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Medical Microbiology Laboratory

Microbiology is the study of
living organisms of microscopic
size which includes bacteria ,
Fungi , Algae , Protozoa and Viruses. It is concerned with the forms, structure , reproduction , physiology , metabolism and classification.

Principle Of Microbiology
Medical microbiology deals with the causative agent of the infectious disease of the human , the ways in which they produce disease in the body and essential information for diagnosis and treatment.

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Medical Microbiology Laboratory

  1. 1. Presented By: Samjhana Joshi Kareena Shrestha Mahanand Yadav Saju Pun Suruchi Thapa Presented To: Tapeshwar Yadav Department of Laboratory Medicine, Nobel College of Medical Sciences, Kathmandu, Nepal
  2. 2. LABORATORY  Laboratory is the place where all the equipments, instruments and chemical (reagents) are available for performing investigative procedures, experimental works and research activities by using biological specimens (whole blood , serum , plasma , urine , stool etc. )  There are various departments of the clinical laboratory -:  Clinical chemistry  Haematology  Microbiology  Blood bank  Support services ( Phlebotomy / Specimen processing)  Urinalysis  Immunology  Serology  Histopathology and Molecular biology etc.
  3. 3. Microbiology  Microbiology is the study of living organisms of microscopic size which includes bacteria , Fungi , Algae , Protozoa and Viruses. It is concerned with the forms, structure , reproduction , physiology , metabolism and classification. Principle Of Microbiology  Medical microbiology deals with the causative agent of the infectious disease of the human , the ways in which they produce disease in the body and essential information for diagnosis and treatment.
  4. 4. Scope Of Microbiology
  5. 5. History of Microbiology  Long ago , the diseases or the death of human beings were believed to be punishment sent from god for human wrong doing.  Some of the scientists who involved in the development of microbiology are listed below :  Robert Hooke divised compound microscope and looked at the slices of cork .  Micrographia was an accurate and detailed record of Hooke’s observation illustrated with magnificient drawing.  Antony Von Leuweenhoek was the first to observe and describe the microorganisms on his own made microscope.  After his invention of microscope, there are number of significant development. Thus he is known as Father of Microbiology.
  6. 6. Golden Era of Microbiology  During this period we see the real beginning of Microbiology as a discipline of biology which began with the work of Louis Pasteur and Robert Koch.  In 1856, Louis Pasteur introduced fermentation techniques , sterilization techniques that is (Steam Sterilization-1000 C , Autoclave, Hot air Oven-1600 ) and Pasteurization technique.  There is number of discoveries in his period. Thus, he is renowned as Father of Modern Microbiology.  Robert Koch discovered Anthrax bacillus, Tubercle bacilli and causative agent of Cholera.  He also introduced the practice of using laboratory animals for the experiments as he formulated Koch’s Postulates .  He also developed pure culture techniques. Therefore, he is known as a Father of Modern Bacteriology. Koch’s Postulates
  7. 7. Various Field of Medical Microbiology  Medical Microbiology 1. Bacteriology 2. Virology 3. Parasitology 4. Mycology 5. Immunology
  9. 9. 1. Bacteriology The subject Microbiology, is hugely dependent in this field. Simply, the study of bacteria is called as bacteriology. Generally, size of bacteria is about 0.2-1.5µm in diameter. Bacteria consists of both DNA and RNA in the form of nucleiod in the nuclear material of it. CLASSIFICATION OF BACTERIA Bacteria can be classified int0 various types they are -:
  10. 10.  On the basis of Morphology A .True Bacteria • Cocci – They are spherical or oval shape on the basis of arrangement of individual organisms they can be descibed as -: a) Monococci(cocci in singles)(e.g. Monococcus spp.) b) Diplococci are pairs of cocci (e.g. Streptococcus pneumoniae and Neisseria gonorrhoeae) c) Streptococci are chains of cocci (e.g. Streptococcus pyogenes). d) Staphylococci are irregular (grape-like) clusters of cocci (e.g. Staphylococcus aureus). e) Tetrads are clusters of four cocci arranged within the same plane (e.g. Micrococcus sp.). f) Sarcina is a genus of bacteria that are found in cuboidal arrangements of eight cocci.
  11. 11.  Bacilli – Bacilli are the rod shaped bacteria divided into various types on the basis of arrangements. I. Diplobacilli II. Streptobacilli III. Pallisades IV. Chinese letter form V. Coccobacilli VI. Comma shaped (vibrio) B. Actinomycetes (actin- ray , mukes – fungus ) – They are like true bacteria but they resemble fungi in that they exhibit branching X- form filaments. C. Spirochetes – They are longer, non branched spiral shaped microorganisms having several coils. Eg- Treponema, Leptospira etc.
  12. 12. D. Mycoplasm – these are bacteria lacking cell wall and highly pleomorphic in nature. Eg . L- form, Spheroplast , protoplast. E. Rickettsiae and Chalamydiae - Very small bacteria which are obligatory in nature.  On the basis of Anatomical features Capsule – Capsulated – Streptococcus pneumoniae Non-capsulated – Streptococcus viridans
  13. 13. Flagella –  Aflagellate (eg. Shigella spp. )  Monotrichous (Single flagella i.e Vibrio )  Lophotrichous (Group of flagella at one end i.e Helicobacter )  Amphitrichous (Single flagella at both end)  Peritrichous ( Flagella all around body i.e Salmonella , Escherichia coli ) Spores  Spore forming – Bacillus spp.  Non spore forming – Escherichia coli
  14. 14.  Based on Staining Gram’s Stain Acid Fast Stain - Acid Fast bacilli ( Mycobacterium tuberculosis ) - Non – Acid Fast bacilli ( Staphylococcus aureus )
  15. 15. STERILIZATION AND DISINFECTION Sterilization It is the process by which medium is freed of all living microorganism either in vegetative form or spore state. Disinfection Substance interfare with membrane function. Eg; Phenol, chloroform, H2O2, Formaldehyde etc. Protein denaturing agent : HCL (modify functional group of protein) . Sterilization Agents  HEAT (Most reliable method of sterilization) A)Dry heat:  It denaturate the protein of bacteria and cause oxidative damage.  It is done by flaming (needle, cotton wool), red heat(Inoculating loop), Incineration (Pathological materials ,solid dressing), Hot air oven (Glasswares) etc.
  16. 16. B)Moist heat Temperature below 100˚c  Vaccine bath- 56˚c for 1 hour for 3 successive day.  Pasteurization of milk. - 63˚c for 30 min (Holder Method) - 72 ˚c for 15 sec (Flash Method) - Inspirator at 80 ˚ -85 ˚C Temperature at 100˚c  Tyndallization (3 successive days ) eg: for media  containing sugar or gelatin. Temperature above 100˚c Steam under pressure- Autoclave for dressing instruments , media , laboratory wares etc under the temperature of 121 ˚ c for 15-20 min at 15 lbs pressure.
  17. 17. C) Filtration : Other method of Sterilization for the heat labile liquid for examples : Serum,toxin , glucose solution. Filters like sintered glass filter, membrane filter etc. D) Radiation: Non ionization (IR ray) – Syringes UV ray – Operating theatres and Laboratories. Ionization – (X-ray,Gamma ray)- For plastic syringes,Catheters, rubber goods etc. E)Gas Vapour Sterilization :  Ethylene Oxide – Plastic goods,culture medias  Aldehyde – Operation Theatre  Hydrogen Peroxide – For contact lense  Halogens – as laboratory disinfectant on surface of bench and in discard spots.  Alcohols - 70% alcohol is used  Phenols - Lysol for disinfection of excreta, Floor of Ward and operation room.
  18. 18.  Laboratory Procedure used in Bacterial Disease Diagnosis : 1. Microbial Culture It is primary method for isolation of infectious bacteria or diseases in the laboratory. These isolated bacterias can also be used for studying its size , structure, morphology , for researches and other various purposes. Culture is done in laboratory by various methods like:  Streak culture  Stroke culture  Carpet culture  Stab Culture  Sweep Plate method
  19. 19. Carpet Culture Streak Culture
  20. 20.  There are 3 types of media which are used for the growth of bacteria using the culture methods.  Solid Culture Media – It contains 1.5-2% of agar base. This media is primarily used to culture bacteria. Some of the examples of this media are Basal media , Enriched media , Differential media , Indicator media, Selective media Transport media etc.  Liquid Culture Media – This media is used for the growth of the cells inside its liquid media (used for detecting Mycobacteria). Eg. Enrichment media ( Thioglycollate broth , Nutrient broth ) etc.  Cell Culture – This method is used to determine the microbe on the cells for the identification of viruses. .
  21. 21. 2. Microscopy Culture techniques will often use a microscopic examination to help in the identification of microbes or bacteria. Various types of microscope are used for this purpose some of them are i. Compound microscope ii. Electron microscope iii. Light microscope iv. Fluorescence microscope v. Dark field microscope.
  22. 22. Staining Methods  Gram’s Stain Danish scientist Hans Christian Gram devised a method to differentiate two types of bacteria based on the structural differences in their cell walls. In his test, bacteria that retain the crystal violet dye do so because of a thick layer of peptidoglycan and are called Gram-positive bacteria. In contrast, Gram-negative bacteria do not retain the violet dye and are colored red or pink. Compared with Gram- positive bacteria, Gram-negative bacteria are more resistant against antibodies because of their impenetrable cell wall. These bacteria have a wide variety of applications ranging from medical treatment to industrial use.
  23. 23. AFB / Ziehl Neelsen Stain It is used to detect the presence of acid fast bacilli like Mycobacterium tuberculosis & Mycobacterium leprae. Interpretation Acid Fast Bacilli – Red or Pink in colour Background – Blue or Green (according to counterstain used)  Other special Stains - Capsule : India Ink - Spore : Moeller stain
  24. 24. Biochemical Tests Fast and relatively simple biochemical test can be used to identify infectious agents. For bacterial identification, the use of metabolic or enzymatic characteristics are common. Acids, alcohol and gases are usually detected in this tests.  Biochemical tests performed are : Catalase test Coagulase test Indole test Bile solubility test Oxidase test Urease test
  25. 25. Serological Method Highly sensitive and specific method which is based upon the ability of an antibody to bind specifically to an antigen. PCR (Polymerase Chain Reaction) For detection and study of microbes. Which is definite accurate and fast.
  26. 26. Medical microbiologists often make treatment recommendations to the patients physician based on the strain of microbes and its antibiotic resistance , the toxicity of antimicrobial drugs and drug allergies the patient has by performing AST. For performing AST, modified Kirby-Bauer method is recommended and Mueller Hinton Agar is used.
  27. 27. Virology It is the study of Viruses which are obligate intracellular parasites, size of viruses is about 20-400 nm. Largest Virus – Pox Virus Smallest Virus – Picorna Virus Viruses either contain DNA or RNA but never both Cancer causing (Oncogenic Virus )  Hepatitis C Virus  Hepatitis B Virus  Human Pappiloma Virus RNA Viruses DNA Viruses Retro viruses, Rhino Viruses Parvo Virus , Pox Virus Hepatitis A,C, D,E Hepatitis B Virus Rota Virus Adeno Virus
  28. 28. Laboratory Procedures Used in Viral Disease Diagnosis : 1. Specimen Collection : Specimen such as Throat swab , Nasal swab , Bronchial washing, Urine , Stool , Rectal swab and CSF etc are used for detection.
  29. 29. 2. Viral Transport Medium : It is used for transportation of Viruses from one place to another. 3. Diagnostic Method in Virology : i. Direct examination : By Ag Detection, Electro microscopy , Light microscopy , Viral genome detection etc. ii. Indirect examination : By cell culture, egg inoculation and animal inoculation. iii. Serology test : They are Ab detection neutralization test , ELISA, Western blot etc.
  30. 30. PARASITOLOGY Parasitology is the area of biology concerned with the phenomenon of dependence of one living organism on another. Medical parasitology deals with the protozoal as well as helminthic parasites, which infect different organ systems of human beings, the disease they produce, various methods of diagnosis and prevention. In lab, smear preparation for microscopy, cultures and serology test is done for detection of parasites.
  31. 31. MYCOLOGY It include the study of fungus and fungal infection. Fungus causes the eczema and allergy. The disease cause by fungal infection is called Mycotic disease. Fungus may infect nail, hair, skin and organs. Fungi is the causative agent of ringworm and tinea. INFECTION OF FUNGI A. Superficial Infection: Infection in skin, hair, nail, eye, ear etc by Microsporum, Trichopytous and Epidermophyton etc. A. Cutaneous Infection: Infect on cutaneous layer which is caused by saprophytic fungi. B. Systemic Infection: Fungal Infection in various organs caused by Histoplasm Capsulatum , Blastomyces Dermatidis etc.
  32. 32. LABORATORY DIAGNOSIS OF FUNGAL DISEASE 1. Microscopy : a. 10 % KOH preparation b. India Ink c. Stain – Giemsa , PAS stain 2. Culture : Commonly used Media is SDA ( Sabouraud Dextrose Agar ) BSA ( Bird Seed Agar for Cryptococcus )
  33. 33. IMMUNOLOGY The term ‘ immunity’ is defined as resistance exhibited by the host against any foreign antigen including microorganisms. This resistance plays a major role in prevention of infectious diseases . Antigen : It is a substance which , when introduced into a body evokes immune response to produce a specific antibody with which it reacts in an observable manner. Antibodies: It is a substance which are formed in the serum and tissue fluids in response to an antigen and react with that antigen specifically and in some observable manner. Antigen- Antibody Reaction Ag-Ab reaction is reversible , occurs at surface and there is no denaturation of Ag and Ab during reaction.
  34. 34. Types of Ag – Ab Reaction 1. Precipitation Reaction : More sensitive for antigen detection. Eg: Biken Test. 2. Flocculation Reaction : Special types of precipitation where precipitation remains suspended instead of sedimentation Eg. VDRL, RPR . 3. Agglutination Reaction : More sensitive than precipitation for antibody detection. Antigen – antibody reaction in which antibody combine with a particulate antigen for eg. Widal Test , Sero-typing etc. 4. Neutralisation Test : When anti toxins combines with its corresponding toxin , neutralization occurs. Eg; Schick Test, Dick Test. 5. Radio Immuno Assay (RIA) : It is used for quantification of hormones , drugs, tumor markers , viral antigen etc. 6. Enzyme Immuno Assay (EIA) : It measures enzyme labelled antigen or antibody. Eg; ELISA, RF (Rheumatoid Factor).
  35. 35. Biological Safety Cabinet(BSC) BSC is an enclosed, ventilated laboratory works place for safety working with materials contaminated with pathogens requiring a defined biosafety level. It protect a laboratory worker and environment from aerosols and air borne particles. Biosafety Cabinet Classification Types of cabinet Agent classification (Biosafety level) Protection Class I Low and moderate risk User only Class II Low and moderate risk User and materials Class III High risk User and environment
  36. 36. Quality Assurance (QA)
  37. 37. THANK YOU