# MALDI-Based Mass Spectrometry in Clinical Testing: Focus on Bacterial Identification
## 4 Sample Preparation Strategies for MALDI-Based Bacterial Identification  
One of the advantages of MALDI-TOF MS is the use of the same basic measurement technique whether the sample contains aerobic bacteria, anaerobic bacteria, or yeast-like fungi. This makes possible to obtain improvements in test efficiency and reduced wastage of test reagents. There are two main methods of using cultured colonies as a spotting method. One is the cell smear method (smear method), in which colonies are directly applied to a MALDI-TOF MS sample plate. The cell smear method identifies Gram-negative rods such as Enterobacteriaceae with high probability, but Gram-positive bacteria, such as _Staphylococcus_ and _Enterococcus_, and yeast are insufficiently identified. This may be due to the insufficient extraction of proteins from the bacterial body due to differences in cell wall structure, resulting in a lack of the spectral information necessary for identification. The identification probability is increased using the formic acid extraction method (on-plate method), in which the colony is applied to a sample plate, and formic acid is subsequently applied to the colony to promote cell wall destruction.  
The cell smear method (direct smear method) is used in routine testing [45; 46; 47; 48; 49]. When evaluating bacteria grown in culture medium, the causative organism is estimated by considering the patient's clinical information, the epidemiological information of the infection, the gram staining results, active inflammatory findings, whether the causative organism was observed, and the number of bacteria that developed.  
The identification probability typically is more accurate when performed using freshly grown colonies. For fast-growing bacteria, colonies that form after one day of culturing are the best substrates; empirically, identification is possible within 2-3 days. For slow-growing bacteria, the timing for identification depends on when a colony is observed. The recommended medium is listed on each instrument for colonies used for identification, but these recommendations differ little from the medium used in daily activities of the typical clinical laboratory. The colony identification method (processing operation on the MALDI sample plate) is shown below.  
\begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Features** & **Content** & **References** \\ \hline High identification accuracy & Equivalent to 16S rRNA gene analysis & [36] \\ \hline Identifies large number of bacterial species & Compared to identification methods based on biochemical properties, the number of species identified is larger and even species with poor biochemical properties can be identified. & [37] \\ \hline Fast identification time & About 10 min & [38] \\ \hline Small number and quantity of reagents & Formic acid, ethanol (if necessary), matrix reagent & [8; 26] \\ \hline Low identification cost & Low running costs and economic benefits, such as shortened hospital stay & [39; 40] \\ \hline Fewer consumables & Only toothpicks for fishing and tips for reagents are used; sample plates used for identification are disposable or reusable depending on the model. & [41] \\ \hline Less waste & Since identification panels for biochemical characterization are not used, less waste is generated. & [42; 43] \\ \hline No distinction by gram staining or morphology & No need to select an identification kit for each strain, as in the case of biochemical characterization. & [44] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Features and advantages of microbial identification by mass spectrometry.