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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
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SPIRAL

Elongated cocci:

COCCOBACILLI
Examples:
Listeria monocytogenes
2. Haemophilus influenzae
1.

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Unusual shapes
Star-shaped Stella
Square Haloarcula
Genetically, most bacteria are monomorphic (one

shape)
A few are pleomorphic based on environmental
conditions

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Pairs: diplococci,

diplobacilli
Packets of four:
tetrads
Packets of eight:
octads
Clusters:
staphylococci
Chains: streptococci,
streptobacilli
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Pallisade arrangement: Corynebacterium diphtheria

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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.

BACTERIA Slides by LDBallares

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.

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PLEOMORPHIC
No cell wall
Has the ability to
exist in variety of
shapes
Example:
Mycoplasma spp.

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LIVING STATE

WET MOUNT
To observe shape &

arrangement of
organism
A drop of bacterial
suspension on slide,
cover it with coverslip
& focus.
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LIVING STATE

HANGING DROP
To observe organisms

motility.
A hanging drop slide w/
concavity at the center
is used.

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FIXED STATE

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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.

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PREPARATION
FOR STAINING

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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.

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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.

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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 (-).

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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
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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.


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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.

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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.

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SPECIAL
STAINS

Heat is required to drive a

stain into endospores.

Are used to

color and
isolate
specific
parts of
microorgani
sms.

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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.

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CULTURE MEDIA anything that possess


nutritional & environmental requirements for
bacterial growth.

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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.

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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.


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CLASSIFICATION OF
CULTURE MEDIA
(According to Physical
State):
Semi-solid medium

contains gelatin or 0.51% agar.


Solid culture medium
contains 2-3% agar.
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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.

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CLASSIFICATION OF CULTURE MEDIA


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

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CLASSIFICATION OF CULTURE MEDIA


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

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CLASSIFICATION OF CULTURE MEDIA


(According to use):
SIMPLE
MEDIUM

Supports the growth &

multiplication of
microorganisms.
It is used for routine cultivation
& maintenance of
microorganisms.
Example: Nutrient Agar

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BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA


(According to use):
ENRICHMENT
MEDIUM

Enhances the propagation of

certain organisms.
Examples: selenite broth,
tetrathionate broth

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CLASSIFICATION OF CULTURE MEDIA


(According to use):
ENRICHED
MEDIUM

Contains nutritive supplements

needed for the growth of some


organisms.
Examples: blood agar plate,
chocolate agar plate

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CLASSIFICATION OF CULTURE MEDIA


(According to use):
DIFFERENTIAL Distinguishes organisms
MEDIUM
growing together by

differences in their cultural


characteristics.
Examples: EMB, Mc Conkey
Agar

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CLASSIFICATION OF CULTURE MEDIA


(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

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Esherichia coli on Eosin Methylene Blue Agar (EMB).


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

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CLASSIFICATION OF CULTURE MEDIA


(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.

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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
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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

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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
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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.

BACTERIA Slides by LDBallares

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)
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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.

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COLONY
MORPHOLOGY

TYPES OF COLONIES:
R or Rough Colonies
granulated in appearance.

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COLONY
MORPHOLOGY

A colony contains millions

of organisms.
Size, shape, color,
elevation & margin are
observed to identify the
bacteria.

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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.
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GROWTH PATTERNS IN PLATES


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
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GROWTH PATTERNS IN PLATES


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
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It refers to the number of cells, not the cell

size.
Growing microbes increases in number.

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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.

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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

BACTERIA Slides by LDBallares

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

BACTERIA Slides by LDBallares

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.

BACTERIA Slides by LDBallares

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.

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REQUIREMENTS FOR
GROWTH:
1.Physical
temperature, pH,
osmotic pressure
2.Chemical Water,
sources of carbon &
nitrogen, minerals,
oxygen & organic
growth factors.
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PHYSICAL
REQUIREMENTS

TEMPERATURE
PSYCHROPHILES cold

loving microbes.
MESOPHILES
moderate-temperature
loving microbes
THERMOPHILES heat
loving microbes.

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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.
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TEMPERATURE RANGE
PHYSICAL
REQUIREMENTS MAXIMUM GROWTH

TEMPERATURE - is the
highest temperature at
which growth is possible.

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BACTERIA Slides by LDBallares

PHYSICAL
REQUIREMENTS

TEMPERATURE
PSYCHROTROPHS
Grow between 0C and 2030C
Cause food spoilage
Also known as moderate
psychrophiles or facultative
psychrophiles

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BACTERIA Slides by LDBallares

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).

BACTERIA Slides by LDBallares

PHYSICAL
REQUIREMENTS

pH
ACIDOPHILIC BACTERIA

remarkably tolerant of
acidity
BASOPHILIC BACTERIA
grows at pH near neutrality.

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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

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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.

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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.

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ISOTONIC SOLUTION
PHYSICAL
REQUIREMENTS In an isotonic environment, water
neither leaves nor enter the cell.
OSMOTIC
PRESSURE

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HALOPHILES
PHYSICAL
REQUIREMENTS halo referring to salt & philic
OSMOTIC
PRESSURE

meaning to love
Bacteria that loves salty
environment.
Example: Vibrio cholera

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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

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BAROPHILES
PHYSICAL
REQUIREMENTS Organisms that thrive deep in
BAROMETRIC
PRESSURE

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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.
BACTERIA Slides by LDBallares

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.

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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.

BACTERIA Slides by LDBallares

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.

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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.

BACTERIA Slides by LDBallares

Microaerophiles need a
CHEMICAL
REQUIREMENTS small quantity of oxygen, but

OXYGEN

large quantities inhibit their


growth or even kill them.

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A. Aerobic
B. Anaerobic

C. Facultative
D. Microaerophilic
E. Aerotolerant

anaerobe

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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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BACTERIA Slides by LDBallares