Wet grinding of invertebrate bulk samples – a scalable and cost-efficient protocol for metabarcoding and metagenomics

DNA metabarcoding is an efficient tool to characterize invertebrate species composition in environmental samples. Most metabarcoding protocols for invertebrate bulk samples start with sample homogenization, followed by DNA extraction, amplification of a specific marker region and sequencing on a high-throughput sequencer. Many of the above-mentioned laboratory steps have been verified thoroughly and best practice strategies exist. Giving the amount of research done to validate almost all laboratory steps of metabarcoding workflows it is surprising that there is no clear recommendation for the basis of almost all metabarcoding studies: the homogenization of samples itself. For homogenization, different devices are used that can be divided into two major categories: bead mills and blenders. While bead-mills accelerate small, hard particles in a closed container or tube to break down specimens into small tissue fragments, blenders work with a rapidly rotating blades that reliably slice specimens as well as other substrate of the sample. Both methods are currently used in metabarcoding studies and have downsides to consider. Bead-mills rely on single-use plastics and therefore produce a lot of waste and are expensive. In addition to that, processing times can go up to 30 minutes making them unsuitable for large scale studies. Blenders on the other hand offer the opportunity to be cleaned and can handle larger sample volumes in shorter time, with an increased risk of cross-contamination. We here aimed to develop a fast, robust, cheap and reliable sample homogenization protocol that overcomes the above-mentioned limitations of both methods, i.e. does not produce difficult to discard waste and avoid single-use plastics while reducing overall costs. We tested the performance of the new protocol using six sorted Malaise trap insect samples and six unsorted stream macroinvertebrate kick-net samples. We used 14 technical replicates of each sample and many negative controls per sample (Fig. 1) to quantify impacts of i) insufficient homogenization and ii) possible sources of cross-contamination. Our results show that homogenization is sufficient after 3 minutes of homogenization. Rinsing the blender with water is sufficient in most cases but leads to low read-numbers in some of the negative controls. These could be further reduced by rinsing the blender with self-made drain-safe sterilization solution based on bleach but far less corrosive than pure bleach. Our results suggest that rinsing 1-2 times for 20 seconds is sufficient to avoid any cross-contamination. The improvements of the protocol in terms of speed, ease of handling, overall reduction of costs as well as the documented reliability and robustness make it an important candidate for sample homogenization after sampling in particular for large-scale and regulatory metabarcoding biodiversity assessments and monitoring programs.

Comparison between devices for homogenization and reduction of soybean grain samples

In the grain market the most diverse equipment is used for homogenization and reduction of the working samples. Thus, this paper aims to analyze the performance of devices used for sample homogenization and reduction for soybean classification. A sample composed of 8% of damaged soybeans with yellow and black coat was previously prepared. Samples were homogenized and reduced in bucket, crate, Boerner, 16:1 multichannel splitter and 4:1 multichannel splitter. The design used was completely randomized with five treatments (homogenizers) and one percentage of damaged grains (8%). Evaluations were subjected to analysis of variance and treatment means were compared to one another by Tukey test (p 0.5) and to the mean of the original sample (8%) by Student s t-test. The devices Boerner, 16:1 multichannel splitter and 4:1 multichannel splitter were similar in the homogenization and reduction of the soybean sample. The crate and bucket showed the worst performance in the grain homogenization because they compromise the result of the product’s qualitative analysis.

Self contained Food Sample Homogenization Filter Bag for Microbial Analysis

The complexity of food materials owing to the diverse matrices and biochemical composition poses challenge to microbiologists especially to identify the microbial contamination at low level. The present study describes the development and evaluation of a ready to use self-contained food sample homogenization bag (All-In-Bag) with the required sterile diluent and an in-built filter for subsequent clarification of the homogenate for microbiological analysis. Three-ply non-foil laminate comprising outer alumina oxide coated polyester film, middle nylon and inner polypropylene layers were used for the outer layers while non-woven polypropylene sheet with of 50 μ to 100 μ size porosity was sandwiched between the laminated sheets to restrain the food debris but allow the microbial cells to pass through across along with the diluent. The homogenization bag along with the diluent was sterilized by thermal (retort) processing with F0 value (lethality value) of 12 to ensure the sterility of diluent during storage. The effectiveness of the All-in-Bag for the homogenisation of different food sample matrices for microbiological analysis was compared with BagPage®+ bag. All-in-Bag withstood the shearing action during sample paddling in the bag mixer/stomacher and no significant difference was observed for both aerobic plate count. Spike and recovery of E. coli from the different food matrices indicating absence of interference for microbial recovery in newly developed All-in-Bag. The All-in-Bag, the first of its kind with 12 months shelf life does away with the requirement of sterile diluent preparation and additional steps for the clarification of the homogenate and thus making microbial food quality analysis easier in places with limited resources.

A Comparative Study of Some Procedures for Isolation of Fruit DNA of Sufficient Quality for PCR-Based Assays

Food fraud has been and still is a problem in the food industry. It is detectable by several approaches, such as high performance liquid chromatography (HPLC), chemometric assays, or DNA-based techniques, each with its own drawbacks. This work addresses one major drawback of DNA-based methods, in particular their sensitivity to inhibitors contained in particular matrices from which DNA is isolated. We tested five commercial kits and one in-house method characterized by different ways of sample homogenization and DNA capture and purification. Using these methods, DNA was isolated from 10 different fruit species commonly used in plant-based foodstuffs. The quality of the DNA was evaluated by UV-VIS spectrophotometry. Two types of qPCR assays were used for DNA quality testing: (i) Method specific for plant ITS2 region, (ii) methods specific for individual fruit species. Based mainly on the results of real-time PCR assays, we were able to find two column-based kits and one magnetic carrier-based kit, which consistently provided fruit DNA isolates of sufficient quality for PCR-based assays useful for routine analysis and identification of individual fruit species in food products.

An Observational Study: Is N-Acetylcysteine Helpful in Performance Improvement of Mycoplasma IST2 Testing through Sample Homogenization?

Background. Culture is still the gold standard for the detection of genital mycoplasma which could cause urogenital infections in humans. Mycoplasma IST2 is a commercial kit widely used for the detection of M. hominis and Ureaplasma species. Its accuracy was partially impaired because clinical specimens are usually mixed with purulent or transparent mucus. We aimed to solve this problem through sample homogenization by N-acetylcysteine (NAC) treatment. Methods. Twenty-two endocervical swab samples were collected from 22 female patients with suspected mycoplasma infection, while 11 of these specimens were with purulent or transparent mucus. Mycoplasma IST2 testing kit was used for mycoplasma culture and AST for the control group and NAC-treated group. Results. Genital mycoplasma was detected in 15 of 22 samples for both groups. The colony number in 6 out of 11 purulent specimens (54.5%) was more than 104 CFU/ml of genital mycoplasma for the NAC-treated group, while only one of 11 (9.1%) for the control group. For the nonpurulent specimens, no significant difference had been found in colony counting of genital mycoplasma between the control group and NAC-treated group (P>0.05). The results of antimicrobial susceptibility testing for the NAC-treated group were highly similar to those for the control group. Conclusions. Our results demonstrate that NAC is helpful in sample homogenization and NAC treatment can improve the detection efficiency of mycoplasma with Mycoplasma IST2 testing.

Size of samples and homogenizers during classification of damaged soybeans

ABSTRACT Grain quality determination involves important stages such as collection of the representative sample, homogenization, and dilution. The interrelation among sampling, homogenization, and working sample size is essential to the reliability of the information generated. Therefore, this work aimed to analyse the performance of mechanical homogenizers used in the commercialization of grains in Brazil, as a function of the size of the working sample masses during grain classification. The samples were homogenized and diluted in Boerner, 16:1 multichannel splitter, and 4:1 multichannel splitter until reaching masses of 0.025, 0.050, 0.075, 0.100 and 0.125 kg to determine the level of damaged grains. A 3 x 4 x 5 factorial design was used, meaning three treatments relative to homogenizers (Boerner, 16:1 multichannel splitter, and 4:1 multichannel splitter), four dilutions (4, 8, 12 and 16% damaged grains), and five grain sample sizes (0.025, 0.050, 0.075, 0.100 and 0.125 kg) with nine repetitions. The means were compared by Tukey test and to the original means of prepared samples (4, 8, 12, and 16%) by Student’s t-test. Working samples can be utilized with masses between 0.025 and 0.125 kg to classify damaged soybeans grains. The devices Boerner, 16:1 multichannel splitter, and 4:1 multichannel splitter are similar in the reduction and homogenization of soybean samples for different levels of damaged grains and sample sizes.

A novel method of sample homogenization with the use of a microtome-cryostat apparatus

A novel method of homogenization of elastic tissues does not cause sample heating and material losses.