Gram Staining PPT

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					ISOLATION, GRAM STAINING AND
  IDENTIFICATION OF BACTERIA
             BY

         DR. TED PASS II
KENTUCKY MICROBIOLOGY LABORATORY
     CERTIFICATION PROGRAM
      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
           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.
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 salts…i.e., Staphylococcus aureus
ISOLATION STREAK TECHNIQUE
ISOLATION OF BACTERIA ON MACCONKEY
FOR GRAM STAINING AND IDENTIFICATION
      Gram-Staining Procedure
The reagents you will need to successfully
 perform this technique are:
•Crystal Violet ( Primary Stain)
•Iodine Solution (Mordant)
•Decolorizer ( 95%Ethanol )
•Safranin ( Counterstain)
•Water (preferably in a squirt bottle)
SMEAR PREPARATION




STEP 1     STEP 2




STEP 3      STEP 4
        STAINING PROCEDURE
   STEP 1: Flood (cover completely) the
    entire slide with Crystal Violet…CV
    attaches to the cell wall of both G+ and G-
    bacteria. Let the crystal violet stand for
    about 60 seconds.
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
      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 specimen…Removes 20% lipids,
    i.e., LPS and CV-I complex (Decolorizing
    the cell) Quickly, rinse the slide with the
    water for 5 seconds.
     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
GRAM NEGATIVE AND GRAM
  POSITIVE ORGANISMS
GRAM POSITIVE COCCI
GRAM NEGATIVE RODS
GRAM POSITIVE COCCI
   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)
ENDOSPORE SLIDE
    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 .
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
                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.
TILT AT ANGLE TO FILL CUPULES
        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.
         •    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
   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.
    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.
ENTEROTUBE INOCULATION
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.
        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.
         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-
    37°C. for 18-24 hours.
POSITIVE REACTIONS

				
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