4. Laboratory methods for identification of microorganisms (Microbiology)

Laboratory methods for identification of microorganisms for B.Sc. Nursing

1. Laboratory methods for identification of microorganisms

2. Identification of unknown bacteria is one of the major responsibilities of the microbiologists. Samples of blood, tissue, food, water and other are examined daily in laboratories throughout the world for the presence of microorganism. With some fundamental knowledge of staining methods, isolation techniques, bacterial nutrition, biochemical activities and growth characterization of bacteria, it becomes easier to identify any unknown bacteria.

3. Collection of samples Body fluids, secretions and biopsy material can all be examined to detect pathogens, antigen or products or the immune response to them. Samples from the environment, e.g. water, food, soil, etc. Some samples should be collected at particular time; for example malaria parasites best sought at the peak fever and a short time afterwards, whereas blood for bacterial culture should be taken as fever for begins to rise.

4. Universal safety and laboratory safety Specimen may contain hazardous pathogens and must be handles with care General precaution is employed to reduce the risk of transmitting blood-borne pathogens Personal protective measures have to be taken in collecting and examining specimens

5. General rule for specimen collection The quantity of specimen must be adequate The sample should be collected from the appropriate region of the body Contamination of the specimen must be avoided by using only sterile equipment and aseptic conditions Always collect specimens prior to administration of antimicrobial drugs Specimen maybe infectious, so take proper precautions like wear gloves, gowns, masks and goggles, whenever necessary. Specimen contains labeled with information about patient name, code number, patient gender, patient age, specimen source, date and time of collection, etc.

6. Collection Skin, nails and hair Mucous membrane Anterior nares or Throat Ear Sputum Urine Faeces Eye swabs Blood Cerebrospinal fluid Pus and wound secretions

7. Specimen handling Immediate after collection, the specimen must be properly labeled and handled. The person collecting the specimen is responsible for ensuring that the name, hospital, registration number, location in the hospital, diagnosis, current antimicrobial therapy, name of attending physician, admission date and types of specimen are correctly and legibly written or imprinted on the culture request form.

8. Specimen transportation Transport the specimen to the clinical laboratory after it has been obtained from the patients is of prime importance. Microbial specimens maybe transported to laboratory by various ways. Special treatment requires for different kind of microorganism . T ransport of blood specimen to the laboratory promptly, or place them in an incubator at 37°C.

9. Specimen transportation Transport of urine specimens to the clinical laboratory must be done as soon as possible. Within 1 hour, specimen should be examined, if time schedule can not be followed, the urine sample must be refrigerated immediately. CSF sample transported to the laboratory within 15 minutes. Specimen for the virus are iced before transport and can be kept at 4 °C for up to 72 hours.

10. Laboratory methods Direct microscopical methods Cultural methods Biochemical methods Serological methods Molecular methods

11. Microscopical methods The microscope has been indispensable in the study of microorganism. The equipment requirement is cheap, reagent cost is low and early result can be obtained. There is no need of multiplication of organism nor to the alive for examination. Microscopy is especially useful for detecting organism that are difficult or dangerous to grow.

12. Microscopical methods Magnification Resolution Parts (Eyepiece lens, Objective lens, Tube, Arm, Base, Illuminator, Stage, Revolving nose piece or Turret, Condenser lens and Diaphragm or iris) Care of microscope Types of microscope

13. Microscopical methods Two important types of microscopy for the diagnosis of infections are Stained preparation Unstained preparation Unstained preparation: Direct examination of unstained preparation is suitable for rapid diagnosis in the laboratory. Many pathogens have a characteristic appearance, e.g., parasites in faeces or bacteria together with white cells in the urine. Unstained preparation are used to study bacterial shape, size, arrangement and motility by using hanging drop method.

14. Hanging drop slide method Objective: To observe living bacteria motility and study the shape, size and arrangement Principle: Many bacteria are show no motion and are termed nonmotile. In an aqueous environment, these same bacteria appear to be moving erratically. This erratic movement is due to Brownian movement. Brownian movement results from the random motion of the water molecules bombarding the bacteria and causing them to move.

15. Hanging drop slide method True motility (self-propulsion) has been recognized in other bacteria and involves several different mechanisms. Bacteria that posses flagella exhibit flagellar motion. Helical shaped spirochetes have axial fibrils (modified flagella that wrap around the bacterium) that form axial filaments. These spirochetes move in a corkscrew and bending-type motion. These type of motility or non motility can be observed over long period in a hanging drop slide.

16. Hanging drop slide method Requirements: Bacterial culture, Cavity slide, cover slip, Vaseline or petroleum jelly, inoculating needle and Bunsen burner Procedure: Apply Vaseline on the edge of the coverslip to provide seal Use the inoculating loop to transfer a small drop of bacterial suspension in the center of a coverslip Invert cavity slide and move slide onto the coverslip. Press gently to form a seal. Turn the slide over and place on the stage of the microscope Examination of hanging drop under low power objective and switch to 90 – 100x objective, using immersion oil

17. Hanging drop slide method

18. Culture methods

19. Biochemical methods Microorganism have been identified by their biochemical characteristics. These characteristics may vary depending on different microorganisms. After the microscopic and growth characteristics of a pure culture of bacterial examined, specific biochemical tests can be performed.

20. Biochemical tests Carbohydrate breakdown Catalase production Citrate utilization Coagulase Decarboxylase and deamines Hydrogen sulfide Indole Oxidase production Nitrate reduction Proteinase production Urease production

21. Serological methods Serological techniques depends on the interaction between antigen and specific antibody. They are useful when the pathogen is difficult or impossible to culture, or dangerous to handle in the hospital laboratories. This process can be divided into two parts: The antigen-antibody interaction The demonstration of this interaction by a testing process

22. Serological methods The antigen-antibody reaction depends on the specific binding between epitopes of the pathogens and antigen-binding sites on the immunoglobulin molecules. The antigen-antibody binding was detected by observing a natural consequences of this interaction: precipitation, agglutination

23. Serological methods Precipitation test: When a soluble antigen is mixed its specific antibody in the presence of electrolytes at a suitable temperature and pH, the antigen-antibody complex forms an insoluble precipitate. This precipitates settle down at bottom of the tube. When precipitate is not sediment and remains as floccules, the reaction is called as flocculation. This is done for both qualitative and quantitative determination of both antigen and antibody. This is sensitive method and able to detect even little antigen level. For Ring test (for anthrax), Slide test (VDRL test- Venereal Disease Research laboratory test for Syphilis)

24. Serological methods Agglutination test: When an antigen or an inorganic particle is mixed with antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. Agglutination reaction is more sensitive than precipitation for detection of antibodies. Slide agglutination test (for identification of cultures of salmonella and shigella) Tube agglutination test (for serological diagnosis of enteric fever, brucellosis and typhus fevers. Widal test Immunofluorescence Radioimmunoassay Enzyme-linked Immunoassay (ELISA) Western blotting