Diagnostics in microbiology

Question 1

non microbiology investigations

Answer 1

Radiology
Haematology
Biochemistry

Question 2

microbiology quality control of pharmaceutical products

Answer 2

avoid contamination
proper waste

Question 3

types of microbiological tests

Answer 3

• rapid tests and immunoassays
  -microscopy - staining can be used to identify gram +/-
  -culture - selective or differential media can be used or enrichment culture
  -biochemical tests - based on growth requirements and enzymatic activities (API strips can be used)
  -molecular testing - no need to isolate bacteria in laboratory setting

Question 4

what should a good diagnostic system have?

Answer 4

sensitivity - no false negatives (how well it can correctly identify individuals with a particular condition)
specificity - no false positives (measure accurately samples that don’t have a particular condition)
simplicity - must be carried out efficiently at the level of routine

Question 5

How do immunoassays work?

Answer 5

recognise a specific epitope of an antigen based on specificity of the antigen- antibody binding

Question 6

how do molecular tests based on nucleic acids work?

Answer 6

e.g. PCR, Hybridisation techniques, sequencing

extract nucleic acids and purification - cell lysis, inactivation of cellular nucleases and separation from nuclear debris

exposure to probe in liquid or solid phase
detection system of recognition between probe and target to give positive or negative

Question 7

why do we use PCR?

Answer 7

allows for rapid amplification of a designed specific fragment of DNA - makes strand easily detectable

Question 8

components of PCR

Answer 8

DNA template
heat resistant DNA polymerase
pair of primers - PCR reverse and forward
deoxynucleotide triphosphates A,C,G,T
Buffer solution
Mg2+ ions

Question 9

PCR 3 steps of cycle

Answer 9

Denaturation - breaking of H bonds at high temp
Annealing - cooling and allowing primers to bind to target sequence
Extension - heated for efficient DNA synthesis

Question 10

3 phases of PCR on graph

Answer 10

1- exponential amplification phase
2-linear phase
3-plateau phase

Question 11

DNA electrophoresis how does it work

Answer 11

Electrical current is used within the gel.
Negatively charged DNA migrates to positive side in gel based on molecular weight of DNA fragments. DNA staining with dye and UV light exposes them within the gel.