LABORATORY PRACTICAL MODULE

COURSE: MICROBIOLOGY
COURSE CODE: MAB 1201

Written by
MICROBIOLOGY TEAM

BACHELOR BIOLOGY STUDY PROGRAM

FACULTY OF MATHEMATICS AND NATURAL SCIENCES
UNIVERSITY OF JEMBER
2023
 FOREWORD

This Microbiology Practicum Manual is a guidebook for working in the Laboratory for
students who take the General Microbiology practical. The main objective is to introduce the basic
concepts of microbiology, laboratory techniques, and procedures.
Therefore, mastering these things well will significantly help students in studying
microbiology, and they will be able to explore various fields of applied microbiology. The
availability of books that are thought of and the readiness of the assistants/lecturers are very
supportive of achieving the intended goal. Likewise, laboratory advice and infrastructure must be
ready to help achieve goals.
On this occasion, we would like to thank all parties who have helped with the material's
content and helped in the technical implementation until the realisation of this book. Suggestions
and criticisms from users of this book are highly expected so that subsequent publications can be
added/improved.

Jember, 25 July 2023

The Team of Course

MICROBIOLOGY LABORATORY PRACTICAL MODULE ii

 LIST OF CONTENTS

FOREWORD …………………………………………………………………………………… ii
LIST OF CONTENTS…………………………………………………………………….……. iii
LIST OF FIGURE ……………………………………………………………………………… iv
I. EQUIPMENT AND MEDIA STERILIZATION …………………………………………… 1
II. ASEPTIC TECHNIQUE ……………………………………………………………………. 3
III. CULTURE TRANSFER TECHNIQUES …………………………………………………. 5
IV. PURE CULTURE ISOLATION TECHNIQUES …………………………………………. 8
V. MORPHOLOGICAL CHARACTERIZATION OF BACTERIA ………………………… 10
Part A. Macroscopic Morphological of Bacteria ………………………..……………
Part B. Microscopic Morphological Characterization: Simple Staining ………… 12
VI. MORPHOLOGICAL CHARACTERIZATION OF ACTINOMYCETES ………………. 17
Part A. Macroscopic Morphological Characterization of Actinomycetes ……… 19
Part B. Microscopic Morphological Characterization of Actinomycetes …….… 20
VII. CHARACTERIZATION OF YEAST CELLS …………………………………………… 21
VIII. MOLD SLIDE CULTURE TECHNIQUE………………………………………………… 23
IX. MORPHOLOGY OF MOLDS AND YEASTS……………………………………………. 26
Part A. Morphology of Thread Molds ………………………..………………………… 26
Part B. Microscopic Observation ………………………..…………………………….. 27
Part C. Yeast Morphology ……………………….……………………………………… 27
Part D. Staining Yeast Spores …………………………….……………………………. 28
X. MORPHOLOGICAL CHARACTERIZATION OF ALGAE ……………………………… 29
XI. PROTOZOA ………………………………………………………………………………... 31
XII. BACTERIOPHAGE OBSERVATION…………………………………………………… 33
REFERENCES ………………………………………………………………………………… 37

MICROBIOLOGY LABORATORY PRACTICAL MODULE iii

 LIST OF FIGURES

Figure 1 Sterilisation method for tools and media ……………………………………….. 1

Figure 2 Laminar Air Flow …………………………………………………………………… 4
Figure 3 Culture transfer technique (subculture) …………………………………….……. 6
Figure 4 Four quadrant technique in the scratch method ………………………………… 8
Figure 5 Characteristics of bacterial culture on media ……………………………………. 10
Figure 6 Shape and arrangement of bacteria ……………………………………..………. 13
Figure 7 Simple staining procedure ………………………………………………………… 14
Figure 8 Microscopic image showing bacterial morphology …………………..…………. 14
Figure 9 Negative staining with nigrosin: basil 1000x.……………………………….…… 15
Figure 10 Negative staining procedure …………………………………………..………… 16
Figure 11 Structure of actinomycete spores ….……………………….……………..……. 19
Figure 12 Yeast colonies growth on media in the laboratory….………………………… 21
Figure 13 The microscopic structure of (a) S. octosporus yeast cells and (b) S. 21
cerevisiae cells forming shoots resulting from vegetative reproduction ………
Figure 14 Yeast structure (a) vegetative reproduction by budding and (b) sexual 22
reproductive structure. ………………………………………………………….…
Figure 15 The diversity of algal body types reflects their habitat.………………………… 29
Figure 16 Wastewater sample enrichment, filtration, and seeding ……………………….. 35

MICROBIOLOGY LABORATORY PRACTICAL MODULE iv

 I. EQUIPMENT AND MEDIA STERILIZATION

Cultivation of microbes in the laboratory requires growth media. This media contains the
nutrients needed for microbial growth. The type of media used is adjusted to the type of microbe
that will be grown, for example, Nutrient Agar media to grow bacteria and Potato Dextrose Agar
media to grow mold. The growth medium can be in the form of solid, semi-solid or liquid media
(broth). Solid or semi-solid media is liquid media (material according to the composition of the
media) which is added with a gelling agent in the form of agar.
In addition to media, microbial cultivation requires equipment (tools) for microbial growth.
Some common equipment used is Erlenmeyer flasks, test tubes, Petri dishes and inoculation
loop. Erlenmeyer flasks, test tubes, and Petri dishes are used for microbial growth media, while
an inoculation loop transfers microbial cultures.
The equipment and growth media used must be sterile (free from microbes) so that the
microbes that grow are actually the expected (target) microbes and not microbes from
contamination (unexpected). Sterilization is an effort to liberate the tools or materials from all kinds
of life forms, especially microbes. Sterilization can be carried out by physical methods such as
heat and filtration or chemical methods (Figure 1). The sterilization method is adjusted to the
nature of the materials and tools being sterilized.

Figure 1. Sterilization Method for Tools and Media (Cappuccino & Welsh, 2019)

Practical Objectives
a. Students can sterilize tools.
b. Students can sterilize media.

Method
Tools and materials
a. Inoculation loop (1)
b. Petri dishes (4 pieces)
c. 100 mL Beaker Glass (1 piece)

MICROBIOLOGY LABORATORY PRACTICAL MODULE 1

d. 20 mL Volumetric flask (1 piece)
e. 1 mL Measuring pipettes (2)
f. Test Tubes (10)
g. Cotton
h. Oven
i. Autoclave
j. Scales
k. Nutrient Broth
l. Bacto agar

Procedures
1. Sterilization of tools
1.1 Sterilization of inoculation loop
Heat the inoculation loop until reddish appears in the flame of a Bunsen burner.
1.2 Sterilization of Petri dishes
a. Wrap 4 Petri dishes each with aluminum foil or doorslag paper.
b. Carry out sterilization using two sterilization methods.
c. A total of two Petri dishes were sterilized using an oven at 160°C for 2 hours (Appendix 1. Oven
Operational Procedures) and two Petri dishes were sterilized in an autoclave at 121°C 1 atm for
15 minutes (Appendix 2. Autoclave Operational Procedures)
1.3 Sterilization of 1 mL glass pipettes.
a. Wrap two glass pipettes each with aluminum foil or doorslag paper.
b. Perform sterilization in two ways. One pipette is sterilized using an oven at 160°C for 2 hours
and one pipette is sterilized using an autoclave at 121°C 1 atm for 15 minutes.

2. Media Sterilization
2.1 Preparation and Sterilization of Nutrient Broth Media (8 g/L)
a. Weigh 0.4 g of nutrient broth using a scale, put it in a 100 mL beaker and add 40 mL of distilled
water, then heat until dissolved using a hot plate and add water to a volume of 50 mL.
b. Put 10 mL into 5 test tubes using a measuring/ volumetric flask and cover with cotton.
c. Next, sterilized using an autoclave.
2.2 Preparation and Sterilization of Nutrient Agar Media
a. Weigh 0.5 g of Nutrient Broth and 0.75 g of Bacto Agar using an electric scale, put it in a 100
mL Beaker and add 40 mL of distilled water.
b. Next, heat the solution until it dissolves using a hot plate and add distilled water to a volume of
50 mL.
c. Put 10 mL of media into 5 test tubes using a measuring/ volumetric flask and cover with cotton.
Next, sterilized using an autoclave.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 2

 II. ASEPTIC TECHNIQUE

The aseptic technique is a series of routine activities taken to prevent cultures, sterile
media and other solutions from contamination by unwanted microbes. Research activities in the
field of microbiology have the main aim of growing microbes, both prokaryotes and eukaryotes,
that are free from contaminants, so aseptic techniques play an important role. The aseptic
technique that is often used in microbiology is the aseptic transfer technique. The aseptic transfer
technique is a method or technique for moving or transferring bacterial/ microbial cultures from
one place to another aseptically so that the contamination of the culture by other microbes does
not occur. This aseptic transfer technique is very essential and the key to the success of microbial
procedures that must be known by someone who wants to carry out a microbiological analysis.

Practical Objectives
Students can carry out laboratory work aseptically in microbiology.

Method
Tools and materials
a. Bunsen burner
b. Laminar Air Flow
c. Test tube
d. Petri Dish
e. Cotton
f. Inoculation loop
g. 70% alcohol solution

Procedures
1. Some examples of aseptic techniques include the following:
a. Clean the workbench with disinfectant before and after work.
b. Limit the exposure duration of the culture or medium to the air.
c. Keep the Petri dish closed.
d. Effectively sterilizes inoculation loops and other equipment that is being used for culture
transfer.
e. Avoid talking while working.

2. Some of the equipment needed to maintain aseptic conditions are as follows:

a. Bunsen burner.
1) The easiest way to create a relatively sterile environment in a laboratory is to use a Bunsen
burner.
2) The role of the Bunsen burner in aseptic technique is to reduce airborne contaminants
suspended in dust particles.
3) Bunsen burners are best for sterilizing loops (inoculation loop), mouths of test tubes and
surfaces of Petri dishes.
b. Laminar Air Flow.
1) Work carried out in laminar air flow will provide a clean workspace.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 3

2) Filtered air eliminates air contaminants entering the work area (Figure 2).

Figure 2. Laminar Air Flow

1. several rules must be known and fulfilled in this aseptic transfer technique, as summarized
below:
a. Before Implementation:
1) Remove all unnecessary items from the desk and workspace.
2) Wear clean and hygienic clothing or a laboratory coat before entering the laboratory.
3) It is recommended to wear a clean and hygienic mask.
4) Wear a clean and hygienic hair cover.
5) Never place tubes and other laboratory equipment outside the laboratory.
b. Before and After Implementation:
1) Wash your hands thoroughly and use antiseptic.
2) Sanitize and disinfect the workspace (laboratory and surroundings) with adequate
disinfectants, including Laminar Air Flow and incubators.
3) Sterilize all tools and materials before use.
c. When Carrying Out Culture:
1) Don't talk.
2) Work near a fire (Bunsen burner) and in Laminar Air Flow.
3) Open the tube or cup over the fire and keep it away from your nose and mouth.
4) Try not to place the lid (cotton cover) of the test tube on the floor/table base or laminar.
5) Tilt the lid of the Petri dish to be opened as a barrier between the culture and your mouth and
nose.
6) Don't open the lid of the Petri dish too wide or for too long. Work quickly.
d. After Culture Implementation:
1) Immediately close all tubes or cups that are still open.
2) Get rid of all equipment or leftover materials that are no longer used.
3) Clean and dry immediately any media spills.
4) Sanitize and re-disinfect the work area (your laboratory).
5) Take off your work clothes and laboratory coat before leaving your work area.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 4

 III. CULTURE TRANSFER TECHNIQUES

The process of transferring microbes from one medium to another is called subculture.
This technique is carried out routinely for the preparation and maintenance of microbial culture
stocks as well as for microbiological analysis needs.
Microbes are always present in the environment, both in the air, equipment and where we
work in the laboratory. The presence of these microbes can potentially be a contaminant, so
proper aseptic technique is very necessary during the subculture process. Several important
steps need to be considered in the subculture process (Figure 3), as follows:
1. Label the tube or Petri dish that you will inoculate with the name of the organism, date and your
initials before you subculture.
2. Hold the culture stock tube and the tube you are going to inoculate in the palm of your hand,
use your thumb to hold and separate the two tubes so that they form a "V" shape in your hand.
3. Sterilize the inoculation loop by holding it in a micro incinerator or the hottest part of a Bunsen
burner until the wire turns red hot. Once the inoculation loop is sterilized, hold it in the circle of
your hand and let it cool for 10-20 seconds. Never place it on your work desk.
4. Open the cap of each tube by holding the first cap on your little finger and the second cap on
your next finger. Hold the cap on the hand holding the inoculation needle. Never place the tube
cap on the work table as this will compromise the aseptic procedure.
5. After removing the tube cap, sterilize the neck and mouth of the tube by passing it through an
incinerator or Bunsen burner flame 2-3 times quickly. Cool the inoculation needle by touching the
inside wall of the tube before removing a small amount of the inoculum sample. You can test
whether the inoculation needle is hot or not on the empty part of the media.
6. Depending on the culture medium, an inoculation loop or needle can be used to obtain inoculum
from the agar culture slant, but be careful when touching the surface of the solid medium in areas
showing growth so as not to gouge the agar.
a. Transfer of culture from slant media to liquid media: take the culture from slant media then
gently shake the inoculation loop or needle into the new medium to release the microbes.
b. Transferring liquid culture to a slant medium: obtain a full circle of liquid culture and place it on
the bottom of the slant medium. Lightly drag the circle onto the surface of the hardened agar in a
straight line or zigzag direction from the bottom of the medium to the top.
c. Transfer the culture from the slant media to the agar medium using the puncture method: take
the inoculum from the slant medium, insert the inoculation needle into the medium in the new tube
in a straight line and quickly pull along the insertion line.
7. After the inoculation process, re-sterilize all equipment including the inoculation loop, test tube
necks and Petri dishes.
8. Replace the test tube lid according to its original position.
9. Re-sterilization of the inoculation loop or needle is carried out to remove remaining microbes.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 5

 Figure 3. Culture transfer technique (subculture) (Cappuccino & Welsh, 2019)

Practical Objectives
Students can carry out culture transfers by applying aseptic microbiology techniques.

Method
Tools and materials
a. Laminar air flow
b. Alcohol 70%
c. Rolled tissue
d. Bunsen burner
e. Inoculation loop
f. Volumetric pipette
g. Nutrient Agar (NA) media, slant in a test tube

MICROBIOLOGY LABORATORY PRACTICAL MODULE 6

h. Nutrient Agar (NA) media in Petri dishes
i. Nutrient Broth (NB) media in a test tube
j. Bacillus sp culture. on the slant agar media
k. Bacillus sp culture. on NB media

Procedures
1. Transferring the culture from liquid media to liquid media
a. Label the new media that you will inoculate.
b. Take 100 µL culture of Bacillus sp. in NB media and inoculate into new NB media in a test tube,
homogenize.
c. Carry out microbiological aseptic activities.
2. Transferring the culture from liquid media to solid media.
a. Label the new media that you will inoculate.
b. Take the liquid culture (100 µL) of Bacillus sp. and inoculate it into NA media in a Petri dish
using the spread plate method.
c. Carry out microbiological aseptic activities.
3. Transferring the culture from solid media to solid media.
a. Label the new media that you will inoculate.
b. Take a culture of Bacillus sp. on slanted NA media and inoculate on new slanted NA media
using the zig-zag streak plate method.
c. Carry out microbiological aseptic activities.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 7

 IV. PURE CULTURE ISOLATION TECHNIQUES

Microbial populations in nature consist of various species that live together. We can
separate the population into pure cultures in the laboratory. This pure culture only contains one
type of organism and is not mixed with others. Pure cultures are prepared to study the morphology
and biochemical properties of these microbes.
The method used to obtain pure culture is by isolation. The isolation technique determines
the success of obtaining a pure culture that is separate from other cultures. There are two ways
of isolation to obtain pure culture, namely:
1. Streak plate method
In principle, this method is to scratch a suspension of material containing microbes on the
surface of the medium. The technique can use the 4 quadrant technique. After incubation, the
scratch marks will grow into separate colonies (Figure 4).

Figure 4. Four quadrant technique in the scratch method (Cappuccino & Welsh, 2019)

2. Spread Method
The spread method consists of two techniques, i.e. spread plate and pour plate.
a. Spread plate is a spreading method by inoculating isolates on solid agar media in Petri dishes
and spreading using a sterile L-shaped glass spreader.
b. Pour plate is a spreading method by inoculating the isolate on solid agar media which melts at
a temperature of around 40°C-50°C, and then is poured into a sterile Petri dish.
The difference between these two is that the microbes that grow using the spread plate technique
are aerobic, whereas with the pour plate method, the growth can be aerobic (on the surface of
the agar) and facultative anaerobic/anaerobic (below the surface of the agar).

Practical Objectives
Students can practice pure culture isolation techniques.

Method
Tools and materials
a. Sterile Petri dishes
b. L-shaped Bent glass
c. Bunsen burner
d. Inoculation loop
e. Rolled tissue
f. Marker pen

MICROBIOLOGY LABORATORY PRACTICAL MODULE 8

g. Vortex
h. Micropipette
i. Yellow/blue tip
j. Mixed culture of isolated microbes
k. 10 mL solid NA media in a test tube
l. 10 mL solid NA media in a Petri dish
m. 70% alcohol solution
n. 5 mL sterile distilled water in a test tube

Procedures
1. Pure culture isolation technique using the four quadrant scratch method.
a. Select a single colony in the mixed culture to isolate.
b. Aseptically, take the colony using an inoculation loop and transfer it to NA media in a Petri dish
with the four-quadrant technique (Figure 1).
c. The first step was carried out by dividing four parts of NA media in a Petri dish as shown using
markers (1, 2, 3, and 4).
d. One colony is taken using an inoculation loop and scratched into area one.
e. Next, the inoculation loop is burned using Bunsen and scratched from area one to two and
further scratches are made on area two without touching area one.
f. This process is done successively until the scratches reach area four.
g. Next, incubated in an incubator at 30°C for two days, then colony growth is observed.
h. The indicator of the success of this process is that a single colony is obtained that is separate
from the others.
2. Pure culture isolation technique using the spread plate method.
a. One colony is taken using an inoculation loop and placed in 5 mL of sterile distilled water and
homogenized using a vortex to obtain a culture suspension.
b. Next, 100 μL of the culture suspension is taken using a micropipette, poured into NA medium
in a Petri dish and spread evenly using an L-shaped bent glass.
c. Next, incubated in an incubator at 30°C for two days, then colony growth is observed.
d. The indicator of the success of this process is that a single colony is obtained that is separate
from the others.
3. Pure culture isolation technique using the pour plate method.
a. A total of 100 μL of culture suspension (from Step 2) was taken using a micropipette and poured
into liquid NA media in a test tube (temperature 40°C-50°C)
b. Then homogenize by rotating the Petri dish to resemble the number eight.
c. Incubate in an incubator at 30°C for two days, then observe the growth of the colony. The
indicator of the success of this process is that a single colony is obtained that is separate from
the others.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 9

 V. MORPHOLOGICAL CHARACTERIZATION OF BACTERIA

Macroscopic Morphology of Bacteria

Bacterial cultures grown in a medium will show different characteristics. The culture
characteristics are used to differentiate bacteria. Bergey's Manual of Systematic Bacteriology
describes the cultural characteristics of all known bacteria. These characteristics are based on
the character of the bacteria on solid nutrient agar media in Petri dishes and tilted media in tubes,
on nutrient broth (NB) and nutrient gelatin (NG) media.

Figure 5. Characteristics of bacterial culture on media (Cappuccino & Welsh, 2019)

The characteristics of the bacterial cultures on various media are presented in Figure 5.
A. Character of bacteria on slant Nutrient Agar media in a test tube
Bacteria are grown on this media by inoculating a loop of bacteria straight on the agar surface.
Bacterial characteristics can be observed in this media.
1. Abundance of growth
Growth can be differentiated between none, minor, moderate, and a lot.
MICROBIOLOGY LABORATORY PRACTICAL MODULE 10
2. Pigmentation
Bacteria can produce pigment that can be seen on the bottom surface of the colony. Some
bacteria produce extracellular pigments that produce color in the media and some bacteria do not
produce pigments.
3. Optical Characteristics
This character is marked by the ability of bacteria to partially transmit. The characteristics are
opaque (no partial transmission), translucent, and transparent (full transmission).
4. Shape
Display of colony growth on the inoculated bacterial culture line.
There are filiform (thread-like growth with smooth edges), echinulate (thread-like growth but
irregular), beaded (nonconfluent to semi-confluent), effuse (spreading growth), arborescent (tree-
like growth), rhizoid (root-like growth).
5. Consistency
The consistency is dry (not moist), buttery (moist and shiny) and mucoid (slimy and shiny).

B. Bacterial characteristics on Nutrient Agar media in Petri dishes.

The character of a colony grown on NA media in a Petri dish can be observed based on the
following characteristics:
1. Size: pinpoint, small, moderate, or large.
2. Colony pigmentation: colony color.
3. Form of colonies:
a. Circular: unbroken, peripheral edge.
b. Irregular: notched, peripheral edge.
c. Rhizoid: like roots, spreading growth.
4. Margin: the appearance of the outer edge of the colony
a. Entire: sharply defined, even.
b. Lobate: marked indentations.
c. Undulate: wavy curve.
d. Serrate: thread-like, the ends spread out.
e. Filamentous: threadlike, spreading edge.
5. Elevation: the degree of colony growth above the agar surface.
a. Flat: elevation not discernible.
b. Raised: slightly elevated.
c. Convex: shaped like a dome (dome-shaped elevation).
d. Umbonate: raised, with elevated convex central region.

C. Character of bacteria grown in Nutrient Broth media

1. Uniform fine turbidity: finely dispersed growth throughout.
2. Flocculent: flaky aggregates dispersed throughout.
3. Pellicle: thick, padlike growth on the surface.
4. Sediment: The concentration of growth at the bottom of the broth culture may be granular,
scaly, or flocculent.
D. Character of bacteria on Nutrient Gelatin media

MICROBIOLOGY LABORATORY PRACTICAL MODULE 11

The gelatin in the media will be degraded by the gelatinase enzyme to form liquid (liquefaction).
The liquefaction pattern of gelatin degradation by bacteria can be distinguished:
1. Crateriform: liquefaction on a surface with a plate shape.
2. Napiform: liquefaction on the surface with round-shaped
3. Infundibuliform: funnel-shaped liquefaction
4. Saccate: elongated, tubular liquefaction
5. Stratiform: the top half of the media undergoes liquefaction

Practical Objectives
Students can describe the characteristics of bacterial cultures in the media.

Method
Tools and materials
a. Stereo microscope
b. Magnifying glass
c. Pure 24-hour cultures of Bacillus cereus, Escherichia coli, Pseudomonas fluorescent,
Staphylococcus aureus and Salmonella sp. in slanted NA medium, NA in Petri dishes and NB
and NG medium.

Procedures
1. Observe the characteristics of the bacterial culture on slanted NA medium, slanted nutrient
agar in a Petri dish (can be aided by magnification using a stereo microscope or magnifying glass)
and NB medium.
2. Take a photo and write down the characteristics of the bacterial culture (see Figure 1) compared
to the characteristics of the bacterial culture in Bergey's Manual of Systematic Bacteriology.

Microscopic Morphological Characteristics: Simple Staining

The principle in simple staining is that we stain a bacterial smear with a single reagent, which
contrasts the organism and the background differently. Basic staining with positively charged
chromogens is preferred because bacterial nucleic acids and specific cell wall components carry
negative charges that are highly attractive and bind to cationic chromogens. The purpose of
simple staining is to explain the morphology and arrangement of bacterial cells (Figure 6).
Methylene blue, crystal violet and carbol fuchsin are the most commonly used basic dyes.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 12

 Figure 6. Shape and arrangement of bacteria (Cappuccino & Welsh, 2019)

Simple staining is a relatively quick and practical testing method for the presence, shape
determination, or determination of the number of bacteria present in a sample. Generally involving
only a single staining step, simple methods are not considered differential or diagnostic and will
have limited utility. However, it is a quick procedure to determine whether a clinical sample has
the presence of foreign bacterial pathogens.

Practical Objectives
Students can carry out simple staining procedures and compare the morphology and arrangement
of bacterial cells.

Method
Tools and materials
a. Bunsen burner
b. Inoculation loop
c. Coloring tray
d. Microscope
e. Lens paper
f. Bibulous paper (highly absorbent)
g. Glass slides
h. 24 hours old of Escherichia coli and Bacillus sp. in slant media
i. 24 hour old Staphylococcus aureus in NB media

MICROBIOLOGY LABORATORY PRACTICAL MODULE 13

j. Alternatively, use a smear prepared in Methylene blue, Crystal violet, and Carbol fuchsin
Reagents

Procedures
1. Prepare a bacterial smear. Note: All smears must be heated before staining. The Simple
Staining in Figure 7 illustrates the following steps: place the slide in a staining tray and drop one
of the dyes using the appropriate exposure time: 1) carbol fuchsin, 15 to 30 seconds; 2) crystal
violet, 20 to 60 seconds; 3) methylene blue for 1 to 2 minutes.

Figure 7. Simple staining procedure (Cappuccino & Welsh, 2019)

2. Wash the dye gently with tap water to remove excess dye. During this step, hold the slide
parallel to the water flow; this can reduce the loss of organisms from the preparation.
3. Dry using bibulous paper, but do not clean the slides.
4. Repeat this procedure with the remaining two organisms, using a different dye for each.
5. Examine all colored slides under immersion oil.
6. Draw and explain the morphology of organisms concerning their shape (e.g., bacilli, cocci, or
spirilla) and sequences (e.g., chains, groups, or pairs). See the photos in Figure 8.

Figure 8. Microscopic image showing bacterial morphology (Cappuccino and Welsh, 2019)
a) Bacillus and dicloxacillin bacteria (rod-shaped) b) Spirilla bacteria (spiral-shaped) c) Coccus
bacteria (round-shaped): Staphylococcus

Characterization of Microscopic Morphology of Bacteria: Negative Staining

MICROBIOLOGY LABORATORY PRACTICAL MODULE 14
The principle of negative staining is using acid dyes such as India ink or nigrosin. Acid dyes with
negatively charged chromogens will not penetrate cells due to the negative charge on the surface
of the bacteria. Therefore, colourless cells are easily seen against a coloured background.
There are two practical applications of negative staining. First, we can see their natural size and
shape because heat fixation is not required and the cells do not experience the distorting effects
of chemicals and heat. Second, we can observe bacteria that are difficult to stain, such as some
spirilla. Since heat fixation is not performed during the staining process, it is essential to remember
that the organisms are not killed, and the slides should be handled carefully. Figure 9 shows
negative staining of bacilli.

Figure 9. Negative staining with nigrosin: basil 1000x (Cappuccino & Welsh, 2019)

The principle of applying negative staining determines whether an organism has a capsule (the
outer layer that makes microorganisms more virulent). However, it can also be used to
demonstrate the formation of spores. This technique is often used to identify fungi such as
Cryptococcus neoformans, a crucial infectious agent found in bird droppings associated with
meningeal and lung infections in humans.

Practical Objectives
Students can perform negative staining procedures and explain the benefits of visualization of
unstained microorganisms.

Method
Tools and materials
a. Bunsen burner
b. Inoculation loop
c. Coloring tub
d. Glass slides
e. Lens paper
f. Microscope
g. Slant agar culture of 1) Bacillus subtilis, 2) Pseudomonas fluorescens, 3) Staphylococcus
aureus, 4) Twenty-four hour old Salmonella typhimurium.
h. Nigrosin Reagent

MICROBIOLOGY LABORATORY PRACTICAL MODULE 15

Procedures
Figure 10 illustrates the steps 1 to 4.
1. Place a small drop of nigrosin close to one end of a clean slide.
2. Using an aseptic technique, place the inoculum of bacterial culture in a drop of nigrosin and
mix.
3. Place the slide against the droplet of the suspended organism at a 45° angle and allow the
droplet to spread along the edge of the slide.
4. Push the slide away from the droplet of suspended organisms to form a thin smear. Air dry.
Note: Do not heat the slide.
5. Repeat steps 1 to 4 to prepare the other culture slides.
6. Examine the slide under oil immersion, and record the observation in a laboratory report.

Figure 10. Negative staining procedure (Cappuccino & Welsh, 2019)

MICROBIOLOGY LABORATORY PRACTICAL MODULE 16

 VI. MORPHOLOGICAL CHARACTERIZATION OF ACTINOMYCETES

Actinomycetes are a group of unicellular organisms with mycelia, which reproduce through
division or by particular spores or conidia. These microorganisms are closely related to actual
bacteria, often considered at a higher taxonomic level as filamentous bacteria. During their
growth, these microorganisms form only substrate (vegetative) mycelium or two types, namely
substrate (vegetative) and aerial mycelium (as the part that forms spores/sporogenous).
At the beginning of the description, actinomycetes were often defined as unicellular
microorganisms, 1 µm in diameter, forming filaments, monopodial branching, rarely dichotomous,
producing colonies with a finger structure. The two generally recognized forms of reproduction
are (a) fragmentation, or formation of conidia, and (b) segmentation. Both types of spores grow
well in the medium and form branched mycelia.
Actinomycetes are generally recognized as a large, heterogeneous group of
microorganisms consisting of several genera and many species. Actinomycetes vary in
morphology, physiology, biochemical activity, and role in natural processes.
The morphology of actinomycetes can be easily observed using slide cultures incubated
in a humid chamber. The mycelium attached to the cover of the object glass, which is placed at
an angle to the growing culture, can be transferred to the object glass and observed at high
magnification.
Morphological characters are still widely used to characterize genera, for example, the
presence or absence of spores in the substrate mycelium or the formation of special spore vesicle
zoospores or spore boxes. The morphological features of actinomycetes include the presence of
mycelium, conidia, sporangia, and other structures.
1. Mycelium
Mycelium can be stable or temporary; if it breaks, the parts and their motility must be observed
(Oerskovia sp. releases flagellate elements). The mycelium formed is either substrate mycelium
and air is formed, or only substrate mycelium (very common), or only air hyphae (very rare-
Sporichthya). The mycelium may form inserted vesicles that do not contain spores
(Intrasporangium) or many spores (Frankia).
2. Conidia
This term is used for asexual spores that are neither intercalary chlamydospores nor
sporangiospores. Actinomycetes form conidia in various ways:
a. Single conidia are found in several genera
The genus Thermoactinomycetes (Group 28), is known for its heat-resistant endospores.
Single non-thermostable conidia were found in the genera Saccharomonospora and
Promicrospora (Group 22), Micromonospora (Group 24.) and Thermomonospora (Group 27).
Meanwhile, genera Frankia, Dactylosporangium, and Intrasporangium members sometimes form
terminal vesicles confused with spores. Also, other organisms, such as actinomadurae, will form
single vesicles when grown under unsuitable conditions.
b. Conidia pair
Longitudinal pairs of conidia are characteristic of the genus Microbispora (Group 26),
which form only in aerial mycelium.
c. Short chains of conidia

MICROBIOLOGY LABORATORY PRACTICAL MODULE 17

 Although it is difficult to determine how long a short chain of conidia is, conidia chains of
up to 20 spores are usually considered short. Representatives of the following genera form chains
of conidia: Nocardia, Pseudonocardia, and Saccaromonospora (Group 22); Streptoverticillium
and Spirochtya (Group 25), Actinomadura and Microtetraspora (Group 26); Streptoallotechus
(Group 27); and Glycomyces (Group 29). These morphological types may also be grouped into
the genera Amycolata and Amycolatopsis (Group 22) and Catellatospora (Group 24). Some
Streptomycetes of the Microellobosporia type form short chains, which are surrounded by a
sheath and can be observed using a light microscope.
d. Long chain of conidia
Several genera of Actinomycetes have long chains of conidia, namely Nocardia,
Nocardiales, Pseudonocardia, Saccharopolyspora, Actimopolyspora, and Amycolatopsis (Group
22); Streptomyces and Streptoverticillium (Group 25); Actinosynnema et al. (Group 27); and
Kibdelsporangium, Kitasatospora, Glycomyces, Saccharothrix (Group 29).
e. Conidia on hyphae fuse into cinemata, releasing motile spores (Actinosynnema, Group 27).
3. Sporangia
Sporangia are spore sacs. The sac-shaped structure of the sporangia is a place where
the spores develop and bind together until they are released so that the sporangia are empty and
become a membrane. The membrane is not integrated into the spore. Sporangia originate from
(a) well-developed aerial hyphae or the surface of colonies with few or no aerial hyphae
(Actinoplanes et al. (Group 24); Planobispora et al. (Group 26); or (b) especially in agar media
(Kineospora, Group 25).
4. Other structures
Some actinomycetes form unusual structures. What has been mentioned are the spores
in synnemata found in the genus Actinosynnema. These organisms in Group 23 form masses of
spores resulting from multiple plane divisions, not from perpendicular divisions in the hyphal axis.
The structures on which the spores lie are called multilocular sporangia.
Some actinomycetes will form spherical structures in aerial hyphae. It is not a condensed
water droplet like a curved chain of spores, or the structure may contain hyphae embedded in an
amorphous matrix (Kibdelosporangium, Group 29).
Sclerotia are globose formed by some Streptomycetes. These skelotia do not contain
spores but rather cells containing lipids. These germinate completely, as pseudosporangia from
kibdelosporangia.
The aerial morphology and surface growth of several genera in groups 22-29 are shown
schematically in the figure below. Morphology is useful for identification of some genera but not
all.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 18

 Figure 11. Structure of actinomycete spores (Li et al., 2016).

Macroscopic Morphological Characterization of Actinomycetes

Practical Objectives
Students can recognize the colony morphology of various actinomycetes genera.

Method
Tools and materials
a. Actinomycete culture
b. Yeast Malt Extract Agar (YMA/ISP-2) media
c. The YMA media is tilted in the test tube
d. Sterile Petri dishes
e. Magnifying glass

Procedure
1. Melt the upright medium in a water bath and aseptically pour it into a sterile Petri dish; wait until
it cools and solidifies.
2. Inoculate a small amount of actinomycetes culture on two plates and one YMA slant agar tube.
3. Incubate both plates upside down at room temperature for 2 to 7 days or more.
4. Observe the changes in the colony, which shows the nature of the colony colour and
pigmentation as well as the shape of the colony, which includes compact, leathery, conical
appearance, dry surface, often covered by air mycelium and observe the presence of spores.
5. Actinomycetes, which produce two types of mycelium, namely substrate or vegetative and air
mycelium, usually form special reproductive cells known as spores or conidia.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 19

Microscopic Morphological Characterization of Actinomycetes

Practical Objectives
Students can describe the microscopic morphology of actinomycetes cells.

Method
Tools and materials
a. Actinomycetes culture
b. YMA cup media
c. Sterile distilled water
d. Sterile Petri dishes
e. Sterile object glass and cover glass
f. Sterile toothpick
g. Microscope
h. Inoculation loop
i. Bunsen burner

Procedure
1. Slide the object glass
a. Cut a thin block of agar from a Petri dish filled with agar, place it in a sterile microscope slide,
then inoculate and cover it with a sterile cover glass.
b. After incubation in a humid chamber, observe the culture slide directly with a microscope,
observing the mycelium in the air and the substrate in the agar.
2. Tilt the glass cover to observe the morphology of actinomycetes
a. Inoculate an agar plate with a small amount of actinomycetes culture and mount the slide cover
at an angle near the scratch.
b. After incubation, attach an object glass cover to the object glass. The top surface faces the
bottom and then drips with water.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 20

 VII. CHARACTERIZATION OF YEAST CELLS

Yeast is a non-filamentous unicellular fungus. The growth characteristics of yeast

resemble bacteria when grown on the surface of artificial laboratory media. The size of yeast
colonies is 5 to 10 times larger than bacteria. Figure 12 illustrates a yeast colony. In microscopic
observation, yeast cells can be ellipsoidal, round, or, in some cases, cylindrical (Figure 13a).
Yeasts differ from fungi, i.e. they do not have aerial hyphae or supporting sporangia.

Figure 12. Yeast colonies growing on media in the laboratory (Cappuccino & Welsh, 2019)

Yeasts reproduce asexually by budding or by fission. Buds form from parent cells that pinch off,
producing daughter cells (Figure 13b). Division occurs in certain yeast species, such as in the
genus Schizosaccharomyces. Division occurs when the parent cell elongates, its nucleus divides,
and the cell divides evenly into two daughter cells.

Figure 13. The microscopic structure of (a) S. octosporus yeast cells and (b) S. cerevisiae cells
forming shoots resulting from vegetative reproduction (Cappuccino and Welsh, 2019)

Some yeasts can also undergo sexual reproduction when two sexual spores conjugate, producing
a zygote, or diploid cell. The nuclei of these cells divide by meiosis, producing four new haploid
nuclei (sexual spores), called ascospores contained in a structure called the ascus (Figure 14b).
When the ascus ruptures, the ascus spores are released, conjugate, and start the cycle again.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 21

Figure 14. Yeast structure (a) vegetative reproduction by budding and (b) sexual reproductive
structure.

Yeast is essential for many reasons. Saccharomyces cerevisiae is known as bread yeast and is
used as a leavening agent in dough. Two main yeast strains, S. carlsbergensis and S. cerevisiae,
are used for brewing beer. The wine industry relies on wild yeasts (present in grapes) for the
spontaneous fermentation of grape juice. Meanwhile, Saccharomyces ellipsoideus is used as a
fermentation inoculum. Yeast's high vitamin content makes it very valuable as a food supplement.

Practical Objectives
Students can describe the growth characteristics and types of reproduction used to identify
various genera of yeast.

Method
Tools and materials
a. Bunsen Lamp
b. Inoculation loop
c. Object glass
d. Cover glass
e. Drop pipette
f. Microscope
g. Whiteboard marker
h. Saccharomyces cerevisiae
i. Candida albicans
j. Lactophenol-cotton-blue solution

Procedure
1. Apply a tube of yeast culture in a few drops of lactophenol-cotton-blue solution placed on a
microscope slide and cover with a cover slip.
2. Examine all yeast wet slide preparations at low and high magnification, noting the shape and
presence or absence of buds.
3. Record observations on the observation table.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 22

 VIII. MOLD SLIDE CULTURE TECHNIQUE

Determining the genus and species of a mould culture is not easy; this requires
identification. The first stage in identifying mould recognizes the morphological characteristics
both macroscopically and microscopically. Macroscopic mould morphology can be seen from the
colour of the colony, the condition of the colony surface, the presence or absence of liquid spots
on the colony's surface, and the presence or absence of radial lines or concentric circles
(zonation). Meanwhile, microscopically, you can see the shape and size of the sporangium,
sporangiospores, and the form of mycelial branching.

Practical Objectives
Students can recognize the morphology of molds microscopically and identify them.

Method
Tools and materials
a. Object glass
b. Cover glass
c. Petri dish containing sterile tissue
d. Ent needle
e. Inoculation loop
f. Fan
g. Tweezers
h. Dropper bottle
i. Painting tub
j. Bunsen burner
k. Microscope
l. Tissue
m. One culture of mold isolate was prepared by the laboratory on an SA plate
n. SA medium
o. 70% alcohol solution
p. Sterile distilled water.

Procedure
1. Aseptically, a small amount of SA agar is placed on both ends of a sterile glass object using an
inoculation loop.
2. Each agar is inoculated with the mold provided using an Ent needle.
3. A sterile cover glass is placed on the top of the agar piece, and then the culture is incubated in
a sterile Petri dish containing a piece of sterile filter paper moistened with sterile distilled water.
Mold will grow on the object glass and cover the glass after incubation for three days.
4. First, dry the bottom surface of the culture slide with tissue. Observe under a microscope, first
with a weak objective lens and then with a strong magnification lens.
5. Note and draw everything observed, such as mycelium branched or not, septumized or not,
smooth or rough, sterigma, conidia, spores, conidiophores, sporangiophores, columella, vesicles,
as in Table 1.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 23

Table 1. Colony morphology and microscopic characteristics of mold (Cappuccino & Welsh, 2019)

MICROBIOLOGY LABORATORY PRACTICAL MODULE 24

 IX. MORPHOLOGY OF MOLDS AND YEASTS

Knowing the name of the genus and species of mold culture is not easy, so an
identification stage is required. The first stage for identification is the introduction of morphological
characteristics, both microscopic and macroscopic morphology. The color of the colony, the
surface of the colony, the presence or absence of radial lines or concentric circles (zonation) and
the presence or absence of liquid dots (exudate drops) on the surface of the colony are characters
that need to be noted and considered in macroscopic colony identification. Furthermore, the
shape and size of the sporangium, sporangiospores, mycelial branching form, branching and
conidial head shape are often accurate considerations for determining the species. A beginner
can certainly have difficulty distinguishing between the Genus Penicillium and the Genus
Paecilomyces. However, by looking at the development of the colony macroscopically and the
nature of the branching of the conidial heads microscopically, the two genera can clearly be
distinguished.
Yeast is a single-celled fungus and does not form mycelium, however, some species can
form pseudo-mycelium. The morphology of yeast is simpler than mold, but its size is larger than
bacteria. Important morphological characteristics of yeast include: cell shape (oval, round like a
sausage, elliptical), cell size, shape and number of buds, shape and number of spores in the
ascus, shape/formation and presence or absence of pseudomycelium are very important for
identification.

Morphology of Thread Molds

Macroscopic Observation
Practical Objectives
Students can recognize the colony morphology and growth characteristics of each type of mold
on the medium.

Method
Tools and materials
a. Sterile Petri dishes
b. Inoculation loop
c. Bunsen burner
d. Pure culture of mold; Aspergillus; Rhizopus; Penicillium; and Mucor
e. PDAs media

Procedure
1. Heat the prepared upright PDA media in a sterile Petri dish until it melts aseptically, and wait
until it cools and solidifies.
2. Transfer a small amount of the culture of each mold species to the surface of the media
3. Incubate for 4-5 days at room temperature
4. Observe every day the color changes that appear on the colony, the condition of the colony's
surface (flat, mountainous like flour, grainy or cotton-like), whether there are radial lines, the
presence of concentric lines or circles (zonation), the presence or absence of exudate drops as
well as the color, whether there is a distinctive odour and the shape and color of the colony.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 25

5. Record the data observed.

Microscopic Observation
Practical Objectives
Students can recognize morphological characteristics microscopically to differentiate one species
from another.
Method
Materials and tools
a. Pure culture of R. oryzae; A. oryzae; Penicillium sp.
b. Object glass and cover glass
c. Lactophenol solution or lactophenol cotton blue
d. Inoculation loop
e. Preparation needle
f. 70% alcohol solution
g. Bunsen burner

Procedure
1. Clean the object glass and cover it with 70% alcohol until they are free from fat, then drop a
few drops of lactophenol solution or lactophenol cotton blue on the surface of the object glass.
2. Take a small amount of the colony with an inoculation loop, place it in a drop of lactophenol
and spread it carefully with the preparation needle. Make sure all the wet mycelium is exposed to
lactophenol.
3. Cover the preparation with a cover glass so there are no air bubbles. Wipe off excess
lactophenol with blotting paper.
4. Observe with a microscope using an objective lens with 10x magnification and then with 40x
magnification. To see the morphology of conidia or spores, use an objective lens with 100x
magnification with immersion oil.
5. Note and draw everything observed, such as whether the mycelium branched or not,
septumized or not, smooth or rough, sterigma, conidia, spores, conidiophores, sporangiophores,
columella, vesicles, etc.

Yeast Morphology
Yeast Cell Staining
Practical Objectives
Students can recognize the various forms of yeast cells and differentiate dead cells from living
cells

Method
Tools and materials
a. Pure culture (72 hours) of Saccharomyces cerevisiae; Candida sp.; Hansenulla sp. in the
medium
b. Methylene blue (MB) solution
c. Object glass and cover glass
d. Inoculation loop

MICROBIOLOGY LABORATORY PRACTICAL MODULE 26

e. 70% alcohol solution (w/v)
f. Bunsen burner

Procedure
1. Clean the slide using 70% alcohol until it is grease-free
2. Drop a little MB on the object glass.
3. Take a small amount of the yeast culture with a loop, place it in the MB drop, and cover it with
a cover slip, ensuring no air bubbles.
4. Observe under a microscope using 40x and 100x magnification using immersion oil.
5. Draw and note cell shape, the presence or absence of budding, the number of shoots in each
cell, and the presence or absence of pseudo mycelium.

Staining Yeast Spores

Practical Objectives
Students can stain, see the various shapes of yeast spores, and count the number of spores in
the ascus.

Method
Tools and materials
1. Object glass.
2. Pure culture (72 hours) of S. cerevisiae; Candida sp.; Hansenulla sp. on carrot slant agar or
carrot slice medium
3. Crystal violet aniline paint solution (paint A)
4. Safranin paint solution (paint B)
5. Acid alcohol solution

Procedure
1. Suspend the pure yeast culture in sterile distilled water, then spread one loop over an object
glass covering an area of approximately 1 cm2.
2. Air dry, fix with a Bunsen burner for 5-7 times over a fire.
3. Cover the culture stain with paint A and heat it for 3 minutes, ensuring it doesn't dry out.
4. Wash with running water.
5. Dissolve with acidic alcohol.
6. Wash with running water
7. Drip with paint B for 10-15 seconds (exactly)
8. Wash with running water and air dry
9. Observe with a microscope at strong magnification with immersion oil
10. Draw and give a complete description of the yeast cells and their spores.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 27

 X. MORPHOLOGICAL CHARACTERIZATION OF ALGAE

Protists can be easily grouped according to their ecological role as algae, protozoa, and fungus-
like protists. The term algae (Latin for "seaweed") applies to about ten groups of protists, most of
which are photosynthetic species. Despite the general features of photosynthesis, algae do not
form monophyletic groups descending from a common ancestor. However, it is polyphyletic (that
is, it has more than one common ancestor), and the details of its phylogeny are still being
researched).
Algae are photosynthetic organisms, eukaryotic and usually do not have multicellular sex organs.
The major groups of algae are differentiated in part by their energy storage products, cell walls,
and colour resulting from the type and abundance of coloured pigments (substances that absorb
light) in their plastids. Biologists often group the algae based on the color of these pigments—for
example, green, brown, and red algae.
Algae are also differentiated based on their cellular organization (Figure 15). Unicellular algae
species appear as single, unattached cells that may or may not be motile. Filamentous algae
species occur as chains of cells attached end to end. These filaments may have few or many cell
lengths and may be unbranched or branched in various patterns. Colonial algae occur as groups
of cells attached in a nonfilamentous manner. For example, a colony may include several to many
cells stuck together as a sphere, flat sheet, or other three-dimensional shape.

Figure 15. The diversity of algal body types reflects their habitat.
(a) The single-celled flagellate genus Chlamydomonas resides in lake phytoplankton. (b) The
colonial genus Pediastrum consists of several cells arranged in a lacy star shape which helps this
algae stay buoyant in the water. (c) Genus Desmidium is filamentous and occurs as rows of
twisted cells. (d) Cladophora is a genus of branching filaments that grow attached to surfaces
near shore large enough to be seen with the naked eye. (e) The relatively large seaweed genus
Acetabularia lives on rocks and coral debris in shallow tropical seas.

Practical Objectives
Students can describe unicellular algae.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 28

Method
Tools and materials
a. Microscope
b. Drop pipette
c. Object glass
d. Cover glass
e. Samples of rice field water, soil, rivers, river soil/sand, and ponds
f. Methylcellulose solution 1-10% (w/v)
g. Lugol's solution

Procedure
1. A drop of water that is thought to contain live unicellular algae is placed on a glass object and
then covered with a glass cover. This procedure is repeated on a new glass object where one
drop of methylcellulose has been placed first. After that, a drop of the same sample water was
added.
2. The preparation is observed with a microscope.
3. Based on the structure, determine the appropriate type of algae
a) Chlamydomonas
1) Preparation showing a Chlamydomonas specimen, if the movement is too fast, make a new
preparation by placing one or two drops of methylcellulose on the slide and adding a drop of water
containing Chlamydomonas.
2) Stir gently and add the cover glass.
3) Note the stigma, which appears as a light-absorbing reddish spot at the anterior end of the cell.
b) Volvox
1. The preparation showing the Volvox specimen is observed with a microscope under low
magnification, observing large, hollow and round colonies.
2. Another structure observed is the presence of flagella on the surface.
c) Diatoms
1) The preparations showing Diatom specimens were observed for their shell types; some were
broken, and some were intact.
2) a clean, insoluble, porous shell mass is another structure observed.
d) Dinoflagellates (supergroup Chromalveolata)
Preparations showing dinoflagellate specimens were observed for flagella and flagellar
longitudinal and transverse grooves.
e) Euglenoids (Excavata Supergroup)
1) The preparation showing the Euglena specimen is observed for the presence of colored eye
spots near the base of the flagella
2) To slow down Euglena, adding a drop of methylcellulose to the preparation is necessary.
3) Another characteristic observed is the movement and changes in the organism's shape.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 29

 XI. PROTOZOA

Protozoa are a large and diverse group of unicellular eukaryotic organisms. Protozoan
taxonomy is continually updated as new technologies allow classification based on molecular
characteristics. For our discussion of protozoa, we follow a more traditional taxonomic scheme,
dividing them into four groups based on locomotion:
1. Sarcodina:
The motility results from the flow of ectoplasm, producing protoplasmic projections called
pseudopods (false legs). Prototypic amoebas include the free-living Amoeba proteus and the
parasite Entamoeba histolytica.
2. Mastigophora:
It has one or more whips, thin structures called flagella. Free-living members include the genera
Cercomonas, Heteronema and Euglena, which are photosynthetic protists that can be classified
as flagellated algae. Parasitic forms include Trichomonas vaginalis, Giardia intestinalis (formerly
called Giardia lamblia), and Trypanosoma species.
3. Ciliophora:
Short, hair-like strands called cilia, whose synchronous beating drives organisms to movement.
A characteristic example of a free-living member of this group is Paramecium caudatum, and an
example of a parasite is Balantidium coli.
4. Sporozoa:
Unlike other members of this phylum, sporozoa do not have locomotor organ cells in their adult
stages; however, immature forms show some movement. All members of this group are parasites.
The most significant members of the genus Plasmodium are malaria parasites in animals and
humans.

Practical Objectives
Students can describe protozoa found in water samples.

Method
Tools and materials
a. Microscope
b. Glasses and covers
c. Pasteur pipette or dropper pipette
d. Well, water, pond water, rice field water, river water, sewer or sewage water
e. Amoeba, Paramecia, Euglena and Stentor preparations (adjusted to availability in the
laboratory).
f. Methylcellulose solution 1-10% (v/v)

Procedure
1. One drop of a pool water sample is taken from the bottom of the water sample and placed in
the middle of a clean glass object.
2. One drop of methyl cellulose was added to the sample to slow down the movement of protozoa.
3. The slide is then covered using a coverslip in the following way to prevent the formation of air
bubbles.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 30

a. Place one edge of the coverslip on the outer edge of the culture drop.
b. Once the culture drop has spread along the inside edge of the coverslip, carefully lower the
coverslip onto the slide.
c. The slide culture is observed under a microscope at the lowest to highest magnification and
observes the presence of different protozoa. Record the results of observations in a report.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 31

 XII. BACTERIOPHAGE OBSERVATION

Bacteriophages are viruses that infect bacteria and can kill the bacterial cells directly or
integrate viral DNA into the chromosomes of the host bacteria. Like other viruses, bacteriophages
also have similar components in the form of a protein coat and nucleic acid in the form of ssRNA,
dsDNA and dsRNA (ss: single strand, ds: double strand); the shape of the nucleic acid strands is
generally linear, circular or segmented.
Judging from the behaviour of bacteriophages, some bacteriophages can insert their
nucleic acid with the nucleic acid of the host bacteria and also be able to directly cause the lysis
of the host bacteria by producing several enzymes that play a role in lysing the bacterial cells.
The results of bacterial lysis by viruses can be observed using Plaque Assay.
Bacterial virus isolates (bacteriophages) can be obtained from various natural sources,
including soil, intestinal contents, raw unprocessed waste, and some insects, such as
cockroaches and flies. Isolation of viruses from this environment is not easy, because phage
particles are usually present in low concentrations. Therefore, isolation requires a series of steps:
1. Collection of samples containing phages in the source.
2. Add enriched susceptible host cell culture to the sample to increase the number of
phage particles for subsequent isolation.
3. After incubation, centrifugation of enriched samples to obtain concentrated particles.
4. The supernatant fluid is filtered through a bacteria-retaining membrane filter.
5. The bacteria-free filtrate is inoculated into the growth area of susceptible host cells
grown on soft agar plate media.
6. Incubation and observation of the culture for the presence of phage particles,
characterised by plaque formation in the area of bacterial growth on the agar plate.

Practical Objectives
Students can observe the formation of plaque by bacteriophages in bacterial cultures.

Method
Tools and materials
a. Erlenmeyer flask
b. Filter membrane
c. Beaker Glass
d. Centrifuge
e. Bunsen burner
f. 1 mL micropipette and tip
g. Test tube rack
h. Label
i. Incubator
j. Sterile Pasteur pipette
k. Five Petri dishes containing Nutrient Agar (NA) media (1.5% agar)
l. Five test tubes containing 3 mL of soft NA media (0.5% Agar)
m. Escherichia coli (24 hours) culture in Nutrient Broth (NB) media
n. T2 coliphage (water sample from Bedadung River)

MICROBIOLOGY LABORATORY PRACTICAL MODULE 32

Procedure
1. Enrichment of Waste Water Samples
a. Add 5 ml of E. coli culture (24 hours) grown in NB media to 45 mL of wastewater sample in a
sterile 250 mL Erlenmeyer flask.
b. Incubated the culture at 37℃ for 24 hours.
2. Filtration and Seeding
a. After being infected with phage, put 50-100 mL of the E. coli culture into microtubes of 2 mL
each and centrifuged at 2500 rpm for 20 minutes.
b. After the centrifuge, the supernatant is carefully poured into a 125 ml beaker.
c. Filter the supernatant with a filter membrane to obtain a bacteria-free supernatant.
d. Melt the NA soft agar media in five test tubes in boiling water and cooled to a temperature of
45℃ so the media remains melted.
e. Label the five reaction tubes containing NA soft agar media with 1, 2, 3, 4, and 5, respectively.
f. Using a micropipette, aseptically add 0.1 mL of E. coli liquid culture to each of the five soft NA
agar tubes.
g. Add 1, 2, 3, 4, and 5 drops of the filtrate to each labelled soft agar tube with a sterile Pasteur
pipette. Mix and pour each tube of softened agar into an agar dish corresponding to the label.
h. The agar in the cup is allowed to harden.
i. All culture dishes were incubated in an inverted position for 24 hours at 37℃.
3. Observation method
a. Observe all culture plates for plaque formation, which indicates the presence of phagecoli
(coliphages) in the culture.
b. Indication of the presence of plaque is marked plus (+) or no negative (-) in each culture in the
practical report table. If there is an indication of plaque, the number is counted.

MICROBIOLOGY LABORATORY PRACTICAL MODULE 33

Figure 16. Wastewater sample enrichment, filtration, and seeding (Cappuccino & Welsh, 2019)

MICROBIOLOGY LABORATORY PRACTICAL MODULE 34

 REFERENCES

Brock, T.D., M.T. Madigan, J.M. Martinko and J. Parker. (2015). Biology of Microorganisms.
Prontice Hall International Inc. 14th ed. The USA.

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