CLASS No.

7
CLASSIFICATION, NOMENCLATURE, AND IDENTIFICATION OF
BACTERIA. ISOLATION OF PURE CULTURE (PART III).

Experiment 7. Continuation of pure culture isolation: defining the purity of

isolated culture. Pure culture identification on a basis of biochemical properties

1. Taxonomy, Classification, Nomenclature, Identification.

It has been estimated that we currently have the capacity to identify
fewer than 10% of the pathogens responsible for causing human disease
because of our inability to culture or target these organisms using molecular
probes. Yet the diversity of even these identifiable pathogens is so great that it
is important to understand the subtle differences associated with each
infectious agent. The reason for understanding these subtleties is significant
because each infectious agent has specifically adapted to a particular mode(s)
of transmission, a mechanism(s) to grow in human hosts (colonization), and a
mechanism(s) to cause disease (pathology). As such, a vocabulary that
consistently communicates the unique characteristics of infectious organisms
to students, microbiologists, and health care workers is critical to avoid the
chaos that would ensue without the organizational restraints of bacterial
taxonomy (the classification of organisms in an ordered system that indicates
a natural relationship). Taxon is a group of organisms that reveal a
determined degree of homogeneity. Classification, nomenclature, and
identification are three separate but interrelated areas of bacterial taxonomy.
Classification is the categorization of organisms with similar traits into
taxonomic groups (taxons). Classification of bacteria requires experimental
and observational techniques; this is because biochemical, physiologic,
genetic, and morphologic properties are often necessary for an adequate
description of a taxon.
Nomenclature refers to the naming of an organism by international
rules (established by a recognized group of medical professionals) according
to its characteristics.
Identification is practical use of a classification scheme to isolate and
distinguish desirable organisms from undesirable ones, verify the authenticity
or special properties of a culture in a clinical setting, and isolate and identify
the causative agent of a disease.
Identification should not be considered as part of taxonomy, although, similar
to taxonomy, it is associated with the description of organisms and is based on
comparison with known taxa. However, the scope and purpose of identification are
much narrower.
 Certain schemes are used to identify and classify bacteria, but identification
and classification schemes can be significantly different. In addition, the
identification scheme for the group can only be developed after this group had
been classified.
In microbiology, as well as in the systematics of higher organisms, the main
taxonomic unit is species – one of the most controversial concepts in bacteriology.
The definition of the concept of "species" in higher plants and animals is based on
clearly expressed morphological and physiological differences, as well as the
impossibility of cross-breeding between representatives of two populations. In the
case of bacteria, these criteria are not suitable, because bacteria are
morphologically rather similar, and do not perform sexual reproduction.
There are several definitions of the concept of "species" in bacteria.
According to the microbiological terminology adopted in Ukraine (DSTU, 1994),
the term "species" is defined as "a taxonomic unit that unites organisms on the
basis of morphological, physiological, biochemical, genetic and other
characteristics."
The category below the rank is the subspecies (subsp) or biovar (biovariant).
These are the populations of microorganisms within the species, which differ in
one or more features. Depending on what characteristics the attention is paid to the
description of subspecies, the following are distinguished:
morphotype (according to morphological characteristics),
pathovar (by type of disease, tissue tropism),
serovar (by antigenic structure),
chemovar (according to physiological and biochemical parameters).
In microbiology, the term "strain" is often used. Various researchers put in
this concept a slightly different meaning. In the classical sense, strain is a pure
culture of a microorganism of any species, isolated at a certain time in a certain
place. The same strain can not be isolated from the same place at another time,
from similar or different source. Often, the strain refers to colonies that have grown
under the isolation of the original culture, or indicate bacterial cultures with certain
deviations from the one studied. In medical practice, same strain can denote
bacteria isolated from different places, for example, with the same spectrum of
resistance to antibiotics. For strains, after the species name, usually indicated is the
abbreviation of the institution, or the collection where it first was described and
where the typical strain of the microorganism is stored.

1.1. Principles of Classification and Nomenclature

1.2. Classification schemes are organized into several descending ranks,
beginning with the most general all-inclusive taxonomic category and ending with
the smallest and most specific category. This means that all members of the highest
category share only one or a few general characteristics, whereas members of the
lowest category are essentially the same kind of organism—that is, they share the
majority of their characteristics. The taxonomic categories from top to bottom are
domain, kingdom, phylum or division, class, order, family, genus, and species.
Thus, each kingdom can be subdivided into a series of phyla or divisions, each
phylum is made up of several classes, each class contains several orders, and so on.
Because taxonomic schemes are to some extent artificial, certain groups of
organisms may not exactly fit into the main categories. In such a case, additional
taxonomic levels can be imposed above (super) or below (sub) a taxon, giving us
such categories as “superphylum” and “subclass.”
Hierarchical system - broad divisions are divided up into smaller divisions:
 Domain
 Kingdom
 Phylum (or “Division”)
 Class
 Order
 Family
 Genus (plural: Genera)
 Species (both singular & plural)

1.2. Nomenclature rules

Many macroorganisms are known by a common name suggested by certain
dominant features. For example, a bird species may be called a “red-headed
blackbird” or a flowering plant species a “black-eyed Susan.” Some species of
microorganisms are also called by informal names, including human pathogens
such as “gonococcus” (Neisseria gonorrhoeae) or fermenters such as “brewer’s
yeast” (Saccharomyces cerevisiae), or the recent “Iraqabacter” (Acinetobacter
baumannii), but this is not the usual practice. If we were to adopt common names
such as the “little yellow coccus,” the terminology would become even more
cumbersome and challenging than scientific names. The method of assigning a
scientific or specific name is called the binomial (two-name) system of
nomenclature. The scientific name is always a combination of the generic (genus)
name followed by the species name. The generic part of the scientific name is
capitalized, and the species part begins with a lowercase letter. Both should be
italicized (or underlined if using handwriting), as follows: Staphylococcus aureus
The two-part name of an organism is sometimes abbreviated to save space, as in
E. coli, but only if the genus name has already been stated. The inspiration for
names is extremely varied and often rather imaginative. Some species have been
named in honor of a microbiologist who originally discovered the microbe or who
has made outstanding contributions to the fi eld. Other names may designate a
characteristic of the microbe (shape, color), a location where it was found, or a
disease it causes.
So the main rules in nomenclature are:
•Genus name + species name
 •Italicized or underlined.
•Genus name is capitalized and may be abbreviated.
•Species name is never abbreviated.
•A genus name may be used alone to indicate a genus group.
•A species name is never used alone.
•eg: Bacillus subtilis (B. subtilis)

1.3. Bergey’s Manual of Systematic Bacteriology

In 1927, David Bergey & colleagues published Bergey’s Manual of
Determinative Bacteriology, a manual that grouped bacteria into phenetic groups,
used in identification of unknowns. It is now in its 9th edition. Bergey’s Manual of
Determinative Bacteriology organizes the prokaryotes into four major divisions
based on cell wall structure:
– Gracilicutes:
gram-negative cell walls and thus are thin-skinned
– Firmicutes:
gram-positive cell walls that are thick and strong
– Tenericutes:
lack a cell wall and thus are soft
– Mendosicutes:
archaea with unusual cell walls
In 1984, a more detailed work entitled Bergey’s Manual of Systematic
Bacteriology was published, still primarily phenetic in its classification. Bergey’s
Manual of Systematic Bacteriology uses both morphological, physiological
characteristics and rRNA sequencing data.

Table. 7.1. Main terms and definitions

The term The definition
Pure culture A culture containing a single kind of microorganism.

The population of A group of organisms of the same species in the same place
microorganisms at the same time.
Species Defined in microbiology as a collection of strains that all share the
same major properties and differ in one or more significant
properties from other collections of strains; defined
phylogenetically as a monophyletic, exclusive group based on
DNA sequence.
Strain A population of cells of a single species all descended from a
single cell; a clone.
Colony A population of bacterial cells of same species which have
grown from one bacterial cell on solid medium.
 2. Scheme for Bacteria Identification
In clinical laboratory, the bacterial culture is indicated in isolation of
organisms in pure culture from clinical specimens and their final identification.
Identification means the correct naming of isolates according to agreed systems of
taxonomy and nomenclature.
Since a species is the “basic unit” of taxonomy, representing a specific,
recognized type of organism, bacteria identification means determining a species
of unknown bacteria.
Precise identification of bacteria is time consuming and contention and is
best carried at in specialized reference centers. For the most clinical purposes what
is required is clear guidance on the likely case of an infection, not a rigorously
accurate, but belated description of what the patient has already recovered (or
possibly died) from. In clinical laboratories, identifying the genus or species of an
organism is more important than understanding evolutionary what species of
bacteria they have/they are just interested in getting well. Their recovery often
depends on the proper identification of the infectious agent.
Colony isolation is essential technique for bacteriological method (“golden”
standard of infectious diseases diagnosis). Every species has specific features of
growth on solid and liquid media. Colony investigation (macroscopic and
microscopic) helps to determine a species (make preliminary diagnosis) and isolate
pure culture (make final diagnosis).

STEP 1. Examination of morphological properties and staining

affinity (Gram staining, or other methods) [Classes 1-4]
Morphology and staining reactions of individual organisms generally
serve as preliminary criteria to place an unknown species in its appropriate
biological group. A Gram stain smear suffices to show the Gram reaction, size,
shape and grouping of the bacteria, and the arrangement of any endospores.
An unstained wet mount may be examined with the darkground
microscope to observe the morphology and delicate spirochaetes; an unstained
wet mount or “hanging drop” preparation is examined with the ordinary
microscope for observation of motility.
To identify mycobacteria, or other acid-fast organisms, a preparation is
stained by the Ziehl- Neelsen method or one of its modifications.
The microscopic features of certain organisms in pathological specimens
may be sufficient for final identification, e.g. Mycobacterium in sputum, or
Treponema pallidum in exudate from a chancre. However, many bacteria share
similar morphological features and further tests must be applied to differentiate
them.
 STEP 2.Studying cultural characteristics [Classes 5-6]
The appearance of colonial growth on the surface of a solid medium,
such as nutrient agar (MPA), is often very characteristic. Attention is paid to
the diameter of the colonies, their outline, their elevation, their translucency
and colour. Changes brought about in the medium (e.g. hemolysis in a blood
agar medium) may also be significant.
The rage of conditions that support growth is characteristic of particular
organisms. The ability or inability of the organism to grow on media
containing selective inhibitory factors. (e.g. bile salts, specific antimicrobial
agent, low or high pH) may be of diagnostic significance.

STEP 3. Revealing Biochemical properties of bacteria

Species that can be distinguished by morphology and cultural characters
may exhibit metabolic differences that can exploited. In general, these methods
fall into the realm of biochemical test because they can determine fundamental
chemical characteristics such as nutrient requirement, products given off
during growth, temperature and gas requirements, and mechanisms for
obtaining energy. It is usual to test the ability of organism to produce acid and
gaseous end-products, when presented with individual carbohydrates (glucose,
lactose, sucrose, mannitol, etc.) as the sole carbon source (table 7.2). Other
tests determine whether the bacterium produces particular end-products (e.g.
indol, hydrogen sulphide) when grow in suitable culture media, and whether it
possesses certain enzyme activities such as oxidase, catalase, urease,
gelatinase or lecithinase.
Sometimes more elaborate procedures may be used the analysis of
metabolic products. For example, gasliquid chromatography is widely used to
recognize characteristic volatile fatty acids and alcohols produced by
anaerobic bacteria.

Table 7.2: Common Microbial Biochemical Tests Used to Differentiate

Among Bacteria
Test Description
1. Carbohydrate The ability to produce acidic metabolic products,
breakdown fermentatively or oxidatively, from a range of
carbohydrates (eg, glucose, sucrose, and lactose) has
been applied to the identification of most groups of
bacteria. Such tests are crude and imperfect in defining
mechanisms, but have proved useful for taxonomic
purposes.

2. Catalase production The enzyme catalase catalyzes the conversion of

hydrogen peroxide to water and oxygen. When a colony
is placed in hydrogen peroxide, liberation of oxygen as
gas bubbles can be seen. The test is particularly useful
 in differentiation of staphylococci (positive) from
streptococci (negative), but also has taxonomic
application to Gram-negative bacteria.
3. Citrate utilization An agar medium that contains sodium citrate as the sole
carbon source may be used to determine ability to use
citrate. Bacteria that grow on this medium are termed
citrate-positive.
4. Coagulase The enzyme coagulase acts with a plasma factor to
convert fibrinogen to a fibrin clot. It is used to
differentiate Staphylococcus aureus from other, less
pathogenic staphylococci.
5. Decarboxylases and The decarboxylation or deamination of the amino acids
lysine, ornithine, and arginine is detected by the effect of the
deaminases
amino products on the pH of the reaction mixture or by the
formation of colored products. These tests are used primarily
with Gram-negative rods.
6. Hydrogen sulfide. The ability of some bacteria to produce H2S from amino
acids or other sulfur-containing compounds is helpful in
taxonomic classification. The black color of the sulfide
salts formed with heavy metals such as iron is the usual
means of detection.
7. Indole The indole reaction tests the ability of the organism to
produce indole, a benzopyrrole, from tryptophan. Indole is
detected by the formation of a red dye after addition of a
benzaldehyde reagent. A spot test can be done in seconds
using isolated colonies.
8. Nitrate reduction Bacteria may reduce nitrates by several mechanisms. This
ability is demonstrated by detection of the nitrites and/or
nitrogen gas formed in the process.
9. O-Nitrophenyl-β-d The ONPG test is related to lactose fermentation. Organisms
galactoside (ONPG) that possess the β-galactoside necessary for lactose
breakdown fermentation but lack a permease necessary for lactose to
enter the cell are ONPG-positive and lactose-negative.
10. Oxidase production The oxidase tests detect the c component of the
cytochrome–oxidase complex. The reagents used
change from clear to colored when converted from the
reduced to the oxidized state. The oxidase reaction is
commonly demonstrated in a spot test, which can be
done quickly from isolated colonies.
11. Proteinase Proteolytic activity is detected by growing the organism
production in the presence of substrates such as gelatin or
coagulated egg.
12. Urease production Urease hydrolyzes urea to yield two molecules of
ammonia and one of CO2. This reaction can be detected
by the increase in medium pH caused by ammonia
production. Urease-positive species vary in the amount
of enzyme produced; bacteria can thus be designated as
positive, weakly positive, or negative.
13. Voges–Proskauer The Voges–Proskauer test detects acetylmethylcarbinol
test. (acetoin), an intermediate product in the butene glycol
pathway of glucose fermentation.

On a basis of information, obtained in steps 1-3, one can make a

conclusion on genus or even genera of bacteria being identified. The larger is
the amount of information obtained, the more efficient identification will be.
Acquisition of information regarding large number of bacterial features is a
basis for Numreical Taxonomy.

STEP. 4 Numerical Taxonomy

Numerical taxonomy use a large number (frequently greater than 100) of
unweighted taxonomically useful characteristics. The Analytical Profile Index
(APITM) is a method commonly used to identify a wide range of
microorganisms. APIs consist of a number of plastic strips, each of which has
about 20 miniature compartments containing biochemical reagents. Almost all
cultivatable bacterial groups and more than 550 different species can be
identified using the results of these API tests. These identification systems
have extensive databases of microbial biochemical reactions. The numerical
clusters derived from these tests identify different strains at selected levels of
overall similarity (usually >80% at the species level) on the basis of the
frequency with which they share traits. The limitation of this approach is that it
is a static system, which does not allow for the evolution of bacteria and
routine discovery of new bacterial pathogens (Figure 7.2).

Figure 7.2 APITM test demonstrating how bacteria can be differentiated using a series
of biochemical tests. Each small compartment contains a dehydrated powder that can be
inoculated from a bacterial culture. After incubation, the colorimetric changes can be scored
numerically to produce a number that matches to a specific bacterial species and genus.
(Courtesy of bioMerieux, Inc.)

STEP 5. Studying Antigenic properties of bacteria [Immunology]

Isolated cultures are investigated by the agglutination test with specific
serum and other serological tests.
Serologic Typing
Clonality with respect to isolates of microorganisms from a common
source outbreak (point source spread) is an important concept in the
epidemiology of infectious diseases. Etiologic agents associated with these
outbreaks are generally clonal; in other words, they are the progeny of a single
cell and thus, for all practical purposes, are genetically identical.
Thus, subtyping plays an important role in discriminating among these
particular microorganisms. Recent advances in biotechnology have
dramatically improved our ability to subtype microorganisms. Hybridoma
technology has resulted in the development of monoclonal antibodies against
cell surface antigens, which have been used to create highly standardized
antibody-based subtyping systems that describe bacterial serotypes. This is an
important tool for defining the epidemiologic spread of a bacterial infection.
Other organisms cannot be identified as unique serotypes. For example, some
pathogens (eg, Neisseria gonorrhoeae) are transmitted as an inoculum
composed of quasispecies (meaning that there is extensive antigenic variation
among the bacteria present in the inoculum). In these cases, groups of
hybridomas that recognize variants of the original organisms are used to
categorize serovariants or serovars, serotypes, serogroups, and serovars. The
designation “sero” simply indicates the use of antibodies (polyclonal or
monoclonal) that react with specific bacterial cell surface structures such as
lipopolysaccharide (LPS), flagella, or capsular antigens. The terms “serotype,”
“serogroups,” and “serovars” all use the specificity of these antibodies to
subdivide strains of a particular bacterial species.

STEP 6. Animal pathogenicity and toxigenicity [Immunology]

Virulence is determined by infecting experimental animal. Biological
tests may be utilized for causative agents of tuberculosis, plaque, tularemia and
demonstration of bacterial toxins.

STEP 7. Phage typing, bacteriocine typing, antibiotic sensitivity

tests [Class 8, Virology]
Phages are viruses that attack bacteria. When this happens the phage
(bacterial virus) will copy itself within the bacteria and either kill the bacteria
or stay in the bacteria in a dormant form. When this happens the phage will
also be copied when the bacteria divides. Bacteria may thus have several
phages sitting in them. Such phages will affect the ability of other phages to
infect the bacteria. In other words, the ability to be infected by different phages
varies between different strains of bacteria, even if they are fairly closely
related.
This fact forms the basis of phage typing. The bacterial strains are
grown and then subjected to attack by a series of different known phages.
Some phages will kill the bacteria (this is clearly visible and therefore
measurable) and others won’t. Depending on which groups of phages can kill
or not kill a bacterial strain (the reaction pattern) the bacteria is given a
The method itself requires that the different phages are available and it’s
therefore a method that can generally only be performed at reference
laboratories. Also it requires substantial technical expertise to perform.
 STEP 8. Nucleic acid–based –analysis. Phylogenetic Classification.
Ribosomal RNA
Several modern analytical and diagnostic tools that focus on the genetic
and molecular characteristics can detect the exact nature of microbial DNA.
The genetic properties of bacteria allow genes to be exchanged among
distantly related organisms. Furthermore, multiplication of bacteria is almost
entirely vegetative, and their mechanisms of genetic exchange rarely involve
recombination among large portions of their genome. Therefore, the concept of
a species — the fundamental unit of eukaryotic phylogeny — has an entirely
different meaning when applied to bacteria. A eukaryotic species is a biologic
group capable of interbreeding, with the ultimate intent to produce variable
offspring. Because bacteria replicate clonally by binary fission, they do not
require a complementary set of chromosomes in order to reproduce.
For the purposes of categorizing bacteria, a species is a genomically
coherent group of individual isolates or strains sharing a high degree of
similarity in many independent features when comparably tested under highly
standardized conditions.
The formal ranks used in the taxonomy of bacteria are listed in Table
7.3.. For practical purposes, only the ranks of the family, genus, and species
are commonly used. There is considerable genetic diversity among bacteria.
Chemical characterization of bacterial genomic DNA reveals a wide range of
nucleotide base compositions among different bacterial strains. The guanine +
cytosine (G + C) content of closely related bacteria is similar, indicating that
genetic relatedness of DNA from similar organisms can be used as a measure
of taxonomic relatedness. The parameter of DNA–DNA similarity based on
the difference in thermal denaturation midpoint has been a gross method for
species delineation.
A more precise method is DNA sequencing. This method has become a
routine procedure, and comparison of the DNA sequences of divergent genes
can give a measure of their relatedness. Genes for different functions, such as
those encoding surface antigens to escape immune surveillance, diverge at
different rates relative to “housekeeping” genes such as those that encode
cytochromes. Thus, DNA sequence differences among rapidly diverging genes
can be used to ascertain the genetic distance of closely related groups of
bacteria, and sequence differences among housekeeping genes can be used to
measure the relatedness of widely divergent groups of bacteria.
 Table 7.2.: Major Categories and Groups of Bacteria That Cause
Disease in Humans as Part of an Identification Scheme Described in
Bergey’s Manual of Determinative Bacteriology, 9th Ed.

8.1.Ribosomal RNA
Ribosomes have an essential role in protein synthesis for all organisms.
Genetic sequence encodings both ribosomal RNAs (rRNA) and proteins (both
of which are required to comprise a functional ribosome) have been highly
conserved throughout evolution and have diverged more slowly than other
chromosomal genes. Comparison of the nucleotide sequence of 16S ribosomal
RNA from a range of prokaryotic sources revealed evolutionary relationships
among widely divergent organisms and has led to the elucidation of a new
kingdom, the Archaebacteria. The phylogenetic tree based on ribosomal
RNA (rRNA) data, showing the separation of bacteria, archaea, and eukaryote
families, is depicted in Figure 7.3, which shows the three major domains of
biological life as they are currently understood. From this diagram, two
kingdoms, the Eubacteria (true bacteria) and the Archaebacteria, are distinct
from the Eukaryotic branch.

Fig. 7.3 A phylogenetic tree based on rRNA data, showing the separation of
bacteria, archaea, and eukaryotes families. The groups of the major known pathogenic
bacteria are designated in grey. The only group of pathogenic bacteria that does not cluster
in this shaded area is the Bacteroides group.

STEP 9 (OPTIONAL). Nonculture methods for the identification of

pathogenic microorganisms
Attempts to estimate total numbers of eubacteria, archaebacteria, and
viruses are problematic because of difficulties such as detection in and
recovery from the environment, our incomplete knowledge of obligate
microbial associations, and the problem of the species concept in these groups.
Nevertheless, estimates indicate that the numbers of uncultured microbial taxa
greatly exceed those of the cultured organisms. However, more recent
estimates suggest that the number of bacterial species in the world is between
107 and 109. Until very recently, microbial identification required the isolation
of pure cultures followed by testing for multiple physiologic and biochemical
traits. Clinicians have long been aware of human diseases that are associated
with visible but nonculturable microorganisms. Scientists are now using a
PCR-assisted approach using rRNA to identify pathogenic microorganisms in
situ. The first phase of this approach involves the extraction of DNA from a
suitable specimen, the use of standard molecular techniques to obtain a clone
library, the retrieval of rRNA sequence information, and a comparative
analysis of the retrieved sequences. This yields information on the identity or
relatedness of the sequences in comparison with the available data base. In the
second phase, proof that the sequences are from cells in the original specimen
is obtained by in situ hybridization using sequence-specific probes. This
approach has been used in the identification of pathogenic microorganisms.
For example, a previously uncharacterized pathogen has been identified as the
Whipple-disease–associated rodshaped bacterium now designated Tropheryma
whipplei. This rRNA approach has also been used to identify the etiologic
agent of bacillary angiomatosis as Bartonella henselae and to show that the
opportunistic pathogen Pneumocystis jiroveci is a member of the fungi.
Undoubtedly, these and other techniques will identify additional etiologic
agents in the future.

Pre-lab questions: (questions to be answered before doing the experiment.

The answers are due at the beginning of the experiment):
1. What are the main principles of biochemical test that reveal the ability of
bacteria to produces nutritional enzymes?
2. What are the main principles of biochemical test that reveal the ability of
bacteria to produces respiratory enzymes?
3. What are the numerical clusters and how they can be derived?
4. What is the basis for dividing bacteria in subgroups on a basis of their
antigenic properties?
5. What is the principle of phage typing of bacteria?
6. What information does the chemical characterization of bacterial DNA
provide?
7. Which steps of bacteria identification use the DNA sequencing?
8. What is the evidence that your culture is pure?
9. Why do we use two tubes in OF test?
10. What is the basis of color alteration in the open/sealed tube?
THE PRACTICAL PART INCLUDED IN YOUR VIDEO LAB.
Experiment 7. Continuation of pure culture isolation: defining the purity of
isolated culture. Pure culture identification on a basis of biochemical
properties.

Materials (per lab group)

• Bunsen burners
• Staining trays and bridges
• Test tubes with sterile water
• Loops
• Slides
• Soap
• Fiber-free cloth
• Fuchsine
• Wash bottles
• Blotting paper
• Immersion oil
• Alcohol
• Microscopes
• Discard jar
• Marker pen
• Forceps
 Test Tube Rack
 Incubator
 Gram staining Kit
 Tubes with Hugh and Leifson (2 per a student, one with media being
covered with liquid paraffin)
 Pure cultures of E. coli and S. aureus on slant MPA
 Experimental procedure

Main objective. Studying the obtained culture of aerobic bacteria. Defining

cultural, morphological, tinctorial (staining affinity) properties, and biochemical
properties of obtained (pure) culture.
PART 1

1. Cultural properties (Growth pattern on slant MPA)

2. Morphological, tinctorial properties
(Gram staining)

Bacterial culture 3. Motility detection

on slant MPA (wet mount)/not performed/

On this stage a conclusion can be drawn on purity of the culture.

If the culture is morphologically uniform and reveals identical staining
properties (tinctorial properties) ONLY IN THAT CASE it can be further
IDENTIFIED on a basis of its biochemical properties.

PART 2 4. Studying biochemical properties of pure culture of aerobic

bacteria: conclusion on ability to ferment glucose under anaerobic
conditions, as well to produce gases.

OF test, Hugh and Leifson media

Aerobic and anaerobic conditions
E. coli S. aureus

This allows to choose a relevant API tets for identification of multiple

biochemical traits of the microorganism

PART 1.
Main task 1. Studying the obtained culture of aerobic bacteria: Defining
cultural, morphological and tinctorial properties provides information on
PURITY of isolated culture.
Step 1. Observe obtained tube with the subinoculated slope agar culture. Try
to determine whether or not the culture is pure (1). Pure culture shows one
type of colonies.
Step 2. Try to determine whether or not the culture is pure (2). Prepare a Gram
stain smear of the culture. If the culture is pure, in Gram staining smears you
observe only one type of bacteria as well in appearance as in staining affinity.
Record your observations with labeled drawing.
Step 3. Fill in the table and draw a conclusion on purity of obtained culture.
Table. Properties of obtained culture
Colony properties
Homogeneity of
growth
Morphological
properties
Morphological
homogeneity
Staining properties
Uniformity of
staining properties
Conclusion on
culture purity:

PART 2.
Maint task 2. Perform the Oxidative fermentative (OF) test. Studying
biochemical properties of pure culture of aerobic bacteria: conclusion on
ability to ferment glucose under anaerobic conditions, as well to produce
gases: sub-inoculating culture in Hugh and Leifson medium. According to
obtained results you can choose a relevant API tets for identification of
multiple biochemical traits of the microorganism. To obtain reliable results a
culture IS TO BE PURE.

THEORETICAL BACKGROUND: The oxidative-fermentative (OF) test was

developed by Hugh and Leifson in 1953. They developed OF media to differentiate between
oxidative bacteria (that produces acid from carbohydrates under aerobic condition only) and
fermentative bacteria (that produces acid both under aerobic and anaerobic conditions).
Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively.
The end products of fermentation are relatively strong mixed acids that can be detected in a
conventional fermentation test medium. However, the acids formed in oxidative degradation
of glucose are extremely weak and less, and the more sensitive oxidation fermentation
medium of Hugh and Leifson’s OF medium is required for the detection. The medium was
made by increasing the amount of glucose above that found in medium used to detect
fermentation and by decreasing the amount of peptone.
 The oxidative-fermentative test determines if microorganisms metabolize glucose by
fermentation or aerobic respiration (oxidatively). During the anaerobic process of
fermentation, pyruvate is converted to a variety of mixed acids depending on the type of
fermentation. The high concentration of acid produced during fermentation will turn the
indicator in OF media from to yellow in the presence or absence of oxygen.
Certain nonfermenting gram-negative bacteria metabolize glucose using aerobic
respiration and therefore only produce a small amount of weak acids during glycolysis and
Krebs cycle. The decrease amount of peptone and increase amount of glucose facilitates the
detection of weak acids thus produced. Dipotassium phosphate buffer is added to further
promote acid detection.
Uses:
OF Test is used to determine if gram-negative bacteria metabolize carbohydrates
oxidatively, by fermentation, or are nonsacchrolytic (have no ability to use the carbohydrate
in the media).
Media
Hugh and Leifson’s OF basal medium or Medium No. 6 (CONTAINS PHENOL RED).
Procedure
Inoculate two tubes of OF test medium with the test organism using a straight wire
by stabbing “half way to the bottom” of the tube.
Cover one tube of each pair with 1 cm layer of sterile mineral oil or liquid paraffin
(it creates anaerobic condition in the tube by preventing diffusion of oxygen), leaving the
other tube open to the air.
Incubate both tubes at 35oC for 48 hours (Slow growing bacteria may take 3 to 4
days before results can be observed)
Interpretation
Acid production is detected in the medium by the appearance of a yellow color. In
the case of oxidative organisms; color production may be first noted near the surface of the
medium.
1. Fermentative result: Acid production on both (open and covered) tubes. The acid
produced changes the pH indicator to yellow. e.g. Escherichia coli
2. Oxidative result: Acid production in the open tube (aerobic) and not the oil-covered
tube (anaerobic) indicates an oxidative result. Nonfermenting bacteria that metabolize
glucose via oxidative metabolism give an oxidative result.
3. Non saccharolytic (Negative OF result): Nonsacchrolytic bacteria give a negative
OF result. The negative result is indicated by no color change in the oil-covered tube and in
some cases an increase in pH (pH 7.6) changing to blue in the top of the open tube. The
increase in pH is due to amine production by bacteria that break down the peptone (protein)
in the medium.
PROCEDURE
Step 1. Take your tube with obtained pure culture or get a tube with a pure
culture on a slope agar from your Instructor if your culture is contaminated.
Step 2. Perform stab inoculation of bacteria from the tube with slant agar into
TWO tubes with Hugh and Leifson media: one open – for aerobic conditions
of growth, and second – covered with sterile mineral oil or liquid paraffin .

1. Place two tubes: one with a slant agar and culture, and one with Hugh
and Leifson media, the agar surface facing upward.
2. Flame the loop.
 3. Remove the caps of both tubes with the little finger of your loop hand
and hold it there.
4. Flame the open end of the tubes by passing it through a flame two or
three times.
5. Holding the loop hand still, move the tube up the wire until the wire tip
is over the desired growth. Touch the loop to the growth and obtain
the smallest visible mass of bacteria.
6. Then, holding the loop hand still, remove the tube from the wire.
7. Place a loop directly into a tube with Hugh and Leifson media.
8. Carefully move the tube over the wire, stab the inoculum down to the
bottom of the deep in a clean, straight stroke.
9. Then, holding the loop hand still, remove the tube from the wire.
10. Flame the tube mouths as before. Keep your loop hand still.
11. Keeping the loop hand still (remember, it has growth on it), move the
tubes to replace the caps.
12. Sterilize the loop as before by incinerating it in the Bunsen burner
flame.
13. Label the tube with your name, date, group number and write the
nature of the inoculated material and organism.
14. Repeat stages 1-13 for second tube.
15. Incubate in the 37 °C incubator for 18-24 hrs.

Step 3. (Performed at Class 8). Interpretation of the results. Check the

ability for anaerobic growth, ability to metabolize glucose in
oxidative or fermentative way, and ability to produce gas.
Open Sealed Interpretation
tube tube
Color
Acid
Gas

Step 4. (Performed at Class 8). Draw the conclusion on biochemical

properties of your bacterial culture, type of API test you should
choose for further identification (for fermenting or non-fermentic
bacteria) AND provide a conclusion on a species you have recovered
from the mix.
Conclusion:
Questions for self-control:
1. Define the term Taxonomy and explain the scope of its application.
2. Define the term Classification and explain the scope of its application.
3. Define the term Nomenclature and explain the scope of its application.
4. Define the term Identification and explain the scope of its application.
5. What are the main principles of Classification?
6. What are the main rules of Nomenclature ?
7. What are the difficulties for defining the term ‘species’ in bacteria?
8. What taxons are added if organism doesn’t fit precisely into main
categories?
9. What are the main rules of bacteria classification by Bergey’s Manuals?
10. Describe all steps for bacteria identification steps.
11. What biochemical reactions are used in identification of bacteria?
12. What is the principle of numerical taxonomy?
13. How bacteria can be identified on a basis of their DNA sequence?
14. How uncultured bacteria samples can be processed and bacteria in them
identified?
15. What is the main principle of OF test?

Literature to get prepared for CLASS 7:

 Cowan M.K, Bunn J. - Microbiology Fundamentals_ A Clinical Approach
(Part I) – pages 24-29.