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 : Professor Kondo Seiichi

Date of submission :

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.

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Figure 4.1.1.

Figure 4.1.2.

Figure 4.1.3.

Figure 4.1.4.

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

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SMEAR 1 SMEAR 2

SMEAR 3

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.)

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Figure 6.1. Figure 6.2.

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)

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