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CLINICAL BACTERIOLOGY (LECTURE) MODULE 3: IDENTIFICATION OF BACTERIA

METHODS OF STUDYING BACTERIA Some of the first steps in identifying a bacterium include examination
The correct identification of microorganisms is important to the of:
Medical Profession and other allied and applied branches of 1. The shape of the individual bacterium
Microbiology. Over the past century, microbiologists have searched 2. Whether the bacteria exist in specific groupings
for more rapid and efficient means of microbial identification. The 3. The colony morphology (the appearance of a “colony”; a
identification and differentiation of microorganisms has principally group of millions of bacteria that arose from one single parent
relied on microbial morphology and growth variables. Advances in cell).
molecular biology over the past 10 years have opened new avenues
for microbial identification and characterization.

Microscopic
Size, shape, and staining characteristics such as Gram stain
can give suggestive information as to the identification of an
organism. Further testing is needed to confirm the organism. Several
special stains are also used to identify unique parts of a bacterial cell
which is used as a presumptive identification of some bacterial
species (ex. Acid Fast Stain for identifying Mycobacterium
tuberculosis).
The size and shape of microorganisms can be readily
determined by microscopic examination of the wet mount. On the
basis of size and shape, one can readily decide whether the organism
in question is a prokaryote, fungus, or protozoan. In a clinical
laboratory, it can sometimes provide all information needed for
diagnosis of certain infections. For example, a wet mount of vaginal
secretion is routinely used to diagnose infections caused by yeast or
by protozoans (Trichomonas). In a clinical laboratory, the gram stain of a specimen by itself is
generally not sensitive and specific enough to diagnose the cause of
most infection, but it is still a useful tool. The clinician can see the
Gram reaction, the shape, and the arrangement of the bacteria and
whether the organism appears to be growing as a pure culture or with
other bacteria or cell of the host. However, most medically important
bacteria do not have distinctive shapes or staining characteristics and
usually cannot be identified by Gram stain alone.
 For example, Streptococcus pyogenes, which causes strep
(Wet mount - yeast cells) (Wet mount -Trichomonas) throat, cannot be distinguished microscopically from other
streptococci that are part of the normal flora of the throat.

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 A Gram stain of a stool specimen cannot distinguish secretion from a male is considered diagnostic for
Salmonella species from E. coli. These organisms must be gonorrhea, the sexually transmitted disease by Neisseria
generally isolated in pure cultures and tested for the gonorrhoeae.
biochemical attributes to provide precise identification.  This diagnosis can be made because this microorganism
is the only gram-negative diplococcus found inhabiting
the normal sterile urethra of a male.

Note:
 Gram-stain:
- most widely used stain in the laboratory
- divides organism into:
(Gram stain of Escherichia coli)  Gram-positive: stains purple to blue
 Gram-negative: stains pink to dark red
In certain cases, the Gram stain gives enough information to  Presumptive Identification: presumes the identification of
start appropriate antimicrobial therapy while awaiting more accurate bacteria based on morphology (structures, stains)
information.  Acid-fast stain: used in mycobacterium tuberculosis, etc.
 For example, a urine sample from an otherwise healthy
woman reveals more than the permissible level of Cultural & Biochemical Characteristics
negative rods per oil-immersion field, the clinician will CULTURE
suspect a urinary tract infection caused by E. coli, the The identification of most prokaryotes relies on analyzing their
common cause of such infection. metabolic capabilities such as the types of sugars utilized or the end
 Likewise, a Gram stain of sputum showing numerous products produced. In some cases, these characteristics are revealed
white blood cells and gram-positive encapsulated by the growth and colony morphology on the cultivation media, but
diplococci is highly suggestive of Streptococcus most often they are demonstrated using biochemical tests.
pneumoniae, an organism that causes pneumonia.
Microorganisms can be grown in a pure culture. They are the
easiest to identify, because it is possible to obtain high number of a
single type of microorganisms.

Even the colony morphology can give initial clues to the

identity of the organisms.

 For example, colonies of the Streptococci are generally fairly

(Gram stain of Streptococcus pneumoniae) small relative to many other bacteria such as Staphylococci.
 Colonies of Serratia marcescens are often red when incubated
In certain cases, the result of a Gram stain is enough to
at 22 ◦ C owing to the production of pigments.
accurately diagnose the infection.  Pseudomonas aeruginosa – often produces a soluble pigment,
 For example, the presence of gram-negative diplococci
which discolors the growth medium. They also have distinct
clustered in the white blood cells in a sample of urethral
fruity odor.
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common causative organism of urinary tract

infections, forms characteristic pink colonies on
MacConkey agar because of its ability to ferment
lactose.
 Other bacteria can also grow and ferment lactose on
this medium; however, colony appearance alone is
not capable enough to conclusively identify E. coli.

(BAP = Strep on the left, Staph on the right) (Serratia marcescens)

(MacConkey Agar)
The use of selective and differential media in the isolation
process can provide additional information that helps to identify an
organism.
 For example, if a soil sample is plated onto a medium that
lacks a nitrogen source and is then incubated aerobically, any
resulting colonies are likely members of the genus
Azitobacter.
 The ability to fix nitrogen under aerobic condition is an
identifying characteristic of these bacteria.

In clinical laboratories, where rapid but accurate diagnosis is

essential, specimens are plated onto media, specially designed to
provide important clues as to identify the disease-causing organism.
 For example, a specimen taken by swabbing the throat
of a patient, complaining of a sore throat, can be
inoculated onto blood agar, a nutritionally rich medium
containing red blood cells.
 This differential medium enables to detect the
characteristic β-hemolytic colonies of Streptococcus  Margin: sides of colony
pyogenes.  Elevation: horizontal view/side view
 Urine sample collected from a patient suspected of  Size: measured using ruler
having a urinary tract infection is plated onto MacConkey  Texture: not touched
agar, which is both selective and differential.  Appearance: same with texture
 MacConkey agar – has bile salts, which inhibit the  Pigmentation: color of colony
growth of most non-intestinal organisms, and lactose o Diffusible: color of colony diffuses with the agar
along with a pH indicator, which differentiates o Non-diffusible: does not diffuse with the agar
lactose fermenting organisms. E. coli, the most

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 Optical property: placed on a printed paper with light source

on top

BIOCHEMICAL CHARACTERISTICS
Biochemical test-based identification systems are familiar
to most microbiologists and require little training to operate. Systems
range from strip cards for specific groups of bacteria (e.g.,
coryneform, bacillus, and enterics) to large plate arrays that may be
automatically scanned for changes due to pH shifts or redox
reactions.

Note:
Biochemical Tests: laboratory tests for bacteria are only until these
tests For example, some species of Streptococcus contain a unique
Microbiology Laboratory: microscopic, culture & biochemical tests carbohydrate molecule as part of their cell wall, which can be used to
are only done to identify pathogens. distinguish them from other species. These carbohydrates, as well as
Molecular diagnostics laboratory: is a separate laboratory and is any distinct protein or polysaccharide, can be detected using
usually used for those microorganisms who are difficult to grow like techniques that rely on the specificity of interaction between
viruses or for research purposes. antibodies and antigens. Methods that exploit such interactions are
called serology.

Note:
Done in the Immunoserology section of the laboratory
Antigen: bacterial antigen
Antibody: produced by the body specific to that antigen; forms
agglutination if positive or you have encountered the bacteria already
Sample: blood (serum)
Principle:
1. Antigen is an identifying marker of the bacteria which is
attached to the polysaccharide. It is an indirect identification of
(IMVC is an example of a biochemical test)
bacteria, because it is specific to that particular bacteria.
2. Immunologic reaction (agglutination or precipitation rxn)
Serologic Methods
occurs when antigen, in the presence of bacteria and antibody,
The protein and polysaccharides that make up a bacterium are produced by the host reacts with each other.
sometimes characteristic enough to be considered identifying
markers. The most useful of these are the molecules that make up
There are a lot of serologic methods like ELISA, Agglutination
surface structures including the cell wall, glycocalyx, flagella, and pili. reactions, Precipitation reactions, Lateral floe immunoassays, Radio-
immunoassays and a lot more. But still the main principle is the
detection of either the antigen or antibody to a specific microorganism.
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The application of molecular technology in medicine is almost

endless, some of the applications of molecular methods are:
1. Classification of organism based on genetic relatedness
(genotyping)
2. Identification and confirmation of isolate obtained from culture
3. Early detection of pathogens in clinical specimen
4. Rapid detection of antibiotic resistance
5. Detection of mutations 6. Differentiation of toxigenic from non-
toxigenic strains
6. Detection of microorganisms that lose viability during
transport, impossible, dangerous and costly to culture, grow
Animal Inoculation
slowly or present in extremely small numbers in clinical
There are some bacteria that cannot be cultured using artificial
specimen.
means. Examples include:
7. Apart from their role in microbiology, these techniques can
• Rickettsia spp. – requires living cells
also be used in identifying abnormalities in human and
• Chlamydia spp. – grows in McCoy cells, HeLa 229, Buffalo
forensic medicine.
Green Monkey Kidney Cells and
Cycloheximide-treated McCoy Cells
MOLECULAR METHODS ARE BROADLY CLASSIFIED AS:
• Treponema pallidum – can be maintained in the laboratory in
 Hybridization methods – better for identification, not as
testicular chancre of rabbits
sensitive as amplification methods
• Mycobacterium leprae
 Amplification methods – improve the sensitivity due to
 Footpad of mice – success is in the low temperature (30
amplification step.
degrees celcius of footpads)
 Sequencing and enzymatic digestion of nucleic acids
 Armadillo – animals with low body temperature
1. Nucleic acid hybridization methods
Most of these bacteria cannot also grow outside of a living host,
Hybridization methods are based on the ability of two nucleic
therefore animal inoculation is required.
acid strands that have complementary base sequence (i.e. are
 Ethical Considerations: Animal testing will require separate
homologous) to specifically bond with each other and form a double-
permits from different agencies in the government like Bureau of
stranded molecule (hybrid).
animals. We also have to follow the animal welfare act in ethical
Since the hybridization requires sequence homology, a
use of animals for laboratory use.
positive hybridization reaction between two nucleic acid strands each
derived from different source indicate genetic relatedness between
Molecular Techniques
the two organisms.
During the past two decades, use of molecular biology in the
Hybridization assays require that one nucleic acid strand is
clinical microbiology laboratory has steadily increased. Advances in
from the known organism while the other is derived from the organism
qualitative, quantitative, and typing methods have resulted in
to be identified or detected. The result of hybridization is expressed as
improved patient care through rapid detection of pathogens or
percent hybridization/ percent similarity/ percent relatedness.
provision of more accurate information than afforded by conventional
methods.
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sequence but also a region capable of hybridizing with

multiple smaller target independent reporter probes.

2. Amplification Techniques
The beginning of molecular diagnostics was initiated at the
end of the eighties with the development of the PCR (Polymerase
Chain Reaction). Amplification methods improve the sensitivity due
to amplification step. These are divided into three categories- target  Sequencing
amplification, signal amplification, and probe amplification. This method involves the determination of nucleotide
 Target amplification sequence in the given DNA molecule.
These systems amplify the target to large numbers. There are two popular methods for sequencing DNA;
Some of these systems are:  Chemical Cleavage Method and
 PCR – thermocycler is required  Chain Terminator Method.
 Isothermal amplification methods: there is no need Both these methods have now been automated and
for thermocycler. Commonly used methods are: the sequence can be read using a computer. Since it is time
 NASBA (Nucleic acid sequence-based amplification) consuming process, it does not much role in diagnostic
 TMA (Transcription mediated amplification) microbiology. This technique can be used to study the
 SDA (Strand displacement amplification) structure of gene, detect mutations, compare genetic
 LAMP (Loop mediated isothermal amplification) relatedness and to design oligonucleotide primers. Many of the
modern molecular tools are based on 16S ribosomal DNA
 Signal Amplification sequence.
These techniques are used to increase the sensitivity
of the probe-based assays. They amplify the signal generated
by the labeled probes. By these procedures, a minimum 103 -
105 nucleic acid targets can be detected.
The simplest signal amplification technique utilizes
multiple reporter molecules attached to a single probe.
Another approach is to use several probes directed
against different regions in the target. This results in multiple
hybridization resulting in greater signal production.
Another system includes an unlabeled target binding
probe that contains not only region complementary to target

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