5 Module 3: Phenotypic Assays
5.1 Module 3.1 – Phenotypic Assays
5.1.1 Instructor Laboratory Preparation Instructions
Materials and Equipment: - Autoclave - Laminar Flow Hood (if available) - 60x15mm Petri Dishes - Agar, Bacteriological - Potato Dextrose Agar - Zinc Sulfate - Alcohol Lamp or Bunsen Burner - Forceps - Scalpel (make sure the handle is metal as the scalpel will be flame sterilized and plastic will melt)
Sterility: If you don’t have a laminar flow hood, you may create a similar environment by setting up 2 torches over your work space. First, clean your work area with bleach, and/or ethanol. Set the torches facing each other with enough space to work underneath. Light the torches and pour media into plates under the flames.
Potato Dextrose Agar (PDA) with Zn Plates for Zn Stress Assay: Suspend 39 grams Potato Dextrose Agar in 1000 ml (1 L) distilled water (or follow the directions on the bottle label). Add the appropriate amount of Zn sulfate to obtain concentrations of 1 mM Zn and 10 mM Zn in the media bottle. Heat to boiling to dissolve the medium completely (in most cases, this step can be skipped as autoclaving will dissolve media). Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
(PDA) Plates for Temperature Assay: Suspend 39 grams Potato Dextrose Agar in 1000 ml (1 L) distilled water (or follow the directions on bottle label). Heat to boiling to dissolve the medium completely (in most cases, this step can be skipped as autoclaving will dissolve media). Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
When the media is cool enough to handle with autoclave gloves, pour 60x15 mm plates in a sterile environment. Do not let it cool too long before pouring as the media will solidify. Each 60x15 mm plate holds approximately 8 mL of media. If plates will be used immediately smaller amounts of media can be poured into plates. If plates will be stored, use larger amounts. Store plates in original Petri dish sleeve in refrigerator (~4 °C) until use.
Working in your sterile environment, place one cellophane disc onto cooled PDA agar in 60x15mm plates. Use sterile forceps to transfer each cellophane disc onto the PDA plate. If you can’t easily remove single discs, try the “slide” technique. Dip your forceps into the container with the discs until you touch a disc. Using the forceps as a probe (not pinched) slide the disc to the side of the container and up the side of the container. Often, you’ll pull out one disc. Sometimes more than one disc will slide up, but it’s usually easy to see the individual discs at this point. You can then use your forceps to pull out one disc. If you grasp one disc with your forceps, you can swirl it in the water in the container to dislodge any other discs that might be attached. Open the lid of the petri dish and lay the cellophane onto the solid agar.
5.2 Module 3.2 – Phenotypic Assays
5.2.1 Phenotypic Assays
Objectives: 1. Determine how fungal isolates react to environmental stressors
Materials: - Student fungal isolates
Procedures: - Observe your fungal cultures and determine if they are pure cultures - We will use these cultures to set up stress tests (Zn assay and temperature assay) and for molecular identification.
5.2.2 Zn Stress Assay
In this lab, we will be investigating the tolerance and growth response of foliar fungal endophytes to varying concentrations of zinc (Zn). Zinc is an essential trace element for many biological processes, but in elevated concentrations, it can become toxic to fungi and other microorganisms. To understand how these fungi cope with different levels of Zn, we will isolate fungal endophytes from plant leaves and subject them to media containing 0 mM, 1 mM, and 10 mM of Zn. By measuring their growth rates over several weeks, we aim to determine the impact of Zn on their growth and survival.
Testing foliar fungal endophytes for Zn tolerance is particularly relevant as these fungi play crucial roles in plant health and ecosystem functioning. Endophytes can enhance plant growth, provide resistance to pathogens, and contribute to nutrient cycling. Understanding their tolerance to heavy metals like Zn is important for ecological studies, especially in areas affected by pollution or where plants are used in phytoremediation efforts. By assessing their ability to thrive in environments with varying Zn concentrations, we can gain insights into their resilience and potential applications in sustainable agriculture and environmental management.
Objectives: 1. Determine if the fungal isolates are tolerant of Zn. 2. Measure how different concentrations of Zn influence fungal growth rates.
Materials: - Pure fungal cultures - PDA 0 mM Zn + antibiotics - PDA 1 mM Zn + antibiotics - PDA 10 mM Zn + antibiotics - Fine tip Sharpie - Culture cutting tool - Burner - Ethanol jar
Procedures: - Label your plates with identifying information - On the bottom of each plate, draw four lines that intersect in the center of the plate (Figure 1) - Cut out three 0.5 cm² chunks from the edge of your pure culture and place one in the center of each of the Zn plates - Close and parafilm your plates - Carefully, flip the plates over and trace the cube using a fine tip Sharpie - Place the plate (unflipped) at room temperature in the dark and observe growth over the next few weeks
5.2.3 Temperature Assays
In this lab, we will explore the effects of temperature on the growth and survival of foliar fungal endophytes. Temperature is a critical environmental factor that influences fungal physiology and ecological dynamics. To investigate how these fungi respond to different thermal conditions, we will isolate fungal endophytes from plant leaves and cultivate them at three distinct temperatures: 4°C, room temperature (approximately 20°C), and 28°C. By measuring their growth rates over several weeks, we will better understand their tolerance to different temperatures.
Objectives: 1. Determine optimal growth temperature. 2. Measure how different temperatures influence fungal growth rates.
Materials: - Pure cultures (2 per student) - PDA with antibiotics (6 per student) - Fine tip Sharpie - Culture cutting tool - Burner - Ethanol jar
Procedures: - Label your plates with identifying information and growth temperatures (2 per temp.) - On the bottom of each plate, draw four lines that intersect in the center of the plate (Figure 6.1) - Cut out three 0.5 cm chunks from the edge of your pure culture and place one in the center of each of the temperature plates - Close and parafilm your plates - Carefully, flip the plates over and trace the cube using a fine-tip Sharpie - Bring your plates to the front of the class and place them (unflipped) in the correct box for incubation
5.2.4 Measuring Fungal Growth
Objectives: 1. Measure growth for Zn stress assay 2. Measure growth for temperature assay
Materials: - Fine tip Sharpie - Three Zn culture plates - Three temperature culture plates - Ruler to measure growth in mm (mm ruler)
Procedures: - On the bottom of the plate, trace the edge of your fungal culture - Measure, in mm, new growth at each of the four edges along the lines - Average the eight measurements to get the average growth and determine the average growth rate (distance/time)