Lab Diagnosis - Prac. Microbiology


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Lab Diagnosis - Prac. Microbiology

  1. 1. Infectious diseases They are clinically evident diseases with the potential of transmission from one person or species to another. They result from the presence of pathogenic microorganisms (very small organisms that are invisible to the naked eye) that are able to cause disease in human beings. Pathogenic micro-organisms include bacteria, viruses, fungi, protozoa, and multicellular parasites. Practical microbiology sessions focus on the diagnosis of these infectious agents.
  2. 2. Laboratory diagnosis of infectious diseases Direct method Indirect (serologic) method Detection of antibodies against the microorganism in the patient’s serum. Detection of:  microorganisms,  their structural components  their products in specimens collected from the patient (e.g. urine, blood, sputum, CSF……etc).
  3. 3. I) DIRECT METHOD 1)Specimen Collection A ’good quality’ clinical specimen. 2) Microscopic Examination: usually before further processing of specimens 3) Microbial Detection: a) Culture technique: b) Non-culture technique
  4. 4. I) DIRECT METHOD 1) Specimen Collection A ’good quality’ clinical specimen  Collecting specimens before the start of antibiotics.  Choosing the appropriate specimen (representing the infection site).  Using sterile containers and avoiding contaminating the specimen.  Transporting the specimen properly to the lab. as early as possible.
  5. 5. Specimen Collection
  6. 6. I) DIRECT METHOD 2) Microscopic Examination: usually before further processing of specimens  stained /unstained (wet)preparations  different types of microscopes Staphylococci in pus
  7. 7. I) DIRECT METHOD 3) Microbial Detection: a) Culture Technique:  Isolation of the organism in pure culture →inoculating the specimen onto appropriate artificial culture media followed by  Identification of the isolate by e.g.     : microscopic examination biochemical reactions reaction with specific antibody (serologic identification of the organism) DNA probes Which of these approaches is used and in what sequence depends upon the type of specimen and organism.  Antibiotic sensitivity After growing the organism in pure culture.
  8. 8. I) DIRECT METHOD 3) Microbial Detection: b) Non-Culture Technique: I. Identification of a specific microbial antigen such as :  a structural component (e.g. cell wall antigen, capsular polysaccharide…etc) or a product (e.g. an exotoxin)  by reacting with specific antibody OR: II. Identification of a specific gene sequence (i.e. nucleic acid of the organism) by the application of different molecular methods (e.g. PCR, DNA probe).
  9. 9. I) DIRECT METHOD  yield more rapid results (minutes or hours) (do not depend on growth and multiplication of the organism) However, antimicrobial susceptibility cannot be determined (although the presence of resistance genes can be determined by molecular methods).  Non-culture techniques are mainly applied if:   a rapid diagnosis is needed The microorganism cannot be cultured on artificial media  A slowly growing micro-organism
  10. 10. II) INDIRECT (SEROLOGIC) METHOD  Serologic diagnosis of infectious diseases involves the use of known microbial antigens to microorganism detect antibodies in the patient’s serum. current (active) infection against the is diagnosed by the detection of one of the following  specific IgM antibodies  rising titre of specific IgG antibodies (4-fold or greater rise)  a single high titre of IgG antibodies in certain diseases Skin tests N.B. based on cell-mediated hypersensitivity may also help in the diagnosis of certain diseases.
  11. 11. Skin Test
  12. 12. Diagnosis of infectious diseases Diagnosis of infectious diseases DIRECT METHOD DIRECT METHOD Specimen Specimen Microscopical ex. Microscopical ex. Culture technique Culture technique Isolation on culture media Isolation on culture media .Identification e.g .Identification e.g ••microscopical.ex. microscopical. ex. ••bioch.Reactions bioch. Reactions ••DNAprobe DNA probe ••serology serology INDIRECT METHOD INDIRECT METHOD (SEROLOGICAL) (SEROLOGICAL) Detection of antibodies in serum Detection of antibodies in serum ••IgM IgM ••risingtitre of IgG rising titre of IgG Non-culture technique Non-culture technique ••detection of specific antigen detection of specific antigen (serology) (serology) •• detection of specific gene detection of specific gene sequence (mol. tech.) sequence (mol. tech.) Antibiotic sensitivity Antibiotic sensitivity
  13. 13. Microscopy
  14. 14. Light Microscope
  15. 15. Stained Preparations Bacterial morphology Unstained preparations Motility
  16. 16. Bacterial Morphology Size Shape Special arrangement Staining affinity Spore formation Capsule formation Motility
  17. 17. Bacterial Shape
  18. 18. Bacterial arrangement Chains. Pairs (diploids). Clusters (group). No special arrangement.
  19. 19. Bacterial arrangement Cocci Pairs Chains Irregular Clusters 22
  20. 20. Bacterial arrangement Bacilli Pairs chains No special arrangement 23
  21. 21. Spore formation  Morphological characters of bacterial spores: * Shape. * Position. * Staining.
  22. 22. Bacterial spores
  23. 23. Bacterial capsule
  24. 24. Staining of Bacteria Bacteria cells are almost colorless and transparent A staining technique is often applied to the cells to color them → Their shape and size can be easily determined under the microscope. 28
  25. 25. Smear preparation S Fixation 30
  26. 26. Smearing out of the sample
  27. 27. Types of Stains 1- simple stain: Single basic dye e.g. Methylene blue All bacteria take the color of the dye 2- Differential stain: Two dyes separated by a decolorizing agent e.g. Gram stain & Ziehl-Neelsen stain 3- Special stain: e.g. Fontana stain
  28. 28. Differential staining Principles of differential stain * Application of the main stain. • Decolourization. *Application of the counter-stain. e.g. Gram stain & Ziehl-Neelsen stain
  29. 29. 1. Gram Stain
  30. 30. Components Primary Stain: Methyl violet + Gram’s iodine Decolourizing agent: 95% ethyl alcohol Counter stain: dil. Carbol fuchsin
  31. 31. Principle Primary Stain Methyl violet + Gram’s iodine All Violet → Decolourizing agent: 95% ethyl alcohol ( violet ) ( Gram + ve) Not decolourized decolourized ( colourless ) (Gram –ve ) Counter stain: dil. Carbol fuchsin→ colourless→pink(Gram -ve )
  32. 32. Gram Staining Technique
  33. 33. Gm+ve cocci & G-ve bacilli
  34. 34. 2. Ziehl-Neelsen Stain
  35. 35. Mycobacterium A 3rd type of cell envelope (high lipids content of cell wall) Not readily stainable with ordinary stains . A strong stain e.g., concentrated carbol fucsin + heat. Resist decolorization by strong mineral acids or acid-alcohol → Acid-fast.
  36. 36. Components Primary Stain: Conc. Carbol fucshin Decolourizing agent: 20% H2SO4 OR 3% HCL in alcohol Counter stain: methylene blue
  37. 37. Principle Primary Stain Conc. Carbol fucshin → All Red Decolourizing agent: 20% H2SO4 OR 3% HCL in alcohol Not decolourized ( red ) ( acid-fast) decolourized ( colourless ) (non acid-fast) Counter stain: methylene blue → Colourless →blue (non acid-fast)
  38. 38. Ziehl-Neelsen Stain Technique 1 2 4 7 3 5 6
  39. 39. Questions
  40. 40. 5.Acid fast bacilli stained by Z-N stain appear: a) Violet b) Blue c) Red d) Colourless e) brown 6.The steps of Gram’s stain is as follows: a) methyl violet / ethyl alcohol / dil. carbol fuchsin / iodine b) methyl violet / ethyl alcohol / iodine / dil. carbol fuchsin c) dil. carbol fuchsin / methyl violet / ethyl alcohol / iodine d)dil. carbol fuchsin / iodine / methyl violet / ethyl alcohol e)methyl violet / iodine /alcohol / dil. carbol fuchsin