MINI-BAERMANN FUNNEL, A SIMPLE DEVICE FOR CLEANING NEMATODE INFECTIVE LARVAE

Abstract

The Baermann filter method is a long-standing, simple technique for recovering nematodes from soil and charcoal coprocultures. Material containing the nematodes is placed on a mesh screen lined with several layers of tissue paper or cheesecloth, and the screen is placed in the mouth of the funnel. Rubber tubing attached to the funnel stem is clamped, and water is added to submerge the material. The filtration material allows the nematodes to swim through while holding back the substrate. Over time the nematodes settle at the clamp in the tubing. After several hours, the clamp is opened and water containing the nematodes is collected. Although recovery of the nematodes is efficient, they are often contaminated with soil or charcoal debris, requiring a secondary cleaning by sedimentation or filtration. Described here is a small, simplified version of the Baermann apparatus that can be used as a secondary cleaning device. The “mini-Baermann” is constructed from materials commonly found in the laboratory. Experiments using infective larvae of 3 nematode species demonstrated that the majority of the larvae applied to the device are collected within 2 hr, and nearly all by 4 hr. Dead larvae fail to pass through the filter and do not significantly impact the passage of living larvae. In addition to removing debris from nematode suspensions, this device can rapidly and efficiently separate living, motile larvae from dead larvae.

The nematode filtering funnel invented in 1917 by Baermann for the isolation of nematodes from the soil is 1 of the most important tools of the nematologist (Baermann, 1917). Since its invention, the Baermann apparatus has undergone a series of minor modifications for specific uses that have enabled its widespread adoption (Cort et al., 1922; Dinaburg, 1942; Beane and Hobbs, 1983). The basic Baermann filter apparatus is composed of a funnel, a rubber tube, a clamp, a 1-mm metal mesh lined sieve, and a piece of filtering material such as cheesecloth or tissue paper. The sieve is lined with filtering material and placed in the funnel. The rubber tubing is attached to the funnel stem and clamped. The material containing the nematodes is added to the sieve, and the funnel is filled with water to submerge the material partially. Active nematodes pass through the filter sieve, leaving the material behind, and settle at the pinched end of the rubber tubing. The nematodes can be harvested by releasing the clamp and collecting the water carrying the clean nematodes into a tube (Dinaburg, 1942).

Though introduced over 100 yr ago, the Baermann apparatus continues to be widely used in parasitology research because of its simplicity, ease of use, low cost, and effectiveness for many nematode species (Cesarz et al., 2019). In addition to being used to isolate nematodes from soil, environmental, and tissue samples, the Baermann technique remains the standard method to recover infective nematode larvae from charcoal coprocultures in the lab. However, recovered nematode larvae are often contaminated by charcoal and fecal debris and require a secondary cleaning procedure such as gradient flotation or pooling with centrifugation, which often only marginally improves the cleanliness of the larvae, before use in vitro or for isolation of nucleic acids. Herein, we describe a simple modification of the Baermann apparatus for secondary cleaning and separation of living from dead larvae of several parasitic nematode species. These “mini-Baermann filters” are easy to assemble from common lab materials, rapid, highly efficient, and yield larvae that are mostly free of culture debris.

Heterorhabditis bacteriophora (strain TT01) was originally provided by Dr. David Clarke (University College, Cork, Ireland), and Steinernema hermaphroditum was a gift from Paul Sternberg (California Institute of Technology, Pasadena, California) (Cao et al., 2021). Both entomopathogens were maintained by infecting Galleria mellonella larvae with 50–100 infective juveniles (IJ) (Lindegren et al., 1993). Emerging IJs were recovered from White traps (White, 1927) on days 10–14 postinfection in 0.01% Tween (H. bacteriophora) or water (S. hermaphroditum), and were stored in flasks at 22 C for up to 4 wk. The A. caninum western Maryland (WMD) isolate was maintained in dogs as described previously (Kitchen et al., 2019). Infective third-stage larvae (L3) were collected from coprocultures using the standard Baermann apparatus and stored in buffer (BU) (Hawdon and Schad, 1991) until needed. Experiments involving vertebrate animals were conducted in strict accordance with the recommendations of the National Institute of Health (USA) Guide for the Care and Use of Laboratory Animals and the United States Department of Agriculture Animal Welfare Act. The animal protocols used in this study (A270, A2021-017) were approved by the Institutional Animal Care and Use Committee of The George Washington University (USA).

The mini-Baermann filter apparatus is shown in Figure 1. To construct the filter, a 15-ml centrifuge was cut at the 10-ml line, and the end containing the taper was discarded. A hole the size of the diameter of the 15-ml tube was cut in the cap of a 50-ml centrifuge tube and the top portion of the cut 15-ml tube was inserted through the hole. A folded Kimwipe tissue paper was wrapped over the cut end of the tube and held in place with a rubber band. Excess tissue paper was trimmed, leaving a neat wrap around the tube. The cap with the protruding filter apparatus was placed into the 50-ml centrifuge tube containing approximately 45 ml of BU or water, depending on the type of nematodes being filtered. The wrapped end of the tube was submerged in at least 1 cm of the liquid, and a solution of larvae to be filtered was added to the 15-ml tube. The apparatus was allowed to stand for the desired incubation time. As the larvae settled, they migrated through the tissue paper, and debris and dead larvae were retained. After the appropriate time, the apparatus was disassembled, and the supernatant aspirated to the 5-ml line. Cleaned nematodes were recovered from the bottom of the 50-ml tube using a pipette (Fig. 2).

Figure 1.

Construction of a mini-Baermann filter apparatus. (A) A 15-ml centrifuge tube is cut at the 10-ml line, and the larger section is discarded. (B) A Kimwipe lab tissue folded into 3 layers is wrapped around the cut end of the short section of the cut tube and secured with a rubber band. Excess paper is trimmed with scissors. (C) A hole of approximately the same diameter as the tube is cut in the cap of a 50-ml centrifuge tube. (D) The 50-ml tube is filled with the desired medium, and the cut tube is pushed through the hole in the cap until the end covered with the Kimwip is submerged. Nematodes to be filtered are added to the 15-ml tube. (E) After the desired incubation period, worms that have migrated through the tissue can be collected from the bottom of the 50-ml tube. The figure was created using Biorender.com.

Figure 2.

Removal of debris by the mini-Baermann apparatus. (A) Centrifuge tube containing Ancylostoma caninum L3 after initial collection and before filtration using the mini-Baermann apparatus. Note the black charcoal debris from the coprocultures at the bottom of the tube. (B) The same L3 following filtration with the apparatus. Note the absence of charcoal contaminants.

To determine the efficiency and rate of filtration, larval recovery at 2-, 4- and 24-hr incubation was measured. Approximately 5,000 nematode larvae in a volume of 500 μl were added to the device. After 2 hr, the device was moved to a second 50-ml tube and incubated for an additional 2 hr (denoted as the 4-hr incubation period), after which the device was moved to a third 50-ml tube and incubated overnight. At the 24-hr time point, the supernatants were aspirated, and the recovered larvae were counted by dilution count. Each experiment was repeated a minimum of 3 times for each species. Means and standard deviations were determined in Microsoft Excel and tested for significance by 1-way analysis of variance with Tukey's multiple comparison test using Prism 9.3 (GraphPad Software, 2021).

As shown in Figure 3 and Table I, an average of 82 ± 17% of the 5,000 H. bacteriophora IJs passed through the filter within the first 2 hr, with nearly all of the remaining IJs from the initial inoculum recovered by 4 hr. Only 3 ± 3% of the initial inoculum was recovered by extending the incubation to 24 hr. Total recovery of the IJs varied from approximately 86% to 115%, indicating variability associated with dilution counting of the larvae added to the apparatus. To account for this, recoveries were also determined based on the total number of IJs recovered, which yielded similar results (not shown). Similar recoveries were seen with the other entomopathogenic nematode, S. hermaphroditum, as approximately 82 ± 16% of the starting number (79 ± 8% of the total recovered) were recovered in the first 2 hr incubation. However, although the majority (83 ± 5.7%) of A. caninum L3 were recovered at 2 hr, the total number of larvae recovered at 24 hr was only 79% of the starting number.

Figure 3.

Recovery of nematode larvae from mini-Baermann apparatus. The apparatus was moved to a new collection tube after each incubation period. The mean ± standard deviation of 3–5 trials is shown. (A) The mean number of Heterorhabditus bacteriophora infective juveniles (IJ) recovered after the addition of approximately 5,000 IJ. (B) The mean number of living H. bacteriophora IJ recovered after the addition of 2,500 living and 2,500 heat-killed IJ. (C) The mean number of living H. bacteriophora IJ after the addition of 5,000 heat-killed IJ. (D) The mean number of Steinernema hermaphroditum IJ recovered after the addition of 5,000 IJ. (E) The mean number of Ancylostoma caninum infective third-stage larvae (L3) recovered after the addition of 5,000 L3. (F) Total recovery of H. bacteriophora (Hba), S. hermaphroditum (She), and A. caninum (Aca) after 24 hr following the addition of approximately 5,000 nematodes to the apparatus. Means were tested for significance by 1-way ANOVA with Tukey's multiple comparison test using Prism 9.3 (GraphPad Software, San Diego, California). Asterisks indicate level of significance: ***P < 0.001, ****P <0.0001, ns, nonsignificant.

Table I.

Recovery of infective nematode larvae of Heterorhabditis bacteriophora, Steinernema hermaphroditum, and Ancylostoma caninum following filtration using the mini-Baermann filter apparatus.

Species	No. of larvae applied	Mean % ± SD recovery of living larvae applied
		0–2 hr	2–4 hr	4–24 hr	Total
H. bacteriophora*	5,000 living	82 ± 17	15 ± 9	3 ± 3	100
	2,500 living/2500 dead	79 ± 15	10 ± 5	3 ± 2	92 ± 14
	5,000 dead	0	0	<1	0
S. hermaphroditum†	5,000 living	82 ± 16	19 ± 6	2 ± 3	103
A. caninum†	5,000 living	65 ± 11	10 ± 6	4 ± 2	79

^*Mean ± standard deviation of 5 trials.

^†Mean ± standard deviation of 3 trials.

Next, we tested whether the filter apparatus could be used to separate living, motile larvae from dead larvae. As shown in Figure 2 and Table I, fewer than 1% of 5,000 heat-killed H. bacteriophora IJs were recovered after 24 hr. Dead larvae (68 total) were only recovered in 1 trial, and only after 24 hr incubation, suggesting that the tissue paper was compromised because of the long incubation. This indicates that only motile larvae can traverse the intact tissue paper. To determine whether dead larvae interfered with the passage of living larvae, 2,500 heat-killed and 2,500 living H. bacteriophora were added to the device, and the recovery was determined at 2-, 4-, and 24-hr as above. In 5 trials, total recovery at 24 hr ranged from 76 to 112% (mean 92 ± 14%) of the 2,500 living IJs added to the device. As in the experiments using only living IJs, the majority (79 ± 15%) of the larvae were recovered in 2 hr, with most of the remaining IJs recovered by 4 hr (10 ± 5%).

The mini-Baermann filter apparatus described herein is a simple, efficient means of separating nematode larvae from debris and dead larvae. The device can be made quickly and inexpensively from common lab materials and is easy to use and clean. Multiple devices can be assembled for use with different species. The vast majority of the larvae added can be recovered by 2 hr, and essentially 100% recovered by 4 hr. This provides a rapid way to recover only living larvae for infections or experiments. The recovery kinetics and efficiencies are similar among the 3 species tested, although the total recovery of hookworm larvae (A. caninum) is slightly lower than that of the entomopathogens studied. This may be a result of the tests being conducted at room temperature, which is closer to the optimal temperatures of the entomopathogens than hookworms. Increasing the incubation temperature to one closer to that of their host might increase hookworm larval recovery by increasing their motility. In summary, the mini-Baermann apparatus quickly separates living larvae from debris and dead larvae and yields a cleaner sample of larvae for experiments and infections.

The authors assert all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The authors would like to thank Sterling Cherise, Indea Rogers, and Julia Zhu for parasite maintenance. This work was supported by the National Institutes of Health grant 5R21AI137771 and the George Washington University Cross-Disciplinary Research Fund. The sponsors had no role in the study design, collection, analysis, or interpretation of data, writing the manuscript, or the decision to submit the manuscript for publication.