UNIVERSITY TUNKU ABDUL RAHMAN

UDEE 2114

MICROBIOLOGY

YEAR 2 SEMESTER 3

NAME: CHEW HUI SIN

ID NUMBER: 09ANB08196
NAME OF GROUP MEMBERS: NGOI JIANG CHIN
HING YI SHIEN
YAP YE VONE
FACULTY: SCIENCE
COURSE: BIOCHEMISTRY
LECTURER’S NAME: Ms TEO KAH CHENG
DATE: 25TH JAN 2011
SESSION: 11:30AM– 01:30 PM
Title:
1. Ubiquity of microorganism
2. The staining of microorganism
Objective:
 To study the bacterial colony morphology and the morphology of individual

bacteria

 To learn the technique in preparation of smear for staining.

 To learn the four technique of staining of microorganisms: Basic staining, acidic

staining, differential staining, and endospore staining.

Result:
Experiment 1

Escherichia coli
White and smooth translucence colonies

Micrococcus luteus
punctiform opaque and smooth colonies with bright yellow, non-diffusable pigment.
Experiment 2
Part A: Direct Staining with Basic Dyes

Escherichia coli
Rod-shaped

Micrococcus luteus
Spherical shape
Part B: Negative or Indirect Staining (Acidic Dyes)

No results were obtained in this part.

Part C: Differential Staining Techniques (Gram Stain)
Escherichia coli
Stained in pink, Gram-negative bacteria

Micrococcus luteus
Stained blue, Gram-positive bateria

Part D: Structural Stains – Endospore Stain

Bacillus Cereus
Red vegetative cell with green endospore.
Discussion
Different bacteria will have different type of colony morphologies and
characteristics that are specific to the genus and species of the organism. From the result
of experiment 1, Escherichia coli form white and translucence colony. While
Micrococcus luteus growth as a yellow opaque colony on nutrient agar. It is smooth and
punctiform on the agar. Micrococcus luteus which carried the meaning of spherical
(coccus) shape and yellow (luteus) in color.
Staining is a method that commonly uses to differentiate the cell and define the cell
structure. Direct staining is a method stain the culture by using basic dye. Basic dye is a
stain that is cationic which are positively charged and so will react with material that is
negatively charged. The cytoplasm of all bacterial cells have a slight negative charge
when grown in a medium of near neutral pH and will therefore attract and bind with basic
dyes. Thus, the culture with basic dye will stain with colour when examine it under a
microscope. On the other hand, acid dye such as Nigrosine are using in this experiment as
an indirect staining. The acidic dyes contain negatively charges in the solution. So, when
it added to the negatively charged culture, the culture will repelled with the dye.
Therefore, the background of the culture was stained but not the cultures itself.

The two method above are use to determine the structure of the culture under the
microscope. For the basic dye, Escherichia coli found to be typically rod-shaped
(bacillus)
Micrococcus luteus has shown us spherical shaped (coccus). We fail to get the result for
the acidic dye part. This might cause by the washed away of the culture during the
staining process. We should practice and masteries more on the preparation of smear.
Gram stain is a type of stain commonly uses to differentiate the bacteria in to 2
different groups (gram-positive and gram-negative) based on the physical properties of
their cell walls. In this experiment, the Escherichia coli has been stained pink color as it
is a gram-negative culture. Whereas, the Micrococcus luteus is a gram-positive culture as
it was stain in blue color. This is because the Micrococcus luteus have a thick mesh-like
cell wall made of peptidoglycan (50-90% of cell wall), which stains purple, whereas
Gram-negative bacteria like Escherichia coli have a thinner layer (10% of cell wall),
which stains pink. Gram-negative bacteria also have an additional outer membrane that
contains lipids, and is separated from the cell wall by the periplasmic space. Crystal
violet (CV) dissociates in aqueous solutions into CV + and chloride (Cl – ) ions. These ions
will penetrate through the cell wall and cell membrane of both Gram-positive and Gram-
negative cells. The CV+ ion interacts with negatively charged components of bacterial
cells and stains the cells purple. The iodine (I – or I3 –) will then interacts with CV+ and
forms large complexes of crystal violet and iodine (CV–I) within the inner and outer
layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that
prevents the removal of the CV-I complex and, therefore, colors the cell in to blue in
color.

When a decolorizer such as alcohol or acetone is added, it interacts with the

lipids of the cell membrane. A Gram-negative cell will lose its outer lipopolysaccharide
membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are
washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-
positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes
become trapped within the Gram-positive cell due to the multilayered nature of its
peptidoglycan. The decolorization step is critical and must be timed correctly. The crystal
violet stain will be removed from both Gram-positive and negative cells if the
decolorizing agent is left on too long. After decolorization, the Gram-positive cell
remains purple and the Gram-negative cell loses its purple color. Counterstain, which is
usually positively charged safranin is applied last to give decolorized Gram-negative
bacteria a pink or red color.

The Vegetative cells are bacteria that are actively growing, metabolizing and
dividing. When vegetative cells are subjected to environmental stresses such as nutrient
deprivation they eventually die. However, some bacteria such as the Bacillus spp. and
the Clostridium spp can circumvent the problems associated with environmental stress by
forming endospores. Endospores are dormant or metabolically inactive forms of a
bacterium that allow it to survive the harsh environmental conditions. Spores are
resistant to heat, UV radiation and chemicals because they are comprised of a tough
proteinaceous covering called keratin. A primary stain such like malachite green is used
to stain the endospores. Because endospores have a keratin covering and resist staining,
the malachite green will be forced into the endospores by heating. In this technique
heating acts as a mordant. Water is used to decolorize the cells; as the endospores are
resistant to staining, the endospores are equally resistant to de-staining and will retain the
primary dye while the vegetative cells will lose the stain. The addition of a counterstain
or secondary stain, safranin is used to stain the decolorized vegetative cells. Thus when
viewing Bacillus Cereus under a microscope we can see When the vegetative cells that
contain endospores should stain pink while the spores should be seen as green ellipses
within the cells.

Reference
1. Education that Works (2010) Micrococcus luteus and M. roseus. Retrieved on 13
February 2011 from Education that Works web site
http://www.sunysccc.edu/academic/mst/microbes/09mlute.htm

2. Today’s Online Textbook of Bacteriology (2008) The Normal Bacterial Flora of

Humans. Retrieved on 13 February 2011 from Today’s Online Textbook of
Bacteriology web site http://www.textbookofbacteriology.net/normalflora.html

3. TheLabRat.com (2005) Micrococcus luteus. Retrieved on 14 February 2011 from

TheLabRat.com web site
http://www.thelabrat.com/restriction/sources/Micrococcusluteus.shtml