4.

1: Microbial Growth and Control

An important aspect within the field of microbiology is the ability to cultivate

microorganisms. Consider the following scenario: You are flying over a field, and you are
trying to see someone on the ground holding a red flag. Would it be easier to see a single flag
or 10 million flags in the same field? The latter would be much easier to detect. The same
principle often applies in microbiology. While many tests are highly sensitive, often times
expanding a given microbial population allows researchers to detect and assess the
microorganism more easily. In the following module, we will first discuss the various
techniques used to promote microbial growth, how to differentiate between samples, and how
researchers safely control its growth.

Growth Media

Growth media is a liquid or solid formulated to support microbial growth. The media
contains essential nutrients to provide the microbe with a source of carbon/energy, which, in
turn, encourages its expansion. The nutrient composition often includes a rich source of
sugars, amino acids, and vitamins. Such media can also be referred to as a nutrient broth and
is commonly used to grow microbes in a suspension. Perhaps the most common nutrient
broth in the lab, LB media (lysogeny broth), is a nutritionally rich liquid known for its ability
to grow a vast array of microbes. However, there are also occasions where a researcher may
want to specifically control what types of microbes can grow. For instance, a researcher may
want to establish conditions where various types of bacteria will grow, while others will not
(selective). On the other hand, a researcher may want to establish conditions where several
types of microbes can be grown simultaneously while being able to distinguish one type from
another (differential). Altogether, these conditions would require specialized media. The two
general classifications of specialized media (selective vs. differential) are expanded on below.

Selective Media

Selective media allows for only the growth of certain microbes and, by extension, restricts
the growth of all others. Such an approach can be accomplished in a variety of ways
including using limiting amounts of nutrients, varying degrees of pH (being either very acidic
or very basic media), or various chemical additives that limit unwanted microbial growth (i.e.
antibiotics). Selective media is often used in medical laboratories for the cultivation of human
pathogens such as Neisseria meningitides, the potential causative agent of meningitis.
Because Neisseria meningitides is a relatively slow-growing organism, other bacteria, mold,
or fungi often outgrow the sample—remember medical samples often contain numerous
unknown microbes! —therefore, additives are included in the growth media to specifically
inhibit such foreign microbes and ensure the isolated growth of Neisseria meningitides.

Differential Media

Differential media distinguishes between two, often related, microbes. For instance, two
microbes Escherichia coli and Salmonella are both Gram-negative but can be distinguished
by the presence (or absence) of lactose fermentation. If grown on the same differential media,
E. coli ferments lactose and turns the culture red. In contrast, Salmonella does not ferment
lactose, and the culture remains white/tan in color. The ability to differentiate within all
Gram-negative (or all Gram-positive) species will be further demonstrated in the Lab section
of this course.

Enriched Media

Enriched media is used to grow fastidious microorganisms—organisms with complex

growth requirements such that if absent will not grow. Thus, enriched medias contain the
essential nutrients required for the growth of this subset of microorganisms. Medias can be a
combination of selective, differential, and enriched.

4.2: Agar Plates

Growth media comes in two forms: liquid and solid. Solid growth media, often contained
within a sterile petri dish, is in its simplest form liquid growth media that has had a hardening
agent added. The addition of a polysaccharide derived from seaweed (algae) extract creates a
solid medium called agar. Agar is used to create a solid, smooth surface on which microbes
can grow. As they grow, microbes form colonies that take on the appearance of individual
isolated dots. However, as the microbe increases in number, the isolates merge into one
another. Once the microbe has grown to cover the entire plate, this is referred to as a lawn. As
we will see below, various forms of agar plates exist in order to aid in the growth and
classification of a broad range of microorganisms.

Note: Outside of the lab you’ve probably come across the cousin of agar, gelatin. Gelatin is
an animal-derived product that gives Jell-O its classic texture and form. Agar is simply the
plant-based form and acts in a very similar way. In both cases, as you increase the amount of
agar/gelatin, the firmer the composition becomes. The dehydrated agar (and other necessary
components) is mixed with water and heated to a high temperature to dissolve the agar into
solution. The high temperature also ensures sterility as it will kill most, if not all, foreign
microbes. Once poured into a sterile petri dish and cooled, the agar plate is a sterile
environment on which microbes can grow.

LB Agar
LB agar, also known as lysogeny broth, is a multi-purpose media capable of growing a wide
variety of microorganisms. LB agar plates are pale yellow in color as shown in Figure 4.1.
LB is classified as a non-selective and non-differential media. LB agar is commonly used for
growing E. coli, a microbe often associated with molecular microbiology applications based
on its ability to easily produce recombinant proteins.
Figure 4.1 Agar plates. LB agar plates are shown with either the top lid in place (left) or tilted off (right). The
agar is melted, sterilized, and poured into a sterile petri dish then allowed to solidify at room temperature.

Trypticase Soy Agar

Trypticase soy agar, also known as TSA or TSAYE, is another multi-purpose media capable
of growing a wide variety of microorganisms. TSA plates are virtually identical in color to
LB plates. TSA media is non-selective and non-differential and serves as the base when
formulating specialized (enriched) medias.

Blood Agar
Blood agar, also known as BAP (blood agar plates), is a derivative of TSA whereby
mammalian blood, often from sheep, is added to the plate composition at a concentration
range of 5-10%. As such, BAP have a distinct red coloration. BAP is an enriched, non-
selective yet differential media. The red blood cells in the media promote the growth of
fastidious microorganisms, such as various strains of Streptococcus. BAP is a differential
media used to detect hemolytic activity. Hemolysis, the lysis (breakdown) of red blood cells,
is classified as alpha, beta, or gamma. When grown on BAP, microbes capable of alpha
hemolysis present as greenish-brown color colonies due to the incomplete (partial) lysis of
red blood cells. Beta hemolysis is classified as the capacity of a microbe to completely lyse
red blood cells. The resulting lysis presents as a distinct zone of clearing around the growing
colony. Gamma hemolysis designates the absence or lack of hemolytic activity, and the
resulting colonies are often white/tan in color growing on the red background color of the
unaffected blood agar plate.

Columbia CNA Agar

Columbia CNA agar is red in color and is an enriched, selective, and differential media. As
the media contains the antimicrobial agents colistin and Nalidixic acid, it suppresses the
growth of Gram-negative bacteria and is, therefore, used for isolation of Gram-positive
microbes. Similar to BAP, the media is also enriched with blood, which allows for
differentiation based on hemolytic patterns.

Chocolate Agar
Chocolate agar, also known as CHOC or as chocolate blood agar (CBA), is an enriched,
non-selective, non-differential derivative of blood agar plates. Unlike blood agar plates that
contain intact red blood cells, chocolate agar plates contain red blood cells that have been
lysed by heat. This cooking of the red blood cells gives the media a dark brown chocolaty
appearance, hence the name. Note: Actual chocolate is never added to the agar. The lysed red
blood cells release various growth factors into the media required for the cultivation of
fastidious, pathogenic bacteria. As such, chocolate agar is also considered an enriched media.
Examples include the Gram-negative bacteria Haemophilus influenzae and Neisseria
meningitidis.

MacConkey Agar
MacConkey agar is pale red in color and is both a selective and differential media. The
presence of crystal violet and bile salts in its formulation restricts Gram-positive bacterial
growth. As such, only Gram-negative microbes can be grown on MacConkey agar. Further,
lactose and the pH indicator neutral red—it is only red when under acidic conditions—are
added to differentiate between lactose fermenters (lac+; red colonies) and non-fermenters
(lac-; white/tan colonies). The base color of the agar plate also often changes color as the
microbes consume the nutrients within the agar. The agar can turn yellow (pH > 8.0) within
the surrounding area of a growing non-fermenting microbe. Similarly, the agar may turn pink
or even a darker red within the region surrounding a lac+ microbe given its level of acid
production during fermentation (pH < 6.8). For example, the Gram-negative/lac+ bacteria
Escherichia coli is often used to demonstrate the formation of vibrant pink colonies. Within
the clinical laboratory setting, where each patient sample may contain hundreds of species of
bacteria, MacConkey agar is used to isolate intestinal pathogenic microbes belonging to the
Enterobacteriaceae family such as Salmonella and Shigella species.

Sorbitol-MacConkey Agar
Sorbitol-MacConkey agar, also known as SMAC, is a variant of MacConkey agar
specifically formulated to detect the presence of the pathogenic strain of Escherichia coli
O157:H7. Under standard enteric (gut) conditions, Escherichia coli is able to ferment both
lactose and sorbitol. However, E. coli O157:H7 is unable to ferment sorbitol. Therefore,
SMAC plates contain sorbitol (instead of lactose) in order to differentiate non-pathogenic
strains of Escherichia coli (red colonies; acidic conditions) from pathogenic O157:H7 strains
(white colonies; neutral to basic conditions).
Eosin Methylene Blue Agar
Eosin Methylene Blue agar, also known as EMB, is red in color and is classified as both a
selective and differential form of media. As the naming suggests, EMB contains eosin and
methylene blue that restricts the growth of Gram-positive bacteria. Microorganisms are
differentiated based on their ability to ferment the lactose present in the media. Similar to
MacConkey agar, non-fermenting (lac-) microbes grow as white/tan colonies, while lactose-
fermenting microbes produce dark (purple to black) colonies on the surface of the agar. Most
noticeably, if Escherichia coli is grown on EMB agar plates, colonies will have a distinctive
metallic green sheen.

Mannitol Salt Agar

Mannitol salt agar, also known as MSA, is red in color and is both a selective and
differential media. MSA is selective for Gram-positive bacteria and can differentiate
members of the Staphylococci family. MSA contains a high concentration of sodium chloride
(~7.5-10%) that prevents the growth of other microorganisms (selectivity) while the presence
of an alternate sugar source, mannitol, along with the pH indicator dye phenol red serves to
differentiate pathogenic from non-pathogenic strains of Staph. Pathogenic strains, such as
Staphylococcus aureus, have the ability to ferment mannitol, thus lowering the pH of the
medium and changing the dye color from red (pH >8.2) to yellow (pH < 6.8).

To illustrate the similarities and differences in agar media, a collective and generalized
representation can be seen in Figure 4.2. Noting the color differences and the associated
properties of each plate will be essential for correctly identifying an unknown pathogen.

Figure 4.2 Selective and Differential Agar Plates. Generalized color depictions of the agar plates described
above are shown. Each plate corresponds with the descriptions found within the text.
4.3: Obtaining a Pure Culture

Now that the various types of growth media and nutrient agars have been outlined, the
question remains: How are these materials used to grow microorganisms? The process of
spreading a bacterial culture onto a petri dish filled with agar is called plating. Plating can be
done using a sterile loop, a sterile swab, or a sterilized wire loop. Each device is simply a
means of spreading the bacteria, most commonly in a simple back-and-forth motion, across
the plate.

Note: The process of plating will be visually demonstrated in the lab section of this course.

The primary advantage of plating a bacterial sample onto agar is that cells are held in place.
Unlike in a nutrient broth where bacterial cells can multiply but are free to move around in
solution, bacteria plated onto agar are fixed in such a way as to support the formation and
visualization of colonies. Colonies are visible to the naked eye but only after the bacterial cell
has multiplied, often a million times over. Thus, each colony is derived from a single cell and
should be free from outside contaminants (other microbes). The process of obtaining an
individual colony (as opposed to a lawn growth pattern) will be discussed in the next section.

To isolate a microbe on an agar plate, microbiologists often utilize a quadrant streak (or
phase-dilution) approach. The purpose of this method is to generate an individual colony so
that a single (pure) bacterial sample can be picked from the plate. Importantly, a pure culture
is free of outside contaminants and can be traced back to a single origin. Once isolated and
expanded, a pure culture can be further examined for its size and shape, motility, Gram
status, biochemical properties, etc.

As outlined below, the sample is spread across the plate in such a way as to establish a
dilution gradient. The resulting gradient should always contain within it the growth of
individual colonies. For a four-phase dilution gradient, the sample is spread into four regions
where each new region is perpendicular to the previous region, such that each region will
become more diluted than the previous. As shown in Figure 4.3 and staying within the first
phase (P1), a sterile loop is first used to streak a sample back-and-forth across a small area of
the plate. The first region will contain the highest concentration of microbial growth. Next, a
new sterile loop will be used to pull some of the sample from the first region into the second
phase (P2), where it is further streaked. The microbial concentration is now diluted because
only a small portion of the bacterial sample from phase one was moved and spread out into
P2. The same process is repeated from P2 to P3, and then from P3 to P4, each time using a
new sterile loop. Thus, each phase should be systematically diluted such that the bacterial
concentration of each phase will be established as P1 > P2 > P3 > P4.

As the process is carried out, a new (or sterilized) loop must be used each time a new phase is
started. Failure to do so would prevent the establishment of a dilution gradient, as the same
bacterial concentration would be spread across both phase regions. Again, each subsequent
region (phase) should contain a lower concentration of microbes such that an individual
colony will be eventually produced. Although individual colonies are most often seen in P4,
depending on the starting concentration in P1 individual colonies may also begin to appear in
P3 or even in P2.

Note: In real-world labs, starting cultures can have various concentrations. To optimize the
growth of single colonies on a plate, researchers routinely modify the phase number and loop
passes used in the dilution streak. If the starting culture has a high concentration (or cell
count), then more dilution is necessary to obtain individual colonies. The number of phases
can be increased (typically to 5) to further dilute the sample. If the starting culture has a low
concentration, then either fewer phases (like reducing to 3 instead of 4) can be used, or the
sterile loop can be passed through the previous quadrant multiple times when streaking out
the next phase. These deviations from the expected protocol are acceptable in practice
provided the resulting gradient contains the growth of individual colonies – if not, the
experiment must be repeated.

Figure 4.3 Agar Streak. A 4-phase dilution streak is shown beginning with the first phase (P1) and ending with
the fourth phase (P4). A sterile loop must be used when switching between phases in order to accurately
generate a dilution series. Single colonies should be evident within P3 or P4.

Once the samples are appropriately streaked onto the media (and the lids placed back onto the
plate), they are inverted to prevent any potential contaminants from settling onto the surface
of the agar and placed in an incubator set at 37°C for ~12-24 hours. Maintaining a
temperature of 37°C is optimal for promoting growth and is the temperature most commonly
used by researchers. Following an overnight (>12 hours) incubation, the regions within each
phase (P1 through P4) are examined and individual colonies identified. Individual colonies
are then isolated, picked, and re-inoculated in nutrient broth (or onto nutrient agar) for the
expansion of the now pure culture to be used for laboratory testing.

Note: Different strains of bacteria vary in their generation time (time it takes them to divide
or double). A short generation time can lead to fast growth, whereas a long generation time
leads to slower growth within a given time frame. Researchers can vary temperature and
incubation time to optimize growth and affect the generation time. Alternative incubation
temperatures may be used to affect microbe growth in a lab setting. Unlike bacteria, other
organisms such as yeast preferentially grow at 30°C. Additionally, because pathogenic
strains of bacteria tend to grow faster than non-pathogenic strains at 37°C, researchers may
set incubators at 25°C to restrict pathogenic growth. Additionally, increasing incubation
time at a particular temperature can encourage more growth, whereas decreasing incubation
time will restrict further cell division and the growth of colonies.

4.4: Bacterial Isolation in Practice

Now that we’ve covered the various types of plates on which microbes can grow and how we
then go about obtaining and isolating a pure culture, the question remains: Why would a
researcher ever want to do so? Let’s look at some real-world situations:

Scenario #1

A patient is brought into the hospital with severe abdominal pain. Upon examination, the
doctor believes the cause of the discomfort to be bacterially related (e.g. food-borne bacterial
illness). The doctor then orders bacterial swab samples to be taken from the patient. Once the
causative agent is identified, the appropriate course of treatment can be given.

Initial Test Results: Gram-negative bacillus and motile

Scenario #2

Two different work crews are in charge of cleaning your workplace. Company 1 is
responsible for cleaning the bathroom sinks and toilets, while Company 2 is responsible for
cleaning the floors. Several office workers have been recently treated for a Staph aureus
infection, and it is believed one of the cleaning crews did not follow the proper sanitary
guidelines while cleaning. The company/source responsible needs to be quickly identified
and dealt with before more people become exposed. Samples are collected from various areas
around the building, as well as from the cleaning tools (mops, sponges, etc.) to test whether
the same or different pathogen is present throughout the workplace.

Initial Test Results:

Company 1 & 2: Gram-positive cocci* and non-motile for all samples
* Test results were inconclusive for the presence of clusters or chains of cocci

In both scenarios one of the most important things to realize is that the samples are being
harvested from within a diverse environment—often many different types of microorganisms
are present within the same area being sampled—some are relevant to the disease, while most
are not. Therefore, the goal is to isolate and grow the potentially pathogenic species while
excluding the non-pathogenic bacteria.

As we discussed at the beginning of this module, often times expanding a given microbial
population is required to assess the microorganism more easily. Since the causative agent in
both scenarios above is unknown and likely in small amounts, the first step is to expand any
potential microbial populations. The easiest way to do this is to inoculate a simple growth
media with the collected sample or streak it onto an agar plate. The culture (or plate) is then
grown at 37°C to encourage microbial growth. Once a bacterial population has been
expanded, samples can be taken and re-streaked onto selective and/or differential media for
further analysis.

Scenario #1

In Scenario #1 the initial test results indicate a motile Gram-negative rod. Now, if the
physician remembers his microbiology he will likely consider the causative agent for
abdominal pain as either Salmonella or E. coli (both are common pathogens of food-borne
illnesses). However, the initial test results are not enough. The physician then orders the
isolated bacterium to be streaked out on EMB agar. The next day he receives a report of a
distinct metallic green growth pattern and immediately knows the causative agent is E.
coli, not Salmonella.

Scenario #2

In Scenario #2, although workers were already being treated for Staph aureus it is unclear if
one, both, or neither of the cleaning companies were responsible for its spread. Initial test
results indicated both companies could potentially be the source as both sets of samples
contained non-motile, Gram (+) cocci—key characteristics of Staph aureus. Unfortunately,
the test results came back with an inconclusive result regarding the exact morphology of the
bacteria isolated from each company. (As a trained microbiologist would know, if any results
came back as decisively Gram-positive cocci chains, this immediately rules out Staph aureus
as it forms Gram-positive clusters). As an alternative strategy, a researcher then streaks the
isolated bacterial samples from each company onto MSA agar. After incubating the samples
overnight at 37°C, Company 1 samples showed only colorless colonies growing on red agar
indicating the presence of non-pathogenic bacteria, while the samples collected from
Company 2 (floors and their cleaning mops and sponges) showed a distinct yellow agar
indicating the presence of Staph aureus.

Obviously, the two scenarios described above are not all-inclusive examples of the
pathogenic (disease-causing) effects of microorganisms. However, they do serve as a general
outline for how to approach unknown samples. As will be discussed in the next module, it is
a diverse microbial world, and, unfortunately, many microbes are also pathogenic.