427L/527L  Laboratory  1:  TECHNIQUES

Both compound and dissecting microscopes are important tools in studying fungi.  Although there are a lot of macroscopic fungi, which can be examined without the help of microscopes, the vast majority cannot be fully appreciated without magnification greater than what our eyes can see.  Today you will be acquainted or reacquainted with microscopes.  Since some of you may have used a microscope before, be patient with those who have not. Many of the fungi we will be observing are much, much larger than bacteria, we therefore do not require the use of oil immersion in this laboratory.

 

I.  MICROSCOPY

 

A.  Review/Familiarization of the different parts of the microscope

Fig. 1.  The compound microscope: its principal parts and their functions.

B.  Care and proper use of microscopes

 

Microscopes are expensive, thus they need to be handled with care and respect to avoid damaging them.  The following general guidelines should be observed.

 

1.         Carry the microscope with one hand beneath the base and one hand on the arm.

2.         Do not tilt the microscope; instead adjust your stool so you can comfortably use the instrument.

3.          Follow the instructions on ÒAdjusting the MicroscopeÓ to set up Kohler illumination and center the condenser.

4.         Observe the slides with both eyes open to avoid eye-strain.

5.         Always focus by moving the lens away from the slide.  Focus slowly and carefully.

6.         When using the low power lens, the iris diaphragm should be barely open so that good     

            contrast is achieved.  More light is needed with higher magnification.

7.         Keep all lenses clean.  Use only lens paper to clean them and do not touch the lenses        

            with your hands.

8.         Keep the stage clean and avoid spilling dyes or any liquid.

9.         Clean the ocular lens carefully with lens paper.  If dust is present, it will rotate as you     

            turn the lens.

10.       After use, remove the slide, put the dust cover on and return the microscope to the           

            designated area.

11.       When a problem does arise with a microscope, obtain help from the instructor.  Do          

            not use another microscope unless yours is declared "out of order."  

 

 

II.  FUNGAL CHARACTERISTICS

Fungi can grow on a variety of substrates. One particularly nutrient rich substrate is the dung of organisms, which often are rich in nitrogen sources and have significant available carbon.  

Today we will plate some environmental samples to attempt to isolate different types of fungi. The samples are:

Rabbit dung

Lizard dung

Saguaro cactus debris

 

1.         Choose two of the three samples, or use ones you have brought in from home. Pick out a few petri dishes to plate specimens. Label plates with date, initials and specimen. For the saguaro debris, use ½ CM plates (1/2 strength corn meal agar). For the other specimens choose any plates, noting which material went on which plate.

            (plates: PDA=potato dextrose agar, MEA= malt extract agar, CM, corn meal agar)

2.         We will incubate the plates at room temperature until Tuesday at which point we will work to isolate and purify strains.

 

FOR TUESDAY:
1. Observe your plates. Write down the characteristics of the cultures you see (eg. fluffy, slimy, dusty, color). How many different fungal specimens can you see?

3.         Use a dissecting microscope to describe the somatic growth of the fungus if possible (septate mycelium, non-septate, yeast-like, etc). Do you need a compound microscope to observe these features?

4.         Look for spores and spore-bearing structures.  Describe these spores and the structures upon which they are bourne (size, single or multi-cellular, clustered, wall thickness, smooth, ornate, etc).

 

III. OBSERVE FIXED SLIDES

Fixed slides with different fungal structures are available to begin to familiarize you with fungi. 
Please look at all of these slides:

- Mucor sporangia (ium)

- Rhizopus sporangia, sexual development and zygospores

- Aspergillus and Penicillium conidiophores

- Peziza apothecium with asci OR

- Morchella apothecium with asci

-Ergot (Claviceps purpurea) stroma

- Coprinus  (longitudinal section)

 

B.  TERMS TO LEARN

 

hypha(e)                                 septate                                    oogonium                               

ascospores                              mycelium/mycelia       coenocytic                              

oospore                                   hyaline                                    macroconidia                          

clamp connections                  perithicium/perithicia  microconidia                           

conidium/conidia                     zoospore                     chlamydospores                     

antheridium                             ascus/asci                    sporangium/sporangia

pycnidium/pycnidia

 

 

V.  QUESTIONS

 

1.         Draw (examine both under 20X and 40X objectives as appropriate) and describe the fungal specimens we will be observing today.

           

2.         What advantages may some of the structures you observed play in the fungus' life cycle?  (eg. Can the spores be readily dispersed by wind or rain?)

 

3.         What is the difference between an asexual spore and a sexual spore?  Give examples of each.

 

4.         What are some of the advantages of producing large amounts of asexual spores?  What are the advantages to producing sexual spores?

 

5.         What is the difference between hypha and mycelium?