The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.
Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.
Pseudomonas syringae is a Gram-negative pathogenic bacterium that infects a wide range of plant hosts. Bacteria enter the host plant through the stomata or physical wounds and proliferate in the apoplast1. Plants have evolved a two-tiered immune response to protect against infection by bacterial pathogens. The first level occurs at the plant cell surface, where pattern recognition receptors on the plant cell membrane perceive highly conserved pathogen-associated molecular patterns (PAMPs) in a process called PAMP-triggered immunity (PTI)2. During this process, the host plant upregulates defense response pathways, including deposition of callose to the cell wall, closure of stomata, production of reactive oxygen species, and induction of pathogenesis-related genes.
Bacteria can overcome PTI by utilizing a type III secretion system to deliver proteins, called effectors, directly into the plant cell3. Effector proteins commonly target components of PTI and promote pathogen virulence4. The second tier of plant immunity occurs within the plant cell upon recognition of the effector proteins. This recognition is dependent on resistance genes, which encode nucleotide-binding site leucine-rich repeat containing receptors (NLRs). NLRs are capable of either recognizing effectors directly or recognizing their activity on a virulence target or decoy5. They then trigger a secondary immune response in a process called effector-triggered immunity (ETI), which is often associated with a hypersensitive response (HR), a form of localized cell death at the site of infection6. In contrast to gene-for-gene resistance associated with ETI, plants can exhibit quantitative partial resistance, which is dependent on the contribution of multiple genes7.
P. syringae pv. tomato (Pst) is the causal agent of bacterial speck on tomato and is a persistent agricultural problem. Predominant strains in the field have typically been Pst race 0 strains that express either or both of the type III effectors AvrPto and AvrPtoB. DC3000 (PstDC3000) is a representative race 0 strain and a model pathogen that can cause bacterial speck in tomato. To combat bacterial speck disease, breeders introgressed the Pto [P. syringae pv. tomato]/Prf [Pto resistance and fenthion sensitivity] gene cluster from the wild tomato species Solanum pimpinellifolium into modern cultivars8,9. The Pto gene encodes a serine-threonine protein kinase that, together with the Prf NLR, confer resistance to PstDC3000 via recognition of the effectors AvrPto and AvrPtoB10,11,12,13,14. However, this resistance is ineffective against emerging race 1 strains, allowing for their rapid and aggressive spread in recent years15,16. Race 1 strains evade recognition by the Pto/Prf cluster, because AvrPto is either lost or mutated in these strains, and AvrPtoB appears to accumulate minimally15,17,18.
Wild tomato populations are important reservoirs of natural variation for Pst resistance and have previously been used to identify potential resistance loci19,20,21. However, current screens for pathogen resistance utilize 4–5-week-old adult plants20,21. Therefore, they are limited by growth time, growth chamber space, and relatively small sample sizes. To address the limitations of conventional approaches, we developed a high-throughput tomato P. syringae resistance assay using 10-day-old tomato seedlings22. This approach offers several advantages over using adult plants: namely, shorter growth time, reduced space requirements, and higher throughput. Furthermore, we have demonstrated that this approach faithfully recapitulates disease resistance phenotypes observed in adult plants22.
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7–14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain 19 (Pst19), and can easily be adapted to other bacterial strains of interest.
1. Preparation and use of biosafety cabinet
2. Preparation of plant media
3. Preparation of plant materials and growth conditions
Figure 1: Developmental stage of typical 10-day-old tomato seedlings. Rio Grande-PtoR tomato seeds were sterilized, plated, and stratified for at least 3 days in the dark at 4 ˚C. The seedlings were grown on 0.5x MS plates for 10 days at 22 ˚C before being flooded. Typically, at 10 days the cotyledons are fully expanded, and the first true leaves are beginning to emerge. Please click here to view a larger version of this figure.
4. Preparation of King's B23 (KB) media
5. Maintenance of bacterial strains and culture conditions
6. Preparation of Pst19 inoculum
7. Preparation of PstDC3000 inoculum
8. Tomato seedling flood method
9. Surface sterilization of cotyledons for bacterial growth assay
10. Bacterial growth assay
Figure 2: Serial dilutions for seedling bacterial growth assays. (A) Macerated leaf tissue from infected plants is diluted prior to colony counting. Dilutions are performed in a 96 well plate (100 is undiluted). Typically, dilutions are made from 10-1 to 10-5. (B) Plating dilutions for bacterial colony counts. A total of 5 µL of each column of the dilution series is plated, from most dilute to most concentrated. After the colonies have fully dried, the plate is incubated at 28 ˚C for 36–48 h. Colonies are counted under a 10x dissecting microscope. Please click here to view a larger version of this figure.
Genotype1 Column A | Tissue Weight (g) Column B | # of Colonies in a spot Column C | Dilution factor for spot2 Column D | Adjusted # of Colonies3 Column E | Dilution factor for serial dilution Column F | Total # of Colonies Column G (cfu/0.01 g)4 | Average # of Colonies (cfu/0.01 g) Column H | Average Log Growth (cfu/0.1 g (log10)) Column I |
Sample 1 | 0.04 g | 10 | 200 | calculated as: (C2 x 0.1 g) / B2 = 25 | 1000 | calculated as: (D2 x E2 x F2) = 5000000 | average for sample 1 through last sample: (ie. average G1:G3) = 7000000 | log of average ie. log(H2) = 6.85 |
Sample 2 | 0.03 g | 15 | 200 | 50 | 1000 | 10000000 | ||
Sample 3 | 0.02 g | 6 | 200 | 30 | 1000 | 6000000 | ||
1Data shown for 3 samples | ||||||||
2Based on plating 5 µL x 200 for 1 mL | ||||||||
3Cotyledons are too small to core so colony counts were normalized to 0.1 g of tissue based on the average mass of one MoneyMaker-PtoS cotyledon (data not shown) | ||||||||
4Adjusted per mL based on volume plated |
Table 1: Sample calculations for seedling bacterial growth assay. Sample calculations demonstrate how to normalize bacterial counts and determine log bacterial growth.
11. Phenotyping for resistance
Figure 3: Schematic representation of a tomato seedling. Different parts of a tomato seedling are depicted, including the hypocotyl, cotyledons, epicotyl, shoot apical meristem, and true leaves. Please click here to view a larger version of this figure.
Figure 4: Schematic representation of expected phenotypes for seedling resistance and death in various genetic backgrounds. (A) Seedlings of Rio Grande-PtoR and the near-isogenic cultivar Rio Grande-PtoS are displayed 7 days after flooding with PstDC3000 (OD600 = 0.005) + 0.015% surfactant. Rio Grande-PtoR displays consistent resistance, and Rio Grande-PtoS displays consistent susceptibility to infection with PstDC3000. These lines give rise to discrete and binary phenotypes. (B) Seedlings of a wild accession, such as Solanum neorickii LA1329, are shown 10 days after flooding with Pst19 (OD600 = 0.0075) + 0.015% surfactant. Seedlings display phenotypic variability but were recorded as binary phenotypes. The amount of phenotypic variability and the method of phenotyping (binary resistance or resistance spectrum) will depend on the particular accession tested. (C) Mapping populations generated by outcrossing wild accessions to susceptible cultivars may display a wider spectrum of phenotypes in F2 segregating populations. In this case, it may be most appropriate to record seedling phenotypes on a spectrum. Highly susceptible seedlings from a mapping population may be phenotyped for death as early as day 7 when flooded with Pst19, and typically show a brown apical meristem, no to very little extension of the epicotyl, and no new, green vegetative growth. The apical meristem of susceptible seedlings may stay green or very light brown for more time, and there may be some extension of the epicotyl and very little vegetative growth, which turns brown and arrests by day 10. Individual seedlings can be phenotyped for resistance based on the amount of new and ongoing vegetative growth by day 14. Seedlings can then be grouped based on the phenotypes described above into different categories of resistance such as weak, medium, or strong resistance. Please click here to view a larger version of this figure.
Detection of PtoR-mediated immunity in cultivars and isogenic lines using the seedling resistance assay
Figure 5 shows representative results for Moneymaker-PtoR and Moneymaker-PtoS cultivars 7–10 days after flooding with PstDC3000. Prior to infection, 10-day-old seedlings displayed fully emerged and expanded cotyledons and emerging first true leaves. The seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control (data not shown) and PstDC3000 at an optical density of 0.005 + 0.015% surfactant. The seedlings were phenotyped 7–10 days after flooding (Figure 5). Individual seedlings from genotypically homogenous lines, such as Moneymaker-PtoR and Moneymaker-PtoS give highly consistent and binary phenotypes in the seedling flood assay. When Moneymaker-PtoR, which carries the Pto/Prf gene cluster (n = 5), was treated with PstDC3000 at the optimal concentration of OD600 = 0.005, resistance due to PtoR-mediated immunity was strong and was typified by new, green vegetative growth in all individuals22. Near-isogenic Moneymaker-PtoS seedlings (n = 5), which cannot recognize the PstDC3000 effectors AvrPto or AvrPtoB, died quickly within 7 days after flooding and characteristically had brown apical meristems, bacterial speck, chlorosis, and no signs of new, green vegetative growth (Figure 5).
Figure 5: Phenotypic characterization of resistance or disease symptoms 7–10 days post-infection in a cultivar. Moneymaker-PtoR and Moneymaker-PtoS tomato seedlings were grown on 0.5x MS plates for 10 days before being flooded with P. syringae pv. Tomato DC3000 (OD600 = 0.005) + 0.015% surfactant. Moneymaker-PtoR seedlings survived (n = 5) and Moneymaker-PtoS seedlings (n = 5) died. The number of surviving seedlings for each genotype out of the total number tested is shown. Scale bar = 1 cm. Please click here to view a larger version of this figure.
Phenotypic screening of wild accessions using the seedling resistance assay
Figure 6 shows representative results for seedlings of susceptible and resistant accessions 10–14 days after flooding with Pst19. Susceptible accessions include RG-PtoR, S. pimpinellifolium LA1375, and S. pimpinellifolium LA1606, and resistant accessions include S. neorickii LA1329. Ten-day-old seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control, and Pst19 at an optical density of 0.0075 + 0.015% surfactant. The seedlings were phenotyped at least 10 days after flooding, as Pst19-infected seedlings died more slowly than PstDC3000-infected seedlings. Mock-inoculated seedlings were green, healthy, and actively growing. This control is important to ensure that the accessions are not sensitive to the concentration of surfactant, and to ensure there is no bacterial contamination. Susceptible accessions (Rio Grande-PtoR [n = 7], S. pimpinellifolium LA1375 [n = 7], and S. pimpinellifolium LA1606 [n = 5]) were dead, had brown apical meristems, and lacked new growth 10–14 days after inoculation with Pst19. In contrast, two S. neorickii LA1329 (n = 3) seedlings displayed a high level of new, green growth and survived infection with Pst19 (Figure 6). Three LA1329 seedlings did not germinate. Typically, 5–7 individuals were screened for each accession in a primary screen to determine the prevalence of resistance in the population. When a more genetically complex wild accession, such as LA1329, is flooded with Pst19, the resistance phenotypes display slightly more variability among individual seedlings, compared to Moneymaker-PtoR treated with PstDC3000. However, the resistance phenotypes were usually less variable than those seen in F2 mapping populations. Thus, binary phenotyping criteria was used for LA1329.
Figure 6: Phenotypic characterization of resistance or disease symptoms 10–14 days post-infection in wild accessions. Rio Grande-PtoR, S. pimpinellifolium LA1606, S. pimpinellifolium LA1375 and S. neorickii LA1329 tomato seedlings were grown on 0.5x MS plates for 10 days, and then flooded with Pst19 (OD600 = 0.0075) + 0.015% surfactant. The number of surviving seedlings for each wild accession out of the total number tested is shown. Scale bar = 1 cm. Please click here to view a larger version of this figure.
Quantitative assessment of bacterial growth using the seedling flood assay
To confirm that the observed resistance in LA1329 to Pst19 resulted in lower bacterial growth, bacterial growth assays were carried out in tomato seedlings. The level of Pst19 growth in Moneymaker-PtoS and S. neorickii LA1329 was determined 4 days post-infection. Moneymaker-PtoS is a near-isogenic line with consistent susceptibility among individual seedlings. Wild accessions such as S. neorickii LA1329 are often more genetically complex. LA1329 displays approximately 60% resistance to Pst19 across the population22. Because seedlings may drop their cotyledons after infection, one seedling was grown on each plate to correlate bacterial growth in the harvested cotyledon with overall seedling survival or death as determined phenotypically at least 10 days after flooding. The bacterial counts on day 4 for each seedling were normalized to 0.01 g of tissue and converted to log growth (CFU/0.01 g(log10)). Log growth for phenotypically resistant LA1329 seedlings (LA1329RES) or phenotypically susceptible seedlings (LA1329SUS) were separately pooled and compared to each other and the susceptible cultivar Moneymaker-PtoS. For example, there was a 1.7 log difference in bacterial growth between LA1329RES (log 6.3) and LA1329SUS (log 8.0), and a 1.6 log difference between LA1329RES (log 6.3) and Moneymaker-PtoS (log 7.9) (Figure 7). Therefore, phenotypic resistance correlated with quantitative resistance in the seedling assays.
Figure 7: Resistant Solanum neorickii LA1329 seedlings support lower bacterial growth than Moneymaker-PtoS or susceptible S. neorickii LA1329. Bacterial counts were determined 4 days post-inoculation from S. neorickii LA1329 (n = 14) and Moneymaker-PtoS (n = 10) seedlings infected with Pst19 and normalization was performed to 0.1 g of tissue. For LA1329, the two phenotypic groups, susceptible (SUS) or resistant (RES), were observed and counted separately. Above the bar * = statistically significant difference determined by a one-factor analysis of variance. A general linear model procedure (p < 0.001) followed by a multiple comparison of means using Tukey's post hoc test was used. Error bars = standard error. The figure indicates one representative experiment. Please click here to view a larger version of this figure.
A protocol for flood inoculation with PstDC3000 or Pst19 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf cluster and complete death on a susceptible cultivar lacking the Pto/Prf cluster22. For a strain such as Pst19, where there are no known resistant varieties, the optical density was optimized to be the lowest possible for consistent and complete plant death22. Uppalapati et al.24 designed a tomato seedling assay to investigate the pathogenesis of PstDC3000 and the virulence function of coronatine. In this virulence assay, infections were performed using bacteria concentrated to an OD600 of 0.124, 20x higher than the optical density of strains used in our resistance assay. Recognition of PstDC3000 effectors AvrPto and AvrPtoB in tomato seedlings carrying the Pto/Prf gene cluster results in ETI and a macroscopic HR22. In the context of a strong immune response such as ETI, a lower bacterial titer was used for PstDC3000 to avoid overwhelming genetic resistance from the Pto/Prf gene cluster22. In addition, these results suggest that a high bacterial concentration could overwhelm weaker immune responses such as PTI or quantitative partial resistance, where multiple genes contribute to the overall phenotype. Surfactant is necessary for the bacteria to adhere to the leaf surface; however, high concentrations can cause chlorosis of the leaf22. We previously tested a range of surfactant concentrations to empirically determine the ideal concentration in 10-day-old tomato seedlings22. When testing new species that may differ in their sensitivity to surfactant, the surfactant concentration should be optimized to identify a concentration that does not cause damage or chlorosis in the absence of bacteria. Appropriate assay conditions will require optimization of a surfactant concentration that does not cause damage, and a bacterial concentration that causes disease in all susceptible controls.
Additional critical parameters for success in the seedling flood assay include using seedlings at specific developmental stages (10-day-old seedlings) (Figure 1), maintaining stable growth chamber conditions (light intensity of about 200 µE m-2 s-1, constant temperature of 22 ˚C, 16 h of light) and performing experiments in a sterile biosafety cabinet. Media volume above 45 mL or below 35 mL may affect consistent death of susceptible controls, because the volume may impact the surrounding microenvironment of the seedlings on the plate. For example, differences in relative humidity inside the sealed plates could affect the infectivity of the bacteria and the ability of the plants to survive infection. Sterile technique is critical, because contamination on the plates may confound the source of death or susceptibility in seedlings. In addition, because plant-pathogen interactions are affected by the circadian clock24,25,26, it is recommended that the plants be infected at a consistent time of the day.
Pst is a foliar pathogen that preferentially colonizes the aerial parts of tomato seedlings, including the cotyledons24 (Figure 3). Therefore, qualitative phenotyping in the seedling flood assay focuses on growth and disease symptoms in aerial portions of the seedling, and tissue for the bacterial growth assay is sampled from the cotyledons for quantitative analysis. After flood inoculation, seedlings may die within 7–10 days after inoculation with PstDC3000 or 10–14 days after inoculation with Pst19, as discussed in section 11. Seedling death is visualized by a brown apical meristem, arrested epicotyl elongation, and/or arrested vegetative growth. If different bacterial strains are used, the timing will have to be empirically determined. In addition, the progression of disease on control plants should be monitored daily after flooding until a consistent time frame from the onset of disease symptoms to seedling death can be identified. Depending on the genotypes and treatments used in the flood assay, seedling phenotypes can be recorded as binary phenotypes or on a disease spectrum (Figure 4). A broader spectrum of phenotypes may be observed when flood inoculating F2 mapping populations from wild tomato accessions crossed to susceptible cultivars (Figure 4C). It may be best to phenotype segregating populations on a disease spectrum depending on how quickly the seedling dies and the degree of new vegetative growth and branching (Figure 4C). The seedling flood assay can also be used in conjunction with the seedling bacterial growth assay to quantitatively assess levels of bacterial growth associated with qualitative phenotypes in individual seedlings (Figure 7). Very large reductions (i.e., ~log 3) in bacterial growth or strong resistance in resistant seedlings of a wild accession compared to a susceptible cultivar suggest that the underlying genetic basis of resistance may be due to ETI22. Smaller reductions in bacterial growth (i.e., ~log 1.7), as observed in LA1329 seedlings, may be due to the contribution of weaker resistance from quantitative trait loci and/or PTI. Thus, the seedling growth assay can be an important tool in further characterizing resistance in wild tomato lines.
Typically, genetic screens have been performed on four- to five-week-old adult tomato plants to identify the genetic basis of P. syringae resistance in wild accessions20,21. Adult tomato plants require much longer growth times, require more space in the growth chamber, and are much larger plants, which means that usually few individuals are screened for each line. The seedling flood assay provides a powerful, alternative approach in the identification of P. syringae resistance in wild tomato accessions. Screening at the seedling stage permits a large sample size to be tested which can be particularly advantageous in detecting resistance in genetically complex populations. Reduced growth chamber space requirements and growth time facilitate a high-throughput approach and rapid detection of natural resistance in wild accessions to emerging pathogens. Furthermore, P. syringae resistance that was identified at the seedling stage in this assay is not restricted to the developmental stage. S. neorickii LA1329 and S. habrochaites LA1253 were initially identified at the seedling stage and also display resistance to Pst19 in adult plants as previously described22.
The seedling flood assay is a versatile protocol that can be modified and optimized to detect host resistance to other P. syringae strains. It could potentially be further applied in the context of different bacterial pathogens of tomato, such as the Xanthomonas species. This method will expedite the search for new sources of disease resistance to bacterial pathogens.
The authors have nothing to disclose.
We thank Jamie Calma for testing the effect of media volume on disease or resistance outcomes. We thank Dr. Maël Baudin and Dr. Karl J. Scheiber from the Lewis Lab for providing constructive comments and suggestions on the manuscript. Research on plant immunity in the Lewis laboratory was supported by the USDA ARS 2030-21000-046-00D and 2030-21000-050-00D (JDL), and the NSF Directorate for Biological Sciences IOS-1557661 (JDL).
3M Tape Micropore 1/2" x 10 YD CS 240 (1.25 cm x 9.1 m) | VWR International | 56222-182 | |
3mm borosilicate glass beads | Friedrich & Dimmock | GB3000B | |
Bacto peptone | BD | 211677 | |
Bacto agar | BD | 214010 | |
Biophotometer Plus | Eppendorf | E952000006 | |
Biosafety cabinet, class II type A2 | |||
BRAND Disposable Plastic Cuvettes, Polystyrene | VWR International | 47744-642 | |
Chenille Kraft Flat Wood Toothpicks | VWR International | 500029-808 | |
cycloheximide | Research Products International | C81040-5.0 | |
Dibasic potassium phosphate anhydrous, ACS grade | Fisher Scientific | P288-500 | |
Dimethylformamide | |||
Dissecting microscope (Magnification of at least 10x) | |||
Ethanol – 190 Proof | |||
Falcon polystyrene 96 well microplates, flat-bottom | Fisher Scientific | 08-772-3 | |
Glass Alcohol Burner Wick | Fisher Scientific | S41898A / No. W-125 | |
Glass Alcohol Burners | Fisher Scientific | S41898 / No. BO125 | |
Glycerol ACS reagent | VWR International | EMGX0185-5 | |
Kimberly-Clark™ Kimtech Science™ Kimwipes™ Delicate Task Wipers | Fisher Scientific | 06-666-A | |
Magnesium chloride, ACS grade | VWR International | 97061-356 | |
Magnesium sulfate heptahydrate, ACS grade | VWR International | 97062-130 | |
Microcentrifuge tubes, 1.5 mL | |||
Microcentrifuge tubes, 2.2 mL | |||
Mini Beadbeater-96, 115 volt | Bio Spec Products Inc. | 1001 | |
Murashige & Skoog, Basal Salts | Caisson Laboratories, Inc. | MSP01-50LT | |
Pipet-Lite XLS LTS 8-CH Pipet 20-200uL | Rainin | L8-200XLS | |
Pipet-Lite XLS LTS 8-CH Pipet 2-20uL | Rainin | L8-20XLS | |
Polystyrene 100mm x 25mm sterile petri dish | VWR International | 89107-632 | |
Polystyrene 150mm x 15mm sterile petri dish | Fisher Scientific | FB08-757-14 | |
Polystyrene 150x15mm sterile petri dish | Fisher Scientific | 08-757-148 | |
Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach) | Staples | 1013131 | |
Rifampicin | Gold Biotechnology | R-120-25 | |
Silwet L-77 (non-ionic organosilicone surfactant co-polymer C13H34O4Si3 surfactant) | Fisher Scientific | NCO138454 | |
Tips LTS 20 μL 960/10 GPS-L10 | Rainin | 17005091 | |
Tips LTS 250 μL 960/10 GPS-L250 | Rainin | 17005093 | |
VWR dissecting forceps fine tip, 4.5" | VWR International | 82027-386 |