LAB 5 - MICROBIOLOGY

( GRAM STAINING )

INTRODUCTION

The gram stain is a differential stain commonly used to differentiates bacteria on the basis of

their cell wall structure. The gram stain is key procedure in the identification of bacteria based

on staining characteristics, and enable the bacteria to be examined using light microscope.

Most bacteria can be divided into 2 groups based on the cell wall structure.

1. Gram positive cell walls have single membrane enclosed by thick, cross link

peptidoglycan. When the dye and mordant complex forms, it adheres firmly to thick

peptidoglycan layer. Alcohol cannot wash out the dye-mordant complex because it

firmly attached in the thick peptidoglycan layer. It has stain dark purple due to

retaining the primary dye ( Crystal Violet ) in the cell wall. Staphylococcus aureus is

the example for gram positive bacteria.

2. Gram negative cell walls have a thin layer of peptidoglycan in the periplasmic space

within its inner and outer lipid membrane. When dye and mordant complex forms, it

does not adhere to the thin layer of peptidoglycan. The dye-mordant complex is easily

removed the peptidoglycan layer with alcohol. It will stain red or pink due to retaining

the counter staining dye ( Safranin ). Escherichia coli is the example for gram negative

bacteria.
OBJECTIVE

 To differentiate between the 2 major categories of bacteria which is gram positive and

gram negative.

 To understand how gram stain affects the gram (+) and gram (-) based on the

structural differences of their cell wall.

METHODOLOGY

1. Firstly, we added 1 drop of sterile water to the slide.

2. Next, we chose staphylococcus aures and smear it to the slide.

3. Then, allow the smear to dry using heat or air dry.

4. We covered the smear with primarily stain ( Crystal Violet ) for 1 min. After 1 minute,

we washed the slide with water.

5. We added the mordant ( iodine ) also for 1 min, after that wash it with water.

6. Using 95% ethanol, we decolorized the smear until the purple color has stopped

leaching off the slide. Wash it immediately with water.

7. We covered the smear with counter staining dye ( Safranin ) for 30 seconds.

8. We washed both the top and bottom of the slide with water.

9. Lastly, using the light microscope to view the smear up to 100×.

RESULT

Gram positive bacteria - staphylococcus aures

Color : Dark Purple

Categories : Gram-positive

Morphology : Cocci
DISCUSSION

In this lab, we want to differentiate between the two major categories of bacteria

which is grap positive and gram negative. We chose S. aures as our experimental species.

S.aures is a facultative anaerobic that can grow without need of oxygen and coccal (round)

bacterium. Although S. aures is not always pathogenic, it is a common cause of skin and

respiratory infections also food poisoning. After we examined the bacteria under light

microscope, we identifying the bacteria to be gram- postive bacteria as it stains purple in

colour.

Gram-positive differ from gram-negative through their cell walls structure and

composition. Gram-positive bacteria have a thick cell walls made up of peptidoglycon layer

beyond the plasma membrane which is cross linked by short chains of peptides. It is then

resulting in the shape and rigidity of the cell wall. The thick layer of peptidoglycan allows to

retain the dye-mordant complex ( crystal violet and iodine ). The alcohol cannot wash out the

dye-mordant complex because it firmly secured in the thick layer of peptidoglycon. As a

result the cells are stain as purple.

CONCLUSION

Staining is a technique used to enhance the clarify of the microscopic image. The most

commonly used staining procedure in microbiological laboratory. Gram stain is a very

important preliminary step in the classification and characterization of bacteria. Gram stain

procedure enables bacteria to retain the color of stains based on the differences in the

composition of their cell walls. From this experiment, we can differentiate between the two

major categories ( gram-positive and gram-negative ).

REFFERENCES

1. Laboratory Manual, Dr Shakinaz Desa

2. http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1

3. http://www.microscopemaster.com/gram-stain.html

4. https://microbeonline.com/gram-staining-principle-procedure-results/