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Lab 6 Objectives
Acid-Fast Staining
(Ziehl-Neelsen Method)
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Background and Purpose:
This staining method is a differential stain used to identify members of
the genera Mycobacterium and Nocardia. These
bacteria, and a few others, have cells walls that contain a waxy, lipoidal
material called mycolic acid. This substance gives these bacteria
unique staining properties that set them apart from most others.
Specifically, when they are stained with carbolfuchsin, these bacteria
retain this primary stain even after treatment with a decolorizing agent called
acid-alcohol. When this occurs, these bacteria are said to be
acid-fast. In contrast, most other bacteria which are readily
decolorized by acid-alcohol are called nonacid-fast. This staining
technique is of diagnostic value for determining the presence of acid-fast
bacteria in sputum or tissues. Notably, this provides presumptive evidence
for diseases such as tuberculosis (Mycobacterium tuberculosis), leprosy (M.
leprae) or nocardiosis (Nocardia spp.). Here in northern
Michigan, acid-fast staining is used to help monitor the prevalence of
bovine tuberculosis (Mycobacterium bovis) among cattle and
white-tailed deer.
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Acid-fast
Mycobacterium smegmatis
growing on
a tryptic soy agar slant |
Nonacid-fast Staphylococcus epidermidis
growing on a tryptic soy agar slant |
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General Procedure:
Using heat, a bacterial smear is first stained with carbolfuchsin
and then decolorized with acid alcohol. After a brief rinsing with water,
the smear is then counterstained with methylene blue.
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Materials:
carbolfuchsin, acid-alcohol, methylene blue,
hot plate (or Bunsen burner and tripod), small beaker (150 ml), staining rack, slide holder,
disposable latex gloves, wash bottle,
bibulous paper
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Motility Determination
(Method 1 - The Hanging Drop Method)
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Background and Purpose:
Several bacteria are capable of true motility. These microbes usually
possess locomotor appendages called flagella. However, spirochetes use axial filaments
that generate a
twisting/flexing movement and the myxobacteria demonstrate a motion known as
gliding.
Each of these active modes of locomotion allow cells to independently move from one
location to another. Bacteria lacking true motility exhibit a passive
vibratory motion call Brownian movement. This erratic, nondirectional movement occurs
when cells are randomly bumped or bombarded by
water molecules.
Knowing whether true motility is present or absent is important in
identifying various groups of bacteria. And, if flagella are present,
knowing their arrangement is a useful characteristic for identification.
One method for determining motility is to microscopically examine live
bacteria. Although the flagella are too slender to be observed with a
light microscope, the movements of these cells can be seen. The
hanging drop method used here is a type of wet mount slide preparation
that permits the observation of living, unstained cells in a fluid medium.
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General Procedure:
A small drop of live bacteria is transferred to the center of a
cover glass. Using petroleum jelly, the cover glass is attached to a
depression slide. After inverting the slide, organisms are observed in a
drop suspended under the cover glass within the concavity of the slide.
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Materials:
depression slide, cover glass, Vaseline (petroleum jelly),
tooth picks
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View
procedure
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View videos of flagellated bacteria exhibiting
both true
motility and Brownian movement.
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View videos of
nonflagellated bacteria exhibiting Brownian movement.
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View
video of flagellated bacteria being moved by water currents
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Motility Determination
(Method 2 - The Tube Method)
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Background and
Purpose: An alternative to
using microscopic examination for motility determination is to employ a
stab technique with a semisoft agar medium in a test tube.
A medium prepared with a 0.4% agar concentration is not enough to inhibit the movement of
motile microbes within the medium. Therefore after one to two days of incubation, motile
microbes will cause the medium to turn cloudy because of their ability to
disperse away from the stab line. In
contrast, the medium inoculated with nonmotile bacteria remains
clear since the bacterial growth is restricted to only a narrow band along where the stab was
made. |
When testing bacteria that pose a higher risk of infection to the worker,
the tube method is preferred over the hanging drop method since the microbes
are contained within a test tube. However, a disadvantage to
the tube method is that it requires a period of incubation before the results
can be determined.
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General Procedure:
Bacteria are introduced
into a semisoft agar medium by performing a stab with an inoculating needle. After incubating
the tube, motility is determined by examining whether or not the bacteria have migrated
away from the stab line and throughout the medium. |
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Materials:
deep tube of semisoft motility medium |
The tube on the left
stabbed with Proteus vulgaris is positive for motility, while the tube on
the right was inoculated with Staphylococcus aureus, a species lacking
motility.
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