Microbiology Lab Exam 2

Catalase Test

• Know what organisms produce catalase

• Know what catalase is and what reaction it performs

• How does hydrogen peroxide damage cells

• How to perform the test and explain how to interpret a positive vs.

negative result

• Only Staphylococcus & Micrococcus species produce catalase

• Catalase is an enzyme that  perform decomposition reaction to break down Hydrogen Peroxide (H2O2) and converts it to oxygen (bubble of oxygen gas) and water

• H2O2 damages DNA and RNA in protein

• Catalase Test Method: transfer bacteria to a glass slide and add hydrogen peroxide,

  bubbling of oxygen gas indicates catalase positive.

Mannitol Salt Agar (MSA)

• Know the importance of Staphylococcus aureus in human health and the

  diseases that it causes.

• Define MRSA and why is this important in human medicine?

• What type of media is Mannitol Salt Agar and what is it made up of?

• What does salt tolerant mean? What is a halophile?

• Which bacteria grow on this media?

  • What is the pH indicator and what color does it change if acid is present?

  • What color would appear if Staph. aureus was grown and why?

  • What color would appear if Staph. epidermidis appear was grown

and why?

• S. aureus is a normal microbiota in the skin surface, and respiratory tract. It can cause

  wide range of infections from skin infection to bacteremia, food poisoning

• Methicillin Resistant Staph. Aureus (MRSA) that are resistant to first line of antibiotics

• MSA contains a high concentration of salt that allows only Staphylococci to grow. It also

  has sugar mannitol & phenol red when fermented release acid to change medium from

  red to yellow

• Halophile: salt tolerant bacteria

• Staphylococcus grow on this media

  • S. aureus colonies’ zone appears yellow (positive)

  • S. epidermidis colonies’ zone appears red (negative)

Staph Latex Agglutination Test

• Which bacterium does it identify?

• Know the steps of the test.

• What is the importance of the ‘control’ reagent and what would indicate

  an invalid test result?

• Know which cellular parts are recognized by the reagents in the Staph

  latex test

• Be able to differentiate between a negative and positive test result.

• Staph Latex test identifies S. aureus

• Add Test Latex Reagent to a drawn-circle on a strip, inoculate bacteria by stirring and

  spreading. Result should appears in 30 seconds

• Control Latex Reagent serves as negative control, agglutination indicates invalid test

• Bound coagulase & Protein A are components found in the cell wall of S. aureus that

  reacts with the reagent.

• Positive result: blue color with red agglutination

Blood Agar Plate (BAP)

• What is an enriched media? Why is blood agar considered a differential media?

• What is hemolysin and which 2 types of bacteria produce it?

• Describe the difference results seen on a blood agar plate between alpha hemolysis, beta hemolysis and gamma hemolysis

• Know the following bacteria and the disease the cause, and the type of hemolysis seen on blood agar

  • Streptococcus pneumoniae

  • Streptococcus pyogenes

  • Enterococcus faecalis

• What is the gold standard for diagnosing strep. throat and

  why?

• Enriched media contains extra nutrient to grow fastidious cells, it is differential because

  some bacteria lyse blood cells changes color of the zone and opacity

• Hemolysins is a toxin produced by Streptococcus & Staphylococcus that breaks down

  red blood cells

• Alpha hemolysis: partial breakdown, appears green and opaque

• Beta hemolysis: full breakdown, appears colorless and transparent

• Gamma hemolysis: no breakdown, no visible changes and opaque

• S. pneumoniae: alpha-hemolysis, causes pneumonia and potential bacteremia and

meningitis.

• S. pyogenes: beta-hemolysis, causes pharyngitis, infect potential soft tissue

• E. faecalis: gamma-hemolysis, causes urinary infections

• Gold standard for collecting sample at back of throat and tonsils and do throat culture blood agar

Strep Latex Agglutination Test

• What bacterial component is detected in this test?

• Know the procedure and how to interpret a positive and negative test

• Carbohydrate antigen on the surface of cell wall

• Procedure

  • Transfer bacteria to test tube,

  • Add 2 drops of reagent 1 & and 2

  • Add 4 drops of reagent 3, this frees carbohydrates from cells wall.Mix suspension

  • Label test card with A and B

  • Use pipette to transfer the substance to test car and test with

    Latex A and Latex B. Agglutination indicates positive result.

  • Strep. pyogenes: positive for Latex A

  • Strep. agalactiae: positive for Latex B

  • Beta Streptococcus species: negative for A/B

MacConkey Agar (MAC)

• Name the 3 most common categories of nosocomial infections

• What is ventilator associated pneumonia?

• Why is it selective? Why is it differential?

• which type of bacteria will grow and do not grow in this media

• What is the pH indicator and what color does it turn if an acid is present?

• What type of bacteria is it most useful to identify?

• Nosocomial: hospital acquired infections, three most common categories are urinary

tract infections, blood infections, and pneumonias.

• Ventor associated pneumonia: nosocomial pneumonia develops by patients who are on

  ventilator

• MAC is selective due to bile salts and crystal violet that inhibits growth of gram-positives.

  It’s differential due to sugar lactose and pH indicator neutral red that detects fermentation.

• The pH indicate is neutral red, if acid is present the area turns pink

• It’s useful to identify gram-negative enteric (gut) bacteria

• E. coli: pink, lactose fermentation

• P. aeruginosa: clear, non-lactose fermentation

Oxidation-Fermentation Test

• What is this test used for and when is it most useful?

• What are the 2 pathways that bacterium metabolize carbohydrates?

• Be able to interpret the pH differences (color) using bromthymol blue

• What is the purpose of the mineral oil?

• Test uses single carbohydrate, and pH indicator bromothymol blue. It’s useful to classify

  gram negative bacteria Enterobacteriaceae vs Pseudomonas

• Bacteria either perform oxidative (CO2 as end product) or fermentative (acid as end

product) metabolisms. Fermentation acidify media more than oxidative bacteria

• Bromothymol blue indicator starts with green color, and below pH 6 will turn yellow,

  and blue if above pH 7.6

• Pseudomonas. aeruginosa performs oxidation only

• E. coi performs both

• Mineral oil excludes exposure to oxygen for fermentation tests.

• Semisolid medium that can test for motility

• Fermentative metabolism: reaction without the presence of oxygen, final product is acid

• Oxidative metabolism: reaction with presence of oxygen, final product is CO2-

• Carbohydrate: reaction used in both oxidative & fermentative metabolisms

• Anaerobic: reaction without oxygen, fermentation is one of several forms of anaerobic

reactions

• Facultative anaerobe: grow in both the presence and absence of oxygen, but its metabolism changes depending on oxygen availability

Oxidase Test

• What is it testing?

• What is the positive result

• Which bacteria test positive/negative

• Cytochrome enzyme reduces oxygen to water, one subtype is called Cytochrome c

  oxidase

• Oxidase reagent will donate an electron to the enzyme, which in turn makes the reagent

  turn dark blue.

• P. aeruginosa is aerobic and is oxidase positive

• Enteric bacteria like E.coli do not have this enzyme results in oxidase-negative