Enzyme-integrated metal-organic framework platform for cascade detection of α-amylase.

As one of the most important industrial enzymes, α-amylase is widely used in food processing, such as starch sugar and fermentation, bringing high added value to industry of more than a trillion dollars. We developed a multi-enzyme system (Glu&Gox@Cu-MOF-74) prepared by embedding α-glucosidase (Glu) and glucose oxidase (Gox) into the biomimetic metal-organic framework Cu-MOF-74 using in situ encapsulation within 15 min at room temperature for efficient and sensitive detection of α-amylase activity. Benefitting from the remarkable peroxidase-mimicking property and rigid skeleton of Cu-MOF-74, the biocatalytic platform exhibited excellent cascade activity and tolerance in various extremely harsh environments compared to natural enzymes. On this basis, a cascade biocatalytic platform was constructed for the detection of α-amylase activity with wide linear range (5-100 U/L) and low limit of detection (1.45 U/L). The colorimetric cascade scheme is important for the sensitive and selective determination of α-amylase in complex fermentation samples, and the detection time is short (∼0.5 h). This work provides new ideas for the detection of α-amylase based on the cascade amplification method.

A magnetic nanozyme platform for bacterial colorimetric detection and chemodynamic/photothermal synergistic antibacterial therapy.

Rapid, convenient, and sensitive detection of bacteria and development of novel antibacterial materials are conducive to accurate treatment of bacterial infection and reducing the generation of drug-resistant bacteria caused by overuse of antibiotics. A dual-function magnetic nanozyme, Fc-MBL@rGO@Fe3O4, has been constructed with broad-spectrum bacterial affinity and good peroxidase-like activity. Detection signal amplification was realized in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) with a detection limit of 26 CFU/mL. In addition, the excellent photothermal properties of Fc-MBL@rGO@Fe3O4 could realize synergistic chemodynamic/photothermal antibacterial therapy. Furthermore, the good bacterial affinity of Fc-MBL@rGO@Fe3O4 enhances the accurate and rapid attack of hydroxyl radical (·OH) on the bacterial membrane and achieves efficient sterilization (100%) at low concentration (40 µg/mL) and mild temperature (47℃). Notably, Fc-MBL@rGO@Fe3O4 has a broad spectrum of antibacterial activity against Gram-negative, Gram-positive, and drug-resistant bacteria. The magnetic nanoplatform integrating detection-sterilization not only meets the need for highly sensitive and accurate detection in different scenarios, but can realize low power density NIR-II light-responsive chemodynamic/photothermal antibacterial therapy, which has broad application prospects.

Integration of multienzyme co-immobilization and biomimetic catalysis in magnetic metal-organic framework nanoflowers for α-amylase detection in fermentation samples.

Multiple enzymes induce biological cascade catalysis is essential in nature and industrial production. However, the shortcomings of enzymes, including unsatisfactory stability, reusability, and sensitivity in harsh microenvironment, have restricted their broader use. Here, we report a facile method for fabricating a cascade system by combining the benefits of immobilized enzymes and biomimetic catalysis based on magnetic metal-organic framework nanoflowers (mMOFNFs). mMOFNFs prepared through the layered double hydroxide-derived strategy exhibited remarkable peroxidase-like activity and accessible amino interface, enabling it to serve not only as a reliable carrier for α-glucosidase and glucose oxidase fixation, but also as a nanozyme participating in cascade. On this basis, a colorimetric biosensor of excellent sensitivity and selectivity for α-amylase detection was constructed with a wide range (2-225 U L-1), low detection limit (2.48 U L-1), and rapid operation (30 min). This work provides a versatile strategy for establishing multi-enzyme cascade systems and rapid analysis of α-amylase.

Bioluminescence assay for rapid detection of live Staphylococcus aureus based on the enrichment of egg yolk antibody modified magnetic metal organic framework immunobeads.

Specific and rapid detection of live Staphylococcus aureus (S.A) in environmental and food samples is critically important for protecting human health. In order to fulfill this purpose, two kinds of novel egg yolk antibody (IgY) immobilized immunomagnetic beads (IMBs; mSiO2-IgY and mMOF-IgY), with core-shell mSiO2 and mMOF as substrate, were prepared for selectively enriching S.A from samples. Furthermore, the IMBs with captured S.A were collected and re-dissolved in 0.5 mL PBS. After that, a cotton swab coated with sodium dodecylsulfate (SDS) was put in the solution to lyse S.A cells and emit ATP bioluminescence of the luciferin/luciferase system. Finally, a portable bioluminescence detector was used for quantification of ATP corresponding to S.A concentration. The results demonstrated that mMOF-IgY can enrich more S.A than mSiO2-IgY and emit a stronger signal. The reasons may be due to the higher immobilization amount of IgY on the IMBs. Under optimal conditions, the calibration line of S.A concentration was 10-105 CFU mL-1 by mMOF-IgY within 30 min. The low detection limit of S.A was 3 CFU mL-1. The results demonstrated that the assay takes much shorter time than plate counting. Its portability and excellent detection capability are suitable for rapid monitoring of specific pathogens in foods.

Protocol for determining protein dynamics using FT-IR spectroscopy.

In biological systems, protein function depends on spatial and temporal changes known as protein dynamics, which can be probed by amide hydrogen/deuterium (H/D) exchange. Here, we present a protocol for determining protein dynamics by Fourier-transform infrared (FT-IR) spectroscopy. We describe steps for protein sample preparation and FT-IR spectra collection. We then detail procedures for spectra analysis. Applications include the effects of protein mutation or protein and metal ion or ligand interactions on the protein H/D exchange rate. For complete details on the use and execution of this protocol, please refer to Yu et al. (2013).1.

Fusion data from FT-IR and MALDI-TOF MS result in more accurate classification of specific microbiota.

Microbes are usually present as a specific microbiota, and their classification remains a challenge. MALDI-TOF MS is particularly successful in library-based microbial identification at the species level as it analyzes the molecular weight of peptides and ribosomal proteins. FT-IR allows more accurate classification of bacteria at the subspecies level due to the high sensitivity, specificity and repeatability of FT-IR signals from bacteria, which is not achievable with MALDI-TOF MS. Previous studies have shown that more accurate identification results can be obtained by the fusion of FT-IR and MALDI-TOF MS spectral data. Here, we constructed 20 groups of model microbiota samples and used FT-IR, MALDI-TOF MS, and their fusion data to classify them. Hierarchical clustering analysis (HCA) showed that the classification accuracy of FT-IR, MALDI-TOF MS, and the fusion data was 85%, 90%, and 100%, respectively. These results indicate that both FT-IR and MALDI-TOF MS can effectively classify specific microbiota, and the fusion of their spectral data could improve the classification accuracy. The FT-IR and MALDI-TOF MS data fusion strategy may be a promising technology for specific microbiota classification.

Rapid detection of Saccharomyces cerevisiae with boronic acid-decorated multivariate metal-organic frameworks and aptamers.

Liquor brewing is a classic solid-substrate fermentation process with a unique brewing microbiome. As one of the most common fungi, Saccharomyces cerevisiae ferments saccharides and has been extensively applied in brewing production. Here, we present the facile fabrication of a selective, sensitive, and integrated fluorescent biosensor for S. cerevisiae detection. The proposed biosensor used aptamer-modified magnetic beads to specifically capture S. cerevisiae, and the enriched fungi were recognized and detected with boronic acid-decorated multivariate metal-organic frameworks. The biosensor allows rapid quantification of S. cerevisiae in the range of 10-106 CFU mL-1, showing excellent specificity and repeatability, and maintaining stable biosensing performance in long-term storage. The analytical ability of the proposed biosensor was successfully verified in distilled yeast and fermented grain samples spiked with S. cerevisiae.

A filter-electrochemical microfluidic chip for multiple surface protein analysis of exosomes to detect and classify breast cancer.

Breast cancer (BC) is a complex disease with high variability and no specific tumor markers available for diagnosis. Exosomes contain rich maternal tumor information and are a novel non-invasive biomarker with the potential for cancer diagnosis and prognosis. However, analysis of exosomal protein markers in blood samples is challenging due to lengthy sample workups and insufficient sensitivity. To address this difficulty, we developed a novel filter-electrochemical microfluidic chip (FEMC) to detect and classify BC directly in whole blood without requiring heavy purification methods. In our system, exosome enrichment was performed using a dual filtration system. The target was directed through a curved channel onto four screen-printed electrodes (SPEs), where it was captured by the previously modified antibodies. Simultaneously, Zr-MOFs encapsulated with a large number of methylene blue molecules (MB@UiO-66) were absorbed on the surface of exosomes due to the high affinity for phosphate groups. This process leads to the amplification of electrical signals. The approach demonstrated that the utilization of BC exosome-associated tumor biomarkers (i.e., PMSA, EGFR, CD81, and CEA), enabled the classification of various BC mouse models samples and clinical BC samples. The entire FEMC assay was completed in 1 h with a limit of detection of 1 × 104 particles/mL. Thus, the FEMC assay can provide real-time detection information, allowing timely and better-informed opportunities for clinical BC diagnosis and typing.

Simple and Rapid Discrimination of Methicillin-Resistant Staphylococcus aureus Based on Gram Staining and Machine Vision.

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinical threat with high morbidity and mortality. Here, we describe a new simple, rapid identification method for MRSA using oxacillin sodium salt, a cell wall synthesis inhibitor, combined with Gram staining and machine vision (MV) analysis. Gram staining classifies bacteria as positive (purple) or negative (pink) according to the cell wall structure and chemical composition. In the presence of oxacillin, the integrity of the cell wall for methicillin-susceptible S. aureus (MSSA) was destroyed immediately and appeared Gram negative. In contrast, MRSA was relatively stable and appeared Gram positive. This color change can be detected by MV. The feasibility of this method was demonstrated in 150 images of the staining results for 50 clinical S. aureus strains. Based on effective feature extraction and machine learning, the accuracies of the linear linear discriminant analysis (LDA) model and nonlinear artificial neural network (ANN) model for MRSA identification were 96.7% and 97.3%, respectively. Combined with MV analysis, this simple strategy improved the detection efficiency and significantly shortened the time needed to detect antibiotic resistance. The whole process can be completed within 1 h. Unlike the traditional antibiotic susceptibility test, overnight incubation is avoided. This new strategy could be used for other bacteria and represents a new rapid method for detection of clinical antibiotic resistance. IMPORTANCE Oxacillin sodium salt destroys the integrity of the cell wall of MSSA immediately, appearing Gram negative, whereas MRSA is relatively stable and still appears Gram positive. This color change can be detected by microscopic examination and MV analysis. This new strategy has significantly reduced the time to detect resistance. The results show that using oxacillin sodium salt combined with Gram staining and MV analysis is a new, simple and rapid method for identification of MRSA.

Metaproteomics profiling of the microbial communities in fermentation starters (Daqu) during multi-round production of Chinese liquor.

Introduction: The special flavor and fragrance of Chinese liquor are closely related to microorganisms in the fermentation starter Daqu. The changes of microbial community can affect the stability of liquor yield and quality. Methods: In this study, we used data-independent acquisition mass spectrometry (DIA-MS) for cohort study of the microbial communities of a total of 42 Daqu samples in six production cycles at different times of a year. The DIA MS data were searched against a protein database constructed by metagenomic sequencing. Results: The microbial composition and its changes across production cycles were revealed. Functional analysis of the differential proteins was carried out and the metabolic pathways related to the differential proteins were explored. These metabolic pathways were related to the saccharification process in liquor fermentation and the synthesis of secondary metabolites to form the unique flavor and aroma in the Chinese liquor. Discussion: We expect that the metaproteome profiling of Daqu from different production cycles will serve as a guide for the control of fermentation process of Chinese liquor in the future.

Rapid and ultrasensitive activity detection of α-amylase based on γ-cyclodextrin crosslinked metal-organic framework nanozyme.

α-Amylase plays a significant part in fermentation and the food industry, as this enzyme effectively regulates the content of different sugars in brewing systems and affects the yield and quality of alcoholic beverages. Nevertheless, current strategies suffer from unsatisfactory sensitivity and are time-consuming or are indirect methods which demand the assistance of tool enzymes or inhibitors. Therefore, they are unsuitable for the low bioactivity and non-invasive detection of α-amylase in fermentation samples. Rapid, sensitive, facile, and direct detection method of this protein remains challenging in actual applications. In this work, a nanozyme-based α-amylase assay was constructed. The colorimetric assay used the interaction between α-amylase and γ-cyclodextrin (γ-CD) which crosslinks MOF-919-NH2. The determination mechanism bases on the hydrolysis of γ-CD by α-amylase, resulting in increased peroxidase-like bioactivity of the released MOF nanozyme. The detection limit was 0.12 U L-1 with a wide linear range (0-200 U L-1) and excellent selectivity. Additionally, the proposed detection method was successfully utilized in distilled yeasts to verify analytical capability in fermentation samples. The exploration of this nanozyme-based assay not only provides a convenient and effective strategy for enzyme activity determination in food industry, but also has promotion significance in clinical diagnosis and pharmaceutical production.

Allosteric regulation of α-amylase induced by ligands binding.

The conformational changes in α-amylase induced by different ligands, including metal ions, substrates, and aromatic compounds in liquor production, were systematically studied using spectroscopy. Fluorescence acrylamide quenching analysis showed that the interaction with active metal cations (K+, Na+, and Ca2+) led to higher exposure of the active sites in α-amylase. In contrast, interactions with substrates (soluble starch, amylose, amylopectin, wheat starch, and dextrin) reduced the degree of exposure of active sites, and the conformation of the enzyme became more rigid and compact. Although the interaction with inhibitory metal cations (Mg2+, Zn2+) and aromatic compounds generated in the brewing process (guaiacol, eugenol, thymol, and vanillin) increased the exposure of active site with a relatively low amplitude, it reduced the enzymatic activity. This finding may be due to the overall structure of the enzyme becoming looser. Structural stability showed that the active cations and substrates increased the stability of the secondary structure of the α-amylase backbone, whereas the inhibitory cations and aromatic compounds reduced the stability of the backbone but increased the compact of domain A and B. Enzymatic assays and molecular docking experiments strongly supported these conclusions. The experimental results may provide a valuable reference for controlling related conditions and improving production efficiency.

MALDI-TOF MS Is an Effective Technique To Classify Specific Microbiota.

MALDI-TOF MS is well-recognized for single microbial identification and widely used in research and clinical fields due to its specificity, speed of analysis, and low cost of consumables. Multiple commercial platforms have been developed and approved by the U.S. Food and Drug Administration. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used for microbial identification. However, microbes can present as a specific microbiota, and detection and classification remain a challenge. Here, we constructed several specific microbiotas and tried to classify them using MALDI-TOF MS. Different concentrations of nine bacterial strains (belonging to eight genera) constituted 20 specific microbiotas. Using MALDI-TOF MS, the overlap spectrum of each microbiota (MS spectra of nine bacterial strains with component percentages) could be classified by hierarchical clustering analysis (HCA). However, the real MS spectrum of a specific microbiota was different than that of the overlap spectrum of component bacteria. The MS spectra of specific microbiota showed excellent repeatability and were easier to classify by HCA, with an accuracy close to 90%. These results indicate that the widely used MALDI-TOF MS identification method for individual bacteria can be expanded to classification of microbiota. IMPORTANCE MALDI-TOF MS can be used to classify specific model microbiota. The actual MS spectrum of the model microbiota was not a simple superposition of every single bacterium in a certain proportion but had a specific spectral fingerprint. The specificity of this fingerprint can enhance the accuracy of microbiota classification.

Protocol for bacterial typing using Fourier transform infrared spectroscopy.

The Fourier transform infrared (FT-IR) signals obtained from bacterial samples are specific and reproducible, making FT-IR an efficient tool for bacterial typing at a subspecies level. However, the typing accuracy could be affected by many factors, including sample preparation and spectral acquisition. Here, we present a unified protocol for bacterial typing based on FT-IR spectroscopy. We describe sample preparation from bacterial culture and FT-IR spectrum collection. We then detail FT-IR spectrum preprocessing and multivariate analysis of spectral data for bacterial typing.

Ultrasensitive Detection of Multidrug-Resistant Bacteria Based on Boric Acid-Functionalized Fluorescent MOF@COF.

The widespread use of antibiotics has made multidrug-resistant bacteria (MDRB) one of the greatest threats toward global health. Current conventional microbial detection methods are usually time-consuming, labor-intensive, expensive, and unable to detect low concentrations of bacteria, which cause great difficulties in clinical diagnosis and treatment. Herein, we constructed a versatile biosensing platform on the basis of boric acid-functionalized porous framework composites (MOF@COF-BA), which were able to realize highly efficient and sensitive label-free MDRB detection via fluorescence. In this design, MDRB were captured using aptamer-coated nanoparticles and the fluorescent probe MOF@COF-BA was tightly anchored onto the surface of MDRB due to interactions between boric acid groups and glycolipids on bacteria cells. Benefitting from the remarkable fluorescence performance of MOF@COF-BA, rapid and specific detection of MDRB, such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB), was realized with a detection range of 20-108 CFU/mL (for both) and limits of detection of 7 CFU/mL (MRSA) and 5 CFU/mL (AB). The feasibility of using the developed platform to selectively detect MRSA and AB from complex urine, human serum, and cerebrospinal fluid samples was also demonstrated. This work provides a promising strategy for accurate MDRB diagnosis, avoiding serious infection using rational antibiotic therapy.

A microfluidic chip-based multivalent DNA walker amplification biosensor for the simultaneous detection of multiple food-borne pathogens.

The rapid, simultaneous, sensitive detection of the targets has important application prospects for disease diagnosis and biomedical studies. However, in practical applications, the content of the targets is usually very low, and signal amplification strategies are often needed to improve the detection sensitivity. DNAzyme-driven DNA walkers are an excellent signal amplification strategy due to their outstanding specificity and sensitivity. Food-borne pathogens have always been a foremost threat to human health, and it is an urgent demand to develop a simple, rapid, sensitive, and portable detection method for food-borne pathogens. In addition, there are various species of pathogens, and it is difficult to simultaneously detect multiple pathogens by a single DNA walker. For this reason, a substrate strand with three rA cleavage sites was cleverly designed, and a multivalent DNA walker sensor combined with the microfluidic chip technology was proposed for the simultaneous, rapid, sensitive analysis of Vibrio parahaemolyticus, Salmonella typhimurium, and Staphylococcus aureus. The developed sensor could be used to detect pathogens simultaneously and efficiently with low detection limits and wide detection ranges. Moreover, the combination of gold stirring rod enrichment and DNA walker achieved double amplification, which greatly improved the detection sensitivity. More importantly, by changing the design of the substrate chain, the sensor was expected to be used to detect other targets, thus broadening the scope of practical applications. Therefore, the sensor can build novel detection tool platforms in the field of biosensing.

Portable ATP bioluminescence sensor with high specificity for live Escherichia coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation.

Live foodborne pathogens proliferate rapidly and do great harm to human health, which requires appropriate methods to supervise. In this work, a portable adenosine triphosphate (ATP) bioluminescence sensor with high specificity for live E. coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation was developed. In brief, the selected phages were directionally immobilized on the poly(diallyldimethylammonium chloride)-modified gold stir bar as the bioreceptor. Following the simple stir bar absorptive extraction and bio-proliferation in the Luria-Bertani medium, the number of captured E. coli O157:H7 exploded. Finally, it was quantified by a portable ATP bioluminescence sensor. Benefitting from the high specificity of phage and simple signal dual-amplification strategy, the proposed biosensor achieved the recognition of live bacteria at strain level with superior sensitivity. Also, the portable signal readout made it suitable for on-site detection. Under optimal conditions, this bioassay provided a detectable range of 102-107 CFU mL-1 with a low detection limit of 30 CFU mL-1 within 30 min. The detection results for real samples demonstrated that there were no differences between the assay and the plate counting method, while the detection time was largely shortened. Furthermore, the assay gives a novel path for the point-of-care test (POCT) of live E. coli strain, which is promising to be extended to other virulent strains measurement with corresponding phages.

Rapid Pathogen Identification in Aqueous Humor Samples by Combining Fc-MBL@Fe3O4 Enrichment and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Profiling.

Prompt clinical diagnosis and antimicrobial therapy are key to managing infective endophthalmitis. The small volume of aqueous humor, low bacterial counts, and empirical medication by physicians make existing diagnostic methods time-consuming and imprecise. Here, we investigated the feasibility of combining Fc-containing mannose-binding lectin-coated Fe3O4 (Fc-MBL@Fe3O4) enrichment with matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) profiling to identify pathogens in aqueous humor. Aqueous humor aspirated from freshly enucleated porcine eyes was treated with different inocula of Staphylococcus aureus, Staphylococcus epidermidis, and Klebsiella pneumoniae. We performed identification directly in aqueous humor samples and after short-term culture of micro-LB broth. Aqueous humor endophthalmitis samples were enriched with Fc-MBL@Fe3O4 and analyzed using MALDI-TOF MS. The identification time and minimum bacterial concentration required for identification were determined. The enrichment efficiency of Fc-MBL@Fe3O4 for different bacteria was greater than (87.5 ± 5.0)%. The objects of direct identification include live bacteria and bacteria treated with antibiotics, which can be completed within 1.5 h. The minimum number of bacteria needed for positive identification was 2.20 × 106 CFU. For micro-LB broth culture, the identification of bacteria can be completed within 6.5 to 9.5 h for aqueous humor samples with an initial bacterial count of tens to hundreds. IMPORTANCE Fc-MBL@Fe3O4 capture not only live bacteria in aqueous humor but also bacteria inactivated by antibiotics. Fc-MBL@Fe3O4 combined with micro-LB broth culture significantly reduced the turnaround time (TAT) by more than half a day by shortening the time required for bacterial identification. Our findings demonstrate that combining Fc-MBL@Fe3O4 enrichment with MALDI-TOF MS identification is a fast, sensitive, and efficient analytical method with great potential for identifying pathogens in aqueous humor samples.

Efficient classification of Escherichia coli and Shigella using FT-IR spectroscopy and multivariate analysis.

Accurate and effective discrimination of E. coli and Shigella is an important clinical issue, and there are many limitations in traditional methods of analysis. FT-IR shows great potential in the classification of bacteria with high specificity and low cost. In this study, we evaluated the efficiency of this technique when combined with multivariate analysis for rapid classification of E. coli and Shigella, which is difficult using traditional analytical methods. Machine learning and statistical tools were employed in combination with FT-IR to classify 14 E. coli and 9 Shigella strains. The classification accuracies for select E. coli and Shigella strains from blood agar were 0.7826, 0.8696, and 0.9565 at the genus, species, and strain levels, respectively. In addition, we used the FT-IR data of select strains from three different culture media for cross-validation, yielding an accuracy of 0.3681 at the strain level. These results indicate that the bacterial culture conditions have a significant impact on the FT-IR patterns. Based on this, an improved strategy for training an ensemble classifier model considering bacterial culture factors was constructed, resulting in almost perfect separation with an accuracy of 0.9394 for strain-level classification. These results show the potential of FT-IR combined with multivariate analysis for more reliable bacterial classification.

Fc-MBL-modified Fe3O4 magnetic bead enrichment and fixation in Gram stain for rapid detection of low-concentration bacteria.

Functional bacterial enrichment magnetic beads (Fe3O4@SiO2@Fc-MBL) and Gram staining were combined for the fast diagnosis of infecting bacteria in meningitis. Fe3O4@SiO2@Fc-MBL has excellent microbial binding ability and can be used for bacterial enrichment from cerebrospinal fluid (CSF). The enriched bacteria are recognized by Gram stain at very low concentrations (10 CFU·mL-1). The feasibility of this method was verified by five common bacteria in meningitis infection (Gram-positive: Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus capitis; Gram-negative: Klebsiella pneumoniae and Escherichia coli). The extraction efficiency of Fc-MBL-modified Fe3O4 magnetic beads was approximately 90% in artificial CSF for the selected bacteria, with the exception of E. coli (~ 60%). The bacteria were successfully recognized by Gram staining and microscopic observation. Fe3O4@SiO2@Fc-MBL acts by capturing and fixing the bacteria in a magnetic field throughout the experiment. Compared with traditional CSF Gram staining, this new method avoids interference by inflammatory cells and red blood cells during microscopic examination. Furthermore, the sensitivity of this method is much better than the centrifugation smear method. The whole process can be accomplished within 30 min. This novel method may have potential as a clinical tool for analysis of bacteria in the CSF.