Bacteria

CELL WALL
PLASMA MEMBRANE
RIBOSOMES
NUCLEOID
CAPSULE
PILI
FLAGELLA
CYTOPLASM
General Structure
Gram (+) Bacteria
Gram (-) Bacteria
Growth & Nutrition
Lecturer:
Susceptibility Testing
Lea D. Ballares, RMT, M. Bio. Ed.
Bacterial Diseases
Slides by LDBallares
BACTERIA Slides by LDBallares
Average size: 0.2-10.0 um in diameter

Basic shapes:
BACILLI
COCCI
SPIRAL
Elongated cocci:
COCCOBACILLI
Examples:
Listeria monocytogenes
2. Haemophilus influenzae
1.
Unusual shapes
Star-shaped Stella
Square Haloarcula
Genetically, most bacteria are monomorphic (one
shape)
A few are pleomorphic based on environmental
conditions
Pairs: diplococci,
diplobacilli
Packets of four:
tetrads
Packets of eight:
octads
Clusters:
staphylococci
Chains: streptococci,
streptobacilli
Pallisade arrangement: Corynebacterium diphtheria
MEDICALLY
IMPORTANT
COCCI
Enterococcus spp.
Neisseria spp.
Streptococcus spp.
MEDICALLY
IMPORTANT
BACILLI
Enterobacter spp.
Escherichia spp.
Klebsiella spp.
Proteus spp.
Salmonella spp.
Shigella spp.
Pseudomonas
aeruginosa
Bacillus spp.
Clostridium spp.
MEDICALLY
IMPORTANT
CURVED/SPIRAL
CURVED BACILLI
Vibrio cholera
Campylobacter
spp.(gull-wing)
SPIROCHETE
Treponema pallidum
Borrelia spp. (Lyme
disease & relapsing
fever)
Some bacteria may lose their
characteristic shape because

of adverse growth conditions
CELL WALL DEFICIENT
BACTERIA shapeless but
revert back to their original
shape when placed under
favorable growth conditions.
PLEOMORPHIC
No cell wall
Has the ability to
exist in variety of
shapes
Example:
Mycoplasma spp.
LIVING STATE
WET MOUNT
To observe shape &
arrangement of
organism
A drop of bacterial
suspension on slide,
cover it with coverslip
& focus.
LIVING STATE
HANGING DROP
To observe organisms
motility.
A hanging drop slide w/
concavity at the center
is used.
FIXED STATE
Adhere organism on
slide & apply stain.
Bacteria are colorless, transparent &
difficult to see.
It is the process
coloring the
microorganisms with a
dye that emphasizes
certain structures.
PREPARATION
FOR STAINING
A thin film of a
solution of microbes
on a slide is a smear.
A smear is usually
FIXED to attach the
microbes to the slide
and to kill the
microbes.
Stains consist of a positive and negative ion.

In a basic dye,
the chromophore
is a cation.
In an acidic dye,
the chromophore
is an anion.
SIMPLE
STAINS
It is made up of an aqueous
solution
To observe bacterial shape &
arrangement.
A mordant may be used to
hold the stain or coat the
specimen to enlarge it.
DIFFERENTIAL GRAM STAIN
STAINS
Developed in 1884 by Danish
Bacteriologist Hans Christian

Gram.
It is one of the most useful
procedures because it divides
the bacteria into 2 large
groups: gram (+) and gram (-).
Color of
Color of
Primary stain:
Gram + cells
Purple
Gram cells
Purple
Crystal violet
Mordant:
Purple
Purple
Iodine
Decolorizing agent:
Purple
Colorless
Alcohol-acetone
Counterstain:
Purple
Red
STEPS
Safranin
DIFFERENTIAL ACID-FAST STAIN
STAINS
Acid Fast stain binds only to
those bacteria that have a

waxy material in their cell
walls.
It is used to identify
Mycobacterium spp.
1. Mycobacterium tuberculosis
2. Mycobacterium leprae
*** Nocardia spp.

DIFFERENTIAL
Cells that retain a basic stain
STAINS
(bacteria that have a waxy

material in their cell walls) in
the presence of acid-alcohol
are called acid-fast bacteria
(stained red).
Nonacid-fast cells (stained
blue) lose the basic stain
when rinsed with acidalcohol, and are usually
counterstained to see them.
SPECIAL
STAINS
Are used to
color and
isolate
specific
parts of
microorgani
sms.
Negative staining is the
process in which the

background & not the organism
is stained.
SPECIAL
STAINS
Heat is required to drive a
stain into endospores.
Are used to
color and
isolate
specific
parts of
microorgani
sms.
SPECIAL
STAINS
Are used to
Flagella staining requires a
mordant to make the flagella

wide enough to see.
color and
isolate
specific
parts of
microorgani
sms.
CULTURE MEDIA anything that possess

nutritional & environmental requirements for
bacterial growth.
3 TYPES OF CULTURE:
Pure Culture made up of one specie of
bacteria
Mix Culture made up of organisms
belonging to different species.
Stock Culture pure culture of
microorganisms as a source of supply in the
industry.
CLASSIFICATION OF
CULTURE MEDIA
(According to Physical
State):
Liquid culture
medium contains no
agar, hardening or
solidifying substances
AGAR polysaccharide extracts of seaweed &
commonly used base medium.

CLASSIFICATION OF
CULTURE MEDIA
(According to Physical
State):
Semi-solid medium
contains gelatin or 0.51% agar.

Solid culture medium
contains 2-3% agar.
CLASSIFICATION OF CULTURE MEDIA

(According to Composition):
Synthetic culture medium exact
composition is known
Non-synthetic culture medium exact
composition is not known.
Tissue culture medium made up of living
cells.

(According to how it is distributed):
PLATED MEDIUM distributed in petri dishes

(According to how it is distributed):
TUBED MEDIUM distributed in test tubes

(According to use):
SIMPLE
MEDIUM
Supports the growth &
multiplication of
microorganisms.
It is used for routine cultivation
& maintenance of
microorganisms.
Example: Nutrient Agar

(According to use):
ENRICHMENT
MEDIUM
Enhances the propagation of
certain organisms.
Examples: selenite broth,
tetrathionate broth

(According to use):
ENRICHED
MEDIUM
Contains nutritive supplements
needed for the growth of some

organisms.
Examples: blood agar plate,
chocolate agar plate

(According to use):
DIFFERENTIAL Distinguishes organisms
MEDIUM
growing together by
differences in their cultural

characteristics.
Examples: EMB, Mc Conkey
Agar

(According to use):
SELECTIVE
MEDIUM
Promotes the growth of
desirable organisms but at

the same time inhibiting the
growth of others.
Examples: Bismuth Sulfide
Agar isolates Salmonella
Esherichia coli on Eosin Methylene Blue Agar (EMB).

EMB is both a selective & differential media. It is
selective for the growth of gram (-) bacilli

(According to use):
SPECIAL
CULTURE
MEDIUM
Specifically prepared to
support the growth of specific

organisms.
Examples:
1. Petragnani medium
Mycobacterium tuberculosis
2. Thayer Martin Neisseria
spp.
GENERAL STEPS IN PREPARING CULTURE MEDIA

WEIGH
INGREDIENTS
DISSOLVE
INGREDIENTS
In plated media: Sterile first
before distribution.
In tubed media: Distribute
first before sterilization.
ADJUST TO
PROPER PH
STERILIZATION
DISTRIBUTION IN STERILE
PETRI DISHES
TECHNIQUES
OF
INOCULATION
INOCULATION
To introduce
microorganisms
into a culture
medium or host.
LIQUID CULTURE MEDIUM
inoculate the organism & shake.

SLANT TUBED MEDIUM
streaking from the bottom with a
zigzag fashion
BUTT MEDIUM stabbing
BUTT/SLANT stab, then
streak
PLATED MEDIUM - streaking
METHODS OF STREAKING PLATED MEDIUM

RADIAL STREAK METHOD place the
inoculum one side of the plate, then bring on

the other side concentric fashion.
OVERLAP STREAK METHOD keeps on
overlapping, used in sensitivity testing.
MULTIPLE STREAK MEDIUM divide the
medium into several division, then streak
separately
METHODS OF STREAKING PLATED MEDIUM

INTERRUPTED STREAK METHOD start
streaking on one side of the plate, stop, then

turn for 180 degrees & streak again
MULTIPLE INTERRUPTED STREAK
METHOD used to obtain pure isolated
colonies.
Works well when the bacteria is present in high

numbers.
A pure culture contains only one species or
strain
A colony is a population of cells arising from
a single cell or spore or from a group of
attached cells
A colony is often
called a colonyforming unit

(CFU)
COLONY
MORPHOLOGY
TYPES OF COLONIES:
S or Smooth colonies
with uniform texture &
homogenicity, associated
w/ virulent organisms
M or Mucoid Colonies
exhibits slimy & watery
appearance, associated w/
capsulated & virulent
organisms.
COLONY
MORPHOLOGY
TYPES OF COLONIES:
R or Rough Colonies
granulated in appearance.
COLONY
MORPHOLOGY
A colony contains millions
of organisms.
Size, shape, color,
elevation & margin are
observed to identify the
bacteria.
GROWTH PATTERNS IN PLATES
SIZE: pinpoint, small,

moderate, or large
PIGMENTATION: color
of colony.
FORM: The shape of
the colony.
(1) Circular: unbroken
peripheral edge.
(2) Irregular: indented
peripheral edge.
(3) Rhizoid: rootlike
spreading growth.

MARGIN: The
appearance of the outer
edge of the colony .
(1) Entire: sharply defined,
even.
(2) Lobate: marked
indentations.
(3) Undulate: wavy
indentations
(4) Serrate: toothlike
appearance
(5) Filamentous:
threadlike, spreading
edge

ELEVATION: The degree
to which the colony
growth is raised on the
agar surface.
(1) Flat: elevation not
discernible.
(2) Raised: slightly
elevated.
(3) Convex. Dome
shaped elevation.
(4) Umbonate. Raised
with elevated convex
region
It refers to the number of cells, not the cell
size.
Growing microbes increases in number.
Bacteria multiply by a
process called binary

fission.
The time required for the
cell to divide or the
population to double is
called generation
(doubling) time.
STAGES OF BINARY FISSION

Bacilli following division
Chromosome division, cell growth
by lengthening
Chromosome divided, cell fully lengthened,
growth of envelope,Chromosomes
segregated
Cross wall completed
Daughter cells separate
Rate of reproduction is equivalent

to death rate due to exhaustion of
nutrients
Bacteria are multiplying at a

constant & maximum rate
Bacteria are adjusting to a new

environment, synthesizing
enzymes & actively metabolizing
The rate of reproduction has

completely stopped due to lack of
nutrients & formation of toxic
products
All organisms, whether they be

NUTRITIONAL
REQUIREMENTS
bacteria, humans, or trees,

need a constant supply of food
in order to live.
Water which is absolutely
essential for cellular function.
Carbon which is the major
structural element in cell
constituents
Energy required for cellular

NUTRITIONAL
REQUIREMENTS
growth.
Nitrogen which is also an
important structural elements,
being a constituent of proteins
& nucleic acids
Traces of other elements
required for life processes.
All organisms, whether they be

NUTRITIONAL
REQUIREMENTS
bacteria, humans, or trees,

need a constant supply of food
in order to live.
FASTIDIOUS organisms that
has demanding nutritional
requirements.
REQUIREMENTS FOR
GROWTH:
1.Physical
temperature, pH,
osmotic pressure
2.Chemical Water,
sources of carbon &
nitrogen, minerals,
oxygen & organic
growth factors.
PHYSICAL
REQUIREMENTS
TEMPERATURE
PSYCHROPHILES cold
loving microbes.
MESOPHILES
moderate-temperature
loving microbes
THERMOPHILES heat
loving microbes.
TEMPERATURE RANGE
PHYSICAL
REQUIREMENTS MINIMUM GROWTH
TEMPERATURE is the
lowest temperature at
which species will grow
OPTIMUM GROWTH
TEMPERATURE is the
temperature at which the
species grows best.
TEMPERATURE RANGE
PHYSICAL
REQUIREMENTS MAXIMUM GROWTH
TEMPERATURE - is the
highest temperature at
which growth is possible.
PHYSICAL
REQUIREMENTS
TEMPERATURE
PSYCHROTROPHS
Grow between 0C and 2030C
Cause food spoilage
Also known as moderate
psychrophiles or facultative
psychrophiles
PHYSICAL
REQUIREMENTS
pH
Most bacteria grow between
pH 6.5 and 7.5.

Molds and yeasts grow
between pH 5 and 6 (greater
pH range compared to
bacteria).
PHYSICAL
REQUIREMENTS
pH
ACIDOPHILIC BACTERIA
remarkably tolerant of
acidity
BASOPHILIC BACTERIA
grows at pH near neutrality.
OSMOTIC PRESSURE is
PHYSICAL
the pressure that is exerted
REQUIREMENTS
OSMOTIC
PRESSURE
on a cell membrane by
solutions inside & outside
the cell.
What are the effects of the
ff. solutions in a bacterial
cell?
1. Hypertonic
2. Isotonic
3. Hypotonic
HYPERTONIC SOLUTION
PHYSICAL
REQUIREMENTS The cell membrane & cytoplasm
shrink away from the bacterial
OSMOTIC
cell wall PLASMOLYSIS.
PRESSURE
Salts & Sugars are added to
certain foods to preserve them.
Bacteria in hypertonic
environment will die as a result of
desiccation.
HYPOTONIC SOLUTION
PHYSICAL
REQUIREMENTS If a bacterial cell is placed in a
hypotonic solution, the fluid
OSMOTIC
pressure w/in the cell increases
PRESSURE
greatly.
If the pressure becomes so great
& cell bursts, cytoplasm escapes
from the cell PLASMOPTYSIS.
ISOTONIC SOLUTION
PHYSICAL
REQUIREMENTS In an isotonic environment, water
neither leaves nor enter the cell.
OSMOTIC
PRESSURE
HALOPHILES
PHYSICAL
REQUIREMENTS halo referring to salt & philic
OSMOTIC
PRESSURE
meaning to love
Bacteria that loves salty
environment.
Example: Vibrio cholera
HALODURIC
PHYSICAL
REQUIREMENTS Organisms that do not prefer
OSMOTIC
PRESSURE
to live in a salty environment

but are capable of surviving
there.
Example: Staphylococcus
aureus
BAROPHILES
PHYSICAL
REQUIREMENTS Organisms that thrive deep in
BAROMETRIC
PRESSURE
ocean & oil wells, where

atmospheric pressure is very
high.
Water in the liquid state is

CHEMICAL
REQUIREMENTS essential for the existence of all
living organisms.
WATER OR
Approximately 75% water is
MOISTURE
present in the cells of every living
organism, including bacteria.
This amount of water is required
to maintain the cell in an active
state, and without liquid water,
living organisms will not be able to
grow or reproduce.
Substances such as sugar and

CHEMICAL
REQUIREMENTS salt makes the water unavailable
for bacteria.
WATER OR
The amount of water available for
MOISTURE
microbial growth is referred to as
Water Activity.
Oxygen is essential for the

CHEMICAL
REQUIREMENTS growth of many bacteria, but
OXYGEN
for others it is lethal.

All bacteria have cell
components that are sensitive
to oxygen and metabolic byproducts of oxygen.
Organisms that live in air
have enzymes that detoxify
these products.
CHEMICAL
Obligate Aerobes absolutely
REQUIREMENTS require oxygen for their
OXYGEN
growth.
Example: Mycobacteria spp.
Obligate Anaerobes are
those that are unable to grow
in the presence of free
oxygen because O2 kills or
inhibits them.
Aerotolerant anaerobe does

CHEMICAL
REQUIREMENTS not require oxygen, grows
OXYGEN
better in the absence of

oxygen, but can survive in the
presence of oxygen.
Facultative anaerobes are
capable of surviving in either
the presence or absence of
oxygen.
Examples: Enterobacteriaceae,
most Streptococci & Staphylococci.
Microaerophiles need a
CHEMICAL
REQUIREMENTS small quantity of oxygen, but
OXYGEN
large quantities inhibit their

growth or even kill them.
A. Aerobic
B. Anaerobic
C. Facultative
D. Microaerophilic
E. Aerotolerant
anaerobe
An atmosphere of 5-10%
CHEMICAL
REQUIREMENTS CO2 is required by some
organisms, referred to as
CAPNOPHILES.
Examples: Neisseria spp.,
Campylobacter spp.,
Haemophilus spp.

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CELL WALL

Lea D. Ballares, RMT, M. Bio. Ed.

BACTERIA Slides by LDBallares

Average size: 0.2-10.0 um in diameter

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

Pallisade arrangement: Corynebacterium diphtheria

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

Some bacteria may lose their

characteristic shape because

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

slide & apply stain.

Bacteria are colorless, transparent &

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

Stains consist of a positive and negative ion.

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

DIFFERENTIAL GRAM STAIN

Developed in 1884 by Danish

Bacteriologist Hans Christian

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

DIFFERENTIAL ACID-FAST STAIN

Acid Fast stain binds only to

those bacteria that have a

*** Nocardia spp.

Cells that retain a basic stain

(bacteria that have a waxy

BACTERIA Slides by LDBallares

Negative staining is the

process in which the

BACTERIA Slides by LDBallares

Heat is required to drive a

stain into endospores.

BACTERIA Slides by LDBallares

Flagella staining requires a

mordant to make the flagella

BACTERIA Slides by LDBallares

CULTURE MEDIA anything that possess

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

commonly used base medium.

contains gelatin or 0.51% agar.

CLASSIFICATION OF CULTURE MEDIA

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA

Supports the growth &

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA

Enhances the propagation of

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA