Experiment. No. 5Isolation 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).

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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 grow into a mound of cells-- a colony.

Methods of culturing microbes

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.

1. Start by washing your down your work or lab benches with a surface disinfectant.

The most commonly used disinfectants for lab use are:

1. 10% bleach (recommended by the CDC)

2. 85% ethanol

1. Turn off any forced air heating or air conditioning units that create strong air

current in your work area.2. 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.

3. 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.

Aseptic Technique Method of handling

material without contamination from the environment

Aseptic – What does it mean?

1. Inoculation Methods

2. 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 techniquesInoculation 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. 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

1. wire glows red

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

IV

III 1/5 I1/10

Rotate plate 90

Flame loopRotate 90

Rotate 90III

1/4

Flame loop

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.

MATERIALS :

1. Agar slope 2. Colonies on agar plate

PROCEDURE :1. 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)2. Sterile the mouth of tubes, replug the test tubes

and flame the loop.3. Label and incubate at 37 for 18-24 hour.℃

4. 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

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Holding Tube

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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pellicle

sediment turbid

contrast

RESULTS:Pellicle , sediment and turbid .

METHODS:1. Flame-sterilize inoculating needle.2. 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).

3. Flame the mouth of the tube and reinsert the cotton plug. Flame-sterilize the needle.

4. Label the tube, incubate for 24 hours at 37 .℃5. Observe the result.

RESULTS:

1. 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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