Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Scribd
Upload
361 views

Lab Report 3

This practical report describes an experiment to identify bacteria based on Gram staining. The objectives were to differentiate between Gram-positive and Gram-negative bacteria by color and shape under a microscope. The methodology described performing Gram staining on bacterial samples and examining them under an oil immersion microscope. Gram-positive bacteria appeared purple and Gram-negative appeared pink. Shapes observed included rods and spheres. The discussion explained how Gram staining works based on differences in bacterial cell wall structure and composition.

Uploaded by

nurul ain
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
361 views

Lab Report 3

This practical report describes an experiment to identify bacteria based on Gram staining. The objectives were to differentiate between Gram-positive and Gram-negative bacteria by color and shape under a microscope. The methodology described performing Gram staining on bacterial samples and examining them under an oil immersion microscope. Gram-positive bacteria appeared purple and Gram-negative appeared pink. Shapes observed included rods and spheres. The discussion explained how Gram staining works based on differences in bacterial cell wall structure and composition.

Uploaded by

nurul ain
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 5

BGY3100

Biology of Microorganisms
First Semester 2022/2023

PRACTICAL REPORT

NAME : NURUL AIN BINTI MOHAMED


SO’ADI
MATRIC NO : 216625
TITLE OF REPORT : IDENTIFICATION OF BACTERIA
BASED ON GRAM STAINING
LAB : 20
LECTURER : DR. NURUL IZZA BINTI ABD GHANI
DEMONSTRATOR : LUQMAN HAKEEM BIN HUZAIRI
INTRODUCTION

Bacteria are small single-celled organisms.Bacteria only can see through oil immersion
objective lens on microscope with 100 magnification.Bacteria has three common shape
which is spherical (coccus),rod-like (bacillus) and curved (vibrio,spirillum or
spirochete).Bacteria have cell walls,fatty acids in their membrane and bacteria lack
histones.The cell wall of bacteria made of peptidoglycan,also known as murein and
consisting of cross-linked polysaccharide chains.Cell wall can maintain the integrity of
cell and shape during growth and protecting cell from mechanical stress.

Gram stain is a frequent method used to distinguish between two major groups of
bacteria based on the components of their various cell wall.The two major types of
bacteria are gram negative and gram positive bacteria.Based on Bergey’s manual of
systematic bacteriology David Bergey created that gram staining properties are generally
considered phenetic and they also play role in phylogenetic classification of microbes.A
Danish physician,Christian Gram developed that gram stain is the most widely employed
staining method in bacteriology in 1984.

As we know,cell wall can differentiate which is a gram positive bacteria and gram
negative bacteria.For gram positive bacteria,the cell wall is thick like a 20nm to 80nm
that composed mainly peptidoglycan.For gram negative bacteria,the cell wall is thin like
for peptidoglycan inner layer is 2nm to 7nm and outer layer of lipid,protein and
lipopolysaccharide are 7nm to 8nm.The cell shape of gram positive bacteria are
spheres,rod or filaments while gram negative bacteria are spheres,ovals,straight or curved
rods,helices or filaments.The purple crystal violet used to stain the cells of gram positive
bacteria is retained by the presence of thick layer of peptidoglycan in cell walls.In
contrast,gram negative bacteria stain pale reddish because their peptidoglycan walls are
thinner and there is outer membrane that protect the peptidoglycan from the stain.Acetone
treatment made the outer membrane permeable.

OBJECTIVES

1.To differentiate between Gram-Positive and Gram-Negative Bacteria baswed on color and shapes
through microscope and to understand function of gram staining.

2.To examine bacteria through microscope and able tomeasure the size of them through compound
microscope using ocular micrometer.
METHODOLOGY

I.Gram Staining

1. The slide was smeared with bacteria by using inoculating loop.


2. The slide was allowed to air dry then was fixed culture by passing the slide rapidly for two or three
times through Bunsen flame.
3. Then it was placed on staining rack.
4. The smeared slide was stained with the basic dye crystal violet-ammonium oxalate solution for 1
minute.
5. The stain was poured off and was washed for 4 seconds in a gentle stream of tap water no more stain
could be seen.Then blotted dry on paper towel lightly.
6. Slide was flooded with Lugol’s iodine solution for 1 to 2 minutes.
7. Lugol’s iodine was poured off and washed in gentle stream of tap water and blotted dry on paper
towel lightly.
8. The slide was decolorized by washing it with 95% ethanol and was blotted dry.
9. Then it was washed thoroughly with water to remove alcohol.
10. It was washed in running tap water.
11. Excess water was blotted and blotted dry on paper towel.
12. The smears was examine under oil immersion (100x) and the bacteria was recorded as Gram positive
or Gram negative.
13. The morphology and relatives size of cells was drew.
14. Grid line was converted into µm.

II.Examination of Bacteria from the Prepared Slides

1. The shapes and the arrangements of bacteria was observed by using microscope under oil immersion
obejective (100x)
2. The bacteria cells was drew and labelled.
3. Then,the grids was used on ocular micrometer,the size of bacteria was measured in terms of number
grid line.
4. This grid line was converted into µm.
DISCUSSIONS

The ability of the bacterial cell wall to retain the crystal violet dye is a basic principle
for gram stain and there is a thick and thin peptidoglycan cell wall.So,this make a
difference in gram staining result which are gram positive bacteria or gram negative
bacteria.
Crystal violet is used as a main stain.Crysal violet dye is positively charged,it sticks to
the both gram positive bacteria and gram negative bacteria cell walls.It also add the
contrast of both gram positive bacteria and gram negative bacteria and giving them a
purple appearance.
Iodine can differentiate either it gram positive or gram negative bacteria.The role of
iodine as a mordant make crystal violet penetrates and adheres to the gram positive
bacteria and the dye remain purple.If its gram negative bacteria the iodine will wash
away the dye and stain colour will be pink.
Alcohol is used to provide the colour differentiation.Alcohol used by rinsing the cells
then it will broke down the lipids removed it.Gram negative bacteria lose the ability to
keep the crystal violet dye,it will turn them to colourless.Ethanol is one of the reagent
that used for gram staining.Since ethanol is non polar solvent,it can easily pass through
the cell wall of gram negative bacteria and dissolve the crystal violet complex.
Safranin is a biological stain that employed in histology and cytology.Safranin also
cheaper and safer lab stain.In some staining procedures, safranin is employed as a
counterstain to colour the cell nuclei red. This is the standard counterstain for endospore
and gram stains.In Gram's staining, the decolorized bacteria are directly stained by the
safranin. Gram-negative bacteria and gram-positive bacteria can be easily separated
with safranin staining.
For comparison,the gram staining results is same to the prepared slide where is the
shape observed is rod shape for Salmonella typhi,Escherichia colli and Bacillus cereus
and the spherical shape for Staphylococcus aureus.
CONCLUSION

At the end of this experiment,we success to differentiate between Gram-Positive and


Gram-Negative Bacteria based on color which are the Gram-Positive Bacteria is purple
stain and the Gram-Negative Bacteria is pink stain.The shapes of both Gram-Positive
Bacteria and Gram-Negative Bacteria are rod and spherical shape.We also got to know
the function of Gram staining which is distinguish Gram-Positive and Gram-Negative
Bacteria based on the components of their various cell wall.
Then,bacteria can be examined through microscope and we able to measure the size of
them through compound microscope using ocular micrometer.

REFERENCES

1. Bacteria. (n.d.).Genome.gov.Retrieved November 2,2022,from https://www.genome.gov/genetics-


glossary/Bacteria
2. Wikipedia contributors.(2022,September 21).Cell wall.Wikipedia.Retrieved from
https://en.wikipedia.org/wiki/Cell_wall
3. Monica Z.Bruckner.(2021,January 14).Gran Staining.Retrieved from
https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
4. Bacteria,Cell, Evolution, & Classification. (2022, October 6). Encyclopedia Britannica.Retrieved
from https://www.britannica.com/science/bacteria/Diversity-of-structure-of-bacteria

You might also like