A Lab Report under the subject of Microbiology. Done as a lab session in Josai University, Japan during a twinning program on 2014. Created by: Annisa Hayatunnufus Bachelor of Pharmacy Management & Science University

1. PHARMACEUTICAL SCIENCES
Subject: MICROBIOLOGY
Lab Report:
Isolation of Pure Culture, Gram-staining,
and Microscopic Observation
Prepared by:
ANNISA HAYATUNNUFUS
ID number:
012014052438 (MSU) – PF14012 (JOSAI)
Lecturer’s Name : ProfessorKondoSeiichi
Date of submission :

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CONTENT
I. Objectives..............................................................................3
II. Introduction.................................................................................................................3
III. Materials & Apparatus.......................................................................................5
IV. Procedures ..........................................................................5
V. Results .................................................................................7
VI. Discussion ...........................................................................8
VII. Reference.........................................................................10

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I. OBJECTIVES
1. Isolating pure culture from contaminated sample and extracting an independent
single colony.
2. Classifying microorganisms into gram-positive or gram-negative through staining
reaction in order to distinguish the permeability of its cell wall for selection of anti-
bacterial agent.
3. Determination of microorganism’s characteristics by microscopic observation.
4. Identification of microorganism species from obtained characteristics.
II. INTRODUCTION
Staining is an auxiliary technique used in microscopic techniques that have the
function to enhance the clarity of the microscopic image. Stains and dyes are widely
used in the scientific field to highlight the structure of the biological specimens, cells,
tissues etc.
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.
Gram staining is a differential staining technique that differentiates bacteria
into two groups: gram-positives and gram-negatives. The procedure is based on the
ability of microorganisms to retain color of the stains used during the gram stain reaction.
Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary
stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain as
purple. After decolorization step, a counterstain is used to impart a pink color to the
decolorized gram-negative organisms.
Figure 2.1. Colour changes that occur at each step in the staining process

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Despite the major classification of these two groups, several bacteria are classified
as the gram-indeterminate bacteria. Also known as gram-variable bacteria, this type of
bacteria do not respond predictably to Gram staining and, therefore, cannot be
determined as either gram-positive or gram-negative. They tend to stain unevenly,
appearing partially gram positive and partially gram negative, or even unstained. Staining
older cultures (over 48 hours) can lead to false gram-variable results, probably due to
changes in the cell wall with aging. Gram-indeterminate bacteria are best stained using
acid-fast staining techniques. Examples include many species of Mycobacterium,
including M. tuberculosis and M. leprae.
The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria based
on staining characteristics, enabling the bacteria to be examined using a light
microscope. The bacteria present in an unstained smear are invisible when viewed
using a light microscope. Once stained, the morphology and arrangement of the bacteria
may be observed as well. Furthermore, it is also an important step in the screening of
infectious agents in clinical specimens such as direct smears from a patient.
The samples of microorganisms that are going to be tested are Escherichia coli,
Stapylococcus aureus, and Candida albicals. E.coli (rod-shape) and S.aureus (spherical
form) are both bacteria. Meanwhile, Candida albicals (yeast or mycelial form) is a fungi
and with S.aureus, both are commonly known as a gram-positive microorganism. Hence,
in the sample, E.coli will be the only microorganism that is classified as gram-negative.

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III. MATERIALS & APPARATUS
MATERIALS APPARATUS
Artificially mixed suspension of bacteria &
fungus (Escherichia coli, Stapylococcus
aureus, Candida albicals)
Simple general tools (tissue, hand gloves,
etc.)
Solid medium (Agar plate)
Simple laboratory tools (test tubes, racks,
pipettes, gas burner, etc.)
Sterilized saline Incubator
Crystal violet solution Platinum loop
Lugol solution Platinum needle
Ethanol Filter paper
Fuchsine solution Slide glass
Distilled water Microscope, lens cleaner, immersion oil
IV. PROCEDURES
1. Isolation of Bacterial Strain (Pure Culture) from Mixed Specimen
- Sterilize the platinum loop and cool it down.
- Take one loop of bacterial suspension.
- Spread on the solid medium (agar plate) as shown on
figure 4.1.1.
- Sterilize the platinum loop and cool it down. Spread
once more on the solid medium (agar plate) as shown
on figure 4.1.2.
- Sterilize the platinum loop again, and then cool it down.
Spread one last time on the solid medium (agar plate)
as shown on figure 4.1.3.
- Sterilize the platinum loop, cool it down, and store it
back. Meanwhile, close the medium with its lid and
incubate for appropriate hours at 37ºC.
- Once the process is done, the medium will be similar to
figure 4.1.4.
Figure4.1.1.Figure4.1.2.Figure4.1.3.Figure4.1.4.

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2. Gram-staining: Preparation of Smear
- Take one loop of sterilized saline on a slide glass.
- Take a small amount of bacterial cells from independent single colony using
platinum needle.
- Suspend the bacterial cells in saline and extend the smear circle approximately
1.5 cm diameter.
- Dry at room temperature.
- Fixation of bacterial cells onto the surface of slide glass by heating (passing the
slide glass through the flame of the gas burner).
- Cool down the slide glass to the room temperature.
3. Gram-staining: Staining
- Apply 4 to 5 drops of crystal violet solution (Gram-staining solution no.1) on the
smear for 2 minutes.
- Wash off the crystal violet solution by adding Lugol solution (Gram-staining
solution No.2)
- Apply 4 to 5 drops of the Lugol solution for 2 minutes.
- Discard the Lugol solution and decolourize with absolute ethanol for 1 to 1.5
minutes.
- Wash the back side of the slide glass with distilled water.
- Apply 4 to 5 drops of fuchsine solution (Gram-staining solution No.3) for 1 minute.
- Wash the back side of the slide glass again with distilled water.
- Remove excess water using filter paper and dry the slide glass.
4. Microscopic observation

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V. RESULTS
SMEAR 1 SMEAR 2
SMEAR 3

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VI. DISCUSSION
I. THEORY
The Gram stain procedure enables bacteria to retain color of the stains, based on
the differences in the chemical and physical properties of the cell wall.
1. Gram positive bacteria
Stain dark purple due to retaining the primary dye called Crystal Violet in
the cell wall. Gram-positive bacteria have a thick mesh-like cell wall which is
made up of peptidoglycan (50-90% of cell wall), which stains purple. The thick
peptidoglycan layer of Gram-positive organisms allows these organisms to retain
the crystal violet-iodine complex and stains the cells as purple (see figure 6.1.)
2. Gram negative bacteria
Stain red or pink due to retaining the counter staining dye called Safranin.
Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell
wall) and lose the crystal violet-iodine complex during decolorization with the
alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink.
They also have an additional outer membrane which contains lipids, which is
separated from the cell wall by means of periplasmic space (see figure 6.2.)
Figure 6.1. Figure 6.2.

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II. IDENTIFICATION
1. SMEAR 1:
a. Smear number 1 has a pinkish color, which classify itself as a gram-negative
microorganism.
b. It has the shape of a rod with identical characteristics for the whole colony
and that it resembles the shape and characteristic of the bacteria in smear 2.
c. It has the same size as smear 2, but slightly bigger than smear 3.
d. Smear 1, same as smear 2, is Escherichia coli.
2. SMEAR 2:
a. Smear number 2 also has a pinkish color, which made it a gram-negative
microorganism.
b. It has the shape of a rod with identical characteristics for the whole colony
and that it resembles the shape and characteristic of the bacteria in smear 1.
c. It has the same size as smear 1, but slightly bigger than smear 3.
d. Smear 1, same as smear 2, is Escherichia coli.
3. SMEAR 3:
a. Smear number 3 has a deep purple color, which classify itself as a gram-
positive microorganism.
b. It has a circular/spherical shape with identical characteristics for the whole
colony.
c. It is slightly smaller compared to smear 1 and 2.
d. Smear 3 is Stapylococcus aureus.

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VII. REFERENCES
 WEBLIOGRAPHY
1. http://amrita.vlab.co.in/?sub=3&brch=73&sim=208&cnt=6
2. http://en.wikipedia.org/wiki/Gram_staining
 TEXT BOOK
1. Black, Jacquelyn (2012). Microbiology: Principles and exploration 8th edition.
John Wiley & Sons. p. 68. ISBN 978-0-470-54109-8 (via Wikipedia)