What are ALL the steps, in order and details, of a Gram Stain.
- Heat fix
- Add crystal violet 30-60sec
- Wash w/ distilled water
- Add Gram’s Iodine 30-60sec
- Wash w/ distilled water
- Add decolorizer 5-10sec
- Wash w/ distilled water
- Add Safranin 40-60sec
- Wash w/ distilled water
- Pat dry
Describe BioSafety level 1
Micro organims that does not cause human disease in healthy adults, standard microbio practices, no special PPE or secondary barriers.
Describe Biosafety level 2
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
Describe Biosafety level 3
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
Describe Biosafety level 4
Max containment, dangerous and exotic agents, high risk of life-threatening disease. Aerosol risk without available therapy or vaccine
What is blood agar, what it contains and why we use it
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
What is BLAST? What does BLAST stand for? What is the purpose?
Basic Local Alignment Search Tool, Algorithm for comparing primary biological sequence information
What is MTM medium and what is it used for?
Modified Thayer Martin; differential for N. gonorrhoeae with suppression of most other gram-negative diplococci, gram-negative bacilli, gram-positive organism, and yeast
What is MSA medium and what is it used for?
Mannitol Salt Agar; selective and differential for pathogenic staphylococci
- S. aureus = yellow colonies
- Staphylococcus spp. = pink/red colonies
What is TSI medium and what is it used for?
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)
What is MR-VP medium and what is it used for?
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
What is a Dichotomous key
Identification of organisms based on a series of choices between alternative characteristics
What is ELISA? What are the principle behind it, color change, antibodies, substrate, antigen
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
What is a catalase test and what is it used for?
Detects presence of catalase in organism; H2o2 Staph for pos and Strept for negative
What is Superoxol Test and what is it used for?
-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
What do you do for a Bacterial Spill?
- Inform people in the immediate area
- Put on PPE
- Cover spill area twice the size of the spill with disinfectant (EtOH)
- Leave on for 20 minutes
- Wipe down contaminated equipment/furniture
- Use forceps or broom to pick up broken glass
What is the “Zone of Inhibition”?
- 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)
What is EMB and what is it used for?
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)
What is Simmons citrate used for?
- 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
What is Christensen Urea agar used for?
- selective media for organisms with urease activity; ability to reduce urea
(ex. Urease positive)
What are the molecular techniques that we use and what for?
Western Blot: proteins
Northern Blot: RNA
Southern Blot: DNA
ELISA: antigen-antibody complex
What is Staphylococcus?
Gram positive clusters; S. aureus ; positive coagulase
Enterobacteria are?
gram negative bacteria
What does MR-VP test for?
depends on glucose fermentation. Methyl red tests for acid end products. Voges test for the production of acetylmetylcarbinol
