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

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Presentation on theme: "CULTIVATION AND IDENTIFICATION OF BACTERIA Doç.Dr.Hrisi BAHAR Istanbul University Cerrahpasa Medical Faculty."— Presentation transcript:

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 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’s contribution)

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

9 Definitions ● Pure Culture ● Pure Culture : a single “strain” of microbe grown in media. ●Strain ● 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 According their production Synthetic media ► Synthetic media Semi- Synthetic media ► Semi- Synthetic media Non - 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 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 ► Nutrients - Energy source - Carbon source - Nitrogen source Mineral salts ► Mineral salts – Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca. A suitable pH ► A suitable pH – 7.2 – 7.4 Accessory growth factors ► 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 1 2 31

24 Agar – 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 98 0 c and solidify at 42 0 c

25 Mueller Hinton Agar for Antibiotic 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 method e 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 proceduresI- Microscopic procedures (wet mount examination ) ► 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 proceduresII- 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

38

39 Identification of bacteria Identification need also to differentiate 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


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