Isolation and Identification of Microorganisms PDF

ISOLATION AND IDENTIFICATION OF MICROORGANISMS Assist. Prof. Dr. Halit Şükür Dr. Hazel Tamakan Yeşilovalı  Most of the diseases are caused by infection by microorganisms. Microorganisms are found in blood, urine, feces, etc. in the continuation of the disease. The fir...

ISOLATION AND IDENTIFICATION OF MICROORGANISMS Assist. Prof. Dr. Halit Şükür Dr. Hazel Tamakan Yeşilovalı  Most of the diseases are caused by infection by microorganisms. Microorganisms are found in blood, urine, feces, etc. in the continuation of the disease. The first objective in the diagnosis of infectious diseases due to microorganisms is to identification the microorganism that makes the disease from the biological materials of the patient in the laboratory.  Disease-causing microorganisms; They are produced in artificial environments called laboratory media, similar to natural habitats. The microorganisms in the samples taken from the patient are isolated from the causative agents in the medium where they are produced. Isolated microorganisms are identified on different media by examining their physiological, morphological and biological properties.  Non-living environments prepared for the production of microorganisms under laboratory conditions, suitable for their development and reproduction, containing the necessary nutrients are called "medium". The vast majority of bacteria and fungi can be produced in such inanimate environments. For this purpose, sterile prepared media are stored in the refrigerator to prevent dehydration and denaturing.  There are hundreds of different media used to produce bacteria. It is necessary to know where the sample to be sown is taken, the m.o. which can be found in the sample and the nutritional requirements of these m.o. For example, blood agar is suitable for the growth of a large number of bacteria, but it is necessary to add enriching or inhibiting substances to the medium to produce certain bacteria.  These are used in the growth of m.o.; A. Live environments -Tissue cultures -Experimental animals -Embryonated chicken eggs *Viruses *Rickettsia spp. *Chlamydia spp. B. Inanimate enviroment -Mediums  A)Live Environments 1. Tissue and Cell Cultures: Tubes, bottles, petri boxes and laboratory containers such as these are produced in appropriate nutrient fluids are used in living tissues. 2. Embryonated chicken eggs: Animal origin of the egg (chicken, duck, turkey), inoculation paths (chorio-allantoic membrane, amniotic cavity, allantoic cavity, yellow sac) and age of embryo; depending on the desired factor. 3. Experimental Animals: In the isolation and identification of bacterial and viral agents, experimental animals are the leading organisms. These are mostly mice, guinea pigs, rabbits, hamsters, chickens and other animals. Depending on the nature of the agent contained in the inoculated material, the species, age, genus and inoculation paths of the animal change. -Conventional Animals: Animals that do not undergo any health check and are used for general purposes. Since no special nutritional and maintenance is applied other than feed and water, they can keep apathogenic and pathogenic agents in their systems and skins. The results of studies on these animals are not reliable. -SPF(Specific Pathogen Free) Animals: The systems of these animals may contain various microorganisms, but are clean for pathogenic microorganisms (Tuberculosis, Salmonellosis and some viral agents). Microbiological investigations of the feed and water to be given for the rearing of these animals should be made and the air should be filtered. The results of studies with these animals are more reliable. -Germ Free) Animals: They do not contain any apathogenic or pathogenic microorganisms, nor should they form antibodies in their blood. Germ-free animals are raised in special air control units (Thord Gustafsson) by cesarean operation. The results are much more reliable.  B)Inanimate Environments: The media are sterile environments that allow the in vitro reproduction and growth of microorganisms (except Rikettsia, Chlamydia and some viruses). Mediums are formulated for the development of microorganisms. These include development of microorganisms, isolation, identification, counting, susceptibility testing, sterility testing, examination of clinical specimens, food, water and environmental controls, obtaining biological products, antibiotic and vitamin analysis, industrial analysis and so on. for different purposes.  MEDİUMS General media: Microorganism has enough carbon and energy resources to produce. Ex: blood agar Broth (liquid medium): ‘bouillon’ used to boil in French. Broth means broth used for the growth of bacteria. Agar (solid medium): Extracted from various seaweeds is a dry, sticky substance. Special media: Selective media: Adding substances such as stain and antibiotics to prevent the growth of unwanted microorganisms. Ex; Crystal Viole added to the media inhibits the growth of Gram positive bacteria; Substances such as potassium tellurite and sodium azide inhibit the growth of Gram negative bacteria. Ex; Brucella agar, Mycoplasma agar Differential media: Provides the identification of breeding colonies according to their color. For example; MacConkey agar, EMB agar Enrichment media: They are mostly liquid media which prevents the growth of unwanted microorganisms and produces much microorganisms. For example; tetrathionate broth. Media used for the determination of biochemical properties: Identification media are used to determine whether the microorganism uses a particular nutrient (eg, lactose), to determine whether a particular nutrient forms metabolites (e.g., indole from tryptophan), which can be determined at the end of metabolism. used with purposes. Ex; TSI Transport media: In order to isolate bacteria that are sensitive to environmental conditions, they must be delivered to the laboratory on their special media. These media are used to protect the activities of bacteria during this transportation. Ex; Amies, Stuart Medium Special Purpose Media: There are various media used for special purposes such as antimicrobial susceptibility and minimal inhibition concentration tests by agar disc diffusion, microbial determination of vitamins and amino acids, preservation (collection) and transportation of pure cultures.  Substances entering the composition of the media:  Agar  Meat extract  Gelatine  Casein hydrolyzate  Yeast extract  Peptone  Serum  NaCl  Carbon resources  Distilled water  * Blood: 5-10% is added to the media. It should be taken sterile and added when the medium has cooled down to 45 ºC.  ISOLATION AND IDENTIFICATION OF MICROORGANISIMS ISOLATION: It is the production of various factors that may be a causative agent, either in vivo or in vitro for diagnostic purposes. IDENTIFICATION: Some characteristics of isolated agents are identified according to their criteria.  DIAGNOSIS A. Anamnesis B. Clinical examination C. Findings of necropsy D. Laboratory diagnosis  D.Laboratory Diagnosis 1.Bacterioscopy: Examine the microscopic morphology of microorganisms by making various staining from the samples. 2.Culture: It is the production of microorganisms at the appropriate temperature and time by planting the solid or liquid media from the materials delivered. 3.Biochemical Tests: Determination of bacterial specific biochemical activities by various tests. 4.Experimental Animals: The suspensions prepared from microorganisms are administered to suitable test animals by appropriate means and used for pure recovery of the agents or to study their effects.  5.Serological Tests: Serum tests are usually based on the detection of antibody titer. 6.Allergic Tests: It is based on the administration of specific allergens to the suspected disease to the animals and monitoring of the reactions that occur. Ex; Glanders, Tuberculosis 7.Antibiotic Susceptibility Tests: The procedure for determining which antibiotics are sensitive to isolated and identified agents is called “antibiogram’.