Transcript Isolation and identification of pyogenic cocci

Experiment Four
Isolation and identification
of pyogenic cocci
Objective of Experiment
• To master primary principle and
method of isolation and identification
pyogenic cocci from clinical
specimens .
• To diagnose clinical disease and
guide us to use medicines .
Morphologic observation
1.Staphylococci
G+, arranged in grape like irregular clusters
2.Streptococci
G+, arranged in chain
3.Pneumococci
G+, Lancet-shaped cell.
The Capsule stain: the capsule appears
colorless in contrast to the blue of the cell
4.Meningococci
G-, kidney-shaped ,arranged in pairs
5.Gonococci
Streptococcus
Staphylococcus
Staphylococcus
Streptococcus
• Diplococcus
N. Gonorrhoeae
• N gonorrhoeae infections have
•
a high prevalence and low
mortality
Acquired by sexual contact and
usually affect
– Mucous membranes of the urethra
in men
– Endocervix in women
– Infection may disseminate to a
variety of tissues
• One of the most common STIs
in the world
PROCEDURE
Smears
(Gram-stain)
Morphological characteristics
(Microscopic examination)
Colonial characteristics
Specimens
Isolation
culture
(blood agar plates)
colonial
morphology
observation
typical
colonies
Hemolytic reaction
Pigmentation
Gram staining
observation with the microsco
Pure
culture
Direct identification
Antibiotic
susceptibility test
Identified method for
STAPHYLOCOCCI
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Gram-stain
Isolation and culture
Pure culture
Direct identification
The antibiotic susceptibility test
Gram-stain
• Morphological characteristics
-Typical staphylococcus cells are
spherical,arranging in irregular
clusters,which just like as clusters of grape.
–The cell is about 1 μm in diameter.
–Typical cells are Gram-
positive,nonmotile and do not form
spore.
Staphylococci are Gram-positive cocci, typically
arranged in clumps or Grape-like clusters
Isolation and culture
• Cultural
– Specimens planted on blood agar
plates give rise to typical colonies in
18 hours at 370C
– Hemolysis and pigment production
may not occur until several days later
and are optimal at room temperature.
•
Isolation and culture
• colonial characteristics
– Colonies of staphylococci on solid
meida are round,smooth,raised,and
glistening.S.aureus forms gray to deep
golden yellow colonies.
– S.epidermidis colonies are gray to white
on primary isolation.
– Various degrees of hemolysis are
produced by S.auerus and occasionally
by other species.
Pure culture
• MATERIALS:
– Agar slope culture
– Colonies on agar plate
• PROCEDURE
•.
– With the flame-sterilized wire inoculating loop,
transfer a small amount of bacteria from the
colony on agar plate. Then streak on the agar
slope.
– Sterile the mouth of tubes, replug the test tubes
and flame the loop.
– Label and incubate at 37℃ for 18-24 hours
Directed identification
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The Coagulase Test
The Dnase Test
The mannitol fermentation test.
The phage typing test
Animal Experiment
The Coagulase Test
• Possession of the enzyme coagulase
which coagulase plasma is an almost
exclusive property of
Staph.aureus.There are two ways of
performing this test
– The Slide Coagulase Test
– The Tube coagulase Test
The Coagulase Test
• Coagulase is an enzyme converting
•
fibrinogen into fibrin promoting blood
clotting. It has been speculated that this
enzyme might be a virulence factor with
the coagulated blood around the bacteria
protecting them from the immune system.
However, coagulase-negative strains are
often as pathogenic as coagulase-positive
strains.
The Slide Coagulase Test
The Tube coagulase Test
positive
negative
The Dnase Test
• Inoculate Dnase agar plates with a loop so
that the growth is in plaques about 1 cm in
diameter.Incubate at 370C overnight.Flood
the plate with 1 N hydrochloric
acid.Clearing around the colonies indicates
Dnase activity.The hydrochloric acid reacts
with unchanged deoxyribonucleic acid to
give a cloudy precipitate.A few other
bacteria,e.g. Serratia,may give a positive
reaction.
The mannitol fermentation
test.
• Inoculate the bacteria into a
mannitol micro-tube,incubate at 370C
for 18h.S.aureus will ferment
mannitol to produce acid,which
causes the medium to turn yellow.
The phage typing test
• This test is used to trace the
infective agent in epidemiology if
necessary.It is usually not done for
routine clinical purpose.
Animal Experiment
Isolation and
identification of
bacteria
Vomit
excrement
remaindered food
Meat soup
media
filter
injection
observation
6--8W
cat
food
poisoning
The antibiotic susceptibility
test
• This test is helpful for the treatment
of S.aureus infection.
• materials
– S.aureus（isolated from the pus of a
patient）.
– Several kinds of filter paper （each
contains different kinds of antibiotics）
– Nutrient agar plate
The antibiotic susceptibility
test
• PROCEDURE
–Streaking
the S.aureus on
(thoroughly covered the plate)
agar
plate
–Put 4 kinds of paper contained different
antibiotics on the plate (each paper are far away
about 2 cm)
–Incubate ar 370C 18-24 hours.
–Observe the results the plates are examined for
the present of zones of inhibition of bacterial
growth around the filter paper.The sensitivity
of the organism is indicated by the diameter of
the zone of growth inhibition.
Hiss’ stain
• Dissection of white mice
• Materials
• Animals:mice died from infection
• Instruments for autopsy:sterile tweezers
and scissors,anatomic board
• Pins,iodine,ethanol,cotton absorbent
gauze balls,3%lysol ,and so on
How to make a smear
• Open the abdomen cavity from pubis and
expose the abdominal viscera .
• Check the intestine
• Put tissue fluid on the slide
Hiss capsule staining
Smear
Dry
1%crystal violet
in alcohol
2mins
Purple black cells of
bacteria and colorless
capsule
Wash with
20%Cuso4
Examine under the
oil immersion lens
Blot dry with
bibulous paper
Pneumococcus