Isolation and identification of pyogenic cocci

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Isolation and identification of pyogenic cocci
Experiment Three
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Objective of Experiment

• To master basic principle and method,which use to
  isolate and identify pyogenic cocci from clinical
  specimens.
• To diagnose clinical disease and guide doctors
  to select proper medicines .

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PROCEDURE
Smears
Morphological characteristics
(Gram-stain)
(Microscopic examination)
Specimens
Isolation culture
(blood agar plate)
stained and observe
typical colonies
Direct identification
Pure culture
Antibiotic susceptibility test
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Identified method for Staphylococci

• Gram-stain
• Isolation and culture
• Pure culture
• Direct identification

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

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

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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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Test of Microbiological experiment

• While a patient is infected by pathogenic
  enterobacteria (Salmonella),how to diagnose with
  microbiological methods in lab?
• (simple procedure)
• While a patient is infected by pyogenic cocci,how
  to diagnose with microbiological methods in
  lab?(simple procedure)
• While a patient is infected by influenza
  virus,how to diagnose with microbiological
  methods in lab?(simple procedure)

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The Coagulase Test

• Coagulase is an enzyme converting fibrinogen into
  fibrin promoting blood clotting.
• It might be a virulence factor with the
  coagulated blood around the bacteria protecting
  them from the immune system.
• 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 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
vomit
excrement
remaindered food
Meat soup media
Isolation and identification of bacteria
filter
injection
68w cat
observation
Food poisoning
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The antibiotic susceptibility test

• This test is helpful for the treatment of
  S.aureus infection.
• materials
• Staphaureus(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
Streaking the staphaureus 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 at 370C for 18-24 hours.
Observe the results the plates The
sensitivity of the organism is indicated by the
diameter of the zone of growth
inhibition.
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Laboratory Diagnosis of Pathogenic
Enterobacterial Infection
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Dimidiation of the enterobacteria according to
the fermentation of lactose

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

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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
• Pathogenetic colonies middle size, red
• Suspect colonies colorless, small,
  opaque

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Biochemical reactions of Salmonella, etc

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

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Antigens of salmonella

• O antigen polysaccharide of LPS
• somatic antigen
• used to divide
  Salmonella to 42 groups
• A-Z groups are
  pathogenic
• stable to heat
  (remains activity at 100 ? )
• H antigen flagella antigen
• divide Salmonella
  tow phase
• special no
  special
• Vi antigen related to the virulence of
  Salmonella
• sensitive to heat (
  lose activity at 60 ?)

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Salmonella
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Serological Identification of Salmonella
Select the specimen
React with A-Z polyvalent antiserum
Agglutination reaction
()
(-)
React with several individual types of anti-O and
anti-H antiserum

• React with anti-Vi antiserum

Identify its group and phage
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Gram Stain
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• Gram stain of
• Nocardia asteroides

acid-fast stain of Nocardia asteroides
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flagella