2-MORPHOLOGY AND DESCRIPTION OF BACTERIA I.ppt

2-MORPHOLOGY AND DESCRIPTION OF BACTERIA I.ppt

1. Microscopy And Morphology
Of Bacteria
Dr. Arvind Nanera

2. INTRODUCTION
•Microorganisms are living
structures of microscopical size .
•These were originally classified
under plant and animal kingdoms .
•This proved to be unsatisfactory ,
therefore a third kingdom Protista was
formed .

3. •PROTISTA
•It is divided into two groups – 1]
EUKARYOTES and 2] PROKARYOTES
•1] EUKARYOTES: Fungi, Algae ,
protozoa, and slime moulds are included
in this group

4. •PROKARYOTES : Bacteria and
blue green algae belong to this group .
•Bacteria are unicellular without
any true branching , except in the higher
bacteria [Actinomycetales] , they don’t
contain chlorophyll but in contrast blue
green algae possess chlorophyll.

5. BACTERIA
•The size of bacteria is measured
in the unit of micron [ micrometre ].
•1 micron or micrometer = 1/1000
mm
•1 millimicron [mmicron] or
nanometre [nm]= 1/1000 micron or oone
millionth of a millimetre
•1angstrom unit A
0
= 1/10 nm
(nanometre)

7. Differences between Prokaryotic
and Eukaryotic Cells
Structure Prokaryotes Eukaryotes
Nucleus
Nuclear membrane Absent Present
Nuceolus Absent Present
Chromosome One More than one
Deoxyribonucleoprotien Absent Present
Division By binary fission By mitosis
Cytoplasm
Mitochondria,golgi apparatus,ER lysosomes,pinocytosis,
All are absent present
Sterols Absent Present
Muramic acid Present Absent

8. MICROSCOPY
•​it is done for the following purpose
•​1 magnification of an object
•​2 maximisation of resolution
•​3 optimisation of the contrast
between structures organisms and
backgrounds
•​following types of microscopes
are being employed for the
structure of bacteria
•​optical or light microscopes
•​Phase contrast microscopes

9. •​3
​dark field / dark ground
microscopes
•​

4 interference microscopes
•​5 fluorescent microscopes
•​6 electron microscopes

10. 1 optical or light microscope
•​light source
•​

a compound lens system
•​objective lenses , conjunction fixed
eyepiece
•​

power , final magnification conjunction
•​Resolving power , wavelength , NA
numerical apperture
•​NA , light gathering power
•​RP
​Resolution power , optimised,
condenser , 1.25
•​RP can be increased by adjusting the
medium
​

11. –​through which light passes between the object
and the objective lens
•​special oils , immersion oils , have a
refractive index similar to glass , thus
use of these oils permits more light to
be incorporated in the image resulting
in improving the resolution power
•​visualisation of bacteria generally
requires the use of immersion oil with
X100 objective
•​this combination results in resolution of
approximately 0.2 microns
•​use of 40 X

16. Phase contrast microscope
–​by improving the contrast , makes
evident the internal structures of cell
which differ in thickness of refractive
index
•​different parts of cell and its surrounding
medium have got different refractive
indices between the object and its
surrounding medium
•​

A special optical system , special
condenser and objective lens converts
difference in phase into difference
in intensity of light , producing light
​

17. •​and dark contrast in the image
•​

a light microscope can be
converted into phase contrast
microscope by using a
special condenser and objective
lens

18. Dark Field Microscope
–​reflected light is used instead of the
transmitted light used in the light
microscope
•​a dark field condenser with a circular
stop is fitted with a light
microscope .
•​this condenser lens system is
arranged in such a way that no light
reaches the eye , unless
reflected from the object
•​the object or bacterium appears self
illuminous against a dark
background

19. –​the contrast gives an illusion of
increased resolution , such as
extremely slender organisms
such as spirochaetes not
visible under ordinary
microscope , can be seen
under the dark field
microscope

20. INTERFERENCE
MICROSCOPE
–​useful ,
•​for revealing cell organelles
–​for quantitative measurements of the
chemical constituents of cells
such as lipids proteins and NA

21. Fluorescent microscope
•​it is a phenomenon that occurs with
an object is impacted by a given wavelength of
light and emits light at a wavelength longer
than the one to which it was exposed .
Specimens are exposed to a light of shorter
wavelength (ultraviolet light ) which results in
emission of longer wavelength visible light
•Due to shorter wavelength of U V light
the resolving power can be proportionately
extended bacteria stained with fluorescent
dyes auramine and rhodamine become visible
as brightly glowing objects in a dark background

22. •fluorescent microscopy has also been
employed for detection of antigen
(direct fluorescent antibody technique )
and antibodies ( indirect fluorescent
antibody method)

23. Electron microscope
•A beam of electrons is employed
instead of the beam of light used in
optical microscope the electron beam
is focussed by circular
electromagnets (magnetic condenser )
, which are analogous to the
lenses of light microscopes .
•​the wavelength of electrons is
approximately 0. 005 nm as
compared to 500nm with visible
light the resolving power of the
electron

24. •should be theoretically 100, 000 times that of
light microscopes but in practice it is
about 0.1 nm
•​SHADOW CASTING : is an important
technique in electron microscopy
•​it is achieved by depositing a thin layer of
metal e.g. platinum on the object
•​such metal coated object held in the path of
the beam scatters the electron and
produces an image which is focused
on fluorescent viewing screen
​

25. •another technique in studying the
fine structures of the object is
negative staining with phosphotungstic
acid

26. •SCANNING ELECTRON
MICROSCOPE : is a recent development
which provides a higher resolution
and three dimensional image of
the object
•​although substantial increase in
resolution power of electron microscope
has led to significant discoveries
•​disadvantages , inability to examine
living cells
•​

to overcome the disadvantage , a
new method is introduced - Freeze
etching

27. •rapid cooling of specimens by deep -
freezing in liquid gas and the
subsequent formation of carbon
platinum replicas of the material .
since such frozen cells may
remain viable it
•​

USED FOR , rapid detection of
virus (non cultivable viruses
•​ultra structural study of various
microorganisms may also be done
in the living state

28. STUDY OF BACTERIA
•A unstained wet preparations
•​these are examined mainly for bacterial
motility , and demonstration of
spirochaetes
•​STAINED PREPARATIONS, lack of contrast ,
•​staining methods produce colour contrast
•​smear first dry and then heat
•​heat kills and then fixes - coagulation
•​stained by appropriate staining technique
​

29. COMMON STAINING
TECHNIQUES
•1 simple staining
•​2 negative staining
•​

3 impregnation method
•​4 differential stains

31. simple stains
•basic dyes , methylene blue , or
basic fuchsin
•​provide colour contrast but impart
the same colour to all the
bacteria
​

32. Negative Staining
•bacteria mixed with dyes such
as India ink or nigrosin
•​background gets stained and
unstained bacteria stand out in
contrast
•​use , demonstration of capsule

33. Impregnation method
•bacterial stains and structures that
are too thin
•​demonstration of bacterial flagella
and spirochaetes
•​as they are too thin to be seen
under light microscope
•​thickened by silver on the surface
to make them visible

34. Differential stains
•impart different colours to different
bacteria or bacterial structures
•​most commonly employed stains
are gram stain , the acid fast
stain and the Alberts stain

35. ALBERT`S STAIN
•staining of corybacterium diphtheria
and other corynebacteria is done by
this technique
•​METHOD
•​the smear is heated gently by flaming
the slide underneath .fix the smear
•​do not overheat
•​cover the smear with Alberts stain for 5
minute

36. •drain off the whole stain without washing
•​pour albert 2 solution without washing
•​blot dry the smear with the help of filter paper
•​REAGENTS , A . Albert or albert stain 1.
​
toludine blue , malachite green ,
​glacial acetic acid ,
alcohol , distill water to make
​
300ML

37. •Albert 2 or Alberts iodine solution
•​

iodine , potassium iodide , distill
water to make
•​microscopic examination of the
smear
•​appear as green coloured bacilli
with bluish black metachromatic
granules these bacilli are
arranged in chainese letter or
cuneiform arrangement
​

38. GRAM STAINING
•The gram staining was originally devised by the
histologist Christian Gram (1884) as a method of staining bacteria in
tissue.
•PRINCIPLE:
•Gram-positive cells have more acidic protoplasm,
which may account for their retaining the basic primary dye more
strongly than Gram- negative bacteria.
• 2. The peptidoglycan of Gram-positive bacteria is
thick and thus able to retain the dye-iodine
complex

39. •3. The high lipid content of Gram-
negative bacteria makes them permeable to
secondary dye after decolourisation with organic
solvent like acetone.
• 4. Iodine makes the protoplasm
more acidic and serves as the mordant, i.e.
iodine combines with dye to form a dye-iodine
complex and fixes the dye in bacterial cell.

40. •5. The Gram-positive cell wall or
cytoplasmic membrane being less permeable,
the dye-iodine complex gets trapped within the
cell.
• 6. The Gram negative cell wall has
increased permeability to acetone or alcohol,
permitting the outflow of complex during
decolourisation

41. •PROCEDURE:
•Cover the fixed smear with pararosaniline dye
such as crystal violet for 1 minute.
•Pour Gram’s iodine for one minute.
•Wash wit tap water.
•Decolourise with an organic solvent such as
ethanol (ethyl alcohol) till no more violet colour comes
off.

42. •Wash the smear with tap water.
•Counter stain with a dye of
contrasting colour such as safranine for 30
seconds.
•Wash the smear with tap water.
•Allow the smear to air dry and
examine under oil immersion objective.

43. •DIFFERENTIATION ON GRAM
STAINING
•TWO BROAD GROUPS
•GRAM POSITIVE COCCI
(COCCI IN CLUSTERS)

44. GRAM POSITIVE COCCI (COCCI
IN CLUSTERS)

45. ACID FAST STAIN
•This was discovered by Ehrlich,
who found that after staining with aniline
dyes, tubercle bacilli resist decolourisation
with acids. The method as modified by
Ziehl & Neelsen, is in common use today.

46. PRINCIPLE:
•Acid fastness has been ascribed to the
high content and variety of lipids, fatty acids and
higher alcohols found in tubercle bacilli.
•A lipid peculiar to acid fast bacilli, a
high molecular weight hydroxyl acid wax
containing carboxyl groups (mycolic acids), is
acid fast in the free state.
• Acid fastness is not a property of lipid
alone but depends also on the integrity of the
call wall.

47. PROCEDURE:
•The carbol fuchsin stain is poured on a
slide containing fixed smear. Gentle heat is
applied to the underside of the slide, by means
of spirit flame, until the stain just commences to
steam. The carbol fuchsin is left on the slide for
5-10 minutes with intermittent heating during that
period. Care must be taken to ensure that the
stain does not dry out, to counteract drying more
solution of stain is added to the slide and the
slide reheated. Heating of the stain is required
for penetration of the dye into cell wall.

48. •Wash in tap water.
•The stained smear is decolourised with
20% sulphuric acid and washed with water. This
step should be repeated till the pink/red colour
stops coming out. In case of lepra bacilli 5%
sulphuric acid is used as M. lepra is less acid
fast. Another alternative for decolourisation is
acid alcohol (3ml HCL and 97 ml ethanol)
•The smear is counterstained with 2%
methylene blue for 1-2 minutes. Malachite green
can also be used as counter stain instead of
methylene blue.

49. •Wash with tap water.
•Allow the smear to air dry and
examine under oil immersion objective.
•The acid fast bacteria retain the
fuchsin (red) colour, while the other take
counter stain.

50. C Morphology of bacteria
•​depending on their shape they are classified as
•​1 .
​cocci ( kokkos meaning berry )
•​2 .
​bacilli ( bacillus meaning rod )
•​coccobacilli , streptobacilli
•​chinese letter or cuneiform pattern
•​comma shaped , spirilla
•​3 . Spirochaetes
•​4 Actinomycetes
•​5 Mycoplasmas
•​6 Rickettsiae and chalmydiae
​

51. Bacterial anatomy
•the outer layer or cell envelope of a
bacterial cell consists of two
components
•​1 , a rigid cell wall
•​2, underlying cytoplasmic or plasma
membrane
•​the envelope encloses the protoplasm
which comprises cytoplasm ,
cytoplasmic inclusions ( mesosomes ,
ribosomes , inclusion granules ,
vacuoles ,) and a single circular
chromosome of deoxyribonucleic acid
DNA

52. •Besides these essential
components , some bacteria may
possess additional structures ,
such as capsule , flagella
and fimbriae

55. 1 cell wall
•it is a tough and rigid structure, surrounding
the bacterium like a shell
•​it is 10 to 25 nm in thickness and weighs about
20 to 25 % of the dry weight of the cell ,
it has following functions
•​accounts for the shape of the cell
•​provides protection to the cell against
osmotic damage
•​confers rigidity upon bacteria
•​it takes part in cell division

56. Differences between cell wall of
GP and GNB
•GPB
•Thickness :
thicker
•Periplasmic
space , absent
•Lipids, absent or
small
•Teichoic acid ,
present
•Peptidoglycan,16-
80 nm
•GNB
•Thinner
•Present
•Present
•Absent
•2nm

62. •it possesses target site for antibiotics
, lysozymes and bacteriophages .
•​it carries bacterial antigens that are
important in virulence and immunity
•​rigid part of the cell wall is a
peptidoglycan which is a mucopeptide
( murein ) composed of N- acetyl muramic
acid and and N - acetyl glucosamine
molecules alternating in chains ,
cross linked by peptide subunits

63. •the cell walls of gram positive
bacteria have simpler chemical nature
than those of gram negative
bacteria
•​1 gram negative cell wall
•​it is a complex structure with the
following component 1 . lipoprotein
layer : connects the peptidoglycan
to outer membrane
•​2 . outer membrane , this contains
certain proteins named as OMP ,
target

64. •sites for phages , antibiotics and
bacteriocins
•​c . ) lipopolysaccharides LPS , this
layer consists of lipid A to which is
attached a polysaccharide
•​it constitutes the endotoxin of GNB
polysaccharide determines a major
surface antigen , the O antigen
•​the toxicity , pyrogenicity , lethal
effects

65. •tissue necrosis of bacteria is
associated with lipidA
•​the periplasmic space : a space in
between the inner and outer membrane
•​it contains various binding proteins for
specific substrates
•​peptidoglycan
•​2 gram positive cell wall
•​a .) Peptidoglycan : this layer in GPB
is thicker 16 to 80 nm than that in
GNB 2nm

66. •b) teichoic acid : Gram positive cell wall
contains significant amounts of teichoic
acid which is absent in GNB
, it constitutes major surface antigens
of gram positive bacteria.
•​they are water soluble polymers
containing ribitol or glycerol polymers
•​TA are of 2 types , cell wall TA,
membrane TA ,
•​cell wall TA , is covalently linked to
peptidoglycan

67. •membrane TA to cytoplasmic
membrane
•​

other components , certain
GPCs also contain antigen such
as protein and polysaccharides

68. Demonstration of cell wall
•the cell wall cannot be seen by light
microscope and does not stain
with simple dyes
•​demonstration of cell wall can be
done by methods such as
•​a ) plasmolysis , bacteria is placed
in hypertonic saline , shrinkage of
the cytoplasm occurs , while cell
wall retains original shape and
size
•​b) Microdissection
•​c) differential staining

69. •d) reaction with specific antibody
•​e) electron microscopy
•​BACTERIA WITH DEFECTIVE CELL
WALL
•​synthesis of cell wall may be inhibited
or interfered by many factors such
as antibiotics , bacteriophages ,
and lysozyme
•​lysozyme , present in many tissue
fluids , lyses susceptible bacteria by
splitting the linkage of peptidoglycan in the
cell wall
​

70. •when lysozyme acts on a GPB in a
hypertonic solution , a protoplast is
formed
•​in case of a GNB , the result is a
spheroplast .
•​bacteria with defective cell wall may
probably have a role in the
persistence of certain chronic infections
such as pyelonephritis
•​bacteria without cell walls or with
deficient cell walls are of 4 types

71. •Mycoplasma , L- forms ,
protoplasts , and spheroplasts
•​1 , Mycoplasma,
•​this is a naturally occuring
bacteria without cell walls
•​classified as an independent
bacterial genus , they dont require
hypertonic environment for
maintenance and are stable in
culture medium
​

72. 2- L- forms
•Kleineberger- Noble , while studying
streptobacillus moniliformis in the lister
institute , London , observed abnormal
forms of the bacteria and named
them L- forms after the lister
institute
•​they develop either spontaneously or in
the presence of penicillin or other
agents that interfere with synthesis of cell
wall
•​these are difficult to cultivate and require
agar containing solid medium having

73. •right osmotic strength
•​they are sometimes spontaneously
formed in patients treated with penicillin
•​

L- forms are more stable than
protoplast and spheroplast
•​Protoplast , derived from GPB , they
contain cytoplasmic membrane and cell
wall is totally lacking .
•​produced artificially by lysozyme in a
hypertonic medium

74. •hypertonic medium is necessary for
their maintenance .
•​SPHEROPLAST : these are derived
from GNB, produced in presence of penicillin ,
they are osmotically fragile
•​must be maintained in hypertonic
culture medium
•​they differ from the protoplast in that
some cell wall material is retained

75. Pleomorphism and Involution
Forms
•certain species of bacteria exhibit
great variation in shape and size of
individual cells called pleomorphism
•​some bacteria show swollen and
aberrant forms in ageing laboratory
culture's and are known as involution
forms.
•​Defective cell wall synthesis is
often responsible for development of these
2 abnormal forms .