Transcript Flame

Experiment. No. 5
Isolation technique with aseptic techniques to cultivate
bacteria
Prepared By
AYMAN SERALKHITIM YOUSIF
M.SC MEDICAL MICROBIOLOGY & IMMUNOLOGY

The process of growing microorganisms in
culture by taking bacteria from the infection
site (in vivo or environment) and grow them
in artificial environment (Culture media ) in
the laboratory (in vitro).
Methods of culturing microbes
1.
Inoculation :
Introduction of a sample into a container of media
to produce a culture of observable growth.
2.
Incubation :
Under conditions that allow growth ( 35- 37 C°).
In the incubator .
3.
Isolation :
Separating one species from another .
If an individual bacterial cell is separated from other
cells and has space on a nutrient surface, it will
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grow into a mound of cells-- a colony.


Inspection
Growth characteristics (color, texture, size) .
Macroscopic
slides (cell shape, size, motility). Microscopic
Identification :
Biochemical tests, immunologic test, DNA analysis

When culturing bacteria or other microorganisms,
it is important to keep your work area as clean as
possible.

This prevents the introduction of other
microorganisms from the environment into your
culture.

The techniques used to prevent contamination are
referred to as sterile techniques.
Start by washing your down your work or
lab benches with a surface disinfectant.
1.
The most commonly used disinfectants for lab
use are:
1.
2.
10% bleach (recommended by the CDC)
85% ethanol
1.
2.
3.
Turn off any forced air heating or air
conditioning units that create strong air
current in your work area.
A small room or closet that can be closed off
is worth the effort to set-up if you will be
doing a lot of microbial culturing.
You can install a UV bulb in a fluorescent
light fixture to surface sterilize your work
bench if you have an enclosed area(?).
Remember to leave the area when you turn
on the UV light source!
5.
All glassware should be cleaned and sterilized
before you begin.
6.
All pipettes, spatulas, and test tube (culture)
racks should also be sterilized.
7.
You can purchase sterile, disposable culture
tubes, petri dishes, and pipettes to minimize the
quantity of glassware that you have to sterilize.
8.
Don’t forget to wash you hands after you finish
cleaning and put on a pair of sterile disposable
gloves before you begin.
9.
Once your work area is clean, your hands are
clean, and your glassware is clean and sterile,
don’t contaminate the work area by placing “dirty
items” such as pencils, pens, notes, or books in
the sterile work area.


1.
2.
Aseptic Technique
Method of handling
material without
contamination from the
environment
Aseptic – What does it
mean?
Inoculation Methods
Inoculation Tools
1- working in 20 cm diameter
around a blue flame (sterile
zone)
2- Never leave a culture dish open,
even for a short time ,When it is
necessary to open a dish, keep
the lid close to the dish, and keep
the lid between your face and the
agar surface.
3- For most bacterial cultures you
will use a sterile loop or needle
to inoculate or to obtain an
inoculums.
4- Flame a loop or needle to red-hot
just prior to use, burning off any
organic material ,Cool the loop
by touching the sterile agar or
liquid surface prior to touching a
culture
Bunsen Burner Flame
Flaming Loop – angle is wrong –
should be inverted
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Inoculation and transfer techniques
 Streak plate ------ Isolation and culture
 Slant inoculation ------- Pure culture
 Liquid medium inoculation ------ Pure culture
 Semisolid medium inoculation ------ Pure culture
Used for the isolation of bacteria in pure
culture from clinical specimens.
 MATERIALS：
1. Bacterial broth (Nutrient broth)
2. Colonies on agar plate
 PROCEDURE:
1. wire glows red

1. Flame your inoculating loop until the
wire glows red.
2. Allow the loop to cool and get a loopful
of the suspension of sample.
3. Pick up your plate and streak the
surface, Flame the loop before
streaking next section.
When streaking, be care not to cut into
the agar and not to be far away from
flame.
3. Streaking surface

PROCEDURE:
4. Cover the Petri dish, invert the plate. Sterilize the loop,
label your name, date et al.
5. Incubate the plate at 37℃ for 18-24 hours.
6. Observe the bacteria colonies.
4. Invert Plate
four-area streak plate technique
I
1/10
I
II
1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
Rotate 90
III
1/4
IV
1- Area of initial inoculation
and the first streak yield
heavy growth .
2- Area of second streak from
area 1 yield less dense
growth.
3-Area of third streak from
area 2 yield weak growth.
4- Area of fourth streak from
area 3 yield single colonies
(discrete colonies ) (pure
colonies )
(pure culture)

Colony- A bacterial population derived from one
bacterial cell. The cells within the colony have
identical, genus, species, genetic and phenotypic
characteristics.

Pure bacteria (Pure Culture ) - derived from a
single colony.

Selection of a pure colony -most important for
bacterial identification





To isolate microorganism from
heterogeneous or mixed population
To study morphology of bacteria
To study role played by specific
microorganisms.
For identification of species
Cultivation and Multiplication

Stroke culture is made in tubes
containing agar slope / slant.

Uses
 Provide a pure growth of bacterium
for slide agglutination and other
diagnostic tests.


1.
2.
3.
4.
MATERIALS :
1. Agar slope
2. Colonies on agar plate
PROCEDURE :
With the flame-sterilized wire inoculating loop,
transfer a small amount of bacteria from the
colony on agar plate. Then streak on the agar
slope. ( DIP & Zeg Zag)
Sterile the mouth of tubes, replug the test tubes
and flame the loop.
Label and incubate at 37℃ for 18-24 hour.
Observe your result.

RESULTS :
There are many similar wet colonies
on the surface.
If there are some other forms, it
indicates culture sample is not pure.
Removing Cap
Flaming Tube
Holding Tube
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

MATERIALS：
1. Nutrient broth
2. Colonies on agar plate
PROCEDURE：
1. Flame-sterilize the wire inoculating
loop.
2. Insert the wire loop containing a small
amount of bacteria into the liquid
culture tube.
3. Scratch the wall of tube over the broth
in order to let bacteria drop into the
liquid.

PROCEDURE：
4. Flame the mouth of the tube and reinsert the cotton plug.
Flame-sterilize the wire loop.
5. Label the tube, incubate at 37℃ for 24 hours
6. Observe the result.
Move the tube/not loop
Film on loop
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Vortex Mixer
Hand Mixing
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RESULTS:
Pellicle , sediment and turbid .
pellicle
contrast
sediment
turbid

1.
2.
3.
4.
5.
METHODS:
Flame-sterilize inoculating needle.
Insert the needle with a small bacteria to the center of the culture,
be care not to touch the bottom of the tube, then draw it out in the
same way. (DIP).
Flame the mouth of the tube and reinsert the cotton plug. Flamesterilize the needle.
Label the tube, incubate for 24 hours at 37℃.
Observe the result.

1.
RESULTS:
Motile bacteria will migrate from the line of
inoculation to form a diffuse turbidity in the
surrounding medium.
2. Non motile bacteria will grow only along the
line of inoculation.
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