Isolation and identification of pyogenic cocci

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Isolation and identification of pyogenic cocci
Experiment Two
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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 .

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PROCEDURE
Morphological characteristics
(Gram-stain)
(Microscopic examination)
Specimens
typical colonies
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Identified method for STAPHYLOCOCCI

• Gram-stain
• Isolation and culture
• Pure culture
• Direct identification
• The antibiotic susceptibility test

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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.

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Staphylococci are Gram-positive cocci, typically
arranged in clumps or Grape-like clusters
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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.

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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.

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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
• .

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Directed identification

• The Coagulase Test
• The Dnase Test
• The mannitol fermentation test.
• The phage typing test
• Animal Experiment

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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

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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.

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The Slide Coagulase Test
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The Tube coagulase Test

Left tube coagulase positive Right tube coagulase
negative
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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.

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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.

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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.

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Animal Experiment
Isolation and identification of bacteria
Vomit
excrement
remaindered food
observation
filter
Meat soup media
6--8W cat
food poisoning
injection
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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

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The antibiotic susceptibility test

• PROCEDURE

• Streaking the S.aureus on agar plate (thoroughly
  covered the 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.

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Dimidiation the enterobacteria according to the
fermentation of lactose

• Lactase fermenters saprophytic and commensal
• Escherichia Klebsiella
  Citrobater
• Enterobacter Enterobacter,
• Non lactase fermenters pathogens
• Salmonella Shigella some
  Citrobacter,
• Proteus Serratia

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Biochemical reactions of Salmonella, etc

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• Species butt slope
  H2S motility
• E.coli AG
  AG -
• Salmonella A -
  /-
• Other Salmonella AG -
  /-
• Shigella A
  - /- -
• butt ferment dextrose slope
  lactose
• Aacid AG acid and gas

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Procedure

Colonial characteristic observation Specimens
isolation Gram Staining
(SS/EMB plate)
Serological identification
TSI

Biochemical reaction

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Specimens

• Different specimens should be taken depending on
  the kind and the process of the disease.
• blood
• bone marrow
• Urine
• stool

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Isolation

• Culture medium S.S agar
• Method streak plate
• Result
• unpathogenetic colonies middle size, red
• Suspect colonies colorless, small,
  opaque

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Escherichia coli
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Shigella sonnei
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Salmonella
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Vibrio cholerae