Lab Final Study Guide

Question 1

What are ALL the steps, in order and details, of a Gram Stain.

Answer 1

1. Heat fix
2. Add crystal violet 30-60sec
3. Wash w/ distilled water
4. Add Gram’s Iodine 30-60sec
5. Wash w/ distilled water
6. Add decolorizer 5-10sec
7. Wash w/ distilled water
8. Add Safranin 40-60sec
9. Wash w/ distilled water
10. Pat dry

Question 2

Describe BioSafety level 1

Answer 2

Micro organims that does not cause human disease in healthy adults, standard microbio practices, no special PPE or secondary barriers.

Question 3

Describe Biosafety level 2

Answer 3

moderate risk agents associated with human disease, skin, and mucosal membrane exposure risk, appropriate for blood and bodily fluids/tissues, BSC and PPE, waste decontamination facility

Question 4

Describe Biosafety level 3

Answer 4

Indigenous and exotic agent with potential for respiratory transmission that have potential to cause serious or lethal infection, autoinoculation and respiratory risk. BSC, PPE, closed lab access, ventilation system, waste decontamination

Question 5

Describe Biosafety level 4

Answer 5

Max containment, dangerous and exotic agents, high risk of life-threatening disease. Aerosol risk without available therapy or vaccine

Question 6

What is blood agar, what it contains and why we use it

Answer 6

Different agar for differing hemolytic types
-alpha hemolysis: turn agar green
-beta hemolysis: turn agar clear
-Gamma hemolysis: no hemolytic activity
Contains 5% sheep blood and general nutrients. We use it for fastidious organisms

Question 7

What is BLAST? What does BLAST stand for? What is the purpose?

Answer 7

Basic Local Alignment Search Tool, Algorithm for comparing primary biological sequence information

Question 8

What is MTM medium and what is it used for?

Answer 8

Modified Thayer Martin; differential for N. gonorrhoeae with suppression of most other gram-negative diplococci, gram-negative bacilli, gram-positive organism, and yeast

Question 9

What is MSA medium and what is it used for?

Answer 9

Mannitol Salt Agar; selective and differential for pathogenic staphylococci

• S. aureus = yellow colonies
• Staphylococcus spp. = pink/red colonies

Question 10

What is TSI medium and what is it used for?

Answer 10

Triple Sugar Iron; differential media for lactose fermenters and H+ production

• Lactose fermenters: color shift from red to yellow
• Thiosulfate anion user: black precipitate (presence of hydrogen sulfide)

Question 11

What is MR-VP medium and what is it used for?

Answer 11

Methyl Red-Voges Proskauer; selective for enteric gram negative bacilli

• VP = detect acetoin in a bacterial broth culture, E.coli (VP neg) from the Klebsiella-Enterobacter groups (VP pos)
• MR = pH indicator; yellow pH6.2

Mixed acid fermentation; MR-neg organisms such as Klebsiella pneumoniae and Enterobacter aerogenes

Question 12

What is a Dichotomous key

Answer 12

Identification of organisms based on a series of choices between alternative characteristics

Question 13

What is ELISA? What are the principle behind it, color change, antibodies, substrate, antigen

Answer 13

Enzyme Linked Immunosorbent Assay; determines the concentration of analyte via antigen antibody interaction

• Antigen: plated in wells and contains varying [antigen]
• Primary Ig: binds to antigen on plated well
• Secondary Ig: binds to Fc site of Primary Ig, has Fc-substarte complex that fluoresces when activated

Question 14

What is a catalase test and what is it used for?

Answer 14

Detects presence of catalase in organism; H2o2 Staph for pos and Strept for negative

Question 15

What is Superoxol Test and what is it used for?

Answer 15

-Presumptive identification of N. gonorrhoeae; is 30% H2o2.

Other Neisseria species and Moraxella catarrhalis are either negative for this test or give a weak delayed reaction

Question 16

What do you do for a Bacterial Spill?

Answer 16

1. Inform people in the immediate area
2. Put on PPE
3. Cover spill area twice the size of the spill with disinfectant (EtOH)
4. Leave on for 20 minutes
5. Wipe down contaminated equipment/furniture
6. Use forceps or broom to pick up broken glass

Question 17

What is the “Zone of Inhibition”?

Answer 17

• Qualitative method used clinically to measure antibiotic resistance
• The diameter of the clear zone determines the resistance/susceptibility to the particular antibiotic, (larger diameter = susceptibility, small or no diameter = resistance)

Question 18

What is EMB and what is it used for?

Answer 18

Eosin Methylene Blue agar; Differential medium used to isolate fecal coliforms (sucrose and lactose fermenters)

• pH indicator: dark purple precipitate at low pH
• Inhibit the growth of the most Gram positive organism (E.coli is EMB positive)

Question 19

What is Simmons citrate used for?

Answer 19

• ability of organisms to utilize citrate as a carbon source

- pH indicator that turns from green to blue when citrate is utilized as the sole carbon source

Question 20

What is Christensen Urea agar used for?

Answer 20

• selective media for organisms with urease activity; ability to reduce urea
  (ex. Urease positive)

Question 21

What are the molecular techniques that we use and what for?

Answer 21

Western Blot: proteins
Northern Blot: RNA
Southern Blot: DNA
ELISA: antigen-antibody complex

Question 22

What is Staphylococcus?

Answer 22

Gram positive clusters; S. aureus ; positive coagulase

Question 23

Enterobacteria are?

Answer 23

gram negative bacteria

Question 24

What does MR-VP test for?

Answer 24

depends on glucose fermentation. Methyl red tests for acid end products. Voges test for the production of acetylmetylcarbinol

Question 25

What does the catalase test for?

Answer 25

production of catalase enzyme which splits hydrogen peroxide and releases oxygen gase

Question 26

What does the oxidase test for?

Answer 26

Tests for the production of the enzyme oxidase