ISOLATION, GRAM STAINING AND IDENTIFICATION OF BACTERIA

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ISOLATION, GRAM STAINING AND IDENTIFICATION OF
BACTERIA

• BY
• DR. TED PASS II
• KENTUCKY MICROBIOLOGY LABORATORY
• CERTIFICATION PROGRAM

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PROCEDURE FOLLOWED TO IDENTIFY BACTERIA

• OBTAIN A PURE CULTURE USING A DIFFERENTIAL MEDIA
  SUCH AS MACCONKEY AGAR
• PREPARE A SLIDE USING GRAM STAIN PROCEDURE
• IDENTIFY BACTERILA ISOLATES USING THE API OR
  ENTEROTUBE
• IDENTIFICATION SYSTEM

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MACCONKEY AGAR
MacConkey agar is probably the most popular solid
differential/selective medium in the world. It is
mainly used in isolation of lactose fermenting,
Gram-negative enteric pathogens and for
inhibiting growth of Gram-positive organisms.
Bacterial colonies that can ferment lactose turn
the medium red. This red color is due to the pH
indicators (Neutral Red) response to the acidic
environment created by the fermentation of
lactose. MAC is decolorized by NLF bacteria...as
they utilize Amino Acids alkaline metabolites
are released and the Neutral Red becomes
colorless.
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Lactose Fermentors vs Non Lactose Fermentors

• Examples of LF
• Escherichia coli
• Klebsiella pneumoniae
• Enterobacter aerogenes
• Citrobacter freundi
• Examples of NLF
• Pseudomonas aeruginosa
• Proteus mirabilis
• Gram Positive bacteria are inhibited by
  Crystal violet and bile saltsi.e.,
  Staphylococcus aureus

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ISOLATION STREAK TECHNIQUE
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ISOLATION OF BACTERIA ON MACCONKEY FOR GRAM
STAINING AND IDENTIFICATION
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Gram-Staining Procedure

• The reagents you will need to successfully
• perform this technique are
• Crystal Violet ( Primary Stain)
• Iodine Solution (Mordant)
• Decolorizer ( 95Ethanol )
• Safranin ( Counterstain)
• Water (preferably in a squirt bottle)
•                             

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SMEAR PREPARATION
STEP 1
STEP 2
STEP 4
STEP 3
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STAINING PROCEDURE

• STEP 1 Flood (cover completely) the entire slide
  with Crystal VioletCV attaches to the cell wall
  of both G and G- bacteria. Let the crystal
  violet stand for about 60 seconds.

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STEP 2 Now, flood your slide with the
iodine solution.

• Rinse the slide with water after 60 sec. At this
  point, the specimen should appear brownish to
  blue-violet in color. Iodine strengthens the bond
  between the CW and the CV

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STEP 3 Add the Decolorizer, Ethanol for 15 sec.

• To be safe, add the Ethanol dropwise until the
  blue-violet color is no longer emitted from your
  specimenRemoves 20 lipids, i.e., LPS and CV-I
  complex (Decolorizing the cell) Quickly, rinse
  the slide with the water for 5 seconds.

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STEP 4 The final step involves applying the
Counterstain, Safranin.

• Flood the slide with the dye as you did in steps
  1 and 2. Let this stand for about a 60 sec.
  Rinse with water for 5 seconds to remove any
  excess dye. Safranin will attach to G- Cell Wall

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GRAM NEGATIVE AND GRAM POSITIVE ORGANISMS
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GRAM POSITIVE COCCI
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GRAM NEGATIVE RODSGRAM POSITIVE COCCI
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The Gram stain differentiates between bacteria
that possess 2 and 20 Lipids in their cell wall
or "cell envelope. The G- CW has an outer
membrane of Lipopolysaccharides LPS)
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ENDOSPORE SLIDE
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API IDENTIFICATION SYSTEM

• This API-20E test strip (from bioMerieux, Inc.)
  is used to identify the enteric gram negative
  rods 20 separate test compartments are on the
  strip, all dehydrated. A bacterial suspension is
  used to rehydrate each of the wells. Some of the
  wells will have color changes due to pH
  differences others produce end products that
  have to be identified with reagents. A profile
  number is determined from the sequence of and -
  test results, then looked up in a codebook having
  a correlation between numbers and bacterial
  species .

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INOCULATION OF AN API STRIP

• Prepare a suspension of the bacteria in the
  saline tube
• Inoculate a large colony (2-3mm diameter)of the
  bacterium (pure culture) into the 0.85 NaCl
  solution.
• Use a McFarland barium sulfate standard 3

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INOCULATION

• Holding the strip at a slight angle up from the
  table top, you will now inoculate the bacterial
  suspension into each well with the sterile
  pipette.
• Touch the end of the pipette to the side of the
  cupule, allowing capillary action to draw the
  fluid into the well as you slowly squeeze the
  bulb. This should eliminate any bubbles forming
  in the wells. Each well should be filled up to
  the neck (see diagram).
• CIT, VP, and GEL have boxes around their names.
  These test wells will be filled all the way up to
  the top of the well.
• LDC, ODC, ADH, H2S, and URE are filled as
  described in step B, but they will then be filled
  up to the top with sterile mineral oil.

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TILT AT ANGLE TO FILL CUPULES
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Incubate the strip in its chamber

• The bottom of the incubation chamber has small
  indented wells in the bottom fill it with water
  just enough to fill these indentations.
• Place the strip into this bottom. There should
  not be so much water that it spills onto the API
  strip.
• Place the top of the incubation chamber over the
  bottom, and label it.
• Place the strip at 35 to 37º C for 18-24 hours.

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INTERPRETATION

• Add the proper reagents to the compartments 1
  drop of Kovac's to the IND (read within a couple
  of minutes)
• 1 drop of Barritt's A and B to VP (a positive
  reaction may take up to 10 minutes)
• 1 drop of FeCl3 to TDA

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• Record results on the diagram handed out to you
  in lab (1, 2, or 4 points for reaction, 0
  points for - reaction). The oxidase test reaction
  should be negative, and is added as the last test
  result.
• Three test reactions are added together at a time
  to give a 7-digit number, which can then be
  looked up in the codebook.

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ENTEROTUBE IDENTIFICATION SYSTEM

• The Enterotube II contains 12 different agars
  enabling the performance of a total of 15
  biochemical tests as well as an enclosed
  inoculating wire.

The Enterotube II contains 12 different agars
enabling the performance
of a total of 15 biochemical tests as well as an
enclosed inoculating wire.
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ENTEROTUBE INOCULATION
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INOCULATION OF ENTEROTUBE

• 1. Remove both caps of the Enterotube II and
  with the straight end of the inoculating wire,
  pick off the equivalent of a colony from your
  unknown plate. A visible inoculum should be seen
  on the tip and side of the wire.
• 2. Inoculate the Enterotube II by grasping the
  bent-end of the inoculating wire, twisting it,
  and withdrawing the wire through all 12
  compartments using a turning motion.

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ENTEROTUBE (contd.)

• 3. Reinsert the wire into the tube (use a turning
  motion) through all 12 compartments until the
  notch on the wire is aligned with the opening of
  the tube. (The tip of the wire should be seen in
  the citrate compartment.) Break the wire at the
  notch by bending. Do not discard the wire yet.

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ENTEROTUBE (contd.)

• 4. Using the broken off part of the wire, punch
  holes through the cellophane which covers the air
  inlets located on the rounded side of the last 8
  compartments. Your instructor will show you their
  correct location. Discard the broken off wire in
  the SHARPS container.
• 5. Replace both caps and incubate the Enterotube
  II on its flat surface at 35-37C. for 18-24
  hours.

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POSITIVE REACTIONS