LAB REPORT

PROGRAM:

AT117

COURSE:

AQU247 (DISEASE AND HEALTH AQUATIC ORGANISM)

PRACTICAL NUMBER:

05

PRACTICAL TITLE:

GRAM STAINING

STUDENT NAME (ID):

SYAFIZ IQMAL BIN RAMLAN

LECTURER NAME:

DR. SHARIFAH RAINA BINTI MANAF

FP FRONT PAGE 2

F FONT & FONT SIZE 1

S SPACING 1
A ALIGNMENT 1
O ORGANIZATION 1
I INTRODUCTION 2
P PROCEDURE 2
R RESULTS 3
D DISCUSSION 3
C CONCLUSION 2
CITATIONS &
Ref 2
REFERENCES
TOTAL MARKS 20

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INTRODUCTION
The most widely used staining procedure in microbiology is the Gram stain, discovered
by the Danish scientist and physician Hans Christian Joachim Gram in 1884 (Colco , 2005).
Gram staining is a well-known technique which are used to differentiate two bacteria large
groups and it is based on their different cell wall constituents. The Gram stain differentiated
their procedures between Gram positive and Gram-negative groups. They differentiate by
colouring these cells red or violet. Gram positive bacteria shows a stain of the colour violet
due to the presence of a thick layer of peptidoglycan in their cell walls, and retains the crystal
violet these cells are stained with. Apart from that, Gram negative bacteria shows stain red,
which is attributed to a thinner peptidoglycan wall, and does not retain the crystal violet
during the process of decolouring (Bruckner, 2021). Gram staining is not used to identify the
presence of archaea, formerly archaeabacteria, since these microorganisms yield widely
varying responses that do not follow their phylogenetic groups (Beveridge, 2001). Gram
staining usually performed on biopsy or body fluid when one suspects an infection. Gram
stains shows the results much faster than culturing, and are mainly important when infection
would make a crucial difference in the patient's treatment and prognosis like synovial
fluid for septic arthritis (Ray & Ray, 2004).

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OBJECTIVES:

1. To differentiate between the two major categories of bacteria: Gram positive and
Gram negative.

2. To understand how the Gram stain reaction affects Gram positive and Gram-negative
bacteria based on the biochemical and structural differences of their cell walls.

MATERIALS:
 
1. Clean glass slides

2. Inoculating loop

3. Bunsen burner

4. Bibulous paper

5. Microscope

6. Lens paper and lens cleaner

7. Immersion oil

8. Distilled water

9. 18 to 24 hours cultures of organisms

REAGENTS:
 
1. Primary Stain         -     Crystal Violet

2. Mordant                 -     Grams Iodine

3. Decolourizer          -     Ethyl Alcohol

4. Secondary Stain    -     Safranin

PROCEDURE

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Procedure:

Part 1: Preparation of the glass microscopic slide

Grease or oil free slides were needed essentially to prepare the microbial smears. Grease or
oil from the fingers on the slides was removed by washing the slides with soap and water.
The slides were then wiped with spirit or alcohol. After cleaning, the slides were and placed
on laboratory towels until ready for use.

Part 2: Labeling of the slides

A circle was drawn on the underside of the slide and glassware-marking pen was used to
clearly designate the area in which the smear was prepared. The slide was labelled with the
initials of the name of the organism on the edge of the slide. Care should be taken that the
label should not be in contact with the staining reagents.

Part 3: Preparation of the smear

Bacterial suspensions in broth: With a sterile cooled loop, a loopful of the broth culture was
placed on the slide and was spread in a circular motion of the inoculating loop to about one
centimetre in diameter. Excessive spreading may result in disruption of cellular arrangement.
A satisfactory smear allowed examination of the typical cellular arrangement and isolated
cells.

Bacterial plate cultures: With a sterile cooled loop, A drop of sterile water or saline solution
was placed on the slide. The loop was again sterilized and cooled before it was picked up to
a very small sample of a bacterial colony and gently stirred into the drop of water/saline on
the slide as an emulsion was created.
Swab Samples: The swab was rolled over the cleaned surface of a glass slide.

Part 4: Heat Fixing

Heat fixing killed the bacteria in the smear, firmly adheres the smear to the slide, the sample
was allowed to more readily take up stains.

The smear was allowed to air dry.

After the smear had air-dried, hold the slide at one end and the entire slide was passed
through the flame of a Bunsen burner two to three times with the smear-side up.

The smear was then stained.

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Part 5: Gram Stain Procedure

The slide was placed with heat fixed smear on staining tray. The smear was gently flooded
with crystal violet and let stand for 1 minute. The slide was slightly tilted and gently rinsed
with tap water or distilled water using a wash bottle. The smear was gently flooded again but
this time with Gram’s iodine and let stand for 1 minute. The slide was tilted slightly and
gently rinsed with tap water or distilled water using a wash bottle. The smear appeared as a
purple circle on the slide. It was then decolorized using 95% ethyl alcohol. The slide was
tilted slightly and alcohol was applied drop by drop for 5 to 10 seconds until the alcohol runs
almost. Do not over-decolorize. Immediately rinsed with water. Flood it gently with safranin
to counter-stain and let stand for 45 seconds. The slide was slightly tilted and gently rinsed
with tap water or distilled water using a wash bottle. The slide was blow dried with bibulous
paper. The smear was viewed using a light-microscope under oil-immersion.

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RESULT

Draw and label examples of Aeromonas hydrophila and Streptococcus agalactiae. (You may
refer from the Internet sources/related sources)

1) Colony 1 : Aeromonas hydrophila

Pink strains
together

DIAGRAM 1.1 Aeromonas hydrophilla

Colony Colour Characteristics Gram

Colony pink Heterotrophic, rod-shaped bacterium Gram
1 mainly found in areas with a warm negative
climate

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2) Colony 2: Streptococcus agalactiae

Whitish grey building-up

strains

DIAGRAM 1.2 Streptococcus agalactiae

Colony Colour Characteristics Gram

Colony Whitish
2 grey It is a round bacterium that has Gram positive
capabilities to form chains

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DISCUSSION
As we can see in DIAGRAM 1.1, It is the most famous species of Aeromonas. It is resistant
to most common antibiotics and cold temperatures and is oxidase- and indole-
positive. Aeromonas hydrophila also has a special symbiotic relationship as gut flora inside
of certain leeches, such as Hirudo medicinalis (Sawyer , 2020). Aeromonas hydrophila are
Gram-negative bacteria, rounded ends with straight rods and has a bacilli to coccibacilli
shape. Usually the size ranges from 0.3 to 1.0 μm in width, and 1.0 to 3.0 μm in length. They
are possible to grow at low temperatures as low as 4 °C. These bacteria are moved by using
a polar flagellum. Due to its unique structure, it is very deadly as it is toxic to most
organisms. It will initiate by entering the body of its victim travelling through the bloodstream
to the first available organ. It produces aerolysin, a cytotoxic enterotoxin that can cause
tissue damage. A. hydrophila is considered to be opportunistic pathogens, meaning they
only infect unhealthy individuals. A. hydrophila is mostly known and considered as a major
fish pathogen and its harm and danger towards humans has been identified for the past few
decades. The genomic insights of aeromonads could be the next step into understanding of
them (Tan, Yin, & Chan, 2015).

In DIAGRAM 1.2, S. agalactiae in general, can also be called group B

streptococcus or GBS which is a gram-positive cocci or round bacterium having the
capability to form chains (as reflected by the genus name Streptococcus. It is a harmless
commensal bacterium being part of the human microbiota colonizing the genitourinary and
gastrointestinal tract of up to 30% of healthy human adults which is asymptomatic carriers.
Moreover, GBS can cause invasive infections that is severe especially in newborns, the
elderly, and people with not many or absence of immune systems (Edward & Baker , 2010).
S. agalactiae is also a well-known veterinary pathogen, because it can cause inflammation
of the udder in dairy cows. The species name agalactiae meaning "of no milk", alludes to this
(Keefe, 1997).

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POST-LAB QUESTIONS
1) Make a flow of the Gram staining steps.

Iodine Counter strain

Fixation Crystal violet Decolorisation
treatment with Safranin

2) Describe the function of each of the following in the Gram stain:

i. Mordant
 mordant is the crystal violet-iodine complex so that the dye cannot be removed
easily
 is used to tensifiy the colour of primary skin
ii. Primary stain
 to impart its color to all cells
iii. Decolorizer
 A decolorizer such as ethyl alcohol or acetone is added to the sample, which
dehydrates the peptidoglycan layer, shrinking and tightening it.
 The large crystal violet-iodine complex is not able to penetrate this tightened
peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.
iv. Counterstain
 It allows the identification of Gram-negative bacteria as well. An alternative
method uses dilute carbofluozide.

3) Which step in the Gram stain is most likely to cause poor results if done incorrectly?
the decolorizer step is the most critical because too much destaining reagent can remove
the dye-mordant complex from the cells, which makes the gram-positive cells appear to be
falsely gram-negative.

4) Why must fresh bacterial cultures be used in a Gram stain?

Gram stain is reliable only on cells from cultures that are in the exponential phase of growth.
Older cultures contain more ruptured and dead cells. Cells from old cultures may stain
Gram negative even if the bacteria are Gram positive.

5) Briefly describe the mechanism of Gram staining.

Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye
during solvent treatment.  Bacteria cell walls are stained by the crystal violet. Iodine is
subsequently added as a mordant to form the crystal violet-iodine complex so that the dye
cannot be removed easily.

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CONCLUSION
I can conclude this lab report by learning how to differentiate the 2 major bacteria categories
which is the Gram-positive and Gram-negative by knowing its function, uses and the
importance of gram staining in identifying pathogens presence in organisms. I have also
learned to understand about how the Gram stain reaction occurs and how does it effects
gram positive and gram negative bacteria based on the biochemical and structural
differences on the cell walls. This is all important for the future as it makes the work easier
once you have known the way to apply it.

REFERENCES

Beveridge, T. J. (2001). "Use of the Gram stain in microbiology". Biotechnic & Histochemistry. , 111-
118.

Bruckner, M. Z. (2021, January 14). MIcrobial Life. Retrieved from Educational Purposes:
https://serc.carleton.edu/microbelife/research_methods/microscopy/
gramstain.html#:~:text=Gram%20staining%20is%20a%20common,these%20cells%20red
%20or%20violet.

Colco , R. (2005). "Gram Staining". Current Protocols in Microbiology. Appendix 3C.

Edward, M., & Baker , C. (2010). "Streptococcus agalactiae (group B streptococcus)". In Mandell GL,
Bennett JE, Dolin R (eds.). Principles and practice of infectious diseases (7th. ed.). Elsevier.,
202.

Keefe, G. (1997). "Streptococcus agalactiae mastitis: a review". Can Vet J., 199-204.

Ray, K. J., & Ray, C. G. (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill, 223.

Sawyer , R. T. (2020). "LEECH BIOLOGY AND BEHAVIOUR Volume II Feeding, Biology, Ecology and
Systematics". Biopharm Leeches.

Tan, W.-S., Yin, W.-F., & Chan, K.-G. (2015). "Insights into the Quorum-Sensing Activity in Aeromonas
hydrophila Strain M013 as Revealed by Whole-Genome Sequencing". Genome
Announcements. , 1-2.

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