Microbiology Week 3

What type of specimen is needed in collection?

highly dependent on the disease and aseptic technique must be used when collecting it to avoid contamination

What are types of specimens collected?

blood, urine, faeces, sputum, vomitus, wound swab, skin scrapings, tissue biopsies, bone marrow, washings/lavage

What are some considerations for sampling?

-some body sites contain normal microbiota and will therefore be present in the sample (upper respiratory swabs, skin scrapings, faeces) -some specimens may be contaminated by normal microbiota during collection (blood, sputum, CSF, urine) -timing

How is timing a consideration for specimen collection?

-samples must be collected as soon as possible (before antimicrobial therapy) -samples will need to be transported to the laboratories and processed -swab <24 hours -blood <2 hours

What is gram staining?

-developed by Hans Christian Gram -classifies bacteria into two groups based on the structure of the cell wall (PIES)

What information can we obtain from gram staining?

-gram reaction -cell arrangement -shape -size

What can growth on culture media be used to obtain?

a presumptive identification or can be used as a confirmatory test (e.g. MCA, MSA, HBA, Cetrimide, Chromogenic)

What is the rapid biochemical tests catalase?

-differentiates between: streptococci (neg) and staphylococci (pos) -clostridium (neg) and (pos)

What is the rapid biochemical tests oxidase?

-detects the presence of cytochrome C oxidase, and enzyme at the end of the ETC -adds e- to oxygen, which in addition with H+ forms water -oxidase test reagent acts as artificial electron acceptor and changes colour -differentiates between gram neg species

What is the rapid biochemical tests Indole?

-differentiates between Enterobacteriaceae -e.coli -pos -klebsiella aerogenes - neg

What is the rapid biochemical tests Coagulase:

-differentiates between S. aureus and other staphylococcus spp. -S. aureus pos is granulated -S. epidermidis neg is smooth

What is the conventional biochemical tests Urease test?

-urease increases ammonia which increase pH (i.e. more alkaline) -phenol red indicator turns bright pink from yellow with increasing pH

What is the conventional biochemical tests Citrate test?

-determines the ability of the organism to utilise citrate as the sole carbon energy source and ammonium salts as the sole nitrogen source -bromothymol blue pH indicator in the medium turns from green to prussian blue, caused by production of pyruvate and CO2

What is the conventional biochemical tests Voges-Proskauer (VP) test?

detects if microbes can produce acetoin from glucose fermentation

What is the conventional biochemical tests Methyl red (MR) test?

determines ability of microbe to ferment glucose via mixed acid fermentation

What is the conventional biochemical tests H2S test?

determines if the microbe can reduce sulfur containing compounds (cysteine or thiosulfate)

What is the conventional biochemical tests Triple sugar iron (TSI) test?

-determines the ability to ferment glucose, lactose, sucrose and produce H2S -used to differentiate between Gram neg rods

What happens if the organism ferments glucose, lactose +/- sucrose in TSI test?

-large amount of acid is produced -pH shift (red -> yellow) occurs in butt and slope -if fermentation produced gas -> bubbles or cracks in agar -H2S may also be produced -> black precipitate

What happens if the organism does NOT ferment glucose, lactose +/- sucrose in TSI test?

-no colour change (medium stays red) -growth (colonies) visible on agar slant

What is the procedure of Vitek 2?

-choose isolate -prepare organism suspension and ensure correct McFarland Standard with DensiCHEK Plus -scan card and isolate barcodes to establish traceability -prepare the ASR dilution -load the cards in VITEK 2 Compact filler

What is the Vitek 2 identification?

-the instrument reads the card every 15 minutes to monitor growth of the microorganism -> specific level of growth is necessary to read the results of the tests -generates a profile of POS/NEG for each test and links to a database to generate an identification

What is antigen detection?

detection of specific structure unique to individual or groups of Microbes

Where are antigens present?

on the surface of microorganisms and may be secreted and detectable in: cerebrospinal fluid, blood, urine, nasal secretions

What are methods for antigen detection?

-agglutination tests -fluorescence antibody assays -enzyme immunoassays

What happens in an agglutination test?

-visible clumping (antibodies linked to polystyrene latex particles -> particles agglutinate in the presence of antigen) -direct agglutination -indirect agglutination (serology)

What is direct agglutination?

soluble antibody (Ab) causing clumping by interaction with cell surface Antigen (Ag)

What is indirect agglutination?

latex beads coated with an antigen clump when mixed with patient serum if serum contains antibodies against the antigen

What is Fluorescence Antibody Assay?

-direct: Ab covalently linked to fluorescent dye -indirect: unlabelled primary Ab detected by fluorescently labelled secondary Ab -tissues

What is Enzyme Linked ImmunoSorbent Assay (ELISA or EIA)?

-covalently bonded enzymes to label Ab or Ag -direct: detects Ag -indirect: detects Ab

How does direct ELISA work?

1. antibodies bound to wells of microtiter plate 2. add patient sample with antigens and wash wells with buffer 3. add antibodies against the antigen that are coated with a conjugated enzyme; wash with buffer 4. add substrate for enzyme 5. measure coloured product; positive will have no colour 6. coloured product is proportional to amount of antigen

What is indirect ELISA?

1. microtiter plates coated with antigens 2. add patient sample containing antibodies; specific antibodies will bind; wash wells with buffer (other antibodies will wash away) 3. add anti-IgG antibodies conjugated to an enzyme (wash with buffer) 4. add substrate for enzyme 5. measure coloured product; positive will have colour, negative will have no colour 6. coloured product is proportional to amount of antibody concentration

What is serology?

-diagnostic identification of antibodies in the serum -the immune system defends against microbes by producing antibodies in response to the presence of their antigens

What is nucleic acid detection?

-detection of Microbial DNA; alive or dead -independent of: pathogen isolation and growth, host immune response -dependent on: species-specific sequences -extracted DNA, sometimes RNA

What are different types of nucleic acid detection?

-conventional PCR -16S rDNA sequencing -real-time PCR and qPCR -reverse transcriptase PCR

How does Conventional PCR work?

-nucleic acid extracted from specimen -PCR amplification in thermocycler -visualised via agarose electrophoresis

How does 16S rDNA sequencing work?

-16S rRNA gene: bacterial speciation and diagnostics -encodes for the small subunit ribosomal RNA -1500 bp long with regions that are highly conserves and others that are hypervariable -universal primers work across most bacterial species

How does Real-time PCR and qPCR work?

-combine amplification and detection steps -quantitative: amplification proportional to microbial gene copies in specimen

How does reverse transcriptase PCR work?

-begins with RNA template -reverse transcriptase enzyme RNA -> cDNA -single stranded cDNA is produced (first strand synthesis) -cDNA then used as template for PCR using specific primers

What is MALDI-TOF MS?

-matrix-assisted laser desorption ionisation time of flight mass spectrometry -the chemical structure of an organism in culture is determined by analysing the mass and the charge of its ions -compared with a database from bacterial species profiles to allow accurate identification of the microorganism -bacteria from a pure culture are added to a well on a slide and a matrix is applied and dried -placed inside the machine -the identity of each specimen is entered into the computer -laser is fired and each colony is tested