Microbial diagnostic tests Flashcards
What does PCR do?
Makes multiple copies of a segment of DNA of interest from a small initial sample
What is used to carry out temperature cycles?
Thermal cycler
How is PCR carried out?
Target DNA, primers, nucleotides and DNA polymerase added to tube
Tube put in thermocycler
Temperature increased to 95 degrees (denatures enzymes and causes strands of DNA to separate)
Taq DNA polymerase is heat resistant
Annealing occurs at 55-65 degrees where primers attach to target DNA
Extension occurs t 72 degrees where DNA polymerase creates DNA strand complementary to target strand
Cycle repeats until have two loose molecules of DNA
Gram stain procedure
Smear bacteria onto slide Heat fix slide Flood slide with crystal violet for 20 seconds Wash with water Flush with iodine for a minute Decolourise with gram's alcohol Wash with water Flush with safranin counterstain for 1 minute Wash again Blot dry
What are gram positive bacteria?
Thick peptidoglycan layer retains crystal violet-iodine complex
What colour do gram positive bacteria turn?
Purple
What are gram negative bacteria?
Thin peptidoglycan layer which doesn’t retain crystal violet but is stained with safranin
What colour do gram negative bacteria turn?
Pink
How to conduct ELISA test
Coat microtiter plate with antigens Add sample Specific antibody can bind to antigen Wash Enzyme labelled antibody Wash Add substrate
How do you treat meningitis?
Antibiotics
How do you rule in/out meningitis?
Lumbar puncture
Which bacterium causes meningitis?
Neisseria meningitidis
When are broad spectrum antibiotics useful?
When we don’t know causative pathogen
Is blood agar selective?
No
When is blood agar culture useful?
Sterile sites or where you are uncertain of cause of infection
