Classification Of Bacteria

Classification of Bacteria
There are different criteria used to classify bacteria which are as follows:

Classification on the basis of Gram Stain and Bacterial Cell Wall

• Gram-positive
Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain,
crystal violet.

• Gram-negative
Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on
addition of ethanol. They are stained pink or red by the counterstain
Classification of Bacteria on the Basis of Shape
There are 4 major types depending on their shapes are as follows:

1. Cocci: These types of bacteria are unicellular, spherical or elliptical shape. Either they may
remain as a single cell or may aggregate together for various configurations.

2. Bacilli: – These are rod shaped or cylindrical bacteria which either remain singly or in pairs.
Example: –Bacillus cereus.

3. Vibrio: – The vibrio are the curved, comma shaped bacteria and represented by a single genus.
Example: – Vibrio cholera.

4. Spirilla: – These type of bacteria are spiral or spring like with multiple curvature and terminal
flagella. Example: –Spirillum volutans.
Classification of Bacteria on the Basis of Mode of Nutrition
 Phototrophs:
•Those bacteria which gain energy from [Link]. Chromatium okenii.
 Chemotrophs:
•Those bacteria gain energy from chemical compounds.
•They cannot carry out photosynthesis. E.g.. Nitrosomonas.
 Autotrophs:
•Those bacteria which uses carbon dioxide as sole source of carbon to prepare its own food.
 Heterotrophs:
Those bacteria which uses organic compound as carbon source.
They lack the ability to fix CO2.
Most of the human pathogenic bacteria are heterotopic in nature.
Some heterotrophs are simple, because they have simple nutritional requirement. However there are some
bacteria that require special nutrients for their growth; known as fastidious heterotrophs.
Classification of Bacteria on the Basis of Temperature Requirement
Bacteria can be classified into the following major types on the basis of their temperatures response as
indicated below:

[Link]:
•Bacteria that can grow at 0°C or below but the optimum temperature of growth is 15 °C or below and
maximum temperature is 20°C are called psychrophiles
•Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid nature to the
cell membrane even at lower temperature.
•Examples: vibrio marinus, Polaromonas vaculata, Psychroflexus.

[Link]:
•Those bacteria that can grow best between (25-40)o C but optimum temperature for growth is 37C
•Most of the human pathogens are mesophilic in nature.
•Examples: E. coli, Salmonella, Klebsiella, Staphylococci.
[Link]:
•Those bacteria that can best grow above 45C.
•Thermophiles capable of growing in mesophilic range are called facultative thermophiles.
•Thermophiles contains saturated fatty acids in their cell membrane so their cell membrane does not become
too fluid even at higher temperature.
•Examples: Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus.

4. Hyperthermophiles:
Those bacteria that have optimum temperature of growth above 80C.
Mostly Archeobacteria are hyperthermophiles.
Monolayer cell membrane of Archeobacteria is more resistant to heat and they adopt to grow in higher
temperature.
Classification of Bacteria on the Basis of Oxygen Requirement

 Obligate Aerobes:
•Require oxygen to live.
•Example: Pseudomonas, common nosocomial pathogen.

 Facultative Anaerobes:
•Can use oxygen, but can grow in its absence.
•They have complex set of enzymes.
•Examples: E. coli, Staphylococcus, yeasts, and many intestinal bacteria.

 Obligate Anaerobes:
•Cannot use oxygen and are harmed by the presence of toxic forms of oxygen.
•Examples: Clostridium bacteria that cause tetanus and botulism.
 Aero tolerant Anaerobes:
•Cannot use oxygen, but tolerate its presence.
•Can break down toxic forms of oxygen.
•Example: Lactobacillus carries out fermentation regardless of oxygen presence.

 Microaerophilic:
•Require oxygen, but at low concentrations.
•Sensitive to toxic forms of oxygen.
•Example: Campylobacter.
Classification of Bacteria on the Basis of pH of Growth
[Link]:
•These bacteria grow best at an acidic pH.
•The cytoplasm of these bacteria are acidic in nature.
•Some Acidophiles are thermophilic in nature, such bacteria are called Thermoacidophiles.
•Examples: Thiobacillus thioxidans, Thiobacillus, ferroxidans, Thermoplasma, Sulfolobus

[Link]:
•These bacteria grow best at an alkaline pH.
•Example: Vibrio cholerae optimum pH. of growth is 8.2.

[Link]:
•These bacteria grow best at neutral pH (6.5-7.5).
•Most of the bacteria grow at neutral pH.
•Example: E. coli
Classification of Bacteria on the Basis of Osmotic Pressure Requirement
 Halophiles:
• Require moderate to large salt concentrations.
• Cell membrane of halophilic bacteria is made up of glycoprotein with high content of negatively
charged glutamic acid and aspartic acids. So high concentration of Na+ ion concentration is required
to shield the –ve charge.
• Ocean water contains 3.5% salt. Most such bacteria are present in the oceans.
• Archeobacteria, Halobacterium, Halococcus.

 Extreme or Obligate Halophiles:

• Require a very high salt concentrations (20 to 30%).
• Bacteria in Dead Sea.

 Facultative Halophiles:
• Do not require high salt concentrations for growth, but tolerate upto 2% salt or more.
Classification of Bacteria on the Basis of Number of Flagella
On the basis of flagella the bacteria can be classified as:
[Link]: – These bacteria has no flagella. Example: Corynebacterium diptherae.

[Link]: – One flagellum is attached to one end of the bacteria cell. Example: – Vibrio
cholerae.

[Link]: – Bunch of flagella is attached to one end of the bacteria cell.

Example: Pseudomonas.

[Link]: – Bunch of flagella arising from both end of the bacteria cell.
Example: Rhodospirillum rubrum.

[Link] : – The flagella are evenly distributed surrounding the entire bacterial cell.
Example: Bacillus.
Classification of Bacteria on the basis of Spore Formation

[Link] forming bacteria:

•Those bacteria that produce spore during unfavorable condition.
•These are further divided into two groups:

i) Endospore forming bacteria: Spore is produced within the bacterial cell.

Examples. Bacillus, Clostridium, Sporosarcina etc.
ii) Exospore forming bacteria: Spore is produced outside the cell.
Example. Methylosinus

[Link] sporing bacteria:

•Those bacteria which do not produce spores.
E.g.. E. coli, Salmonella.
CULTIVATION OF BACTERIA
CULTIVATION OF BACTERIA
A microbial culture/cultivation is a method of multiplying microorganisms by letting them reproduce
in predetermined culture media under controlled laboratory conditions.
Microbial cultures are used to determine the type of organism, its abundance in sample being tested
or both.
Culture media is an artificial media that provide the nutrients required for bacterial growth.
Uses: . Isolation and identification of micro-organisms
. Performing anti-microbial sensitivity tests
Common ingredients of culture media
. Peptone
. Meat extract
. Yeast extract
. Mineral salts
. Carbohydrates
. Agar
. Water
Peptone: Hydrolyzed product of animal and plant proteins: Free amino acids, peptides and
proteoses(large sized peptides).
It provides nitrogen; as well carbohydrates, nucleic acid fractions, minerals and vitamins.
Meat extract: supply amino acids, vitamins and mineral salts.
Yeast extract: It is bacterial growth stimulants.

Mineral salts: these are:

Sulfates as a source of sulfur.
. Phosphates as a source of phosphorus.
. Sodium chloride
. Other elements

Carbohydrates: Simple and complex sugars are a source of carbon and energy.
.Assist in the differentiation of bacteria.
Eg. Sucrose in TCBS agar differentiates vibro species.
Lactose in MacConkey agar differentiates enterobacteria.
Agar: It is an inert polysaccharide of seaweed. It is not metabolized by micro-organism.
Property
• It has . high gelling strength
• high melting temperature(90-95 o c)
• low gelling temperature
• It forms firm gel at 1.5% W/V concentration.
• It forms semisolid gel at 0.4-0.5% W/V concentration.
Uses: . Solidify culture media
. May provide calcium and organic ions to inoculated bacteria.

Water Deionized or distilled water must be used in the preparation of culture media.
Types of culture media based on functional use or application
1. Basic media
2. Enriched media
3. Selective media
4. Differential media
5. Transport media
6. Enrichment media
7. Anaerobic media
1. Basic /Simple / All purpose media
It is a media that supports the growth of micro-organisms that do not require special nutrients.
Uses :
. To prepare enriched media
. To maintain stock cultures of control bacterial strains
. To subculture pathogenic bacteria from selective/differential medium prior to performing biochemical or
serological tests.
Eg. Nutrient Broth , Nutrient Agar
2. Enriched media
Media that are enriched with whole blood, lyzed blood, serum, special extracts or vitamins to
support the growth of pathogenic bacteria.
Example
• Blood Agar
• Chocolate Agar

3. Enrichment media
Fluid media that increases the numbers of a pathogen by containing
enrichments and/or substances that discourage the multiplication of
unwanted bacteria.
Example
• Selenite F broth media
• Alkaline peptone water
4. Selective media
Media which contain substances ( E.g.. Antibiotics) that prevent or slow down the growth of
bacteria other than pathogens for which the media are intended.
Example
• Modified Thayer –Martin Agar
• Salmonella-Shigella( SS) agar
5. Transport media
Media containing ingredients to prevent the overgrowth of commensals and ensure the survival of
pathogenic bacteria when specimens can not be cultured soon after collection.
Example
• Amies transport media
• Stuart media
• Kelly-Blair media
Differential media
Media to which indicator substances are added to differentiate bacteria.
Example
• TCBS Agar differentiates sucrose fermenting yellow colonies of Vibrio cholera to non-sucrose
fermenting blue colonies other Vibrio species.
Most differential media distinguish between bacteria by an indicator which changes color when acid is
produced following carbohydrate fermentation.
Anaerobic media
Anaerobic bacteria need special media for growth
because they need low oxygen content, reduced
oxidation-reduction potential, and extra nutrients.
Media for anaerobes may have to be supplemented
with nutrients like hemin, and vitamin K. Such media
may also have to be reduced by physical or chemical
means. Boiling the medium serves to expel any
dissolved oxygen. Adding 1% glucose, 0.1%
thioglycollate, 0.1% ascorbic acid, 0.05% cysteine, or red
hot iron filings can reduce the medium. Before using,
the medium must be boiled in a water bath to expel any
dissolved oxygen and then sealed with sterile liquid
paraffin.
Example
• Robertson Cooked Meat (RCM)
Classification of culture media based on consistency
1. solid culture media
2. semisolid culture media
3. Fluid culture media
1. Solid culture media
. Plate cultures in petri dishes
. stab/slope cultures in tubes and bottles
Uses: Description of bacterial colonies
• size : diameter in mm
• Out line : circular, entire, wavy, indented
• Elevation: flat, raised, low convex and dome shaped.
• Transparency: transparent, opaque, and translucent.
• Surface: smooth (mucoid) and shiny, rough and dull.
• Color: colorless, white, pink, and pigmented
• changes in medium Eg. Hemolysis in Blood Agar Blackening of medium due to hydrogen sulfide
production.
2. Semisolid culture media
Uses:
• as an enrichment media
• as motility media

3. Fluid culture media

Bacterial growth in fluid media is shown by a turbidity in the medium.
Uses :
• as an enrichment media
• as biochemical testing media
• as blood culture media
Choice of culture media
The selection of culture media will depend on:
1. The major pathogens to be isolated, their growth requirements and the features by which
they are recognized.
2. Whether the specimens being cultured are from sterile sites or from sites having normal
microbial flora.
3. The cost, availability and stability of media.
4. The training and experience of laboratory staff in preparing, using and controlling culture
media.
Isolation and purification
of
Microorganisms
Isolation of Microorganisms:
Microorganisms occur in natural environment like soil. They are mixed with several other forms of life.
Many microbes are pathogenic. They cause a number of diseases with a variety of symptoms,
depending on how they interact with the patient. The isolation and growth of suspected microbe in
pure culture is essential for the identification and control the infectious agent.

The primary culture from natural source will normally be a mixed culture containing microbes of
different kinds. But in laboratory, the various species may be isolated from one another. A culture
which contains just one species of microorganism is called a pure culture.

The process of obtaining a pure culture by separating one species of microbe from a mixture of other
species, is known as isolation of the organisms.
Methods of Isolation and purification
There are special techniques employed to obtain pure cultures of microorganisms. In few cases it is possible to
secure pure culture by direct isolation or direct transfer. This can be done only in those situations in which pure
culture occurs naturally.

Following isolation methods are employed to isolate microbes from mixed cultures:

1. Streaking

2. Plating

3. Dilution

4. Enriched procedure, and

5. Single cell technique.

. Streaking:
This is most widely used method of isolation. The technique consists of pouring a suitable
sterile medium into sterile petri plate and allowing the medium to solidify. By means of a
sterile loop or straight needle or a sterile bent glass-rod a small amount of growth preferably
from a broth culture or bacterial suspension is streaked back and forth across the surface of
agar until about one third of the diameter of the plate has been covered.
The needle is then flamed and streaking is done at right angles to and across the first streak.
This serves to drag bacteria out in a long line from the initial streak. When this streaking is
completed the needle is again flamed and streaking is done at right angles to the second
streak and parallel to the first.
Inoculating wire loop
Plating:
It includes diluting of a mixture of microorganisms until only a few hundred bacteria are left in
each milliliter of the suspension. A very small amount of the dilution is then placed in a sterile
petri plate by means of a sterile loop or pipette. The molted agar medium is cooled to about
45°C and is poured into plate. The microorganism and agar are well mixed. When the agar is
solidified the individual bacterium will be held in place and will grow to a visible colony.
Dilution:
This method is used for the microorganisms which cannot be easily isolated by streaking or
plating method. Sometimes when several organisms are present in a mixture, with one
organism predominating, the predominating form may be isolated by this method. For
example, when raw milk is allowed to sour at room temperature it will, at the time of curding,
have a mixture of microorganisms with high percentage of Streptococcus lactis.

If 1 ml of the sour milk is taken into a tube containing 9 ml. of sterile milk (in which no
organisms are present) then 1 ml. of this mixture is transferred with a sterile pipette into a
second tube of sterile milk and the procedure is repeated i.e. from second to third tube, third
to fourth tube until a series of about 10 tubes are inoculated. By this serial dilution, the
chances are that a pure culture of S. lactis will be obtained
Enrichment Procedure:
This procedure involves the use of media and conditions of cultivation which favor the growth of
the desired species. For example, when a man suffers with typhoid, the intestinal discharge
posses small number of Salmonella typhus when compared with E. coli and other forms. It is
almost impossible to isolate the typhoid organisms because they represent only a fraction of a
per cent of the total microorganisms present. The media are therefore derived, which allow the
rapid growth of the desired organisms, at the same time inhibiting the growth of other
microorganisms.
Single Technique:
This is one of the most ideal and difficult method of securing pure culture. In this method a
suspension of the pure culture is placed on the under-side of a sterile cover-glass mounted over a
moist chamber on the stage of the micros­cope.
While looking through the microscope, a single cell is removed with the help of sterile
micropipette and transferred to a small drop of sterile medium on a sterile cover-glass and is
mounted on a sterile hanging drop slide, which is then incubated at suitable temperature. If the
single cell germinates in this drop, few cells are transferred into a tube containing sterile culture
medium which is placed in the incubator to obtain pure culture originated from single cell.
Instrument used for this technique is called micromanipulator.
MICROMANIPULATOR
A micromanipulator is a device which is used to physically interact with a sample under a
microscope, where a level of precision of movement is necessary that cannot be achieved
by the unaided human hand. It may typically consist of an input joystick, a mechanism for
reducing the range of movement and an output section with the means of holding a micro
tool to hold, inject, cut or otherwise manipulate the object as required.
Micromanipulator are used in conjugation with microscope. It is the method used in
isolation of pure culture.
Method:
Micromanipulator have been built, which permit one to pick out a single cell from mixed
culture.
The instrument is used in conjugation with microscope to pick a single cell from a hanging
drop preparation.
The cell is drawn into the micropipette by gentle suction and then transferred to a large drop
of sterile medium on another sterile coverslip.
When the number of cells increases in that drop as a result of multiplication, the drop is
transferred to a culture tube having suitable medium.
This yields a pure culture of microorganism.
Characterization/Identification of
Microorganism

50
Strategies used for the Identification

When grown on a variety of media, microorganisms will exhibit the difference in their
morphological characteristics and will also show different microscopic appearances. These
differences form the basis for characterization and identification of microorganisms.

Microorganism are broadly characterized based on following features

• Microscopic examination
• Metabolic capabilities
• Biochemical tests
• Nucleic acid analysis

51
 Microscopic Examination

An important initial step in identifying a microorganism is to determine

its size, shape, and staining characteristics. Microscopic examination
gives information very quickly and is sometimes enough to make a
presumptive identification

Gram staining
a) Differentiate between Gram +ve and –ve

b) Have limitations i.e. can not identify the Streptococcus pyogenes

c) Can not differentiate between Salmonella and [Link] of stool samples

Acid Fast Strain

• Mycobacterium –Tuberculosis
52
Metabolic Capabilities
• Culture Characteristics
The identification of most prokaryotes relies on analyzing their metabolic capabilities such as the types of sugars
utilized or the end products produced. In some cases these characteristics are revealed by the growth and
colony morphology on cultivation media, but most often they are demonstrated using biochemical tests.

o Serratia marcescens –red pigment at 22 oC

o Pseudomonas auroginosa – green pigment

o Streptococcus pyogenes –Blood agar –β hemolysis

o [Link] –UTI –Pink colonies on MacConkey agar –lactose fermentation

PSEUDOMONAS AUROGINOSA
Morphological characteristics
Based on morphology following characteristics of a colony are noted
1. Size: in millimeter
2. Shape: circular / irregular
3. Surface : smooth, rough, granular
4. Elevation : flat, low convex, high convex, raised, umbonate, umbulate
5. Edge/margins: entire, undulate, lobate, crenated, fimbricate, ciliate
6. Opacity : opaque, translucent, transparent
7. Colour of colony
8. Consistency : mucoid, friable
9. Other properties : hemolysis, pigmentation, swarming
• Biochemical Tests
Growth characteristics on culture media can give clues as to the identity of an
organism, but biochemical tests are generally necessary for a more conclusive
identification. for conclusive identification

Catalase –present in all aerobes

tetramethyl-p-phenylenediamine
CATALASE
CITRATE GELATINASE HYDROGEN
SULFIDE INDOLE TEST
PRODUCTION
Lysin Decarboxylase Methyl Red
Oxidase Phenylalanin
Deaminase
Other Methods
a) Serology

b) FAME

Serology

Identification of the molecules that make up the surface structures

i.e. cell wall, capsule, flagella, and pili.

This method rely on the specificity of interaction between

antibodies and antigens

69
FAME
Bacteria differ in the type and relative quantity of fatty acids that make up their
membranes; thus, the cellular fatty acid composition can be used as an identifying marker.
Gram negative – cytoplasmic and outer membrane
Cells grown –treated – sodium hydroxide and methanol –fatty acids and volatile methyl
esters –GC-MS
 Genotypic Characteristics For The Identification

a) DNA probe and PCR

b) Probe-Single-stranded piece of nucleic acid, usually DNA,

Labelled with a dye/radioisotope

c) Complementary to the sequence of interest

d) Fluorescence in situ hybridization (FISH)-16sRNA

e) PCR of specific DNA

f) Sequencing Ribosomal RNA Genes-16s, SSU

g) Using rDNA to identify Uncultivated 71

ANTIBIOGRAMS
Antibiotic susceptibility patterns, or antibiograms, can
distinguish different strains. As with phage typing, this
method has now largely been replaced by molecular
techniques. To determine the antibiogram, a culture is
uniformly inoculated onto the surface of a nutritional
agar medium. Paper discs, each of which has been
impregnated with a given antibiotic, are then placed on
the agar. During incubation, the organism will multiply to
form a visible film of cells. A clear area, indicating lack of
growth, will form around each antibiotic disc that inhibits
the organism.