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_Applying Staining Techniques to View and Identify Bacteria _
By: Matt
Abstract The main objective of this lab was to identify different bacteria by
simple, negative, and gram staining. To view each bacteria cell, the bacteria
was transferred aseptically to a slide, and they were then viewed by using oil
immersion, by a light microscope. From this lab, it was determined that E.
coli and B. megaterium are gram negative and B. subtilis and S. Marcesans are
gram positive. Introduction The purpose of this lab was to view the different
characteristics of bacteria by applying various staining techniques. It is
important to know the make up if a certain bacteria so an antibiotic may be
engineered to destroy the bacteria. From the gram stain, it was possible to
determine which bacteria was gram positive or gram negative. This is important
because gram-negative bacteria are generally more toxic (due to the
lipopolysaccaride) are resistant to antibiotics than the gram-positive
bacteria. Methods The materials used for this lab were: 1. A light microscope
2. Four glass slides 3. And inoculating loop 4. A Bunsen Burner 5. Bacteria
(E.coli, S. megaterium, B. subtilis, and S. marcesens) 6. Alcohol 7. Staining
bowl 8. Methylene blue, oil, nigrosin, crystal violet, iodine, safranin
9. Distilled water Three different staining procedures were then used for all
four types of bacteria. The directions for each staining process can be found
on pages 18-19 of the lab manual. For simple stain, bacteria was removed with
a sterile inoculating loop and placed on a glass slide. Methylene blue was
then applied until the slide was covered. Distilled water was then poured on
the slide until the methylene blue was removed. The slide was allowed to dry.
Next, oil was placed on the slide so that the oil objective lens on the light
microscope was employed. The bacterium was viewed and a sketch was made. For
the negative stain, a loopful of bacteria was placed on a slide. A drop of
Nigrosin solution was placed next to the bacteria, and the blood smearing
technique learned in the first lab was applied to cover the slide with
nigrosin solution. A sketch was then made. Gram staining was more complicated.
First, two separate types of bacteria were used. One type was placed on one
slide, and the other bacteria were placed on the opposite side. Both bacteria
were also placed in the middle of the slide so they could be viewed together.
Crystal violet, iodine and safranin were all placed on the slide and washed
off with distilled water. Once dried, oil was applied to the slide, and it was
ready to be viewed. Again a sketch was made. Results The sketches were drawn
on a separate piece of paper and then revised. A table is providedE.coli B.
subtilis S. marcesans S. megaturiun Size Small, rod-shaped Small,
rod-shaped Round, very small Round, small clumps
Elevation Convex Flat Convex Convex Surface Shiny Rough Red, smooth Smooth
Color White, shiny Pale yellow Reddish-orange Light purple
Edge Wrinkled Round Round Round Discussion The basic purpose of this lab
exercise was to view the different characteristics of certain types of
bacteria. Using the oil immersion lens accomplished this task. The importance
of the lens is that it can get so close to the slide (it is basically touching
it) and gives the experimenter a great view of the desired object. The
different staining techniques were very useful. The simple stain just stains
the cells, so the basic shape of the cell is viewed. With the negative stain,
the background is stained, so the elevation, color, and surface are more
easily observed. The gram stain, dyed the bacteria either red or purple by
using the iodine, methylene blue, crystal violet, and safranin, helped
determine the difference between the gram negative or gram-positive bacteria.
If the gram stain is negative it will have a lipopolysaccharide cell wall, if
positive, it is a peptidoglycan cell wall. Once this is known, scientists can
engineer antibiotics that disrupt the bacterias process of protein synthesis.
Some different examples of this are erythromycin and tetracycline. References
Appendix- Gene Transfer in the Environment. Cambell, Neil. Biology. 4th
Edition. Benjamin/Cummings Pub. Co. 1996 Abstract The main objective of this
lab was to identify different bacteria by simple, negative, and gram staining.
To view each bacteria cell, the bacteria was transferred aseptically to a
slide, and they were then viewed by using oil immersion, by a light
microscope. From this lab, it was determined that E. coli and B. megaterium
are gram negative and B. subtilis and S. Marcesans are gram positive.
Introduction The purpose of this lab was to view the different
characteristics of bacteria by applying various staining techniques. It is
important to know the make up if a certain bacteria so an antibiotic may be
engineered to destroy the bacteria. From the gram stain, it was possible to
determine which bacteria was gram positive or gram negative. This is important
because gram-negative bacteria are generally more toxic (due to the
lipopolysaccaride) are resistant to antibiotics than the gram-positive
bacteria. Methods The materials used for this lab were: 1. A light microscope
2. Four glass slides 3. And inoculating loop 4. A Bunsen Burner 5. Bacteria
(E.coli, S. megaterium, B. subtilis, and S. marcesens) 6. Alcohol 7. Staining
bowl 8. Methylene blue, oil, nigrosin, crystal violet, iodine, safranin
9. Distilled water Three different staining procedures were then used for all
four types of bacteria. The directions for each staining process can be found
on pages 18-19 of the lab manual. For simple stain, bacteria was removed with
a sterile inoculating loop and placed on a glass slide. Methylene blue was
then applied until the slide was covered. Distilled water was then poured on
the slide until the methylene blue was removed. The slide was allowed to dry.
Next, oil was placed on the slide so that the oil objective lens on the light
microscope was employed. The bacterium was viewed and a sketch was made. For
the negative stain, a loopful of bacteria was placed on a slide. A drop of
Nigrosin solution was placed next to the bacteria, and the blood smearing
technique learned in the first lab was applied to cover the slide with
nigrosin solution. A sketch w
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