Experiment #4 The Gram Stain
START
Obtain:
1. Pure Broth Cultures (12-18h): staphylococcus
(Se), Serratia Marcescens (Sm).
2. Streak Plate from Experiment 3
3. Staining Solutions (Hucker’s crystal violet,
Gram’s iodine solution, 95% ethyl alcohol,
Safranin)
4. Demonstration slides: Serratia Marcescens
(Sm) and Staphylococcus (Se))
Step 1: Choose a scenario for the gram staining
procedure. Use a sharpie to draw three dime sized
circles on the slide and label. Work aseptically &
flame between samples
Step 2: Flame loop and place 2-3 drops onto the slide for
each smear. Flame loop again and allow it to cool,
removing a small quantity of bacteria cells from the streak
plate (Less than a loopful).
Step 3: place cells in the water on the slide,
rotating the loop in a circular motion, spread
out into a thin smear. (for solid cultures place
2-3 loopfuls of water onto slide before adding
cultures)
Step 4: Flame loop again, repeat steps
2-4 (only if completing scenario A).
Agitate broth culture smears and aseptically
obtain a loopful of broth, smearing it in
appropriate circle. (see page 47 Figures 2.5, 2.6)
Step 5: Place slide on staining rack, ensuring its
level. Cover smears with 1-3 drops of crystal
violet stain for 1 minute. Do not allow it to dry.
Pour excess into pan and rinse with DI water.
Step 6: Cover smear with 1-3 drops of Grams
iodine. Allow it to sit for 1 minute then gently
wash with DI water
Trinity R Copeman
Sept. 29 2025
Step 7: Hold slide at 45 degree
angle over the pan, apply alcohol
dropwise at the top of the slide
allowing it to run over the smear and
off the slide. Repeat until the alcohol
running off the slide no longer has
colour. (maximum of 4-5 drops)
Step 8: Place slide on staining rack
over the pan and rinse off alcohol
with DI water. Counter stain by
covering smear with 1-3 drops of
safranin for 1 minute. Then rinse with
DI water. Gently blot dry the slide
and let air dry.
Step 9: Make microscopic
examinations of each stained smear,
10x
40x
oil immersion 100x
(Page 37, Figure 1.2). No need to
draw results however you will be
identifying morphology of the
cultures.
→
→
DO NOT:
Make smears too thick (difficult to observe)
Overheat the smears (two seconds of heating)
Avoid overdoing decolorization - keep washing
and drying the same between steps
End of experiment: Clean oil immersion from lense
and slide with kimwipe. Put away microscope and
write down observations and results.
FINISH
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