1. Isolation technique with aseptic techniques to cultivate bacteria
Experiment. No. 5
Isolation technique with aseptic techniques to cultivate bacteria
Prepared By
AYMAN SERALKHITIM YOUSIF
M.SC MEDICAL MICROBIOLOGY & IMMUNOLOGY

2. Cultivation
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).

3. 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 ( 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. 3 3

4. Methods of culturing microbes
Inspection
Growth characteristics (color, texture, size) . Macroscopic
slides (cell shape, size, motility). Microscopic
Identification :
Biochemical tests, immunologic test, DNA analysis

5. Sterile Technique

6. Sterile Technique
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.

7. Sterile Technique
Start by washing your down your work or lab benches with a surface disinfectant.
The most commonly used disinfectants for lab use are:
10% bleach (recommended by the CDC)
85% ethanol

8. Sterile Technique (2)
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!

9. Sterile Technique (3)
All glassware should be cleaned and sterilized before you begin.
All pipettes, spatulas, and test tube (culture) racks should also be sterilized.
You can purchase sterile, disposable culture tubes, petri dishes, and pipettes to minimize the quantity of glassware that you have to sterilize.

10. Sterile Technique (4)
Don’t forget to wash you hands after you finish cleaning and put on a pair of sterile disposable gloves before you begin.
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.

11. Aseptic Technique Aseptic Technique
Method of handling material without contamination from the environment
Aseptic – What does it mean?
Inoculation Methods
Inoculation Tools

12. Aseptic technique involves:
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

13. Sterilizing Instruments
Bunsen Burner Flame
Flaming Loop – angle is wrong – should be inverted 13

14. Inoculation and transfer techniques
Streak plate Isolation and culture
Slant inoculation Pure culture
Liquid medium inoculation Pure culture
Semisolid medium inoculation Pure culture

15. 1-Streak Plate Method
1. wire glows red
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

16. 1-Streak Plate Method 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 hours.
6. Observe the bacteria colonies.
4. Invert Plate

17. Streak-plate method

18. 1. Streak-plate technique
four-area streak plate technique I
1/10 II I
1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
III
1/4 IV
Rotate 90

19. Results of Streak plate technique
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)

20. Colony- (clone)
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

21. Advantages of pure culture techniques
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

22. Morphological and pure culture studies22

23. Use of a Plate Counter for Estimating Microbial Populations

24. 2- Slant inoculation or STROKE CULTURE (Agar Slope Method )
Stroke culture is made in tubes containing agar slope / slant.
Uses
Provide a pure growth of bacterium for slide agglutination and other diagnostic tests.

25. 2- Slant inoculation or STROKE CULTURE (Agar Slope Method )
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 hour.
Observe your result.

26. 2- Slant inoculation or STROKE CULTURE (Agar Slope Method )
RESULTS :
There are many similar wet colonies on the surface.
If there are some other forms, it indicates culture sample is not pure.

27. Aseptic Transfer
Flaming Tube
Removing Cap
Holding Tube 27

28. Slant observation

29. 3- Liquid medium inoculation (Liquid Medium Culture )
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.

30. 3- Liquid medium inoculation (Liquid Medium Culture)
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.

31. Broth to Broth Transfer
Move the tube/not loop
Film on loop 31

32. Mixing Broth Cultures
Vortex Mixer
Hand Mixing 32

33. Pellicle , sediment and turbid .
RESULTS:
Pellicle , sediment and turbid .
pellicle
contrast
turbid
sediment

34. 4- Semisolid medium inoculation (Semisolid medium Culture )
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. Flame-sterilize the needle.
Label the tube, incubate for 24 hours at 37℃.
Observe the result.

35. 4- Semisolid medium inoculation (Semisolid medium Culture )
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.

36. 36 36