UNIVERSITY TUNKU ABDUL RAHMAN

UDEE 2114

MICROBIOLOGY

YEAR 2 SEMESTER 3

NAME: CHEW HUI SIN ID NUMBER: 09ANB08196NAME OF GROUP MEMBERS: NGOI JIANG CHIN

HING YI SHIEN YAP YE VONE

FACULTY: SCIENCECOURSE: BIOCHEMISTRYLECTURER’S NAME: Ms TEO KAH CHENG DATE: 25TH JAN 2011SESSION: 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 coliWhite 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 coliRod-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 coliStained in pink, Gram-negative bacteria

Micrococcus luteusStained 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