BACTERIAL ISOLATION &

IDENTIFICATION
Mikrobiologi & Parasitologi TA. 2018/2019
ERNANIN DYAH W.
 ▪ Innoculation
▪ Isolation
Culturing
Microbes: ▪ Incubation
The Five “I’s ▪ Inspection
▪ Identification
 ▪ Innoculation: Producing a pure culture
▪ Innocula: The sample from which microorganisms will
be isolated
▪ Introduce bacteria into a growth medium using “aseptic
technique” to prevent contamination.
▪ Tools: Bunsen burner, loop. Needle, etc.

Innoculation
Innoculation
 ▪ Isolation: The separation of individual organisms from
the mixed community
▪ Pure culture: isolation on general and special
“differential media”

▪ Colony on media, one kind of microbe, pure culture

Isolation
Isolation
Isolation
 ▪ Many colonies? Use a needle, pick one, and redo streak
plate

Isolation
 ▪ Choose the proper medium

Isolation
 ▪ Incubation: Allow organisms to grow under the optimal
conditions
▪ Temperature, with or without oxygen etc

Incubation
 ▪ Inspection: Observation, description
▪ Colony Morphology, Microscopic examination (Grams
stain)
▪ Systematic recording of “DATA”

Inspection
 ▪ Identification: Correlating data from all
observations to ID organism to species
▪ Methods
1. Phenotypic
▪ Morphology
▪ Gram reaction, size, shape, motility, pigments, etc.
Identification ▪ Biochemical reactions
▪ Resources: flow charts, Bergey’s manual etc.
2. Genotypic
▪ Pattern or finger print based techniques
▪ Ex: Polymerase Chain Reaction (PCR)
▪ Sequence based techniques
▪ Ex: 16S rRNA
 ▪ Ex. Gram – bacilli, ferments lactose, green sheen on
EMB: E.coli

Identification
 ▪ Gram + cocci, grape like clusters, golden yellow
colonies, catalase +, coagulase +, resistant to Methicillin
(MRSA)
▪ Staphylococcus aureus

Identification
 ▪ Staining bacteria cells for microscopic
examination makes it possible:
▪ to define their cell size, shape, arrangement;
▪ to study their chemical properties,and structures.
Staining ▪ These characteristics can be use for bacterial
Bacteria Cells identification
 ▪ Simple stain – one dye
▪ Differential stain – complex procedure, see difference
between cells
▪ Grams + and (-)
Staining ▪ Acid fast + and (-)
Bacteria Cells ▪ Negative – acid dye stains background and cells are white
(cell wall repels stain)
▪ Capsule – modified negative stain to show capsule layer
Overview of a
bacterial
staining
procedure
Preparation of
the heat-fixed

sample 1
smear
 ▪ Simple stains use a single basic dye (e.g. crystal violet,
methylene blue, safranin) to color bacterial cells so that
their size, shape and arrangement can be observed

Simple Stain
Simple Stain

Fracisella tularensis
Causitive agent of Rabbit fever
Methylene blue stain
Simple Stain
Sacharromyces cerevisiae
(Brewer’s yeast)
Methylene blue stain
Simple Stain

Bacillus anthracis (anthrax)

Crystal violet stain
Simple Stain

Campylobacter jejuni (traveler’s diarrhea)

Fuchsin stain
 ▪ Differential stains, such as the Gram stain and the acid-
fast stain, differentiate bacteria based on the chemical
composition of their cell wall.
▪ Differential stain use two dyes instead of one: the first
stain is the primary stain, the second is the counterstain.

Differential ▪ A decolorization step occurs between the application of

the primary stain and counterstain.
Staining ▪ Depending on the composition of the cell wall, bacteria
will either retain the primary stain during
decolorization or lose the primary stain and take up the
counterstain.
 ▪ Hans Christian Gram was a
Danish bacteriologist.
▪ He developed the Gram stain
as a means to differentiate
pneumococci from Klebsiella
pneumonia in 1884.
Gram Staining ▪ It remains one of the most
important staining techniques
in microbiology today.
▪ The Gram stain is often the first
test performed in the
identification of bacteria.
Gram Staining
Procedure
Gram Staining
Gram + & -
Cell Walls
Gram Staining

Staphylococcus aureus,1mm
Gram Staining

Escherichia coli, 1x3 mm

Gram Staining

Gram stain of a mixture of Staphylococcus

aureus and Escherichia coli
 Neisseria gonorrhoeae

Gram Staining

Gram Stain of pus smear Gram stain of yogurt

 ▪ Paul Ehrlich was a
German physician.
▪ He developed the
acid-fast stain in 1882
as a means of staining
the tubercle bacillus,
The acid-fast Mycobacterium
tuberculosis.
stain
▪ His original method
has undergone
modifications by Ziehl
and Neelsen that are
still used today.
 ▪ The acid-fast stain distinguishes different types of
bacteria based on the wax content of their cell wall.
▪ Bacteria with a high wax content retain the primary
stain carbolfuchsin when decolorized with acid-alcohol.
These are acid-fast bacteria.
The acid-fast ▪ Bacteria with a low wax content lose carbolfuchsin
stain when decolorized with acid-alcohol and take up the
counterstain methylen blue. These are non acid-fast
bacteria.
▪ This stain is important in distinguishing acid-fast
bacteria of the genus Mycobacterium.
 ▪ Stain with carbolfuchsin……….…5
min. with heat
▪ Wash off with tap water
▪ Decolorizer Acid-Alcohol (3% HCl-
Ethanol 95%)
Acid-fast Stain
Procedure ▪ Wash off with tap water
▪ Counterstain with methylene
blue………2 min.
▪ Wash off with tap water
▪ Blot dry with bibulous paper
 Acid Fast
staining of
Mycobacterium
Negative Stain
Special Stain:
Capsule
Special Stain:
Flagella

Special stain: flagella staining

(carbolfuchsin and a mordant)
Special Stain:
Flagella

Special stain: flagella staining (carbolfuchsin

and tannic acid)
Special Stain:
Endospore
Special stain:
endospore
Endospore
Identification
Identification
Media

▪ Biochemical Test: Based on the characteristization of

metabolic pathways of the bacteria
 Fermentation of Carbohydrates

Biochemical test
 Indole Production

Biochemical
test: IMViC
 Mixed Acid Fermentation: MR/VP

Biochemical
test: IMViC
 Citrate Utility Test

Biochemical
test:
IMViC
 Catalase activity

Biochemical test
 Oxydase Test

Biochemical test
 Coagulase Test

Biochemical test
 Urease Test

Biochemical test
 DNase Test

Biochemical test
Biochemical
Test
 “IF YOU DON'T LIKE BACTERIA,
YOU'RE ON THE WRONG PLANET”
-Stewart Brand-