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Chapter 12  Laboratory Skills  239


               Care must be taken when preparing the specimen      When dry, place beside microscope with bottle of
             slide. A common mistake is applying too much inoculum   immersion oil. If using paper records place the patient
             on the slide, resulting in uneven staining. Swabbed sam-  record beside slide. Notify veterinarian or technician
             ples are rolled onto the slide, one pass per line is all that   when slide is ready to read.
             is needed. If getting the sample from an agar plate, a
             sterile wire is used to select the desired bacterial colony
             and a tiny bit of the colony is mixed with a drop of sterile
             water or saline on the slide.                         Learning Exercise
               To stain the slide, start by setting up the stains each in   Write the steps for the Gram stain in your refer-
             their own dropper bottles or Coplin jars. If in dropper   ence book.
             bottles you will need a staining rack. PPE will be gloves
             and goggles.

             1.  Fix the sample on the slide by passing it briefly over
                a hot flame. This is usually accomplished with a   Inoculation of Media
                Bunsen burner. Fixing adheres the sample to the   Inoculating media is the transferring of a sample or
                slide and preserves the morphology (shape) of the   specimen onto media that will support bacterial growth.
                bacteria.                                       Culturing (growing) bacteria may take 24–48 hours in
             2.  Place the staining rack over the sink and lay the   an in‐house incubator. Blood agar is commonly inocu-
                fixed slide on top of it in a position in which it won’t   lated initially because many types of pathogens will grow
                fall off.                                       on it. It contains 5% sheep blood. If the specimen is
             3.  The first stain applied is the crystal violet. Cover   going to be worked up in‐house, the first swab taken is
                the entire slide with this stain and allow it to   used to inoculate the blood agar plate. The quadrant
                remain on the slide for 30 seconds. Gently rinse   streak method is commonly used to end up with isolated
                with tap water.                                 colonies of bacteria growing in the last quarter of the
             4.  Apply iodine solution in the same manner and let it   plate. Isolated colonies are necessary so they can be
                sit on the slide for 30 seconds. Gently rinse with tap   picked out from other colonies as cross‐contamination
                water.                                          from different bacteria can complicate the identification
             5.  Apply the de‐colorizer to the slide for approxi-  procedures.
                mately 10 seconds or until no more purple coloring   Lift the lid using the free hand (Figure 12.22). Only
                washes away. (Do not overdo.) Gently rinse with tap   tip one side of the lid upward enough to slide the culture
                water.                                          swab between the upper and lower portions of the plate
             6.  Apply Safranin stain and let it sit on the slide for   without touching either part. Removing the lid com-
                30 seconds. Gently rinse with tap water.        pletely will allow room‐air contaminants to settle onto
             7.  Air dry by propping the slide upright or blot dry   the media. The swab is swept across one‐quarter of the
                using blotting paper. This is different from Kem   plate, with a back and forth motion. Flame a wire loop,
                Wipes and lens paper.                           rotate the plate a quarter turn, slide the loop into the

             Staining in Coplin jars                            previous quadrant then sweep it back and forth in the
                                                                second quadrant. The process is repeated, flaming
             1.  Use forceps or a spring‐type clothes pin to move the   the loop and streaking material from each quadrant of
                slide from jar to jar. Insert the slide and hold it still   the plate. In the fourth quadrant, a single squiggled
                for the prescribed time.                        streak is used. Care must be taken to not put too much
                Move the slide according to sequence and times   inoculum (sample) on the plate initially as it may run
                listed below. Drain the end of the slide on the edge   across the surface as the plate is tilted. Flaming the wire
                of each jar before moving to reduce cross‐contami-  between streaks is essential to thin out the sample
                nation of solutions.                            enough to get single colonies.
                •  Crystal violet stain: 30 seconds                After streaking, the plate is labeled and placed
                •  Tap water rinse                              upside down in the incubator to avoid condensation on
                •  Iodine solution: 30 seconds                  the agar surface which would cause the bacteria to mix.
                •  Tap water rinse                              Enter the date and time that the culture was set up in
                •  95% ethanol (decolorizer): 10 seconds        the patient’s file and laboratory log. If there is no
                •  Tap water rinse                              growth after 24 hours, the blood agar plate is re‐incu-
                •  Safranin counterstain: 30 seconds            bated for another 24 hours. If there is still no growth,
                •  Tap water rinse                              then the specimen is reported as “no growth.” If there
             2.  When finished dry in the same manner as described   is growth, representative colonies from the plate will be
                earlier.                                        selected for Gram staining. Based on the Gram stain
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