From lung and brain tissue to eye and nasal swabs, the bacteriology section cultures and identifies microorganisms from a variety of samples submitted to ADDL for testing. When a sample is received in bacteriology, an analyst plates the sample on the appropriate media required for growth based on the sample type and the animal’s history. After an incubation period, which can vary in length and environmental conditions depending on the suspected bacteria, a highly trained analyst will examine the culture media for any significant organisms which can then be identified by Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF).
MALDI-TOF is a mass spectrometry technique that uses a laser to identify a bacterium based on its protein spectral “fingerprint.”
Bacterial cells, from a single pure colony, are placed on a stainless-steel plate (target) and allowed to dry before being covered with matrix, a mixture of organic solvents and acids. The applied matrix plays an important role in MALDI-TOF by absorbing the energy of the laser so the internal bacterial proteins (analytes) remain intact as they are released from the bacteria cell (desorption). Additionally, matrix transfers a positive charge (ionization) to the analytes so they are released from the cell and into an electrostatic field. The analytes are accelerated through the electrostatic field before being separated in a vacuum tube based on time-of-flight, the time it takes an analyte to reach the detector at the end of the tube. Time-of-flight is dependent on the mass-to-charge ratio of the analyte with the lightest arriving to the detector first. This data is used to create a spectral profile “fingerprint” for the organism, which is compared to a database containing spectral “fingerprints” of over 9,000 bacterial, yeast, and fungal organisms to identify the organism’s genus and species. Roughly, 90-95% of microorganisms isolated in the Bacteriology Section can be identified using MALDI-TOF as described above. Some bacteria (such as Mycobacterium species), yeast, and fungal samples require more processing before being applied to the target for analysis due to their cell wall compositions.
Before the use of MALDI-TOF in diagnostic laboratories, bacterial identification involved growing pure cultures of suspicious bacteria before any analyses can begin. Depending on the growth rate of the organism, this process could take days or even weeks. Bacteria can be identified by observing their appearance on agar media, cellular shape and cell wall characteristics, and metabolic properties. Culture media, staining dyes, and biochemical media are expensive, and the latter require an additional incubation period before their metabolic activity can be observed.
By using MALDI-TOF, results can be sent to veterinarians as soon as a single pure colony of bacteria is observed; with some pathogens, results can be available one day after a sample is received.
MALDI-TOF has reduced turn-around time for bacterial identification and is more cost-effective than traditional biochemical testing or automated systems that utilize biochemical reactions for identification. Delays in bacterial identification can occur when there is a high amount of contamination, as only pure bacterial colonies can currently be identified using MALDI-TOF. Additionally, infrequently isolated organisms that are not currently in any MALDI-TOF database may require more traditional bacterial identification tests or molecular techniques such as DNA sequencing. As more veterinary diagnostic laboratories utilize MALDI-TOF for bacterial identification, the databases will continue to grow, allowing for more and more organisms to be identified rapidly. Researchers continue to develop more applications using MALDI-TOF, some of them include identification of mixed bacterial cultures, identifying bacteria directly from samples, and determining antimicrobial resistance patterns.
Dominika Jurkovic, Ph.D., Laboratory Scientist 3, Bacteriology Section