Download Gram Stain Lab Prokaryotic Cell Wall Differentiation

Gram Stain Lab
Prokaryotic Cell Wall Differentiation
Honors BioMed 1
Redwood High School
Name:
10/05
 Background
The Gram stain is a very useful stain for identifying and classifying bacteria. The Gram stain is a differential stain that
allows you to classify bacteria as either gram positive or gram negative. As a reference figure 27.5 from Campbell can be
found at the bottom of page 3 of this lab. The staining technique consists of the following steps:
1. Apply primary stain (crystal violet). All bacteria are stained purple by this basic dye.
2. Apply mordant (Gram's iodine). The iodine combines with the crystal violet in the cell wall to form a crystal
violet-iodine complex called the CV-I.
3. Apply decolorizing agent (95% ethanol or acetone alcohol). The primary stain is washed out (decolorized) of
some of the bacteria, while others are unaffected.
4. Apply secondary stain or counterstain (safranin). This basic dye stains the decolorized bacteria pink.
The most important determining factor in the procedure is that bacteria differ in their rate of decolorization. Those that
decolorize easily are referred to as gram-negative and appear pink, whereas those that retain the primary stain are called
gram-positive and appear dark blue/purple to almost black.
Bacteria stain differently because of chemical and physical differences in their cell walls. Gram-positive cells consist of
many layers of peptidoglycan. The CV-I molecular complex is larger than the crystal violet or iodine molecules that initially
entered the cell wall and cannot pass through the thick peptidoglycan. In gram-negative cells, the alcohol dissolves the outer
lipopolysaccharide layer, and the CV-I complex washes out through the thin layer of peptidoglycan.
Examination of Gram-stained organisms usually provides a starting point for classifying, identifying, and characterizing
bacteria. Such information helps to determine the source of microbes isolated as contaminants, for example, in the industrial
production of foods and pharmaceuticals. The presence of gram-positive cocci indicates that shedding of normal human flora
is the likely source of contamination; the presence of gram-positive, endospore-producing bacilli on the other hand suggests
environmental sources. A gram stain can steer decisions on how best to remove or destroy the contaminant and how to
prevent future contamination by the intruding microbe.
This differential staining technique is also a fundamental step in the diagnosis and treatment of disease. The Gram stain
of clinical material taken directly from an infected patient can rapidly provide valuable information about the
microorganism(s) causing the disease. This information helps guide the selection of subsequent tests needed to identify the
bacteria. For example, identifying gram-positive cocci or gram-positive bacilli requires a different battery of tests than are
used for the identification of gram-negative bacilli.
Gram stains of clinical specimens are especially important in determining the most effective antibiotic for critically ill
patients who require immediate therapy. Penicillin, for example, is more effective against most gram-positive bacteria than
against most gram-negative bacteria
 Procedures
1. Prepare a heat-fixed smear of the bacteria to be stained.
2. Cover the smear with crystal violet for an exposure time of 60 seconds.
3. Rinse the smear with distilled water.
4. Cover the smear with Gram's iodine for an exposure time of 60 seconds.
5. Rinse the smear with distilled water.
6. Decolorize the smear using the prescribed decolorizer (an alcohol). Allow the decolorizer to drip
onto the slide and run across the smear until the exact moment that the run-off turns clear. This is a
critical step. Do not over decolorize. The degree of alcohol decolorization you will need depends on
the thickness of the smear. Good judgment and some experience are the only ways you will be able
to determine how long to decolorize.
7. Immediately rinse the smear with distilled water.
8. Cover the smear with the safranin counterstain for an exposure time of 45 seconds.
9. Rinse the smear with distilled water.
10. Gently blot the slide dry with bibulous paper.
11. Observe the smear at 1000X (using immersion oil), and identify the bacteria as either gram-positive
or gram-negative.
* Some common sources for error:
1. The loop was too hot - destroying the bacterium during the heat-fixing step.
2. Excessive heat, or not enough heat, was applied during heat fixing.
3. Decolorizing agent was left on the smear too long.
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Gram Stain Lab
Gram Stain Lab
10/04
 Observations/Data
Bacterial Species
Morphology
Pictorial
Gram (+) or (-)
B. megaterium
large, lab-only bacterium
159 1:B
E. coli
occurs in the lower part of the
intestines (colin) of warm
blooded animals 420 1:B
S. epidermidis
member of the normal flora of
the surface of human skin, and
the mucous membranes of the
nasopharynx 1019 2:B
Figure 27.5
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Gram Stain Lab