Lab #2 Objectives

Alison Kobylski
BIOL 2730 C1603
Pathogenic Microbiology Lab #2 Objectives

1. Define:
a. Roccal: green disinfectant liquid that we use in lab
b. Pathogen: infectious living organisms
c. Wet mount slide: a microscope slide of a liquid specimen covered with a
cover glass
d. Yeast: microscopic fungus, single oval cells, reproduce by budding
e. Budding: method of asexual reproduction among yeast
f. Mold: multicellular masses of filamentous fungal growth
g. Hyphae: individual filaments, each generally compromised of more than
one cell
h. Mycelium: entire mass of intermeshed hyphae
i. Colony: sometimes circular body of fungal growth that is visible to the
naked eye
j. Reproductive spore: means of both reproduction and dissemination of
molds since they are readily carried by air currents
k. Septa: divide the filaments into separate cells
l. Petri plate (dish): special covered dish in which the mold is cultured
m. Medium: solid or liquid substance in which an organism lives or is
cultured
n. Agar: provides a body to the medium, slightly thicker than gelatin, still
quite soft
o. Smear: thin film of microbial cells on a microscope slide
p. Fixing: process of adhering a smear to a slide using heat
q. Simple staining: staining cells with a single dye so they can be more
readily observed
r. Basic dye: has a positively charged chromophore, will be chemically
attracted to any negatively charged substance (ex: bacterium)
s. Acidic dye: has a negatively charged chromophore, not useful in staining
bacteria
t. Chromophore group: staining group
u. Bacilli: rod-shaped bacteria
v. Endospore: clear, unstained bodies inside the bacilli, the survival forms for
the cells
w. Bacterial spore: released endospores scattering throughout the bacilli
2. Describe the steps in the preparation of a wet mount slide and name
the three different specimens used.
a.
 Place two drops of the suspension in the middle of the clean
microscope slide
 Return lid onto suspension and leave transfer pipet in beaker after
use
 Immediately cover the specimen on the slide with a cover glass
b.
 Large motile protozoa
 Small motile protozoa
Alison Kobylski
BIOL 2730 C1603
 Non-motile medium-sized algae
3. State the scientific name of the yeast studied in the laboratory.
a. Saccharomyces cerevisiae
4. Name the medium upon which the mold was cultured.
a. Sabouraud Agar
5. Name the stain routinely employed on fungal specimens.
a. Lactophenol cotton blue
6. List two methods by which the mold specimen was examined.
a. Creating a stained slide using lactophenol blue and observing it under the
microscope
b. Observing the mold on the agar while still on the Petri plate
7. Name the specimen in which moving organisms were seen.
a. Hay infusion
8. List the steps in the proper transfer of microbes from a culture to a
slide using an inoculating loop.
a. Light the laboratory burner
b. Flame the metal part of the inoculating loop in the flame of the burner
until it is glowing hot
c. Wait for the inoculating loop to cool, without waving or blowing on it
9. Describe the procedures for the preparation of smears from both
liquid and solid (agar) cultures.
a. Liquid
 Mark one end of the microscope slide with a #1
 Shake the culture vial from side-to-side and sterilize the inoculating
loop by flaming, then let it cool
 Transfer a loopful of the liquid to the #1 end of the slide using the
flat side of the loop
 Flame the loop again, let it cool, and then get another loopful of the
specimen and put it in the same spot as the last loopful
 Mix the loopfuls together and spread it out to be about the size of a
tack
 Sterilize the inoculating loop again and set it aside
 Let the smear air dry, either on its own or with a slide warmer (do
not blow on it)
b. Solid (Agar)
 Mark the other side of the microscope slide with a #2
 Sterilize the inoculating loop
 Open the water squeeze bottle, get a loopful and place it on the slide
where its marked #2
 Sterilize the loop again, get another loopful of water, transfer it to
the #2 end of the microscope slide
 Flame sterilize the inoculating loop and let it cool
 Open the Petri plate with the bacterium culture
 Gently touch the flat side of the inoculating loop to the visible
culture on the agar
Alison Kobylski
BIOL 2730 C1603
 Touch the loop to the water on the microscope slide, swirling it
around to mix and spread the microbes to about the size of a dime
 Let the smear air dry, either on its own or with a slide warmer (do
not blow on it)
10. Describe the process of heat fixing a microbial smear.
a. Let the smear air dry either on its own or using a slide warmer
b. Pass the slide through the laboratory burner with the smear side up, 3-4
times with a rapid succession
c. The slide should be warm to the touch, but not hot
11. Name the bacterium employed in the smear and simple staining
exercises.
a. Bacillus megaterium
12. List the materials and steps employed in simple staining.
a. Materials
 1 microscope (clean before using)
 2 heat fixed smears of Bacillus megaterium from previous exercise
b. Steps
 Place the smeared slide on the rack in the staining pan with the
smear side up
 Cover both smears with 1-2 drops of methylene blue and let sit for 1
minute
 Rinse the slide with water from the squeeze bottle, be sure to
remove all residual stain in one rinse
 Remove the slide from the rack and place it under the fold of a
paper towel and gently pat the paper towel to the slide to remove
excess water (be sure not to rub, otherwise the specimen will be
removed)
13. Describe the difference in appearance of organisms or slide from
broth and agar cultures.
a. Hay infusion: spiral, small bacteria; small and large protozoa that move
quickly; non-moving, round, green algae
b. Yeast suspension: small and round cells with nuclei and some budding, no
motility
c. Mold: long, thin filaments (hyphae), with segments (septa), round spores
d. Bacillus (liquid): rod-like cells that are stained, some round endospores,
less abundant amounts
e. Bacillus (agar): stained rod-like cells, some round endospores, more
abundant amounts

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Alison Kobylski

BIOL 2730 C1603

Pathogenic Microbiology Lab #2 Objectives

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