1. specimen will be allowed to air

1.       Why
is staining important in the process of identifying bacteria?   

Staining is important in order to
identify bacteria and other microbes because cells, due to their high water
content, are naturally translucent. Light will pass through them. Contrast is
needed in order to visualize and identify cells. Staining is the simplest way
to increase contrast in order to visualize cells.

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2.       Describe
the difference between differential and simple stains.

A simple stain,
using a single staining agent, provides contrast to a biological specimen,
allowing a normally transparent biological sample to be visualized with a light
microscope. While a single stain allows cells to be differentiated from the
background, it does not give very much information about the cellular
structures or properties; cells of different species can have different
physical and chemical properties. These differences can be exploited using
staining techniques so that cellular differences can be easily visualized under
a bright field microscope. Differential stains use two or more different types
of stains to show the different parts of the microorganism. 

3.       Prior
to staining the bacteria must be heat fixed to the slide.  Why is this process important?  Briefly describe this process.

In order to stain
microbiological specimens, the organisms must be adhered to the microscope
slide. The easiest way to adhere organisms to a glass slide is to heat the
slide. Prior to heat fixing, organisms must be applied to the glass slide. This
is done using the wet mount technique. Once applied, the specimen will be
allowed to air dry (5-15 min.). Once air dry, the slide must be heated to
adhere the cells to the slide. Ignite a Bunsen burner. Quickly pass the center
of the slide, where the specimen is located, over the flame 2 to 3 times. The
slide should be warm. The temperature can be tested by moving the slide close
to the back of the hand; do not touch. If there is warmth from the slide, it
has been heated sufficiently. If the slide is over-heated, or the specimen was
not allowed to air dry, the cells will burst, ruining the specimen.


4.       a.  Name the components of the Gram Stain and
briefly describe the function of each of the components.

Primary stain: The basic dye crystal violet is
applied as the primary stain to a heat-fixed specimen. Crystal violet is
soluble in water, and when applied to a specimen smear, will quickly make its
way through the cell walls of bacteria.


Mordant: After the dye has been allowed to
absorb and rinsed off, a mordant (Gram’s iodine) is applied to the specimen.
The mordant helps the crystal violet to “set” into the bacterial cell wall (a
mordant helps fix a dye).


Decolorizer: After the mordant has been given
time to work, with the excess being rinsed off, a decolorizing agent (ethanol
or acetone) is used to remove the primary stain. If the stain has not set, then
crystal violet will be washed off by the decolorizing agent. If it has set,
primarily due to the action of the mordant, then the cell will remained colored
with crystal violet.


Counter Stain: Once
the decolorizing agent has been removed, a counter stain, safranin, is applied
to the specimen. After decolorization, cells that are unable to retain crystal
violet become colorless, therefore the counter stain will colorize the cell.
Safranin does not colorize cells with crystal violet.


Which step is most likely to cause poor results if done incorrectly?

Decolorizer; the decolorizing agent
can eventually remove even a set-in stain. Thus, it is critical that the
decolorizing agent is only in contact with the specimen for a brief period of


5.       Name
the components of the Acid-Fast Stain and briefly describe the function of each
of the components. 


Primary Stain: The primary stain
used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains
phenol, which helps the stain penetrate the waxy cell wall.  This is
further assisted by the addition of heat. 


Decolorizer: The smear is then
rinsed with a very strong decolorizer, acid alcohol, which strips the stain
from all non-acid-fast cells but does not permeate the cell wall of acid-fast


Counter Stain: The decolorized
non-acid-fast cells then take up the counterstain, methylene blue.


b.  Name the medically important
bacteria for which the acid-fast stain is used.

The acid-fastness of Mycobacteria
is due to the high mycolic acid content of their cell walls, which is responsible
for the staining pattern of poor absorption followed by high retention.