General Structure

Gram (-) Bacteria

Growth & Nutrition
Gram (+) Bacteria
Susceptibility Testing
Bacterial Diseases
Gram positive bacteria have walls containing relatively large
amounts of peptidoglycan = a starch
Staphylococcus epidermidis, Streptococcus
pyogenes, Clostridium tetani, Bacillus anthacis
(ANTHRAX)

Gram negative species have walls containing small amounts of
peptidoglycan and a lipopolysaccharide = a fat/sugar combo

Escherichia coli, Salmonella typhi, Vibrio cholerae and
Bordetella

Gram negative bacteria are harder to control with antibiotics
Average size: 0.2-10.0 um in diameter
Basic shapes

Cocci
Bacilli
Spiral
Elongated cocci: Coccobacilli
Examples:
1. Listeria monocytogenes
2. Haemophilus influenzae

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
Medically
Important Bacilli
Medically
Important
Curved/Spiral
Enterococcus spp.
Neisseria spp.
Streptococcus spp.
Enterobacter spp.
Escherichia spp.
Klebsiella spp.
Proteus spp.
Salmonella spp.
Shigella spp.
Pseudomonas
aeruginosa
Bacillus spp.
Clostridium spp.
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.


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
1. Lag phase: When the cells are adjusting to their new
environment. During this phase, cellular metabolism is
accelerated, the cells are increasing in size, there is no
cell division and therefore no increase in numbers.
2.Logarithmic (log)/Exponential phase: Under
optimum nutritional and physical conditions, the
physiologically robust cells reproduce at a uniform and
rapid rate by binary fission
3. Stationary phase: During this stage, the number of
cells undergoing division is equal to the number of cells
that are dying. There is no further increase in cell number
and the population is maintained at its maximum level
for a period of time.
4. Decline or death phase: Because of the continuing
depletion of nutrients and buildup of metabolic wastes,
the microorganisms die at a rapid and uniform rate.
Nutritional Requirements
All organisms, whether they be 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 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 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.

Temperature
Psychrophiles cold loving microbes.
Mesophiles moderate-temperature loving
microbes
Thermophiles heat loving microbes.
Temperature Range
Minimum Growth Temperature : is the lowest
temperature at which species will grow
Optimum Growth Temperature : is the temperature
at which the species grows best.
Maximum Growth Temperature : is the highest
temperature at which growth is possible
Temperature Psychrotrophs
Grow between 0C and 20-30C
Cause food spoilage
Also known as moderate psychrophiles or facultative
psychrophiles

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).
Acidophilic Bacteria: remarkably tolerant of acidity
Basophilic Bacteria: grows at pH near neutrality.



Osmotic Pressure is the pressure that is exerted 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
The cell membrane & cytoplasm shrink away from the bacterial cell
wall Plasmolysis.
Salts & Sugars are added to certain foods to preserve them.
Bacteria in hypertonic environment will die as a result of
desiccation.


Hypotonic Solution
If a bacterial cell is placed in a hypotonic solution, the
fluid pressure w/in the cell increases greatly.
If the pressure becomes so great & cell bursts,
cytoplasm escapes from the cell Plasmoptysis.


Isotonic Solution
In an isotonic environment, water neither leaves nor
enter the cell.


Halophiles
halo referring to salt & philic meaning to love
Bacteria that loves salty environment.
Example: Vibrio cholera

Haloduric
Organisms that do not prefer to live in a salty
environment but are capable of surviving there.
Example: Staphylococcus aureus


Barometric Pressure
Barophiles
Organisms that thrive deep in ocean & oil wells,
where atmospheric pressure is very high.
Physical Requirements
Water in the liquid state is essential for the existence of all
living organisms.
Approximately 75% water is 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 salt makes the water unavailable
for bacteria.
The amount of water available for microbial growth is referred
to as Water Activity.



Oxygen is essential for the growth of many bacteria,
but for others it is lethal.
All bacteria have cell components that are sensitive
to oxygen and metabolic by-products of oxygen.
Organisms that live in air have enzymes that
detoxify these products.
A. Aerobic
B. Anaerobic
C. Facultative
D. Microaerophilic
E. Aerotolerant anaerobe

Obligate Aerobes absolutely require oxygen for their 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 not require oxygen, grows 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 small quantity of oxygen, but large
quantities inhibit their growth or even kill them.
Chemical Requirements
Carbon Dioxide
An atmosphere of 5-10% CO2 is required by some
organisms, referred to as Capnophiles.
Examples: Neisseria spp., Campylobacter spp.,
Haemophilus spp.

Living State
To observe shape &
arrangement of organism
A drop of bacterial
suspension on slide,
cover it with coverslip &
focus.


Wet Mount
Living State
To observe organisms
motility.
A hanging drop slide w/
concavity at the center is
used.


Hanging Drop
Fixed State
Adhere organism on
slide & apply stain.


It is the process coloring the microorganisms with a dye that
emphasizes certain structures.
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.
Preparation For
Staining
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
Stains
Gram Stain
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 (-).
Steps
Color Of
Gram + Cells
Color Of
Gram Cells
Primary stain:
Crystal violet
Purple Purple

Mordant:
Iodine
Purple Purple
Decolorizing agent:
Alcohol-acetone
Purple Colorless
Counterstain:
Safranin
Purple Red
Acid-fast Stain
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.

Cells that retain a basic stain (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 acid-alcohol, and
are usually counterstained to see them.

Special Stains
Negative staining is the process in
which the background & not the
organism is stained.
Are used to
color and
isolate specific
parts of
microorganisms
.

Special Stains
Heat is required to drive a stain
into endospores.

Are used to color
and isolate
specific parts of
microorganisms.

Special Stains
Flagella staining requires a
mordant to make the flagella
wide enough to see.

Are used to color
and isolate
specific parts of
microorganisms.

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.
Agar polysaccharide extracts of seaweed & commonly
used base medium.
Esherichia coli on Eosin
Methylene Blue Agar (EMB).
EMB is both a selective &
differential media. It is
selective for the growth of gram
(-) bacilli
Weigh Ingredients
Dissolve Ingredients
Adjust To Proper PH
Sterilization
Distribution In Sterile
Petri Dishes
In plated media: Sterile first before
distribution.

In tubed media: Distribute first before
sterilization.
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
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 colony-forming unit (CFU)

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

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