
End of lecture