Download Bacterial Agglutination

Bacterial Agglutination
Abad, Mary Raina Angeli Z.
Advincula, Janina Karla D.C.
Al Dhaheri, Sara Ali
Anacan, Keight Arren R.
Objectives


perform bacterial agglutination test
explain the principle of serological identification
of bacteria


interpret the result based on agglutination
differentiate serological identification of bacteria
from culture identification
Materials and Methods
Materials

glass slides

70% alcohol

saline solution

pasteur pipets

heat-killed E.coli

E.coli anti serum
Methods
1. Wash slides thoroughly with soap and water.
Rinse with 70% ethanol and air dry.
2. Mark two circles at the opposite ends of the slide
3. Using a Pasteur pipets, place a drop of saline
solution on one end and anti-serum on the other.
4. Add a drop of heat-killed E.coli on each circle.
Rock gently back and forth for two seconds.
Test Procedure
LATEX KIT + BACTERIAL SUSPENSION
Put 1 drop of saline
solution into a circle of a
E.coli
latex
test
kit
agglutination slide.
Mix the latex reagent and
the bacterial suspension
with a clean mixing stick
and rock the slide gently
two or three times.
Examine for agglutination
within a maximum of 2
minutes.
Using another
dropper/pippetor, dispense
1 drop of the bacterial
suspension in the same
circle in the slide close to
the E.coli latex drop.
LATEX KIT + COLONY
SUSPENSION
Using an inoculating loop,
remove a colony from the
selected agar plate and
emulsify the colony in the
drop of saline to produce a
heavy smooth suspension.
Suspensions should only
be made from colonies
with morphologies
resembling E. coli.
Mix with an inoculating
loop. Rock the slide
gently up to 2 minutes
and observe for
autoagglutination or
clumping.
DESIRED
RESULT
Reagent
Control
Saline soln. + E. coli latex
Agglutination
Positive
Control
Positive control + E. coli latex
No
agglutination
Serologic Test
1. Bacterial suspension + E. coli latex
2. Bacterial colony + E. coli latex
Agglutination
Definition of Terms




Agglutination - the clumping of particles. The
word agglutination comes
from
the Latin agglutinare, meaning "to glue to."
Serology – a science that studies bloodserum
and immune responses
that are found
in it.
O antigen – carbohydrate antigen within the
wall of microorganism
H antigen – flagellar components that also
act as an antigen
Principle behind Bacterial
Agglutination



Bacterial agglutination is a highly specific
identification of microorganisms that can be
obtained by serological techniques.
In vitro, antigens and antibodies combine and
produce reactions that are visibly measured.
The chemical compositions of antigens differ;
therefore, each antigen will react only with a
complimentary antibody that is unique to that
antigen.
In gram-negative bacilli, the carbohydrate
antigens within the wall of the organism are
called “O” antigens.
O from German “Ohne” = body
Each species has a different array of O antigens that
can detected in serological tests.
In like manner, those bacilli that are motile also
contain characteristic flagellar protein
components called “H” antigens.
H from German “hauch” = motility
Antigen-antibody reaction can be demonstrated in
several ways:
▪ Latex agglutination- are coated with polyvalent antisera
against a wide range of antigens, when mixed with the
suspension of microorganism latex particles rapidly
agglutinate to form visible clumps
▪ Coagglutination – the aggregation of particulate antigens
combined with agglutinins with more than one specificity.
▪ Enzyme-linked assays. – any enzyme immuno assay
using an enzyme-labeled immunoreactant and an
immunosorbent
These tests depend on linking antibody to a particle or
enzyme in order for a positive
reaction to be observed.
A simple test, which detects O and H antigens
of gram-negative enteric bacilli (usually
Salmonella and Shigella species and
Escherichia coli), is the Latex agglutination
test.
When the unknown organism isolated in culture
is mixed with an antiserum that contains
antibodies specific for its antigenic makeup,
agglutination (clumping) of the bacteria
occurs.

If the antiserum does not contain specific
antibodies, no clumping is seen.

A control test in which saline is substituted for
the antiserum must always be included to be
certain that the organism does not clump in
the absence of the antibodies
✔NOTE: In latex agglutination testing, the antibody
has been conjugated to
 a latex particle in order to enhance the visibility of
the agglutination reaction.
Latex bead
Homospecific antibody