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
Is Maconky culture selective?
Yes
Characteristics of maconky agar
Entrobacteriacae
Restricts growth of gram positive and support understanding of coliform present
Can have lactose fermentive side and lactose non-fermentive side
Characteristics of chromogenic candida agar
Green indicates candida albicans
Useful for non-sterile sites where you know that causative bacteria belong to one family
What are the three types of streptococcus?
Alpha beta and gamm
What does alpha streptococcus indicate?
Partial haemolysis
What does beta streptococcus indicate?
Complete hemolysis
What does gamma streptococcus indicate?
No hemolysis
What is a DNA template?
dsDNA of interest, separate from sample
What is DNA polymerase?
Thermostable enzyme that doesn’t denature at high temperatures
What are oligonucleotide primers?
ssDNA complementary to 3’ end of sense and anti-sense strands of target sequence
What are the 4 deoxynucleotide triphosphates?
dATP, dTTP, dGTP, dCTP
What doe the deoxynucleotide triphosphates do?
Provide energy for polymerisation and building blocks for DNA synthesis
What does ELISA do?
- Quantification of a desired marker in biological sample
- Marker can be antibody or antigen
- Antigen on bottom of plate, antibodies added. Sometimes add antihuman IgG with conjugate enzyme. Add dye that is cleaved by enzyme, the color change is detected by electric plate reader
Advantages of microscopy
Quick
Visualise bacteria, fungi, protozoa and metazoa
Disadvantages of microscopy
Not definitive identification
No information on antibiotic sensitivity
Can’t detect viruses
Advantages of cultures and confirmatory tests
Confirmed identification
Information on antibiotic sensitivity
Bacteria and fungi
Disadvantages of cultures and confirmatory tests
Slow
Commensal bacteria may interfere
Some organisms difficult to culture
Can’t culture viruses, protozoa and metazoa on agar
Advantages of PCR
Quick
Confirmed identification
Detects full range of microorganisms
Automated
Disadvantages of PCR
Each PCR looks for one organism only
No information on antibiotic sensitivity
Advantages of antibody detection
Detects whether antibodies are raised against full range of microorganisms
Automated
Disadvantages of antibody detection
Slow - antibodies will only be present once immune system has responded
Antibodies may be result of previous infection
