Cell Wall Plasma Membrane Ribosomes Nucleoid Capsule Pili Flagella Cytoplasm

CELL WALL PLASMA MEMBRANE RIBOSOMES NUCLEOID CAPSULE PILI FLAGELLA CYTOPLASM
General Structure Gram (+) Bacteria Gram (-) Bacteria Growth & Nutrition
Lecturer:
Lea D. Ballares, RMT, M. Bio. Ed.
Susceptibility Testing
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 (-).
STEPS
Primary stain: Crystal violet Mordant: Iodine Decolorizing agent: Alcohol-acetone Counterstain: Safranin
Color of Gram + cells Purple Purple
Color of Gram cells Purple Purple
Purple Purple
Colorless Red
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
Negative staining is the
color and isolate specific parts of microorgani sms.
process in which the background & not the organism is stained.
SPECIAL STAINS
Are used to
Heat is required to drive a
stain into endospores.
SPECIAL STAINS
Are used to
Flagella staining requires a
mordant to make the flagella wide enough to see.
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.


(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 ADJUST TO PROPER PH STERILIZATION
In plated media: Sterile first
before distribution. In tubed media: Distribute first before 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.
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
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
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 The rate of reproduction has completely stopped due to lack of nutrients & formation of toxic products
Bacteria are adjusting to a new environment, synthesizing enzymes & actively metabolizing
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

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.
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.
TEMPERATURE
PSYCHROTROPHS Grow between 0C and 2030C 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).
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.

Cell Wall Plasma Membrane Ribosomes Nucleoid Capsule Pili Flagella Cytoplasm

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CELL WALL PLASMA MEMBRANE RIBOSOMES NUCLEOID CAPSULE PILI FLAGELLA CYTOPLASM

BACTERIA Slides by LDBallares

Average size: 0.2-10.0 um in diameter Basic shapes:

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

shape) A few are pleomorphic based on environmental conditions

BACTERIA Slides by LDBallares

Pallisade arrangement: Corynebacterium diphtheria

BACTERIA Slides by LDBallares

MEDICALLY IMPORTANT COCCI

MEDICALLY IMPORTANT BACILLI

MEDICALLY IMPORTANT CURVED/SPIRAL

BACTERIA Slides by LDBallares

Some bacteria may lose their

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

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

BACTERIA Slides by LDBallares

slide & apply stain.

BACTERIA Slides by LDBallares

Bacteria are colorless, transparent &

BACTERIA Slides by LDBallares

PREPARATION FOR STAINING

BACTERIA Slides by LDBallares

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.

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

DIFFERENTIAL GRAM STAIN

Developed in 1884 by Danish

BACTERIA Slides by LDBallares

Color of Gram + cells Purple Purple

Color of Gram cells Purple Purple

BACTERIA Slides by LDBallares

DIFFERENTIAL ACID-FAST STAIN

Acid Fast stain binds only to

*** Nocardia spp.

Cells that retain a basic stain

BACTERIA Slides by LDBallares

Negative staining is the

color and isolate specific parts of microorgani sms.

process in which the background & not the organism is stained.

BACTERIA Slides by LDBallares

Heat is required to drive a

stain into endospores.

color and isolate specific parts of microorgani sms.

BACTERIA Slides by LDBallares

Flagella staining requires a

mordant to make the flagella wide enough to see.

color and isolate specific parts of microorgani sms.

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA

medium contains no agar, hardening or solidifying substances

commonly used base medium.

CLASSIFICATION OF CULTURE MEDIA

CLASSIFICATION OF CULTURE MEDIA (According to Composition):

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA

BACTERIA Slides by LDBallares

CLASSIFICATION OF CULTURE MEDIA