The research work was divided into two sections. In the first section sample collection and serological study were performed. In the second section isolation of Mycoplasma organisms was done. 1. The major works of the present research: Sero-surveillance was carried out to identify the extent of infection (Mycoplasmosis) present in the field with commercial MG antigen. A total of 612 sera samples were collected from 57 poultry farms of 11 districts (Savar, Gajipur, Mymensingh, Manikgonj, Tangail, Sherpur, Bogra, Joypurhat, Khulna, Barisal and Comilla) of Bangladesh. The farms were randomly selected. The sera samples were subjected to Rapid Serum Agglutination (RSA) test using commercial antigen. The test was being carried out at room temperature (20-25?) within 72 hours of collection of serum. For isolation of the organism, samples consisting of infra orbital sinus, trachea, lungs, thoracic and abdominal air sacs were collected aseptically following postmortem examination of the sick or dead birds. After taking the samples into the laboratory, these were grinded by pastle and mortar followed by resuspending in Mycoplasma broth. One drop of representative samples was inoculated onto Mycoplasma agar media and few drops of the samples were inoculated in Mycoplasma broth. Then both the media were incubated for 3-5 days in 5% CO2 incubator. 2. Materials: (1) Cotton swab: Packed sterilized cotton swabs were taken and tubes were used for collection of swabs from air sacs, trachea, lungs, nasal cavity and infra-orbital sinus. (2) Glassware and other appliances: The different types of sterilized glassware and appliances used for the experiment were, Test tubes, Falcon tubes, Syringe and Needle, Eppendrof tubes, PCR tube, Petri dishes, Conical flasks, Pipettes, Incubator, Balance, Micropipette, Spirit lamp, Hand gloves and Bacteriological loop, Micro centrifuge machine, Cylinder, Candle, Vortex machine, Water bath, Scissors, Scalpel, Handle and blade, Ice-boxes. (3) Media for culture: (1) Mycoplasma broth base (Oxide Ltd. Bahingstoke, Hampshire, England): This is a selective medium for Mycoplasma which was used to grow Mycoplasma organisms and it also inhibits the growth of other gram positive and gram negative bacteria. (2) Mycoplasma broth base (Oxide Ltd. Bahingstoke, Hampshire, England): This medium was used as a selective media for Mycoplasma organisms in which Mycoplasma grows easily and shows typical colony characteristics. (3) Mycoplasma selective supplement-G (Oxide Ltd. Bahingstoke, Hampshire, England): This medium was used as a supporting media to enhance the growth of Mycoplasma. (4) Horse serum: Horse blood was selected from the horse of BAU, clinics and CDIL, Dhaka. 120 ml blood was collected in 6 Falcon tubes, 20 ml in each. Falcon tubes were placed in slanting position and after half an hour blood stamp were broken by uaing a stirrer and were then left for four hour. Horse serum was used to compare the growth pattern of Mycoplasma organisms in Mycoplasma agar supplemented with Horse serum with that of supplemented with Mycoplasma supplement-G. (4) Nobilis MG Antigen (Bengal Overseas Ltd.): Nobilis MG Antigen was used for the Rapid Serum Agglutination (RSA) test. 3. Methods: (1) Cleaning and sterilization of required glassware and plastic ware: During course of the research work, properly cleaned and sterilized glassware and plastic ware were used. New and previously used glassware and plastic ware were dipped in 2% sodium hypochlorite solution and left there until cleaned. All sterile glassware and plastic ware were kept in dust free place for further use. (2) Rapid Serum Agglutination (RSA) test using commercial antigen: Rapid Serum Agglutination (RSA) test was done using commercial antigen. The test was carried out at room temperature (20-25? ) within 72 hours of collection of serum. The samples that showed agglutination or clumping within two minutes were recorded as positive for RSA test. (3) Clinical history: Case history was taken from the owner of farm. It was recorded that the affected chickens (broiler and layer) were gradually emaciated; reduced feed comsumption; had tracheal rales. Nasal discharge and coughing; and reduce egg production in laying flocks. (4) Necropsy and collection of samples: Routine necropsy was done and gross lesions were recorded. Dead birds were opened along the mid ventral line, viscera removed and examined grossly. Tissue samples from trachea, lungs, liver, swabs from lung exudates, tracheal exudates, and air sacs were collected aseptically in Mycoplasma broth.(5) Preparation of culture media: (1) Mycoplasma broth base (Oxide Ltd. Bahingstoke, Hampshire, England): 25.5 grams of Mycoplasma broth base powder was added to 1 liter of distilled water and autoclaved at 121? for 15 minutes.It was cooled to 50 ?. The mixture was mixed well and dispensed into 10 ml volumes in sterile screw cap test tubes. (2) Mycoplasma broth base (Oxide Ltd. Bahingstoke, Hampshire, England): 35.5 grams powder of Mycoplasma agar base was added to 1000 ml distilled water in a flask and autoclaved at 121? for 15 minutes for dissolving the medium completely. The medium was then allowed to cool down at 50?. (6) Inoculation of different inoculums of media: The Mycoplasmal broth medium was used for the primary growth of organisms from collected samples. Then these were streaked on Mycoplasma agar and further subcultured on these media for obtaining pure culture of the organisms. Inoculated plates were incubated at 37? in CO2 incubator for 5-7 days. Increased humidity in the atmosphere was obtained by the inclusion of wet cotton in the container to enhance the growth. After 2 or 3 passages they produced typical colony on Mycoplasma media.