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Classification and identification of microorgasnisms
Laboratory procedures employed in the
identification of bacteria
1.
2.
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6.
Isolation of organism in pure culture
Bacterial colony morphology
Microscopic morphology and Staining reaction
Biochemical test
Serological procedure
Antibiotic sensitivity
Isolation of organism in Pure Culture
• Pure culture (axenic culture)
– Population of cells arising from a single cell
- the approach used for the isolation of organism depends
upon the source of clinical specimen
Blood, spinal fluid and closed abscesses may yield almost
pure bacterial culture
specimen of sputum, stool, materials from the skin and
body orifices usually contains mixture of organism
- Spread plate, streak plate, and pour plate are
techniques used to isolate pure culture
Laboratory Cultivation
 Cultivation is the process of growing microorganisms by
taking bacteria from the infection site by some means of
specimen collection and growing them in the artificial
environment of the laboratory
 For the in vitro environment of the bacteria, required nutrients
are supplied in a culture medium
 culture - organisms that grow and multiply in or on a culture
media
Culture Medium
- is a liquid or gel designed to support the growth of microorganisms
- 2 major types of growth media:
- those used for cell culture, which use specific cell types derived
from plants or animals
- microbiological culture, which are used for growing
microorganisms such as bacteria or yeast
-The most common growth media for microorganisms are
nutrient broths and agar plates
- specialized media are sometimes required for microorganism and cell
culture growth
Based on Chemical Composition
Complex Media
- Contain some ingredients of unknown composition and/or conc.
- is a medium that contains:
• carbon source such as glucose for bacterial growth
• water
• various salts needed for bacterial growth
• a source of amino acids and nitrogen (e.g., beef, yeast extract)
- Nutrient media contain all the elements that most bacteria need
for growth and are non-selective, so they are used for the
general cultivation and maintenance of bacteria kept in
laboratory culture collections
Defined or Synthetic Media
-All components and their concentrations are known
Functional Types of Media
Supportive or general purpose media
- Support the growth of many microorganisms
- E.g., Tryptic soy agar
Enriched media
- General purpose media supplemented by blood or other special
nutrients
• Blood agar is an enriched medium in which nutritionally rich whole blood
supplements the basic nutrients
• Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown
and gives the medium the color for which it is named
Selective media
- Favor the growth of only selected microorganisms and inhibit growth of others
• eosin-methylene blue agar (EMB) that contains methylene blue
– toxic to Gram (+) bacteria, allowing only the growth of Gram (-) bacteria
• blood agar (used in strep tests), which contains beef heart blood that becomes
transparent in the presence of hemolytic Streptococcus
• MacConkey agar for Gram-negative bacteria
• Mannitol Salt Agar (MSA) which is selective for Gram (+) bacteria and differential for
mannitol
Alpha Hemolytic Streptococci
Gamma Hemolytic Streptococci
Beta Hemolytic Streptococci
Incomplete lysis of RBC’s
Complete lysis of RBC’s
No lysis of RBC’s
Differential media
– Distinguish between different groups of microorganisms based
on their biochemical characteristics growing in the presence
of specific nutrients or indicators (such as neutral red, phenol
red, eosin y, or methylene blue) added to the medium to
visibly indicate the defining characteristics of a microorganism
Ex.
• Blood agar – differentiates hemolytic versus non-hemolytic bacteria
• MacConkey agar - lactose fermenters versus non-fermenters
• Eosin methylene blue (EMB), which is differential for lactose and sucrose
fermentation
• Mannitol Salt Agar (MSA), which is differential for mannitol fermentation
Bacterial colony morphology
• Bacteria grow on solid media as colonies
• colony is defined as a visible mass of microorganisms all
originating from a single mother cell, when inoculated into
appropriate medium containing 2% agar and incubated
18-24 hours in a favorable atmosphere
• therefore a colony constitutes a clone of bacteria all genetically alike
• Ideally, the colony is the progeny of one, or at most, a few bacteria
• A colony will usually contain millions of bacterial cells
• Colony morphology can sometimes be useful in bacterial identification
• Colonies are described as to such properties as size, shape, texture,
elevation, pigmentation, effect on growth medium
To identify the following colonial characteristics/culture characteristics:
WHOLE SHAPE OF COLONY
EDGE/MARGIN OF COLONY
ELEVATION OF COLONY (turn the place on end to determine height)
CHROMOGENESIS (pigmentation)
- Some bacterial species form an array of pigments: white, red, purple, etc.
• Some pigments are contained within the cell (i.e., probably not water soluble)
• Some pigments readily diffuse throughout the medium (i.e, water soluble)
• Some pigments fluoresce in UV light
OPACITY OF COLONY:
transparent (clear), opaque,
translucent (almost clear, but distorted vision–like looking through frosted glass
iridescent (changing colors in reflected light)
CONSISTENCY:
butyrous (buttery), viscid (sticks to loop, hard to get off)
brittle/friable (dry, breaks apart)
EMULSIFIABILITY OF COLONY:
Is it easy or difficult to emulsify? Does it form a uniform suspension, a granular
suspension, or does not emulsify at all?
SURFACE OF COLONY:
smooth, mucoid/glistening, rough, dull (opposite of glistening),
rugose (wrinkled)
Smooth - colonies gives the appearance of homogeneity and uniform
texture without appearing as liquid or as mucoid colonies
characteristically isolated from fresh wild type organism such as
gram- negative enterobacteria Ex. Salmonella, Shigella
Mucoid - colonies exhibits a water-like glistening confluent
appearance commonly seem among organism which from
slime layer or capsule. Ex. Kleb. pneumoniae, S. pneumoniae
Rough – colonies are granulated and rough in appearance, usually
produced by mutant strain that lacks surface protein and
polysaccharide of freshly isolated wild-type parent organism
Microscopic morphology
• Provide presumptive identification of an organism
Bacterial Morphology
• Bacterial cell is a fundamental unit of any living organism
• All its functions are genetically controlled and performed by that
particular cell structure whether it be physiologic or
biochemical
• Bacteria and other microorganism are usually transparent, which
makes the study of the morphologic detail difficult when they
are examined in the natural state
• Routinely used to determine: shape
arrangement
staining reaction
I. Bacterial Shape and Arrangement

Bacterial Shape
•
determined by the configuration of the cell wall
detected by brightfield microscopy of stained smear
Bacterial Arrangement
•

 is the result of the number of plane division the organism may undergo
and how the cell remain attached afterwards
 divides only across their short axis

3 conventional forms :
Spherical (cocci)
Rod (bacilli)
Spirals
Spherical (Cocci)

Shape:

round like a ball, perfect sphere or globe

Variations :
1. Ovoid shape - both sides rounded ends are pointed
Ex. Streptococcus
2. Lancet-shape - one end is pointed, other end is flat
Ex. Pneumococcus
3. Coffee-bean shape - flat on one side, opposite
side convex or appear as letter “D” form
Ex. Neisseria
Arrangements:
1. Singly – occurs as a single spherical cell
2. Chain – “ streptococci”
- common among ovoid-form resulting from one
plane division with daughter cells remained attached
to one another to form a chain
Ex. Streptococcus pyogenes
3. Pairs – “diplococci”
- common with lancet-shaped and coffee-bean
shaped spherical resulting from one plane division
with daughter cell separating
Ex. Streptococcus pneumoniae
Neisseria gonorrheae
4. Clusters – “staphylococci”
- common with spherical resulting from many plane
division with daughter cell in grape-like agglomeration
Ex. Staphylococcus aureus
5. Tetrads – (Packets of 4)
- result from 2 plane division with daughter cell
separating from one another to form group of 4 cells
Ex. Micrococcus tetragenous
6. Sarcinae – (Packets of 8)
- results from many plane division producing cubical
packets of 8 cells
Ex. Sarcina lutea
Rods (Bacilli)
 Shape
 cell appears longer than wide or cylindrical form
 both sides parallel and ends are convex
 varies in actual form depending on the species
 divides only across their short axis
 Variations :
1. Clubbed/drumstick shaped – swollen on one end
Ex. Clostridium diphtheriae/C. tetani
2. Corset-shape – both sides swollen, ends flat or
concave
Ex. Bacillus anthracis
3. Fusiform – both sides parallel, ends pointed
Arrangements:
1. Singly – occurs as a single rod
2. Chain – result from one plane division with daughter
cell remain attached to one another
Ex. Bacillus anthracis
3. Palisade – arrangement like fence due to slipping
movement of daughter cells (side-by-side)
Common among clubbed shaped rods
Ex. Mycobacterium tuberculosis
4. Chinese-letter – common with clubbed-shaped rods
resulting from a snapping post division
movement of the daughter cells (V shape)
Ex. Corynebacterium diptheriae
5. Packets of cigarette – arrangement like bundles
Ex. Mycobacterium leprae
6. Serpentine – commonly seen with virulent strain
of Mycobacterium tuberculosis
Intermediate forms
Coccobacilli
- when a rod is short & wide/plump
- these form is intermediate between a spherical
and
rod
Ex. Haemophilus, Brucella
Vibrio
- a gently curve bacteria (comma-shaped)
- it is an intermediate between a rod and a spiral
Ex. Vibrio cholerae
Spirals
 bacteria with more than one somatic curve
 may be regarded as bacillary forms trusted in the form of a helix
 no characteristic cell arrangement
 most occurs singly
 different specie vary in size, length, rigidity and amplitude of their
coils
 2 types :
1. Flexible – spirals that can contract and relax & move by creeping
movement
Ex. Spirochetes
 2. Rigid – spirals that cannot contract and relax & move by
rotation or corkscrew-like motion
Ex. Spirillum
SPIRILLUM
- whose long axis remains rigid when in
motion
Ex. Campylobacter jejuni
SPIROCHETE
– whose long axis bends when in motion
Genus Treponema
– char. tightly coil w/ cork screw appearance
Ex. Trepanema pallidum
Genus Leptospira
– less tightly coiled w/ sharp hook-like bends
Ex. Leptospira interrogans
Genus Borrelia
– much less tightly coiled w/c has the
appearance of extremely long undulating
bacillary pores
Ex. Borrelia recurrentis
II. Bacterial size
 all linear measurements in microbiology are expressed in
•



metric units
the basic unit of the metric system is the meter “m”
centimeter cm (1/100th of a m)
- the largest unit of length used for measuring microorganism
micrometer µm
- visible only with high powered microscope
- unit of measurement most frequently used in microbiology
1µm = 1/1000 of a mm
 Cocci = 0.4-2µm
 Bacilli = 0.2-4µm in width by o.5-20µm in length
 Spirals = 1-4µm in length
 nanometer nm - commonly used to measure virus
 Angstrom – smallest unit of measurement
III. Bacterial
Staining Reaction
Staining – procedure that applies colored chemicals called dyes to
specimen in order to facilitate identification
Stains - salts composed of a positive and negative ion, one of which
is colored (chromophore – color bearing ion), which imparts
a color to cell or cell parts by becoming affixed to them
through a chemical reaction
Basic (cationic) Dyes - chromophore is the positive ion dye
Acid (anionic) Dyes - chromophore is the negative ion dye
Bacteria are slightly negative, so are attracted to the positive chromophore
of the BASIC DYE
 Preparing smears for staining
1. Smear preparation
- depends on the physical state; if in liquid state spread the
smear out
- Bacteria on slide
2. Air Dry
- preserve the morphology of the bacteria
- allow the smear to adhere to the slide
3. Bacteria are HEAT FIXED to the slide
Heat Fixation
- simultaneously kills the specimen and secures it to the slide
- preserve various cellular component in a natural state with
minimal distortion
4. Stain is applied
Staining – coloring the microorganisms with a dye
Types of Staining:
1. Simple Staining
- employs one dye
- most common: methylene blue, crystal violet,
carbol fuchsin,safranin
- sufficient to determine size, shape & arrangement
- most cells will stain the same color with the dye used
Appearance of
organisms
Background
Positive Staining
Negative staining
Colored by dye
Clear and colorless
Not stained
(generally white)
Stained
(dark gray or black)
2. Differential Staining
- employs the use of more than one dye added in several steps
and stained structures are differentiated by color as well as
shape
- it is based on the relative affinity of different bacterial cells for
the stains used
- enables microbiologist to differentiate one group from another
a) Gram staining - differentiate gram (+) from gram (-) bacteria
b) Acidfast staining - differentiate acidfast from non-acidfast
bacteria
Gram-staining
 Hans Christian Gram (1884), a Danish doctor, accidentally
stumbled on a method which still forms the basis for the
identification of bacteria; which divided almost all bacteria into
two large groups
 The reagents needed:
 Crystal Violet (Primary Stain)
 Iodine Solution (Mordant)
 Mordant - intensifies the stain or coats a structure to make it
thicker and easier to see after it is stained
- Increase the affinity of a stain to the specimen
 Decolorizer (ethanol is a good choice, mixture of acetone & alcohol)
 Safranin (Counterstain)

Counterstain – gives contrasting color to the primary stain
Gram Staining
STEP 1: Make a smear. Mounted and heat fixed.
STEP 2: Flood the entire slide with crystal violet (primary
stain) for 1min. Then rinse with the water.
STEP 3: flood the slide with the iodine solution (mordant)
for 1min. Then rinse with water for 5 seconds. The bacteria
become deeply stained and appear deep purple in color due
to crystal violet-iodine-complex formation
Step 4: addition of the decolorizer, 95% ethanol.
Rinse with water.
Gram (+) cells : purple dye is retained
Gram (-): purple dye is readily removed and appears colorless
STEP 5: Flood the slide with the counterstain, safranin
Again, rinse with water.
Gram (+) cells will incorporate little or no counterstain and will
remain purple in appearance
Gram (-) bacteria take on a pink/red color
PRINCIPLE:
 Gram reaction is based on the structure of the bacterial cell wall
 Gram-positive bacteria
 the peptidoglycan appears to act as a permeability barrier preventing
loss of crystal violet-iodine-complex
 When gram-positive bacteria are treated with alcohol, the alcohol
causes coagulation and dehydrateion of the thick layer of
peptidoglycan resulting in shrinkage of pores preventing CVI-complex
from escaping and the bacteria remain deep purple
 Reaction to Gram staining is also believed to be asso. With protein
complex Magnesium ribonucleate which is absent in Gram (-) org.
 Gram Negative bacteria
 peptidoglycan is very thin in gram (-) bacteria and has larger pores
 Alcohol readily penetrates the lipidrich outer layer of the cell wall and
extracts enough lipid thus increasing the porosity further
 alcohol more readily removes the deep purple CVI-complex from
gram (-) bacteria thus becomes decolorized
 The outer membrane is then permeabilized by the decolorizer, and
the pink safranin counterstain is trapped by the peptidoglycan layer
Divides bacteria into 2 groups
 Gram (+) : violet
 Gram (-) : red
Dictome of Gram Staining
 All COCCI are Gram Positive except Neisseria group,
Moraxella (Branhamella) catarrhalis and Veilonella
 All BACILLI are Gram Negative except the acid fast
organisms (Mycobacterium, Nocardia) , Sporeformers
(Bacillus, Clostridum) and Corynebacterium species
 Spirals are difficult to stain but when stained, they are
Gram Negative
Acid Fast Staining
 Acid-fast stain is a useful differential staining procedure that
specifically stains all members of the genera mycobacteria
 The walls of certain bacteria contain long chain fatty acids
(mycolic acid) lending the property of resistance to decolorization
of basic dyes by acid alcohol; thus called “acid fast”
 The high lipid and wax content of the mycobacterial cell walls is
thought to be the reason for such impermeability
 2 methods
 Ziehl-Neelsen method
 The procedure utilizes heat and phenol (carbolic acid) to help the
penetration of the dye, carbol fuchsin, to the inside of mycobacterial
cells, which are impermeable to basic dyes in routine stains like in
Gram staining
 Cold Kinyoun technique
 Instead of heat, this technique uses increasing the concentration of
phenol or the inclusion of a detergent in the stain
 Divides bacteria into 2 groups
 Acid - Fast organism: red
 Non Acid – Fast organism: blue
 The reagents needed
1. Primary stain: Carbol fuchsin
2. Decolorizer: Acid Alcohol
3. Counterstain: Methylene Blue
Acid - Fast Staining (Ziehl-Neelsen method)
STEP 1: Make a smear. Mounted and heat fixed
STEP 2: Flood the entire slide with Carbol Fuchsin.
STEP 3: Using a Bunsen burner, heat the slides slowly until
they are steaming. Acid fast organisms have a very
hydrophobic surface which resist entry of dyes. Heat is used to
enhance penetration and retention of dye
Maintain steaming for 5 minutes by using low or intermittent
heat (i.e. by occasionally passing the flame from the Bunsen
burner over the slides) Then rinse the slide with water.
STEP 4: Flood the slide with 3% acid-alcohol and allow to
decolorize for 5 minutes. Throughout the 5 minutes, continue to
flood the slides with 3% acid-alcohol until the slides are clear of
stain visible to the naked eye. Rinse the slide thoroughly with
water and then drain any excess from the slides.
STEP 5: Flood with the counterstain, Methylene Blue Keep
the counterstain on the slides for 1 minute. Rinse with water.
3. Special Staining
- used to color and isolate specific structure of a microorganism like
capsule, flagella, inclusion granule, endospore and etc.
Positive Staining
Capsule
Flagella
Endospore
Negative staining
Biochemical Test
 various species of organism exhibits characteristic pattern
of substrate utilization, metabolic product formation and
sugar fermentation
 Enzyme based test – based on its reaction with a substrate

Catalase, oxidase, indole, urease
 Reactions in glucose fermentation broth
 Reactions in lactose fermenation broth
 Starch hydrolysis of test strains
 Nitrate Broth reactions
 60% of common pathogens can be identified by
metabolic test
Serological procedure
 Antigen and antibody determination
 Serological Tests
 Use group specific antiserum isolated from the plasma of
animals that have been sensitized to the organism
 The antiserum contains antibody proteins that react with
antigens on the unknown organism.
 Procedures: agglutination, precipitation test, hemagglutination
inhibition, complement fixation, ELISA, RIA, Western blot assay
 Advantages:
 Highly specific
 Does not usually require the organism to be isolated into pure
culture
 Can be used to identify organisms that can’t be grown on
medium
Antibiotic sensitivity
 antibiotic sensitivity is a term used to describe the susceptibility
of bacteria to antibiotics
 Antibiotic susceptibility testing (AST) is usually carried out to
determine which antibiotic will be most successful in treating a
bacterial infection in vivo
 Methods of testing:
 Broth dilution
 The lower the dilution, the greater the antibiotic content
 Agar dilution
 Disk diffusion
 the Kirby-Bauer test for antibiotic susceptibility, called the disc
diffusion test, is a standard that has been used for years

 The bacterium is swabbed on the agar
and the antibiotic discs are placed on top
 The antibiotic diffuses from the disc into
the agar in decreasing amounts the further
it is away from the disc
 Bacteria are not able to grow around antibiotics
to which they are sensitive
 If the organism is killed or inhibited by the
concentration of the antibiotic, there will be
NO growth in the immediate area around the disc:
called the zone of inhibition
The zone sizes are looked up on a standardized chart to
give a result of sensititive, resistant, or intermediate
 Many charts have a corresponding column that also gives the MIC
(minimal inhibitory concentration) for that drug