Skill Development Lab 3.0 Fun with Fungi Microscope Skills, Refining Observation Techniques Bioprospecting Stream „ Freshman Research Initiative „ by Dr. Marsha J Lewis

Name/uteid: Assigned:

February 22 , 2012

Due:

March 7 , 2012

Submission:

Assessments: Turn in @ Group Meeting 4pm

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Discussion Board Post: 4pm on Blackboard Reading:

Lab Manual Chapter 8 and textbook Chapter 9.1

Item

Possible

1.0 Microscope Assessment

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2.0 Systematic Observations (macroscopic

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Earned

observations recorded) 3.0 Direct Mount and Staining (observations

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recorded) 4.0 Slide Culture and Staining (observations

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recorded and compared and contrast with direct mount method) 5.0 Discussion Boar Post: your write up should

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be complete with Fungi Obseration Form comments and Specimen Preparation method choice and why. Lab Notebook (LN) (LN can be checked at anytime

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while this lab is in progress: looking for pre-lab complete in proper timeframe, results, format, observations, and details. See Appendix 2 in text and Chapter 3 in Lab Manual)

Late Penalty (-10 every day late) Final Grade

120 1

Table of Contents Skill Development 3.0 Required Reading to properly prepare for lab:  Read textbook Chapters 9.1 and 9.2  Read Bioprospecting Lab Manual Chapter 8. 1.0

Conventionl Light Microscopy and Microscope Assessment 1.1 Print this assessment form and use for your oral assessment in lab.

2.0

Systematic Observations 2.1 In lab work

3.0

Direct Mount and staining of fungal specimen 3.1 Execute protocol 3.2 In lab work and document in lab notebook

4.0

Slide Culture and staining of fungal specimen 4.1 Execute protocol 4.2 In lab work and document in lab notebook

5.0

Discussion Board Post: Culture Observation Form and Sample Preparation choice

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Objectives This is your third Skill Development Lab (you are half way there!). You will continue to use and further develop your aseptic technique into a good, routine habit. After completing this lab, you should feel comfortable working with a conventional light microscope. You will be able to prepare a sample for microscopic inspection and apply a stain. You will also develop a system to record your observations of microscopic and macroscopic characteristics of fungi. This will assist you in determining if you have a viable fungal culture and appreciate the complexity and diversity of the fungi kingdom! These tools will then permit you to apply the techniques to increasingly complex and interesting experiments that you will encounter in your own research project. At this time, it is important that you thoroughly read the assigned Bioprospecting Lab Manual and textbook chapters. Introduction Record answers to Questions posed in this Skill Development Lab in your Lab Notebook. Preparing your lab notebook for data entry and to answer questions posed here is considered pre-lab work! 1.0 Conventional light microscopy In this lab we will use a compound light microscope. Please read the Lab Manual, Chapter 8 for background relevant to how a microscope works and the different types available. The lab manual also contains details describing each component. In this lab exercise I will just refresh your memory with a labeled diagram of the microscope parts. All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path. The components are listed

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below in the order the light travels through them. In addition, the vast majority of microscopes have the same 'structural' components: These entries are numbered according to Figure 1. 

Ocular lens (eyepiece) (1)



Objective turret or Revolver (to hold multiple objective lenses) (2)

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Objective (3)

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Focus wheel to move the stage (4 coarse adjustment, 5 - fine adjustment)

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Frame (6)

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Light source, a light or a mirror (7)

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Diaphragm and condenser lens (8)

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Stage (to hold the sample) (9)

Figure 1: Components of a conventional light microscope.

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There are only a few ABSOLUTE rules to observe in caring for the microscopes you will use. Taken care of, these instruments will last many decades and continue to work well. Please report any malfunctions immediately to your instructor. 1. ALWAYS use two hands to carry the scope - one on the arm and one under the base - NO EXCEPTIONS! NEVER carry the scope upside down, for the ocular can and will fall out. 2. Use lens paper to clean all lenses when necessary.. DO NOT EVER, NOT NOW, NOT EVER, USE ANYTHING BUT LENS PAPER TO CLEAN THE LENSES. Other papers are too impure and will scratch the optical coating on the lenses. 3. Always use the proper focusing technique to avoid ramming the objective lens into a slide - this can break the objective lens and/or ruin an expensive slide. 4. Always turn off the light when not using the scope. 5. Always replace the cover on the microscope when you put it away

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Microscope Assessment Instructions: Please check each box as the student addresses the subject. You can prompt the student for subject-areas that are not covered - the student should then be able to elaborate on their own. Topics: ______ Turn on the microscope and discuss how the lenses would be cleaned.

The student will demonstrate how to turn on the microscope and discuss (not actually!) how to clean the lenses stating that kim-wipes and solvents will NEVER be used, only specifc lens tissues that are moistened with specific lens cleaner (typically ammonia/ethanol mix) ______ Identify the following parts of the microscope: objective, objective nosepiece, stage, specimen coarse and fine focus knobs, oculars, focusing collar.

The student will point out each part of the microscope and indicate where the sample will be mounted. ______ Demonstrate how to adjust the microscope for comfortable viewing

The student should demonstrate how to adjust the height, and the interpupillary distance in the binocular headpiece, ______ The student should be able to properly focus a test slide.

By describing the test slide accurately the student demonstrates they have successfully adjusted the microscope and are able to find objects. Student EID: _________Name: _______________________________________________ Research Staff Signature: ________________________________ Date: ______________

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2.0 Systematic Recording of Observations: Microscopic and Macroscopic It is convenient to have a standard observation form in a group situation to document microscopic and macroscopic observations of fungal growth. A standard observation form ensures that details are consistently recorded and fungi can be consistently identified as this lab group’s record of observations accumulates. In order to complete the form (Figure 2), standard observations will need to be defined and described. First, a fungal sample is observed macroscopically, that is without a microscope. We did this in the previous lab. Now, we will formalize our observations. During the macroscopic observation the following are noted: form, elevation, margin, surface, opacity, and color (pigmentation) on underside and topside of growth. The fungi radius is measured (cm) and radial changes in pigmentation are noted at specific measurements. Once the macroscopic evaluation is complete, a microscopic evaluation is initiated. The conventional light microscope does not lend itself to a very detailed microscopic examination of fungi. However, you will be able to distinguish between filamentous, unicellular fungi and may be able to discern if the hyphae contains septate junctions. You will also be able to determine if bacterial contamination is evident. Different stains will help you identify whether you have a filamentous fungi or bacteria by the degree of staining and relative size. Bacteria are significantly smaller than fungi. Print the Fungi Observation Form and record your macroscopic observations of your assigned specimen. Tape the observation form into your lab notebook.

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Macroscopic Observation Form: Characteristic Select best description or cross out: Overall Form

Comments Diameter (cm):

Circular, Irregular, Filamentous, or Rhizoid Elevation Margin

Surface Opacity Color

Smooth or Coarse Transparent , Translucent , or Opaque Top-side:_______________Under-side:___________________ Does the color change radially? If yes, describe. Microscopic Observation Form: Characteristic Select best description or cross out: Gram Stain Red (gram negative bacteria) or Blue (gram positive bacteria or fungi) Lactophenol Cotton Blue (stains chitin in fungus wall) Septate junctions

Comments

Non-septate (i.e. Coenocytic hyphae)

Ascomycetes (Aspergillus) fruiting bodies 10X (the above three images from MCC Microvision)

Ascomycetes (Thermomyces lanuginosus):

40X

100X

(the above two images from: S. Shrivastava, et al: Correlative characterization of changes in hyphal morphology during xylanase production in submerged culture by Thermomyces lanuginosus SS-8. The Internet Journal of Microbiology. 2008 Volume 4 Number 2)

Other stain: ___________ Figure 2: Fungi Observation Form: Assists researchers to provide consistent observations.

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3.0 Direct Mount and Staining for Microscopic Observation In this first exercise, you will perform a direct mount of your specimen using fungal tape. The direct mount method with “sticky tape” is an efficient and facile method. Unfortunately, sometimes this method can disrupt your specimen and relevant features may become undistinguishable. Lactophenol Cotton Blue (LPCB) Stain is formulated with lactophenol, which serves as a mounting fluid, and cotton blue. Organisms suspended in the stain are killed due to the presence of phenol. The high concentration of the phenol deactivates lytic cellular enzymes thus the cells do not lyse and cell integrity is maintained. Cotton blue is an acid dye that stains the chitin present in the cell walls of fungi. The General Procedure for a Direct Mount and LPCB staining. (Bioprospecting Laboratory Protocol can also be used as reference). 1. Wear new gloves when handling each individual sample to reduce the risk of cross-contamination. 2. Label the end of a microscope slide using a carbon or grease pencil with a fungal ID number, date, and your initials. 3. Place a drop of lactophenol cotton blue in the center of the slide. 4. Remove a precut piece of Fungi-Tape from the box being careful not to contaminate the adhesive surface. 5. Bend the tape-strip (without creasing), adhesive-side out, between your thumb and index finger so that the tape forms the shape of a “U” (Figure 3).

Figure 3. Correctly forming the tape in the shape of a “U”

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6. Sample fungal growth by touching the fungal growth with the sticky side of the tape. 7. Lightly touch the adhesive surface of the tape-strip, at the bottom of the “U”, to an area of fungal growth. If only a small area is transferred to the tape, use a different portion of the same tape “U” to touch another area of fungal growth. DO NOT attempt to obtain more than 3 lifts per tape strip (Figure 4).

Figure 4. Fungal growth sample shown on tape

8. Align the tape-strip containing the fungal sample, adhesive-side down, over the microscope slide. Ensure that the edges of the tape-strip do not protrude beyond the edges of the microscope slide when laid flat, and do not remove any portion of the tape-strip from the glass slide once it has adhered (Figure 5).

Figure 5. Align the tape-strip on the microscope slide.

9. Lightly wipe over the top surface of the tape-strip using a kim-wipe to consistently adhere the strip to the slide. Make sure to wipe very lightly as we do not want to crush the conidia or separate the conidia from the conidiophores. 10. Examine the preparation under low (10X) and high (40X), dry magnification for the presence of characteristic mycelia and fruiting structures. To use the 100X magnification setting you will have to observe with the objective submerged in oil. 10

11. Record your observation in the Fungi Observation Form used previously (and already taped in your lab notebook) to record your macroscopic observations.

4.0 Slide Culture and Staining for Microscopic Observation In this exercise, you will make a slide culture of your specimen for microscopic observation. The slide culture is the best method for observing unknown fungi. Important structures are not disturbed using this method and if the conditions are right, you may also be able to observe sporulation characteristics that are important for identifying specific fungal species. The General Procedure for a Slide Culture and LPCB staining. (Bioprospecting Laboratory Protocol can also be used as reference). 1. First, you will need to establish your slide culture from your fungal specimen. The protocol is simple and takes less than 30 minutes, however, you need to permit time for fungal growth (i.e. incubation), which can take 2 to 5 days. 2. Assemble the materials you will need to initiate a slide culture: YM or PD agar plate (depends on your specimen), something to cut the agar (you can use a cover slip, a test tube to cut out a circular plug, or a scalpel), one or two microscope slides, one or two cover slips, and some autoclaved filter paper. 3. Label the end of a microscope slide using a carbon or grease pencil with a fungal ID number, date, and your initials. 4. In an aseptic environment, extract a piece of agar from the agar plate. Figure 6. Illustrates using a cover slip to cut out squares of agar. Cut as many squares or remove as many circular plugs as you will need (One piece is sufficient for this exercise. The agar plate can be used again by another person. Thus, it is important that you are careful not to contaminate the plate and return it to storage promptly and correctly.) Carefully remove the agar piece with the same instrument you used to cut it.

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Figure 6. These pictures show a person cutting agar pieces using a cover slip.

5. Place the agar piece onto a clean microscope slide (Figure 7). You can place one or two pieces of agar per slide.

Figure 7. The agar piece is placed on a clean microscope slide.

6. Place the slide on a clean petri dish to prevent contamination and preserve moisture during incubation. Raise the slide from the bottom of the petri dish or else the surface tension between the microscope slide and the petri dish that is created by moisture accumulation during incubation will make it difficult to remove the slide later. You can use whatever is convenient to raise the slide: filter paper pieces, wooden stir rods, etc.(see Figure 8).

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Figure 8. The microscope slide is placed in a petri dish and raised off the dish bottom . 7.

Use a sterile instrument (an inoculation loop, needle, toothpick, etc.) and inoculate the agar piece by touching hyphae to each edge of the agar piece as shown in Figure 9.

Figure 9. Transfer the fungus to the sides of the agar block. It is not necessary to smear the entire side, rather just touch the center of each agar block side. 8.

After inoculating the agar piece, place a clean cover slip on the surface of the agar piece. Don’t forget this very important step!!! (Figure 10)

Figure 10. Don’t forget the cover slip!

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9.

Add a few drops of sterile water to the petri dish to prevent the agar piece from drying out over time (especially if your fungus is slow growing!). Seal your plate with Parafilm™ (cellulose tape) (Figure 11).

Figure 11. Final preparations for incubation of your slide culture. 10.

The slide culture is now ready for incubation. Incubate the slide at the appropriate temperature and time for your assigned specimen. Fungi can overgrow the agar piece very quickly, so monitor the growth visually daily. (Record these observations in your lab notebook!)

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After sufficient growth, you will examine your slide culture microscopically. To examine the slide culture, remove the slide from the petri dish and gently remove the cover slip from the agar piece. The fungus will partially adhere to the cover slip (see Figure 12).

Figure 12. A slide culture after successful incubation. 12.

Place a drop of lactophenol cotton blue stain onto a clean microscope slide and place the cover slip from the slide culture onto the stain (growth down!) 14

13.

Gentle wipe any excess stain from the edges of the microscope slide with a cleanwipe.

14.

Examine the slide under the microscope under different magnifications. Print out another Fungi Observation Form (microscopic observation only) and complete your microscopic observations. Tape the form in your lab notebook. In your lab notebook, compare and contrast your microscopic observations from your direct mount and slide culture.

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If something goes wrong, you can observe the same culture by carefully removing the agar piece from the slide. You will find that some fungal growth adhered to the microscope slide as well. You can then stain the growth and place a clean cover slip on it and observe microscopically.

5.0 Discussion Board Post The Blackboard discussion board is an excellent resource for communicating issues to other students: basically, learning from each other’s successes and mistakes. Feel free to initiate a discussion thread at any time. Communication amongst lab members is important enough to warrant assigning discussion board posts for points! In your first posting please address the two topics below in a succinct manner. Your post should not be more than 1 to 2 brief paragraphs. 1. Is the lab’s “Fungi Observation Form” missing anything? If so, be specific and tell me what should add (or remove)! 2. Do you prefer the direct mount or the slide culture preparation method for investigating a fungal specimen microscopically? Why?

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