Isolation and identification of pyogenic cocci

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Isolation and identification of pyogenic cocci
Experiment Four
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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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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
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• Streptococcus
• Staphylococcus

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• Staphylococcus

Streptococcus
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• Diplococcus

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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

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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

positive 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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Hiss stain

• Dissection of white mice
• Materials
• Animalsmice died from infection
• Instruments for autopsysterile tweezers and
  scissors,anatomic board
• Pins,iodine,ethanol,cotton absorbent gauze
  balls,3lysol ,and so on

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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

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Hiss capsule staining
1crystal violet in alcohol
Smear
Dry
2mins
Purple black cells of bacteria and colorless
capsule
Wash with 20Cuso4
Blot dry with bibulous paper
Examine under the oil immersion lens
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Pneumococcus