Final Lab Practical Review2.txt

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Final Lab Practical Review2.txt
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  1. Activity 15: Transformation Experiment (Handout)
    •Principle behind the transformation experiment
  2. •Bacterial transformation refers to the uptake of naked DNA molecules by bacterial cells. In this experiment, we will simulate this classic experiment and manipulate bacterial cells so that they will exhibit new genetic traits.
  3. Activity 15: Transformation Experiment (Handout)
    •Clinical significance of transformation behind bacteria
    •Some bacteria has the natural ability to take up naked DNA molecules from the environment, in other words they are “competent.” Many others including E. coli, are not naturally competent, but they can be made competent via laboratory manipulation. The ability to manipulate E. coli to be transformed competent, and hence take up naked DNA, revolutionized the field of molecular biology. For example, human insulin genes have been introduced into E. coli, and strains of this bacterium now human insulin or humulin.
  4. Activity 15: Transformation Experiment (Handout)
    •Characteristics of E coli. Strain and plasmid used in the experiment
    • •During this experiment we introduced a new genetic trait and an observable phenotype into a commonly used strain E. coli by transforming the cells with the plasmid, pGREEN that confers ampicillin resistance to the wild strain converting it into a transformant (mutant strain) cell. The plasmid pGREEN not only carries the ampr gene for ampicillin resistance it also carries a GFP gene for a green florescent protein which will produce a green color (“glow”) in transformed cells.
    • •Detection of transformed bacteria is facilitated by the observation of colonies on ampicillin agar plates which will “glow” do to the expression of the GFP gene. This is a positive mutant selection procedure because we eliminate the wild ampicillin susceptible strain using ampicillin as the selective agent.
  5. Activity 15: Transformation Experiment (Handout)
    •Purpose of CaCl2 reagent, alternation between warm and cold incubations, recovery step
    • •CaCl2 reagent: To neutralize the cell
    • •Alternating between hot and cold incubations: Creates pores so plasmid can enter the cell
    • •Recovery step: Closes pores so plasmid cannot escape
  6. Activity 15: Transformation Experiment (Handout)
    •Interpretation of results on LB/amp and LB plates with –pAMP and +pAMP samples and satellisism?
    • (+) control(+) control (-) control(experimental)
    • -pGREEN on LBA +pGREEN on LBA -pGREEN on LBA + Ampicilli +pGREEN on LBA + Ampicillin
  7. Activity 15: Transformation Experiment (Handout)
    •Why will we use +pGREEN LB/amp with transformed cells for the transformation experiment?
    It is the experimental plate for the experiment
  8. Activity 15: Transformation Experiment (Handout)
    Why will we use +pGREEN LB plate with transformed cells for the transformation experiment?
    • •It is one of the positive controls for the experiment.
    • •45 uL = 0.045 mL
  9. Activity 15: Transformation Experiment (Handout)
    Quiz Questions:
    • •Transformation involves the acquisition of soluble DNA or plasmids from the environment by a bacterial cell that has lyses.
    • •ThepGREENplasmid used in this lab contains two genes:amprandGFP
  10. Activity 15: Transformation Experiment (Handout)
    Lab Questions:
    •Correlate your results and determine which plate/s showed transformation? Explain why this is so
    • Experimental plate showed transformation. We took a plasmid and made it enter a bacterial cell. The new genotypes (ampr and GFP) resulted in two new phenotype traits: 1. ampicillin resistance (beta lactomase) and green fluorescent protein.
    • *ampr resulted in phenotype ampilicillin resistance (beta lactomase)
    • *genotype GFP resulted in phenotype green fluorescent protein
  11. Activity 15: Transformation Experiment (Handout)
    •What is the purpose of using the –pGREEN LB + ampicillin plate? Of the –pGREEN LB plate?
    • • –pGREEN LB + ampicillin plate: this is the (-) control group the ampicillin should kill the E. coli and there should be no growth. (Testing for contamination)
    • •–pGREEN LB plate: This (+) control group and the E. coli should grow throughout the medium. (testing for growth)
  12. Activity 15: Transformation Experiment (Handout)
    •What possible explanations are there for getting Ampicillin resistant colonies from the “no plasmid” sample (-pGREEN LB + ampicillin plate)?
    •Contamination
  13. Activity 15: Transformation Experiment (Handout)
    What factors are likely to influence the transformation efficiency of E. coli?
    •Contamination•Not properly heat shocked•Additional NaCl•Did not add plasmid•Kept it on ice for to long•Etc
  14. Activity 15: Transformation Experiment (Handout)
    •What is the purpose of using the +pGreen LB ampicillin plate? Of the +pGREEN LB plate?
    • •+pGreen LB ampicillin plate: This is the experimental group – to show expression of beta lactamase and green fluorescent protein.
    • •+pGREEN LB plate: This is the (+) control group and the E. coli should grow throughout the medium
  15. Activity 15: Transformation Experiment (Handout)
    •Transformation
    •Transformation – in the microbial genetics, the transfer of genetic material contained in “naked” DNA fragments from a donor cell to a competent recipient cell. (*Cell lyses and DNA leaves the cell – other cells can pick up this DNA)
  16. Activity 15: Transformation Experiment (Handout)
    •Transduction
    The transfer of genetic material, from one bacterium to another by means of a bacteriophage vector.
  17. Activity 15: Transformation Experiment (Handout)
    •Conjugation
    • In bacteria, the contact between donor and recipient cells associated
    • with the transfer of genetic material such as plasmids. Can involve
    • special sex (pili). Also a form of sexual recombination in ciliated
    • protozoa.
  18. Activity 15: Transformation Experiment (Handout)
    •Using the results of the laboratory, discuss the relationship between genotype and phenotype and between wild and mutant type.
    • •Genotype – The genetic makeup of an organism. The genotype is ultimately responsible an organisms phenotype, or expressed characteristics. (genetic make-up: cannot see it)
    • •Phenotype - The observable characteristics of an organism produced by the interaction between its genetic potential (genotype) and the environment. (expression of genotype: can see it)
    • •Wild type – (aka wild strain) the natural non-mutated form of a genetic train
    • •Mutant type – (aka mutant strain) a subspecies of microorganisms that has undergone a mutation, causing expression of a trait that differs from other members of that species.
    • *ampr resulted in phenotype ampilicillin resistance (beta lactomase)
    • *genotype GFP resulted in phenotype green fluorescent protein
  19. Activity 15: Transformation Experiment (Handout)
    •Why is bacterial transformation useful to genetics and molecular biologists?
    Study other genes and proteins from other types of cells and microbes
  20. Activity 15: Transformation Experiment (Handout)
    •Plasmid exchange and transformation in bacteria is rampant in nature as well as in clinical settings. What impact does this phenomenon have on human disease and healthcare?
    Bacterial cells in the body can become drug resistant
  21. Activity 15: Transformation Experiment (Handout)
    Faint green growth TNTC
    • Plasmid caused two new traits (phenotypes) Produced Beta Lactamase to result in ampicillin resistant protein and GFP caused protein to be fluorescent green.
    • LB plate with transformed cells +pGREEN
    • (positive control)
  22. Activity 12A: Polymerase Chain Reaction (Handout)
    Microorganisms used:
  23. •Staphylococcus aureus
  24. Activity 12A: Polymerase Chain Reaction (Handout)
    •Clinical applications of PCR
    •PCR is an indispensable technique used in medical and biological research for a variety of applications including: sequencing, diagnosis of heredity and infectious disease, and identification of genetic fingerprints (used in forensic sciences and paternity tests).
  25. Activity 12A: Polymerase Chain Reaction (Handout)
    •Principle involved in PCR
    • •One of the most important technical developments in the past 20 years in molecular biology is the polymerase chain reaction (PCR). PCR is based on the enzymatic amplification of a desired fragment of DNA.
    • •*We will be using PCR and gel electrophoresis to identify MRSA.
  26. Activity 12A: Polymerase Chain Reaction (Handout)
    •Advantages of PCR over conventional methods
    • •In particular, PCR-based tests are able to detect the presence of pathogenic agents earlier than serologically-based methods, as patients can take weeks to develop antibodies against an infectious agent. Earlier detection of infection can mean earlier treatment and an earlier return to good health.
    • Advantages
    • Disadvantages
    • Faster
    • Cost
    • More specific
  27. Activity 12A: Polymerase Chain Reaction (Handout)
    •Three steps involved in one cycle, temperature used, and end result.
    • •1. Denature - unzip
    • •2. Anneal - primer puts down start sequence
    • •3. Taq Polymerase – extends DNA
    • *Starts with one and ends with 2 – keeping going approximately 30x this = 1 million.
  28. Activity 12A: Polymerase Chain Reaction (Handout)
    •The purpose of thermocycle
    • •Thermocyle - An apparatus which changes temperatures according to precise programming.
    • •The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
  29. Activity 12A: Polymerase Chain Reaction (Handout)
    •What is Taq polymerase
    •Use of the thermostableTaq polymerase– extends the DNA (make more DNA).
  30. Activity 12A: Polymerase Chain Reaction (Handout)
    •What is the purpose of the primers
    •The use of primers – anneals DNA – this puts down the start sequence.
  31. Activity 12A: Polymerase Chain Reaction (Handout)
    •What is the mecA gene, its gene product and its occurrence in MRSA
    • •The mecA gene is a gene found in bacterial cells. The most commonly known carrier of the mecA gene is the bacterium known as MRSA. The mecA gene allows a bacterium to be resistant to antibiotics such as Methicillin, and other penicillin-like antibiotics. The mecA gene does not allow the ring like structure of penicillin-like antibiotics to attack the enzymes that help form the cell wall of the bacterium (transpeptidases), and hence the bacteria is allowed to replicate as normal.The mecA gene encodes the protein PBP2A (Penicillin binding protein 2A).
    • PBP2A has a low affinity for beta-lactams such as Methicillin, and this
    • enables transpeptidase activity in the presence of beta-lactams to
    • allow cell wall synthesis. It is also found in Staphylococcus aureus and
    • Streptococcus pneumoniae resistant to penicillines. *Confers to drug
    • resistance because of the gene product PBP2A (Penicillin binding protein
    • 2A).
  32. Activity 12A: Polymerase Chain Reaction (Handout)
    Quiz Questions:
    •What diagnostic technique involves denaturation, annealing, and extension?
    PCR•PCR which stand for Polymerase Chain Reaction amplifies the DNA to around 1 million copies.•Antibiotic Resistance can also be detected by Kirby-Bauer.•Taq Polymerase is the enzyme responsible for the amplification of DNA in this lab.
  33. Activity 12A: Polymerase Chain Reaction (Handout)
    Quiz Questions:•
    What is the name of the gene of Methicillin Resistant Staphylococcus aureus (MRSA) that you are detecting in this lab?
    It can be identified as a mecA, 533 bpband by using gel.
  34. Activity 12A: Polymerase Chain Reaction (Handout)
    Lab Questions:
    •What is the role of each of the following in PCR:•Taq DNA polymerase
    Extension (making more DNA)
  35. Activity 12A: Polymerase Chain Reaction (Handout)Lab Questions:•What is the role of each of the following in PCR: •Primers
    Annealing (putting down the start sequence)
  36. Activity 12A: Polymerase Chain Reaction (Handout)Lab Questions:•What is the role of each of the following in PCR:
    •94 C
    Denature (apply heat)
  37. Activity 12A: Polymerase Chain Reaction
    What are the advantages and disadvantages of using PCR technique versus Kirby-Bauer Method?
    • PCR is faster, more specific and highly sensitive compared to the Kirby-Bauer but the Kirby Bauer is much more cost efficient.
    • You can use the following test to identify MRSA:•PCR (3-4 hours)•Kirby-Bauer (24 hours)•Gel electrophoresis
  38. Activity 12A: Polymerase Chain Reaction
    Interpretation:
    • Agarose gel electrophoresis of PCR product:
    • •The presence of the 533bp band, corresponding to mecA gene, on the gel indicates that the strain tested is methicillin resistant (MRSA).
    • •The absence of the mecA gene indicates a non methicillin resistant strain
  39. Activity 13: Staphylocci (Ex 52)
    •Clinically important members of this genera
    • Date: 10/25/2011,
    • •There are 19 species of staphylococci listed in the Bergey’s Manual, with the most medically important being:
    • •S. aureus
    • •S. epidermidis
    • •S. saprophyticus.
    • •A significant characteristic separates S. aureus from the other two species: S. aureus can coagulate plasma, which is often correlated with its pathogennicity. However, the non-coagulase-producing organisms S. epidermidis and S. saprophyticus do cause infections of the cerebrospinal fluid, prosthetic joints, and vascular grafts.
  40. Activity 13: Staphylocci (Ex 52)
    •Sources of these microbes in the environment and human host
    • •Collectively, the Staphylococci and streptococci are referred to as the pyogenic (pus –forming) gram positive cocci.
    • •The staphylococci were originally isolated from pus in wounds but were subsequently demonstrated to be part of the normal flora of the nasal membranes, hair follicles, skin and perineum of healthy individuals. Most of the human population is carriers of staphylococci.
    • •This is especially important in hospitals where 90% of the personnel can be carriers and where these bacteria are responsible for many nosocomial (hospital-acquired) infections. (example MRSA)
  41. Activity 13: Staphylocci (Ex 52)
    •Steps of Experiment:
    • •Day 1: Specimen collection
    • •Inoculate m-staphylococcus broth (MSB)
    • •Day 2: Primary Isolation procedures (Taking MSB test tubes to inoculate MSA and SM110)
    • •Inoculate Mannitol Salt Agar (MSA)
    • •Inoculate Staphylococcus medium 110 (SM110)
    • •Day3: Plate Evaluations and Coagulase
    • •Catalase Test
    • •Bacitracin Test (for Beta)
    • •Optochin Test (for Alpha)
    • •Salt tolerance Test (for Gamma)
    • •Bile Esculin Agar (for Gama)
    • •Day 4: Confirmation
    • •Read Results:
    • •Read Rabbit Plasma
    • •Read Blood Agar
  42. Activity 13: Staphylocci (Ex 52)
    Table 52.1 Differentiation of Three Species of Staphylococci: S. Aureus
  43. Activity 13: Staphylocci (Ex 52)
    Blood Agar (BA)
    Hemolytic Reaction
  44. Activity 13: Staphylocci (Ex 52)
    Rabbit Plasma
    Coagulase
  45. Activity 13: Staphylocci (Ex 52)
    •Principle involved in inoculating m-staphylococcus broth (MSB)
    •What type of organism grow in this medium?
    Halotolerant organisms (salt tolerant organisms) such as Staphylococcus aureus

    •What in the medium allows the organisms to grow? 10% NaCl
  46. Activity 13: Staphylocci (Ex 52)•Principle involved in inoculating m-staphylococcus broth (MSB)
    •What in the medium allows the organisms to grow?
    10% NaCl
  47. Activity 13: Staphylocci (Ex 52)•Principle involved in inoculating m-staphylococcus broth (MSB)
    •How do you classify this medium?
    MSB is enriched selective medium
  48. Activity 13: Staphylocci (Ex 52)
    •Gram Stain of Staphylococcus
    •What type of gram reaction, morphology and arrangement for Staphylococcus?
    • Gram (+), cocci clusters
  49. Activity 13: Staphylocci (Ex 52)•Gram Stain of Staphylococcus
    •Why does Staphylococcus stain this particular color with the gram stain?
    • It is based on the chemical and physical properties of their cell walls.
    • Primarily, it detects peptidoglycan, which is present in a thick layer
    • in Gram positive bacteria. A Gram positive results in a purple/blue
    • color while a Gram negative results in a pink/red color.
  50. Activity 13: Staphylocci (Ex 52)
    •Principle involved in catalase test, reagents used and its applications
    • •Used to test for presence of enzyme catalase.
    • •2 H2O2 2 H2O2 + O2
    • •Procedure: (1) streak tryptic soy agar slant with organism using wire loop. (2) Incubate at optimum temperature for 24-48 hours. (3) Place a few drops of 3% H2O2 on the slant
    • •Interpretation: Positive – Bubbling (O2 gas is liberated from the H2O2). Negative – no bubbling
  51. Activity 13: Staphylocci (Ex 52)
    •Usage of MSA and S1110 Agar and typical colony morphology of members of this genera on these media
    • •Mannitol Salt Agar (MSA)
    • •MSA Is a selective and differential media
    • •Which plate showed growth of Staphylcoccus?
    • and
    • •Which plate(s) show an organism that ferments mannitol?
  52. Activity 13: Staphylocci (Ex 52)•Usage of MSA and S1110 Agar and typical colony morphology of members of this genera on these media
    •Which plate(s) show an organism does ferment mannitol?
  53. Activity 13: Staphylocci (Ex 52)
    •What is the name of the selective agent in MSA?
    NaCl
  54. Activity 13: Staphylocci (Ex 52)
    What is the name of the differential agent in MSA?
    Mannitol
  55. Activity 13: Staphylocci (Ex 52)
    What is the name of the pH indicator in MSA?
    Phenol red
  56. Activity 13: Staphylocci (Ex 52)
    Staphylococcus Medium 110 (SM110):What is the advantage of using this medium in the microbiology lab?
    SM110 also contains NaCl and mannitol, but it lacks phenol red. Its advantage over MSA is that is favors colony pigmentation by different strains of S. aureus. Since this medium lacks phenol red, no color change takes place as mannitol is fermented.
  57. Activity 13: Staphylocci (Ex 52)
    How is this medium (SM110) different from MSA?
    Does not have a pH indicator therefore colony pigmentation occurs.
  58. Activity 13: Staphylocci (Ex 52)
    •What is the gram reaction of the organism that can grow on MSA?
    Gram (+)
  59. Activity 13: Staphylocci (Ex 52)
    •Hemolytic reactions on Blood Agar of members of this genera
    •Which plate shows beta hemolysis?
  60. Activity 13: Staphylocci (Ex 52)
    Blood Agar
    •What toxin is responsible for plate E for the staphylococcus lab? Alpha toxin
    • Alpha toxin
    • •What organism produces this toxin for the staphylococcus lab? S. aureus
    • •Which plate shows alpha hemolysis?
  61. Activity 13: Staphylocci (Ex 52)
    Blood Agar
    •What organism produces this toxin for the staphylococcus lab? (Alpha Toxin)
    S. aureus
  62. Activity 13: Staphylocci (Ex 52)
    Blood Agar
    •What is the enriched in this medium?
    5% sheeps blood
  63. Activity 13: Staphylocci (Ex 52)
    Blood Agar
    •What is the differential agent in this medium?
    .5% NaCl
  64. Activity 13: Staphylocci (Ex 52)
    •Principle involved in coagulase test, reagents used in and its applications
    •Which test tube shows the production of coagulase?
  65. Activity 13: Staphylocci (Ex 52)
    •What is the purpose of the coagulase test?
    Is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci.
  66. Activity 13: Staphylocci (Ex 52)
    What organisms is positive for this test? (coagulase test)
    S. aureus
  67. Activity 13: Streptocci (Handouts)
    •Clinically important members of this genera
    •Streptococcus pyogenes & Streptococcus pneumonia
  68. Activity 13: Streptocci (Handouts)
    •Throat culture and normal throat microbiota
    •Species of Streptococci are the predominant microorganisms encountered in routine throat cultures. Streptococcus pyogenes, for instance, is the major cause of bacterial sore throat or acute pharyngitis. The throat culture procedure being performed here is intended for the recovery of this bacterial species.
  69. Activity 13: Streptocci (Handouts)
    •Steps of Experiement:
    • •Day 1:
    • •Throat Culture
    • •Day 2:
    • •Hemolysis
    • •Day 3:
    • •Catalase Test
    • •Bacitracin Test
    • •Optochin Test
    • •Bile Esculin Test
    • •Salt Toleramce Test
    • •Day 4:
    • •Results
  70. Activity 13: Streptocci (Handouts)
    •Gram Stain of Streptococci
    •What is the Gram reaction, morphology, and arrangement for Streptococcus?
    Gram (+), cooci, chains
  71. Activity 13: Streptocci (Handouts)
    Gram Stain of Streptococci
    •Why does Streptococcus stain this particular color with the Gram stain?
    • t is based on the chemical and physical properties of their cell walls.
    • Primarily, it detects peptidoglycan, which is present in a thick layer
    • in Gram positive bacteria. A Gram positive results in a purple/blue
    • color while a Gram negative results in a pink/red color.
  72. Activity 13: Streptocci (Handouts)
    •Hemolytic reactions on Blood Agar of members of this genera
    •Which plate shows beta hemolysis?
    • Complete hemolysis, giving a clear zone with a clean edge around the colony.
  73. Activity 13: Streptocci (Handouts)•
    Hemolytic reactions on Blood Agar of members of this genera
    •What toxin is responsible for plate E for the staphylococcus lab?
    Alpha toxin
  74. Activity 13: Streptocci (Handouts)
    •Hemolytic reactions on Blood Agar of members of this genera
    •What organism produces this toxin for the staphylococcus lab?
    S. aureus
  75. Activity 13: Streptocci (Handouts)•Hemolytic reactions on Blood Agar of members of this genera
    Which plate shows alpha hemolysis?
    Incomplete hemolysis, producing cloudy zone of greening around the colony due to production of methemoglobin.
  76. Activity 13: Streptocci (Handouts)•Hemolytic reactions on Blood Agar of members of this genera
    •Which plate shows gamma hemolysis?
    No hemolysis or no change in the blood agar around the colony.
  77. Activity 13: Streptocci (Handouts)
    •Hemolytic reactions on Blood Agar of members of this genera
    •What is the name of the differential agent in this medium?
    5% NaCl
  78. Activity 13: Streptocci (Handouts)•Hemolytic reactions on Blood Agar of members of this genera
    •What is the enriched agent in this medium?
    5% Sheeps blood
  79. Activity 13: Streptocci (Handouts)
    •Principle involved in catalase test, reagents used and its applications
    • •*Catalase test: Streptococcus Lab with Staphylococcus aureus as an control
    • •Perform catalase test on the Brain Heart Infusion Agar (BHIA) slant cultures of your isolate by adding 3% H2O2
    • •Why did we use this medium in the Streptococcus lab? Because it is a highly nutritious general-purpose growth medium for fastidious microorganisms, such as streptococci
    • •How do you classify this medium? As an enriched non-selective medium for the isolation and cultivation of most anaerobic bacteria and other fastidious microorganisms
    • •Principle - Used to test for presence of enzyme catalase.
    • •2 H2O2 2 H2O2 + O2
    • •Procedure: (1) streak tryptic soy agar slant with organism using wire loop. (2) Incubate at optimum temperature for 24-48 hours. (3) Place a few drops of 3% H2O2 on the slant
    • •Interpretation: Positive – Bubbling (O2 gas is liberated from the H2O2). Negative – no bubbling
  80. Activity 13: Streptocci (Handouts)•
    Principle involved in catalase test, reagents used and its applications
    •Why did we use this medium in the Streptococcus lab?
    • Because it is a
    • highly nutritious general-purpose growth medium for fastidious
    • microorganisms, such as streptococci
  81. Activity 13: Streptocci (Handouts)
    •Principle involved in catalase test, reagents used and its applications
    •How do you classify this medium?
    • As an enriched non-selective medium
    • for the isolation and cultivation of most anaerobic bacteria and other
    • fastidious microorganisms
  82. Activity 13: Streptocci (Handouts)•
    Principle involved in catalase test, reagents used and its applications
    •Principle and Procedure
    • •Principle - Used to test for presence of enzyme catalase. •2 H2O2 2 H2O2 + O2•
    • Procedure:
    • (1) streak tryptic soy agar slant with organism using wire loop. (2)
    • Incubate at optimum temperature for 24-48 hours. (3) Place a few drops
    • of 3% H2O2 on the slant•Interpretation: Positive – Bubbling (O2 gas is liberated from the H2O2). Negative – no bubbling
  83. Activity 13: Streptocci (Handouts)•
    •When would the catalase test be used?
    The catalase test is commonly used to differentiate streptococci (negative) for staphylococci (positive)

    •What is the name of the end products in a positive test result? Water (2 H2O) and Oxygen gas (O2)
  84. Activity 13: Streptocci (Handouts)•
    Catalase
    •What is the name of the end products in a positive test result?
    Water (2 H2O) and Oxygen gas (O2)
  85. Activity 13: Streptocci (Handouts)•
    Catalase Test
    •What reagent do you use for this test?
    3% H2O2(Hydrogen peroxide)
  86. Activity 13: Streptocci (Handouts)
    •Principle involved in Bacitracin disks tests and Streptococcus group identified with it.
    •This test is used to differentiate beta-hemolytic Streptococcus pyogenes from other beta-hemolytic Streptococci. Streptococcus pyogenes is the most common cause of acute pharyngitis and its presumptive identification is based on its susceptibility Bacitracin, an antibiotic that inhibits cell wall synthesis.
  87. Activity 13: Streptocci (Handouts)
    Bacitracin disks tests
    What hemolytic colonies do we perform this test for?
    Beta
  88. Activity 13: Streptocci (Handouts)
    Bacitracin disks tests
    •What does the zone of inhibition need to record for a positive test result?
    >/= 10mm
  89. Activity 13: Streptocci (Handouts)
    Bacitracin disks tests
    •What is the name of the organism that is susceptible to bacitracin?
    Streptococcus pyogenes
  90. Activity 13: Streptocci (Handouts)
    •Principle involved in Optochin disks tests and Streptococcus group identified with it
    •Principle - the Optochin test is used to presumptively differentiate Streptococcus pneumonia from other Alpha-hemoltic Streptococci. Streptococcus pneumonia is the only streptococcus that is susceptible to Optochin.

    • •What does the zone of inhibition need to record for a positive test result? >/= 14mm
    • •What is the name of the organism that is susceptible to optochin? Streptococcus pneumonia
  91. Activity 13: Streptocci (Handouts)
    Optochin disks tests
    What hemolytic colonies do we perform this test for?
    • Alpha
  92. Activity 13: Streptocci (Handouts)
    Optochin disks tests
    •What is the name of the organism that is susceptible to optochin?
    Streptococcus pneumonia
  93. Activity 13: Streptocci (Handouts)
    Optochin disks tests
    •What does the zone of inhibition need to record for a positive test result?
    >/= 14mm
  94. Activity 13: Streptocci (Handouts)
    •Bile Esculin Agar: typical reactions observed
    • •Principle - This test is commonly used for the presumptive identification of Enterococci and members of Group D Streptococcus (Non-Enterococcus).
  95. Activity 13: Streptocci (Handouts)
    •Bile Esculin Agar
    •What test tube is showing a positive test result?
    The second test tube
  96. Activity 13: Streptocci (Handouts)
    •Bile Esculin Agar:
    •What hemolytic colonies do we perform this test for?
    Gamma
  97. Activity 13: Streptocci (Handouts)
    •Bile Esculin Agar:
    •If the test is positive, do you need to perform an additional test to determine the identity of your unknown organism?
    Yes the Salt Tolerant Test
  98. Activity 13: Streptocci (Handouts)
    •Salt tolerant test for some members of this genera: media used and typical reactions observed
    •Principle - This test is based on the ability of Enterococci to grow in 6.5% salt broth
  99. Activity 13: Streptocci (Handouts)
    •Salt tolerant test
    •What hemolytic colonies do we perform this test for? What test tube is showing a positive test result?
    • Gamma• Second test tube (yellow)
  100. Activity 13: Streptocci (Handouts)
    •Salt tolerant test
    What test tube is showing a positive test result?
    • Second test tube (yellow)
  101. Activity 13: Streptocci (Handouts)
    •Salt tolerant test
    •What is the name of the pH indicator in this medium?
    • Bromcresol purple pH indicator
    • •If both the Bile Esculin test and this test record positive, what is the identity of your unknown organism? Enterococcus spp. (Enterococcus faecalis)
    • •Flow chart for Pyogenic Gram Positive Cocci
    • •In lab packet
  102. Activity 13: Enterics (Ex 54)
    •Clinically important members of Family
    • Enterobacteriaceae
    • •Salmonella and Shigella
  103. Activity 13: Enterics (Ex 54)
    •Distinguish important groupings of enteric bacteria: enterics, califorms, and E. coli = indicator organism for fecal contamination
    • •Enterics – The gram negative, facultatively anaerobic bacilli that reside in the large instestine belong to a large group of bacteria, the Enterobacteriaceae.
    • •Califorms – Enteric bacilli that are lactose fermenters
    • •E. Coli –
  104. Activity 13: Enterics (Ex 54)
    •Sources of these microbes: environmental, animal and human hosts
    • •Salmonella - Salmonella infections are zoonotic (can be transferred from animal) and can be transferred between humans and nonhuman animals. Many infections are due to ingestion of contaminated food
    • •Shigella - thrives in the human intestine and is commonly spread both through food and person-to-person contact. Shigella is the third most common pathogen transmitted through food
  105. Activity 13: Enterics (Ex 54)
    •Selective and differential media used: MAC – selective agents, differential agents, pH indicators incorporated in these media
    • •MacConkey Agar (MAC)
    • •Type of media - Classified as biochemical media
    • •Selective agents – Biosalts and crystal violet (to inhibit most gram positive organisms)
    • •Differential agents – is used to distinguish organisms from each other based on a reaction that occurs as they grow. Lactose positive and lactose negative
    • •pH indicators – neutral red
    • •What is the Gram reaction of organisms that grow on MAC? Gram (-)
    • •Which plate(s) show an organism that ferments lactose? Lactose positive organisms (E. coli) produce acid when they grow. This acid causes the pH to drop. Neutral red is absorbed and the colony turns red as in the second plate.
    • •Which plate(s) show an organism that does not ferment lactose? Lactose negative organisms (salmonella & shigells) do not produce acid when they grow on MAC. Lactose negative organisms remain colorless and translucent as in the first plate.
  106. Activity 13: Enterics (Ex 54)
    •Triple Sugar Iron Agar (TSI)
    •Type of media
    • •Type of media – Differential biochemical media
    • •Selective agents & Differential agents - this is a key medium for use in beginning the identification of a Gram-negative bacilli of the enteric group. It contains glucose (0.1%), lactose (1%), sucrose (1%) and peptone (2%)as nutritional sources. Sodium thiosulfate serves as the electron receptor for reduction of sulfur and production of H2S. Detects fermentation of sucrose, lacotse, glucose, as well as production of hydrogen sulfide and/or gas.
    • •pH indicators – Phenol red
    • •Reading TSI:
    • •1. Slant (A – yellow: this means fermented (+) sucrose and or glucose) or (K – red/pink: this means fermented (-) in the slant)
    • •2. Butt (A-yellow: this means glucose fermented) or (K-pink/red: this means fermented (-) in the butt)
    • •3. Gas (G – for presence of gas which is bubbling or lifting at the butt) or (no G for no bubble or lifting at the butt)
    • •4. H2S (S when there is black at the butt – ferrous sulphide is produced) or (no S if there is not black in the butt)
    • •What color indicates that fermentation has occurred? Yellow
  107. Activity 13: Enterics (Ex 54)
    •Triple Sugar Iron Agar (TSI)
    •Selective agents & Differential agents -
    • this is a key medium for
    • use in beginning the identification of a Gram-negative bacilli of the
    • enteric group. It contains glucose (0.1%), lactose (1%), sucrose (1%)
    • and peptone (2%)as nutritional sources. Sodium thiosulfate serves as the
    • electron receptor for reduction of sulfur and production of H2S.
    • Detects fermentation of sucrose, lacotse, glucose, as well as production
    • of hydrogen sulfide and/or gas.
  108. Activity 13: Enterics (Ex 54)
    •Triple Sugar Iron Agar (TSI)
    •pH indicators
    – Phenol red
  109. Activity 13: Enterics (Ex 54)
    •Triple Sugar Iron Agar (TSI)
    Reading TSI:
    • 1. Slant (A – yellow: this means fermented (+) sucrose and or glucose)
    • or (K – red/pink: this means fermented (-) in the slant)•2. Butt
    • (A-yellow: this means glucose fermented) or (K-pink/red: this means
    • fermented (-) in the butt)•3. Gas (G – for presence of gas which is
    • bubbling or lifting at the butt) or (no G for no bubble or lifting at
    • the butt)•4. H2S (S when there is black at the butt – ferrous sulphide
    • is produced) or (no S if there is not black in the butt)
  110. Activity 13: Enterics (Ex 54)
    •Triple Sugar Iron Agar (TSI)
    •What color indicates that fermentation has occurred?
    Yellow
  111. •Motility Medium Triphenyl Tetrazolium Chloride (MTM)
    • •Type of media – General purpose media
    • •Which tube(s) shows a negative result for motility? Last two
    • •Which tube(s) shows a positive result for motility? First two
    • •What is percentage of agar in this medium? .4% agar
    • •What is the name of the indicator dye for this medium? Triphenyl-Tetrazolium chloride
    • •What is the name of the red precipitate in a motile positive test tube? Formazan
  112. •Urea Broth (UB)
    • •Type of media – Biochemical media
    • •pH indicators – phenol red
    • •Which tube(s) show the production of urease? Second tube (intense pink color)
    • •What is the name of the substrate in this medium? Urea
    • •What is the name of the product(s) produced in a positive urease result? 2 ammonia & CO2
  113. •Simmons Citrate Agar (CIT)
    • •Type of media – Enriched biochemical medium
    • •pH indicators - Bronthynol blue
    • •Which test tube shows the utilization of citrate? Second tube (blue) - Positive- alkaline pH causes media to change from green to prussian blue Negative- no color change
    • •What is the name of the carbon source in this medium? Sodium citrate
    • •What is the name of the Nitrogen source in this medium? Ammonium salts
    • •What is the name of the pH indicator in this medium? Bronthynol blue
    • •What is the name of the end product that creates an alkaline pH in a positive test result? In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result (the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.
  114. •Tryptone Broth (TRP)
    • •Type of Media – Differential biochemical medium
    • •Which tube(s) shows indole production? The last tube (red on top)
    • •What enzyme is secreted in this positive test result? Tryptophanase
    • •What reagent is used for this medium? Kovacs reagent
  115. •Colony appearance of lactose and non-lactose fermenters on these media; clinical significance of detecting LF and NLF colonies; examples of organisms that are LF and NLF.
    • •MAC
    • •Only gram (-) grow on this plate. Gram (+) do not grow on this plate.
    • •Clinical significance of detecting LF and NLF colonies -
    • •Lactose fermenters – show up purple on media, gram (+), example: ecoli
    • •Non-lactose fermenters (NLF) – non-color on media, gram (-), examples: Salmonella and Shigella
    • •Lactose positive means that the organism can use lactose as an energy source.
    • •Lactose negative means that the organism can not use lactose as an energy source.
    • •Principle involved in the use of TSI as a screening test for enterics: components of the medium – sugars, pH indicator, H2S indicator incorporated in this medium; inoculation technique, incubation time (why only 18-24 hours?);
    • •Interpret TSI results (Refer to table given out as handout);
    • •Reading TSI:
    • •1. Slant (A – yellow: this means fermented (+) sucrose and or glucose) or (K – red/pink: this means fermented (-) in the slant)
    • •2. Butt (A-yellow: this means glucose fermented) or (K-pink/red: this means fermented (-) in the butt)
    • •3. Gas (G – for presence of gas which is bubbling or lifting at the butt) or (no G for no bubble or lifting at the butt)
    • •4. H2S (S when there is black at the butt – ferrous sulphide is produced) or (no S if there is not black in the butt)
    • •What color indicates that fermentation has occurred? Yellow
    • •Give the readings for tubes below
  116. •Principles involved in the use of Motility, UB, CIT, Indole test (TRP): components of these media, pH indicators, inoculation techniques, special reagents used if any; interpretation of reactions in these media (Consult table of Biochemical Test handout or the Bergey’s Manual)
    • •Motility test
    • •Medium Used – Motility Medium with TTC
    • •Medium description – light pink color - semisolid
    • •Principle of this test – Used to determine whether or not an organisms is motile
    • •Components of this media – The medium contains 0.4% agar and 2,3,5 – Triphenyl-Tetrazolium Chloride (TTC). Motile bacteria will be able to swim through the medium and will reduce TTC to form a red precipitate called formazan
    • •Inoculation techniques – 1. Use a needle to inoculate by making a single stab about two-thirds down and then pull the needle out along the same path. 2. Incubate at the optimum temperature for 24-48 hours.
    • •Special reagents used if any - none
    • •Interpretation of reactions in these media – Motile: the tube will appear red and cloudy and usually the organism will spread over the top of the medium and or throughout. Non-motile: the organisms will grow along the streak line only; the medium will NOT be cloundy.
  117. •Urease Test
    • •Medium Used – Urea Broth
    • •Medium description – soft orange color - liquid
    • •Principle of this test – Used to determine the ability of an organism to split urea to form ammonia (an alkaline end product) by the action of the enzyme, urease.
    • •Components of this media - The medium also contains the pH indicator phenol red, which turns an intense pink at alkaline pH.
    • •Inoculation techniques – 1. Inoculate urea broth with the organisms using a wire loop (Dip and swirl). 2. Incubate at optimum temperature for 24-48 hours.
    • •Special reagents used if any – None
    • •Interpretation of reactions in these media – Positive – intense pink color. Negative – no color change. (*Note: continue incubation of negative tubes for a total of 7 days to check for slow urease producers.)
  118. •Citrate Utilization
    • •Medium Used – Simmons Citrate Agar (CIT)
    • •Medium description – green semi-solid slant
    • •Principle of this test – Used to determine if any organism is capable of using citrate as the sole source of carbon with production of enzyme citrase.
    • •Components of this media – The medium contains sodium citrate as the carbon source, and ammonium salts as the nitrogen source, with bronthyml blue as the pH indicator. An organis,s that uses citrate breaks down the ammonium which creates an alkaline pH.
    • •Inoculation techniques – 1. Stab/ streak (stab and fish tail slant) Simmons Citrate Agar slant with straight wire inoculum of organisms. 2. Incubate at optimum tempertarure for 24-48 hours.
    • •Special reagents used if any - none
    • •Interpretation of reactions in these media – Positive: alkaline pH causes medium to change from green to Prussian blue. Negative: no color change
  119. •Indole Test
    • •Medium Used – Tryptone Broth
    • •Medium description - Liquid
    • •Principle of this test – Used to determine the ability of an organisms to split indole from the amino acid tryptophan using enzyme tryptophanase.
    • •Components of this media -
    • •Inoculation techniques – 1. Inoculate broth with the organism using a wire loop (dip & swirl). 2. Inoculate at optimum temperature for 24-48 hours. 3. Add 10-12 drops of Kovacs Reagent.
    • •Special reagents used if any - Kovacs Reagent.
    • •Interpretation of reactions in these media Positive – red layer forms on the surface of the medium. Negative – yellow layer on the surface of the medium
  120. Notes:
    •Enrichment medium - Selects the microorganism of highest growth rate among all the microbial population able to grow under the conditions provided
  121. •Selective medium -
    Favors the growth of a particular organism
  122. •Differential Medium -
    Distinguishes between different groups of bacteria

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