General methods of studying microorganisms cultivation, isolation, purification and characterization

General Methods of Studying Microorganisms: Cultivation, Isolation, Purification and Characterization

1. General Methods Of Studying Microorganisms: Cultivation, Isolation, Purification And Characterization.

2. Aseptic technique Aseptic technique refers to a procedure that is performed under sterile conditions. AIM: To prevent the access of micro-organisms during the preparation and testing.

3. In The Microbiology Lab We Use Aseptic Technique To: Prevent contamination of the specific microorganism we are working with. Prevent contamination of the room and personnel with the microorganism we are working with.

4. SOURCES OF CONTAMINATION 1. The Atmosphere 2. The Breath 3. The Hands 4. Clothing 5. The Hair 6. The Working Surface 7. Equipment

5. Sample Collection for the isolation of Microbe s Different samples can be collected by using different techniques . Different types of samples are using for the isolation of microbes. Examples A ). Clinical Samples Clinical sample should be collected from the active site of infection and precautions should be taken to exclude surface contamination of the sample. We can use different body fluids or tissue of the body for the isolation of the microbes. Body fluids , i.e : Blood, Urine, Saliva, Pus, Sputum, CSF ( Cerebrospinal fluid), Stool, Semen, Synovial fluid, Pericardial fluid, Peritoneal fluid, Pleural fluid, Vaginal discharge and body Tissue etc.

6. B) Soil Samples C) Water Samples Fresh Water Samples Waste Water Samples D) Food Samples C) Milk Samples pasteurized  Milk Un pasteurized  Milk E) Air etc…

7. CULTURE What is a culture? Population of microorganisms grown under well defined conditions. i.e Nutrients Temperature pH Humidity Time ( Incubation period )

8. C ontinue……. Culture methods The specimens received in the laboratory and Streaking on the plated culture media . The appropriate culture media is selected depending upon the bacteria suspected . The following precautions need to be taken into consideration when the culture methods are processed. Optimal atmospheric conditions ( To provide specific Incubation period ) Optimal temperature ( Most of the bacteria requires a temperature of 37°C for optimal growth ) Growth requirement of the bacteria ( Different bacteria have different growth requirements )

9. Medium Medium is the plural of media. Different types of medium are use for the bacterial culturing . Medium forms Have two forms Liquid ( broth ) media . ( Liquid media are used for growth of pure batch cultures) Solid media ( semi-solid). (solidified media are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. Have two types Selective media ( support / growth specific microbe) D ifferential   media ( Can support & growth both gram positive & negative bacteria)

10. Streaking .

11. Incubation Allow the cultured plate in the incubator at 37°C for 24 hrs for growth. After 24 hrs observe the growth of bacteria on cultured plate.

12. Continue…. After 24 hrs observe the growth of bacteria on cultured plate. Bacterial Growth on the petri-plate

13. isolation The process of screening a pure culture by separating one type of microbes from a mixture is called Isolation .

14. purification What is pure culture? A culture containing only one species of microbe is called pure culture . Importance To isolated a single colony. To isolated desire colony. To get a single colony/ species from a mixed growth culture.

15. Continue….. In this process a growth culture medium is prepared, a single bacterial colony is picked from growth plate by using sterile wire loop and streak on the culture plates. These culture plates will keep in the incubator at 37°C for 24 hrs, for growth. After 24 hrs, billions colonies of a single bacterial strain will appear on the culture plates.

16. Characterization & IDENTIFICATION Morphological Identification Morphology: shape , size, color of colony and Gram staining Biochemical Identification Different types of biochemical test are perform for the identification of gram (+) and gram (-) bacteria . i.e. Catalase , Coagulase, Urease, Oxidase Activities, TSI, carbohydrate fermentation and Indole production tests.

17. Summery of the chapter

18. SAMPLE COLLECTION From any sources ISOLATION Pure Culturing technique are use for the isolation of bacterial strain and nutrient agar media IDENTIFICATION I dentification by two methods.

19. IDENTIFICATION M orphological I dentification Biochemical Identification Morphology , shape, size, color of colony and Gram staining. Catalase, Coagulase, Urease, Oxidase Activities, TSI, carbohydrate fermentation and Indole production tests.

20. End Amjad Khan Afridi Date: 20/12/2018