MALDI TOF Objectives

Describe Tube Biochemical.

• fermentation tubes → color change

• lots of tests to identify a single organism

Describe API.

• standardized inoculum added to each cupule

Describe Vitek.

• analyzes samples for life

• uses cards with sugars and substrates

Describe Microscan.

• microtiter plate, sugar/substate, color change

What is Genotypic Identification?

• looks for genome expressed proteins

• uses organisms unique molecular signal identified from its genome-expressed proteins to determine its identity

What is Phenotypic Identification?

• observable physical / biochemical characteristics

  • colony morphology

  • microscopic appearance

  • biochemical reactions

  • growth requirements

What are examples of Genotypic Platforms?

• Molecular probes

• PNA FISH

• multiplex PCR / gene expert / microarrays

• Next generation sequencing

• Proteomics

• Metabolomics

Describe molecular probes?

• genetic platform targets DNA

  • have to have specific target

Describe PNA FISH.

• genetic platform

• detects RNA

Describe Multiplex PCR/gene expert/ microarrays

• genotypic platform

• amplification technique

• needs specific target

• allows you to ramp up if you ONLY have a small amount of organisms

Describe Next generation sequencing.

• 16 rRNA

• can find needle in a haystack

• conserved DNA

Describe Proteomics.

• MALDI-TOF

• Spec

• looks at proteins

Describe Metabolomics.

• growing field

• looks at single molecules

What is MALDI-TOF

• matrix assisted laser

• desorption / ionisation time of flight

• spectrometry

• gives us peaks that we use for ID

What is the X-axis in MALDI-TOF?

• mass to charge ratio / molecular weight

What is the Y-axis in MALDI-TOF?

• intensity

What occurs in the Target Prep step of MALDT-TOF?

• small amount of bacterial colony placed on metal target plate

• matrix solution (with formic acid / acetyl nitrate) is added on top and allowed to dry

• matrix crystalizes with microbial proteins

• mixture absorbs laser energy and protects sample from direct destruction during ionization

Describe TOF treatment step of MALDT-TOF.

• laser pulse strikes sample matrix → matrix vaporize/ ionize sample proteins

• generated ions are accelerated in an electric field towards a detector

• lighter ions move faster, time of flight is proportional to mass-to charge ratio

• instrument records mass spectrum, showing peaks that represent protein fingerprint of microorganism

Describe the ID step of MALDT-TOF

• spectrum compared to reference database

• software matches pattern of peaks to identity of species

What is the principle of the preparation of MALDI-TOF

• mix analyte with molar excess of matrix

• desorption of bulk portions of analyte by a short pulse of laser light

Describe the principle of Pattern recognition-biotyper in MALDI-TOF.

• score based pattern matching

• reference spectra created from a set of individual measurements contain information

• unknown microorganism is matched against each main spectrum in library

• ranking according to matching score, threshold for ID

What is the purpose of formic acid?

• breaks open cells

What is the purpose of ethanol-formic acid?

• used for organisms with thick cell walls when other procedures are NOT enough

What are clinical applications of MALDI-TOF.

• resistance detection

  • detects antibiotic resistance enzymes (B-lactamases) by detecting hydrolysis of antibiotic substrates

Discuss the financial impact of MALDI-TOF for clinical labs.

• cheaper, pays well

Discuss MALDI-TOF-MS

• at its core, it is a continuation of what we have done in microbiology lab that is, pattern recognition for identification

What is the purpose of acetyl nitrate?

• concentrates proteins