Bacteriology Laboratory Organization

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Bacteriology Laboratory Organization

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Bacteriology Laboratory Organization

  1. 1. Bacteriology Laboratory Organization and Skills Dr.T.V.Rao MD10/14/2012 Dr.T.V.Rao MD 1
  2. 2. Bacteriology Laboratory Bacteriology Laboratory makes the Backbone of any Hospital and without which no hospital can function to the Minimal needs, All the Microbiologists and Lab professionals need the basic skills and safety for effective functioning of services
  3. 3. Before staring, be familiar with Normalpathogenic, and opportunistic pathogens• Normal Flora• Opportunis tic Pathogens• Pathogens
  4. 4. Microbiology and the Role of the Microbiologists• Microbiology – study of microorganisms (simple forms of life visible only with a microscope)• Microorganisms –Normal flora –Pathogenic
  5. 5. Microbiology and the Role of the Medical TechniciansMedical technicians can be Assists physician / Microbiologists Obtains specimens Prepares specimens for direct examination Prepares specimens for transportation to reference laboratory If office has a POL, performs microbiologic procedures
  6. 6. Classification and Naming of Microorganisms• Classification by structure –Subcellular – DNA or RNA surrounded by a protein coat – viruses –Prokaryotic – simple cell structure with no nucleus or organelles – bacteria –Eukaryotic – complex cell structure with nucleus and specialized organelles – protozoans, fungi, parasites10/14/2012 Dr.T.V.Rao MD 6
  7. 7. Bacteria: Classification and Identification (cont.)• Special groups – Chlamydia – Mycobacteria – bacilli • Cell wall structure with a cell wall that differs from other differs from most bacteria bacteria • Live and grow within other living cells – Rickettsia • Very small • Live and grow within – Mycoplasmas – other living organisms completely lack the such as mites and ticks rigid cell wall10/14/2012 Dr.T.V.Rao MD 7
  8. 8. BacteriaSingle-celled prokaryotic organismsReproduce rapidlyClassification Shape Ability to retain dyes Ability to grow with / without air Biochemical reactions10/14/2012 Dr.T.V.Rao MD 8
  9. 9. Bacteria: Classification and Identification (cont.)• Ability to retain certain dyes – Gram’s stain – Acid-fast stain• Ability to grow in presence or absence of air – Aerobes – grow best in the presence of oxygen – Anaerobes – grow best in the absence of oxygen• Biochemical reactions10/14/2012 Dr.T.V.Rao MD 9
  10. 10. Bacteria: Classification and Identification• Shape – Coccus – spherical, round, or ovoid – Bacillus – rod-shaped – Spirillum – spiral-shaped – Vibrio – comma-shaped10/14/2012 Dr.T.V.Rao MD 10
  11. 11. All Microbiologists should be familiar with :• Clinically significant bacteria – Morphological characteristics – Biochemical characteristics – Signs and symptoms they cause in the host they are infecting – Virulence factors – Pathophysiology of infection
  12. 12. How Infections Are Diagnosed Steps to diagnosis and treatment 1. Examine the patient Presumptive diagnosis May or may not need additional tests 2. Obtain specimen(s) Label properly Include presumptive diagnosis10/14/2012 Dr.T.V.Rao MD 12
  13. 13. How Infections Are Diagnosed (cont.)3. Examine specimen directly • Wet mount • Smear4. Culture specimen Culture medium – contains nutrients Examine culture visually and microscopically
  14. 14. Before starting the work ..Different media are used to culturemicroorganisms, be certain that you are using theappropriate media for your organism.Always use sterile technique to preventcontamination.Choose the type of media (liquid or plate) appropriatefor your investigation or application.Sterile liquid culture tubes and media plates canbe prepared in advance and stored in therefrigerator for later use (2 weeks for liquidculture tubes, 2 months for media plates).
  15. 15. Before starting work …Liquid culture tubes, solid slant tubes, and petriplates can be used to culture microbes.Media and lab materials should be sterilized priorto use; an autoclave or a pressure cooker can beused in the sterilization process.Serial dilution and plate counttechniques are used to estimatemicrobial populations fromenvironmental or commercialcultures.
  16. 16. Specimen CollectionMust be collected correctly If not, may not grow in culture Contaminants may be mistakenly identified Patient may receive incorrect or harmful therapy10/14/2012 Dr.T.V.Rao MD 16
  17. 17. Specimen Collection (cont.)• Devices – Use appropriate collection device or specimen container – Sterile swabs – absorbent material on the tip• Collection and transporting systems – Sterile, self-contained – Transport medium – Aerobic or anaerobic
  18. 18. Specimen Collection: GuidelinesAvoid causing Obtain sufficient harm, discomfort, or quantity of specimen undue embarrassment Obtain specimen priorCollect from to the start of appropriate site antimicrobial therapyObtain specimen at Label correctly correct timeUse appropriate devices10/14/2012 Dr.T.V.Rao MD 18
  19. 19. Specimen Collection (cont.)Throat culture specimens Swab back of throat in the area of the tonsils Avoid touching any structures in the mouth Prepare culture plate or prepare correctly for transport to laboratory
  20. 20. Specimen Collection (cont.)Urine specimen Sputum Clean-catch specimen midstream to Specimen from minimize contaminants lungs Process within 60 Avoid minutes or contaminating refrigerate specimen with saliva10/14/2012 Dr.T.V.Rao MD 20
  21. 21. Specimen Collection (cont.)Wound specimen Stool Specimens Swab wound or Technique varies lesion Bacterial infection Do not touch outside Protozoal or parasitic of wound infection Instruct patient in correct collection procedure10/14/2012 Dr.T.V.Rao MD 21
  22. 22. Transporting Specimens to an Outside Laboratory Many offices send cultures to an outside lab Three main objectives  Follow proper collection procedures and proper collection device  Prevent deterioration of specimen  Protect anyone handling specimen10/14/2012 Dr.T.V.Rao MD 22
  23. 23. Direct Examination of SpecimensEnables physician to initiate treatment immediately Potassium hydroxideWet mounts (KOH) mounts Nacl mixed with Used if a fungal infection of the specimen of glass slide skin, nails, or hair is Presence of pathogen suspected and movement of KOH dissolves keratin microorganism that can mask presence of a fungus10/14/2012 Dr.T.V.Rao MD 23
  24. 24. Preparation and Examination of Stained Specimens Quick, tentative • Gram’s stain diagnosis – Moderate- Differentiation complexity test between types of – Bacteria either retain infections or lose purple color • Gram-positive bacteria • Gram-negative bacteria10/14/2012 Dr.T.V.Rao MD 24
  25. 25. Procedure for Making a ‘Smear’• Using aseptic technique remove a colony from a plate or cells from your slant. Be carefully to gently touch the surface of your culture with the inoculating loop.• Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide• Add a drop of water in the middle• Mix again• Let Air dry10/14/2012 Dr.T.V.Rao MD 25
  26. 26. Making a Smear • Wash the glass slide thoroughly with soap and water then rinse with 95% alcohol to sterilize. • 2. Allow the slide to dry properly. • 3. Pass the clean slide over a flame with its face down to further sterilize it. (Make sure to hold it by its edge) • 4. Draw a small circle on the slide so you can put your bacteria on the back of the marked area.10/14/2012 Dr.T.V.Rao MD 26
  27. 27. Smear Preparation• Smear Preparation• Only a small amount of bacterial culture should be used.• Thick smear causes overcrowding of a large number of cells.• Two different media require two different techniques• Liquid Medium/ Broth Culture• 1. Take the loop and hold it in the flame at 45o until it turns red. Your loop is inoculated now. Let it cool for a few minutes.10/14/2012 Dr.T.V.Rao MD 27
  28. 28. Procedure for Making a ‘Smear’ • Run the slide through the flame until the slide is warm ( The frosted side should be down) This fixes the bacteria to the slide • Let the slide cool • Place in the metal tray or in the rack10/14/2012 Dr.T.V.Rao MD 28
  29. 29. Media Types• General Purpose Media• Enriched Media• Selective Media• Differential Media10/14/2012 Dr.T.V.Rao MD 29
  30. 30. Culturing Microorganisms• There are two basic culture techniques used in microbiology: 1. Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium.  Liquid medium is best when you want to rapidly increase the concentration of the organism or when you want to grow10/14/2012 motile cells. Dr.T.V.Rao MD 30
  31. 31. Culturing Microorganisms • There are two basic culture techniques used in microbiology: 2. Culture Plates: Liquid medium is solidified using agar (Agarose) and poured as a thin layer in the bottom of a culture dish (also sometimes called petri plate)  Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate individual colonies from environmental samples.10/14/2012 Dr.T.V.Rao MD 31
  32. 32. Culturing Specimens in the Laboratory• More common to send specimens for culture to outside labs• Culturing involves placing a sample of specimen on a culture medium – Medium – nutrients – Place in incubator for growth – colony develops as microorganism multiplies
  33. 33. Sterile TechniqueWhen culturing bacteria or other microorganisms, it is important to keep your work area as clean as possible.This prevents the introduction of other microorganisms from the environment into your culture.The techniques used to prevent contamination are referred to as sterile techniques.10/14/2012 Dr.T.V.Rao MD 33
  34. 34. Organise your Work area10/14/2012 Dr.T.V.Rao MD 34
  35. 35. Sterile Technique1. Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are: 1. 10% bleach (recommended by the CDC) 2. 85% ethanol10/14/2012 Dr.T.V.Rao MD 35
  36. 36. Aseptic Technique • First requirement for study of microbes – pure cultures, free of other microbes • Maintain a clean environment; work close to the flame10/14/2012 Dr.T.V.Rao MD 36
  37. 37. Sterile Technique1. Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are: 1. 10% bleach (recommended by the CDC) 2. 85% ethanol10/14/2012 Dr.T.V.Rao MD 37
  38. 38. Culturing Specimens (cont.)• Culture media – Liquid, semisolid, or solid forms – Contains agar – Selective or nonselective10/14/2012 Dr.T.V.Rao MD 38
  39. 39. Holding the Inoculating loop10/14/2012 Dr.T.V.Rao MD 39
  40. 40. Media Types• General Purpose Media: • Supports the growth of many microorganisms • i.e. Nutrient agar• Enriched Media: • Has special nutrients to encourage the growth of fastidious heterotrophs • i.e. Blood Agar• Selective Media: • Favors the growth of one type of microorganisms and inhibits the growth of others • Luria + penicillin Agar• Differential Media: • Distinguishes between different groups of bacteria on the basis of biochemical characteristics • i.e. Eosin Methylene Blue Agar 10/14/2012 Dr.T.V.Rao MD 40
  41. 41. Inoculation of Culture Plates and Tubes Clean and surface sterilize your work area as detailed in the section on Sterile Technique. Use either disposable inoculation loops or a metal loop that can be heat sterilized to inoculate plates, slants, and liquid culture tubes.If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media or the sterile culture plate before the placing the loop in your live culture. Failure to cool the loop will kill your active microbial cultures!10/14/2012 Dr.T.V.Rao MD 41
  42. 42. Sterility of the Loop Important in Culture Work10/14/2012 Dr.T.V.Rao MD 42
  43. 43. Inoculating Petri Plates Step 1:Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture. Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture. Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.10/14/2012 Dr.T.V.Rao MD 43
  44. 44. Culturing Specimens (cont.)• Inoculating a culture plate – Transfer some of the specimen onto a culture plate – Label the plate correctly – Qualitative analysis – determination of type of pathogen – Quantitative analysis – number of bacteria present in sample10/14/2012 Dr.T.V.Rao MD 44
  45. 45. Inoculating Petri PlatesStep 4: Holding the petri dish lid at an 30- 45 angle, work the inoculating loop from the outside of the plate toward the center in a zig-zag pattern that covers approximately 25% of the plate surface .10/14/2012 Dr.T.V.Rao MD 45
  46. 46. Inoculating Petri PlatesStep 5: Turn the petri plate 90 to the right, dragging the inoculation loop through the last section of the plate, moving from the outside to the inside in a zig-zag motion.Step 6: Repeat this process twice more until the entire plate surface is covered.NOTE: If you are trying to isolate individual colonies, each turn of the dish will give you fewer microbes so that you can distinguish individual colonies.10/14/2012 Dr.T.V.Rao MD 46
  47. 47. Procedure for Transferring Microorganisms to a Slant• 1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer• 2. Using the pinkie finger of your dominant hand twist the red cap from the tube. Hold in your pinkie and do not place it on the counter• 3. Pass the mouth of the culture tube across the flame• 4. Direct the inoculating needle into the broth.• 5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack• 6. Pick up the slant in your non dominant hand 4710/14/2012 Dr.T.V.Rao MD
  48. 48. Procedure for Transferring Microorganisms to a Slant• 7. Twist off the red cap• 8. Flame the mouth of the slant tube• 9. Direct the inoculating needle into the tube and “ stab” the agar in the base( butt)• 10. Withdraw on the entry line and when you reach the surface make a simple streak along the face.• 11. Flame the mouth of the tube and replace the cap.• 12. Flame your inoculating needle and replace in your rack.10/14/2012 Dr.T.V.Rao MD 48
  49. 49. Culturing Specimens (cont.)• Inoculating a culture plate – Transfer some of the specimen onto a culture plate – Label the plate correctly – Qualitative analysis – determination of type of pathogen – Quantitative analysis – number of bacteria present in sample10/14/2012 Dr.T.V.Rao MD 49
  50. 50. Triple Streak Method10/14/2012 Dr.T.V.Rao MD 50
  51. 51. Optimal results with Scientific Streaking10/14/2012 Dr.T.V.Rao MD 51
  52. 52. Streak plate method of isolation10/14/2012 Dr.T.V.Rao MD 52
  53. 53. Colony Morphology10/14/2012 Dr.T.V.Rao MD 53
  54. 54. Read Colony Morphology • Colony morphology • Color • Shape • Margin • Elevation10/14/2012 Dr.T.V.Rao MD 54
  55. 55. Determining Antimicrobial Sensitivity• An outside lab Procedure reports Filter paper containing – Sensitive – no growth antimicrobial agents – Intermediate – little placed on inoculated growth agar plate – Resistant – Incubated for 24 hours overgrown Evaluate effectiveness of agent10/14/2012 Dr.T.V.Rao MD 55
  56. 56. Microorganism Categories • How are microorganisms categorized? –By genetics to show how they are related –By tissues they infect to show how they cause disease –By pathogenicity and communicability (also known as their Biosafety Level)10/14/2012 Dr.T.V.Rao MD 56
  57. 57. Biosafety is a Concern for all Microbiologists10/14/2012 Dr.T.V.Rao MD 57
  58. 58. Biosafety Level 1 Standard Microbiological Practices• Restrict or limit access when working• Prohibit eating, drinking and smoking in the laboratory• Pipetting by mouth strictly forbidden 10/14/2012 Dr.T.V.Rao MD 58 2.3
  59. 59. Biosafety Level 1 Standard Microbiological Practices10/14/2012 Dr.T.V.Rao MD 59 2.3
  60. 60. Standard practices also include:• Keep work areas uncluttered and clean• No food in lab refrigerator• Minimize splashes and aerosols• Decontaminate work surfaces daily• Maintain insect & rodent control program10/14/2012 Dr.T.V.Rao MD 60
  61. 61. Decontamination•Sterilization•Disinfection
  62. 62. Decontamination Chemical • Types –Liquids, i.e. chlorox, hydroge n peroxide –Gases, i.e. ethylene oxide10/14/2012 Dr.T.V.Rao MD 62
  63. 63. Decontamination Chemical• General Lab Use - Hypochlorite Solutions –Large Spills/Large Organic Load • undiluted from bottle –Small Spills/Virus Inactivation • 10% - 1:9 –General Surface Disinfection • 1% - 1:99 10/14/2012 Dr.T.V.Rao MD 63
  64. 64. In case of a spill • Wear disposable gloves • Cover large blood spill with paper towels and soak with 1% (10000 ppm) of household bleach and allow to stand for at least 5 minutes • Small spill - wipe with paper towel soaked in 1% bleach • Discard contaminated towels in infective waste containers • Wipe down the area with clean towels soaked in a same dilution of household bleach10/14/2012 Dr.T.V.Rao MD 64
  65. 65. • Programme Created by Dr.T.V.Rao MD for Medical Microbiologists in the Developing World • Email • doctortvrao@gmail.com10/14/2012 Dr.T.V.Rao MD 65

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