Cultivation, Microscopy and Immuniology Flashcards
what is clinical microbiology
-Very simple strategy
-Collect specimen from patient
-Examine specimen for evidence of pathogen
-Find pathogen in specimen by e.g
Direct microscopy
-Find evidence of pathogen by e.g
Immune response or Biomarker signature (protein, lipid, etc.)
-Arrive at a diagnosis
what are the factors of clinical specimens
-Material has to be appropriate to the clinical condition= Diarrhoeal symptoms suggest that faeces be collected and Blood, urine, CSF, biopsy tissue etc. as appropriate
-Aseptic collection
-Collect an appropriate quantity
-Collect specimen before treatment is initiated
-Fast transport from collection to laboratory analysis
what are some clinical collection materials
-Swabs =skin, wounds, nose, ear etc.
-Needles= Blood, tissue fluids, Cerebrospinal Fluid
-Sterile cups= mucus, stool
-Catheter = urine, blood
-Intubation= extraction of fluids from hollow organs
what do we use to determine if a pathogen is present in a sample
-Microscopy
-Laboratory Culture of microorganisms
-Direct detection= Immunology, nucleic acid technology and analytical chemistry
what traditional methods do microbiology labs rely on
culture, phenotypic, immunological and biochemical tests
what samples are anaylsed in a clincal lab
blood, faeces, sputum, skin swabs, wound swabs etc
what are the best diagnostic tests to use
sensitivity and specificity
what is sensitivity
-Sensitivity refers to a test’s ability to designate an individual with disease as positive.
-A highly sensitive test means that there are few false negative results, and thus fewer cases of disease are missed.
-A sensitive test for an infectious disease should also be able to work with relatively low numbers of pathogens present in the specimen.
what is the equation for sensitivity
-ability of a test to detect a true positive.
-Sensitivity = True positive / True positive + false negative x100
what is specificity
-The specificity of a test is its ability to designate an individual who does not have a disease as negative.
-A highly specific test means that there are few false positive results.
what is the equation for specificity
-Specificity = ability of a test to exclude a true negative.
-Specificity = True negative / True negative + false positive x100
what is desirable in a test
a test that is both highly sensitive and highly specific. This is frequently not possible
what is looked at in diagnosis using microscopy (direct…)
-Direct examination of sample
-Variety of formats
what are the advantages of microscopy
-Rapid
-Cheap
-adaptable according to sample
-Specificity possible
(antibody/nucleic acid staining)
what are the disadvantages of microscopy
-Limited specificity
-No recovery of organism for further analysis.
-Poor sensitivity
-Expensive for viruses
what occurs in diagnosis using a laboratory culture
-Inoculation of specialised medium with specimen
-Incubate for specified time
-Recover individual colonies
(possibly grow a new culture from a single colony to ensure working with a pure culture of the organism of interest)
-Look at morphology and Gram reaction
-Undertake additional biochemical tests to obtain an ID
what mediums are used in laboratory culture strategy
selective, differential and chromogenic mediums
what tests are used in laboratory culture strategy
gram stain, biochemical, immunological and molecular tests
what is general purpose media examples
Tryptone Soy Agar
Brain-Heart Infusion Agar
what is an example of selctive media
Vogel and Johnston Agar
what is examples of differential media
MacConkey Agar
Blood agar
what is chromogenic media
-Most recent development
-Species-specific chromogens developed to take advantage of what we understand about bacterial metabolism.
-Beta-galactosidase
-Beta glucuronidase
what is chromogen
“a chemical compound, itself without colour, that can be transformed into a coloured compound, or can react with another material to form a coloured compound”
it is transformed by a specific enzyme and this enzyme is only found in one particular species
why do we use Chromogens?
-Improve overall specificity of a diagnostic medium.
-Positive colonies are easy to identify.
-Reduce number of sub-cultures required.
what are the advantages of culture
-Unequivocal demonstration of presence of pathogen
-Pathogen is available for further antimicrobial susceptibility testing
-Established technology and standards
-Relatively inexpensive
-Low technology
-Less to go wrong (hopefully)
what are the disadvantages of culture
-slow= It can take days to identify a bacterial pathogen and to determine the most appropriate antibiotics
-Technically challenging in certain instances= e.g Chlamydia spp. + Mycobacterium tuberculosis
-Impossible to culture some pathogens= e.g Treponema pallidum A bacterium causing syphillis
-Culturing methods assume that all pathogens are culturable all of the time and does not take into account either= injured organisms and viable but non-culturable organisms.
what is the equation for the biochemical testing of bacteria
-Catalase test
-2 H2O2 –> Catalase –> 2 H2O + O2
S. aureus +
Streptococcus -
explain bacteria and fermenting sugars
-Many bacteria have different abilities to ferment sugars
-Specific sugar and pH indicator in the broth e.g glucose, galactose, mannose etc.
what are the colours produced when bacteria ferment sugar
-Purple: no fermentation
-Yellow: fermentation occurs and acid produced
-Yellow: fermentation occurs and both acid and gas produced
what is API
-API: Miniaturised biochemical tests on a strip.
-Can easily see that these three species are different
-Different patterns
-ID based on database
What is the advanatages of Immunodiagnostics
-Recognition of species
-Fast assays
-Very flexible
what is Immunodiagnostics used for
-Direct analysis of sample to detect presence of pathogen
-Used to confirm identification when pathogen isolated
-Lots of simple systems in use= Agglutination and ELISA
what test is used in Immunodiagnostics
Agglutination Test
what is the Agglutination Test
-Simplest test available to see Visible clumps on a slide
-Widely used for confirmation
tests on organisms isolated on an agar plate
-S. aureus on Mannitol Salt Agar
-Remove colony
-Mix with anti-S. aureus serum
-Shake on slide
-Look for agglutination
what is ELISA
Enzyme-linked immunosorbent assay
what does the name of ELISA suggest
-Name suggests three components
-Antibody= Allows for specific detection of antigen of interest and If antigen is specific to a bacterium then we can identify the bacterium.
-Solid phase (sorbent)= Allows one to wash away all the material that is not specifically captured
-Enzymatic amplification= Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader
what is Alkaline Phosphatase
-One of the most widely used reporter systems in ELISA and Histochemistry
-A general purpose Hydrolyase enzyme that removes phosphate groups under alkaline conditions
-Can act on many different substrates
what is used in the lateral flow test strip
-Conjugation of Particles
-Conjugate Pad
-Test Strip
-Absorbent pad (wicking)
what is the advantage of the lateral flow test strip
rapid diagnosis
what are the main elements in clincal diagnostic microbiology
-Specimen subjected to a number of treatments
-Microscopy
-Laboratory Culture of microorganisms
-Direct detection= Immunology, Nucleic acid technology and Analytical chemistry
-In practice more than one set of techniques are used together to give a more accurate result.
