Lab 3

Question 1

If a gram positive cocci isolate is catalase negative, then you have to use _______ to conduct antibiotic sensitivity test

Answer 1

Blood Mueller-Hinton agar plate

Question 2

Catalase

Answer 2

2H2O2 + catalase —> 2H2O + O2 (bubbles)

• distinguishes between streptococci (neg) and staphylococci (pos)
• formation of gas bubbles indicates a positive test

Question 3

Catalase method

Answer 3

Place a drop of hydrogen peroxide on a slide

• obtain growth from a plate and place in the drop
• do NOT cool a hot loop or needle in blood containing media before performing this test
• do NOT stir, watch for bubbles originating from growth

Question 4

Colistin-nalidixic acid agar

Answer 4

Precludes growth of gram neg organisms

Only gram positive organisms will grow on CNA agar

Question 5

Coagulase test (slide test)

Answer 5

Cell bound coagulase

• place a drop of human or rabbit plasma on a glass slide
• remove growth from a colony and emulsify it by mixing it onto the slide next to the plasma
• mix organism and plasma
• if organism possesses cell bound coagulase, clumping will occur within 30 seconds
• negative test results in homogenization of growth in suspension

Question 6

Coagulase test (tube test)

Answer 6

Cell free coagulase test

• inoculate 0.5 ml or a few drops of rabbit plasma from a fresh staphylococcus culture
• incubate at 37 C
• coagulation of plasma within 24 hrs = positive test
• -> plasma checked for coagulation after 4 hrs of incubation, if result is neg at 4-8 hrs then a full 24 hr incubation is required before reporting a neg result
• -> if result is positive at 4-8 hrs, then further incubation is not needed

Question 7

Coagulase explaination

Answer 7

Staph and intermedius produce coagulases (enzymes) that activate clotting of plasma = gelling in the tube test or fibrin formation in the slide test

• some strains of bacteria are neg on slide and pos on tube test
• rabbit plasma works best

Question 8

Cytochrome C oxidase test method

Answer 8

• rub a loopful of growth from agar culture on filter paper containing oxidase reagent or impregnate filter paper with reagent
• reagent is quickly oxidized upon exposure to light/air
• ascorbic acid is added as an antioxidant
• cytochrome oxidase C is a part of the oxidative phosphorylation pathway

Question 9

Cytochrome c oxidase result

Answer 9

Positive: dark purple within 30 seconds
Negative: yellowish reaction or delayed reaction of purple (more than 30 seconds)

Question 10

Triple sugar iron agar (TSI) method

Answer 10

Stab base and streak slant with inoculating needle –> leave cap loose and incubate at 37 C

• use for presumptive identification of gram negative rods and the basis for additional tests
• needs to be read at 24 hrs (longer incubation times lead to alkaline results)

Question 11

TSI results

Answer 11

• incubation of slant is aerobic conditions
• incubation of base is anaerobic conditions
• incubate for 24 hrs
• K: alkaline (red)
• A: acid (yellow)
• G: gas (bubbles or fracturing of media)
  +/- H2S positive: presence of black precipitant in agar
  -/-: H2S negative

Question 12

TSI differential chemicals

Answer 12

• dextrose 1 part
• lactose 10 parts
• sucrose 10 parts
• FeSO4
• phenol red

Question 13

TSI interpretation

Answer 13

• orange-red: uninoculated
• yellow (acid): glucose and/or sucrose and/or lactose fermented
• red (alkaline): glucose, sucrose, and lactose not fermented or exhausted
• blackening in base: H2S produced
• gas bubbles in base: aerogenic (gas producing) organism

Question 14

TSI explaination

Answer 14

Iron traps H2S produced from sulfur containing precursors to form black iron sulfide, and gas produced in depths of agar shows up as bubbles or disruption of the agar