Mushroom Bioremediation Lab Notes (Weeks 1–2)

Overview of the Mushroom Bioremediation Lab (Weeks 1–2)

  • This lab guides a group-based experiment to inoculate a cardboard substrate with oyster mushroom mycelium and test the mushroom’s ability to decompose a chosen substrate (bioremediation).
  • The study tracks mycelial growth over the semester and compares effectiveness on different test substances.
  • Emphasis on practical skills: microscopy, stereoscope use, mushroom cultivation techniques, prediction making, observations, and evaluation of bioremediation performance.

Fungi: basic concepts and terminology

  • Fungi are a separate kingdom from plants and animals.
    • Approximately 150,000150{,}000 described species (including yeasts, molds, mildews, mushrooms).
    • New DNA sequencing data suggests a much higher range, possibly 3extto133 ext{ to } 13 species? (note: the handout frames this as a striking estimate for unknown diversity).
  • Historical classification vs. evolutionary relationships:
    • Fungi were historically classified as plants, but are evolutionarily closer to animals.
  • Fungal body plan:
    • The body of a fungus is the mycelium, composed of hyphae, thin filaments that grow toward and around food, secreting enzymes to digest and absorb nutrients.
  • Reproductive structures:
    • The mushroom is the spore-producing reproductive structure, called the fruiting body; it is only a small part of the larger organism (analogous to a fruit on a plant).
  • Key figures referenced in hands-on activities (Figure 1 and Figure 2 in the handout):
    • Figure 1: Labeled fungi – conceptual image.
    • Figure 2: Life cycle of oyster mushroom (life mostly in a haploid stage; see below).

Fungal life cycle and reproduction (Week 2 focus on oyster mushroom)

  • Reproductive cycle complexity varies among fungi.
  • Oyster mushroom life cycle:
    • Most of its life is in a haploid stage (one set of chromosomes).
    • Our lab propagates fungus asexually by transferring mycelium pieces to fresh substrate.
  • Haploid vs. diploid:
    • Humans are diploid (two sets of chromosomes); many fungi spend life primarily as haploid.
  • Saprotrophic fungi (like oyster mushrooms):
    • Nature’s primary recyclers; decompose dead organic matter to feed themselves.
    • Capable of breaking down lignin, a polyphenolic component that provides wood with rigidity.
    • This activity helps recycle nutrients and return them to soil.
  • Bioremediation:
    • Fungi can break down pollutants and transform them into less harmful substances or nutrients, aiding soil health and ecosystem recovery.
    • The lab explores the limits of mushroom bioremediation across multiple weeks.

Week 1: Microscopy focus and pre-lab preparations

  • Week 1 activity: Examining preserved fungi with a compound microscope and prepared slides
    • Prepared slides are professionally mounted specimens used for practice.
    • Goals:
    • Practice using a microscope and become familiar with fungal structure.
    • Observe differences in illumination requirements for various specimens.
    • Sketch or photograph specimens in OneNote.
    • Study tip: There will be a microscope skill check next week; practice focusing, refocusing, and using all microscope parts.
  • Week 1 lab questions (to answer after the microscope exercise):
    • a) Is the image always best with highest illumination?
    • b) Is the same level of illumination best for all magnifications?
    • c) Which magnifications require the most illumination for best clarity and contrast?
    • d) What happens to the image when the diaphragm is closed?
  • Handling and storage reminders for the microscope:
    • Return slides to original holding racks when instructed by the TA.
    • Return the plastic ruler to the storage bin.
    • Turn off the light source, ensure no slide remains on the stage, and coil the power cord.
    • Return the microscope to its cabinet and match the label number on the arm with the parking space label.

Week 1: Bioremediation experiment preparation (group setup and materials)

  • Lab grouping:
    • Groups of 44 students.
  • Materials provided per group:
    • One 900extmL900 ext{ mL} glass jar with lid
    • Cardboard substrate to grow mushrooms
    • Box cutter or scissors for breaking up cardboard
    • Strainer
    • Autoclave tape
  • Experimental prep steps:
    1. Record or photograph the cardboard source to track potential additives that might affect mycelial acceptance.
    2. Break cardboard into pieces of about 2.5extcm2.5 ext{ cm} ext(approximately1extinch)ext{(approximately } 1 ext{ inch)} or smaller squares to fill roughly 23\frac{2}{3} of the jar.
    3. Soak cardboard pieces in tap water for 2−5extminutes2{-}5 ext{ minutes} (record exact time).
    4. Drain excess water using the strainer and place cardboard pieces in the jar.
    5. Label the jar with a section number, date, and group initials (handout notes on making labels: include a folded tab to facilitate removal).
    6. Loosely close the jar and attach autoclave tape (with tab) to the lid and jar to allow steam and heat to penetrate during sterilization.
    7. Autoclave or sterilize the jar for use in Week 2 when introducing mycelium and the test object.
    8. Decide on a test substance to inoculate (biological or hydrocarbon-based, about a fist-sized piece, easily fit in the jar) and bring it to the next lab.
    9. Bring the test substance to Week 2 for setup.
  • Practical note:
    • Autoclave tape color change indicates sterility status (tape color changes as sterilization completes).

Week 2: Bioremediation experimental setup

  • Week 2 objective and materials:
    • Spawn: grain pieces with alive oyster mushroom mycelium
    • Test substances: bring a couple of potential materials (one backup)
  • Experimental procedure (Week 2):
    1. State your prediction and the reasoning behind your choice; record in your notebook.
    2. Locate your jar and observe autoclave tape color change indicating sterility.
    3. Add a second label with the name of your chosen substance and the date of introduction.
    4. If your group chooses to moisten or surface-sterilize the object with Ethanol, note it; otherwise add the object as is.
    5. Combine the spawn with cardboard inside the jar and add the chosen substance, ensuring contact by submerging it in the cardboard.
    6. Close the lid tightly and take a picture of the jar (refer to Figure 3 in the handout).
    7. Place the jar on the rack with others in your section.
    8. Plan to examine the jar next week to assess whether mycelium colonized the cardboard and which food sources were chosen.
    • If mycelium does not colonize, discuss with your TA the possibility of resetting the jar using a different cardboard source, as experimental alterations may be necessary.
    1. Clean up the workspace and remove lab coat.
  • Figure reference:
    • Figure 3: Mushroom experiment jar image.

Week 2: Examining live oyster mushrooms with a stereoscope

  • Objective: Compare live mushrooms to preserved specimens and learn stereoscope operation.
  • Equipment: Labomed Luxeo Stereoscope (specific model shown in Figure 5).
  • Procedure for visualizing mycelium/mushrooms on a plate:
    1. To visualize white mushrooms on a Petri plate, cover the bottom light with black paper to create contrast, then place a mushroom over it.
    2. Turn on the top light.
    3. Adjust the eyepieces and diopter adjustment similarly to the microscope practice from Part I.
    4. Rotate the zoom knob to minimum magnification (smallest value on the knob).
    5. Use the focus knob to bring the specimen into focus; adjust as needed to view the entire specimen.
    6. Sketch what you see and compare live specimens to preserved ones.
    7. Return the stereoscope to its storage in the same manner as the microscope.

References used in this handout

  • Hyde, K. D. The numbers of fungi. Fungal Diversity 114, 1 (2022).
  • Strete, Dennis; Vodopich, Darrell S. Photo Atlas for General Biology (2001).

Practical and conceptual takeaways

  • Bioremediation with fungi leverages natural decomposition pathways to transform pollutants into less harmful substances or nutrients, contributing to soil health and ecosystem restoration.
  • The laboratory emphasizes both fundamental skills (microscopy, stereoscopy, sterile technique, labeling) and scientific thinking (predictions, observations, documentation, troubleshooting).
  • Flexibility and troubleshooting are important: if colonization fails, be prepared to adjust the substrate, cardboard source, or experimental conditions in consultation with the TA.
  • Ethical and real-world relevance: understanding fungal roles in nutrient cycling and pollutant degradation highlights sustainable approaches to environmental remediation.