Lecture 2: Basic Micro
- Created by: chandanee
- Created on: 02-01-19 17:58
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- Basic Micro
- Role of medical microbiologist
- ID OG
- Provide advice
- Manage outbreaks
- Advise on new healthcare
- Trends in infection
- Generate local epidemiological data
- Monitor theatre
- Samples/Specimen
- Enrichment culture
- 1) Direct examination/microscopy
- Define - reveals pathology which is indicative of a condition
- Staining - Gram Stain += thicker layer, purple, e.g. Bacillus Gram - = thinner layer, pink e.g. E. coli. Fixation then crystal violet then iodine then decolourise then safranin. Problem = intracellular, phagocytes may clump, some bacteria stain poorly, rods appear as cocci, mycobacteria difficult to stain
- Acid fast:
- 2) Culture - differential/selective
- Biochemical Tests
- API ***** = ID enteric bacteria, 550 species, ONPG, CIT, URE, BGal
- Rapid Staph Plus = ID Staph Plus. Cultures: blood, urine, sputum, gastric, stool.
- Non-Selective = permits growth of many MO's. Diifferential = discriminates between MO's e.g. blood agar. Selective = contains inhibitors e.g. CCFA. Adv = definite ID, can then isolate. Disadv= 16-24 hours, skilled microbiologist
- Cytotoxicity = e.g. C difficile = difficult to diagnose, toxins detected by stool samples, Adv = sens and spec Disadv = slow turn around, variability due to cell lines, experience, variability in protocols and interpretation.
- Biochemical Tests
- 3) Ab detection/ serology
- 2 approaches: recognise the Ag or detect the Ab. Common tests = slide agglutination e.g. meningitis, Rapid tests
- ELISA detects viruses and cell surface antigens or toxins. Capture Ab, directed against the protein, linked to a solid support, specimen added if viral Ag present, captured by bound Ab, bound Ag then detected by 2AB linked to an enzyme
- GLC: anaerobes HPLC: all mycobacteria Adv: rapid, isolation not needed, minimal training for rapid tests. Disadv: false positives from cross reacting Ag, false negs from evolving Ag.
- 4) Genetic
- Hybridisation: Virochip gDNA > labelled DNA> clean up >pipette sample >hybridise wash and scan. PCR/RTPCR Adv= rapid, lab culture not needed, sens and specific. Disadv = quant? OG viable?
- 1) Direct examination/microscopy
- Enrichment culture
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- New Methods
- xTAG Gi pathogen panel (GPP) Chips = detect and ID bacterial, viral and parasitic all in one test >90% pathogens. Signs of gastroenteriditis in patients, 5 hours.
- LLMDA: Rapid ID of multiple species in same samples, within 24 hours, contains probes fitted onto a slide. 2000 viruses and 900 bacteria. Fluoresce and read by scanner.
- Isothermal NA amplification/LAMPPCR = rapid, sens, amplification is extensive. Constant temp unlike most PCRs. 60-65C so no thermocyclers needed. "loop" primers accelerate reaction. Detection by photometry by increasing quant of Mg pyrophosphate. Useful for low income countries. Less sensitive than PCR - useful for diagnostic/detection
- Mass Spec Matrix (MALDITOF) used for early ID of bacteria in blood, UTIs, cerebrospinal fluid, respiratory tract infections Peptide mass fingerprints also
- NGS = faster sequencing at lower cost, template > amp> sequencing - may involve SNPs, new genes, mutations, degree of expression
- Bacteria or virus - new tool? Use GENES to ID between viruses and bacteria in respiratory infections, blood tests, helps cut back on antibiotic usage, 87% accuracy, long time for results (10-12 hours) and could become a home based kit.
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- Synthetic Biology
- Toehold switch synthetic riboregulator: sample collection > RNA extraction > RNA amp >toehold dertection >visual readout
- Application: designer probiotics, urogenital healthcare, anti-tumour, inhibition of angiogenesis, sensing disease, managing obesity, diagnosis, antimicrobials
- Factors to consider: Sens =no of MOs + result Spec= tend to discrimate between MOs. Rapid = C. diff = toxin test Also, reproducibility, differentiation, ease, cost, platform
- Beyond Diagnosis: 1. Antimicrobial susceptibility test - which drugs? dose? Disc diffusion - zone of inhibition 16-24hr incubation 2. Epidemiology - different cases attributed to same strain. Study of incidence, distribution, disease control, outbreaks, trends, evolution
- Role of medical microbiologist
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