An Optimized Protocol for Candida albicans Infection in Schmidtea mediterranea to Study Fungal Pathogenesis and Host Defense

Summary

This protocol provides an optimized, detailed guide for using the planarian Schmidtea mediterranea as a model system to study host-pathogen interactions during fungal infection. The method builds on the previous procedure for infecting planarians with the human fungal pathogen Candida albicans, providing detailed guidance to enhance reproducibility and experimental consistency.

Abstract

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes most humans. Although typically a benign commensal, dysbiosis caused by antibiotic use, immune dysfunction, or epithelial barrier disruption can trigger fungal overgrowth and infection, ranging from superficial mucosal disease to life-threatening systemic candidiasis. New preclinical infection models are needed to dissect C. albicans pathogenesis in vivo across distinct infection stages and with different measurable host outcomes. The planarian Schmidtea mediterranea was previously established as an invertebrate host for studying host-pathogen interactions during C. albicans infection. S. mediterranea relies entirely on conserved innate immune mechanisms capable of overcoming infection with pathogenic microorganisms, including bacteria and fungi. Planarians’ remarkable regenerative capacity and accessible stem cell populations make this organism a tractable model to analyze early immune responses, tissue repair, and pathogen clearance in vivo. This model supports simultaneous analysis of fungal virulence and host transcriptional responses, providing valuable insights into infection dynamics. Here, an updated protocol with detailed modifications, standardized procedures, and optimized steps for infecting S. mediterranea with C. albicans has been presented, designed to enhance reproducibility and enable systematic studies of fungal pathogenesis and host defense.

Introduction

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes 50-70% of humans^1,2,3. Typically, a harmless commensal, C. albicans can overgrow under conditions such as microbiota imbalance, antibiotic use, immunosuppression, epithelial barrier disruption, or the presence of medical devices, leading to diseases that range from superficial mucosal infections to life-threatening systemic candidiasis^4,5. In immunocompromised individuals – including those with HIV/AIDS, patients undergoing chemotherapy or immunosuppressive therapies, individuals with chronic illnesses, or those in intensive care units – mortality rates can exceed 70%, underscoring the significant public health burden of invasive candidiasis^6,7,8. Globally, invasive fungal infections affect more than 6.5 million people annually, with direct healthcare costs in the United States alone exceeding $8 billion per year for Candida-related infections^9,10. Despite available antifungal treatments, morbidity and mortality rates associated with C. albicans infections have remained largely unchanged for decades, highlighting the urgent need to better understand host-pathogen interactions in fungal disease.

C. albicans employs a diverse repertoire of virulence factors that support colonization, persistence, and pathogenesis across a range of host environments. These include adhesion to host tissues, immune evasion, cell-wall remodeling, secretion of hydrolytic enzymes, morphological plasticity, and biofilm formation^{11,12,13,14,15,16}. Collectively, these traits allow C. albicans to adapt to diverse host niches, modulate immune responses, and resist antifungal therapies^{16,17,18,19,20,21}. Elucidating how these processes shape infection outcomes – including host morbidity, mortality, and pathogen clearance – is essential for developing more effective preventive and therapeutic strategies.

To investigate these host-pathogen dynamics, we established a model system using the planarian Schmidtea mediterranea as a host^22,23,24. This model serves as a versatile platform for studying the multisystem host response to fungal infection, leveraging a soaking-based infection method that enables synchronous exposure of animals to C. albicans. The following section presents updated step-by-step procedures that refine this protocol, improve reproducibility, and facilitate consistent implementation across laboratories.

Planarians are free-living invertebrates with exceptional regenerative capacity, capable of rapidly replacing tissues lost to injury or infection²⁵. They lack an adaptive immune system and rely entirely on conserved innate defenses – including pattern recognition receptors, antimicrobial peptides, mucus secretion, and phagocytic cells – to clear bacterial and fungal pathogens within days^26,27,28,29. Because innate immunity represents the first line of defense across eukaryotes and remains understudied outside mammalian systems²⁷, planarians offer an opportunity to examine these processes in vivo with cellular and molecular resolution. Their regenerative ability is driven by adult pluripotent stem cells, known as neoblasts, which constitute approximately 30% of adult cells and contribute to recovery after infection^23,30. These features make S. mediterranea a powerful model for dissecting pathogen virulence and host defense at genetic, cellular, tissue, and organismal scales, enabling us to address questions that are often difficult to study using traditional mammalian systems.

In addition, planarians’ small size (typically a few millimeters in length), low cost, and ease of maintenance³¹ support large-scale experiments while avoiding the financial, ethical, and regulatory constraints inherent to vertebrate models. Like C. albicans, S. mediterranea is genomically tractable, with a fully sequenced and annotated genome, and it supports advanced molecular and cellular techniques – including transcriptional profiling, high-resolution immunohistochemistry and histology, and robust RNA interference (RNAi)^{32,33,34,35,36,37,38}. These tools enable parallel analysis of host and pathogen responses during infection.

Several alternative preclinical models have been used to study fungal infections. Invertebrate hosts such as Galleria mellonella (wax moth), Caenorhabditis elegans (roundworm), and Drosophila melanogaster (fruit fly), as well as the vertebrate Danio rerio (zebrafish), each provides distinct experimental advantages but also has notable limitations. These systems often rely on survival as the primary endpoint because infections rapidly induce host death, preventing detailed assessment of morbidity, recovery, and multisystem responses – features that the planarian model readily captures.

Mammalian models such as Mus musculus (mouse), Rattus norvegicus (rat), Oryctolagus cuniculus (rabbit), and Cavia porcellus (guinea pig) more closely recapitulate human physiology but are limited by high costs, small cohort sizes, ethical and regulatory requirements, and the challenge of distinguishing innate from adaptive immune responses. Moreover, decades of fungal pathogenesis research in mice have focused predominantly on late-stage systemic infection^{39,40,41,42,43}, often overlooking early events such as epithelial barrier disruption and mucosal overgrowth – the most common routes of fungal disease initiation in humans^44,45,46.

Studying host-pathogen interactions is therefore essential for understanding the biological processes that underlie fungal virulence, host defense, and disease progression. Invasive fungal infections represent an escalating global threat, with multiple species now exhibiting resistance to all major antifungal drug classes. More than 1 billion people are estimated to be affected by fungal infections, and factors such as climate change, widespread antimicrobial use, and the increasing number of immunocompromised individuals are accelerating the emergence of antifungal-resistant pathogens^47,48,49,50. Understanding how pathogenic fungi colonize and interact with their hosts is critical for developing new preventive and therapeutic strategies.

The following describes a revised and standardized systemic infection protocol for S. mediterranea-C. albicans interactions, designed to promote reproducibility and to enable systematic investigation of fungal pathogenesis and host defense mechanisms. The original protocol was followed in prior publications^22,23,24. Table 1 summarizes the key updates incorporated into this revised version, including improvements to fungal inoculation procedures, reduced well volumes, defined animal inclusion criteria, determination of infectious and lethal doses, and updated animal husbandry recommendations. This workflow provides detailed guidance for culturing C. albicans, infecting planarians through soaking exposure, and assessing fungal virulence and host responses using qualitative and quantitative readouts under symptomatic and lethal-dose conditions (Figure 1). The optimized procedures presented here incorporate variables empirically identified as key determinants of infection dynamics and host outcomes, thereby improving reproducibility and enabling systematic assessment of host–pathogen interactions.

Protocol

This protocol builds on the previous methods^22,23,24 and establishes clear, standardized procedures to enhance reproducibility and enable systematic investigation of fungal pathogenesis and host responses in S. mediterranea. The key protocol refinements include prespecified animal inclusion and exclusion criteria, standardized microbial preparation, and comprehensive assessment of host endpoints.

All procedures must be performed using appropriate sterile and aseptic biosafety level 2 techniques. Standard laboratory personal protective equipment (PPE), including gloves, a lab coat, and closed-toe shoes, should be worn at all times. Procedures involving the planarian S. mediterranea were conducted in accordance with institutional guidelines for the ethical use of invertebrates.

All protocol refinements are summarized in Table 1. All reagents, critical equipment, and media, buffers, and immunostaining recipes are listed in the Table of Materials and Table 2.
NOTE: All procedures involving the invertebrate animal model S. mediterranea should be conducted to ensure the highest standards of animal welfare. S. mediterranea should be housed in optimized conditions simulating natural habitats. Experiments should be designed to minimize potential pain or distress to ensure ethical treatment throughout the study.

1. Fungal culturing

1. C. albicans strain information
   1. The standard C. albicans lab strain SN250⁵¹ was used as the wild-type strain for all experiments, and is publicly available at the Fungal Genetics Stock Center (http://www.fgsc.net/). SN250 is derived from the clinical isolate SC5314, originally collected from a blood culture of a patient with disseminated candidiasis⁵².
   2. C. albicans strains are maintained as 25% glycerol stocks at −80°C.
2.  Culturing C. albicans strains for infection
   1. Streaking from cryogenic stock
      1. Using a sterile applicator, streak the desired strain from −80°C stock onto yeast peptone extract (YPD) (1% yeast extract, 2% peptone, 2% glucose; pH 6.8) agar plates.
      2. Incubate statically at 30°C for 72 h.
3. Preparing overnight cultures
   1. Inoculate a single colony into 4 mL liquid YPD in sterile borosilicate glass culture tubes (18 mm × 150 mm) using a sterile applicator stick.
   2. Include a negative control tube containing uninoculated YPD under identical conditions.
   3. Loosely cap the tubes to allow aeration and incubate overnight (16 h) at 30 °C with shaking at 220 rpm (orbital) in a shaking incubator.
      NOTE: If microbial growth occurs in the negative control, abort the experiment and restart with new media and sterile technique.
4. Harvesting and washing cells
   1. Using aseptic technique, transfer cultures to labeled 15 mL centrifuge tubes.
   2. Centrifuge cultures for 5 min at ~3,000 × g to pellet the cells.
   3. Carefully remove the YPD supernatant without disturbing the pellet.
   4. Resuspend the pellet in planarian water (refer to Table 2 for composition) in a volume equal to that of the YPD used.
   5. Mix thoroughly by vortexing.
   6. Repeat steps 1.4.2-1.4.4 twice more to remove the growth media entirely.
   7. After the final wash, resuspend the cell pellet in fresh planarian water by vortexing.
5. Quantifying C. albicans cell concentration
   1. Dilution for OD measurement
      1. Prepare three sterile 1.7 mL microcentrifuge tubes with a 1:20 dilution (50 µL culture + 950 µL planarian water) or an alternative chosen dilution factor. Mix thoroughly.
      2. Prepare a blank tube containing 1000 µL planarian water.
   2. Spectrophotometry
      1. Set the spectrophotometer to measure optical density at 600 nm (OD₆₀₀).
      2. Blank the spectrophotometer with planarian water.
      3. Measure OD₆₀₀ for each diluted sample and record values.
      4. Average the three readings and multiply by the dilution factor to determine the OD₆₀₀ of the stock culture. Convert OD₆₀₀ to cell concentration.
         ​NOTE: The optical density conversion factor should be determined empirically for each spectrophotometer. Based on the specific instrumentation used, an OD₆₀₀ of 1 corresponds to approximately 2 ×10⁷ C. albicans cells/mL⁵³.
6. Determining experimental inoculum concentrations
   1. Prepare the desired inoculum concentration for each experimental condition.
      NOTE: Perform at least three biological replicates to determine the effective dose for the planarian colony, particularly for infectious or lethal endpoints. A representative calculation for preparing infectious and lethal inoculum concentrations is provided in Supplementary File 1.

2. Planarian species and maintenance

1. Planarian species and maintenance
   1. Use the CIW4 asexual clonal line of the planarian S. mediterranea for all experiments.
   2. Maintain planarian colonies in clear food-grade plastic containers containing 1,500–2,000 mL of 1× planarian water.
   3. Keep containers in darkness at 20 °C with lids loosely closed to allow gas exchange.
   4. Do not treat planarians with antibiotics.
2. Colony maintenance and feeding
   1. Feed colonies fresh strained beef liver purée once per week.
   2. Clean containers twice per week.
   3. Maintain planarian density at 200–400 animals per container. Previous work demonstrates detailed procedures on colony preparation and long-term maintenance³¹.
3. Pre-experiment conditioning
   1. Prior to experimental infection, starve planarians for 7–12 days to standardize metabolic and size conditions.
   2. Use groups of 5–10 animals per well for each experimental condition. Ensure equal numbers of animals across all conditions, including positive and negative controls.
4. Animal size selection
   1. Select planarians approximately 5 mm in length, corresponding to ~5 × 10⁵ host cells per animal⁵⁴.
   2. Avoid using planarians smaller than 5 mm, as animals of this size may include individuals with blastemas or incomplete development that can produce inconsistent results.
   3. Avoid using planarians larger than 5 mm, as larger animals are less suitable for fluorescence immunostaining.
   4. Use planarians approximately 5 mm in length when establishing infectious dose (ID₅₀) and lethal dose (LD₅₀) conditions.
      NOTE: To facilitate consistent size selection, print a 5 mm × 5 mm reference grid and place it inside a plastic binder sleeve beneath the transparent planarian container. Ensure planarians are fully extended before measuring length.
5. Health and inclusion criteria
   1. CRITICAL STEP: Examine all planarians under a stereomicroscope prior to use.
   2. Exclude animals with visible damage or injury, including the following conditions:
      1. Presence of blastemas (tissue lacking pigmentation) or incomplete pigmentation development.
      2. Abnormal pigmentation, such as significantly darker regions.
      3. Missing or damaged tissue.
      4. Lesions or scars.
      5. Open wounds.
      6. Morphological abnormalities or deformities.
      7. Any chronic or non-healing condition.
   3. Exclude animals with developmental abnormalities.
      1. Bifurcated or duplicated head or tail regions.
      2. Misshapen or asymmetric body morphology.
      3. Incorrect number of photoreceptors (only one bilateral pair is acceptable).
      4. Abnormally positioned pharynx.
      5. Defective ciliation resulting in impaired motility or irregular gliding behavior.
   4. Include only animals that are approximately 5 mm in length, fully developed, and free of visible injuries or abnormalities.
6. Plate preparation
   1. Transfer healthy planarians meeting all inclusion criteria into 2 mL of fresh planarian water per well in a non–tissue culture-treated 6-well polystyrene plate.
      NOTE: Transfer animals one day prior to infection to allow acclimation to the new environment and population density.
   2. Assign wells for the following experimental conditions:
      1. Negative controls: uninfected or mock-treated animals.
      2. Positive controls: animals infected with wild-type C. albicans.
   3. Remove and replace all water with 2 mL of fresh planarian water to ensure uniformity across wells.

3. Infection setup 

1. Prepare inoculation (see Supplementary File 1 for sample calculation)
   1. Using aseptic technique, gently remove all water from the 6-well plate by tilting the plate toward the user.
   2. Add the calculated volume of planarian water to each well.
      NOTE: Proceed one well at a time to prevent animals from drying out.
   3. Add the calculated volume of C. albicans culture to each well.
      NOTE: Avoid exposing animals to air for prolonged periods, as drying can cause stress and alter infection outcomes.
2. Experimental plate incubation conditions
   1. Place the inoculated 6-well plate in a dark, static location at room temperature.
   2. Maintain experimental plates in a separate area from uninfected planarian colonies to prevent cross-contamination.
   3. Record the date and time of inoculation (designated as time 0 h).
      NOTE: Avoid high-traffic or vibration-prone areas (e.g., drawers, instrument surfaces, or busy benchtops), as movement can stress animals and confound results.
   4. Incubate planarians with C. albicans for up to 72 h, depending on the desired endpoint.
3. Resuspension of fungal cells
   1. At each time point, gently resuspend settled C. albicans cells without disturbing the planarians.
      NOTE: Time (hours [h] or days [d] post-infection) refers to the elapsed time since the initial exposure (time 0).
   2. At 24 h post-infection (hpi)
      1. Using a sterile 3 mL transfer pipette, tilt the plate slightly toward the user.
      2. Aspirate the planarian water and gently dispense it into an area of the well free of animals to mix the settled fungal layer.
      3. Repeat this step 5–10 times per well to break up the settled fungal layer, avoiding direct contact with planarians.
      4. Observe fungal settlement against a dark background for best visibility before and after the resuspending steps.
      5. Resuspend uninfected control wells under identical conditions.
   3. At 48 hpi
      1. Repeat the resuspension procedure described above.
      2. Perform all resuspension steps within ± 1 h of the designated timepoint.
4. End of exposure (72 hpi)
   1. At 72 hpi, gently transfer planarians to a new 6-well plate containing 2 mL of fresh planarian water per well.
   2. Minimize transfer of fungal culture by using the smallest possible volume of liquid.
   3. Once animals are transferred, remove all water and replace it with fresh planarian water.
      ​NOTE: Handle animals and any tissue fragments gently to avoid mechanical injury or additional stress.

4. Host endpoint assessment

1. Recording host outcomes
   1. Begin assessing host outcomes at 1-day post-infection (dpi) and continue daily until the desired experimental endpoint.
   2. Quantify host damage using the standardized 0–3 scoring system (Figure 2).
      Score 0: No change from uninfected controls (healthy, asymptomatic).
      Score 1: Mild symptom(s).
      Score 2: Severe symptom(s).
      Score 3: Death.
2. Criteria for host symptom evaluation
   1. Score 0 (Asymptomatic):
      1. Identify animals that display normal morphology, pigmentation, behavior, and responsiveness with no observable changes relative to uninfected controls.
   2. Score 1 (Mild):
      Assign a mild score when at least one of the following symptoms is observed relative to asymptomatic controls.
      1. Reduced movement speed or abnormal gliding behavior.
      2. Partial or complete loss of phototactic response.
      3. Sustained or repetitive body contractions.
      4. Abnormal or non-uniform pigmentation (lighter or darker regions).
      5. Regression of head or tail tissue.
   3. Score 2 (Severe):
      Assign a severe score when one or more of the following symptoms are present while animals remain alive.
      1. Eye film or loss of one or both eyes.
      2. Complete head loss or notable anterior tissue damage.
      3. Major tissue loss in the pre-pharyngeal region.
      4. Fragmentation into multiple body regions (cephalic, pharyngeal, or posterior segments).
      5. Open wounds with or without mucus secretion or internal leakage.
      6. Partial tissue lysis resulting in loss of structural integrity.
      7. “C-shaped” body posture, paralysis, or severe motility impairment.
   4. Score 3 (Death):
      Identify death based on one or more of the following criteria.
      1. Complete tissue or whole-animal lysis.
      2. Total loss of response to stimuli.
      3. Failure to recover or regenerate.
         NOTE: Figure 2 provides representative images and defining features for each score to ensure consistent host phenotype classification.

5. Determining host survival after fungal exposure

1. At 3 dpi, gently transfer planarians to a new 6-well plate containing 2 mL of fresh planarian water per well, avoiding transfer of residual fungal culture.
2. Observe animals using light microscopy (≥ 10x objective).
3. Record host outcomes using the standardized scoring sheet provided in Supplementary File 2.
4. Determining live animals
   1. Identify living animals based on one or more of the following criteria.
      1. Negative phototaxis.
      2. Response to gentle transfer pipette perturbation.
      3. Intact body structure.
      4. Stable attachment to the plate.
5. Determining dead animals
   1. Identify dead animals based on one or more of the following criteria.
      1. Complete lysis or disintegration (residual debris only).
      2. Transparent or eviscerated body.
      3. Absence of movement or response to stimuli.
      4. Failure to regenerate (absence of blastema formation).
6. Removal of dead animals
   1. Remove dead animals and debris only after confirming non-viability under microscopy.
   2. CRITICAL STEP: Remove dead animals immediately to prevent cross-contamination or altered survival measurements.
   3. If debris or fungal carryover remains, transfer surviving animals again to a fresh plate containing clean planarian water to prevent C. albicans reseeding or biofilm formation.
      NOTE: Place 6-well plates over a dark background during visual inspection to improve contrast and facilitate phenotype assessment.

6. Fungal burden assessment

1. Colony-forming unit (CFU) quantification
   1. At the desired infection timepoints, collect planarians (5–10 animals per condition per day). Include matched uninfected/mock controls and wild-type infected controls.
   2. Gently rinse wells two times with 2 mL of fresh planarian water to remove background C. albicans cells.
   3. Transfer planarians into sterile and labeled 1.7 mL or 5.0 mL microcentrifuge tubes and remove all residual water.
   4. Add 300 µL of fresh planarian water to each tube.
   5. Homogenize animals using a sterile pestle until no large fragments remain (approximately 30–45 s).
   6. Perform serial dilutions of the homogenate (e.g., 1:10, 1:100, 1:1000).
   7. Plate 100–200 µL of each dilution onto YPD agar plates supplemented with 50 µg/mL each of ampicillin, rifampicin, streptomycin, and neomycin. Spread evenly using sterile glass beads or a sterile cell spreader.
   8. Allow plates to remain upright on the benchtop for 5–10 min after plating to permit absorption of liquid into the agar.
   9. Prepare triplicate plates for each dilution and condition.
   10. Incubate plates at 30 °C for 48 h.
   11. Count CFUs and multiply by the dilution factor.
   12. Normalize CFU values to the number of planarians to calculate average CFUs per planarian per condition. For example, if 100 colonies are observed at a 10× dilution from a homogenate of 5 animals, the calculated value is 200 CFUs per animal.

7. Immunofluorescence anti-Candida staining

1. General staining considerations
   1. Perform all steps with gentle orbital rocking (100–140 rpm) at room temperature unless otherwise specified. See Supplemental File 1 for antibody details and reagent sources.
      CAUTION: Formaldehyde, sodium dodecyl sulfate (SDS), and hydrogen peroxide are hazardous chemicals and must be handled in accordance with institutional safety regulations. Formaldehyde is toxic and classified as a carcinogen; SDS is an irritant; and hydrogen peroxide is a strong oxidizer that may cause burns. Wear appropriate personal protective equipment (lab coat, nitrile gloves, closed-toe shoes, and safety goggles). Perform procedures involving volatile reagents in a certified chemical fume hood and dispose of waste according to institutional Environmental Health and Safety guidelines.
2. Sample collection
   1. Collect animals from each experimental condition and time point.
   2. Transfer animals into labeled 20 mL scintillation vials containing 2 mL of planarian water.
   3. Remove all liquid, rinse once with 2 mL of fresh planarian water, then all liquid once more.
3. Fixation and permeabilization
   1. Sacrifice animals in 7.5% N-acetylcysteine (NAC) in 1× PBS for 3 min.
   2. Remove the solution and fix animals in 4% formaldehyde in 0.3% PBSTx for 15 min.
   3. Rinse samples two times with 1× PBS.
   4. Permeabilize samples in 1% SDS for 15 min.
   5. Rinse samples three times with 1× PBS.
4. Bleaching
   1. Bleach samples in 6% hydrogen peroxide in 1× PBS under a bright LED light for 4–12 h.
      NOTE: If bleaching cannot proceed immediately, store fixed samples in 1× PBS at 4 °C for up to 1 week or dehydrate samples in 100% methanol for long-term storage.
5. Blocking and antibody incubation
   1. Transfer animals to 24-well plates containing 1× PBS.
   2. Block nonspecific binding in 2.5% PBS-TB (PBS containing Triton X-100 and bovine serum albumin) for 4 h at room temperature or 8 h at 4 °C.
   3. Incubate samples with primary anti-Candida antibody (1:500 in PBS-TB) for 4 h at room temperature or 8 h at 4 °C.
   4. Remove primary antibody and wash samples eight times every 20 min with 0.3% PBSTx (total ~2.5 h).
   5. Incubate samples with Alexa-568-conjugated anti-rabbit secondary antibody (1:800 in PBS-TB) for 4 h at room temperature or 8 h at 4 °C.
      NOTE: Protect samples from light to prevent photobleaching. Antibody solutions may be reused.
   6. Remove secondary antibody and wash samples eight times every 20 min with 0.3% PBSTx.
6. Optional slide mounting
   1. Place animals onto a microscope slide with all animals oriented anterior side up.
   2. Remove excess water using a transfer pipette.
   3. Carefully absorb remaining liquid from the edges using the edge of a paper towel.
   4. Add 150–175 µL of Gelvatol mounting medium (Table 2 for composition) dropwise until animals are fully covered.
   5. Carefully place a coverslip over the sample, avoiding air bubbles.
   6. Allow Gelvatol to solidify for at least 2 h before imaging.
   7. Store slides in a cool, dark location for long-term preservation.
7. Imaging
   1. Image samples using yellow-green excitation (~578 nm) and observe orange-red emission (~603 nm).
   2. Use a 2x objective lens to visualize whole-animal staining patterns.

8. Troubleshooting

1. If infection outcomes are inconsistent, perform a dose-titration curve using wild-type SN250 or another baseline strain in increments of 5–10 × 10⁷ cells/mL.
2. Perform at least three biological replicates containing 5–10 animals per group.
3. Include negative and positive control groups to identify potential issues such as cross-contamination, aseptic technique failure, strain viability problems, or poor baseline animal health.
4. After completing the dose-titration experiment, pool replicates to determine median survival per group.
5. Analyze survival differences using Mantel–Cox log-rank pairwise analysis.
6. Use Kaplan–Meier survival analysis to evaluate time-to-death differences between strains or treatments.
7. For sublethal or infectious dose conditions, analyze symptomatic host outcomes (e.g., mild or severe phenotypes or fungal colonization) instead of survival.
8. Compare host responses across dose groups to determine the appropriate infectious or lethal dose range for subsequent experiments.

Results

An updated protocol for infecting planarians with C. albicans (Figure 1) and systematically quantifying host outcomes (Figure 2) is presented. This refined workflow supports robust assessment of multiple infection parameters, including morbidity (symptomatic disease severity), survival (lethality), fungal burden (CFUs), and spatial-temporal dynamics of pathogen colonization (fluorescence immunostaining). Unlike most invertebrate infection models, which ...

Discussion

Varying the infectious dose of wild-type C. albicans produces reproducible, dose-dependent differences in morbidity in S. mediterranea, underscoring the model’s sensitivity for quantitative assessment of fungal virulence. This protocol outlines strategies for establishing baseline infectious and lethal doses within laboratory-maintained planarian colonies and presents criteria for confirming successful systemic infection via soaking, integrating host health scoring with fungal burden measurements ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.7 mL microcentrifuge tubes	VWR/Genemate	490004-436-BOXOF500	Clear, box of 500 pieces
20 mL scintillon vials	Fisher Scientific	12-100-006	Clear glass bottles for immunostaining
24-well non-treated polystyrene plates	Fisher Scientific	08-772-51	Immunostaining
5.0 mL microcentrifuge tubes	Eppendorf/Sigma Aldrich	EP0030119487-200EA	Clear, box of 200 pieces
6-well non-treated polystyrene plates	Fisher Scientific	08-772-49	Infection assays
Agar	VWR	89405-066	Bacteriological grade for YPD medium
Applicator sticks	Thermo Fisher/Puritan	22-029-491	C. albicans culturing
BioRender	BioRender Inc.	N/A	Figure design software used to generate schematic illustrations
Calcium chloride (CaCl2)	Sigma-Aldrich	C5080-500G	Planarian water/1x Montjuïc salts
Candida albicans polyclonal antibody anti-Rabbit, IgG	Thermo Fisher	PA1-27158	Immunostaining
Centrifuge tubes	VWR	89039-664	C. albicans harvesting
Culture tubes borosilicate glass 18x150mm	VWR	47729-583	C. albicans culturing
Cuvettes	Fisher Scientific	14-955-127	To measure optical density of cultures
Dextrose (D-glucose)	Fisher Scientific	D16-3	YPD medium
Glycerol	Fisher Scientific	G33-4	Molecular biology grade to supplement for cryogenic storage of C. albicans
Graphing and data visualization tool	GraphPad/Prism		Statistical software and vizualization tool. Verison 10.6.1.
Graphing tool	R		Statistical software. Verison 4.4.3.
Incubator Shaker	Eppendorf	EPM1282-0004-1EA	New Brunswick Innova 44/44R for C. albicans culturing
Magnesium chloride (MgCl2)	Sigma	M8266-100G	For planarian water  (1x Montjuïc salts)
Magnesium sulfate (MgSO4)	Sigma-Aldrich	M7506-500G	For planarian water  (1x Montjuïc salts)
Microscope Slides	VWR	16004-368
Multizoom microscope	Nikon		Nikon AZ100
N-Acetyl-L-cysteine	Sigma Aldrich	A7250-50G	Mucolytic and sacrificial reagent  fixation
Paramaldehyde, 16% solution, molecular biology grade	Electron Microscopy Sciences	15710	Fixative reagent
Pellet Pestle Cordless Motor	Fisher Scientific	12-141-361	Planarian homogenization
Peptone, bacteriological grade	HIMEDIA/VWR	89129-480	YPD medium
Pestles	Fisher Scientific	12-141-368	RNase-free disposable pellet for planarian homogenization
Petri Dishes, 100 x 15 mm	VWR	25384-342
Poly(vinyl alcohol)	Sigma-Aldrich	P-8136	For Gelvatol
Potassium chloride (KCl)	Sigma Life Science	P9541-500G	Planarian medium/water  (1x Montjuïc salts)
Secondary Antibody Goat anti-Rabbit, IgG, Alexa Fluor 568	Fisher Scientific	A-11011	Immunostaining
SN250	Fungal Genetics Stock Center		Wildtype C. albicans strain (https://www.fgsc.net/)
Sodium Azide	Fisher Scientific	S227-100	For Gelvatol
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S5761-1KG	Planarian medium/water  (1x Montjuïc salts)
Sodium chloride (NaCl)	Sigma Life Science	S3014-500G	Planarian medium/water  (1x Montjuïc salts)
Spectrophotometer	Agilent	26354	BioTek Epoch 2 Microplate to measure optical density
Static Incubator	Fisher Scientific	15-015-2634	C. albicans culturing
Transfer pipettes	Fisher Scientific	13-711-9CM	Sterile disposable graduated pippettes for transferring planarians
Tris Base	Sigma-Aldrich	252859	For Gelvatol
Yeast extract	Thermo Fisher	212750	YPD medium