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2. Objectives of today’s class
T
o define the term prokaryote &eukaryote
T
o understand different shapes of bacteria
T
o describe bacterial anatomy in detail
T
o explain about bacterial growth curve
T
o know the factors affecting bacterial growth
01/31/18 2
3. Introduction
• L
iving organisms classified under3 kingdoms:
1.Plant kingdom
2.Animal kingdom
3.P
rotista
• Under Protista- Microorganismsareclassified as
i.P
ROK
ARYOT
E
S (Bacteria& Blue-green algae)
ii.E
UK
ARYOT
E
S (Fungi, Protozoa, slimemolds& other algae)
01/31/18 3
4. P
rokaryotes
• B
acteria are single celled microorganism containing a single
circularchromosome
• B
acteria lack Nuclearmembrane, nucleolus, deoxyribo-
nucleoprotein.
• M
itotic division is absent in bacteria
• Cytoplasmic streaming, pinocytosis, M
itochondria, L
ysosomes,
Golgi apparatus, E
ndoplasmic reticulum are absent in the
bacterial cytoplasm.
• Chemical composition (Sterols-, M
uramic acid+)
• B
acteria do not contain chlorophyll; B
acteria do not show true
branching. E
xception: Actinomycetales (H
igherbacteria)
01/31/18 4
5. P
rokaryotic Cells
• M
uch smaller(microns) and more simple than eukaryotes
• prokaryotes are molecules surrounded by a membrane and cell
wall.
• they lack a true nucleus and don’t have membrane bound
organelles like mitochondria, etc.
• large surface-to-volume ratio : nutrients can easily and rapidly
reach any part of the cells interior
7. Differences between prokaryotic & eukaryotic cells
Character Prokaryotes E
ukaryotes
Nucleus Nuclear
membrane
Absent Present
Nucleolus Absent Present
Chromosome One circular One ormore paired and
linear
Cell division B
inary fission M
itosis
Cytoplasmic
membrane
Structure and
Composition
fluid phospholipid bilayer,
lacks sterols
fluid phospholipid bilayer
containing sterols
F
unction Incapable of endocytosis
(phagocytosis and
pinocytosis) and exocytosis
Capable of endocytosis
and exocytosis
8. Differences between prokaryotic & eukaryotic cells
Character Prokaryotes E
ukaryotes
Cytoplasm M
itochondria Absent P
resent
L
ysosomes Absent P
resent
Golgi apparatus Absent P
resent
E
ndoplasmic
reticulum
Absent P
resent
Vacuoles Absent P
resent
Ribosomes 70 S 80 S
9. Differences between prokaryotic & eukaryotic cells
Character Prokaryotes E
ukaryotes
Cell W
all Present Animals &P
rotozoans –
Absent
P
lants, F
ungi &Algae -
P
resent
Composition Peptidoglycan –
complex carbohydrate
Cellulose orchitin
L
ocomotor
organelles
F
lagella F
lagella/Cilia
10. Size of bacteria
• Unit of measurement used in bacteriology is micron or
micrometre (µm)
• One micron is equal to one thousandth of millimetre
• One nanometre (nm) ormillimicron (mµ) is equal to
one thousandth of a micron orone millionth of a
millimetre
• One Angstrom unit (Å) is equal to one tenth of a
nanometre
01/31/18 10
11. L
imit of resolution
• L
imit of resolution with the unaided human eye is 200
microns (0.2mm)
• B
acteria are even much smallerand can be visualized
only undermagnification.
• B
acteria of medical importance generally measure 0.2-
1.5µm in diameterand about 3-5µm in length
01/31/18 11
12. M
icroscopy
• L
eeuwenhoek – hand ground lenses – F
irst observed bacteria
• P
arts of a microscope: E
ye piece (5x/
10x), Arm, B
ase,
M
echanical stage, L
ight source, Condenser, Diaphragm,
Objectives (Scanner, L
P
, H
P
, OI), Cylindrical tube length,
Stage adjustor(H
orizontal, vertical), F
ine adjustment and
coarse adjustment knob.
• M
agnification power
• Resolution power
• T
otal magnification: Objective x E
yepiece
• Refractive index of lens (glass), air, water&oil
• F
ocal length of lens of human eye (Range:16-25cm)
01/31/18 12
13. T
ypes of M
icroscopes
a. Optical orlight microscope
b. P
hase contrast microscope
c. F
luorescent microscope
d. E
lectron microscope
e. Dark field /Dark Ground microscope: 2 types namely
polarisation &interference microscopes
01/31/18 13
14. Staining techniques
• Simple stains providecolour contrast but impart samecolour
to all bacteria. Ex: Methyleneblue, Basic fuchsin.
• Negative staining provideuniformly coloured background
against which theunstained bacteriastand out in contrast. Ex:
Indiaink or Nigrosin.
• Impregnation methods for Cellsand structurestoo thin to be
seen under ordinary microscopethat may berendered visibleif
they arethickened by silver impregnation on thesurface. Used
for demonstration of Spirochetesand Bacterial flagella.
01/31/18 14
15. Staining techniques – Contd…
• Differential stains: Stainsimpart different colour to
different bacteriaor itsstructures. Ex: Gram stain & Acid fast
stain.
01/31/18 15
16. Shape of B
acteria
• Cocci – spherical/oval shaped majorgroups
• B
acilli – rod shaped
• Vibrios – comma shaped
• Spirilla – rigid spiral forms
• Spirochetes – flexible spiral forms
• Actinomycetes – branching filamentous bacteria
• M
ycoplasmas – lack cell wall
17. Arrangement of bacteria: Cocci
Cocci in pair – Diplococcus
Sarcina – groups of eight
Tetrad – groups of four
Cocci in chain - Streptococci
Cocci in cluster - Staphylococci
Coccus
23. CE
L
LW
AL
L
• Outermost layer, encloses cytoplasm
1. Confers shape and rigidity
2. 10 - 25 nm thick
3. Composed of complex polysaccharides (peptidoglycan/mucopeptide) -
formed by N acetyl glucosamine (NAG) & N acetyl muramic acid
(NAM
) alternating in chains, held by peptide chains.
24. • Cell wall –
4. Carries bacterial antigens – important in virulence &immunity
5. Chemical nature of the cell wall helps to divide bacteria into two broad
groups – Gram positive &Gram negative
6. Gram +ve bacteria have simplerchemical nature than Gram –ve
bacteria.
7. Several antibiotics may interfere with cell wall synthesis e.g. P
enicillin,
Cephalosporins
25. Gram positive cell wall
The Gram-positive cell wall is composed of a thick, multilayered peptidoglycan sheath outside of the
cytoplasmic membrane. Teichoic acids are linked to and embedded in the peptidoglycan, and
lipoteichoic acids extend into the cytoplasmic membrane
26. Gram negative cell wall
The Gram-negative cell wall is composed of an outer membrane linked to thin, mainly single-layered
peptidoglycan by lipoproteins. The peptidoglycan is located within the periplasmic space that is created
between the outer and inner membranes. The outer membrane includes porins, which allow the passage of
small hydrophilic molecules across the membrane, and lipopolysaccharide molecules that extend into
extracellular space.
28. Differences between Gram positive &Gram
negative
Property of bacteria Gram Positive
Gram Negative
Thickness of wall 20-80 nm 10 nm
Number of layers in wall 1 2
Peptidoglycan content >50% 10-20%
Teichoic acid in wall + -
Lipid & lipoprotein content 0-3% 58%
Protein content 0% 9%
Lipopolysaccharide 0 13%
Sensitive to penicillin Yes Less sensitive
Digested by lysozyme Yes Weakly
29. Cytoplasmic (P
lasma) membrane
• T
hin layer5-10 nm, separates cell wall from cytoplasm
• Acts as a semipermeable membrane: controls the inflow and outflow
of metabolites
• Composed of lipoproteins with small amounts of carbohydrates
30. OtherCytoplasmic Components
• Ribosomes – protein synthesis
• M
esosomes –
1. M
ultilaminated structures formed as invaginations of plasma membrane
2. P
rincipal sites of respiratory enzymes
3. Coordinate nuclear&cytoplasmic division during binary fission
4. M
ore prominent in Gram +ve bacteria
• Intracytoplasmic inclusions – reserve of energy &phosphate forcell
metabolism e.g. M
etachromatic granules in diphtheria bacilli
31. Nucleus
• No nucleolus
• No nuclearmembrane
• Genome –
• single, circulardouble stranded DNA.
• H
aploid
• Divides by binary fission
32. Additional Organelles
1. P
lasmid –
• E
xtranucleargenetic elements consisting of DNA
• T
ransmitted to daughtercells during binary fission
• M
ay be transferred from one bacterium to another
• Not essential forlife of the cell
• Confercertain properties e.g. drug resistance, toxicity
33. Additional Organelles
2. Capsule &Slime layer–
• Viscous layersecreted around the cell wall.
• P
olysaccharide /polypeptide in nature
a) Capsule – sharply defined structure, antigenic in
nature
• P
rotects bacteria from lytic enzymes
• Inhibits phagocytosis
• Stained by negative staining using India Ink
• Can be demonstrated by Quellung reaction (capsule
swelling reaction)
a) Slime layer– loose undemarcated secretion
34. Additional Organelles
3. F
lagella –
L
ong (3 to 12 µm), filamentous surface appendages
Organs of locomotion
Chemically, composed of proteins called flagellins
T
he numberand distribution of flagella on the bacterial surface are characteristic fora
given species - hence are useful in identifying and classifying bacteria
F
lagella may serve as antigenic determinants (e.g. the Hantigens of Gram-negative
enteric bacteria)
P
resence shown by motility e.g. hanging drop preparation
35. T
ypes of flagellararrangement
Polar/ Monotrichous – single flagellum at one
pole
Lophotrichous – tuft of flagella at one pole
Peritrichous – flagella all over
Amphitrichous – flagella at both poles
Amphilophotrichous – tuft of flagella at both ends
36. Flagella
• Long unbranched sinuous filaments present on motile bacteria
• Made of 3 parts
• Filament, hook, and basal body
• Filament is external and hook basal body complex embedded in the cell
envelope
• Hook and basal body are antigenically different
37.
38. • Detachment of flagella does not affect the viability of cell
• 3-20 m long, uniform diameter
μ
• M
ade up of a protein flagellin
• F
lagella of different genera - similarchemical composition they
are antigenically different
• F
lagellarantibodies help in serodiagnosis
42. Shape & position of bacterial spore
Oval central
Spherical central
Oval sub terminal
Oval sub terminal
Oval terminal
Spherical terminal
Free spore
Non bulging
Bulging
43. Capsule/ slime layer
• B
acteria secrete viscid material
• organised into capsule - pneumococcus
• L
oose undemarcated secretion (slime layer) -
leuconostac
• M
icrocapsules – too thin to be seen underlight
microscope
• B
oth capsule and slime layermay be present
44. •Slime layer/
capsule
• polysaccharide orpolypeptide
• mucoid colonies on culture media
•Slime layer
• little affinity forgram stain
•Capsule
• stained by negative staining
• Capsularmaterial antigenic.
• Serological tests with anti capsularserum
• Quellung reaction – Neufeld in 1902
48. • F
ine hairlike appendages
• Shorterand thinnerthan flagella projecting from cell membrane
• P
resent on both motile and non motile bacteria
• M
ade up of pilin
• 8 morphological types known
• Classified as common orsex pili
• Conferadhesive property to the cell
• Disappearfollowing subcultures
49. Properties
• Agglutinate red blood cells of guinea pigs, horses and pigs
strongly
• H
uman and sheep cells weakly
• Detected by hemagglutination
• Antigenically similarand may cause cross reaction in
serological tests
50. Sex pili
• L
ongerand fewer
• F
ound on male bacteria
• Attach to female bacteria
forming hollow conjugation
tubes through which genetic
material is transferred
51.
52. Spores
• H
ighly resistant resting stages
• E
ndospores – formed inside cell
• Due to deletion of exogenous nutrients /unfavourable environment
• Young spores are seen attached to parent cell
• Appearas unstained area in gram stain
• Special spore stain can be used
59. Pleomorphism & involution forms
• Variation in shape and size of bacteria – pleomorphism
• Aberrant forms in ageing cultures – involution forms
• Due to defective cell wall synthesis
60. L forms
• Swollen cells and aberrant morphological forms observed by
Kleinberger Nobel at Lister institute, London
• Develop spontaneously or in the presence of agents that interfere
with cell wall synthesis
• May be stable or unstable
• Resemble mycobacteria
62. Pleomorphism & Involution forms
• P
leomorphism – great variation in shape &size of individual cells
e.g. Proteus species
• Involution forms – swollen &aberrant forms in ageing cultures,
especially in the presence of high salt concentration e.g. plague
bacillus
• Cause – defective cell wall synthesis
63. Bacterial Taxonomy
• Includes three components:
1. Classification : orderly arrangement
2. Identification of an unknown unit
3. Nomenclature: naming theunits
64. Bacterial Taxonomy: Classification
• Orderly arrangement :
• Kingdom – Division – Class– Order – Family – Tribe– Genus– Species
Phylogenetic classification – representsabranching treelikearrangement. One
characteristic being used for division at each branch or level
Molecular or Genetic classification – based on thedegreeof genetic relatednessof
different organisms
Intraspeciesclassification – based on biochemical properties(biotypes), antigenic
features(serotypes), bacteriophagesusceptibility (phagetypes)
65. Bacterial Taxonomy: Nomenclature
• T
wo kinds of name are given to bacteria
• Casual /common name – forlocal use, varies from country to country
e.g. “typhoid bacillus”
• Scientific /International Name – same all overworld, consists of two
words (in Italics) e.g. Salm
onellaty
phi,
Staphy
lococcus aureus
66. What is Physiology?
• Study of the mechanical, physical and biochemical functions
of living organisms.
67. Oxygen requirements
• Aerobic respiration is the release of energy from glucose or
anotherorganic substrate in the presence of Oxygen.
• Obligateaerobes-Pseudomonas
• Microaerophilic –Streptococcus
69. Carbondioxide – factor affecting growth
• Small amount of CO2 is produced by most bacteria and used
endogenously formetabolism
• Capnophiles – bacteria which require a higherCO2 tension (5 –
10%) to grow.
• eg: B
rucellae, meningococcus , gonococcus
70. Bacterial vitamins
• Growth factors to be provided exogenously forgrowth.
• E
ssential
• Accessary
• H
.influenzae requires accessary factors X (haemin) and V
(NAD) present in blood to grow.
71. Effect of light
• Bacteria usually grow well in the dark.
• Phototrophs – need light to grow
• Pigmented bacteria like cyanobacteria
72. Reaction
AUTOTROPHS HETEROTROPHS
PHOTO CHEMO PHOTO CHEMO
ENERGY
FIXING
Light
energy
Inorganic
compnds
Light
energy
Organic
compnds
CARBON
FIXING
CO2 CO2 Organic
compnds
Organic
compnds
Cyanobacteria
Green sulphur &
Purple sulphur
bacteria
Nitrosomonas
Nitrobacter
Non Purple sulphur
Non green sulphur
bacteria
Most bacteria
All fungi
protozoa
74. • L
ag phase - adaptation
- build necessary enzyme &metabolic intermediates
• L
og/exponential phase – division by binary
fission
numberof cells
• Stationary phase – cell division stop due to depletion of nutrients &
accumulation of toxic products
• P
hase of decline – population decrease due to cell death
76. E
.coli M
.leprae
20 minutes 20 days
• At extremes we eitherhave a mass progeny (4000 tones) in
24 hours in a continuous culture (or) no replication at all.
• In laboratories it is always a batch culture where the nutrients
are limited and toxic products accumulate.
• GE
NE
RAT
ION T
IM
E
77. Bacterial count
• T
otal count – total numberof bacteria in the sample
• Direct counting
• Coultercounter
• Viable count – measures the numberof living organisms
• Dilution methods
• P
lating methods
82. Osmotic effect and mechanical stress
• Tolerant to osmotic variations esp Gram positive bacteria
• Sudden transfer from concentrated solution to distilled water –
plasmoptysis
• Mechanical stress like grinding, vigorous shaking with glass beads or
exposure ultrasonic vibration – rupture of cell
