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Aseptic Technique & Pure Culture

In natural environments, microorganisms usually exist as mixed


populations. However, if we are to study, characterize, and identify
microorganisms, we must have the organisms in the form of a pure
culture.
A pure culture is one in which all organisms are descendants of the
same organism. A culture in which only one strain or clone is present.
Techniques for obtaining pure cultures from a mixed population will
be described later.
In working with microorganisms we must also have a sterile nutrientcontaining-medium in which to grow the organisms. Anything in or on
which we grow a microorganism is termed a medium.
A sterile medium is one which is free of all life forms. It is usually
sterilized by heating it to a temperature at which all contaminating
microorganisms are destroyed.

Finally, 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.

In most cases, bacterial physiology only can be


studied in pure cultures.
A pure culture is usually obtained from a mixed
culture. 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.

Obtaining pure culture from a mixed


culture

Pure culture

For this reason, and to protect against disease,


strict sterile procedures must be used.

ASEPTIC TECHNIQUE

Sterilizing Instruments
Bunsen Burner Flame

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 aseptic transfer


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Flame the loop.


Without setting the loop down, open the first 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.
Insert the loop into the culture medium, then withdraw it.
Flame the mouth of the first culture tube again, and replace the cap.
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.
Insert the loop into the second culture tube and spread the culture suspension
(on the loop) inoculum into/onto the second culture medium.
Flame the mouth of the second culture tube, then replace the cap.
Flame the loop and set on the bench.
When in doubt about the sterility of an instrument or container, sterilize it.

Aseptic Transfer
Removing Cap

Flaming Tube

Holding Tube

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Mixing Broth Cultures


Vortex Mixer

Hand Mixing

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Broth to Broth Transfer


Move the tube/not loop

Film (culture) on loop

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Plate inoculation
Invert Plate

Streaking Plate

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Video:
Aseptic technique
Streak plate

The 6 Is in microbiology techniques

1. Inoculate
2. Incubate
3. Isolation
4. Inspection
5. Information gathering
6. Identification

An overview of some general laboratory techniques carried out by

An Overview of Microbiology Techniques

1. Inoculation
Introduction of a small sample inoculum
into a container of media to produce a
culture of observable growth

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Culturing Microorganisms
Obtaining Pure Cultures
Cultures composed of cells arising from a single
progenitor
Progenitor is termed a CFU

Aseptic technique prevents contamination of


sterile substances or objects
Two common isolation techniques
Streak plates
Pour plates

2012 Pearson Education Inc.

Figure 6.9 Streak plate method of isolation-overview

Streaking Plate

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Figure 6.10 Pour plate method of isolation-overview

2. Incubation
Temperature-controlled chamber
Microbe multiplies and produces
macroscopically observable growth

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3. Isolation
If an individual bacterial cell is separated
from other cells and has space on a nutrient
surface, it will grow into a mound of cells-a colony.
A colony consists of one species.

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Figure 6.8 Characteristics of bacterial colonies-overview

4. Inspection
Inspection observation; macroscopic and microscopic
Pure culture grows only single known species of
microorganisms
Mixed cultures hold two or more identified species or
microorganisms
Contaminated culture once pure or mixed culture that
has unwanted microbes growing

macroscopic and microscopic appearance,


biochemical tests, genetic characteristics,
immunological testing
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Various conditions of cultures

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5. Information gathering

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6. Identification

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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 colonies= 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
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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

Remember

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Bacteria
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