Transcript Sterile Technique & Pure Culture Concept Why

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.
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
• 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. Are everywhere!
2. On every surface of the body
3. Including digestive tract
4. Harmless
5. Beneficial
6. Pathogenic
7. 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
• growth inhibitors
• liquid/solid
• temperature
• source of energy
• sources of carbon, nitrogen, ...
• Aseptic technique:
• 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 form:
2.
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