biochemical & spectroscopic techniques

Diagnostic methods: biochemical reactions:

  • catalase → differentiates between isolates that can produce catalase to decompose hydrogen peroxide (H2O2 → H2O + O2) (bubbles forming)

  • oxidase → differentiates between isolates that possess cytochrome c as a respiratory enzyme

→ these tests can be performed in less than a minute and require no incubation time

  • coagulase - detection of coagulase, AKA ‘clumping factor’

→ used to differentiate between different species of staphylococci

→ commercially available kits contain an antibody to the clumping factor

→ positive reaction indicated by agglutination

  • amino acid metabolism - detects specific enzymes

  • carbohydrate oxidation / fermentation - used to create a biochemical profile

→ results are combined together in assimilation panels

→ often require an incubation period

→ data base will tell the probability for the identification of the organism…

(1) dealing with live organisms, some organisms will have undergone mutations to be able to suddenly be able to metabolise that sugar or amino acid

(2) results out of the data base are only as good as the data base itself - if you put a code in which is not already in the data base it will not be recognise → importance of microbiologists in this process

Alternatives to biochemical tests - spectrometry:

  • mass spectrometry is an analytical tool useful for measuring the mass-to-charge ratio (m/z) of one or more molecules present in a sample

  • the measurements can often be used to calculate the exact molecular weight of the sample components as well

  • typically mass spectrometers can be used to…

→ identify unknown compounds via molecular weight determination

→ to quantify known compounds

→ to determine structure and chemical properties of molecules

  • we can apply these principles to identifying colonies of microorganisms

4 mains steps for spectrometry:

  1. Ionisation → atom or molecule or colony is ionised by knocking one or more electrons off to give a positive ion

  2. Acceleration → ions are accelerated so that they all have the same kinetic energy

  3. Deflection → ions are then deflected by a magnetic field according to their masses (lighter ions = greater deflection)

  4. Detection → beam of ions passing through the machine is detected electrically

Matrix Assisted Laser Desorption Ionisation- Time Of Flight mass spectrometry (MALDI-TOF):

→ ionisation of a single colony into its constituent proteins

→ measure time they take to travel through a vacuum to the detector (based upon size)

→ can plot size against time and compare with known values on a database - based upon probability

Focus on laboratory investigation of blood culture samples:

  • detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory

  • samples are taken when patients show symptoms of possible bacteraemia or septicaemia

  • patients often started on broad-spectrum antibiotics that is often changed as results become available

  • automation is commonplace

  • samples are treated as urgent, samples must be transported to the laboratory without delay and incubated as soon as possible

  • blood cultures should only be taken by individuals who have been trained specifically in order to reduce the risk of contamination by skin organisms

  • 2 types of samples can be sent:

  1. a ‘pair’ of blood culture bottles made up of an aerobic and anaerobic bottle - together this pair will provide appropriate conditions for most bacterial growth

  2. if you are unable to gain enough blood, minimum 5-8ml then it is advisable to use a ‘pediatric’ bottle

‘flagging’:

  • fluorescent technology in detecting the growth of organisms in the blood culture bottles

  • if microorganisms are present in the cultured vials, they metabolise nutrients in the culture medium, releasing carbon dioxide into the medium

  • a dye in the sensor at the bottom of the culture bottles with react with CO2

  • this changes the amount of light that is absorbed by a fluorescent material in the sensor

  • the measurement is interpreted by the system according to preprogrammed positivity algorithms

blood culture reporting:

  • blood culture positives will come ina series of updated results;

→ the gram stain

→ the organism identification from basic biochemical tests alongside Mass Spec result

→ preliminary sensitivity

→ final report

Gram stain:

  • when the analyser detects the change - ‘flags’ positive

  • the bottle is removed from the system and a gram film is made to look for the presence of microorganisms

  • culture media is inoculated - this is in some part dependent upon the organisms seen in the gram film

  • antibiotics therapy may be changed at this point

  • antimicrobial susceptibility testing is also performed directly from the bottle

Initial identification:

  • after 24 hours incubation on the culture media, the colonial morphology can be determined

  • mass spectrometry can be performed - same day result

  • antibiotic therapy may be changed again at this point

  • biochemical tests are put up - some same day results, others require a further incubation period

  • repeat antimicrobial susceptibility testing is performed - requires a further incubation

Final report:

  • once all the biochemical results have been interpreted, a final identification and antimicrobial susceptibility can be reported

  • this may lead to antimicrobial treatment being changed once again (the most appropriate treatment for the causative agent)