Micro Lab Exam II

  1. objectives of the medical lab technologists
  2. Examples of Infectious disease symptoms:
    • 1. Diffuse redness and swelling of the throat or tonsils
    • 2. Serous or purulent discharge from wounds or mucous membranes
    • 3. Accumulation of pus in abscesses or body cavities which often results in pain, swelling, and increased heat to the area
    • 4. Cough or increased sputum production
    • 5. Burning on urination
    • 6. Dysentery (inflammation of the intestinal mucous membrane characterized by abdominal pain, and diarrhea with the passage of mucous or blood)
  3. The Four Cardinal Signs of Inflammation
    • 1. Pain
    • 2. Redness
    • 3. Heat (fever)
    • 4. Swelling
  4. Basic Rules for Specimen Collection
    • 1. Culture specimen must be material from the actual infection site and must be collected w/minimum of contamination
    • (General aseptic technique & procedures should be used including sterile sample containers and other tools to prevent contamination from the environment or patient)
    • 2. Optimal times must be established for specimen collection for the best chance of recovery of the causative organisms.
    • 3. A sufficient quantity of specimen must be obtained to perform the culture techniques requested.
    • 4. Appropriate collection devices, specimen containers, and culture media must be used to ensure optimal recovery of microorganisms
    • 5. Whenever possible, obtain cultures prior to the administration of antibiotics.
    • 6. Culture container must be properly labeled.
  5. Proper Collection of Microbiology Specimens
    A proper collection of throat, nasal, and nasopharyngeal swabs
    • 1. Throat swabs should be collected from the posterior oropharynx taking care not to touch the patient's teeth, inner cheeks, or lips.
    • 2. Nasal swabs should be collected form the anterior nares.
  6. Throat Nasal (proper collection)
    • 1. Nasopharyngeal
    • 2. Oropharygeal swabs
    • Use only sterile dacron or rayon swabs with plastic shafts.
    • Do not use calcium alginate swabs or swabs with wooden sticks, as they may contain substances that inactivate some viruses and inhibit PCR testing
  7. Proper collection of Throat, Nasal
    1. Nasopharyngeal swabs
    • Insert swab into the nostril parallel to the palate.
    • Leave the swab in place for a few seconds to absorb secretions
    • Swab both nostrils
  8. Proper collection of Throat, Nasal
    2. Oropharyngeal swabs
    Swab the posterior pharynx and tonsillar areas, avoiding the tongue
  9. Proper Collection of Sputums and Lower Respiratory Secretions
    • 1. Expectorated sputum is the usual specimen submitter from patients with indication of pneumonia and tuberculosis
    • 2. Sputum specimens collected early in the morning, after awakening, are the best
    • 3. Patients who may have trouble producing sputums may have to be given respiratory therapy treatments to yield sufficient sputum
  10. Proper collection of Urine Specimens
    • 1. "Clean catch" urine specimens are the most common type of urine specimen submitted
    • 2. Catheterized urine specimens may be used for culture
    • 3. Free flowing urine from the catheter tubing should be obtained with a syringe.
    • 4. Occasionally, a suprapubic puncture of the urinary bladder is necessary to obtain a valid urine specimen, especially on infants when anaerobes are present
  11. Proper Collection
    • 1. Culture for Neisseria gonorrhae- should be collected from the endocervical canal
    • 2. In case of vafinitis, wet mounts should be made of the vaginal discharge shortly after collection. (Cue cells and trophozoites)
    • 3. Specimens for suspected endometritis should be collected with a narrow catheter and a collection swab inserted through the catheter into the uterus without touching the cervix
  12. Ear and eye (proper collection of specimenst)
    1. Eye
    2. Ear
    • 1. A swab of eye discharge and conjuctival scrapings are the 2 most common specimens submitted for culture from an eye
    • (The most common pathogen is Haemophilus influenzae-aegyptuis)
    • 2. Collection of cultures from the ear canal is usually done by a physician. Usually ear cultures are ordered for the infections of the outer ear.
    • (fungus or Pseudomonas aeruginosa)
    • 3. It is not usually possible to collect specimens from cases of otitis media (infections of the middle ear, unless the ear drum is ruptured)
  13. Cerebrospinal Fluid (proper collection)
    • Lumbar spinal puncture is the procedure used by physicians to obtain CSF for culture
    • 1. Once the appropriate location is palpated, local anesthetic is infiltrated under the skin and then injected along the intended path of the spinal needle
    • 2. A spinal needle is inserted between the lumbar vertebrae L3/L4 or L4/L5 and pushed in until there is a "give" that indicates the needle is past the dura mater
    • 3. Usually the last tube filled in the tube which should be refrigerated prior to culture because suspect organisms are sensative to cold (Neisseria meningtidis and Haemophilys influenzae)
  14. Blood Cultures
    • Disinfection and timing of the blood specimen are important aspects of blood collection
    • It is best to collect blood on a fever spike, and in the absence of a fever spike, approximately an hour apart
  15. Surgically removed tissue, biopsies, and autopsies
    • 1. A piece of tissue should always be ground in a tissue grinder or minced with a sterile scalpel blade.
    • 2. Biopsy specimens are usually needle punch obtained by fiber optic instrumentation.
    • 3. Autopsy specimens are usually collected quite after death, and contamination with gram negative flora from the gut is often a problem
  16. Wounds and abscesses
    Best specimen can be obtained with a syringe and needle from deep in the wound after thorough cleansing of the surface of the wound with surgical soap or alcohol
  17. Stool Specimens
    • 1. The common stool specimens are Campylobacter, Salmonella, and Shigella. Shigella is extremely sensitive to pH changes.
    • 2. Buffered glycerol saline is acceptable for both Shigella and Camphlobacter
  18. Two Main Objectives of Medical Microbiologists
    • 1. Isolate and identify the organism responsible for the patient's infection
    • 2. Perform a susceptibility rest on the isolated organism to determine which antibiotics would be effective against that particular organism
  19. Golden Rule
    Always follow the Microbiology Procedure Manual of your hospital for proper collection and transport of the different microbiology specimens
  20. Specimens and Tools
    • Sterile*:
    • 1. Blood
    • 2. Cerebrospinal Fluid
    • 3. Tissue fluids

    • *sterile needle aspiration
    • *antiseptic technique is used at punture site
  21. Specimens and Tools
    • Nonsterile*:
    • 1. Urine
    • 2. Feces
    • 3. Sputum

    *need to be refrigerated as samples are prone to deterioration-- overgrowth of normal flora could prevent isolation of pathogen
  22. Additional Sources of Specimens*:
    • 1. Vagina
    • 2. Ear
    • 3. Eye
    • 4. Ear canal
    • 5. Nasal cavity
    • * taken by swab
  23. The Five I's
    • 1. Inoculation (note specimen collection is first)
    • 2. Incubation
    • 3. Isolation (a susceptibility test should be done simultaneously, as soon as isolated colonies are available)
    • 4. Inspection
    • 5. Identification
  24. 1. Inoculation
    • Placing of part of a specimen into a container with culture medium, which provides bacteria with the required nutrients for growth (see selective, differential and non-differential medias).
    • With a sterile loop, sterile needle, or sterile swab, avoiding contamination
    • The technique and culture medium used will depend on the specimen being cultured
  25. 2. Incubation
    • Involves the placing of the inoculated medium in a temperature controlled chamber (incubator)
    • Optimum temperature for most pathogens is 35-37oC
    • Bacteria require from 18hrs. to several days for good mature growth
    • Most common pathogens grow in 20-24hrs.
  26. 3. Isolation
    • Separation or spreading of bacteria in an effort to produce isolated (discrete) colonies
    • Isolated colonies are required to perform identification procedures and susceptibility tests
  27. Isolation Techniques
    • 1. 4 Quadrant streaking
    • 2. Colony Count streaking
    • 3. Pour Plate technique
    • 4. Z streak
  28. Isolation pics
    Image Upload 1
  29. 4. Inspection
    • Cultures are observed for obvious growth characteristics (color, texture, size) in terms of colonies
    • Slides are made to assess microscopic details (cell shape, size, motility)
    • Staining may be used to determine more information
  30. Inspection (reading plates)
    • After proper incubation of clinical specimens, bacterial cultures are examined for the presence of possible pathogens
    • Because diff organisms produce colonies that are slighty different, this step is base in recognition by experience of the characteristic colonies of common pathogens
    • From inspection you would determine if you need to subculture an organism, or if u have a pure culture, or the # of diff organisms present if it's a mixed culture
  31. Inspection and normal flora
    • Inspection may involve being able to differentiate pathogens from organisms that are considered normal flora or from environmental contaminants
    • Normal Flora: the diff organisms that inhabit diff areas of the body, generally in a permanent basis and which, under normal circumstances, do not cause disease
    • Inspection also involves being familiar with the patterns of growth that bacteria exhibit in the diff types of culture media
    • Proper inspection will allow you to determine the proper method for identification
  32. 5. Identification
    • Purpose is to determine the type of organism and the level of species using
    • 1. Biochemical tests
    • 2. Antibiotic discs
    • 3. Immunological tests
    • 4. Genetic analysis
  33. Culture Media: providing nutrients in the laboratory
    • Physical states of Media (classifying media by their physical state):
    • Liquid media:
    • 1. Broths
    • 2. Infusions
    • Nutrient broths: contains beef extract, peptones, carbohydrates, minerals and other nutrients
    • Other types of liquid broths are methylene blue milk, litmus milk, and fluid thioglycolate
  34. Liquid Media
    defined as water based solutions that do not solidify at temperatures above freezing and flow freely when the container is tilted.
  35. Presence-absence broths for coliforms
    This broth contains lactose and bromocresol purple dye. Coliforms convert lactose into lactic acid, which lowers the pH. Note Pseudomonas and E. Coli.
  36. 2. Semisolid media
    • Culture media with a clot-like consistency
    • mainly used for determining the motility of bacteria
    • motility can also be determined by direct microscopic observation
  37. 3. Solid Media
    • (liquefiable) It provides a solid surface in which bacteria
    • can discrete colonies
    • it contains agar as the solidifying agent, whihc is a complex polysaccharide derived from seaweed
    • Agar melts at temperatures close to the boiling point of water and re-solidifies when cool down to about 42o C
    • Agar is not a digestible nutrient for microorgaisms
  38. 3. Solid media (nonliquefiable)
    • These are agars that do not melt which include:
    • 1. Rice grains (fungi)
    • 2. Cooked meat media (anaerobes)
  39. Chemical Content of Media
    (Classifying media by their chemical content)
    • 1. Synthetic
    • 2. Complex (non-synthetic) media
  40. Synthetic (chemically defined media)
    • have their molecular content specified by an exact formula
    • synthetic media is mainly used in microbiology research
  41. 2. Complex (non-synthetic) media
    • media that have chemically defined components, but they also have one or more components that are not chemically definable (not represented by a chemical formula), like peptones, extracts from yeasts or plants, or digests from protein or soybean
    • the exact chemical composition to the molecular level is not known
    • most media used in clinical microbiology are complex media
    • can be liquid (nutrient broths), semi-solid or solid (nutrient agar)
    • can be prepared in the lab or ordered from manufacturing companies
  42. Media to Suit Every Function (diff types of microbio culture media)
    • 1. Selective Media
    • 2. Non-selective Media (General Purpose Media)
  43. Media to Suit Every Function
    1. Selective Media
    media that contain one or more agents that allow or encourage (selects) the growth of target organisms, while inhibiting the growth of undesired organisms
  44. Media to Suit Every Function
    2. Non-Selective Media (General Purpose Media)
    • allows the growth of most organisms of clinical significance
    • they are designed to grow a broad spectrum of organisms and not to inhibit the growth of any
    • may grow some organisms of no clinical significance
    • 1. TSA 5% Blood Agar
    • 2. Chocolate Agar
  45. Media to Suit Every Function
    2. Non-Selective Media (General Purpose Media)
    TSA 5% Blood Agar
    • Main Components:
    • 1. trypticase soy extracts
    • 2. intact sheep red blood cells (5%)
    • Selective:
    • growth of most bacteria of clinical significance
    • except for (1) Neisseria gonorrhea and (2) Haemophilus spp.
    • Differential:
    • hemolytic organisms (organisms capable of producing hemolysis) and not hemolytic
  46. TSA 5% Blood Agar pic
    Image Upload 2
  47. 3. Non-Selective Media (General purpose media)
    Chocolate agar
    • it has a similar composition as blood agar but it also has extra enrichment ingredients (isovitaleX, NAD, extra hemoglobin)
    • extra nutrients make this medium the most "non-selective" medium
    • even allows the growth of Neisseria and Haemophilus species
  48. 4. Selective and Differential media
    • most differential media used in clinical microbio laboratories is both selective and differential
    • Selective:
    • will encourage the growth of target organisms while inhibiting the growth of unwanted ones
    • Differential:
    • will allow to differentiate between diff groups of bacteria present in the same culture plate
    • grow several types of microorganisms but are designed to bring out visible diffs among those microorg.
    • Differences show up as variations in colony size or colors, in media color changes, or in the formation of gas bubbles and precipitates.
  49. Columbia colistin-naladixic acid (CNA) agar with 5% sheep blood
    • Selective: for gram (+) bacteria
    • Differential: differentiate between hemolytic and non-hemolytic organisms
    • *similar to Phenylethanol (PEA) agar
  50. Mannitol Salt Agar
    • Selective: gram (+)
    • Differential: differentiate between mannitol fermenters and nonmannitol fermenters
    • Image Upload 3
  51. MacConkey agar
    • Contains:
    • 1. Bile salts
    • 2. Crystal violet
    • 3. Lactose
    • 4. Neutral red
    • Selective: gram (-)
    • Differential:
    • lactose fermenters: appear as dark colonies
    • non-lactose fermenters: beige, clear, very pale pink colonies
    • Image Upload 4
  52. Hektoen Enteric (HE) agar
    • contains:
    • 1. bile salts in a higher concentration
    • 2. lacotse
    • 3. bromothymol blue
    • Selective: gram (-) mainly pathogens (Shigella and Salonella)
    • Differential:
    • lactose fermenters- yellow to orange colonies
    • non-lactose fermenters- clear or green colonies
    • H2S produccers (colonies with a black center)
    • Image Upload 5
    • Image Upload 6
  53. Eosin Methylene Blue
    • contains:
    • 1. Peptone
    • 2. Lactose
    • 3. Sucrose
    • 4. Dyes esoin Y and
    • 5. Methylene blue
    • Selectives: gram (-)
    • presumptive for coliforms
    • Differential:
    • pink mucoid color- lactose fermentation w/little acid production
    • purple/black with a green sheen- ferments lactose with acid production
    • Image Upload 7
  54. CHROMagar
    • detection of urinary tract pathogens
    • general nutrient agar for isolation of various microorganisms
    • instant palette of colors to obtain a larger spectrum of full differentiation of species
    • CHROMagar Orientation when supplemented with various antibiotics in detecting increasingly important nosocomial and multiple resistant microorganisms
    • Image Upload 8
  55. Enriched (Enrichment) media
    • Contains: variety of nutrients and special growth factors (vitamins, amino acids) that make it "rich" in nutrients
    • Designed to ensure the recovery of fastidious organisms or when they are present in very small numbers or maybe coming from a patient being treated
    • Ex:
    • 1. Thioglycollate broth
    • 2. Cooked meat broth
  56. Biochemical tests
    Simple (non incubated) enzymatic biochem tests
    do not require incubation and are generally used to narrow down the identification to a group of genera, or to a specific genus
  57. Biochemical tests
    Biochemical Enzymatic Testing (Incubated)
    • exposing or inoculating the organism to be identified into a specific substrate (or several diff substrates) and observing if it uses them or not
    • A typical biochem test includes (at the very least) the 3 previously mentioned components:
    • 1. Substrate 2. Peptones 3. pH indicator
  58. Utilization of substrates diagram text
    • 1. Fermentation                         
    •                       enzyme

    Carbohydrate   ------->  organic acid & alcohol

    2. Decarboxylation or deamination


    Substrate    ------>     Alkaline product
  59. enzyme pic diagram
    Image Upload 9
  60. Summary
    • 1. Microbe is cultured in a medium w/ a special substrate and then tested for a particular end product.
    • 2. Presence of the end product indicates that the enzyme is expressed in that species: its absence means it lacks the enzyme for utilizing the substrate in that way.
  61. Examples of simple biomedical tests:
    • 1. Catalase Test
    • 2. Oxidase test
    • 3. Bacitracin discs ("A" disc)
    • 4. Optochin disc ("P" disc)
    • 5. Novobiocin disc
    • 6. Automated Systems
  62. Catalase test
    • Differentiate:
    • Staphylococcus and the genus Streptococcus
    • Staphylococcus is catalase (+)
    • Streptococcus is catalase (-)
  63. Coagulase
    • Coagulase is a plasma clotting enzyme secreted by Staphylococcus aureys- allows for clot formation
    • 1. (+) test for coagulase indicates presenece of Staphylococcus aureus
    • 2. (-) test may indicate presence of Staphylococcus epidermis or S. saprohyticus
  64. Colony on cotton swab turns pink-red if positive
    • 1. PYR (+) presence of the presence of enterococcus
    • 2. PYR (-) non-hemolytic streptococcus
    • Image Upload 10
  65. Spot indole test-color change of pink
    Distinguishes Escherichia, Citrobacter, Proteus and Providencia
  66. Examples of simple biochemical tests:
    Oxidase Test
    • Oxidase positive colonies produce purple and black colonies
    • distinguishes: Staphylococcus
  67. Antibiotic identification discs
    • some bacteria are identified based on the fact that they have been (traditionally) sensitive (susceptible or "killable") by a particular antibiotic
    • Others are identified because they are generally resistant (capable of surviving) the effects of a particular anitbiotic.
  68. Bacitracin discs ("A" disc)
    Bacitracin discs ("A" disc) identifies Steptococcus pyrogenes as it is sensitive to it (won't grow int the presence)
  69. Optochin disc ("P" disc)
    • Optochin disc ("P" disc)
    • identifies Streptococcus pneumoniae as it is sensitive to it
  70. Novobiocin disc
    Staphylococcus saprophyticys as this organisms is resistant
  71. Automated Systems
    • employ instruments that incubate and automatically read the results of the biochem tsts fo the organism to be identifies. The computer module of the instrument generates a numeric code based on the tests results.
    • Finally, the instrument looks up the coode in the catalog stored in its computer database and completes the identification
  72. Staphylococcus Aureus
    • Size: medium
    • Pigmentation: cream
    • Elevation and margin: raised
    • Hemolysis: beta-hemolytic
  73. Staphylococcus Epidermis
    • Size: small
    • Pigmentation: white
    • Elevation and margin: circular raised
    • Hemolysis: non-hemolytic
  74. Streptococcus pyogenes
    • size: pinpoint
    • Pigmentation: clear
    • Elevation and Margin: circular convex
    • Hemolysis: Beta-Hemolytic
  75. Steptococcus Agalactiae
    • Size: pinpoint
    • Pigmentation: Cream
    • Elevation and Margin: circular, convex
    • Hemolysis: Beta-hemolytic
  76. Enterococcus Faecalis
    • Size: pinpoint
    • Pigmentation: crean
    • Elevation and Margin: circular convex 
    • Hemolysis: Non-hemolytic
  77. Streptococcus Pneumoniae
    • Size:
    • Pigmentation: green
    • Elevation and Margin: 
    • Hemolysis: Alpha-Hemolysis
  78. Steptococcus Viridans
    • Size: pinpoint
    • Pigmentation: green
    • Elevation and Margin:
    • Hemolysis: Alpha-Hemolysis
  79. Staphylococcus Saprohyticus
    • Size: small
    • Pigmentation: white
    • Elevation and Margin: circular convex
    • Hemolysis: Non-hemolytic
  80. E. Coli
    • Size: pinpoint
    • Elevation and Margin: raised circular
    • Lactose reaction: yes
    • (on MacConkey agar only)
  81. Klebsiella Pneumoniae
    • Size: medium
    • Elevation and Margin: convex, circular
    • Lactose: yes pink
    • (on MacConkey agar only)
  82. Proteus Mirabilis
    • Size: small
    • Elevation and Margin: circular, convex
    • Lactose reaction: no
Card Set
Micro Lab Exam II
microbiology lab exam II