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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