Experiment 1 Simple Staining

Experiment Inventory

Materials Labware
Crystal Violet 100 mL Beaker
6 Drops Sterile Saline Forceps
Nutrient Agar 2 Sterile Cotton Swabs
Candle Disposable Gloves
Matches Reusable Metal Inoculation Loop
*2 mL Distilled Water Sterile Transfer Pipette
*Bleach 1/2 Sheet of Bibulous Paper
*Isopropyl Alcohol (2) 5 cm Petri Dish
*Sink or Disposable Plastic Container Sterile Plate Spreader
*Microwave or Boiling Water Bath 6 Glass Microscope Slides
*Permanent Marker Wax Pencil

Note: You must provide the materials listed in *red. 9 cm Petri Dish

Parafilm®

*Hot Pad

*Lab Notebook (optional)

*Scissors

EXPERIMENT 1: SIMPLE STAINING

Simple staining quickly assesses bacterial shape and size. This experiment uses crystal violet to help visualize and understand
microbial structures more accurately.

PROCEDURE

Note: If you are keeping a lab notebook, record the date, time, and experiment title on a fresh page before
you begin.

Prepare Agar Plates

1. Loosen or remove the cap on the nutrient agar bottle.

2. Place the bottle in a microwave. You will need to remove the bottle from the microwave and swirl the contents every 10
seconds to evenly distribute the heat. If you do not have a microwave, place the bottle in a heat-safe bowl, create a water
bath by pouring boiling water into the bowl (around the bottle), and heat until the entire bottle of agar is liquefied.
 Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling.
Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. Once boiling has
stopped, use a hot pad to protect your hands and remove the bottle from the microwave. Use caution when
removing the bottle from the microwave as it will be HOT!

3. With a hot pad protecting your hands, remove the agar bottle from the microwave. Use caution when removing the bottle
from the microwave as it will be HOT!

4. Gently swirl the bottle a final time to mix the solution.

5. Pour the agar solution into the bottom half of two Petri dishes. Use the agar sparingly. You only need enough to coat the
bottom of the petri dish.

Note: Approximately 5 mL should be enough agar to cover the entire bottom of the dish. If not, continue to
add agar in approximately 1 mL increments until the bottom of the petri dish is completely covered. The agar
should be only 1 or 2 millimeters deep. If too much agar is poured, there will be no space under the cover for
microbial growth.

6. Place the lids onto the dishes slightly off-center to create a small space to allow condensation to evaporate and allow the
agar to solidify undisturbed. This should take approximately 30 – 60 minutes. If you will not be using the dishes immediately,
you can store them upside down in the refrigerator after they have fully gelled. Remove them from the refrigerator, and allow
them to sit at room temperature for at least 1 hour prior to use.

Inoculate Prepared Plates

1. Select two surfaces from which to collect microorganisms. Examples of possible sources include shoes, a table, teeth,
mouth or throat, bathroom doors, and shopping carts. Avoid surfaces that are frequently cleaned with antibacterial
detergents and soaps.

2. Put on a pair of gloves, and begin collecting your samples by rubbing a cotton swab on the first surface you selected. Be
sure to get good coverage on the cotton swab, and use a new cotton swab for each sample.

Note: It helps if the cotton swab has been lightly moistened with distilled water prior to collecting your
samples. The water helps promote visible, well-developed bacterial growth.

3. Remove the lid from the agar plate, but hold it closely over the top of the plate to use as a shield to prevent airborne
contamination of the plate.

4. Carefully streak the cotton swab onto the gelled medium (agar). Ensure that you do not press too hard when streaking the
swab on the agar to prevent cutting into the agar surface.

5. Place the lid onto the agar plate, and seal it with a strip of Parafilm® (hold one end of the Parafilm® firmly against the side of
the Petri dish, and stretch it to the other side to cover the entire perimeter).

6. Use a permanent marker to label the bottom the Petri dish with the source of the sample (e.g., if you collect your sample
from a table, label the dish as “Table”).

7. Repeat steps 2 – 6 for one additional surface, using a new sterile swab and a new agar plate.

8. Let the plates incubate upside-down in a warm location for three days, or until visible colony growth has formed.

Smear the Slide

Note: You will prepare a total of six slides in this experiment. You will use only two slides for this experiment.
The other four will be used in Experiments 2 and 3.
 1. Use the wax pencil to label the end of each slide with the sample source and a future treatment. You will need two slides for
each treatment (Simple, Negative, and Gram), with ideally one slide per treatment for each source. For example, for the
simple staining procedure you will need one slide labeled “Simple: Table” and another slide labeled “Simple: Shoe.” Place
the two “Negative” slides in one-half of the 9 cm Petri dish, cover, and safely store.

2. Use the wax pencil to make a circle in the center of the four remaining microscope slides.

3. Use a pipette to transfer one drop of sterile saline into each circle (4 drops total).

4. Find two well-developed and isolated bacterial colonies on the Petri dish for the two different sample sources.

5. Sterilize your inoculation loop with your candle.

a. Pour 70% isopropyl alcohol into a 100 mL beaker to a depth of 2 – 4 cm. Place the cap back on the bottle, and position it
far out of the way.

b. Light your candle, and set it aside. Be very cautious that your candle is safely positioned away from the beaker of
isopropyl alcohol.

c. Dip the inoculation loop into the isopropyl alcohol for 10 seconds. Once you’ve put the inoculation loop in the alcohol,
keep it angled down so that no alcohol drips back onto your hand.

d. Without touching the inoculation loop to anything, carefully pass the end of the inoculation loop through the flame several
times.

e. Extinguish the flame when complete.

6. Use your sterile inoculating loop to transfer a small amount of one of the bacterial colonies to the saline drop within the circle
on the corresponding slide. Mix gently.

7. Repeat Steps 5 – 6 for three more colonies, using the remaining three slides. Allow the samples to air-dry.

8. Light the candle. Pick up one slide with the forceps, and fix the sample by passing the slide, smear side up, through the
candle flame two or three times (Figure 8). Repeat this process until all of the
slides have been heat-fixed. Extinguish your flame when done.

9. You will use the Simple slide for the rest of this procedure. Place the Negative
and Gram slides in the other half of the 9 cm Petri dish, cover, and safely store
for future experiments.

Stain the Slide (simple technique)

1. Over a sink or disposable plastic container, place several drops of crystal violet
onto the smear on one of the “Simple” slides so that the smear is completely
covered (Figure 9). Let the sample incubate in the dye for 1 minute. Take a
photograph of this step. Submit your photograph to your instructor at the end of
the lab.
Figure 8: Pass the slide smear side up over the
2. Gently rinse the slide with distilled water for 30 seconds. candle to heat fix the smear.

Note: If the stream of water is too strong, you may accidentally

wash off your smear even though it has been heat-fixed.

3. Use one half piece of bibulous paper to blot the excess water from the slide.
Take caution not to disturb the sample.

4. If you have a microscope available, observe the stained slide under increasing
magnification, and record what you see at each magnification in Table 1,
focusing on morphology and arrangement of the cells using terms learned in
this lab. If no microscope is available, refer to Figure 10 for your observations,
and take a photograph of your slide if required by your instructor. If keeping a
lab notebook, print out Table 1, and tape it into your lab notebook or re-create it
by hand.
Figure 9: Pour crystal violet over the smear.
 5. Place your slide in a disposable plastic container, and pour the bleach over the surface until the sample is completely
covered/saturated. Allow the sample to soak in the bleach for approximately 20 minutes, and then pour the bleach down the
sink with running water.

6. Wrap the slide in Parafilm® and dispose of it in the trash.

Figure 10: Isolated bacteria simple-stained with crystal violet.

Data Sheet Experiment 1 Data Sheet

Table 1: Experiment 1 Staining Observations

Stain Used:

Observations: