introduction

1. Mr.R.R.Patil
Dr.Shivajirao Kadam College of Pharmacy,
Kasabe digraj, Sangli

2. Introduction
 Bacteria defined as microscopic single celled organism that
can penetrate into healthy tissues & start multiplying into
vast numbers.
 These are unicellular, free living small microorganism
which are visible under the light microscope.
 Those are belongs to kingdom prokaryotae (Monera).
 They occur in water, soil, air & all natural environments.

3.  The size & shape vary between the
dimensions of 0.75 to 4.0 µm.
 The cocci diameter near about 1 µm & bacilli
are 1 to 8 µm.
 They are found in spherical shape i.e
coccoid forms or as cylindrical form i.e rod
shaped forms.

4. Shape of Bacteria
 On basis of shape, bacteria are classified as follows…
1. Cocci
2. Bacilli
3. Vibrios
4. Spirilla
5. Spirochetes
6. Actinomycetes
7. Mycoplasams

5. Cocci
 Cocci are small, spherical or oval in shape
 In greek ‘kokkos’ means berry
 E.g. Micrococcus

6. Bacilli
 They are rod in shapes.
 It is derived from greek word ‘Bacillum’ meaning stick.
 Some of the bacilli the length of the cell may be equal
to width those are called coccobacilli
 E.g. Bracella

7. Vibrios
 Vibrios are comma in shaped, curved rods
 E.g. Vibrio comma

8. Spirilla
 Those are longer rigid rods with several curves or coils.
 Those are helical in shape & rapid bodies.
 E.g. Spirillum ruprem.

9. Spirochetes
 They are slender & flexuous spiral forms.

10. Acetinomycetes
 These are branching filamentous bacteria.
 The characteristics shape due to the presence of rigid
cell wall.
 E.g. Streptomyces species.

11. Mycoplasma
 Those are cell wall deficient bacteria
 So that, they do not have stable morphology.
 They occur as round or oval bodies with interlacing
fillaments.

12. Arrangement of Bacteria
Coccoid cells to exhibit growth in aggregates. Accordingly that
assembly they again exist in following five manners:
 As pairs or diplococci.
 As group of four systematically arranged in a cube or sarcinae.
 As unorganized array like a bunch of grapes or staphylococci.
 As chain like a string of beads or streptococci.
 In that cocci divided into two planes & remain in group of four
that is tetrads.

14.  Arrangement of groupings formed by bacilli species
are limited & those split across their short axis.
 They may appear as pair those called Diplobacilli e.g.
klebisella pneumoniae

15.  Some bacilli species are found in chain like structure
those called streptobacilli e.g Bacillus subtilis.

16.  Some bacilli species are found in chain like structure
but have much large area of contact between the
adjacent cells those are called trichomes e.g.
Saprospira species

17. Structure of Bacterial cell

18. Flagella
 Flagella are long, slender, thin-hair like structure.
 Flagella attached with cytoplasm.
 They play important role in bacteria for motility.
 They have 0.01 to 0.02 µm in diameter.
 They have 3 to 20 µm in length.
 Flagella found in both Gram-positive & negative bacteria.
 Few coccal forms, most bacilli & almost all of the spirilla
& vibrios are motile by flagella.
 They can be seen by compound microscope with special
staining technique & can be seen easily under electron
microscope & dark filled microscope.

19. Flagella seen in bacterial species in different
manners..
 Monotrichous : Single polar flagella e.g. Vibrio cholera
 Lophotrichous : two or more flagella at only one end e.g
Pseudonomas fluorescens
 Amphitrichous : single flagella or more flagella at both
end e.g Alcaligenes fecales
 Peritrichous : several flagella present all over the surface
e.g. Salmonella typhi

20. Periplasmic flagella or endoflagella or axial fibris :
 This type flagella present in some helical bacteria i.e.
(spirochetes)
 That type of bacteria showing their motility only in highly
viscous media.
 In that type of bacteria flagella like structure present within the
cell.
 E.g. Treponema pallidum.
Gliding motility:
In that type of bacteria showing their motility when they are
contact with solid surface. E.g. Cytophaga species

21. Parts of flagella
Three main parts present in flagella those are…
 Filament
 Hook
 Basal body

22. Structure of flagella

23. Fimbriae
 Fimbriae are similar structure like flagella but not involved in
motility.
 It is shorter than flagella (3 µm).
 Fimbriae can be distributed over the entire surface of the cell.
 Fimbriae act primarily as adhesions & allow to microorganism
to attach to surface.
 They responsible for haemagglutination & cell clumping in
bacteria.

24. Pili
 Pilis are morphologically & chemically similar to fimbriae.
 But they are present in small in numbers compatibly
fimbriae.
 Pilis joins to the bacterial cell for transfer of DNA
(bacterial conjugation) from one cell to another cell.
 So pili also called as sex pili or fertility pili (F-pili).

25. Capsules & Slime layer
 Many bacteria secrets EPS (extracelluar polysaccharides) that
are associated with the exterior of the bacterial cell.
 The EPS contains 2% carbohydrate & 98% water so, they
produce gummy exterior to the cell.
 Morphologically two extreme forms exist…
i. Capsules
ii. Slime
 Capsules: which forms rigid, tightly & closely associated with
cell
 Slimes: which are loosely associated with cell.

26. Function of capsule & slime
 They protect from desiccation.
 They provide a protection barrier against the penetration
of biocides.
 They protect against engulfment by phagocytes &
protozoa.
 They may promote the stability of bacterial suspension by
preventing the cells from aggregation & settling.
 They may promote attachments of bacteria to surface.

27. Cell wall
 Cell wall gives definite shape to the bacteria.
 Cell wall situated between the capsule & cytoplasmic
membrane.
 It is about 20- 30 nm in thickness.
 In the cell wall contains diaminopimelic acid (DAP),
murmaic acid & teichoic acid.
 These substance joined together to formed a complex
polymer structure known as peptidoglycan or murein or
mucopeptide.

29.  Peptidoglycan is a large macromolecules containing glycan
(polysaccharide) chains that are cross-linked by short peptide
bridge.
 The glycan chain act as a backbone to peptidoglycan.
 Those short peptide bridge composed of alternating residues
of N-acetyl muramic acid (NAM) & N-acetyl glucosamine
(NAG).
 Each molecule of NAM attached a tetrapeptide.
 Tetrapeptide consisting of the amino acids L-alanine, D-
alanine, D-glutamic acid & lycine or diaminopimelic acid
(DAP).

31.  This glycan tetrapeptide repeat unit is cross -linked to
adjacent glycan chain.
 This adjacent glycan chain occurs through a direct peptide
linkage or a peptide interbridge.
 The type & number of cross linking amino acids vary from
organism to organism.

32. Cell wall structure of Gram-positive &
Gram-negative bacteria
 On the staining technique bacteria are divided into two
large groups…i. Gram-positive
ii. Gram- negative
 This staining technique are called as Gram staining
technique.
 In that gram staining technique, the bacterial film
treating with crystal violet & iodine solution & then
washed with alcohol solution.

33.  After washing with alcohol solution the gram negative
organism cells appears the colourless while, gram positive
organisms are retain the dye.
 When both gram positive & negative cells are treated with
different colour dye e.g carbol fuchsin (red in colour).
 That time, gram negative cells appears red & gram
positive appears purple.
 On that it reflects that both have different cell wall
structure.

34. Gram-positive cell wall structure
 Gram positive bacterial cell wall consist of a single type of
molecules.
 Cell wall thick near about 20 to 80 nm.
 In that present of 60 to 80 % peptidoglycan.
 Gram positive walls frequently contains acidic polysachrides
are called teichoic acids.
 Teichoic acid are either ribitol phosphate or glycerol phosphate
molecules that are connected by phosphodiester bridge.

35.  In some gram positive bacteria glycerol-teichoic acids are
bound to lipids membrane and termed as lipoteichoic
acid.
 Those lipoteichoic acid create infection by killing
bacteria & shows inflammation.

36. Cell wall structure of Gram-positive
bacteria

37. Gram-negative cell wall structure
 Gram negative cell wall are multilayered & complex type
structure.
 Gram negative cell wall consist 10 to 20 % peptidoglycan.
 In that second layer found outside the peptidoglycan layer.
 This layer is asymmetrical & contains proteins,
lipoproteins, phospholipids & lipopolysaccharide (LPS).

38.  This outer layer is attached to peptidoglycan & the other end is
fixed in the outer membrane.
 In the inner leaf of the outer layer conatins phospholipids & it’s
outer layer composed with LPS (lipopoysaccharide), a
polysaccharide-lipid molecule.
 In gram negative cell, the LPS is an important molecule
because it determine the antigenicity & it is extremely toxic to
animal cell.
 In the LPS molecules contains three regions
i. lipid A
ii. Core polysaccharide
iii. O-specific polysaccharide

39.  Lipid A linked to core polysaccharide by the molecule
KDO (ketodeoxyoctonate) & other end of core
polysaccharide is the O-polysaccharide.
 In the O-polysaccharide or O-antigen usually contains
six-carbon sugars as well as one or more usually deoxy
sugars such as abequose.

40.  In the lipid A components are gives toxic & pathogenic
properties to the gram-negative bacteria.
 Gram negative bacterial outer membrane is relatively
permeable to small molecules but not for enzymes or
large molecules.
 The region between the outer surface of the cytoplasmic
membrane & the inner surface of the outer membrane is
called the periplasm.

41. Cell wall structure of Gram-negative
bacteria

42. Cytoplasmic Membrane
 Cytoplasmic membrane is thin near about 5 to 10 nm.
 Biochemically, the cytoplasmic membrane is fragile,
phospholipid bilayer with proteins distributed
randomly throughout.
 In the phospholipids bilayer most of the proteins are
tenaciously held & are called integral proteins.

43.  Other proteins are loosely attached those are called
peripheral proteins.
 The phospholipids molecules are arranged in two parallel
rows, called a phospholipid bilayer.
 Each phospholipid molecule contains a polar head & tail.
 Polar head composed of a phosphate group & glycerol.
 The non-polar tails are interior of the bilayer.
 Prokaryotic plasma membrane are less rigid than
eukaryotic due to lack of sterols.

44. Functions of cytoplasmic
membrane
 They including in transportation of nutrients.
 It provides mechanical strength to the bacterial cell.
 It helps in DNA replication.
 It contains the enzymes involved in the biosynthesis of
membrane lipids & various macromolecules of the
bacterial cell wall.

45. Structure of cytoplasmic membrane

46. CYTOPLASM
 In the bacterial cytoplas is a type of suspension, in that contains
organic, inorganic solute in a viscous water.
 It contains the nucleus, ribosomes, proteins & other water
soluble components & reserve material.
 The cytoplasm bacteria differ from that of higher eukaryotic
microorganisms in not containing endoplasmic reticulum, golgi
apparatus, mitochondria & lysosomes.
 In most of the bacteria also contains extrchromosomal DNA (i.e
DNA are not connected to chromosome) is also present.

47. Ribosomes
 Ribosomes are most important structure in bacterial
cytoplasm.
 They involved in protein synthesis.
 Ribosomes numbers varies with the rate of protein
synthesis.
 If greater the number of ribosomes then the greater the
protein synthesis.
 They have 200 Ao in diameter.
 They are characterised by their sedimentation properties.

48.  These bacterial ribosomes are called as 70 S ribosomes.
S= svedberg unit..unit of sedimentation.
 After sedimentation carried in ultra-centrifuge & then placed
in low concentration of magnesium that time 70 S ribosomes
dissociated into 50 S & 30 S particles.
 Each 50 S particles contain…one molecule of 23 S RNA, one
molecule of 5 S RNA & 32 different proteins.
 And, each 30 S particles contains…one molecule of 16 S RNA &
21 different proteins.
 During protein synthesis these ribosomes are associated with
the m-RNA & such association are called polysomes.

49. Mesosomes
 In most of the bacteria, particularly in Gram-positive bacteria
the growth condition depending upon the membrane appears
to be infolded at more than one point.
 Such infoldings are called mesosomes.
 Mesosomes presents in two types…
In central (septal) mesosomes & peripheral (lateral) mesosomes.

50. Structure of mesosomes

51.  Central mesosomes present deep into the cytoplasm & locate
near the middle of the cell.
 These are involved in the DNA segregation & in the formation
of cross walls during cell division.
 The peripheral mesosomes are not present at central location
& are not associated with nuclear material.
 Mesosomes are also called as chondroids & are visible only
under electron microscope.
 Larger numbers of mesosomes have a higher respiratory
activity e.g. Azotobacter.

52. Nucleus
 Nucleus appears oval or elongated bodies & generally present
one per cell.
 The genome consists of a single molecule of double stranded
DNA arrangement in a circle.
 It may open under certain conditions to form a long chain
about 1000 µm in length.
 In bacterial nucleus does not contains nuclear membrane,
nucleous & deoxyribonucleoprotein.
 The bacterial chromosome is haploid & replicated by simple
fission instead of mitosis as in an eukaryotic cell.

53. Spores
 Many bacterial species produce spores inside the cell & outside
the cell.
 Inside the spores are called endospores & outside the spores
are called exospores. E.g Bacillus anthracis, Bacillus subtilis
etc.
 Spores are extremely resistant to desiccation, staining,
radiation, disinfecting chemicals & heat.
 Each bacterial spore on germination forms a single vegetative
cell.
 They remain viable for long time & help bacteria to survive for
long period under unfavourable condition.

54.  Endospores are thick-walled, highly refractile bodies that are
produced one per cell.
 All the endospores contain large amount of DPA (dipicolinic
acid).
 It occurs in combination with large amount of calcium, which
is present in central part of the spore (core).
 That calcium & DPA complex play important role in the heat
resistant of endospores.
 Endospores consists of a core or envelope or protoplast.
 In the core or protoplast consist of DNA & ribosomes, t-RNA &
enzymes.
 The spore envelop consist of the inner membrane, outer
membrane, cortex & spore coat.
 In some species have the outer layer called exosporium which
bears ridges & fold.

56. SPORULATION
 The process of endospore formation is known as
sporulation it may take 4 to 8 hrs in a vegetative cell.
 Sporulation process….
 Firstly..a newly replicated bacterial chromosomes & small
portion of cytoplasm are isolated by an ingrowth of the
plasma membrane called a spore septum.
 The situated septum derived from the cytoplasmic
membrane is then formed by a process of invagination
which divides into a forespore & sporangium.
 The forespore is subsequently encircled by a dividing
septum as a double layered membrane.
 Between the two layers is laid a spore cortex & outer layer is
transformed into spore coat which consists of several layer.

58. Bacterial reproduction & growth
kinetics
1.Multiplication & divisional cycle
 Many bacterial cell multiply by binary fusion.
 Means, each individual cell increase in size until it is large
enough to divide into two identical daughter cells.
 At that time of separation each daughter cell must be capable
of growth & reproduction.
 While each daughter cell will automatically contain mRNA,
rRNA, ribosomes, enzymes, cytochromes etc. from mother
cell.

59. During DNA replication process the bacterial
chromosome which is circular in shape that attached to
the cytoplasmic membrane where, it is able to uncoil the
DNA.
The process of DNA replication proceeds at a fixed rate &
it’s depend on temperature. Therefore the time taken to
copy of an entire chromosome depend on the number of
base pairs within it & the growth temperature.
 e.g Escherichia coil growing at 370C will take 45 mints for
replication of chromosome.

60.  These copies of chromosome must segregates to opposite sides
of the cell before cell division can proceed.
 Cell division occurs differently in Gram-positive & Gram-
negative bacteria.
 In Gram positive cells have rigid cell wall in that develop cross
wall that divides the cell into two equal halves.
 Construction & cross-wall formation takes approximately 15
mints. to complete.
 In Gram negative cells do not have rigid cell wall so that divide
by a process of construction followed by membrane fusion.

61. In DNA replication, chromosome segregation (C-phase) &
cell divison (D-phase) occur sequentially in slow-growing
cells with generation times of greater than 1 hr & are the
final events of the bacterial cell.
Cells are able to replicate faster than once every hour by
initiating several rounds of DNA replication at a time.
Thus partially replicated chromosomes become segregated
into the newly formed daughter cells.

62. 2. Population growth
Population growth means an orderly increase in all
cellular constituents.
Increase of mass may not really reflects growth because
there is only increase in the size & weight of the cell.
All actively growing cells mainly multiply by the binary
fission.
In the binary fission one specific cell undergoes division
to give rise to the formation of two cells.

63.  Thus, population growth increases, geometrically..
1  2  22  23  24 …. 2n
Where, n = number of generations.
Assuming that there is no cell death at all, each succeding generation
shall give rise to double its population.
Thus, the total population N at the end of a specific given time period
is expressed as….
N = 1 x 2n
But, under practical condition, it is difficult to inoculate only one
bacteria so formula as..
N = N0 x 2n
No is the number of bacteria inoculated at time zero.

64. Taking logarithm on both sides…
log N = log No + n log 2
n = log N – log No
log 2
n = log N – log No
0.301
n = 3.3 (log N – log No )
If we know the initial population & the population after
growth then we can calculate the number of generation by
using the above formula.

65. Growth curve of bacteria
 Growth curve of the bacteria obtained by inoculating a
small number of bacterial cells into a suitable culture
medium & counting the bacterial samples at regular
intervals.
 When logarithms of the number of bacteria vs times on
graph paper the specific curve found that resulting curve is
called bacterial curve.
 The resulting curve has four distinct phases…
1. Lag phase
2. Log or logarithmic or exponential phase
3. Stationary phase
4. Death or decline phase

66. 1. Lag phase:
 Lag phase is the phase or period between inoculation & the
starting the multiplication of bacteria.
 In that phase bacterial cells are adjust to adopt the new
environment of the medium.
 In that phase bacterial cell are synthesised the enzymes,
coenzymes & other essential molecules.
 In that phase cells are metabolically & physiologically very
active but do not divide.
 The length of the lag phase depend upon nature of medium,
species of microorganism & other factors.

67. 2. Log phase or Exponential phase:
 In that phase bacteria cells rapidly grow.
 Means in that phase bacteria multiply at their maximum
rate & their number increases exponentially.
 The time required for one bacterial division during this
phase is known as generation time.
 The number of bacteria present in each generation period
is almost twice that in previous period.
 At this phase the log of number of cells plotted against
time result in a straight line.
 The generation time (g) can be determined from the
number of generations (n) that occurs in particular time
(t)
g = t/n
t/n = t / 3.3 (log N – log N0 )

68. 3. Stationary phase:
 In stationary phase cells is maintained a balance between cell
division & cell death.
 In that phase multiplication is reduced because depletion of
nutrition, accumulation of toxic waste products, very high
conc. of cells & low partial pressure of oxygen.
 At this phase cells consumed reserve food materials, proportion
of ribosomes may be degraded & enzymes may still be
synthesised.
 At this phase the log of number of cells plotted against time
result in a no change in line.

69. 4. Death or decline phase :
 In that phase, the number of bacterial cells decreases exponentially,
essentially the inverse of growth during the log phase.
 A variety of conditions involved to bacterial death but mostly
responsible are depletion of nutrition & accumulation of toxic waste
products etc.
 At this phase the log of number of cells plotted against time result in
decline the line.

70. Synchronous growth
 A synochronous or synochronized culture is a microbial culture
or cell culture that are all in the same growth stage.
 Thus, the entire population is kept uniform with respect to
growth & division.
 But practically it is not possible to determine a single bacterial
cell to obtain the information about growth behavior.
 Synchronous culture provides the entire cell crop in the same
stage.
 Synchronized culture provides information on measurement
made on such culture are equivalent to the measurement made
on individual cells.

71.  A synchronous population can be generated either by
physical separating cells in the same stage of division or
by forcing a cell population an identical, physiological
condition by change in the environment.
 physical separation done by centrifugation, filtration or
by periodic changes in nutritional & environmental
conditions produce synchronously dividing cell
populations.
 The synchrony is generally lost after a few generation.

72. Continuous growth
 In that technique microbial population is maintained in the
log phase or exponential phase of growth in a constant
environment.
 It is necessary maintained population for research & industrial
process.
 This technique or condition also known as steady state growth.
 In this apparatus fresh medium flows into growth chamber at a
controlled rate.
 The rate of growth is then controlled by regulating the inflow
rates. Hence in a chemo stat apparatus, indefinite growth at
any constant are can be maintained.

73. Growth & Genetic exchange
 Three major process involved in genetic exchange….
Transformation
Transduction
Conjugation

74. Transformation
 The early work of Fred Griffith in 1928 on the transfer of virulence in
the pathogen Streptococcus pneumoniae .
 The stage for the research that first showed that DNA was the
genetic material.
 Griffith found that if he boiled virulent bacteria and injected them
into mice, the mice were not affected and no pneumococci could be
recovered from the animals.
 When he injected a combination of killed virulent bacteria and a
living nonvirulent strain, the mice died; moreover, he could recover
living virulent bacteria from the dead mice.
 Griffith called this change of nonvirulent bacteria into virulent
pathogens transformation.

75. Transduction
 Defined as: a phenomenon causes genetic recombination in bacteria
wherein DNA is carried from one specific bacterium to another by a
bacteriophage.
 There are group of viruses are called bacteriophage. Bacteriophage
have bacterial cells as their hosts.
 These bacteriophage inject viral DNA into the bacterial cell & after
that viral DNA is then replicated & transcribed at the expense of the
host & assembled into new viral particles.
 Normally the host cell lysed in order release the viral progeny but in
exceptional conditions they enter in a replication cycle of the viral
DNA & become incorporated by recombination into the chromosome
of the bacterium.
 This is known as temperate phage.

76.  Then, viral DNA forms part of the bacterial chromosome
& will be copied to all daughter cells.
 As well as temperate phage will be active once again at a
low frequency & phasing between temperate & lytic forms
ensures the long-term survival of the virus.

77. Conjugation
 Conjugation is a natural process representing the early stages in a true
sexually reproductive process.
 In that transcribed to produce singular viral elements, which cannot
assemble or lyse the host cell. Such DNA strand are known as
plasmids.
 Plasmids are circular & can either be integrated into the main
chromosome, in which case they are replicated along with
chromosome & passed to daughter cells or they are separate from it &
can replicate independently.
 The simplest form of plasmid is F-factor (fertility factor).
 This can be transcribed at the cell membrane to generate F-pilus
within the cell envelop & cells containing an F-factor are designed F+

78.  The F-pilus is a hollow appendage that is capable of
transferring DNA from one cell to another.
 In its simplest form an unassociated F-factor will simply
transfer a copy to a recipient cell & such a transfer process
is known as cojugation.

79. Nutritional Requirements
Bacteria required the nutrition's, pH, oxygen & temperature
for growth & multiplication process.
 So, for cultivation of microorganism required elements
such as sodium, potassium, magnesium & iron.
 As well as in media required contains of source of carbon,
nitrogen, hydrogen, oxygen & phosphorus.
 Bacteria can be classified depending upon nutritional
requirements…such as carbon, energy, electron etc.

80.  Source of energy:
Energy obtained from sunlight are called phototrophs bacteria
e.g. Rhodospirillum rubrum.
Energy obtained from chemical reaction those called
chemotrophs bacteria e.g. Escherichia coli or E-coli.
 Source of electrons:
All bacteria required electrons for metabolism.
Lithotrops : In that type of bacteria species use the inorganic
compounds as electron donor e.g pseudomonas pseudoflava.
Organotrophs : In that type of bacteria species use the organic
compounds as electron donor e.g Escherichia coli or E-coli.

81. Photolithotrophs : some phototropic bacteria use
inorganic compound (H2S) as source of electron.
e.g. Chromatium okenii.
Photoorganotrophs: some phototropic bacteria use
organic compound such as fatty acids & alcohols as
electron donors e.g Rhodospirillum rubrum.
Chemolithotrophs: some chemotrophic bacteria use
inorganic compound as source of electron.
e.g. Nitrosomonas europaea.
Chemoorganotrophs: some chemotrophic bacteria use
organic compound such as sugar &amino acids as electron
donors e.g Escherichia coli or E-coli.

82.  Source of carbon: microorganism required carbon for
synthesizing cell components.
Autotrophs: some species use CO2 as the major source of
carbon these microorganisms are called autotrophs.
e.g. Chromatium okenii.
Heterotrops: some species use organic compounds as a
source of carbon such species are called heterotrophs.
e.g. Escherichia coli or E-coli.

83.  Nitrogen:
Nitrogen is the major component of protein & nucleic acids, so
that bacteria can use nitrogen from the atmosphere or from
inorganic compounds such as nitrites, nitrate.
 Sulphur:
Sulphur is needed for synthesis of amino acids.
 Phosphorus:
Phosphorus usually supplied in the form of phosphate is an
essential component of nucleotides, nucleic acid etc.

84.  Water:
It is the major essential nutrient as it account for about 80
to 90% of the total weight of cell.
 Mineral salts:
Bacteria require salts, particularly the anions such as
phosphate & sulphate & the cations as sodium,
potassium, magnesium, iron & calcium. These are present
in the natural environment or may be added in cultural
media.

85. Cultural Media
 Cultural or bacteriological media are mainly used for growth,
isolation, purification, maintenance & identification of
microorganisms.
 Nutrient agar is mainly used for growth of many bacterial
species.
 Common ingredients present in cultural media are..
 Water
 Peptone
 Yeast extract
 Meat extract
 Agar

86. Classification of cultural media
 Depending on physical state (media consistency)
1. Solid media (1.5 to 2.5% agar) e.g. Nutrient agar.
2. Semisolid media (0.2 to 0.5% agar) e.g. Nutrient broth cont.
0.5% agar.
3. Liquid media (absence of agar) e.g. fluid thioglycollate broth.
 Depending on oxygen requirement
1. Aerobic media e.g. MacConkey’s broth.
2. Anaerobic media e.g. Robertson’s cooked meat medium.

87.  Depending on chemical composition
1. Simple or basal media
2. Synthetic or defined media
3. Non-synthetic or undefined or complex media
 Depending on functional type
1. Enriched media
2. Enrichment media
3. Selective media
4. Indicator media
5. Differential media
6. Sugar media
7. Transport media
8. Assay media
9. Storage media

88. Depending on chemical composition
media
 Simple or basal media: In that type of media peptone water &
nutrient broth is added & used in the study of common
bacteria. Addition of 2%agar to nutrient broth forms a nutrient
agar medium which is solid basal medium.
 Complex or non-synthetic or undefined media: In that type
of media included biological origin such as blood or milk or
yeast those provides cultivation of bacteria.
 Synthetic or defined media: Those media are prepared from
pure chemical substance. These media are used for research
purpose & used in metabolic studies of specific
microorganisms.

89. Depending on functional type media
 Enriched media : In that type of media included of substance
such as blood, serum & egg to basal medium. E.g. blood agar
(streptococcus).
 Enrichment media: specific substance added in the
liq.medium which inhibits the unwanted bacteria & favors only
wanted bacteria such media are called enriched media. E.g
tetrathionate broth.
 Selective media : same as enrichment media but only in solid
form & added to a solid medium. E.g MacConkey’s agar (E-
coli).

90.  Indicator media: When bacterial species growth in them that
time it change in colour. E.g. Wilson & Blair medium.
 Differential media: these media distingwish between types of
bacteria. E.g. MacConkey’s medium shows lactose fermenter as
red colonies & non lactose as white or plae colonies.
 Assay media: these type of media used for the assay of
antibiotics, amino acids, vitamins & also used for testing of
disinfectants.
 Storage media: used for help in preservation & storage of
bacteria for long time of period. e.g. Dorset’s egg medium.

91. Counting Chamber method
 Counting chamber method or Haemocytometer mehtod.
 Counting method used for determination of cell numbers
or count the bacteria.
 This is the simple & rapidly counting technique with
minimum equipment required.
 In that technique take, a minute drop of the culture is
placed on Neubar’s slide.
 Neuber’s slide is tiny, shallow, rectangular glass slide with
accurately ruled into squares that are 1/400 mm2.
 In counting method, cells can be determined by using a
phase contrast microscope.

92. Total no. of
bacterial cells/mm3 = No. of cells counted x Dilution
area counted x Depth of fluid
Cells counted in 5 squares & each square divide into 16 small
squares & each square is equal to 1/400 mm2.
So, area of 5 square = 5 x 16
=80 squares.
= 80/400 mm2
= 1/5 mm2

93. It is a special microscopic slide with a counting chamber
1/10 mm deep so that,
Volume of liq. Over a one square = 1/10 mm
Dilution = 1:200
=200
So formula..
No.of bacterial cells counted = N x 200
1/5 x 1/10
= N x 200 x 50
= N x 10,000 cells/mm3

94. Identification Procedure
 Identification of microorganism means in that study
included for determination of morphological structure of
microorganisms.
 In the morphological studies depending upon the no.of
factors such as, stain studied, nature of culture media,
temp. & duration of incubation.
 By using differential staining technique was present on that
we can easily determined the nature of microorganism, the
size of microorganism, shape of microorganism & also
determined their nature of microorganism with using
microscope.

95.  Stain is an organic compounds which contains benzene
ring with chromophore & auxochrome group.
 Different techniques was used for visualisation,
differentiation & separation of bacteria in terms of
morphology.
 Staining different techniques are…
1. Simple staining
2. Negative staining
3. Gram staining
4. Acid-fast staining
5. Spore staining
6. Capsule staining
7. Other staining

96. Simple staining
 By using simple staining technique, we can easily
determine their morphology & arrangement of
bacterial cell.
 In the simple staining technique used the single stain
e.g. crystal violet, methylene blue, carbol fuchsin
safranin etc.
 In that used the basic stain with a positively charged
chromogen.
 When, positively charged chromogen react with
bacteria, having a negative charge on nucleic acid &
certain cell wall components.
 That time, negative charge components strongly binds
with positively charged chromogen.

97. Negative staining
 In negative staining technique use the acidic stain e.g.
nigrosin or eosin.
 Acidic stain are negative charge so they not penetrate into
the cell but, it deposit the around the cell.
 So that, unstained cells are easily observed against the
coloured background.
 Advantage of that staining tech. compare with simple
technique that, it doesn’t required heat fixation & in that
technique determine the natural size & shape of
microorganism can be seen.

98. Gram staining
 Gram staining technique discovered by Dr. Christian
Gram in 1884.
 By that technique use for not just for determination of
morphology but also use for the differentiae in
between Gram-positive & Gram negative cell.
 Gram positive cell retain the violet stain.
 But gram negative cell decolourised & appears the red
colour in some species e.g E-coli, salmonella typhi,
vibrio cholerae, klebsiella pneumoniae etc.

99. Acid-fast staining
 Acid fast staining technique used for differentiate between
acid-fast & non-acid fast bacteria.
 Mycobacterium species & actinomycetes bacterial species
containig mycolic acid & other waxy material in their cell
wall.
 Such bacterial species do not get stained with ordinary
staining technique. So, ZNCF (Zhiel-Neilson Carbol
Fuchsin) stain is used with steam heating.
 The acid fast bacteria appear pink & non acid bacteria
appears blue.

100. Spore staining
 Spore staining technique used for detection of spore
carrying bacteria & type of spores.
 For spore staining use Dorners method.
 In Dorners method, carbol fuchsin is used as primary
satin.
 After heating the slide with stain for 5 to 10 minutes,
wash it & perform negative staining procedure.

101. Capsule staining
 Capsule staining technique used for determination of
capsule outside the cell. For performing, using the
positive staining & negative staining technique.
 By positive staining the crystal violet is applied & also
applied copper sulphate solution (20%) which is
created osmotic difference, which causes diffusion of
stain towards the outer surface.
 After removing stain, the capsular layer as light violet
colour against deep violet cell.
 In negative staining, stains the cell & capsule are
visible colourless. By negative staining technique it can
easily visible.

102. Characteristics of Staphylococcus
 Staphylococcus means staphyle (bunch of grapes) &
kokkos (grain or berry) was derived from Greek words.
 Classification:
1. On the basis of pigment production:
i. Staphylococcus aureus: golden yellow colnies
ii. Staphylococcus albus: white colnies
iii. Staphylococcus citreus: lemon yellow colnies
2. On the basis of pathogenecity:
i. Pathogenic species :e.g. Staphylococcus aureus
ii. Non-pathogen species: e.g. Staphylococcus epidermidis
3. Baird-parker classified Staphylococcus into subgroup I
to VI

103. Staphyloccous aureus

104.  Staphylococcus aureus are spherical