PArT 1

Basic Laboratory Techniques

for Isolation, Cultivation,
and Cultural Characterization
of Microorganisms
LearnIng OBjeCTIves

Once you have completed the experiments in this section, you should be
familiar with
1. The types of laboratory equipment and culture media needed to develop and
maintain pure cultures.
2. The types of microbial flora that live on the skin and the effect of hand
washing on them.
3. The concept of aseptic technique and the procedures necessary for
successful subculturing of microorganisms.
4. Streak-plate and spread-plate inoculation of microorganisms in a mixed
microbial population for subsequent pure culture isolation.
5. Cultural and morphological characteristics of microorganisms grown in pure
culture.

Introduction medium. Basically, all culture media are liquid,

semisolid, or solid. A liquid medium lacks a solidi-
Microorganisms are ubiquitous. They are found fying agent and is called a broth medium. A broth
in soil, air, water, food, sewage, and on body sur- medium is useful for the cultivation of high num-
faces. In short, every area of our environment is bers of bacterial cells in a small volume of medium,
replete with them. The microbiologist separates which is particularly helpful when an assay
these mixed populations into individual species requires a high number of healthy bacterial cells.
for study. A culture containing a single unadultera- A broth medium supplemented with a solidifying
ted species of cells is called a pure culture. To agent called agar results in a solid or semisolid
isolate and study microorganisms in pure culture, medium. Agar, an extract of seaweed, is a com-
the microbiologist requires basic laboratory appa- plex carbohydrate composed mainly of galactose,
ratus and the application of specific techniques, and is without nutritional value. Agar serves as
as illustrated in Figure P1.1. an excellent solidifying agent because it liquefies
at 100°C and solidifies at 40°C. Because of these
Media properties, organisms, especially pathogens, can
The survival and continued growth of microorgan- be cultivated at temperatures of 37.5°C or slightly
isms depend on an adequate supply of nutrients higher without fear of the medium liquefying. A
and a favorable growth environment. For survival, completely solid medium requires an agar concen-
most microbes must use soluble low-molecular- tration of 1.5% to 1.8%. A concentration of less than
weight substances that are frequently derived from 1% agar results in a semisolid medium. A semi-
the enzymatic degradation of complex nutrients. solid medium is useful for testing a cell’s ability to
A solution containing these nutrients is a culture grow within the agar at lower oxygen levels and

17
 Broth
Media Semisolid Agar slant
Solid Agar deep
Autoclave Agar plate
Bunsen burner
Microincinerator
Culture tubes
Petri dishes
Equipment Wire loops and needles Transfer instruments
Pipettes
Waterbaths
Cultivation chambers
Incubators
Refrigerators

Streak plate
Pure culture techniques Pour plate–loop dilution Isolation of pure cultures
Spread plate

Figure P1.1 Laboratory apparatus and culture techniques

for testing the species’ motility. A solid medium area for microorganism growth while minimizing
has the advantage that it presents a hardened the amount of medium required. Similar tubes
surface on which microorganisms can be grown that, following preparation, are allowed to harden
using specialized techniques for the isolation of in the upright position are designated as agar
discrete colonies. Each colony is a cluster of cells deep tubes. Agar deep tubes are used primarily
that originates from the multiplication of a single for the study of the gaseous requirements of
cell and represents the growth of a single species microorganisms since gas exchange between
of microorganism. Such a defined and well­isolated the agar at the butt of the test tube and the exter­
colony is a pure culture. Also, while in the lique­ nal environment is impeded by the height of the
fied state, solid media can be placed in test tubes, agar. Liquid agar medium can also be poured into
which are then allowed to cool and harden in a Petri dishes, producing agar plates, which provide
slanted position, producing agar slants. These are large surface areas for the isolation and study of
useful for maintaining pure cultures. The slanted microorganisms. The various forms of solid media
surface of the agar maximizes the available surface are illustrated in Figure P1.2.

Side view Front view

(a) Agar slants (b) Agar deep tube (c) Agar plate

Figure P1.2 Forms of solid (agar) media

18 Part 1
 160° to 180°C for 11/2 to 3 hours; for
Dry (hot air)
empty glassware, glass pipettes, and glass syringes

Free-flowing steam at 100°C (intermittent

sterilization); for thermolabile solutions (e.g.,
sugars, milk)
Heat Moist (wet heat)
Autoclave, steam under pressure, temperatures
above 100°C; for culture media, syringes,
thermostable solutions, etc.

Cellulose-acetate membrane filters Removal of organisms from thermolabile solutions

Filtration with pore sizes in the range of 8.0 µm by passage through filters that retain bacteria; note,
to less than 0.05 µm viruses are not removed by this procedure

Ethylene oxide Plastic dishes and pipettes

Chemicals
Beta-propiolactone Living tissues

Radiation Ionizing Plastic pipettes and Petri dishes

Figure P1.3 Sterilization techniques

In addition to nutritional needs, the environ­ sleeve­like caps (Morton closures) made of metal,
mental factors must also be regulated, including such as stainless steel, or heat­resistant plastics.
proper pH, temperature, gaseous requirements, and The advantage of these closures over the cotton
osmotic pressure. A more detailed explanation is plug is that they are labor­saving and, most of all,
presented in Part 4, which deals with cultivation of slip on and off the test tubes easily.
microorganisms; for now, you should simply bear in Petri dishes provide a larger surface area for
mind that numerous types of media are available. growth and cultivation. They consist of a bottom
dish portion that contains the medium and a larger
top portion that serves as a loose cover. Petri
Aseptic Technique dishes are manufactured in various sizes to meet
Sterility is the hallmark of successful work in the different experimental requirements. For routine
microbiology laboratory, and sterilization is the purposes, dishes approximately 15 cm in diameter
process of rendering a medium or material free are used. The sterile agar medium is dispensed to
of all forms of life. To achieve sterility, it is manda­ previously sterilized dishes from molten agar deep
tory that you use sterile equipment and employ tubes containing 15 ml to 20 ml of medium, or from
aseptic techniques when handling bacterial a molten sterile medium prepared in bulk and con­
cultures. Using correct aseptic techniques mini­ tained in 250­, 500­, and 1000­ml flasks, depending
mizes the likelihood that bacterial cultures will on the volume of medium required. When cooled
be contaminated, and reduces the opportunity for to 40°C, the medium will solidify. Remember that
students to be exposed to potential pathogens. after inoculation, Petri dishes are incubated in an
Although a more detailed discussion is presented inverted position (top down) to prevent condensa­
in Part 9, which describes the control of microor­ tion formed on the cover during solidification from
ganisms, Figure P1.3 is a brief outline of the routine dropping down onto the surface of the hardened
techniques used in the microbiology laboratory. agar. For this reason, Petri dishes should be labeled
on the bottom of the dish. This makes it easier to
Culture Tubes and Petri Dishes read the label and minimizes confusion if two Petri
dish covers are interchanged. Figure P1.4 illustrates
Glass test tubes and glass or plastic Petri dishes
some of the culture vessels used in the laboratory.
are used to cultivate microorganisms. A suitable
Built­in ridges on tube closures and Petri dishes pro­
nutrient medium in the form of broth or agar may
vide small gaps necessary for the exchange of air.
be added to the tubes, while only a solid medium
is used in Petri dishes. A sterile environment is
maintained in culture tubes by various types of Transfer Instruments
closures. Historically, the first type, a cotton plug, Microorganisms must be transferred from one
was developed by Schröeder and von Dusch in the vessel to another or from stock cultures to various
nineteenth century. Today most laboratories use media for maintenance and study. This transfer

Part 1 19
 A B C D E

(b) Petri dish

A. Bacteriological tube D. Metal closure

B. Screw cap E. Nonabsorbent cotton
C. Plastic closure

(a) Test tube rack with tubes showing various closures (c) DeLong shaker flask with closure

Figure P1.4 Culture vessels

is called subculturing and must be carried out (commonly called a “pipetter”) with a disposable,
under aseptic conditions to prevent possible single­use plastic tip is useful for transferring small
contamination. volumes of liquid (less than …1 ml).
Wire loops and needles are made from inert Figure P1.5 illustrates these transfer instru­
metals such as Nichrome or platinum and are ments. Your instructor will demonstrate the proper
inserted into metal shafts that serve as handles. procedure for using pipettes.
They are extremely durable instruments and are
easily sterilized by incineration in the blue (hottest)
portion of the Bunsen burner flame. A wire loop is
Pipetting by mouth is not permissible!
useful for transferring a small volume of bacteria
Pipetting must be performed with mechanical
onto the surface of an agar plate or slant. A needle
pipette aspirators.
is used primarily to inoculate a culture into a broth
medium or into an agar deep tube.
A pipette is another instrument used for
aseptic transfers. Pipettes are similar in function
to straws; that is, they draw up liquids. They are Cultivation Chambers
glass or plastic and drawn out to a tip at one end, The specific temperature requirements for growth
with a mouthpiece forming the other end. They are discussed in detail in Part 4. However, a prime
are calibrated to deliver different volumes depend­ requirement for the cultivation of microorganisms
ing on requirements. Pipettes may be sterilized is that they be grown at their optimum tempera­
in bulk inside canisters, or they may be wrapped ture. An incubator is used to maintain optimum
individually in brown paper and sterilized in temperature during the necessary growth period.
an autoclave or dry­heat oven. A micropipette It resembles an oven and is thermostatically

20 Part 1
 Loop
No etched ring
on mouthpiece
Needle Etched ring (to deliver)
on mouthpiece
(blow out)

TD 1 IN 1/100 ml
Identification
and graduations

0.1 ml: major

division

0.01 ml each:
Shaft minor divisions

Handle

Final few drops

must be blown
out to deliver
indicated volume

(a) Transfer (b) Transfer (c) Blow-out (d) To-deliver

needle loop pipette pipette

Mechanical Pipette Aspirators

(f) Plastic (g) Rubber

(e) Micropipette pump bulb

Figure P1.5 Transfer instruments

Part 1 21
controlled so that temperature can be varied Many laboratories also use shaking incuba­
depending on the requirements of specific micro­ tors that utilize dry air incubation to promote
organisms. Most incubators use dry heat. Moisture aeration of the broth medium. This method has
is supplied by placing a beaker of water in the a distinct advantage over a shaking waterbath
incubator during the growth period. A moist envi­ since there is no chance of cross contamination
ronment retards dehydration of the medium and from microorganisms that might grow in the
thereby avoids misleading experimental results. waterbath.
A thermostatically controlled shaking
waterbath is another piece of apparatus used to
cultivate microorganisms. Its advantage is that Refrigerator
it provides a rapid and uniform transfer of heat A refrigerator is used for a wide variety of purposes
to the culture vessel, and its agitation provides such as maintenance and storage of stock cultures
increased aeration, resulting in acceleration of between subculturing periods, and storage of ster­
growth. The primary disadvantage of this instru­ ile media to prevent dehydration. It is also used as
ment is that it can be used only for cultivation a repository for thermolabile solutions, antibiotics,
of organisms in a broth medium. serums, and biochemical reagents.

22 Part 1
 E xP E R IMEnT

Culture Transfer Techniques

1
4. Uncap each tube by grasping the first cap with
LearnIng OBjeCTIves
your little finger and the second cap with your
Once you have completed this experiment, next finger and lifting the closure upward.
you should be able to Note: Once removed, these caps must be kept
in the hand that holds the sterile inoculating
1. Carry out the technique for aseptic
loop or needle; the inner aspects of the caps
removal and transfer of microorganisms point away from the palm of the hand. The
for subculturing. caps must never be placed on the laboratory
2. Correctly sterilize inoculating instruments bench because that would compromise the
in a microincinerator or the flame of aseptic procedure.
a Bunsen burner. 5. After removing the caps, flame the necks and
3. Correctly remove and replace the test mouths of the tubes by briefly passing them
tube closure. through the opening of the microincinerator or
through the Bunsen burner flame two to three
times rapidly. The sterile transfer instrument
is further cooled by touching it to the sterile
inside wall of the culture tube before removing
Principle a small sample of the inoculum.
Microorganisms are transferred from one medium 6. Depending on the culture medium, a loop or nee­
to another by subculturing. This technique is of dle is used for removal of the inoculum. Loops
basic importance and is used routinely in prepar­ are commonly used to obtain a sample from a
ing and maintaining stock cultures, as well as in broth culture. Either instrument can be used to
microbiological test procedures. obtain the inoculum from an agar slant culture
Microorganisms are always present in the by carefully touching the surface of the solid
air and on laboratory surfaces, benches, and medium in an area exhibiting growth so as not to
equipment. These ambient microorganisms gouge the agar. A straight needle is always used
can serve as a source of external contamination when transferring microorganisms to an agar
and interfere with experimental results unless deep tube from both solid and liquid cultures.
proper aseptic techniques are used during a. For a slant­to­broth transfer, obtain inoculum
subculturing. Described below are essential from the slant and lightly shake the loop or
steps that you must follow for aseptic transfer needle in the broth culture to dislodge the
of microorganisms. The complete procedure microorganisms.
is illustrated in Figure 1.1.
b. For a broth­to­slant transfer, obtain a loop­
1. Label the tube to be inoculated with the name full of broth and place at the base of an agar
of the organism and your initials. slant medium. Lightly draw the loop over the
2. Hold the stock culture tube and the tube to hardened surface in a straight or zig­zag line,
be inoculated in the palm of your hand, secure from the base of the agar slant to the top.
with your thumb, and separate the two tubes c. For a slant­to­agar deep tube transfer,
to form a V in your hand. obtain the inoculum from the agar slant.
3. Sterilize an inoculating needle or loop by Insert a straight needle to the bottom of the
holding it in the microincinerator or the hottest tube in a straight line and rapidly withdraw
portion of the Bunsen burner flame, until the along the line of insertion. This is called a
wire becomes red hot. Once sterilized, the stab inoculation.
loop is held in the hand and allowed to cool 7. Following inoculation, remove the instrument
for 10 to 20 seconds; it is never put down. and reheat or reflame the necks of the tubes.

23
 PROCEDURE

1 Label the tube to be inoculated with the 2 Place the tubes in the palm of your hand, secure
name of the organism and your initials. with your thumb, and separate to form a V.

3 Flame the needle or loop 4 With the sterile loop or needle 5 Flame the necks of the tubes by
until the wire is red. in hand, uncap the tubes. rapidly passing them through
the flame once.

6 Slant-to-broth transfer: Obtain Broth-to-slant transfer: Obtain a loopful Slant-to-agar deep transfer: Obtain
inoculum from slant and dislodge of broth and place at base of slant. inoculum from slant. Insert the needle to
inoculum in the broth with a slight Withdraw the loop in a zigzag motion. the bottom of the tube and withdraw
agitation. along the line of insertion.

7 Flame the necks of the tubes by 8 Recap the tubes. 9 Reflame the loop or needle.
rapidly passing them through
the flame once.

Figure 1.1 Subculturing procedure

24 Experiment 1
8. Replace the caps on the same tubes from Equipment
which they were removed.
Microincinerator or Bunsen burner, inoculating
9. Resterilize the loop or needle to destroy any loop and needle, and glassware marking pencil.
remaining organisms.
In this experiment, you will master the
manipulations required for aseptic transfer of Procedure Lab One
microorganisms in broth­to­slant, slant­to­broth,
1. Label all tubes of sterile media as described in
and slant­to­agar deep tubes. You will be using a
the Laboratory Protocol section on page 15.
positive and a negative control to test your ability
to maintain aseptic techniques while transferring 2. Following the procedure outlined and
cultures. The technique for transfer to and from illustrated previously (Figure 1.1), perform
agar plates is discussed in Experiment 2. the following transfers:
a. Broth culture “A” to a nutrient agar slant,
nutrient agar deep tube, and nutrient
broth.
C L I n I C A L A P P L I C AT I o n b. Broth culture “B” to a nutrient agar slant,
Aseptic Inoculation and Transfer nutrient agar deep tube, and nutrient
broth.
It is mandatory that microbiology laboratory workers
learn and perfect the skill of inoculating bacterial c. S. marcescens agar slant culture to a
specimens on agar plates, in liquid broth, or in nutrient agar slant, nutrient agar deep
semisolid medium, and be able to subculture the tube and nutrient broth.
organism from one medium to another. A sterile 3. Incubate all cultures at 25°C for 24 to
inoculating needle or loop is the basic instrument 48 hours.
of transfer. Keep in mind that, transferring bacterial
cultures requires aseptic or sterile techniques at all
times, especially if you are working with pathogens.
Do not contaminate what you are working with and
Procedure Lab Two
do not contaminate yourself. 1. Examine all cultures for the appearance of
growth, which is indicated by turbidity in
the broth culture and the appearance of an
orange­red growth on the surface of the slant
and along the line of inoculation in the agar
deep tube.
AT T h E B E n C h 2. Record your observations in the chart
provided in the Lab Report.
3. Confirm your results with the instructor to
Materials determine the negative control tube.

Cultures
Twenty­four–hour nutrient broth and nutrient agar
slant cultures of Serratia marcescens and a sterile Tips for SucceSS
tube of nutrient broth. The nutrient broth tubes
1. It is imperative that you maintain sterility and
will be labeled “A” and “B,” and the contents will
utilize aseptic techniques at all times. If you
be known only by the instructor.
allow a contaminating organism into your
bacterial culture, you will see a positive growth
Media in media that was inoculated with the negative
Per student: three nutrient broth tubes, three nutri­ control.
ent agar slants, and three nutrient agar deep tubes.

Experiment 1 25
 E xP E R IMEnT

Name:
1
Date: Section: Lab report

Observations and results Culture “a”

Nutrient Nutrient Agar Nutrient Agar
Broth Slant Deep
Growth
(+) or (–)

Orange-red
pigmentation
(+) or (–)

Draw the
distribution of
growth.

Observations and results Culture “B”

Nutrient Nutrient Agar Nutrient Agar
Broth Slant Deep
Growth
(+) or (–)

Orange-red
pigmentation
(+) or (–)

Draw the
distribution of
growth.

Experiment 1: Lab Report 27

Observations and results S. marcescens
Nutrient Nutrient Agar Nutrient Agar
Broth Slant Deep
Growth
(+) or (–)

Orange-red
pigmentation
(+) or (–)

Draw the
distribution of
growth.

1. Explain why the following steps are essential during subculturing:

a. Flaming the inoculating instrument prior to and after each inoculation.

b. Holding the test tube caps in the hand as illustrated in Figure 1.1 on page 24.

c. Cooling the inoculating instrument prior to obtaining the inoculum.

d. Flaming the neck of the tubes immediately after uncapping and before
recapping.

28 Experiment 1: Lab Report

2. What are ambient microorganisms? Why should they not be present during
subculturing?

3. Explain why a straight inoculating needle is used to inoculate an agar

deep tube.

4. There is a lack of orange­red pigmentation in some of the growth

on your agar slant labeled S. marcescens. Does this necessarily
indicate the presence of a contaminant? Explain.

5. Upon observation of the nutrient agar slant culture, you strongly

suspect that the culture is contaminated. Outline the method you
would follow to ascertain whether your suspicion is justified.

Experiment 1: Lab Report 29

 E xP E R IMEnT
Techniques for Isolation
of Pure Cultures 2
In nature, microbial populations do not segregate
themselves by species, but exist with a mixture
Principle
of many other cell types. In the laboratory, The techniques commonly used for isolation of
these populations can be separated into pure discrete colonies initially require that the number
cultures. These cultures contain only one type of organisms in the inoculum be reduced. The
of organism and are suitable for the study of resulting diminution of the population size ensures
their cultural, morphological, and biochemical that, following inoculation, individual cells will
properties. be sufficiently far apart on the surface of the
In this experiment, you will first use one of the agar medium to separate the different species.
techniques designed to produce discrete colonies. The following are techniques that can be used
Colonies are individual, macroscopically visible to accomplish this necessary dilution:
masses of microbial growth on a solid medium 1. The streak-plate method is a rapid qualitative
surface, each representing the multiplication isolation method. It is essentially a dilution
of a single organism. Once you have obtained technique that involves spreading a loopful
these discrete colonies, you will make an aseptic of culture over the surface of an agar plate.
transfer onto nutrient agar slants for the isolation Although many types of procedures are per­
of pure cultures. formed, the four­way, or quadrant, streak
is described. Refer to Figure 2.1, which
schematically illustrates this technique.
PA RT A Isolation of Discrete a. Place a loopful of culture on the agar
Colonies from a Mixed Culture surface in Area 1. Flame the loop, cool it
by touching an unused part of the agar
surface close to the periphery of the plate,
LearnIng OBjeCTIve and then drag it rapidly several times
across the surface of Area 1.
Once you have completed this experiment,
you should be able to b. Reflame and cool the loop, and turn the
Petri dish 90°. Then touch the loop to a
1. Perform the streak-plate and/or the corner of the culture in Area 1 and drag
spread-plate inoculation procedure it several times across the agar in Area 2.
to separate the cells of a mixed culture The loop should never enter Area 1 again.
so that discrete colonies can be c. Reflame and cool the loop and again; turn
isolated. the dish 90°. Streak Area 3 in the same
manner as Area 2.

Turn Turn Turn

plate 90. plate 90. plate 90.

2 3 4
Flame 1 Flame Flame
loop. loop. loop. 2
1 3
1

1 2

Figure 2.1 Four-way streak-plate technique

31
 yet to master the necessary lab skills that
would allow them to use the rapid method
Heavy confluent listed above. This alternative method involves
growth spreading a loopful of culture over the surface
of an agar plate that has the quadrants laid out
Heavy growth visibly for quick reference. Refer to Figure 2.3,
which illustrates this technique.
Discrete colonies
a. Using a marker, draw two bisecting lines
on the bottom of the Petri dish to divide
the plate into 4 equal parts. Label each
Light growth quadrant 1 through 4, starting with the
top right quadrant and labeling counter­
clockwise. Sterilizing the loop at the points
Figure 2.2 Four-way streak-plate inoculation indicated is to dilute the culture due to
with Serratia marcescens fewer organisms available to be streaked
into each area, resulting in the final desired
separation.
d. Without reflaming the loop, again turn
the dish 90° and then drag the culture b. Turn the Petri dish over and place a loopful
from a corner of Area 3 across Area 4, of culture on the agar surface in quadrant 1.
using a wider streak. Don’t let the loop Using the edge of the loop and holding
touch any of the previously streaked the loop at a shallow angle so as not to
areas. The flaming of the loop at the points gouge the agar, quickly spread the bacteria
indicated is to dilute the culture so that throughout the quadrant.
fewer organisms are streaked in each area, c. Reflame and cool the loop, and turn
resulting in the final desired separation. the Petri dish 90°. Then touch the loop
A photograph of a streak­plate inoculation into an area that has been streaked in
is shown in Figure 2.2. quadrant 1 and drag it across the agar
2. An alternative streak­plate method is for into quadrant 2, repeat this twice without
students new to the laboratory who have flaming the loop.

(a)
Label bottom of dish View through agar

2 1

4
3 2
1
4 3

(b)

3 4
1 2 1 2
2

2 1
4 3 4 3
1

Figure 2.3 Alternate streak-plate method

32 Experiment 2
 d. Reflame and cool the loop and again turn the
dish 90°. Streak the bacteria into quadrant 3
C L I n I C A L A P P L I C AT I o n
in the same manner used for quadrant 2. Isolation of Cultures as a Diagnostic
e. Reflame and cool the loop and again turn the Technique
dish 90°. Streak the bacteria into quadrant 4 The isolation of pure cultures is the most important
in the same manner used for quadrant 3. diagnostic tool used in a clinical or research
3. The spread-plate technique requires that laboratory to uncover the cause of an infection
a previously diluted mixture of microorgan­ or disease. Before any biochemical or molecular
isms be used. During inoculation, the cells techniques may be used to identify or characterize
are spread over the surface of a solid agar the causative organism, an individual bacterial
medium with a sterile, L­shaped bent glass rod colony must be isolated for testing. The isolation
while the Petri dish is spun on a “lazy Susan” of Staphylococcus aureus from cultures taken from
turntable. The step­by­step procedure for this abscesses or Streptococcus pyogenes from a throat
technique is as follows: culture are two examples of clinical applications
a. Place the bent glass rod into a beaker and of this technique.
add a sufficient amount of 95% ethyl alcohol
to cover the lower, bent portion.
b. Place an appropriately labeled nutrient agar
plate on the turntable. With a sterile pipette,
place one drop of sterile water on the
center of the plate, followed by a sterile
loopful of Micrococcus luteus. Mix gently
AT T hE BE nCh
with the loop and replace the cover.
c. Remove the glass rod from the beaker, and
pass it through the Bunsen burner flame Materials
with the bent portion of the rod pointing
downward to prevent the burning alcohol Cultures
from running down your arm. Allow the 24­ to 48­hour nutrient broth cultures of a
alcohol to burn off the rod completely. mixture of one part Serratia marcescens and
Cool the rod for 10 to 15 seconds. three parts Micrococcus luteus and a mixture
d. Remove the Petri dish cover and spin the of one part Escherichia coli and ten parts
turntable. Micrococcus luteus.
e. While the turntable is spinning, lightly touch Sources of mixed cultures from the environ­
the sterile bent rod to the surface of the ment could include cultures from a tabletop,
agar and move it back and forth. This will bathroom sink, water fountain, or inside of an
spread the culture over the agar surface. incubator. Each student should obtain a mixed
culture from one of the environmental sources
f. When the turntable comes to a stop, replace
listed above.
the cover. Immerse the rod in alcohol and
reflame.
g. In the absence of a turntable, turn the
Media
Petri dish manually and spread the culture Three Trypticase™ soy agar plates per designated
with the sterile bent glass rod. student group for each inoculation technique to
be performed.
4. The pour-plate technique requires a serial
dilution of the mixed culture by means of
a loop or pipette. The diluted inoculum is Equipment
then added to a molten agar medium in a Microincinerator or Bunsen burner, inoculating
Petri dish, mixed, and allowed to solidify. loop, turntable, glassware marking pencil, culture
The serial dilution and pour­plate procedures tubes containing 1 ml of sterile water, test tube
are outlined in Experiment 18. rack, and sterile cotton swabs.

Experiment 2 33
Procedure Lab One Tips for SucceSS
1. Following the procedures previously described, 1. an isolation plate has isolated distinct,
prepare a spread­plate and/or streak­plate individual colonies. If your technique results
inoculation of each test culture on an in isolated colonies in a quadrant that was not
appropriately labeled plate. the last one to be streaked, that is okay. The
2. Prepare an environmental mixed culture. point of using this method is to get those indi-
vidual colonies somewhere on the plate.
a. Dampen a sterile cotton swab with sterile
2. Pay attention to how well you sterilize your
water. Wring out the excess water by
loop and maintain your aseptic technique.
pressing the wet swab against the walls
If the loop is not properly sterilized between
of the tube.
streaks, or your aseptic technique is not
b. With the moistened cotton swab, obtain maintained, the resulting plate will not exhibit
your mixed­culture specimen from one of a decrease in bacteria leading to individual
the selected environmental sources listed colonies. With that in mind, if a plate you have
in the section on cultures. streaked or poured does not exhibit a decrease
c. Place the contaminated swab back into in bacterial colonies area-to-area, you may
the tube of sterile water. Mix gently and let want to re-examine your technique for main-
stand for 5 minutes. taining sterilization.
d. Perform spread­plate and/or streak­plate
inoculation on an appropriately labeled
plate.
3. Incubate all plates in an inverted position PART B Isolation of Pure
for 48 to 72 hours at 25°C.
Cultures from a Spread-Plate
or Streak-Plate Preparation
Procedure Lab Two
1. Examine all agar plate cultures to identify the LearnIng OBjeCTIve
distribution of colonies. In the charts provided Once you have completed this experiment,
in Part A of the Lab Report, complete the
you should be able to
following:
1. Prepare a stock culture of an organism
a. Draw the distribution of colonies appearing
on each of the agar plate cultures. using isolates from mixed cultures
prepared on an agar streak plate and/or
b. On each of the agar plate cultures,
spread plate.
select two discrete colonies that differ in
appearance. Using Figure 3.1 on page 42
as a reference, describe each colony as
to its
Form: Circular, irregular, or spreading. Principle
Elevation: Flat, slightly raised, or markedly Once discrete, well­separated colonies develop
raised. on the surface of a nutrient agar plate culture,
each may be picked up with a sterile needle and
Pigmentation.
transferred to separate nutrient agar slants. Each
Size: Pinpoint, small, medium, or large. of these new slant cultures represents the growth
2. Retain the mixed­culture plates to perform of a single bacterial species and is designated as
Part B of this experiment. a pure or stock culture.

34 Experiment 2
 C L I n I C A L A P P L I C AT I o n Media
Four Trypticase™ soy agar slants per designated
Transferring a Colony of Bacteria student group.
Daughter Cells
For identification of a bacterial pathogen, a discrete Equipment
bacterial colony must be transferred from a streak Microincinerator or Bunsen burner, inoculating
or spread plate to the new testing media. This needle, and glassware marking pencil.
new culture will consist of daughter cells that are
genetic and metabolic clones of the original bacte-
rial cells that were transferred to the plate. This Procedure Lab One
will allow for identification of the unknown bacte-
1. Aseptically transfer, from visibly discrete
rial species through its biochemical and molecular
colonies, the yellow M. luteus, the white
characteristics.
E. coli, the red S. marcescens, and a discrete
colony from the environmental agar plate
specimen to the appropriately labeled agar
slants as shown in Figure 2.4.
2. Incubate all slants at 37°C for 18 to 24 hours.

AT T h E B E n C h
Procedure Lab Two
1. In the chart provided in Part B of the Lab
Materials Report, complete the following:
a. Draw and indicate the type of growth of
Cultures each pure­culture isolate, using Figure 3.1
on page 42 as a reference.
Mixed­culture, nutrient agar streak­plate and/or
spread­plate preparations of S. marcescens and b. Observe the color of the growth and record
M. luteus, M. luteus and E. coli, and the environ­ its pigmentation.
mental specimen plate from Part A. c. Indicate the name of the isolated organisms.

Experiment 2 35
 PROCEDURE

1 Flame the straight needle until the entire 2 After isolating a discrete colony on the agar streak
wire is red. plate, touch the straight needle to the surface of
the selected colony.

3 Uncap the agar slant and pass the neck of the 4 Inoculate the slant by drawing the needle upward
tube rapidly over the Bunsen burner flame. in a zigzag motion along the surface of the agar.
Do not dig into the agar.

5 Flame the neck of the tube and recap. 6 Flame the inoculating needle.

Figure 2.4 Procedure for the preparation of a pure culture

36 Experiment 2
 E xP E R IMEnT

Name:
2
Date: Section: Lab report

Observations and results

PART A: Isolation of Discrete Colonies
from a Mixed Culture
STREAK-PLATE TECHNIQUE
S. marcescens and M. Iuteus M. Iuteus and E. coli

Draw the colonies that

appear on each agar plate.

Colony description: Isolate 1 Isolate 2 Isolate 3 Isolate 4

Form
Elevation
Pigmentation
Size

Experiment 2: Lab Report 37

 ENVIRONMENTAL SPECIMEN
Spread-Plate Technique Streak-Plate Technique

Draw the colonies that

appear on each agar plate.

Colony description:
Form
Elevation
Pigmentation
Size

PART B: Isolation of Pure Cultures from a Spread-Plate

or Streak-Plate Preparation

Draw the distribution of

growth on the slant
surface.

Type of growth
Pigmentation
Name of organism

38 Experiment 2: Lab Report

review Questions
1. Why is it important to use a sterilized loop between streaks when preparing
a streak­plate?

2. Observation of a streak­plate culture shows more growth in Quadrant 4 than

in Quadrant 3. Account for this observation.

3. Describe the way in which you can isolate an individual colony from a
spread­plate or a streak­plate that holds multiple colonies.

4. Outline the differences between a streak plate and a spread plate.

Experiment 2: Lab Report 39

 E xP E R IMEnT
Cultural Characteristics
of Microorganisms 3
3. Optical characteristics: Optical charac­
LearnIng OBjeCTIve
teristics may be evaluated on the basis of
Once you have completed this experiment, the amount of light transmitted through the
you should be able to growth. These characteristics are described as
opaque (no light transmission), translucent
1. Determine the cultural characteristics of
(partial transmission), or transparent (full
microorganisms as an aid in identifying transmission).
and classifying organisms into taxonomic
4. Form: The appearance of the single­line
groups.
streak of growth on the agar surface is
designated as:
a. Filiform: Continuous, threadlike growth
with smooth edges.
b. Echinulate: Continuous, threadlike
Principle growth with irregular edges.
When grown on a variety of media, microorgan­ c. Beaded: Nonconfluent to semiconfluent
isms will exhibit differences in the macroscopic colonies.
appearance of their growth. These differences, d. Effuse: Thin, spreading growth.
called cultural characteristics, are used as a
e. Arborescent: Treelike growth.
basis for separating microorganisms into taxo­
nomic groups. The cultural characteristics for all f. Rhizoid: Rootlike growth.
known microorganisms are contained in Bergey’s 5. Consistency:
Manual of Systematic Bacteriology. They are
a. Dry: Free from moisture.
determined by culturing the organisms on nutri­
ent agar slants and plates, in nutrient broth, and b. Buttery: Moist and shiny.
in nutrient gelatin. The patterns of growth to be c. Mucoid: Slimy and glistening.
considered in each of these media are described
below, and some are illustrated in Figure 3.1. nutrient Agar Plates
These demonstrate well­isolated colonies and are
nutrient Agar Slants evaluated in the following manner:
These have a single straight line of inoculation
1. Size: Pinpoint, small, moderate, or large.
on the surface and are evaluated in the following
manner: 2. Pigmentation: Color of colony.
3. Form: The shape of the colony is described
1. Abundance of growth: The amount of
as follows:
growth is designated as none, slight, moderate,
or large. a. Circular: Unbroken, peripheral edge.
2. Pigmentation: Chromogenic microorganisms b. Irregular: Indented, peripheral edge.
may produce intracellular pigments that are c. Rhizoid: Rootlike, spreading growth.
responsible for the coloration of the organisms 4. Margin: The appearance of the outer edge
as seen in surface colonies. Other organisms of the colony is described as follows:
produce extracellular soluble pigments
a. Entire: Sharply defined, even.
that are excreted into the medium and also
produce a color. Most organisms, however, are b. Lobate: Marked indentations.
nonchromogenic and will appear white to gray. c. Undulate: Wavy indentations.

41
 Crateriform Napiform Infundibuliform Saccate Stratiform

(a) Gelatin liquefaction Uniform fine turbidity Flocculent growth

Forms

Circular
Rhizoid

Irregular Flat
Entire

Raised

Lobate Undulate

Convex
Pellicle Sediment
Serrate
Filamentous
Umbonate
Margins Elevation

(b) Colonies on agar plates (c) Growth in broth media

Filiform Echinulate Beaded Effuse Arborescent Rhizoid

(d) Growth on agar slants

Figure 3.1 Cultural characteristics of bacteria

42 Experiment 3
 d. Serrate: Toothlike appearance.
e. Filamentous: Threadlike, spreading AT T hE BE nCh
edge.
5. Elevation: The degree to which colony
growth is raised on the agar surface is
described as follows:
Materials
a. Flat: Elevation not discernible. Cultures
b. Raised: Slightly elevated. Twenty­four–hour nutrient broth cultures of
c. Convex: Dome­shaped elevation. Pseudomonas aeruginosa BsL -2 , Bacillus
d. Umbonate: Raised, with elevated convex cereus, Micrococcus luteus, and Escherichia coli.
central region. Seventy­two– to 96­hour Trypticase™ soy broth
culture of Mycobacterium smegmatis.
nutrient Broth Cultures
These are evaluated as to the distribution and Media
appearance of the growth as follows: Per designated student group: five each of nutri­
ent agar slants, nutrient agar plates, nutrient broth
1. Uniform fine turbidity: Finely dispersed tubes, and nutrient gelatin tubes.
growth throughout.
2. Flocculent: Flaky aggregates dispersed Equipment
throughout.
Microincinerator or Bunsen burner, inoculating
3. Pellicle: Thick, padlike growth on surface. loop and needle, and glassware marking pencil.
4. Sediment: Concentration of growth at the
bottom of broth culture may be granular, flaky,
or flocculent. Procedure Lab One
1. Using aseptic technique, inoculate each of the
nutrient Gelatin appropriately labeled media listed below in
This solid medium may be liquefied by the the following manner:
enzymatic action of gelatinase. Liquefaction a. Nutrient agar slants: With a sterile needle,
occurs in a variety of patterns: make a single­line streak of each of the
1. Crateriform: Liquefied surface area is cultures provided, starting at the butt and
saucer­shaped. drawing the needle up the center of the
slanted agar surface.
2. Napiform: Bulbous­shaped liquefaction at
surface. b. Nutrient agar plates: With a sterile loop, pre­
pare a streak­plate inoculation of each of the
3. Infundibuliform: Funnel­shaped.
cultures for the isolation of discrete colonies.
4. Saccate: Elongated, tubular.
c. Nutrient broth cultures: Using a sterile
5. Stratiform: Complete liquefaction of the loop, inoculate each organism into a tube
upper half of the medium. of nutrient broth. Shake the loop a few
times to dislodge the inoculum.
d. Nutrient gelatin: Using a sterile needle,
prepare a stab inoculation of each of the
C L I n I C A L A P P L I C AT I o n
cultures provided.
Examining Colony Growth Characteristics 2. Incubate all cultures at 37°C for 24 to 48 hours.
to Aid Identification
Bacterial species each have a characteristic
pattern of colony growth in a liquid culture or on
Procedure Lab Two
a solid medium. While not truly a diagnostic tool, 1. Before beginning observation of all the cul­
recognition of these patterns of characteristics will tures, place the gelatin cultures in a refrigera­
aid in a clinical lab setting by helping to minimize tor for 30 minutes or in a beaker of crushed
the list of potential bacterial species to test for. ice for a few minutes. The gelatin culture will
be the last to be observed.

Experiment 3 43
2. Refer to Figure 3.1 on page 42 and the descrip­ Record your observations in the chart pro­
tions presented in the introductory section vided in the Lab Report.
of Experiment 3 while making the following c. Nutrient broth cultures: Observe each of the
observations: nutrient broth cultures for the appearance
a. Nutrient agar slants: Observe each of of growth (flocculation, turbidity, sediment,
the nutrient agar slant cultures for the or pellicle). Record your observations in the
amount, pigmentation, form, and consis­ chart provided in the Lab Report.
tency of the growth. Record your obser­ d. Nutrient gelatin: Remove gelatin cultures
vations in the chart provided in the Lab from the refrigerator or beaker of crushed
Report. ice, and observe whether liquefaction of
b. Nutrient agar plates: Observe a single, the medium has developed and whether the
well­isolated colony on each of the nutri­ organism has produced gelatinase. Record
ent agar plate cultures and identify its size, your observations in the chart provided in
elevation, margin, form, and pigmentation. the Lab Report.

44 Experiment 3
 E xP E R IMEnT

Name:
3
Date: Section: Lab report

Observations and results

nutrient Agar Slants
NUTRIENT AGAR SLANT CULTURES
M. Iuteus P. aeruginosa M. smegmatis E. coli B. cereus

Draw the distribution of

growth on the slant
surface.

Amount of growth
Pigmentation
Form
Consistency

nutrient Agar Plates

NUTRIENT AGAR PLATES
M. Iuteus P. aeruginosa M. smegmatis E. coli B. cereus

Draw distribution
of colonies.

Size

Elevation

Margin

Form

Pigmentation

Experiment 3: Lab Report 45

nutrient Broth Cultures
NUTRIENT BROTH CULTURES
M. Iuteus P. aeruginosa M. smegmatis E. coli B. cereus

Draw the distribution of

growth.

Appearance of growth

nutrient Gelatin
NUTRIENT GELATIN CULTURES
M. Iuteus P. aeruginosa M. smegmatis E. coli B. cereus

Draw liquefaction
patterns.

Liquefaction (+) or (–)

Type of liquefaction

46 Experiment 3: Lab Report