lab practical 2

• describe the source of specificity in the rapid strep test


  detects group A streptococcal (GAS) antigens that bind to anti-GAS antibodies to form a dye-colored antigen/antibody complex that travels to test region where it will indicate a positive result; test is invalid if control region doesn't show dye
• describe the source of specificity in the rapid staph test


  latex beads coated with human fibrinogen and IgG agglutinate after reacting with enzyme coagulase and Protein A produced by S.aureus; coagulase converts fibrinogen to fibrin producing clots and protein A binds to the Fc region of IgG antibodies that induce agglutination
• What protein binds to the IgG antibodies on the latex coated beads for the Staphylococcus test

  Protein A
• Immuno-based assays are tests that utilize products of the immune system to specifically detect the presence of a substance. These products are typically called ____________. The substance being detected is called _____

  antibodies; antigens
• Explain the principle behind two common immune-based rapid testing formats and compare the advantages and disadvantages of both.

  rapid strep has the benefit of ease of use, specificity, and speed, because no bacteria culture is needed, but comes at the cost of sensitivity; meaning these tests can provide false positive/negative results.

  rapid staph require bacterial cultures to be isolated and grow before performing test but they are high in specificity and and reduce time and resources by bypassing multiple culture-based tests
• What is agglutination?


  aggregation or clumping of particles
• Why is the TEST region placed before the CONTROL region on the rapid Strep test?

  What is the purpose of the control line?

  - ensures that both the sample has traveled across the test region and that the antibodies used in the test strip have not been destroyed

  - ensure that the test was conducted appropriately
• interaction that takes place at the test portion of a rapid test strip

  
• explain the principle of a positive rapid staph test


  protein a/coagulase from sample (isolated colony) bind to antibodies and agglutinate latex beads coated with human fibrinogen and IgG on test region after mixing

  
• explain the principle of a positive rapid group A strep test


  antigens from collected sample binds to anti-GAS colored+dye antibodies and travel to test region and control region where both regions will color a line
• which strip shows a positive result

  1

  
• which strip shows a negative result

  3
• which strip shows an invalid result

  2
• which one shows a positive staph test

  sample 1

  
• During a catalase test, bubbles indicate what?


  presence of catalase enzyme that converts hydrogen peroxide into water and molecular oxygen (gas)
• Catalase is a(n) __________ that converts hydrogen peroxide into water and

  _________ gas.

  enzyme; oxygen
• Streptococcal/Enterococcal species utilize the enzyme __________, to

  detoxify hydrogen peroxide

  peroxidase
• On the image below, which catalase test is positive? Which is negative? How

  do you know? What bacterial species is shown on the left? Which bacterial

  species is shown on the right

  -sample on the right is catalase positive, indicated by the presence of bubbles (oxygen gas) as enzyme catalase detoxifies hydrogen peroxide into water and molecular oxygen (gas).

  -the sample on the left may be streptococcus/enterococcus as they use a different enzyme to detoxify hydrogen peroxide that does not release oxygen gas as a product

  - the sample of the right is staphylococcus as it uses catalase that detoxify hydrogen peroxide

  
• what tests are used to identify catalase positive bacteria


  novobiocin sensitivity, dnase agar, mannitol salt, agar, pyr

  (NDMP: no drinking maybe pray)
• What antibiotic is used to help identify catalase positive organisms? What

  antibiotic is used to help identify catalase negative organisms

  catalase positive: Novobiocin

  catalase negative: Bactrim (SXT)
• what is the purpose of novobiocin sensitivity test in catalase positive bacteria


  novobiocin is an antibiotic that inhibits DNA replication by interfering with the enzyme DNA gyrase and is often used to differentiate staphylococcus species
• what is the purpose of a DNAse agar for catalase positive bacteria


  differentiation of organisms based on their ability to secrete exoenzyme DNase which hydrolyzes DNA into nucleotides
• what is the purpose of a PYR test for catalase positive bacteria

  differentiate streptococcus and staphylococcus by PYR aminopeptidase presence which cleaves a colorless substrate that if cleaved by PYR will release b-naphthylamine as a product and react with the PYR reagent to produce a red product.

  if pyr positive, the b-naphthylamine reacts with pyr reagent to produce a red color

  if negative, it will be light orange
• what is the purpose of a Mannitol Salt agar for catalase positive bacteria


  select for halotolerant organisms such as members of the staph genus; and differentiate organisms that are able to ferment mannitol and lower the pH causing phenol red indicator to turn yellow
• what tests are used to identify catalase negative bacteria


  SXT sensitivity, BEA slant, hemolytic pattern on blood agar, PYR

  (SBHP: some baylor houses pray)
• what is the purpose of an SXT sensitivity test for catalase negative bacteria?

  identify susceptibility
• what is the purpose of a bea slant for catalase negative bacteria?

  differentiate organisms that are able to hydrolyze carbohydrate esculin into glucose and esculetin which reacts with ferric ammonium citrate to form black/grey precipitate; more than 50% of slant should be black for it to be positive
• what is the purpose of hemolytic pattern on blood agar for catalase negative bacteria?


  differentiate among streptococcus species by beta/alpha/gamma hemolysis; used also to test the susceptibility of streptoccoccus species to Bactrim
• what is the purpose of a PYR test for catalase negative bacteria?


  differentiate streptococcus and staphylococcus by PYR aminopeptidase presence which cleaves a colorless substrate that if cleaved by PYR will release b-naphthylamine as a product and react with the PYR reagent to produce a red product.

  if pyr positive, the b-naphthylamine reacts with pyr reagent to produce a red color

  if negative, it will be light orange
• What would a DNase plate look like after adding HCL when it is positive?

  The polymerized DNA will form a clouded complex when exposed to a strong acid (1M HCl). Bacteria that secrete DNase will break down the DNA into smaller fragments. These smaller fragments will not form a cloudy complex in the presence of an acid, and will result in clear zone around the bacterial growth.

  
• What would a DNase plate look like after adding HCL when it is negative?


  Bacteria that do not secrete DNAse will still grow on this media, but the media will remain cloudy around the growth following addition of HCl.

  
• The MSA plate is both selective and differential. What organisms does this plate select for?


  halotolerant organisms capable of growing in 7.5% NaCl
• If the bacteria grows on an MSA plate and turns the media yellow, what

  information does this tell you about the organism?

  it is halotolerant and is able to ferment mannitol and produce acid fermentation that lowered the pH and caused the phenol red indicator to turn yellow
• List the six tests used to differentiate the catalase positive Gram positive cocci:

  • Sensitivity to the antibiotic ____________

  • ___________________ characteristics and

  __________________morphology

  • Ability to secrete ______________________

  • Ability to grow in ____________

  • Ability to ferment in _____________________

  • The presence of the _______________________


  novobiocin

  growth

  colony

  enzyme DNAse

  7.5% NaCl

  sugar alcohol mannitol

  aminopeptidase PYR
• List the four tests used to differentiate catalase negative Gram positive cocci:

  • Sensitivity to the antibiotic ______________

  • __________ pattern

  • Ability to hydrolyze _________________ in the presence of bile

  • The presence of the ________________________

  Bactrim (SXT)

  hemolytic

  esculin

  aminopeptidase PYR
• DNAse agar interpretation


  left: positive, smaller dna fragments form clear zone around bacterial growth

  right: negative, cloudy media around bacterial growth

  
• msa agar interpretation


  left: halotolerant, postive for mannitol fermentation

  right: halotolerant, negative for mannitol fermentation

  
• PYR test interpretation


  red: positive, aminopeptidase cleaved colorless substrate that reacted with PYR reagent and produced red product

  yellow: negative, no presence of aminopeptidase

  
• bea test interpretation


  left: positive, organism hydrolyzed esculin into glucose and esculetin which reacted with ferric ammonium citrate in media to form black precipitate

  right: negative, organism is unable to hydrolyze esculin

  
• blood agar hemolytic pattern interpretation (SXT sensitivity was also performed here)


  a: beta

  b: alpha

  c: gamma