Introduction to Clinical Bacteriology

It is an adaptation of microbiological techniques to the study of etiological agents of infectious disease.
It is related to the laboratory diagnosis of human and animal infections and the role of the laboratory in both the management of infectious diseases and the elucidation of the epidemiology of infections.

1. Amjad Khan Afridi Lecture #01

2. INTRODUCTION It is an adaptation of microbiological techniques to the study of etiological agents of infectious disease. It is related to the laboratory diagnosis of human and animal infections and the role of the laboratory in both the management of infectious diseases and the elucidation of the epidemiology of infections.

3. Diagnosis of different bacterial diseases is called clinical bacteriology . Bacteria are transmitted to humans through air, water, food, or living vectors. The principal modes of transmission of bacterial infection are contact, airborne, droplet, vectors, and vehicular.

4. LABORTORY DIAGNOSIS Specimen Selection ( according to the diseases ) Specimen Collection Specimen Transportation Specimen Processing Morphological Identification of bacteria isolates (A) Culture characterizes (B) Biochemical Testing Principles of Laboratory Diagnosis of Infectious Diseases

5. Specimen Collection Proper specimen collection, container labeling, and culture requests are the responsibility of the ordering physician. The volume is important because infecting organisms that are present in small numbers may not be detected in a small sample. The quantity material must be adequate Specimens are selected on the basis of signs and symptoms, and this should be representative of the disease process 5

6. Specimen Transport Specimens should be transported to the laboratory as soon after collection as possible because some microorganisms survive only briefly outside the body. Transport media: Stabilize conditions and prevent drying. In general, they are buffered fluid or semisolid media containing minimal nutrients and are designed to prevent drying, maintain a neutral pH, and minimize growth of bacterial contaminants. 6

7. Cont … Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful. Meaningful specimens to diagnose bacterial infections must be protected before antimicrobial drugs are administered.

8. Bacteria Identification Since the discovery of microorganisms a few centuries ago, scientists have been looking for better ways to identify bacteria. Some methods are old and time tested, while some methods are new and genetically based . Either way, a very specific work flow is required to identify bacteria.

9. Staining of Bacteria Bacteria cells are almost colorless. A staining techniques is used to determine the shape and size of bacterial cells under microscope. 9

10. Microscopic Examination Microscopic examination is usually done by way of the gram stain procedure. This staining method will differentiate gram-positive (purple) bacteria from gram-negative (pink) bacteria. This is based on the composition of the cell wall of the organism. Common gram-positive bacteria are staphylococcus and streptococcus . While, gram-negative bacteria include E. coli and Klebsiella .

11. Cont … In addition, microscopic morphology that is useful in identification can be noted, for example staphylococci are gram-positive cocci in clusters. So the gram stain appears microscopically as purple spheres in a formation resembling a group of grapes.

12. Media Selection Based on the specimen source (urine, blood, sputum, stool, wound, etc.) and the likely organisms that would be present, the appropriate bacteriological media are selected to grow the organism for further work-up. In addition, selecting the appropriate temperature and atmospheric conditions (aerobic versus anaerobic) will allow bacterial growth. Colony Morphology How the bacterial colony appears on bacteriological growth media offers key characteristics about the bacteria. Characteristics like size, color, odor and the ability to lyse (break apart) blood cells are all important identification criteria for determining what the unknown bacteria is.

13. Biochemical test Biochemical tests are the tests used for the identification of bacterial species based on the differences in the biochemical activities of different bacteria. Bacterial physiology differs from one type of organism to another. The ability of bacteria to form organic compounds by metabolizing certain carbohydrates and related compounds is a widely used method for the identification of microorganisms. Many commercially prepared tests kits are available to identify bacteria using biochemicals and enzy mes .

14. Antibiotic susceptibility test Susceptibility is a term used when microbe such as bacteria and fungi are unable to grow in the presence of one or more antimicrobial drugs. Susceptibility testing is performed on bacteria or fungi causing an individual's infection after they have been recovered in a culture of the specimen. Testing is used to determine the potential effectiveness of specific antibiotics on the bacteria and/or to determine if the bacteria have developed resistance to certain antibiotics. The results of this test can be used to help select the drug(s) that will likely be most effective in treating an infection.

15. Serological Methods Serologic methods for identifying bacteria are frequently used in the laboratory. This typically involves testing an unknown antibody against a known antigen bound to a latex particle or similar structure. After mixing the two together and rotating, a visible agglutination (clumping) will appear if positive for the organism tested. Other tests look for antibody neutralization, inhibition and other end products.

16. Molecular Methods The latest and most specific methods available in the laboratory are molecular methods. These tests are based on the ability to detect, identify and characterize microorganisms based on their DNA or RNA. Polymerase chain reaction (PCR) is a common molecular method used to identify bacteria.

17. Microbiologic Examination Culture: Isolation of infectious agents frequently requires specialized media. Nonselective ( noninhibitory ) media permit the growth of many microorganisms. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms. Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic probes are also used.

18. Microbiologic Examination Antimicrobial Susceptibility: Microorganisms, particularly bacteria, are tested in vitro to determine whether they are susceptible to antimicrobial agents. Serodiagnosis: A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis. Direct Examination and Techniques: Direct examination of specimens reveals gross pathology. Microscopy may identify microorganisms. Immunofluorescence, immuno-peroxidase staining, and other immunoassays may detect specific microbial antigens. Genetic probes identify genus- or species-specific DNA or RNA sequences.