Laboratory procedures employed in the

identification of bacteria

6.Isolation of organism in pure culture

7.Bacterial colony morphology
8.Microscopic morphology and Staining reaction
4. Biochemical test
5. Serological procedure
6. 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

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
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
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:
•Singly – occurs as a single rod
•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
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 :
7. 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

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
Positive Staining Negative staining

Appearance of Colored by dye Clear and colorless

organisms

Background Not stained Stained

(generally white) (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
 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
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
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 Negative staining

Capsule

Flagella

Endospore
 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