Like archeans, bacteria are prokaryotic cells.
This means that they are single-celled organisms without a nucleus membrane
(nuclear envelope). While they are very small, they are diverse and vary in
shape and size.
Before preparing for microscopy,
bacteria are grown in culture media. This helps enhance cell division and thus
increase their numbers.
Some of the media used include:
Basal Media (e.g. nutrient broth and peptone water) - This
is used for such bacteria as Staphylococcus that do not require special
enrichment for growth
Selective Media (e.g. Lowenstein-Jensen
media) - This type of media is used to grow specific types while
Transport Media (e.g. Amies medium)
Transport media are particularly useful in instances where the sample has to be
preserved. This also prevents the bacteria from overgrowing before it is
transported to the desired destination (laboratory etc).
Enriched Media (e.g. with added blood) - Enriched media
contain a special component added to the media to favor the growth of given
bacteria. For instance, the addition of blood in a given media supports the
growth of Streptococci.
Using the marking pencil,
mark a circle at the center of a clean slide to mark a spot for the smear
Using either a clean
dropper or the inoculating loop (make sure to flame the loop using the Bunsen
burner) place a drop of distilled water in the marked spot on the slide (if the
media being used is broth, then distilled water is not necessary)
Pass the inoculating loop
through the flame again and allow to cool before scooping (the surface of the
culture) a small amount of the sample from the tube or Petri dish with the
culture (to prevent contamination of the remaining sample, pass the tips of the
tube through the flame before covering it with the lid)
Gently mix the sample with
the drop of water on the slide to create a smear
Place the slide on the
drying rack and allow it to dry completely (air dry)
Fix the smear by passing
the slide over the flame several times (about 3 times) - Fixing enhances stain
penetration into the cells
Bacteria Staining Procedure
Place the slide on a
staining rack and cover with either of the following stains (gram stains) for
Run a gentle stream of
water along the slide to remove excess stain
Wipe away excess water and
stain on the edges of the slide using a absorbent paper
View under the microscope
starting with low power (for high power, add immersion oil)
Depending on the sample under investigation,
students will have the opportunity to observe and identify the size and shape
of the bacteria. They are categorized according to their shape (Morphology)
and the how they stain (gram positive and gram negative bacteria).
There are several types based on
their general appearance (shape) including:
Cocci (coccus) bacteria are some of the most
common bacteria. They are spherical (or ovoid at times) in shape and are
divided into; diplococcus (occur in pairs such as Neisseria spp), streptococcus
(occur as a long chain or cells such as Streptococcus pneumoniae) and
staphylococcus where they occur in clusters (e.g. staphylococcus
saprophyticus). Cocci may also occur in tetras or in packets of 8 to
form a structure that appears like a cube such as the sarcina bacteria.
Unlike coccis bacteria, bacillus will appear as elongated
rods (rod-like) when viewed under the microscope. In most cases, the bacilli occur as single cells (e.g. Mycobacterium tuberculosis), but may occur
in pairs (diplobacillus) or form chains commonly refered to as streptococcus
(e.g. Bacillus cereus).
Whereas vibrio bacteria appear comma shaped, spirilla are the
type that appear spiral in shape. While some people may confuse the
two when viewed under the microscope, they are different when students
compare them under high magnification.
Example of vibrios includes vibrio
vulnificus and Vibrio harveyi while some examples of Spirilla include
members of the Campylobacter species.
* Spirilla have flagella (on both
ends), which they use to move in water or aquatic environment. This allows them
to move faster in such environments compared to other bacteria. On the other
hand, Vibrio have a flagellum on one end that allows them to move
Some of the other types of bacteria based on
spiral bacterial) e.g. Spirochaetales – Spirochetes have an axial
filament that allow them to move in a type of motion that has been described as
Mycoplasmas (lack cell wall
and may appear as elongated cells with a thicker central section or spherical)
e.g. Mycoplasma hominis
Bacteria can also be grouped according to how
Gram negative are the type of bacteria
that do not retain the primary stain. During decolorization, these bacteria
lose the crystal violet stain (primary stain) because they have a thin
Peptidoglycan layer. However, they take up the counter stain (safranin) and will appear reddish or pink when viewed under the microscope.
of gram-negative include Salmonella spp, Escherichia coli (E. coli)
and the Enterobacteriaceae spp.
Unlike gram-negative, gram positive
bacteria have a thick peptidoglycan layer that allows them to retain the
primary stain/dye (crystal violet stain). Fixing with heat allows the stain to
penetrate the layer, which is then retained even when the cells are washed
When viewed under the microscope, gram-positive appear
purple or bluish in color. Examples of gram-positive include;
Staphylococcus epidermidis, Bacillus cereus and Clostridium botulinum among
The main methods of reproduction among bacteria
Binary fission - Binary fission is the main method of
reproduction. The process starts with the cell growing in size and
then splitting into two separate cells.
Before the cell splits into two, the
genetic material has to be copied and separated into two copies that move to
the polar ends of the cell before the cell cytoplasm separate followed by cell splitting.
Budding - When viewing under the microscope, students may be
able to see a small protrudence on the surface of some of the cells (bud). For
some bacteria, budding is a means of reproduction.
This involves the formation
of a bud on one end of the cell surface followed by replication of the genetic material.
A copy of the material gets in to the bud as it enlarges and ultimately breaks
off and separates from the parent bacterial cell.
Formation of endospores - This is a form of
asexual reproduction among gram positive bacteria particularly during extreme, unfavorable
During such conditions, part of the protoplast is transported near to the chromosome and ultimately surrounded by a touch cell wall forming the
endospore. This allows the endospore to survive extreme conditions.
conditions (presence of moisture, nutrients and the right temperature) the
tough wall is broken down allowing a new bacteria cell to grow from the
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