Transcript bacterial identification methods

BACTERIAL
IDENTIFICATION
METHODS
C ONTENT

Purification of cultures

Morphological and pure culture
studies

Biochemical tests
P URIFICATION OF CULTURES
Reason to purify cultures.

To characterize an individual species.

To study the morphology and physiology of
individual bacterial species

To study their biochemical behavior and
response.
To purify, pure cultures techniques can be
used.Method:

Streak plate method

Pour plate method

Spread plate method
I MPORTANT PROCEDURE !!
Need to have a control procedure to
avoid contamination.

Specimen collection

Preparation of media

Microbiological tecniques

Staining and reagents

Equipment used.
S PECIMEN C OLLECTION

Applied the sterile techniques

Use correct media for transportation and stock.

The transport media used to preserve and ensure the
viability of bacteria during the transportation period

Important! Label your specimen.

Crucial for cerebrospinal fluid, blood culture and fecal
specimens, etc.
U SING STERILE
6
TECHNIQUES

Bacteria are everywhere

Media used for bacteria growth  welcoming
for many bacteria

We only want specific ones to grow  Sterile
techniques

Sterile remain sterile as long as doesn’t touch
anything that isn’t sterile

Also avoid prolonged exposure to air
7
A SEPTIC T ECHNIQUE :
These are various techniques that are used to
minimize the introduction of microorganisms into
media especially during transfer processes, such as :
 pouring of media into Petri dishes
 inoculation of cultures
These techniques include:
 cleaning the bench top work areas with
disinfectant solution
 washing hands before starting work
 other specific techniques that will be
demonstrated in the lab.
S TERILE TECHNIQUES : WHAT CAN
YOU DO IN THE LAB ?
8

Wash your hands

Keep your bench clean

Wear gloves

Flame loop, neck of tube

Keep cap facing down

Work quickly and efficiently

Limit talking when opening
cultures
P REPARATION
OF MEDIA

The media should be packed well to prevent
from leakage and breaks, protected from
moisture and sunlight and excessive heat

The expiry date should be noted and the
instruction of storage should be followed

The mix bacterial colonies should be sub
cultured until the culture are purified

the bacterial colony characteristic should
only derive from a single colony
C ULTURE
Plate
Slant
10
MEDIA
Broth
Deep
M ORPHOLOGICAL AND PURE CULTURE
STUDIES

Morphological studies:
- Sizes, shapes, cell arrangement, cell wall, surface
adherents or
appendages,flagella,pili,endospores,ribosomes.
- Macroscopic examintation

Techniques used in the study:
- Microscopic examintion
- Staining techniques
M ORPHOLOGICAL AND PURE
CULTURE STUDIES
I SOLATION OF P URE
B ACTERIAL CULTURES
Divide into 3 groups:
Selective media
Differental media
Enrichment media
S ELECTIVE
MEDIA

Prepared by the addition of specific subtances to a
culture medium that will permit growth of one
bacteria while inhibiting the growth of others.

Contain antimicrobial agents such as crytal violet,bile
salts,sodium azide,antibiotic and e.t.c.
Salmonella-Shigella Agar- media contain bile salts
(inhibits many coliform bacteria).Produce colorless
colonies (unable to ferment lactose)
Mannitol Salt Agar -Isolation of Staphylococci.
Bismuth Sulfite Agar-Isolation for Salmonella
typhi.Reduces the sulfite to sulfide results in black
colonies and with metallic sheen.
D IFFERENTIAL

MEDIA
The incorporation of certain chemicals into a medium
may result in diagnostically useful growth or visible
change in the medium after incubation.
Eosin Methylene Blue(EMB)- Differentiate between
lactse and non-lactose fermenters.
Mac Conkey Agar-contain crystal violet and bile
salts.Use for selection of Enterobacteriaceae and
related gram negative rods.
Hektoen Enteric Agar-High concenration of bile
salts.Inhibit Gram positive bacteria and retards the
growth of many coliform strains.
I N M AC C ONKEY
AGAR
I N M AC C ONKEY
AGAR
E NRICHMENT
MEDIA

These are routinely employed in a laboratory e.g. nutrient
broth, nutrient agar, infusion broth,blood agar.

They support the growth of fastidious bacteria.
IN
NUTRIENT AGAR
P URE
COLONY
I N B LOOD
AGAR
H EMOLYSIS

Destruction of erythrocytes nd hemoglobin in
medium.

Can be divided into 3 categories:alpha hemolysis,
beta hemolysis and gamma hemolysis
Alpha hemolysis-greenish to brownish discolouration
around the colonies. (Streptococous
gordonii,Streptococcus pneumoniae)
Beta hemolysis-complete lysis of blood cell resulting
in clearing effect around the growth of
colony.(S.aureus)
Gamma hemolysis-no change in the
medium.(Enterococcus faecalis)
B IOCHEMICAL

Catalase test

Oxidase test

Coagulase test

Sugar fermentation test

MRVP test

Indole test

Citrate test

Motility test

H2S test

Litmus milk test
TESTS
C ATALASE
TEST

Produce bubble just after
attaching the bacteria to the
reagent

To differentiate staphylococci
and streptococci
O XIDASE
TEST

Have 2 methods:Filter paper/Sterile swab

To help identify Vibrio, Neisseria, Pasteurella and
Pseudomonas sp.

Oxidase enzymes oxydize phenylenediamine.

Deep purple colour on reagent paper
O XIDASE
TEST
C OAGULASE
 To identify S.aureus
 The enzyme
coagulase clots
plasma
 Tube : fibrin clot
 Slide: clumping of
bacterial cells
TEST
S UGAR FERMENTATION TEST

Glucose test

Maltose test

Sucrose test

Lactose test

Some will appear with gas
production
V OGES -P ROSKAUER
TEST

To differentiate
enterobacteria

Organism ferments glucose
with acetoin production.
Acetoin is oxidised to
diacetyl which reacts with
creatine.

Brick red colour develop
slowly

Eg: E.coli (-)

Klebsiella sp. (+)
M ETHYL R ED
TEST

To differentiate
Enterobacteria.

Detect the production of
sufficient acid during
fermentation of glucose in
buffered medium to give a
colour change of indicator

Brick red medium
I NDOLE
TEST

Using Kovac reagent.

To differentiate Gram negative rods,
especially E.coli .

Demonstrates the ability of certain
bacteria to decompose amino acid
tryptophan to indole which
accumulates in the medium.

Reddening of strip or medium
I NDOLE TEST USING OTHER
REAGENT
C ITRATE

Test the ability of organism to utilise citrate as a
sole carbon source and ammonium salt for
nitrogen.Result in alkalinization in the medium
with colour change indicator.

Use Koser’s liquid citrate medium.

Differentiate Enterobacteria from other bacteria.

Positive result : Blue and turbid medium
TEST
M OTILITY
TEST
LITMUS TEST

Medium consisting of LACTOSE,CASEIN and the pH indicator azolitmin.

It is used to differentiate members within the genus Clostridium. It
differentiates Enterobacteriaceae from other Gram-negative bacilli
based on enterics' ability to reduce litmus.

The skim milk provides nutients for growth. The protein is casein and
the lactose is for fermentation.

Azolitmin is purple between pH of 4.6 and 8.2. It turns pink when pH
reaches 4.5 and blue at a pH of 8.3.

Because of this, litmus milk can give quite unreliable results .
Thus, you would be advised to use litmus milk as a confirmatory test but
not a definitive test (except as a last resort).
T RIPLE
SUGAR ION

Triple Sugar Iron medium is a differential medium that
can distinguish between a number of Gram-negative
enteric bacteria based on their physiological ability (or
lack thereof) to:

a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S.
T ERMS
FOR TODAY
 Culture collection centre.
ATCC
American type culture Collection Centre
NCTCC
National Collection of Type Culture
NCIM
Natonal Collection of Industrial and Marine Bacterial
NCDO
National Collection of Dairy Organism
T HE END