CULTIVATION AND IDENTIFICATION OF BACTERIA Doç.Dr.Hrisi BAHAR Istanbul University

1. CULTIVATION AND IDENTIFICATION OF BACTERIA Doç.Dr.Hrisi BAHAR Istanbul University Cerrahpasa Medical Faculty

2. WHAT IS CULTIVATION OF BACTERIA ►The survival of microorganisms in the laboratory, as well as in nature, depends on their ability to grow under certain chemical and physical conditions ►Cultivation of bacteria is to obtain a bacterial growth under certain chemical and physical conditions .

3. BACTERIAL GROWTH ► An increase in population number of a bacteria by reproduction ► Not an increase in cell size ► Most bacteria reproduce by Binary Fission ◌ The cell doubles in size ◌ Replicate the chromosome ◌ Forms a septum in the center ◌ Synthesizes a cell wall at the septum ◌ Daughter cells separate

4. PURPOSE OF CULTURING BACTERIA ◌ Isolation of bacteria ◌ Understand properties of bacteria ◌ To create antigen for laboratory use ◌ To test for antibiotics sensitivity ◌ Estimate viable counts ◌ Maintain stock cultures ◌ Typing with bacteriophages and bacteriocins susceptibility

5. A CULTURE MEDIA A culture media is a combination of nutrients used to growth organisms outside of their natural environment. Culture media are employed in the ►Isolation and maintenance of pure cultures of bacteria ►Identification of bacteria according to their biochemical and physiological properties.

6. Major Contribution to Culture Media is provided by

7. Agar - Agar (Frau Hesse’scontribution)

8. Why we need culture media ? ►Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. ►The chemicals and elements of this environment that are utilized for bacterial growth are referred to asnutrientsornutritional requirements. ►In the laboratory, bacteria are grown inculture mediawhich are designed to provide all the essential nutrients in solution for bacterial growth.

9. Definitions ● Pure Culture : a single “strain” of microbe grown in media. ●Strain: A microbial culture which is the descendent of a single cell originally isolated from the environment. ●Aseptic technique:Method of hendling material without contamination from the environment.

10. Classification of Culture Media According their production ► Synthetic media ► Semi- Synthetic media ► Non - Synthetic media According their usage ◌MINIMAL MEDIUM ◌ALL-PURPOSE MEDIUM ◌SELECTIVE MEDIUM ◌DIFFERENTIAL MEDIUM

11. A MINIMAL MEDIUM ● A minimal medium is one which supplies only the minimal nutritional requirements of a particular organism . ● These media vary in composition according to the minimal nutritional requirements of the particular species under study

12. AN ALL- PURPOSE MEDIUM ● This medium is rich in a wide variety of nutrients (including many growth factors) and will, therefore, support the growth of a wide range of bacteria ◌Chocolate Agar ◌ Nutrient Agar ◌ Brain Heart Infusion Agar ◌ Blood Agar

13. Blood culture – ‘Liquid Medium’

14. Chocolate agar

15. A SELECTIVE MEDIUM ● This media supports the growth of desired organisms while inhibiting the growth of many or most of the unwanted ones ◌MacConkey Agar. ◌Salmonella,shigella medium

16. Salmonella Shigella agar

17. Lowenstein Jensen Medium - cultivation of Mycobacterium tuberculosis

18. A DIFFERENTIAL MEDIUM ●This medium is one which allows two or more different types of organisms to grow. It allows identification of bacteria based on specific properties. ●It contains dyes and/or other components upon which different organisms act in various ways to produce a variety of end products or effects, often detected by variations in color. ◌Glucose Fermentation Broth ◌Three sugar iron agar

19. Three sugar iron Agar

20. Anaerobic Culture Methods Anaerobic jar • Anaerobic jar Figure 6.5

21. Basic requirements of culture media ►Nutrients - Energy source - Carbon source - Nitrogen source ►Mineral salts – Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca. ►A suitable pH – 7.2 – 7.4 ►Accessory growth factors - Tryptophan for Salmonella typhi - X & V factors for H. influenzae

22. Major elements, their sources and functions in bacterial cells.

23. According to physical properties, media can be classified as three types: 1. solid medium ( 1.5～2.5% of agar)agar plate 2. semi-solid medium (0.3～0.5% of agar) 3. liquid medium 2 1 3 1

24. Agar – Agar ► Solid medium is made by adding agar ► Agar is obtained from Sea weeds of New Zealand ► Agar contain long chain of polysaccharides, Inorganic salts and protein like substances ► Melts at 980c and solidifyat 420c

25. Mueller Hinton Agar forAntibiotic Testing is a solid medium

26. Liquid Medium ► Difficult to identify all types of organisms ► Suitable for isolation of bacteria from Blood culturing or bacteria existing in small number.

27. IDENTIFICATION OF BACTERIA ►The identification process started by isolating pure colony by Quadrant streaking method on an appropriate culture media for the type of bacteria being targeted to be isolated. This means: ►To get suitable culture media that will allow the growth of certain species and inhibit others or ►To follow the standard and proper culturing method that enables us to isolate a pure colony. 27

28. Quadrant streaking methode method Figure 4.2

29. Identification of bacteria 29

30. Identification of bacteria ●Once we have got a pure colony the next step is to identify the bacterial species. 30

31. Identification of bacteria We start to accumulate information about the organism like: ► Morphological characteristics ► Culture characteristics and ► Physiological ( biochemical) characteristics

32. Bacterial Identification procedures I- Microscopic procedures II- Macroscopic procedures • I- Microscopic procedures ►Examination of unstained preparations (wet mount examination )for bacterial motility. ►Staining a preparation(Gram,E.ZN,GIEMSA) Gram staining will determine: a- Bacterial Gram reaction b-Bacterial morphology c- Bacterial strain arrangement

33. 33

34. Bacterial Identification procedures • II- Macroscopic procedures 1- Gram reaction confirmation test • On a clean slide one or two colonies are mixed with two or three drops of 4% sodium hydroxide solution: • Gram negative bacteria will produce a mucoid line when strengthen while gram positive will not produce it. 34

35. Bacterial Identification procedures 35

36. Identification of bacteria 2- Culture appearance : a- The appearance of bacterial colonies b- The effect of colony on culture media are often characteristic of the bacterial species. 36

37. Identification of bacteria • a- The appearance of the colonies is described using conventional terms and may include: • Shape of the colony • Size of the colony • Elevation of the colony • Surface of the colony • Color of the colony • Opacity of the colony • Consistency of the colony

39. Identification of bacteria Identification need also todifferentiate species following their aspects like: 1-Requirement of oxygen 2-The need of co2 3-Capacity to form pigments 4-Power of hemolysis

40. Identification of bacteria • b- The colony effects on culture media: • 1- Pigment production: • Pigment producing organisms will have colored colonies .The development of the color will depend on the type of the medium. 2-Beta hemolyses 40