FACULTY OF PHARMACY

UiTM CAWANGAN PULAU PINANG KAMPUS BERTAM

P H D 2 1 3

MICROBIOLOGY
A LABORATORY MANUAL

PRACTICAL 1
Basic Laboratory Techniques for Isolation and
Cultivation of Microorganisms.

2 0 2 2 E D I T I O N
O C T 2 0 2 2 – F E B 2 0 2 3
 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

PRACTICAL 1

Basic Laboratory Techniques for

Isolation and Cultivation of
Microorganisms.

LEARNING OBJECTIVES
At the end of the session, students should be able to:
1. Understand the types and usage/ function of laboratory equipment and culture media
needed to develop and maintain pure culture media.
2. Understand the concept of sterility and the procedures necessary for subculturing of
microorganisms.
3. Apply aseptic techniques in preparing agar plates to prevent contamination.
4. Perform streak-plate and spread-plate inoculation of microorganisms in a mix
microbial population for subsequent pure culture isolation
5. Describe cultural and morphological characteristic of microorganisms grown in pure
culture.

INTRODUCTION

Bacteria are found everywhere, on the countertops, in the air, and on your skin. Aseptic, or
sterile, technique is necessary to prevent the contamination of bacteria cultures you are
working with and to avoid the further contamination of countertops, the air, and your skin.
Aseptic technique is also used in the lab when working with other microorganisms such as
viruses and fungi.

Aseptic technique in the lab involves handling materials in such a way as to prevent
contamination. Contamination not only refers to keeping one's self from accidental exposure
but also to prevent the introduction of anything into your work that does not belong. Bacteria
will grow on practically any source of organic food which provides carbon compounds to be
respired for energy, and nitrogen compounds to be incorporated into proteins for growth.
These substances are normally provided dissolved in water. However, in nature, bacteria
can break down solid and insoluble substances by releasing enzymes into the substrate in
which they are growing. These substances are thus broken down or digested to simpler
substances and the process is called extracellular digestion because it takes place outside
the bacterial cells.

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 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

Sterilization:
Sterility is the hallmark of successful work in the microbiology laboratory. To achieve sterility,
it is mandatory that you use sterile equipment and sterile techniques. Sterilization is the
process of rendering a medium or material free of all forms of life.

Media:
The survival and continued growth of microorganisms depend on an adequate supply of
nutrients and a favorable growth environment. Most microbes must use soluble
lowmolecular-weight substances that are frequently derived from the enzymatic degradation
of complex nutrients. A solution containing these nutrients is a culture medium (plural =
media). Media are utilized in several different forms.

The two normal media used in bacteriology are a clear soup-like liquid nutrient broth, usually
in tubes, and nutrient agar, which is set into a jelly by the addition of a seaweed extract
called agar, and when melted poured into glass or plastic Petri dishes - also known as
"plates". A standard carbon source is glucose, and nitrogen is often provided by peptones
(partially digested proteins), or inorganic salts. Minerals and vitamins may also be provided,
according to the growth requirements of the bacteria. Combinations of chemicals (buffers)
may be used to keep the pH stable. Measured amounts of the concentrates are added to
water, and dissolved to reconstitute the media. Sometimes, substances are mixed into
media, in order to suppress growth of other types of bacteria. There are many such selective
media. All media in this practical session will be sterilized in a device called an autoclave,
which uses steam under pressure to destroy all known infectious agents

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 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

EXPERIMENT 1: MEDIA PREPARATION

MATERIALS

Media
2 bottles of 20 mL sterile molten agar (nutrient agar)

Equipment
Sterile Petri Plates
Bunsen burner

METHODS

1. Label all sterile Petri plate as described in Laboratory Protocol section.

2. Remove the cap of the universal bottle containing molten agar and flame the mouth
of the bottle by rapidly passing it through the flame once
3. Slightly lift the lid of the plate just high enough to allow the plate to be poured, and
quickly half filled the dish with molten agar (Refer to Figure 1.1). Try to minimize the
introduction of bubbles.
4. Replaced the lid of the Petri plate. Swirled the plate gently to ensure even distribution
of the molten agar, and then left the plate to stand on the bench for at least 20
minutes to solidify.
5. Once all the plates are poured, re-flame the bottle’s mouth and returns the cap to the
bottle.
6. Allow the plate to set (solidified). Seal the Petri plates with tape and store at 37ºC for
24 to 48 hours. (Remember to keep the plate inverted (upside down) all the while).

(a) (b)

Figure 1.1: (a) Removal of a bottle cap aseptically. Note how the rest of the hand is
free to manipulate a pipette or plate lid. (b) Pouring agar into a plate. Note how the lid
shields the agar from airborne contamination.

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 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

EXPERIMENT 2: CULTURE TRANSFER TECHNIQUE

MATERIALS

Cultures
24 hours nutrient agar cultures

Media
Nutrient broth
Nutrient agar slant
Nutrient agar deep tube

Equipment
Bunsen burner
Inoculating loop and needle

METHODS

1. Label all tubes of sterile media as described in Laboratory Protocol section.

2. Plate to Broth Transfer: Holding your loop like a pencil, insert the loop into the
flame as illustrated in Figure 2.1. The orientation of the loop wire in the flame should
be at 30-degree angle for proper incineration. Keep the wire in the flame until it is
red-hot, and then move the adjacent non-wire part of the loop lightly through the
flame. The wire will now be sterile, and the non-wire part will have any dust burned
off that might have fallen into the media during the transfer procedure. Allow the loop
to cool for a few seconds in the air before touching it to your culture or medium.

Figure 2.1: Proper flaming of a loop. Note how the loop handle is held by only
the thumb and first two fingers and the loop is inserted into the hottest part of
the flame.
3. Slightly lift the lid of the nutrient agar culture and touch the flamed and cooled loop to
the surface of the selected colony. Remove a loop full of a small but visible amount of
bacterial growth with the loop. Replaced the lid of the Petri plate.
4. Remove the cap of the nutrient broth tube, and then flame the neck of the tube by
rapidly passing it through the flame once.
5. Insert the loop (with culture) into the broth without touching the sides of the tube,
swirl it around to get the inoculum off the loop and into the broth and then remove the
loop. Make sure that the inoculum has been transferred to the broth and is not still
adhered to the loop. Dislodge the culture by slight agitation.
6. Pass the top of the culture tube through the flame, replace the tube cover, and return
the tube to a rack. Re-flame the inoculating loop.

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 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

7. Broth to Slant Transfer: Flame the inoculating loop until the entire wire is red. While
holding the sterile loop, pick up the culture tubes (prepared in steps 2 to 6) with free
hand. Shake the tube gently from side to side.
8. Remove the cap of the culture tube, and then flame the neck of the tube by rapidly
passing it through the flame once.
9. Insert the sterile loop into the culture without touching the sides of the tube, and then
remove it, carrying a loopful of culture.
(a) (b)

Figure 2.2: Transferring a culture. (a) Removal of a tube cap while manipulating a
loop; (b) Obtaining inoculum from a broth tube while maintaining sterility of the cap
(note cap in hand).
10. Pass the top of the culture tube through the flame, replace the tube cover, and return
the tube to a rack.
11. Pick up the labeled sterile nutrient agar slant. Remove its cover, (if it's a glass tube,
pass the lip of the tube through the flame).
12. Insert the loop (with culture) to base of the slant; withdraw the loop in a zigzag
motion.

Figure 2.3: Inoculation using zigzag motion.

13. Pass the top of the culture tube through the flame, replace the tube cover, and return
the tube to a rack. Re-flame the inoculating loop.
14. Broth to Deep Tube Transfer: Flame the inoculating needle until the entire wire is
red. While holding the sterile needle, pick up the culture tubes (prepared in steps 2 to
6) with free hand. Shake the tube gently from side to side.
15. Follow steps 8 to 10.
16. Pick up the labeled sterile nutrient agar deep tube. Remove its cover, (if it's a glass
tube, pass the lip of the tube through the flame).

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PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

17. Insert the needle (with culture) to the bottom of the tube and withdraw along the line
of insertion.

Figure 2.4: Inoculating deep agar tube for determining motility.

(A) Uninoculated tube of semisolid agar. (B) Same tube being inoculated by stabbing the
inoculating wire into the medium. (C) Pattern of growth of a non-motile organism, after
incubation. (D) Pattern of growth of a motile organism, after incubation.
18. Pass the top of the tube through the flame, replace the tube cover, and return the
tube to a rack. Re-flame the inoculating needle.
19. Incubate all cultures at 37⁰C for 24 to 48 hours.
20. Examine all cultures for the appearance of growth, which is indicated by turbidity in
the broth culture and the appearance on the surface of the slant and along the line of
inoculation in the agar deep tube.
21. Record all observation in the space provided.

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 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

EXPERIMENT 3: ISOLATION TECHNIQUE

MATERIALS

Cultures
24 hours nutrient agar cultures
Media
Nutrient agar
Equipment
Bunsen burner
Inoculating loop

METHODS

1. Label all sterile Petri plate as described in Laboratory Protocol section.

2. Flame the loop and wire until it become red-hot and cool it by touching an unused
part of the agar surface close to the periphery of the plate.
3. Slightly lift the lid of the nutrient agar culture and touch the flamed and cooled loop to
the surface of the selected colony. Remove a loop full of a small but visible amount of
bacterial growth with the loop and then drag it rapidly several times across the
surface (Area 1). Replaced the lid of the Petri plate.
(a) (b)

Figure 3.1: Streak plate isolation technique (a) Selection of colony from starter plate
(b) First streak on Area 1.
4. Ref-lame and cool the loop and turn the Petri plate 90º. Then touch the loop to a
corner of the culture in Area 1 and drag it several times across the agar (Area 2). The
loop should never enter Area 1 again.
5. Ref-lame and cool the loop again turn the plate 90º. Streak Area 3 in the same
manner as Area 2.
(c) (d)

Figure 3.1: Streak plate isolation technique (c) Second streak on Area 2 (d) Third
streak on Area 3.

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 PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

6. Without re-flaming the loop, again turn the plate 90º and then drag the culture from a
corner of Area 3 across Area 4, using a wider streak. Don’t let the loop touch any of
the previously streaked area. The flaming of the loop at the points indicated is to
affect the dilution of the culture so that fewer organisms’ area streaked in each area,
resulting in the final desired separation.

Figure 3.2: Four-way streak plate inoculation

7. Seal the plate and incubate it in an inverted position at 37º for 24-48 hours.
8. After 24 – 48 hours incubation, examine the colonies formed thoroughly and describe
them in detail.

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 APPENDIX 1

Appendix 1: Bacterial colony morphology description on agar plate

 Colony characterization on agar plates

Characteristic Description

Size of colony Pinpoint, small, moderate or large

Pigmentation Colour of colony

Form Shape of colony:

• Circular – unbroken, peripheral edge
• Irregular – indented, peripheral edge
• Rhizoid – rootlike, spreading growth
Margin Appearance of the outer edge of the colony:
• Entire – sharply defined, even
• Lobate – marked indentations
• Undulate – wavy indentation
• Serrate – toothlike appearance
• Filamentous – threadlike, spreading edge
Elevation Degree to which colony growth is raised on the agar surface:
• Flat – elevation is not discernible
• Raised – slightly elevated
• Convex – dome-shaped elevation
• Umbonate – raised, with elevated convex central region
PHD213 – MICROBIOLOGY: A LABORATORY MANUAL – PRACTICAL 1

Appendix 2: Growth characterization on broth cultures

Characteristic Description

Uniform fine turbidity Finely dispersed throughout

Flocculent Flaky aggregates dispersed

throughout

Thick, pad-like growth on

Pellicle
surface

Concentration of growth at the

Sediment
bottom of broth culture may be
granular, flaky, or flocculants

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 Appendix 3: Growth characterization on agar slants
Characteristic Description
Abundance of growth The amount of growth is designated as none, slight, moderate or large
Pigmentation Colour of colony. Chromatogenic microorganisms may produce intracellular pigments that are responsible for the
colouration of the organisms as seen in surface of colonies. Other organisms produce extracellular soluble pigment
that are excreted into the medium and that also produce a colour. Most organisms. However, are nonchromogenic and
will appear white or gray.
Form Appearance of the single-line streak of growth on the surface of agar:

1. Filiform – continuous, threadlike growth with smooth edges

2. Echinulate - continuous, threadlike growth with irregular edges
3. Beaded – nonconfluent to semiconfluent colonies
4. Effuse – thin, spreading growth
5. Arborescent – treelike growth
6. Rhizoid – rootlike growth
Margin Appearance of the outer edge of the colony:
• Entire – sharply defined, even
• Lobate – marked indentations
• Undulate – wavy indentation
• Serrate – toothlike appearance
• Filamentous – threadlike, spreading edge
Elevation Degree to which colony growth is raised on the agar surface:
• Flat – elevation is not discernible
• Raised – slightly elevated
• Convex – dome-shaped elevation
• Umbonate – raised, with elevated convex central region