1) What does MALDI-TOF stand for?
Matrix-Assisted Laser Desorption & Ionisation- Time Of Flight.
2) What is the purpose of adding a drop of FORMIC ACID to the sample?
This is a rapid extraction method. It helps to break down the cell wall of the bacterium thus exposing the proteins and facilitating their ionisation. It is not always necessary, but can help to improve the “hit rate”
3) What is the purpose of adding the MATRIX?
The matrix serves two functions. it absorbs a high amount of UV light, thus facilitating the ionisation process. They are also acidic in nature, so that it can easily excite photon and ionise the target for analysis.
4) Outline on a step by step basis how MALDI-TOF works?
- Energy in the form of laser is fired at the matrix/sample crystals.
- Laser energy is absorbed by the matrix and converted to heat energy. Energy desorbs matrix and analyte molecules and ionises the sample.
- The positive ions are accelerated through a vacuum by an applied electrical field of known strength.
- The time taken for the positive ions to reach the detector depends on their mass/charge ratio (m/z)
- Different ions of differing mass charge ratios will thus give rise to a spectrograph.
- For bacterial identification by mass spectrometry the ions analysed are proteins. Each bacterial species has a different protein composition, thus giving rise to a specific mass spectrograph.
- The mass spectrograph produced by a sample is then compared with many thousands stored in a spectrograph database to see which one it most closely matches. Thus an identification is acheived.
5) Is MALDI-TOF better for Gram negative or Gram positive bacteria?
Slightly better for Gram negative bacteria, possibly because of the thinner cell wall and thus easier access to proteins.
6) When might you need to perform a FULL EXTRACTION on the sample?
For identifying yeasts, for mucous organisms, for organisms where conventional testing has failed to give an ID and you are still really keen to get an ID.
7) What organisms can MALDI-TOF mix up?
Does not discriminate between Streptococcus pneumoniae and Streptococcus mitis. Does not discriminate between E.coli and Shigella species. Finds it difficult to discriminate between Klebsiella oxytoca and Raoultella species.
8) What are your options if MALDI-TOF fails to give an ID?
- Repeat the process again.
- Use manual laser firing to try and target sample/matrix crystals within the spot
- Perform a full extraction of the sample.
- Re-incubate the plate to get more growth/fresher cultures.
- Use an alternative means of identification.
9) What test controls are required for the MALDI-TOF?
The recommended control requirements vary depending on the instrument used.
BioMerieux’s Vitek MS requires a control to be run with each batch.
Bruker’s Microflex LT does not have specified control requirements but leaves it up to individual laboratories/accreditation authorities to determine (though it does require a monthly calibration).
10) Why is thorough cleaning of the testing plate important?
This question is only relevant if you are using re-usable plates. In our experience if the target plates are insufficiently cleaned the Maldi-tof hardware can pick up the previous spot and give the identification of the sample that was on that spot on the previous one. Worthwhile checking if you think an identification doesn’t fit.