Republic of the Philippines

Romblon State University

Romblon, Philippines

Laboratory Activity 3
Microscopic Examination of Mold and Bacteria
Laboratory Goals
o Learn the difference between simple and differential stains.
o Become familiar with staining and observing yeast.
o Examine different molds, and identify morphologies of mycelium and hyphae.

Learning Concept

Bacterial Mount
Prior to observing anything under a microscope, the sample must be prepared on a microscope
slide. This is known as mounting. For a standard compound light microscope, bacteria are usually
prepared as a dried film, which is heat fixed and stained. Samples do not have to be stained for
observation using a phase contrast microscope; therefore, a simple wet mount can be used.

Simple Stain
A simple stain will stain all cells in a sample with the same color, which aids in observing cellular
morphology or performing a direct microscopic count of cell numbers. Methylene blue will be used in
this laboratory exercise. This is a basic stain. Basic stains tend to have a high affinity toward acidic cell
wall components.

Gram Stain
This stain is named after Christian Gram, who developed it in the 1800s. This stain differentiates
between two broad classes of organisms, which differ in their cell wall compositions. Gram-positive
(Gram+) cells have thick peptidoglycan layers. Gram-negative (Gram−) cells have thin peptidoglycan
layers and a lipopolysaccharide (LPS) layer. In this staining procedure, crystal violet is the primary stain.
Like methylene blue, this stain is alkaline and has a high affinity to cell components. The mordant is
iodine. The purpose of the mordant is to combine with the dye to form an insoluble complex between
the dye and cellular components. This complex, when present on certain cells (Gram+), will be resistant
to the decolorization step. In the decolorization step, ethanol is used to remove excess dye from the
slide as well as dye from certain cells (Gram−). The counterstain, safranin, will be used to stain
decolorized cells a reddish pink color.
The Gram stain is a step-by-step procedure. Reproducibility is based upon strict adherence to
staining times and careful attention to technique. Errors can be incorporated by poor techniques, as
explained in the following examples:
1. Overheating the film while heat fixing: If the cells break open due to overheating, the
Gram+ cells may lose the crystal violet complex and appear as Gram−.
2. Too many cells in the film: This may cause irregular staining and decolorization may be
incomplete. This could cause Gram- cells to appear as Gram+.
3. Decolorization is the critical step: If too much time is taken, you will decolorize the Gram+
cells. If too little time is taken, you will not completely remove the crystal violet from the
Gram−cells.
4. Age of the culture: Old cultures may not stain accurately.
5. Iodine solution can deteriorate over time: The solution should be dark golden yellow in color.

Microscopic Observation of Fungi

Filamentous fungi are usually identified by basic structures that can be observed by light
microscopy. Filamentous fungi have long filaments or tubes called “hyphae.” Within the hyphae, fungi
may or may not have “septa” or cross-walls. Intertwined hyphae are called a “mycelium.” Within the
mycelium, a portion remains on the substrate, and reproductive shoots grow into the air. Filamentous
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fungi can grow asexually (imperfect form) or sexually (perfect form). Within a fungi genus, the structures
will vary between the perfect and imperfect forms. The image below represents the nonsexual growth
of fungi that you will be looking at in this lab.

Rhizopus

Mucor
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The genera Aspergillus and Penicillium belong to the Deuteromyces phylum of fungi. All
members of this group have branching septate hyphae and rarely have a perfect (sexual) form. They
both produce “conidiospores” on top of aerial structures known as “conidiophores.” The conidiospores
are produced in long chains and are attached to the conidiophore via a structure called a “phialid”.
In general, members of the genus Penicillium have a narrow head of conidiospores, while
Aspergillus members usually have a spherical head of conidiospores. The “foot cell” is unique to the
genus Aspergillus, and this three-pronged structure is found at the base of the conidiophore where it
meets the mycelium. Mucor and Rhizopus are genera of the Zygomycetes class of fungi. These
organisms are nonseptate and produce asexual “sporangiospores” or sexual “zygospores.” The fruiting
bodies of these two genera are similar: the aerial structure is called a “sporangiophore” with a large
globular “sporangium” that contains large amounts of sporangiospores under a membrane. When
mature, the spores are released after the rupture of the sporangial membrane. Mucor and Rhizopus
are differentiated based upon the organization of the mycelium. Generally, members of the Mucor
genus develop sporangiophores randomly in any direction from the branching mycelium. Members of
the Rhizopus genus have a more organized structure, with nodes in which the aerial sporangiophores
grow up and structures called “rhizoids” develop growing down. Some members of Rhizopus develop
with the rhizoids positioned directly below the sporangiophores and other members may have the
rhizoids growing downward elsewhere from the mycelium.
To get the best view of fungi structures, they should be propagated on a small amount of solid
media mounted on a glass slide. In this lab, we will be using a quick method, or “the old transparent
tape trick,” to obtain enough hyphae with which to observe the fungal structures.
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Laboratory Procedures
Materials: microscope slides, inoculating loop, clear tape, tongs, water bottle
Solutions: methylene blue, crystal violet solution, iodine solution, alcohol (95% ethanol),
safranin solution

Preparation of “Heat-Fixed” Bacterial Smears

1. Make a smear of a culture on a microscope slide.

a. If from a plate, place one loop of water on the slide. Flame a loop, and select a single
colony for examination. Place the colony in the water on the slide, and spread it into a
thin film (approximately 1cm2).
b. If from broth, place one loop of culture on the slide, and spread it into a thin film
(approximately 1cm2).
2. Allow the smear to dry completely. If this is not allowed to dry, you will later lose your bacterial
smear. Slides placed at the base of a lit burner will dry faster.
3. Place the slide into a clothespin. Quickly bring the slide through the flame once or twice. The
slide should not be burning hot. If you overheat the slide at this step, you can change the
staining characteristics of the organisms.

Methylene Blue Staining

Perform methylene blue staining on each bacterial culture.

1. Prepare a heat-fixed film as described in the previous section.

2. Cover the film with methylene blue solution for 1 to 2 min.
3. Tilt the slide to allow the excess stain to run off into the staining tub, and wash gently with the
water bottle.
4. Allow the slide to air dry without blotting or blot gently with a lint-free laboratory wipe.
5. Examine the stained bacteria with your microscope using the 40× objective.

Gram Stain

Gram stain the mixed culture and a colony from your streak plates from Laboratory 1 or from those
provided by your instructor.

1. Cover the heat-fixed film with crystal violet solution and leave for 1 min.
2. Tilt the slide to allow excess crystal violet to run off into a staining pan. Rinse with water bottle
gently for 1 to 3 sec.
3. Make sure the excess water is drained off, then cover the film with iodine solution and leave it
on for 1 minute. Drain the iodine solution and rinse with water as above. Allow the excess
water to run off.
4. Flood the film with alcohol (95% ethanol) for less than 30 seconds to remove the purple/blue
color. (This step is critical and should be performed with extreme care. It can also be done by
holding the slide at an angle and adding about 5 to 10 drops of the alcohol, drop-wise, until a
purple/blue color no longer streams from the film.)
5. Rinse immediately with water.
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6. Cover the film for 30 to 60 sec with safranin solution.

7. Rinse it with water, blot gently, and allow the film to air dry.
8. Observe the stained film under the microscope.
9. Record the Gram reaction of each stained sample. For identification purposes, the cellular
morphology (such as long, short, or irregular rods or chains, clumps or individual cocci) should
be recorded for each sample.

Observation of Molds

1. Record colony characteristics, such as color and texture.

2. Use a dissecting microscope to observe the mass of mycelia at the edge of a colony.
3. Take a small piece of tape and roll it into a loose circle with the sticky edge facing out.
4. In a biological safety hood, pick up a small amount of mycelia from the edge of a colony.
5. Uncurl the tape and place it on a microscope slide, with the mycelia (sticky side) facing down.
The tape will act as a coverslip.
6. At the microscope, start at 100×(10×objective) or 20×(2×objective) magnification, and look for
sections of mycelium. Tape slides will have large amounts of loose spores, so you will need to
search the slide for an area with mycelium. Once you find some mycelia, look for structures
described in the images presented.
Usually you can get a better view of filamentous fungi at lower magnifications, but you can
increase the magnification to 400×(40×objective) to obtain more detail.
7. Make drawings in your notebook and label parts of each fungi.

Laboratory Notes

1. Record the cellular morphology and the Gram reaction of each bacterial culture.
2. Record the colony morphology and specific morphology of hyphae and fruiting bodies of the
filamentous fungi.

Laboratory Questions

1. Focus a slide on an object (any slide, any objective). Use the mechanical stage to move the
slide slightly to the left. Repeat this while looking through the microscope. What happens?
Why?
2. What are the fundamental differences between Gram+ and Gram− bacteria?
3. What might occur if the heat fixation step is skipped?
4. What might occur if decolorization is too long?
5. What might occur if decolorization is too short?
6. What is the difference between a simple stain and a differential stain?
7. Is the Gram stain a simple stain or a differential stain?