Cultivation, Microscopy and Immuniology

Question 1

what is clinical microbiology

Answer 1

-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

Question 2

what are the factors of clinical specimens

Answer 2

-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

Question 3

what are some clinical collection materials

Answer 3

-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

Question 4

what do we use to determine if a pathogen is present in a sample

Answer 4

-Microscopy
-Laboratory Culture of microorganisms
-Direct detection= Immunology, nucleic acid technology and analytical chemistry

Question 5

what traditional methods do microbiology labs rely on

Answer 5

culture, phenotypic, immunological and biochemical tests

Question 6

what samples are anaylsed in a clincal lab

Answer 6

blood, faeces, sputum, skin swabs, wound swabs etc

Question 7

what are the best diagnostic tests to use

Answer 7

sensitivity and specificity

Question 8

what is sensitivity

Answer 8

-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.

Question 9

what is the equation for sensitivity

Answer 9

-ability of a test to detect a true positive.

-Sensitivity = True positive / True positive + false negative x100

Question 10

what is specificity

Answer 10

-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.

Question 11

what is the equation for specificity

Answer 11

-Specificity = ability of a test to exclude a true negative.

-Specificity = True negative / True negative + false positive x100

Question 12

what is desirable in a test

Answer 12

a test that is both highly sensitive and highly specific. This is frequently not possible

Question 13

what is looked at in diagnosis using microscopy (direct…)

Answer 13

-Direct examination of sample
-Variety of formats

Question 14

what are the advantages of microscopy

Answer 14

-Rapid
-Cheap
-adaptable according to sample
-Specificity possible
(antibody/nucleic acid staining)

Question 15

what are the disadvantages of microscopy

Answer 15

-Limited specificity
-No recovery of organism for further analysis.
-Poor sensitivity
-Expensive for viruses

Question 16

what occurs in diagnosis using a laboratory culture

Answer 16

-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

Question 17

what mediums are used in laboratory culture strategy

Answer 17

selective, differential and chromogenic mediums

Question 18

what tests are used in laboratory culture strategy

Answer 18

gram stain, biochemical, immunological and molecular tests

Question 19

what is general purpose media examples

Answer 19

Tryptone Soy Agar
Brain-Heart Infusion Agar

Question 20

what is an example of selctive media

Answer 20

Vogel and Johnston Agar

Question 21

what is examples of differential media

Answer 21

MacConkey Agar
Blood agar

Question 22

what is chromogenic media

Answer 22

-Most recent development
-Species-specific chromogens developed to take advantage of what we understand about bacterial metabolism.
-Beta-galactosidase
-Beta glucuronidase

Question 23

what is chromogen

Answer 23

“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

Question 24

why do we use Chromogens?

Answer 24

-Improve overall specificity of a diagnostic medium.
-Positive colonies are easy to identify.
-Reduce number of sub-cultures required.

Question 25

what are the advantages of culture

Answer 25

-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)

Question 26

what are the disadvantages of culture

Answer 26

-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.

Question 27

what is the equation for the biochemical testing of bacteria

Answer 27

-Catalase test -2 H2O2 --> Catalase --> 2 H2O + O2 S. aureus + Streptococcus -

Question 28

explain bacteria and fermenting sugars

Answer 28

-Many bacteria have different abilities to ferment sugars -Specific sugar and pH indicator in the broth e.g glucose, galactose, mannose etc.

Question 29

what are the colours produced when bacteria ferment sugar

Answer 29

-Purple: no fermentation -Yellow: fermentation occurs and acid produced -Yellow: fermentation occurs and both acid and gas produced

Question 30

what is API

Answer 30

-API: Miniaturised biochemical tests on a strip. -Can easily see that these three species are different -Different patterns -ID based on database

Question 31

What is the advanatages of Immunodiagnostics

Answer 31

-Recognition of species -Fast assays -Very flexible

Question 32

what is Immunodiagnostics used for

Answer 32

-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

Question 33

what test is used in Immunodiagnostics

Answer 33

Agglutination Test

Question 34

what is the Agglutination Test

Answer 34

-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

Question 35

what is ELISA

Answer 35

Enzyme-linked immunosorbent assay

Question 36

what does the name of ELISA suggest

Answer 36

-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

Question 37

what is Alkaline Phosphatase

Answer 37

-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

Question 38

what is used in the lateral flow test strip

Answer 38

-Conjugation of Particles -Conjugate Pad -Test Strip -Absorbent pad (wicking)

Question 39

what is the advantage of the lateral flow test strip

Answer 39

rapid diagnosis

Question 40

what are the main elements in clincal diagnostic microbiology

Answer 40

-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.