The World of Microbes: A simplified module of General Microbiology

BIO 211 – General Microbiology

1. Title of the Module

Chapter 3: Isolation and Cultivation of Microorganisms

2. Introduction
The study of microorganisms generally requires isolating and cultivating them in a
culture media. Culture media or growth media is a liquid, semis-solid, or solid mixture
used to support the growth of different microorganisms. This chapter will introduce you
to the different types of culture media, how to isolate microorganism into pure culture
using different isolation techniques, their cultivation and proper storage.

3. Learning outcome

At the end of this lecture, the students will be able to:

1. Enumerate and describe each culture media;
2. Compare and explain the principles behind the different isolation techniques;
3. Describe the basic culture preservation techniques; and
4. Explain the importance of culture collection and list some examples.
4. Learning Content
Most microbes are single-celled undifferentiated organisms, but some are
multicellular, and some can even form complex cell structures. Microorganisms usually
exists in complex communities (Figure 1), and their activities are controlled by interactions
with other microbes, with other organisms, and with their environments. The science of
microbiology is everything about microorganisms, who they are, what they do, and how
they work.

Figure 1. a) Bacterial community in the depth of a small Michigan lake, b) bacterial

community in a sewage sludge, and c) microbial community scraped from a
human tongue.
Source: Madigan et al. (2019). Brock Biology of Microorganisms. 15th Ed.
Microbiologists use several tools to study microorganisms. The discovery of the
microscope gave birth to microbiology, and microscopy is foundational to microbiology.
Microbiologists have already developed several ways to visualize microorganisms. Aside
from microscopy, isolation, and cultivation of microorganisms are essential to
microbiology. A microbial culture is a group of cells cultivated in or on a nutritional
media. A medium (plural, media) is a liquid, semi-solid, or solid nutritional combination
that includes all the necessary nutrients a microbe needs for growth. The term “growth”
is used in microbiology to describe the increase in cell population that occurs as a result
of cell division. On a solid nutritional media, a single microbial cell can develop and divide
into millions of cells, forming a visible colony on nutrient medium (Figure 2).

Figure 2. Microbial colonies. a) Colonies of Photobacterium, a bioluminescent (light-

emitting) bacteria grown in a culture medium. b) A single colony of
approximately 2 mm that contain more than 10 million individual cells. c)
Photobacterium under scanning electron microscope.
Source: Madigan et al. (2019). Brock Biology of Microorganisms. 15th Ed

A. Types of Culture Media

Culture medium can be classified in three ways: based on consistency, nutritional

component, and functional use.

1. Consistency

a. Liquid media. A liquid media has no solidifying agent. Turbidity, surface

pellicle, or granular deposits are indication of bacterial growth in liquid media.
However, culturing bacteria in liquid media has some disadvantages. Bacterial
properties are not apparent, and the presence of more than one type of bacteria
cannot be recognized.
b. Solid media. Certain solidifying agents can be added to any liquid medium to
make it solid. The most common solidifying agent is agar. It is an unbranched
polysaccharide derived from the cell membranes of red algae. It melts at 95⁰C
and solidifies at 42⁰C. Agar has no nutritional properties, not hydrolyzed by
most bacteria, and typically lacks growth- promoting or -retarding substances.
 Agar is in powered form and is commonly used at concentration of 1.5%-2.0%
to make a solid agar medium.
c. Semi-solid media. Sometimes, media consists of both liquid and solid media.
To make a semi-solid medium, agar is commonly used at concentration of
0.10%-0.50%.

2. Nutritional component

There are three types of culture media based on nutritional component: simple,
complex, and synthetic (or defined). Bacteria that can grow with few or minimal
nutrients are said to be non-fastidious, whereas those that need more nutrients
are considered as fastidious.

a. Most non-fastidious microorganisms can be grown on simple medium like

peptone water and nutrient agar.
b. Ingredients in complex media, such as blood agar, are difficult to estimate and
not all components are chemical defined like potato infusion (of plant origin),
beef extract (of animal origin), and yeast extract (of microbial origin).
c. Synthetic or defined media, such as Davis & Mingioli medium are specially
produced media for research purposes where each media component is well
known.

3. Functional use or application

a. General purpose media can support most or almost all types of microbial
species. Examples are nutrient agar (NA) that supports the growth of bacteria
and potato dextrose agar (PDA) which is best for the cultivation and
enumeration of yeasts and molds.
b. Differential media, also known as indicator media, are used to identify one
microbial species from another that are grown on the same medium. To visually
show the defining properties of a microbe, this media can use the biochemical
characteristic of a microorganism growing in the presence of a particular
nutrients or indicators (such as phenol red, neutral red, or methylene blue)
added to the medium.

For example, a certain substrate (carbohydrate) is added to a medium and a

mixture of bacteria is inoculated on it, only the bacteria that can ferment the
carbohydrate generates acid. A pH indicator in the medium detects the change
in pH, and the bacteria that can ferment the sugar shows a distinct color. This
includes the Eosin Methylene Blue Agar (EMBA).

Figure 3 shows the growth of Escherichia coli on EMBA (left) and on NA (right).
EMBA is both a selective and differential medium. It contains the dyes eosin
and methylene blue, which inhibit the growth of Gram-positive bacteria and
therefore select for Gram-negative bacteria. It also contain the carbohydrate
lactose, which allows the differentiation of Gram-negative bacteria based on
their ability to ferment lactose which produce larger, mucoid colonies, often blue
 or black in the center. On the other hand, lactose non-fermenter produces
either colorless or light pink colonies.

a) b)

Figure 3. Escherichia coli grown in a) Eosin Methylene Blue Agar (EMBA) and
b) nutrient agar.
Source: https://microbenotes.com/escherichia-coli-e-coli/

The most commonly used media for growth and identification of Gram-negative
bacteria is MacConkey’s agar (Figure 4) (especially members from
Enterobacteriaceae). Bile salts (selective agent), peptone and neutral red (pH
indicators), lactose (sugar), agar and water are some of the components of the
medium. Lactose-fermenting bacteria produce pink colonies while lactose-
nonfermenting bacteria form colorless colonies.

Figure 4. MacConkey’s agar.

A) colonies produce by lactose
fermenters and B) colonies produce
by non-lactose fermenters.
Source: https://paramedicsworld.com/microbiology-
practicals/preparation-macconkey-agar-medium-mac-
principle-composition/medical-paramedical-studynotes

c. Selective media are used for the growth of selected microorganisms. For
example, a microbe that is resistant to a particular antibiotic, such as ampicillin
or tetracycline, that antibiotic can be added to the medium to prevent or inhibit
the growth of the non-resistant cells. In addition, dyes, chemical, adjustments
of pH, or combination of these can all be used to make a selective medium.
 Bacillus Cereus Agar Base (Figure 5) is an example of a selective medium. It
is used with Polymyxin B and Egg Yolk Emulsion for the isolation and
presumptive identification of Bacillus cereus. This medium differentiates B.
cereus from other bacteria based on resistance to polymyxin, lack of mannitol
fermentation, and presence of lecithinase. Bacteria that ferment mannitol
produce acid products and form colonies that are yellow. Bacteria that produce
lecithinase hydrolyze lecithin and a zone of white precipitate forms around the
colonies. B. cereus is typically mannitol-negative (blue colonies) and
lecithinase positive (zone of precipitation around colonies).

Figure 5. Bacillus Cereus Agar.

Bacillus cereus forms blue
colonies with white precipitate.
Source:
https://www.thermofisher.com/blog/food/bacillus-
cereus-selective-media-for-unpasteurized-food-
samples/

d. Enrichment media are used to increase the number of microorganisms with

unusual physiological characteristics. This includes the addition of special
nutrients like blood in blood agar.

Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies
to induce hemolysis when grown on blood agar (Figure 6) is used for the
classification of certain microorganisms. This is particularly useful in classifying
streptococcal species. A substance that causes hemolysis is a hemolysin.
When alpha hemolysis (α-hemolysis) is present, the agar under the colonies
is dark and greenish. Streptococcus pneumoniae and a group of oral
streptococci (Streptococcus viridans or viridans streptococci) display α-
hemolysis. This is sometimes called green hemolysis because of the color
change in the agar. Other synonymous terms are incomplete hemolysis and
partial hemolysis. The α-hemolysis is cause by hydrogen peroxide produced
by the bacterium, oxidizing hemoglobin to green methemoglobin. Beta
hemolysis (β-hemolysis), sometimes called as complete hemolysis, is a
complete lysis of red blood cells in the media around and under the colonies;
the area appears lightened and transparent. Streptococcus pyogenes display
β-hemolysis. If an organism does not induce hemolysis, it is said to display
gamma hemolysis (γ-hemolysis), the agar under and around the colony is
unchanged (this is also called non-hemolytic). Enterococcus faecalis display γ-
hemolysis.
 Figure 6. Blood agar. Hemolysis of Streptococci.
Source: https://microbenotes.com/hemolysis-of-streptococci/

e. Assay media are used for investigative procedure in laboratory medicine,

pharmacology, environmental biology, and molecular biology for quantitatively
measuring or qualitatively assessing the presence or amount of the functional
activity of the target microorganism.

Figure 7. The Kirby-Bauer Test against Staphylococcus aureus.

Source: https://www.pngwing.com/en/free-png-tiziy/download
 The disk-diffusion method, also known as the Kirby-Bauer test (Figure 7), is
the most commonly used antibiotic susceptibility test for assessing which
antibiotics to use while treating an illness. In this method, the inhibition of
bacterial growth is measured under standard conditions. For this test, a culture
medium, especially Mueller-Hinton agar (MHA), is evenly and aseptically
inoculated with the test organism (in this case the Staphylococcus aureus),
followed by the placing the filter paper discs impregnated with a specified dose
of an antibiotic on the MHA medium.

On the MHA plate, the organism will grow while the antibiotic on the paper
discs “works” to stop it. If the organism is sensitive to the antibiotic, there will
be no growth around the paper disc with the antibiotic. As a result, a "zone of
inhibition" may be observed and quantified to assess an organism's sensitivity
to an antibiotic. The organism can be categorized as Resistant (R),
Intermediate (I), or Susceptible (S).

B. Isolation Techniques

These are the specific methods used to obtain pure cultures of microorganisms:

1. Plating technique
a. Streak plating is a microbiological technique for isolating a pure strain from a
single species of microorganism, most often bacteria. The organism can be
studied, identified, or tested by taking samples from the resulting colonies and
growing a microbial culture on a new plate. A sterile material, such as cotton swab
or, more often, an inoculating loop, is used for streaking. This is dipped in an
inoculum containing a high concentration of bacterial cells, such as broth or
patient specimen. The inoculum is streaked in each quadrant of the agar surface
in such a way that it “thins out” the sample (Figure 8). The kind of growth media
used depends on which type of microorganism is being cultured or selected for.

Figure 8. Streak plate method. Successful streaks lead to individual colonies of

microbes.
Source: https://microbenotes.com/streak-plate-method-principle-methods-significance-limitations/
b. Spread plate method involves plating a liquid sample containing the bacteria so
that they can be counted and isolated easily. A successful spread plate will
produce a countable number of isolated bacterial colonies equally distributed
throughout the petri plate. A measured sample containing the bacteria is
transferred onto a petri plate with growth medium using a sterile pipette (Figure
9.1). This method uses a tool called a plate spreader or L-rod, flame-sterilized
using ethanol and alcohol lamp. Flame-sterilized spreader is used to spread the
liquid around the plate by gently pushing the spreader back and forth on the
medium (Figure 9.2). The lid of the plate was used to spin the plate to get an even
spread. Spread plating is continuously done until all the liquid forms an even layer
on the surface of the growth medium (Figure 9.3).

Figure 9. Spread plate technique.

Source: https://microbeonline.com/spread-plate-technique/

c. Pour plate method is typically used when counting the quantity of colony-forming
bacteria present in a liquid specimen. Because the sample is combined with the
molten agar medium, it is possible to utilize a larger volume than with the spread
plate. As shown in Figure 10, a sterile pipette is used to place a predetermined
volume of inoculum (usually 1 ml) from a broth/sample in the center of a sterile
Petri dish. Agar that has been melted and cooled (about 15mL) is then put into the
Petri plate holding the inoculum and thoroughly mixed. The plate is inverted and
incubated at 37°C for 24-48 hours after the agar has solidified. Microorganisms
can grow on the surface as well as within the media.

Figure 10. Pour plate technique.

Source: https://microbenotes.com/pour-plate-technique-procedure-significance-advantages-limitations/
2. Enrichment

The goal of this method is to create favorable conditions for the growth of the
organism of interest while being unfavorable to other competing microorganisms. For
instance, if you want to isolate thermophilic bacteria (one that prefers to grow at a
high temperature, such as 55⁰C), incubate the sample at high temperature.
Organisms that cannot survive that temperature will die or simply stop growing,
whereas thermophiles will multiply and become a large proportion of the overall
bacterial population in the sample over time. This is an example of enrichment
through physical conditions modification.

Modifying the nutritional content of the culture medium can also be used
for enrichment (Figure 11). For example, a number of microorganisms found in soil
are capable of converting N2 into ammonia. These processes are the primary source
of nitrogen in the biosphere. Organisms capable of this type of nitrogen
transformation can be isolated by incubating a soil sample in a culture medium that
contains all the required nutrients for growth except nitrogen. Those bacteria that can
"fix" nitrogen and produce their own nitrogenous nutrients for growth will have a
selection advantage over those that can't, and their numbers will increase. Only
nitrogen-fixing bacteria will thrive in the medium since nitrogen is likely to be present
in small amounts as a contaminant in the soil or in the other culture components.
They will, however, have a significant advantage and will eventually outgrow the other
microorganisms present in the sample.

Figure 11. Enrichment procedure of isolating of an organism from natural environment.

Source: https://www.reddit.com/r/dankmemes/comments/8odhig/cultural_enrichment_creates_colonies_its_just/
 Another example of modifying culture condition is the isolation of bacteria that can
break down a xenobiotic (man-made, unnatural substance) compound, such as a
pesticide. Pesticides are organic substances (mostly C) that are used to kill pests.
Bacteria use organic substances as a source of building materials and energy. Only
those bacteria that can utilize the pesticide as the sole carbon source will significantly
grow in the medium, while others will not. Eventually, the culture will have a
high number of pesticide-degrading microorganisms.

3. Serial dilution
It is used if the desired microorganism is present at a higher number than any other
microorganism. As you make a lower dilution to a higher dilution, the number of
microorganisms become diluted. Dilution (d) can be computed as:

𝑣𝑜𝑙𝑢𝑚𝑒 𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟𝑟𝑒𝑑
𝑑= 𝑥 𝑝𝑟𝑒𝑣𝑖𝑜𝑢𝑠 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒

Hence, Figure 12 showed that the dilution of the first tube is 10-1, the second tube is
10-2, third tube is 10-3, fourth tube is 10-4, and the fifth tube is 10-5.

Figure 12. Procedure of serial dilution.

Source: https://microbenotes.com/serial-dilution/

Serial dilution is usually combined with spread or pour plating. In Figure 12, each tube
is being plated on a solid medium and as you go to a higher dilution (10-5) the number
of microorganisms decreases. There are less microorganisms present in the higher
dilution and more isolated colonies (2 colonies) are obtained.
4. Single cell isolation

This is one of the most desirable and difficult methods of ensuring pure culture. This
method involves placing a suspension of pure culture on the surface of a sterile cover
slip mounted on the microscope stage.

Briefly, a single cell is removed with a sterile micropipette while looking through the
microscope, a small drop of sterile media is transferred on a sterile cover-glass cover
slip, and mounted on a sterile hanging drop slide, which is then incubated at
optimum temperature (Figure 13). If a single cell grows in this drop, a few cells are
moved to a tube containing sterile culture medium and placed in an incubator to
obtain pure culture from a single cell.

Figure 13. Single cell isolation technique.

Source: https://v3.pubpub.org/pub/fiber-microprobe-for-selective-electroporation

5. Membrane filtration technique

Membrane filters have a specified uniform porosity (usually 0.45 µm) that is
small enough to trap microorganisms. Using a filter funnel and a vacuum system,
sample is passed through the membrane through the process of membrane filtration.
Any organisms present in the sample are concentrated on the surface of the
membrane filter.

The membrane with the trapped bacteria, is then placed in a petri plate with
culture medium. During incubation, the passage of nutrients through the
membrane filter promotes the growth of organisms on the upper surface of the
membrane in the form of colonies. The formed isolated colonies can be simply
transferred to a confirmatory medium.

The membrane filter technique is a reliable and widely used method for detecting
microbial contamination in fluid samples. It requires less preparation than many
other standard procedures and is one of the few approaches that allows for
microbial isolation and enumeration. Membrane filters are widely used to sterilize fluid
materials in the laboratory and in industry.
 Figure 14. Method of membrane filtration technique.
Source: https://biologyreader.com/membrane-filtration-method.html

C. Maintenance and Preservation of Pure Cultures

Once a microorganism has been isolated and cultivated in pure culture, it is vital to
preserve the pure culture that is free of contamination in order to retain the microbe's
viability and purity. Pure cultures are typically inoculated onto or into a fresh medium
(subculturing) in laboratories to allow the continued development and viability of
microorganisms. To avoid contamination, the transfer is always done under aseptic
conditions.

Because repetitive subculturing tedious and time consuming, it becomes difficult to

keep a large number of viable pure cultures for an extended period of time.
Furthermore, there is a possibility of contamination as well as genetic mutations. As
a result, advanced methods that do not require repeated subculturing are currently
being used. Refrigeration, paraffin technique, cryopreservation, and lyophilization
(freeze-drying) are some of these approaches.

1. Periodic transfer

Strains can be kept viable by making a fresh culture from the previous stock
culture on a regular basis. The culture medium, storage temperature, and time
interval at which the transfers are performed differ according on the species and
must be determined beforehand. The temperature and kind of medium chosen
should promote a slow rather than a fast rate of microbial growth, so that the
time between transfers can be as long as possible. In a media like nutrient agar,
heterotrophs (microorganisms that use organic carbon for growth) remain viable
for weeks or months.

The disadvantage of this method is that it does not prevent changes in a strain's
properties due to the generation of variations and mutants.
2. Oil Overlay

This is the simplest and most cost-effective way to keep viable cells
of bacterial and fungal cultures. This procedure involves pouring sterile liquid
paraffin over the culture's slant and storing it in upright position at room
temperature. The paraffin layer maintains anaerobic conditions and prevents the
medium from becoming dehydrated. This condition allows microbes or pure
culture to remain inactive, allowing the culture to be stored for months or even
years (varies with species).

The advantage of this procedure is that we may use an inoculating needle to take
some of the growth under the oil, inoculate a fresh medium, and keep the original
culture. The method's simplicity makes it appealing, although changes in a strain's
traits can still occur.

3. Lyophilization

Sublimation removes water and other solvents from a frozen product during the
freeze-drying process. Sublimation is the process by which a frozen liquid
transforms a solid to a gaseous state without passing through the liquid phase.

Slow rates of cooling is preferred because it allows vertical ice crystal formations,
which allows for more efficient water sublimation from the frozen product.
Because freeze-dried items are hygroscopic, they must be kept dry during
storage. The microbial cells are dehydrated, and their metabolic processes are
inhibited under these conditions; as a result, the microorganisms stay dormant
and remain viable for years. Pure cultures that have been lyophilized or freeze-
dried are then sealed and kept in refrigerators at 4°C in the dark.

The most common procedure employed by culture collection centers is freeze-

drying. Several kinds of bacteria preserved by this technique have remained
viable and unchanged in their properties even after 30 years.

4. Liquid nitrogen

Cryopreservation (freezing in liquid nitrogen at -196°C or in the gas phase above

liquid nitrogen at -150°C) aids in the long-term storage of pure cultures.

In this procedure, microbial cultures are rapidly frozen in liquid nitrogen at -196°C
in the presence of stabilizing chemicals like glycerol or dimethyl sulfoxide (DMSO),
which limit cell damage and enhance cell survival by preventing ice crystal
formation.

This liquid nitrogen method has proven successful with many species that cannot
be kept by lyophilization, and most species can survive for 10 to 30 years without
 changing their properties under these conditions; nevertheless, this method is
expensive.

5. Drying

In this method, samples are grown on sterile paper disk saturated with nutrient,
then the disks are allowed to air dry and stored aseptically and maintained at drying
temperature of 45°C. Due to this high temperature, it is best recommended for
spore- and cyst- formers microorganisms. It can also be used for microorganisms
that are sensitive to freeze-drying.

Other ways for drying includes: First is by dropping bacterial suspension in gelatin
which are placed n sterile petri plate and dried off over phosphorus pentoxide using
a vacuum. Secondly, bacteria in small ampoules are dried from liquid state using
vacuum pump, desiccant, and water bath. Lastly, organisms are dried over calcium
chloride in vacuum and stored in the refrigerator.

D. Culture collections

Microbial cells can be kept in culture collections which are organizations that
maintain authentic pure cultures of microorganisms. They provide the ‘type’ strains
to microbiologists worldwide.

Examples of culture collections:

1. American Type Culture Collection (ATCC) Maryland
2. National Collection of Type Cultures (NCTC) London
3. Japan Collection of Microorganisms (JCM)
4. Philippine National Collection of Microorganisms (PNCM)

5. Teaching and Learning activities:

You may use separate paper for your answers. Scan or capture your work and submit it
in pdf or picture file with file name YourSURNAME_FirstName_Activity3. Deadline of
submission will be on October 10, 2021.

Answer briefly and concisely. Write your answer in not more than 5 sentences.

1. Can you spread plate 10 mL inoculum in a petri plate with culture medium? Why
or why not? (5 pts)
2. In what instances would the pour plate method be more appropriate than the
spread plate method? (5 pts)
3. Can humans exist in chemically defined media for 48 hrs? (5 pts)
6. Recommended learning materials and resources for supplementary reading.

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)
/6%3A_Culturing_Microorganisms/6.3%3A_Culturing_Bacteria/6.3A%3A_Culture_
Media

https://www.micro.iastate.edu/video/microbiology-004-spread-plate-method

7. Flexible Teaching Learning Modality (FTLM) adopted

Modules are available at https://classroom.google.com/u/1/c/MzkyOTUwODc0OTU0

8. Assessment task

1. Classify culture media.

2. What is microbial colony and how it is formed?

9. References:

Madigan, M. T., Bender K. S., Buckley D. H., Sattley, W. M. & Stahl D. A. (2019). Brock
biology of microorganisms (15th edition). Harlow: Pearson Education Limited.

Madigan, MT, Bender, KS and Buckley, DH. (2013). Brock Biology of Microorganisms.
15th Edition. Pearson Education Inc.

Pelczar, M.J., Chan, E.C.S. and Krieg, R.N. (2012). Microbiology. 10th Edition. McGraw-
Hill.

http://shs-manual.ucsc.edu/policy/kirby-bauer-antibiotic-sensitivity

https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/
Microbiology_Labs_I/09%3A_Kirby-Bauer_(Antibiotic_Sensitivity)

http://www.clt.astate.edu/dgilmore/Research%20students/enrichment_culture.htm