CHAPTER 8

IDENTIFICATION OF
MICROORGANISMS
8.1 MORPHOLOGICAL OF BACTERIA
WHAT IS MORPHOLOGY ?
• A BRANCH OF BIOLOGY DEALING WITH THE
STUDY OF THE FORM AND STRUCTURE OF
ORGANISMS AND THEIR SPECIFIC STRUCTURAL
FEATURES.

• BACTERIAL MORPHOLOGY MEANS SYSTEMATIC

STUDY OF EXTERNAL CHARACTERS OF BACTERIA.
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 BACTERIAL MORPHOLOGY

• MORPHOLOGY OF BACTERIAL CELL DEALS WITH STUDY OF :

(I) SIZE OF BACTERIA.
(II) SHAPE OF BACTERIA.
(III) ARRANGEMENT OF BACTERIA.

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Size of bacteria

 Unit for measurement:

Micron or micrometer, µm
1 µm = 10-3 mm
 Size : Varies with kinds of bacteria
and also related to their age and
external environment

 Cocci: sphere, 1 µm 4

 Bacilli: rods, 0.5-1 µm in width

 Spiral bacteria: 1-3 µm in length and 0.3-0.6 µm in width
Insert figure 1.7
measurements

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 SHAPE OF BACTERIA
• BACILLUS (ROD-SHAPED)
- If the cells are rod or cylindrical in shape it is called bacilli.
COCCUS (SPHERICAL)
- If the cells are spherical or ball shape then the cells are called as cocci
shape bacteria.
• SPIRAL
- A bacteria which is twisted two or more time along the axis is called a spiral
form bacteria.
• Spirillum
• Vibrio
• Spirochete
 ARRANGEMENT OF BACTERIA
• Arrangement of cells is dependent on pattern of division and how cells
remain attached after division:
• Cocci:
• Singles
• Diplococci – in pairs
• Tetrads – groups of four
• Sarcinae – groups of eight
• Irregular clusters
• Chains

• Bacilli:
• Chains 7

• Palisades
 Cocci that remain in pairs after
dividing are called diplococci.

Cocci that remain in chains after

dividing are called streptococci.

Cocci that divide in two planes and

remain in groups of four are called
tetrads.

Cocci that divide in three planes

and remain in groups cube like
groups of eight are called sarcinae.

Cocci that divide in multiple planes

and form grape like clusters or
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sheets are called staphylococci.

 Most bacilli appear as single
rods. Diplobacilli appear in
pairs after division.

Streptobacilli appear in chains

after division.

Some bacilli are so short and fat

that they look like cocci and are
referred to as coccobacilli.
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Vibrios look like curved rods.

Spirilla have a helical shape and

fairly rigid bodies.

Spirochetes have a helical shape

and flexible bodies.
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8.2 USE OF STAINS IN CLASSIFICATION

The cell envelope

• External covering outside the cytoplasm
• Composed of two basic layers:
• Cell wall and cell membrane
• Maintains cell integrity
• Two generally different groups of bacteria demonstrated by
gram stain:
• Gram-positive bacteria: thick cell wall composed
primarily of peptidoglycan and cell membrane

• Gram-negative bacteria: outer cell membrane, thin

peptidoglycan layer, and cell membrane 13
Insert figure 4.12
Comparative cell envelopes

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 STRUCTURE OF CELL WALLS
• Determines cell shape, prevents lysis (bursting) or
collapsing due to changing osmotic pressures
• Peptidoglycan is primary component:
• Unique macromolecule composed of a repeating
framework of long glycan chains cross-linked by
short peptide fragments

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 GRAM-POSITIVE CELL WALL
• Thick, homogeneous sheath of
peptidoglycan
• 20-80 nm thick
• Includes teichoic acid and
lipoteichoic acid: function in cell
wall maintenance and enlargement
during cell division; move cations
across the cell envelope; stimulate a
specific immune response

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 GRAM-NEGATIVE CELL WALL
• Composed of an outer membrane and a thin
peptidoglycan layer
• Outer membrane is similar to cell membrane bilayer
structure
• Outermost layer contains lipopolysaccharides
and lipoproteins, LPS
• Endotoxin that may become toxic when
released during infections
• May function as receptors and blocking
immune response
• Contains porin proteins in upper layer –
regulate molecules entering and leaving cell
• Bottom layer composed of phospholipids and
lipoproteins
• Single, thin sheet of peptidoglycan
• Protective structure while providing some flexibility
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and sensitivity to lysis
• Periplasmic space surrounds peptidoglycan
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 THE GRAM STAIN
 Differential stain that distinguishes cells with a gram-
positive cell wall from those with a gram-negative cell
wall
◦ Gram-positive - retain crystal violet and stain purple
◦ Gram-negative - lose crystal violet and stain red from
safranin counterstain
 Important basis of bacterial classification and
identification
 Practical aid in diagnosing infection and guiding drug
treatment

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8.3 BIOCHEMICAL TESTS USED FOR
THE IDENTIFICATION OF BACTERIA
WHAT ARE BIOCHEMICAL TEST?

• Biochemical tests are the tests used for the identification of

bacteria species based on the differences in the biochemical
activities of different bacteria.
• Bacterial physiology differs from one species to the other.
Biochemical test can be used to differentiate even closely
related microorganisms.
• The microbe is cultured in a media with a special substrate
and tested for an end product. 22
Example of biochemical test used for identification of bacteria

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 8.3.1 SUGAR FERMENTATION
Sugar fermentation test
• Objective:
To determine the ability of microbes to ferment
carbohydrates with the production of acid and/or gas
• Principles:
Sugars are metabolized through different metabolic
pathways depending on types of microbial species
and aerobic or anaerobic environment.
If fermenting bacteria are grown in a culture media
containing the carbohydrate, they may produce organic
acids as by products of the fermentation.
These acids are released into the medium and so, lower pH of medium.
If a pH indicator such as “phenol red” or “bromocresol blue” is included
in the medium, the acid production will change the medium from its 24
original color to yellow.
GLUCOSE BROTH WITH DURHAM TUBES
• This is a differential medium. It tests an organism's ability to
ferment the sugar glucose as well as its ability to convert the end
product of glycolysis, pyruvic acid into gaseous by products.
• This is a test commonly used when trying to identify gram-negative
enteric bacteria, all of which are glucose fermenters but only some
of which produce gas.
• This medium also contains the ph indicator, phenol red. If an
organism is capable of fermenting the sugar glucose, then acidic
by products are formed and the pH indicator turns yellow.
• The end product of glycolysis is pyruvate. Organisms that are
capable of converting pyruvate to formic acid and formic acid to H2
(g) and CO2 (g), via the action of the enzyme formic hydrogen
lyase, emit gas.
• This gas is trapped in the durham tube and appears as a bubble
25 at

the top of the tube.

 GLUCOSE BROTH WITH DURHAM TUBES
Shigella Pseudomonas Proteus
dysenteriae aeruginosa mirabilis
Changes of color:
Proteus mirabilis (far right) and Shigella
dysenteriae (far left) are capable of fermenting
glucose. Pseudomonas aeruginosa (center) is a
nonfermenter.

Gas formation:
Proteus mirabilis (far right) is gas producer.
Notice that Shigella dysenteriae (far left) ferments
glucose but does not produce gas.

Note - broth tubes can be made containing sugars other

than glucose (e.G. Lactose and mannitol). Because the
same pH indicator (phenol red) is also used in these
fermentation tubes, the same results are considered
positive (e.g. A lactose broth tube that turns yellow after
incubation has been inoculated with an organism 26 that can
ferment lactose).
 MANNITOL SALT AGAR (MSA)
• The differential ingredient in MSA is the sugar mannitol.
• Organisms capable of using mannitol as a food source will
produce acidic by products of fermentation that will lower the
pH of the media.
• The acidity of the media will cause the pH indicator (phenol
red) to turn yellow.

Staphylococcus aureus is capable of

fermenting mannitol (left side of plate)
while Staphylococcus epidermidis is
not (right side of plate).
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 MACCONKEY AGAR
• The differential ingredient in MacConkey agar is the sugar
lactose.
• Organisms capable of using lactose as a food source will
produce acidic by products of fermentation that will lower the
ph of the media.
• The acidity of the media will cause the ph indicator (neutral
red) to turn a bright pinky colour.

Thus organisms capable of lactose

fermentation such as Escherichia coli,
form bright pinky-red colonies (left side of
plate). While Salmonella is not (right side
of plate).
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Notes: MacConkey agar is commonly used to differentiate between the Enterobacteriaceae.

 METHYL RED TEST (MR TEST)
Objective: To differentiate E. coli and E.
aerogen and to determine the ability of
microbes to oxidize glucose with production
and stabilization of high content of acid and
product.

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 8.3.2 PROTEOLYSIS
TESTS FOR PROTEOLYTICS ACTIVITIES
• Objective:
To determine the ability of microbes to performed proteolysis
process.

• Principles:
Proteolysis is a process in which a protein is broken down
partially, into peptides or completely into amino acids by
proteolytic enzymes, present in bacteria and in plants but most
abundant in animals.
For biochemical characterization of proteolytic strain, tests like
indole production test, nitrate reduction test, citrate utilization test
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& urease test were performed as standard protocols.
 INDOLE PRODUCTION TEST
Objective: To determine the ability of microbe to degrade the
amino acid tryptophan

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Interpretation:
If urea is present in the medium,
then it will be degraded which
creates alkaline condition in the
medium which result in colour
change from reddish pink to deep
pink.

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6.3.3 HYDROLYSIS
STARCH TEST
• Differentiates bacteria based on their ability to hydrolyze
starch with the extra cellular enzyme amylase.
• In order to interpret the results of the starch hydrolysis
test, iodine must be added to the agar. The iodine reacts
with the starch to form a dark brown color.
• Thus, hydrolysis of the starch will create a clear zone
around the bacterial growth.

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Starch test procedure:
1) Inoculate a starch plate with the organism to be tested.
2) Incubate at optimum temperature for at least 48 hours.
3) Flood plate with iodine, observed results.
4) Blue color indicates no hydrolysis, while a clear zone
indicates hydrolysis.

Bacillus subtilis is positive for starch

hydrolysis (on the left). The organism
shown on the right is negative for 35

starch hydrolysis.
 HYDROLYSIS OF CASEIN
• Casein (a protein) is broken down
by protease into peptones and
amino acids.
• During the degradation process,
polypeptide bonds are broken.
Once the bonds are broken, amino
acids are produced.
• Inoculate a milk agar plate by
streaking once across the surface &
incubate at the optimum growth
temperature.
• A clear zone surrounding streak 36

line of agar indicates a (+) result.

• Organism that gives (+) result is Bacillus subtilis,
Clostridium spieces. (-) Results are indicative that
organism does not cause a clear zone. An example of
organism displaying (-) result is Escherichia coli.

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 HYDROLYSIS OF CASEIN
• Properties of skim milk agar:
- skim milk agar contents- casein, lactose and other
nutrients, which support growth of lactobacilli.
- Gives the white colour to milk.

• As seen from the above, we can infer if bacteria present in

milk, it is able to break down the casein in milk. Milk may
have abnormal particles that are not white in colour.
• Application of this biochemical test is in food testing.

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 HYDROLYSIS OF GELATIN
• Another protein commonly found in food products
is gelatin.
• It is broken down by gelatinase into smaller
polypeptides, peptones and amino acids that can
cross the cell membrane and be utilised by the
organism.
• Stab inoculate a tube of nutrient gelatin and
incubate at 20-250c for up to 30 days.
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• Property of gelatin agar:
- when gelatin is broken
down via hydrolysis, it
cannot solidify anymore,
the areas of solid gelatin
media where the
organism grows, will turn
into liquid. Even if you
refrigerate this medium,
the media will remains
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liquid.
6.4 DESCRIPTION OF SOME MICROORGANISMS
MOLDS & YEASTS, VIRUSES, ALGAE

MOLDS & YEASTS

 Macroscopic
Mushrooms
Fungi
KINGDOM
FUNGI
Microscopic Molds,
Fungi Yeasts

• 100,000 species
• The fungi are a group of eukaryotic microorganisms. Most are
multicellular (i.e. Mushrooms, molds) and some are unicellular
(i.e. Yeast)
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 MICROSCOPIC FUNGI
• Exist in two morphologies:
• Yeast – round ovoid shape, asexual reproduction
• Hyphae – long filamentous fungi or molds
• Some exist in either form – dimorphic – characteristic of
some pathogenic molds

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Dimorphic fungi are fungi which are able to grow in two
different forms.
- As molds at room temperature
- As yeast on incubation at 370C @ at body temperature

Examples: Histoplasma capsulatum

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Understanding the structure of Fungus

- Simplest fungus: Unicellular budding

yeast
- Hypha : is a long, branching
filamentous structure of a fungus.
Hyphae may be septate @ non-septate
(coencytic hyphae)
- Mycelium : tangled mass of hyphae is
called mycelium.

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 MOLD VS YEAST
MOLD YEAST

Definition a type of fungi that grows in multicellular A type of fungi that contains
filaments called hyphae. These tubular branches only a single cell.
have multiple, genetically identical nuclei, yet
form a single organism, known as a colony
Uses Some molds are used in food production, for Ethanol production, baking.
example, Penicillium is used in the production of
cheese, Neurospora in the production of oncom,
which is made from the by-product of tofu.
Health Can cause allergic reactions and respiratory Can cause infection in
Hazards problems individuals with compromised
immune systems.
Habitat Typically found in damp, dark or steam-filled Very common. Can be found on
areas. fruit and berries, in the
stomachs of mammals and on
skin, among other places.
Reproduction Reproduce through small spores, which can be Most reproduce asexually
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either sexual or asexual. through mitosis. Most common

form called “budding.”
 FUNGAL NUTRITION

• All are heterotrophic

• Majority are harmless saprobes living off dead
plants and animals
• Some are parasites, living on the tissues of
other organisms, but none are obligate;
mycoses – fungal infections
• Growth temperature 20o-40oc
• Extremely widespread distribution in many
habitats
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 FUNGAL ORGANIZATION
• Most grow in loose associations or colonies
• Yeast – soft, uniform texture and appearance
• Filamentous fungi – mass of hyphae called mycelium;
cottony, hairy, or velvety texture
• Hyphae may be divided by cross walls – septate
• Vegetative hyphae – digest and absorb nutrients
• Reproductive hyphae – produce spores for
reproduction

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 FUNGAL IDENTIFICATION

• Mycology is the branch of biology

concerned with the study of fungi.
• Isolation on specific media
• Macroscopic and microscopic observation
of:
• Asexual spore-forming structures and
spores
• Hyphal type
• Colony texture and pigmentation
• Physiological characteristics
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 ROLES OF FUNGI
• Adverse impact
• Mycoses (tinea versicolor), allergies, toxin
production
• Destruction of crops and food storages
• Beneficial impact
• Decomposers of dead plants and animals
• Sources of antibiotics, alcohol, organic acids,
vitamins
• Used in making foods and in genetic studies

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6.4.2 VIRUSES
WHAT IS VIRUS?
• A virus is a small infectious agent that replicates only
inside the living cells of other organisms. Viruses can
infect all types of life forms, from animals and plants to
microorganisms, including bacteria and archaea.

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• Size range
Most <0.2 μm;
requires electron
microscope

• Virion – fully
formed virus (the
nucleic acid
surrounded by a
protein coats)
able to establish
an infection

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 GENERAL STRUCTURE OF VIRUSES
Capsids
• All viruses have capsids - protein coats that enclose and protect their
nucleic acid.
• Each capsid is constructed from identical subunits called capsomers
made of protein.
• The capsid together with the nucleic acid are nucleoscapsid.
• Some viruses have an external covering called envelope; those lacking an
envelope are naked

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 GENERAL STRUCTURE OF VIRUSES
Viral envelope
• Mostly animal viruses
• Acquired when the virus leaves the host
cell
• Exposed proteins on the outside of the
envelope, called spikes, essential for
attachment of the virus to the host cell
Functions of capsid/envelope
• Protects the nucleic acid when the virion is outside the
host cell
• Helps to bind the virion to a cell surface and assists
the penetration of the viral DNA or RNA into a suitable
host cell 60
NUCLEIC ACIDS
• Viral genome – either DNA or RNA but
never both
• Carries genes necessary to invade host
cell and redirect cell’s activity to make new
viruses
• Number of genes varies for each type of
virus – few to hundreds

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 GENERAL STRUCTURE OF VIRUSES

• Complex viruses: atypical viruses

• Poxviruses lack a typical capsid
and are covered by a dense layer
of lipoproteins.

• Some bacteriophages have a

polyhedral nucleocapsid along
with a helical tail and attachment
fibers.
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POXVIRUSES
-The largest and most complex
viruses.

- Oval or "brick-shaped" particles

200-400nm long.

-could infect both vertebrate and

invertebrate animals.

-Virions are complex, which consist

of:
* Core
* Lateral bodies
* Outer membrane
* Envelope
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 VIRAL REPLICATION
• A virus cannot replicate on its own :
• It must attach to and enter a host cell
• It then uses the host cell’s energy to synthesize protein,
DNA, and RNA

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 6.4.3 ALGAE
KINGDOM PROTISTA
• Algae (Photosynthetic organisms)
• Protozoa

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 ALGAE
 Photosynthetic organisms
 Kelps, seaweeds, euglenoids, green algae, diatoms,
dinoflagellates, brown algae, and red seaweeds
 Microscopic forms are unicellular, colonial, filamentous.
 Macroscopic forms are colonial and multicellular.
 Contain chloroplasts with chlorophyll and other
pigments
 Cell wall
 May or may not have flagella

70
 ALGAE
 Most are free-living in fresh and marine water –
plankton.
 Provide basis of food web in most aquatic habitats
 Produce large proportion of atmospheric O2
 Dinoflagellates can cause red tides and give off
toxins that cause food poisoning with neurological
symptoms.
 Classified according to types of pigments and cell
wall
 Used for cosmetics, food, and medical products
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 Major Groups Of Algae

Algae Group Common Name Morphology Cell Wall Major habitat

Chlorophyta Green Algae Unicellular & Cellulose Freshwater, soils,

Leafy a few marine

Euglenophyta Euglenoids Unicellular No wall present Freshwater, a few

flagellated marine
Dinoflagellata Dinoflagellata Unicellular Cellulose Mainly marine
flagellated
Chrysophyta Golden Brown Unicellular Two over-lapping Freshwater,
Algae components made marine, soil
of silica
Phaeophyta Brown Algae Filamentous to Cellulose Marine
leafy,
occasionally
massive and
plantlike
Rhodophyta Red Algae Unicellular, Cellulose Marine
filamentous to
leafy
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 PROTOZOA

 65,000 species
 Vary in shape, lack a cell wall
 Most are unicellular; colonies are rare
 Most are harmless, free-living in a moist habitat
 Some are animal parasites and can be spread by
insect vectors
 All are heterotrophic
 Feed by engulfing other microbes and organic matter
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PROTOZOA CLASSIFICATION

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STRUCTURE OF PROTOZOA

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Euglena
Toxoplasma gondii
Plasmodium falciparum
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