PART 5 – BASIC LABORATORY TECHNIQUES FOR ISOLATION,

CULTIVATION, AND CUTURAL 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 concept of aseptic technique and the procedures necessary for successful
subculturing of microorganisms.
3. Streak-plate and spread-plate inoculation of microorganisms in a mixed microbial
population for subsequent pure culture isolation.
4. Cultural and morphological characteristics of microorganisms grown in pure culture.

Introduction

Microorganisms are ubiquitous. They are found in soil, air, water, food, sewage, and on body
sur- faces. In short, every area of our environment is replete with them. The microbiologist
separates these mixed populations into individual species for study. A culture containing a
single unadulterated species of cells is called a pure culture. To isolate and study
microorganisms in pure culture, the microbiologist requires basic laboratory apparatus and the
application of specific techniques, as illustrated in Figure P5.1.

Figure P5.1 Laboratory apparatus and culture techniques

Media

The survival and continued growth of microorganisms depend on an adequate supply of

nutrients and a favorable growth environment. For survival, most microbes must use soluble
low-molecular- weight substances that are frequently derived from the enzymatic degradation of
complex nutrients. A solution containing these nutrients is a culture medium Basically, all
culture media are liquid, semisolid, or solid. A liquid medium lacks a solidifying agent and is
called a broth medium. A broth medium is useful for the cultivation of high numbers of bacterial
cells in a small volume of medium, which is particularly helpful when an assay requires a high
number of healthy bacterial cells. A broth medium supplemented with a solidifying agent called
agar results in a solid or semisolid medium. Agar, an extract of seaweed, is a com- plex
carbohydrate composed mainly of galactose, and is without nutritional value. Agar serves as an
excellent solidifying agent because it liquefies at 100°C and solidifies at 40°C. Because of these
properties, organisms, especially pathogens, can be cultivated at temperatures of 37.5°C or
slightly higher without fear of the medium liquefying. A completely solid medium requires an
agar concentration of 1.5% to 1.8%. A concentration of less than 1% agar results in a semisolid
medium. A semi- solid medium is useful for testing a cell’s ability to grow within the agar at
lower oxygen levels and for testing the species’ motility. A solid medium has the advantage that
it presents a hardened surface on which microorganisms can be grown using specialized
techniques for the isolation of discrete colonies. Each colony is a cluster of cells that originates
from the multiplication of a single cell and represents the growth of a single species of
microorganism. Such a defined and well isolated colony is a pure culture. Also, while in the
liquefied state, solid media can be placed in test tubes, which are then allowed to cool and
harden in a slanted position, producing agar slants. These are useful for maintaining pure
cultures. The slanted surface of the agar maximizes the available surface area for
microorganism growth while minimizing the amount of medium required. Similar tubes that,
following preparation, are allowed to harden in the upright position are designated as agar deep
tubes. Agar deep tubes are used primarily for the study of the gaseous requirements of
microorganisms since gas exchange between the agar at the butt of the test tube and the exter-
nal environment is impeded by the height of the agar. Liquid agar medium can also be poured
into Petri dishes, producing agar plates, which provide large surface areas for the isolation and
study of microorganisms. The various forms of solid media are illustrated in Figure P5.2.

In addition to nutritional needs, the environ mental factors must also be regulated, including
proper pH, temperature, gaseous requirements, and osmotic pressure
 Figure P5.2 Forms of solid (agar) media

Aseptique Technique

Sterility is the hallmark of successful work in the microbiology laboratory, and sterilization is the
process of rendering a medium or material free of all forms of life. To achieve sterility, it is
mandatory that you use sterile equipment and employ aseptic techniques when handling
bacterial cultures. Using correct aseptic techniques minimizes the likelihood that bacterial
cultures will be contaminated, and reduces the opportunity for students to be exposed to
potential pathogens. Figure P5.3 is a brief outline of the routine techniques used in the
microbiology laboratory.

Figure P5.3 Sterilization techniques

Culture Tubes and Petri Dishes

Glass test tubes and glass or plastic Petri dishes are used to cultivate microorganisms. A
suitable nutrient medium in the form of broth or agar may 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 closures. Historically, the first type, a cotton plug, was developed by Schröeder
and von Dusch in the nineteenth century. Today most laboratories use sleeve like caps (Morton
closures) made of metal, such as stainless steel, or heat resistant plastics. The advantage of
these closures over the cotton plug is that they are laborsaving and, most of all, slip on and off
the test tubes easily.

Petri dishes provide a larger surface area for 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 dishes are manufactured in various sizes to meet different experimental requirements. For
routine purposes, dishes approximately 15 cm in diameter are used. The sterile agar medium is
dispensed to previously sterilized dishes from molten agar deep tubes containing 15 ml to 20 ml
of medium, or from a molten sterile medium prepared in bulk and contained in 250, 500, and
1000ml flasks, depending on the volume of medium required. When cooled to 40°C, the
medium

will solidify. Remember that after inoculation, Petri dishes are incubated in an inverted position
(top down) to prevent condensation formed on the cover during solidification from dropping
down onto the surface of the hardened agar. For this reason, Petri dishes should be labeled on
the bottom of the dish. This makes it easier to read the label and minimizes confusion if two
Petri dish covers are interchanged. Figure P4.4 illustrates some of the culture vessels used in
the laboratory. Built in ridges on tube closures and Petri dishes pro vide small gaps necessary
for the exchange of air.

Transfer Instruments
Microorganisms must be transferred from one vessel to another or from stock cultures to
various media for maintenance and study. This transfer is called subculturing and must be
carried out under aseptic conditions to prevent possible contamination.

Wire loops and needles are made from inert metals such as Nichrome or platinum and are
inserted into metal shafts that serve as handles. 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 useful for transferring a small volume of bacteria onto the surface of an agar plate or
slant. A needle 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 glass or plastic and drawn out to a tip at one end,
with a mouthpiece forming the other end. They are calibrated to deliver different volumes
depending on requirements. Pipettes may be sterilized in bulk inside canisters, or they may be
wrapped individually in brown paper and sterilized in an autoclave or dry heat oven. A
micropipette (commonly called a “pipetter”) with a disposable, single use plastic tip is useful for
transferring small volumes of liquid (less than ...1 ml). Figure P4.5 illustrates these transfer
instruments. Your instructor will demonstrate the proper procedure for using pipettes.

Cultivation Chambers

Requirement for the cultivation of microorganisms is that they be grown at their optimum
temperature. An incubator is used to maintain optimum temperature during the necessary
growth period. It resembles an oven and is thermostatically controlled so that temperature can
be varied depending on the requirements of specific micro organisms. Most incubators use dry
heat. Moisture is supplied by placing a beaker of water in the incubator during the growth
period. A moist environment retards dehydration of the medium and thereby avoids misleading
experimental results.

A thermostatically controlled shaking water bath is another piece of apparatus used to cultivate
microorganisms. Its advantage is that it provides a rapid and uniform transfer of heat to the
culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth.
The primary disadvantage of this instrument is that it can be used only for cultivation
of organisms in a broth medium. Many laboratories also use shaking incubators that utilize dry
air incubation to promote aeration of the broth medium. This method has a distinct advantage
over a shaking water bath since there is no chance of cross contamination from microorganisms
that might grow in the water bath.

Refrigerator

A refrigerator is used for a wide variety of purposes such as maintenance and storage of stock
cultures between subculturing periods, and storage of sterile media to prevent dehydration. It is
also used as a repository for thermolabile solutions, antibiotics, serums, and biochemical
reagents.
 Figure P5.5 Transfer instruments

Activity 11 – Culture Transfer Techniques

Learning Objectives
Once you have completed this experiment, you should be able to:

1. Carry out the technique for aseptic removal and transfer of microorganisms for
subculturing.
2. Correctly sterilize inoculating instruments in a microincinerator or the flame of a Bunsen
burner.
3. Correctly remove and replace the test tube closure.

Principle`

Microorganisms are transferred from one medium to another by subculturing. This technique is
of basic importance and is used routinely in preparing and maintaining stock cultures, as well as
in microbiological test procedures. Microorganisms are always present in the air and on
laboratory surfaces, benches, and equipment. These ambient microorganisms can serve as a
source of external contamination and interfere with experimental results unless proper aseptic
techniques are used during subculturing. Described below are essential steps that you must
follow for aseptic transfer of microorganisms. The complete procedure is illustrated in Figure
11.1.

1. Label the tube to be inoculated with the name of the organism and your initials.
2. Hold the stock culture tube and the tube to be inoculated in the palm of your hand, secure
with your thumb, and separate the two tubes to form a V in your hand.
3. Sterilize an inoculating needle or loop by holding it in the microincinerator or the hottest
portion of the Bunsen burner flame, until the wire becomes red hot. Once sterilized, the
loop is held in the hand and allowed to cool for 10 to 20 seconds; it is never put down.
4. Uncap each tube by grasping the first cap with your little finger and the second cap with your
next finger and lifting the closure upward. Note: Once removed, these caps must be kept in
the hand that holds the sterile inoculating loop or needle; the inner aspects of the caps point
away from the palm of the hand. The caps must never be placed on the laboratory bench
because that would compromise the aseptic procedure.
5. After removing the caps, flame the necks and mouths of the tubes by briefly passing them
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 a small sample of the inoculum.
6. Depending on the culture medium, a loop or needle is used for removal of the inoculum.
Loops are commonly used to obtain a sample from a broth culture. Either instrument can be
used to obtain the inoculum from an agar slant culture by carefully touching the surface of
the solid medium in an area exhibiting growth so as not to gouge the agar. A straight needle
is always used when transferring microorganisms to an agar deep tube from both solid and
liquid cultures.
a. For a slant to broth transfer, obtain inoculum from the slant and lightly shake the
loop or needle in the broth culture to dislodge the microorganisms.
b. For a broth to slant transfer, obtain a loop full of broth and place at the base of an
agar slant medium. Lightly draw the loop over the hardened surface in a straight or
zigzag line, from the base of the agar slant to the top.
c. For a slant to agar deep tube transfer, obtain the inoculum from the agar slant.
Insert a straight needle to the bottom of the tube in a straight line and rapidly
withdraw along the line of insertion. This is called a stab inoculation.
7. Following inoculation, remove the instrument and reheat or reflame the necks of the tubes.
8. Replace the caps on the same tubes from which they were removed.
9. Resterilize the loop or needle to destroy any remaining organisms.
In this experiment, you will master the manipulations required for aseptic transfer of
microorganisms in broth to slant, slant to broth, and slant to agar deep tubes. You will be using
a positive and a negative control to test your ability to maintain aseptic techniques while
transferring cultures. The technique for transfer to and from agar plates is discussed in the next
activity.

Figure 11.1 Subculturing procedure

CLINICAL APPLICATION

Aseptic Inoculation and Transfer

It is mandatory that microbiology laboratory workers learn and perfect the skill of inoculating
bacterial specimens on agar plates, in liquid broth, or in semisolid medium, and be able to
subculture the organism from one medium to another. A sterile inoculating needle or loop is the
basic instrument 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 do not contaminate yourself.

AT THE BENCH

Materials

Culture: Twenty four–hour nutrient broth and nutrient agar slant cultures of Escherichia
coli and a sterile tube of nutrient broth. The nutrient broth tubes will be labeled “A” and
“B,” and the contents will be known only by the instructor.

Media: Per student: three nutrient broth tubes, three nutrient agar slants, and three
nutrient agar deep tubes.

Equipment: Microincinerator or Bunsen burner, inoculating loop and needle, and

glassware marking pencil.

Procedure Lab One

1. Label all tubes of sterile media as described in the Laboratory Protocol section.
2. Following the procedure outlined and illustrated previously (Figure 5A.1), perform the
following transfers:
a. Broth culture “A” to a nutrient agar slant, nutrient agar deep tube, and nutrient
broth.
b. Broth culture “B” to a nutrient agar slant, nutrient agar deep tube, and nutrient
broth.
c. E. coli agar slant culture to a nutrient agar slant, nutrient agar deep tube and
nutrient broth.
3. Incubate all cultures at 25°C for 24 to 48 hours.

Procedure lab Two

1. Examine all cultures for the appearance of growth, which is indicated by turbidity in the
broth culture and the appearance of an orange red growth on the surface of the slant
and along the line of inoculation in the agar deep tube.
2. Record your observations in the chart provided in the Lab Report.
3. Confirm your results with the instructor to determine the negative control tube.

Tips for Success

  It is imperative that you maintain sterility and utilize aseptic techniques at all
times. If you allow a contaminating organism into your bacterial culture, you will see a
positive growth in media that was inoculated with the negative control.

Activity 11 – Culture Transfer Techniques

Lab Report

Name: _________________________________ Score: ___________

Date: _________________ Section: __________

Observations and Results Culture “A”

Observations and Results Culture “B”

Observations and Results E. coli

Review Questions

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

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

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

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

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. 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.

Activity 12 - Techniques for Isolation of Pure Cultures

In nature, microbial populations do not segregate themselves by species, but exist with a
mixture of many other cell types. In the laboratory, these populations can be separated into
pure cultures. These cultures contain only one type of organism and are suitable for the study
of their cultural, morphological, and biochemical properties.

In this experiment, you will first use one of the techniques designed to produce discrete
colonies. Colonies are individual, macroscopically visible masses of microbial growth on a solid
medium surface, each representing the multiplication of a single organism. Once you have
obtained these discrete colonies, you will make an aseptic transfer onto nutrient agar slants for
the isolation of pure cultures.

Part A – Isolation of Discrete Colonies from a Mixed Culture

Learning Objective

Once you have completed this experiment, you should be able to

1. Perform the streak-plate and/or the spread-plate inoculation procedure

to separate the cells of a mixed culture so that discrete colonies can be isolated.

Principle

The techniques commonly used for isolation of discrete colonies initially require that the number
of organisms in the inoculum be reduced. The resulting diminution of the population size
ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of
the agar medium to separate the different species. The following are techniques that can be
used to accomplish this necessary dilution:

1. The streak-plate method is a rapid qualitative isolation method. It is essentially a dilution

technique that involves spreading a loopful of culture over the surface of an agar plate.
Although many types of procedures are per formed, the four way, or quadrant, streak is
described. Refer to Figure 12A.1 and a three-phase streak plate method (Figure 12A.4)
which schematically illustrates this technique.

Figure 12A.1 Four-way streak-plate technique

a. Place a loopful of culture on the agar 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,
and then drag it rapidly several times across the surface of Area 1.

b. Reflame and cool the loop, and turn the Petri dish 90°. Then touch the loop to a
corner of the culture in Area 1 and drag it several times across the agar in Area
2. The loop should never enter Area 1 again.

c. Reflame and cool the loop and again; turn the dish 90°. Streak Area 3 in the
same manner as Area 2.

d. Without reflaming the loop, again turn the dish 90° and then drag the culture from
a corner of Area 3 across Area 4, using a wider streak. Don’t let the loop touch
any of the previously streaked areas. The flaming of the loop at the points
indicated is to dilute the culture so that fewer organisms are streaked in each
area, resulting in the final desired separation. A photograph of a streak plate
inoculation is shown in Figure 12A.2.

Figure 12A.2 Four-way streak-plate inoculation with Serratia marcescens

2. An alternative streak plate method is for students new to the laboratory who have yet to
master the necessary lab skills that would allow them to use the rapid method listed above.
This alternative method involves spreading a loopful of culture over the surface of an agar
plate that has the quadrants laid out visibly for quick reference.

Figure 12A.3 Alternate streak-plate method

a. Using marker, draw two bisecting lines on the bottom of the Petri dish to divide the
plate into 4 equal parts. Label each quadrant 1 through 4, starting with the top right
quadrant and labeling counter clockwise. Sterilizing the loop at the points indicated is
to dilute the culture due to fewer organisms available to be streaked into each area,
resulting in the final desired separation.
b. Turn the Petri dish over and place a loopful of culture on the agar surface in quadrant
1. Using the edge of the loop and holding the loop at a shallow angle so as not to
gouge the agar, quickly spread the bacteria throughout the quadrant.
c. Reflame and cool the loop, and turn the Petri dish 90°. Then touch the loop into an
area that has been streaked in quadrant 1 and drag it across the agar into quadrant
2, repeat this twice without flaming the loop.
d. Reflame and cool the loop and again turn the dish 90°. Streak the bacteria into
quadrant 3 in the same manner used for quadrant 2.
e. Reflame and cool the loop and again turn the dish 90°. Streak the bacteria into
quadrant 4 in the same manner used for quadrant 3.
3. The spread-plate technique requires that a previously diluted mixture of microorganisms be
used. During inoculation, the cells are spread over the surface of a solid agar medium with a
sterile, L shaped bent glass rod while the Petri dish is spun on a “lazy Susan” turntable. The
step by step procedure for this technique is as follows:
a. Place the bent glass rod into a beaker and 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 with the loop and replace the cover.
c. Remove the glass rod from the beaker, and pass it through the Bunsen burner flame
with the bent portion of the rod pointing downward to prevent the burning alcohol
from running down your arm. Allow the alcohol to burn off the rod completely. Cool
the rod for 10 to 15 seconds.
d. Remove the Petri dish cover and spin the turntable.
 e. While the turntable is spinning, lightly touch the sterile bent rod to the surface of the
agar and move it back and forth. This will spread the culture over the agar surface.
f. When the turntable comes to a stop, replace the cover. Immerse the rod in alcohol
and reflame.
g. In the absence of a turntable, turn the Petri dish manually and spread the culture with
the sterile bent glass rod.

Figure 12A.4 – Three-phase streak plate

CLINICAL APPLICATION

Isolation of Cultures as a Diagnostic Technique

The isolation of pure cultures is the most important diagnostic tool used in a clinical or research
laboratory to uncover the cause of an infection or disease. Before any biochemical or molecular
techniques may be used to identify or characterize the causative organism, an individual
bacterial colony must be isolated for testing. The isolation of Staphylococcus aureus from
cultures taken from abscesses or Streptococcus pyogenes from a throat culture are two
examples of clinical applications of this technique.

AT THE BENCH

Materials

Culture: Materials24 to 48hour nutrient broth cultures of a mixture of one part E. coli and
three parts Micrococcus luteus and a mixture of one part Escherichia coli and ten parts
Micrococcus luteus. Sources of mixed cultures from the environment could include
 cultures from a tabletop, bathroom sink, water fountain, or inside of an incubator. Each
student should obtain a mixed culture from one of the environmental sources listed
above.

Media: Three Trypticase soy agar plates per designated student group for each
inoculation technique to be performed.

Equipment: Microincinerator or Bunsen burner, inoculating loop, turntable, glassware

marking pencil, culture tubes containing 1 ml of sterile water, test tube rack, and sterile
cotton swabs.

Procedure Lab One

1. Following the procedures previously described, prepare a spread-plate and/or streak-

plate inoculation of each test culture on an appropriately labeled plate.
2. Prepare an environmental mixed culture.
a. Dampen a sterile cotton swab with sterile water. Wring out the excess water by
pressing the wet swab against the walls of the tube.
b. With the moistened cotton swab, obtain your mixed culture specimen from one of
the selected environmental sources listed in the section on cultures.
c. Place the contaminated swab back into the tube of sterile water. Mix gently and
let stand for 5 minutes.
d. Perform spread plate and/or streak-plate inoculation on an appropriately labeled
plate.
3. Incubate all plates in an inverted position for 48 to 72 hours at 25°C.

Procedure Lab Two

1. Examine all agar plate cultures to identify the distribution of colonies. In the charts
provided in Part A of the Lab Report, complete the following:
a. Draw the distribution of colonies appearing on each of the agar plate cultures.
b. On each of the agar plate cultures, select two discrete colonies that differ in
appearance. Describe each colony as to its:
 Form: Circular, irregular, or spreading.
 Elevation: Flat, slightly raised, or markedly raised.
 Pigmentation.
 Size: Pinpoint, small, medium, or large.
2. Retain the mixed culture plates to perform Part B of this experiment.

PART B – Isolation of Pure Cultures from a Spread-Plate or Streak-Plate

Preparation

Learning Objective

Once you have completed this experiment, you should be able to:
1. Prepare a stock culture of an organism using isolates from mixed cultures prepared on an
agar streak plate and/or spread plate.

Principle

Once discrete, well separated colonies develop on the surface of a nutrient agar plate culture,
each may be picked up with a sterile needle and transferred to separate nutrient agar slants.
Each of these new slant cultures represents the growth of a single bacterial species and is
designated as a pure or stock culture.

CLINICAL APPLICATION

Transferring a Colony of Bacteria Daughter Cells

For identification of a bacterial pathogen, a discrete bacterial colony must be transferred from a
streak or spread plate to the new testing media. This new culture will consist of daughter cells
that are genetic and metabolic clones of the original bacterial cells that were transferred to the
plate. This will allow for identification of the unknown bacterial species through its biochemical
and molecular characteristics.

AT THE BENCH

Materials

Cultures: Mixed culture, nutrient agar streak plate and/or spread plate preparations of
S. marcescens and M. luteus, M. luteus and E. coli, and the environmental specimen
plate from Part A.
Media: Four Trypticase soy agar slants per designated student group.

Equipment: Microincinerator or Bunsen burner, inoculating needle, and glassware

marking pencil.

Procedure Lab One

1. Aseptically transfer, from visibly discrete colonies, the yellow M. luteus, the white
E. coli, the red S. marcescens, and a discrete colony from the environmental agar plate
specimen to the appropriately labeled agar slants.
2. Incubate all slants at 37°C for 18 to 24 hours.

Procedure Lab Two

1. In the chart provided in Part B of the Lab Report, complete the following:
a. Draw and indicate the type of growth of each pure culture isolate.
b. Observe the color of the growth and record its pigmentation.
c. Indicate the name of the isolated organisms.
 Figure 12B.1 Procedure for the preparation of a pure culture

Activity 12 – Techniques for Isolation of Pure Cultures

Lab Report

Name: _________________________________ Score: ___________

Date: _________________ Section: __________

Observations and Results

Part A: Isolation of Discrete Colonies from a Mixed Culture

Part B: Isolation of Pure Cultures from a Spread-Plate or a Streak-Plate Preparation
Review Questions

1. Why is it important to use a sterilized loop between streaks when preparing a streak-plate?

2. Observation of a streak-plate culture shows more growth in Quadrant 4 than in Quadrant

Account for this observation.

3. Describe the way in which you can isolate an individual colony from a spread-plate or a
streak-plate that holds multiple colonies.

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