STAIN, X100)✦Sporangiophores emerge from a foot cell with the shape of an inver ted “T” (arrow). This is a wet mount of A.
flavus. Notice all the conidia in the field.
✦ Materials
Per Student Group
✦agar slant of Saccharomyces cerevisiae
✦Potato Dextrose Agar or Sabouraud Dextrose Agar plate culture of Aspergillus spp.(with lid taped on)
✦Potato Dextrose Agar or Sabouraud Dextrose Agar plate culture of Penicillium spp.(with lid taped on)
✦Potato Dextrose Agar or Sabouraud Dextrose Agar plate culture of Rhizopus spp.(with lid taped on)
✦Gram’s iodine stain or Methylene Blue
dissecting microscope
✦prepared slides of:
⽧Aspergillus spp. conidiophore
⽧Candida albicans
⽧Penicillium spp. conidiophore
⽧Rhizopus spp.sporangia
⽧Rhizopus spp.gametangia
Procedure
Yeasts
1 Using an inoculating loop and aseptic technique, make a wet mount slide of Saccharomyces cerevisiae as illustrated in Figure 3-19, and stain with iodine or Methylene Blue. Observe under high-dry and oil immersion. Identify vegetative cells and budding cells. Sketch representative cells in the space provided on the Data Sheet.
2 Observe prepared slides of Candida albicans.
Identify vegetative cells and budding cells. Sketch representative cells in the space provided on the Data Sheet.
Molds
1 Obtain the plate culture of Rhizopus. Do not re move the lid. Uncovering the organism will spread spores and contaminate the laboratory.
a. Examine the colony morphology, and sketch a representative colony in the space provided on the Data Sheet. Record the coloron both the front (obverse) and reverse surfaces. Also record the colony textureas glabrous (leathery), velvety, yeast-like, cottony, or granular (powdery), and the colony topographyas flat, rugose (with radial grooves), folded, crateriform, verrucose (warty, rough) or cerebriform (brain-like).
b. Examine the colony under the dissecting micro- scope and identify hyphae, rhizoids, and sporan- gia. Sketch and label representative structures in the space provided on the Data Sheet.
2 Examine prepared slides of Rhizopussporangia using medium and high-dry powers. Identify the following: sporangiophores, sporangia, and spores.
Sketch and label representative structures in the space provided on the Data Sheet.
3 Examine prepared slides of Rhizopusgametangia using medium and high dry power. Identify the following: progametangia, gametangia, young zygosporangia, mature zygosporangia. Sketch and
12-20 PENICILLIUM NOTATUMCOLONY ONSABOURAUD
DEXTROSEAGAr ✦The green, granular sur face with radial furrows and a white apron are typical of the genus.
12-21 PENICILLIUMCONIDIOPHORE(X1000)✦Penicillium species produce a characteristic brush-shaped conidiophore (penicillus). Metulae (M), phialides (P), and chains of spherical conidia (C) are visible.
C P M
label representative structures in the space provided on the Data Sheet.
4 Obtain the plate culture of Penicillium. Do not remove the lid from the plate or you will spread spores and contaminate the laboratory.
a. Examine the colony morphology and sketch a representative colony in the space provided on the Data Sheet. Record the coloron both the front (obverse) and reverse surfaces. Also record the colony textureas glabrous (leathery), velvety, yeast-like, cottony, or granular (powdery), and the colony topographyas flat, rugose (with radial grooves), folded, crateriform, verrucose (warty, rough) or cerebriform (brain-like).
b. Examine the colony under the dissecting micro- scope. Identify hyphae and conidia. Sketch and label representative structures in the space pro- vided on the Data Sheet.
5 Observe prepared slides of Penicillium conidiophores.
Identify the following: hyphae, conidiophores, and chains of conidia. Sketch and label representative structures in the space provided on the Data Sheet.
6 Obtain the plate culture of Aspergillus. Do not remove the lid from the plate or you will spread spores and contaminate the laboratory.
a. Examine the colony morphology and sketch a representative colony in the space provided on
the Data Sheet. Record the coloron both the front (obverse) and reverse surfaces. Also record the colony textureas glabrous (leathery), velvety, yeast-like, cottony, or granular (powdery), and the colony topographyas flat, rugose (with radial grooves), folded, crateriform, verrucose (warty, rough), or cerebriform (brainlike).
b. Examine the colony under the dissecting micro- scope. Identify hyphae and conidia. Sketch and label representative structures in the space pro- vided on the Data Sheet.
7 Observe prepared slides of Aspergillusconidiophores.
Identify hyphae, conidiophores, and conidia. Sketch and label representative structures in the space pro- vided on the Data Sheet.
References
Collins, C. H., Patricia M. Lyne, and J. M. Grange. 1995. Chapter 51 in Collins and Lyne’s Microbiological Methods,7th ed. Butterworth- Heineman, Oxford, United Kingdom.
Fisher, Fran, and Norma B. Cook. 1998. Chapter 2 in Fundamentals of Diagnostic Mycology.W. B. Saunders, Philadelphia.
Forbes, Betty A., Daniel F. Sahm, and Alice. S. Weissfeld. 2002. Chapter 53 in Bailey & Scott’s Diagnostic Microbiology, 11th ed. Mosby-Year Book, St. Louis.
Koneman, Elmer W., Stephen D. Allen, William M. Janda, Paul C.
Schreckenberger, and Washington C. Winn, Jr. 1997. Chapter 19 in Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. J. B.
Lippincott, Philadelphia.
✦ Theory
The background material concerning fungi is contained in Exercise 12-1.
✦ Application
Classical fungal taxonomy has relied on the appearance and arrangement of sexual spores, as well as certain features of the hyphae. Because sporangia are so delicate, it is difficult to transfer a portion of mycelium to make a wet mount showing an intact sporangium with its spores.
(For an example of what these wet mounts frequently look like, see Figure 12-19 and notice all the scattered, detached spores!) By growing the fungus on a micro- scope slide, intact sporangia can be observed without disturbing them.
✦ In This Exercise
You will grow a mold in a slide culture and observe its sexual spores.
✦ Materials
Per Class
✦Fume hood or cell culture hood
✦Aerosol disinfectant in hood Per Student Group
✦Sabouraud Agar slant or plate culture of mature (sporing) Penicillium spp.(Figures 12-20 and 12-22A) (This can be located in the fume or cell culture hood and one culture should serve the entire class.)
✦Sabouraud Agar slant or plate culture of mature (sporing) Aspergillus spp.(Figures 12-19 and 12-22B) (This can be located in the fume or cell culture hood and one culture should serve the entire class.)
✦5 mL tube of sterile, molten Sabouraud Agar (stored in a 50°C water bath until needed)
✦50 mL beaker with alcohol
✦50 mL flask of sterile water
✦Bunsen burner
✦petroleum jelly
✦sterile toothpicks or wooden applicators
✦jar of disinfectant large enough to immerse the micro- scope slides when finished
Per Student
✦sterile Petri dish
✦glass rod bent to about 30 degrees in a way it will fit into the Petri dish (a bendable drinking straw will also work)
✦filter paper cut to fit inside the Petri dish
✦sterile transfer pipettes
✦depression microscope slide
✦cover slips
✦forceps
✦inoculating (or dissecting) needle
✦scalpel
✦gloves
Procedure
Lab One
1 Place a folded paper towel on your desk. It should be about the size of a magazine. Put all the working materials on the towel: Petri dish, glass rod, depres- sion slide, cover slip, forceps, inoculating needle, and sterile transfer pipettes.
2 Place the filter paper inside the Petri dish. Set the glass rod on top of the filter paper (Figure 12-23).
12-2 Fungal Slide Culture
12-22 SLIDECULTURESILLUSTRATINGCONIDIOPHORES✦ APenicillium. BAspergillus.
A B
3 Saturate (but don’t flood) the filter paper with sterile distilled water using a sterile transfer pipette. Replace the lid.
4 Using a sterile transfer pipette, rinse the depression slide with alcohol. Tap the excess alcohol off on the paper towel. Then, holding the slide with a forceps pass it throughthe flame and let the alcohol burn off. Do NOT perform this step over the paper towel or the alcohol because a drop of flaming alcohol can start a fire. After it cools, place it on the paper towel with the depression upwards.
5 Repeat Step 4 with the cover slip. Be sure not to heat the cover slip too much or it will melt or crack.
6 Using the toothpick or wooden applicator, apply a bead of petroleum jelly around the two sides and bottom of the depression on the slide (Figure 12-24).
This is made easier if the petroleum jelly is slightly warm. Make sure it is applied so its size matches the cover slip’s size.
7 Dip the forceps in alcohol, then pass them through the flame. Let the alcohol burn off and set them aside on the paper towel. Do NOT perform this step over the paper towel or the alcohol because a drop of flaming alcohol can start a fire.
8 Using a sterile transfer pipette, place a drop of molten Sabouraud Agar in the depression of the slide. Immediately place the cover slip over the agar using the sterile forceps. Gently press it down to seal the edges. Allow the agar to solidify (Figure 12-25).
9 Once the agar has solidified, gentlyslide the cover slip away from the unsealed edge so about two- thirds of the agar is exposed.
10 Dip the scalpel blade in alcohol, then pass the blade through the flame. Let the alcohol burn off. Be sure not to catch the scalpel’s handle on fire! Also, do NOT perform this step over the paper towel or the alcohol because a drop of flaming alcohol can start a fire.
11 Gently make a straight cut across the diameter of the agar with the scalpel. Remove the top (uncovered) half.
12-24 PETROLEUMJELLYSEAL✦Use a wooden applicator or toothpick to make a bead of petroleum jelly about the size of the cover slip on three sides of the slide’s depression. This is easier to do if the petroleum jelly is slightly warm. In this photo, blue food coloring has been added to the petroleum jelly so it is easier to see.
12-25 AGAR IN THEWELL✦ Shown here is the solidified agar in the well and covered with the cover slip.