Professional Documents
Culture Documents
Bacteriology: Basics
Bacteriology: Basics
Basics
Morphology, Classification, Staining
Methods
Dr.T.V.Rao MD
Introduction:
Microorganisms several classes of living
beings
Based on the organization of their cellular
structures, all living cells can be divided into
two groups: eukaryotic and prokaryotic
Eukaryotic cell types - Animals, plants, fungi,
protozoans, and algae
Prokaryotic cell types - bacteria & blue green
algae
Dr.T.V.Rao MD 2
Antioni van Leeuwenhoek
Leeuwenhoek is called
"the inventor of the
microscope"
Created a simple
microscope that could
magnify to about 275x, and
published drawings of
microorganisms in 1683
Could reach magnifications
of over 200x with simple
ground lenses
Dr.T.V.Rao MD 3
How a Microscope Works with..
Ocular Lens Objective Lens
(Magnifies Image) (Gathers Light,
Magnifies
And Focuses Image
Body Tube Inside Body Tube)
Dr.T.V.Rao MD
(Image Focuses)
Dr.T.V.Rao MD
phase-contrast microscope
fluorescence microscopes
compound microscopes
image formed by action of 2 lenses 5
The Compound Microscope
The Optical System
Objective Lens: the lens closest to the
specimen; usually several objectives are
mounted on a revolving nosepiece.
Parafocal: when the microscope is focused with one
Dr.T.V.Rao MD
objective in place, another objective can be rotated
into place and the specimen remains very nearly in
correct focus.
Eyepiece or Ocular Lens: the lens closest to the
eye.
Monocular: a microscope having only one eyepiece
6
Binocular: a microscope having two eyepieces.
Phase Contrast Microscopy
light rays through objects of different
change in phase, not intensity
special ring-shaped condenser diaphragm
special glass disc in objective
change phase differences to intensity
differences
can view transparent
objects as dark on light
background (without staining)
Right; human brain glial
cells Dr.T.V.Rao MD 7
The Bright-Field Microscope
Produces a dark image against a
brighter background
Has several objective lenses
Dr.T.V.Rao MD
par focal microscopes remain in focus
when objectives are changed
total magnification
product of the magnifications of the
ocular lens and the objective lens 8
Fluorescence Microscopy
Illuminate specimen with UV visible fluorescence
(filter removes harmful UV)
View auto-fluorescent objects (e.g., chloroplasts)
Stain with specific fluorescent dyes, which absorb in
region 230-350 nm & emit orange, yellow or
greenish light
Images appear coloured against a dark background
Dr.T.V.Rao MD 9
Schematic of typical animal (eukaryotic) cell, showing subcellular
components.
Dr.T.V.Rao MD 10
Background Information
Prokaryotes
Dr.T.V.Rao MD 12
Differences between prokaryotic & eukaryotic cells
Character Prokaryotes Eukaryotes
Ribosomes 70 S 80 S
Dr.T.V.Rao MD 14
Differences between prokaryotic &
eukaryotic cells
Dr.T.V.Rao MD 15
Prokaryotic Cells
Much smaller (microns) and more simple
than eukaryotes
Prokaryotes are molecules surrounded by
a membrane and cell wall.
They lack a true nucleus and dont have
membrane bound organelles like
mitochondria, etc.
Large surface-to-volume ratio : nutrients
can easily and rapidly reach any part of
the cells interior
Dr.T.V.Rao MD 16
Size of Bacteria
Unit of measurement in
bacteriology is the micron
(micrometre, m)
Bacteria of medical importance
0.2 1.5 m in diameter
3 5 m in length
Dr.T.V.Rao MD 17
Eukaryotic cell Prokaryotic cell
(e.g. animal) Gram + Flagellum
Rough endoplasmic Cell membrane Nucleoid Cell wall
reticulum
Nucleus
Gram -
Pili
Granule
Capsule
Cytoplasm
Cell (inner) membrane Outer membrane
Mitochondria
Ribosomes
Dr.T.V.Rao MD
Cell wall 18
Shapes of Bacteria
Cocci spherical/ oval shaped major groups
Bacilli rod shaped
Vibrios comma shaped
Spirilla rigid spiral forms
Spirochetes flexible spiral forms
Actinomycetes branching filamentous
bacteria
Mycoplasmas lack cell wall
Dr.T.V.Rao MD 19
Bacteria Have One of Three Cellular
Shapes
Rods (bacilli)
Coccoid-Shaped
Spirilla
Dr.T.V.Rao MD 20
Arrangement of bacteria: Cocci
Coccus
Dr.T.V.Rao MD 21
Reproduction
Prokaryotic cell division is
binary fission.
Single DNA molecule that
first replicates.
Attaches each copy to a
different part of the cell
membrane.
Cell begins to pull apart.
Following cytokinesis, there
are then two cells of
identical genetic
composition.
Dr.T.V.Rao MD 22
Arrangement of bacteria: Bacilli
Dr.T.V.Rao MD 23
Other shapes of bacteria
Comma shaped
Spirilla
Spirochetes
Dr.T.V.Rao MD 24
Anatomy of a Bacterial Cell
Dr.T.V.Rao MD 25
Anatomy of A Bacterial Cell
Outer layer two components:
1. Rigid cell wall
2. Cytoplasmic (Cell/ Plasma) membrane
present beneath cell wall
Cytoplasm cytoplasmic inclusions,
ribosomes, mesosomes and nucleus
Additional structures plasmid, slime
layer, capsule, flagella, fimbriae (pili),
spores
Dr.T.V.Rao MD 26
Structure of Bacteria
All cells have 3 main components:
DNA (nucleoid)
genetic instructions
surrounding membrane (cytoplasmic
membrane)
limits access to the cells interior
cytoplasm, between the DNA and the
membrane
where all metabolic reactions occur
especially protein synthesis, which occurs
on the ribosomes
Bacteria also often have these features:
cell wall
resists osmotic pressure
flagella
movement
pili
attachment
capsule
protection and biofilms Dr.T.V.Rao MD 27
Typical shapes of bacteria
Dr.T.V.Rao MD 29
Characteristic grouping (or not grouping)
Dr.T.V.Rao MD 30
The Cell Envelope
Dr.T.V.Rao MD 31
GRAM POSITIVE
Lipoteichoic acid Peptidoglycan-teichoic acid
Cytoplasmic membrane
Cytoplasm
Thin peptidoglycan celll wall layer. Thick peptidoglycan celll wall layer.
Dr.T.V.Rao MD 34
Cell Envelope
The cell envelope is all the
layers from the cell
membrane outward,
including the cell wall, the
periplasmic space, the outer
membrane, and the capsule.
All free-living bacteria
have a cell wall
periplasmic space and
outer membrane are
found in Gram-negatives
the capsule is only found
in some strains
Dr.T.V.Rao MD 35
Structure & Function
of Cell Components
CELL WALL
Outermost layer, encloses cytoplasm
1. Confers shape and rigidity
2. 10 - 25 nm thick
Dr.T.V.Rao MD 38
Transport Across the Cell Membrane
Basic rule: things spontaneously
move from high concentration
to low concentration (downhill).
This process is called diffusion.
Getting many molecules into the
cell is simply a matter of opening
up a protein channel of the proper
size and shape. The molecules
then move into the cell by
diffusing down the concentration
gradient. Passive transport, or
facilitated diffusion.
Dr.T.V.Rao MD 41
Gram negative cell wall
Dr.T.V.Rao MD 43
Summary of the differences between
Gram positive & Gram negative bacteria
Protein content 0% 9%
Lipopolysaccharide 0 13%
Dr.T.V.Rao MD 47
Additional Organelles
1. Plasmid
Extra nuclear genetic elements consisting
of DNA
Transmitted to daughter cells during
binary fission
May be transferred from one bacterium
to another
Not essential for life of the cell
Confer certain properties e.g. drug
resistance, toxicity
Dr.T.V.Rao MD 48
Additional Organelles
2. Capsule & Slime layer
Viscous layer secreted around the cell
wall.
Polysaccharide / polypeptide in nature
Peritrichous monotrichous
(or amphi, or lophotrichous
Dr.T.V.Rao MD 52
FLAGELLA
Dr.T.V.Rao MD 54
Additional Organelles
4. Fimbriae/ Pili
Thin, hairlike appendages on the surface of many Gram-
negative bacteria
10-20 long, acts as organs of adhesion (attachment) - allowing
bacteria to colonize environmental surfaces or cells and resist
flushing
http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/nopili.html
Dr.T.V.Rao MD 55
Additional Organelles
5. Spores
Highly resistant resting
stages formed during
adverse environment
(depletion of nutrients)
Formed inside the
parent cell, hence called
Endospores
Very resistant to heat,
radiation and drying and
can remain dormant for
hundreds of years.
Formed by bacteria like
Clostridia, bacillus
Dr.T.V.Rao MD 56
The cycle of spore formation and germination
Spherical terminal
Free spore
Dr.T.V.Rao MD 58
Spores
Some bacteria can form very tough
spores, which are metabolically
inactive and can survive a long time
under very harsh conditions.
Allegedly, some bacterial
spores that were embedded
in amber or salt deposits for
25 million years have been
revived. These experiments
are viewed skeptically by
many scientists.
Dr.T.V.Rao MD 59
Spores
Spores can also survive very high or low temperatures and
high UV radiation for extended periods. This makes them
difficult to kill during sterilization.
Anthrax
Spores are produced only by a few genera in the Firmicutes:
Bacillus species including anthracis (anthrax) and cereus
(endotoxin causes ~5% of food poisoning)
Clostridium species including tetani (tetanus), perfringens
(gangrene), and botulinum (botulism: food poisoning from
improperly canned food)
Dr.T.V.Rao MD 60
Pleomorphic & Involution forms
Pleomorphism 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
Dr.T.V.Rao MD 61
Bacterial Taxonomy
Includes three components:
1. Classification : orderly
arrangement
2. Identification of an unknown
unit
3. Nomenclature : naming the
units
Dr.T.V.Rao MD 62
Bacterial Taxonomy: Classification
Orderly arrangement : Kingdom
Division Class Order Family Tribe Genus
Species
Phylogenetic classification represents a branching
tree like arrangement. One characteristic being used
for division at each branch or level
Molecular or Genetic classification based on the
degree of genetic relatedness of different organisms
Intraspecies classification based on biochemical
properties (biotypes), antigenic features (serotypes),
bacteriophage susceptibility (phage types)
Dr.T.V.Rao MD 63
Bacterial Taxonomy: Nomenclature
Two kinds of name are given to
bacteria
Casual / common name for local use,
varies from country to country
e.g. typhoid bacillus
Scientific / International Name same
all over world, consists of two words (in
Italics)
e.g. Salmonella typhi, Staphylococcus
Dr.T.V.Rao MD 64
Microscopy helps to Measure
and Observe the Bacteria
Measurement
Microorganisms are very small
Use metric system
Metre (m) : standard unit
Micrometre ( m) = 1 x10-6 m
Nanometre (nm) = 1 x10-9 m
Angstrom () = 1 x10-10 m
Dr.T.V.Rao MD 65
Why we should be Stain Bacteria
Bacteria have nearly the same refractive
index as water, therefore, when they are
observed under a microscope they are
opaque or nearly invisible to the naked
eye.
Different types of staining methods are
used to make the cells and their internal
structures more visible under the light
microscope. Dr.T.V.Rao MD 66
What is a Stain
A stain is a substance that adheres to a cell, giving
the cell color.
The presence of color gives the cells significant
contrast so are much more visible.
Different stains have different affinities for
different organisms, or different parts of
organisms
They are used to differentiate different types of
organisms or to view specific parts of organisms
Dr.T.V.Rao MD 67
Simple staining
Methylene blue, Basic fuchsin
Provide the color contrast but impart the
same color to all the organisms in a smear
Loffler's ethylene blue: Sat. solution of M. blue
in alcohol - 30mlKoH, 0.01% in water -
100mlDissolve the dye in water, filter. For
smear: stain for 3. For section: stain
Dr.T.V.Rao MD 68
Simple Staining Easier to Perform
But has Limitations
Dr.T.V.Rao MD 69
Differential Stains
Differential Stains use two or more stains
and allow the cells to be categorized into
various groups or types.
Both techniques allow the observation
of cell morphology, or shape, but
differential staining usually provides
more information about the
characteristics of the cell wall
(Thickness). Dr.T.V.Rao MD 70
Gram staining
Named after Hans
Christian Gram,
differentiates
between Gram-
positive purple and
Gram-negative pink
stains and is used to
identify certain
pathogens.
Dr.T.V.Rao MD 71
Gram staining - Principles
Gram staining is used to determine gram status to
classify bacteria broadly. It is based on the composition
of their cell wall. Gram staining uses crystal violet to
stain cell walls, iodine as a mordant, and a fuchsin or
safranin counterstain to mark all bacteria. Gram status
is important in medicine; the presence or absence of a
cell wall will change the bacterium's susceptibility to
some antibiotics.
Gram-positive bacteria stain dark blue or violet. Their
cell wall is typically rich with peptidoglycan and lacks
the secondary membrane and lipopolysaccharide layer
found in Gram-negative bacteria
Dr.T.V.Rao MD 72
Gram Staining Steps
1. Crystal violet acts as the primary stain. Crystal violet may also
be used as a simple stain because it dyes the cell wall of any
bacteria.
2. Grams iodine acts as a mordant (Helps to fix the primary
dye to the cell wall).
3. Decolorizer is used next to remove the primary stain (crystal
violet) from Gram Negative bacteria (those with LPS imbedded
in their cell walls). Decolorizer is composed of an organic
solvent, such as, acetone or ethanol or a combination of both.)
4. Finally, a counter stain (Safranin), is applied to stain those cells
(Gram Negative) that have lost the primary stain as a result of
decolorization
Dr.T.V.Rao MD 73
Structure and Reactivity to Gram
Staining.
Dr.T.V.Rao MD 74
Dr.T.V.Rao MD 75
Gm+ve cocci & Gm-ve bacilli
Dr.T.V.Rao MD 76
GRAM-POSITIVE BACTERIA
GRAM-POSITIVE BACTERIA
are characterized by having
as part of their cell wall
structure peptidoglycan as
well as polysaccharides
and/or teichoic acids. The
peptidoglycans which are
sometimes also called
murein are heteropolymers
of glycan strands, which are
cross-linked through short
peptides.
Dr.T.V.Rao MD 77
What are Gram Negative Bacteria
Gram-negative bacteria are those bacteria that
do not retain crystal violet dye in the Gram
staining protocol. In a Gram stain test, a counter
stain (commonly safranin) is added after the
crystal violet, coloring all Gram-negative bacteria
with a red or pink color. The test itself is useful in
classifying two distinct types of bacteria based on
the structural differences of their cell walls. On
the other hand, Gram-positive bacteria will retain
the crystal violet dye when washed in a
decolorizing solution.
Dr.T.V.Rao MD 78
Gram negative bacteria
On most Gram-stained
preparations, Gram-
negative organisms will
appear red or pink because
they are counterstained.
Due to presence of higher
lipid content, after alcohol-
treatment, the porosity of
the cell wall increases,
hence the CVI complex
(Crystal violet -Iodine) can
pass through. Thus, the
primary stain is not
retained. Dr.T.V.Rao MD 79
Gram Negative Bacteria
Also, in contrast to most
Gram-positive bacteria,
Gram-negative bacteria
have only a few layers
of peptidoglycan and a
secondary cell
membrane made
primarily of
lipopolysaccharide
Dr.T.V.Rao MD 80
ACID FAST STAINING
Dr.T.V.Rao MD 81
Acid-Fast Stain
Acid-fast cells contain a
large amount of lipids and
waxes in their cell walls
primarily mycolic acid
Acid fast bacteria are
usually members of the
genus Mycobacterium or
Nocardia
Therefore, this stain is
important to identify
Mycobacterium or Nocardia
Dr.T.V.Rao MD 82
Ziehl-Neelsen stain
Ziehl-Neelsen staining is used to stain
species of Mycobacterium tuberculosis
that do not stain with the standard
laboratory staining procedures like Gram
staining.
The stains used are the red colored
Carbol fuchsin that stains the bacteria
and a counter stain like Methylene blue
or Malachite green.
Dr.T.V.Rao MD 83
Acid-Fast Organisms
Primary stain binds cell wall mycolic acids
Intense decolorization does not release
primary stain from the cell wall of AFB
Color of AFB-based on primary stain
Counterstain provides contrasting
background
Dr.T.V.Rao MD 84
AFB Staining Methods
Zeihl
Neelsens-hot
stain
Kinyouns-cold
stain
Modifications
Dr.T.V.Rao MD 85
ALBERTS STAINING FOR
C.diptheria
Dr.T.V.Rao MD 86
Diphtheria is Serious Disease
When you suspect Diphtheria
In all cases of suspected
cases of Diphtheria, stain
one of the smears with
Gram stain
If Gram stained smear
shows morphology
suggestive of C.diptheria,
proceed to do Albert
staining which
demonstrates the presence
or absence of
metachromatic granules.
Dr.T.V.Rao MD 87
Appearance of C.diptheria
C.diptheria are thin Gram positive bacilli, straight
or slightly curved and often enlarged (clubbing) at
one or both ends and are arranged at acute
angles giving shapes of Chinese letters or V shape
which is characteristic of these organisms (Fig 1).
Present in the body of the bacillus are numerous
metachromatic granules which give the bacillus
beaded or barred appearance. These granules are
best demonstrated by Alberts stain.
Dr.T.V.Rao MD 88
Albert staining
Albert stain I Albert stain II
Toluidine blue 0.15 gm
Malachite green 0.20
Iodine 2.0 gm
gm Potassium
Glacial acetic acid 1.0 iodide 3.0 gm
ml
Alcohol(95%) 2.0 ml Distilled water
Distilled water 100 ml 300 ml
Dr.T.V.Rao MD 89
Albert staining Procedure
Cover the heat-fixed smear with Albert
stain I. Let it stand for two minutes.
Wash with water.
Cover the smear with Albert stain II.
Let it stand for two minutes.
Wash with water, blot dry and
examine.
Dr.T.V.Rao MD 90
How the C.diptheria appear
To demonstrate
metachromatic
granules in
C.diptheria. These
granules appear
bluish black whereas
the body of bacilli
appear green or
bluish green.
Dr.T.V.Rao MD 91
Programme created by Dr.T.V.Rao MD
from several resources in world wide
web, and Thankful for Dr. Ekta
www.medmicrobes from basic
programme on Bacterial cell
Email
doctortvrao@gmail.com
Dr.T.V.Rao MD 92