Identifying Yeasts by MALDI-TOF: CDC's Example Laboratory Procedure

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is one of the easiest and most accurate ways to identify yeasts. CDC's Mycotic Diseases Laboratory has published an example standard operating procedure for identifying yeast isolates on the Bruker Biotyper MALDI-TOF platform, using a custom extraction optimized for species in both the Bruker database and CDC's MicrobeNet database.

It is an example, not a controlled document. Each laboratory is responsible for adapting it to its own regulatory, quality and safety requirements and for checking that it is still current; the methods may change without notice. Trade names are for identification only and are not an endorsement.

What you need

Equipment: a biological safety cabinet and a Bruker Biotyper MALDI-TOF mass spectrometer.

Reagents and supplies:

• 100% ethanol, sterile, labeled "molecular biology reagent";
• sterile, cell-culture-grade water for injection (WFI);
• Sabouraud dextrose agar (Emmons) plates and slants, and CHROMagar Candida chromogenic agar;
• a reusable polished steel MALDI target plate with 96 sample positions;
• Bruker HCCA matrix (α-cyano-4-hydroxycinnamic acid), prepared by adding 250 µL of Bruker Standard Solvent and vortexing until clear;
• 70% formic acid, diluted with sterile cell-culture-grade water;
• Bruker Bacterial Test Standard (BTS), prepared to the manufacturer's instructions and stored at −18°C or colder;
• Bruker Standard Solvent;
• 1 µL culture loops and 1.5 mL microcentrifuge tubes.

Safety

Follow all institutional safety procedures. At minimum wear gowns, gloves and safety glasses at all times, and do all setup in a biological safety cabinet. Formic acid is flammable, toxic and corrosive; HCCA irritates the skin and seriously irritates the eyes.

Preparing samples

• Use an overnight pure culture for routine identification. Slow growers such as Candida haemulonii may need several days.
• Plates can be kept at room temperature for 1–5 days. The medium (Sabouraud or CHROMagar) and growth temperature (25°C or 37°C) make little or no difference.
• Do not use organisms stored at 4°C or colder — it degrades the spectra and their reproducibility.

Quality control: in every run, include pure cultures of Candida auris and Candida parapsilosis, in duplicate, as positive controls.

Extraction and spotting

Before starting, record on a sample-key spreadsheet where each specimen will go on the 96-spot target plate. Work in the biological safety cabinet, and do everything in duplicate.

1. Label two 1.5 mL tubes per sample, including the control strains, and add 50 µL of sterile WFI water to each.
2. With a sterile 1 µL loop, transfer one loopful of organism into each tube (more than one isolated colony if colonies are small), and close the lid.
3. Add 50 µL of 100% ethanol to each tube, and pipette up and down 10 times with a pipette set to 50 µL to mix.
4. Spot 1 µL of each suspension onto the target plate and let it air-dry for two minutes.
5. Overlay each spot with 1 µL of 70% formic acid and air-dry for two minutes.
6. Once a week, add 1 µL of BTS to two empty spots — without formic acid — and let it dry.
7. As soon as the spots are dry, immediately overlay each with 1 µL of HCCA matrix — any delay lowers the quality of the run — and air-dry for two minutes.
8. The target must be completely dry before it goes into the instrument, and must be run within 24 hours. Check the sample key against the original sample numbers.
9. Run the samples on the Bruker Biotyper and interpret the spectra against the Bruker database.

A stainless-steel MALDI target plate. CDC.

Reading the results

• Scores: Bruker accepts any score above 2.0 as a species identification. A laboratory may validate a lower acceptable score in-house.
• Controls first: at least one of each control strain's duplicates must score 2 or higher with the correct identification, with no conflict between duplicates. If the controls score below 2 or are misidentified, the run cannot be interpreted and extraction must be repeated.
• Samples: a Candida species is identified when one or both duplicates score 2 or higher.
• No identification: repeat the extraction with a new subculture. If the second attempt also fails, identify the isolate by another method.

Optional: checking spectra against MicrobeNet

Labs can also compare spectra with CDC's MicrobeNet database: request an account on the MicrobeNet website and log in, convert the main spectra to an XML file in the Bruker software, and drag and drop the file onto the site. The top 10 matches are shown.

Sources

• Centers for Disease Control and Prevention, Mycotic Diseases Laboratory: "Identification of Yeasts Using MALDI-TOF" (example test procedure).