Simple staining quickly assesses bacterial shape and size.

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 sec-

onds 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: You must provide the materials listed in *red.

 

 

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 pro-

tect 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.

Note: Approximately 5 mL should be enough agar to cover the entire bottom of the dish. If not, continue to add agar in ap-

proximately 1 mL increments until the bottom of the petri dish is completely covered. The agar should be only 1 or 2 millime-

ters 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

7. 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 deter-

gents and soaps.

8. 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 sterile, deionized water prior to collecting your samples.

The water helps promote visible, well-developed bacterial growth.

9. 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 contam-

ination of the plate.

10. 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.

11. 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).

12. 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”).

13. Repeat steps 7 – 11 for one additional surface, using a new sterile swab and a new agar plate.

14. 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.

15. 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.

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

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

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

19. 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 isopro-

pyl 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.

20. Use your sterile inoculating loop to transfer a small amount of one of the bacte-

rial colonies to the saline drop within the circle on the corresponding slide. Mix

gently.

21. Repeat Steps 18 – 20 for three more colonies, using the remaining three slides.

Allow the samples to air-dry.

22. 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 7). Repeat this process until all of the slides have been heat-fixed. Ex-

tinguish your flame when done.

23. 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)

24. 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 8). 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 7: Pass the slide smear side up over the can- dle to heat fix the smear.

Figure 8: Pour crystal violet over the smear.

 

 

25. Gently rinse the slide with deionized water for 30 seconds.

Note: If the stream of water is too strong, you may accidentally wash off your smear even though it has been heat-fixed.

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

27. 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 mi-

croscope is available, refer to Figure 9 for your observations, and take a photograph of your slide if required by your instruc-

tor. If keeping a lab notebook, print out Table 1, and tape it into your lab notebook or re-create it by hand.

28. Place your slide in a disposable plastic container, and pour the 10% bleach solution 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.

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

Figure 9: Isolated bacteria simple-stained with crystal violet.

 

 

Data SheetExperiment 1 Data Sheet Table 1: Experiment 1 Staining Observations

Stain Used:

Observations:

 

1. 1 Loosen or remove the cap on the nutrient agar bottle:
2. 2 Place the bottle in a microwave You will need to remove the bottle from the microwave and swirl the contents every 10 sec:
3. 3 With a hot pad protecting your hands remove the agar bottle from the microwave Use caution when removing the bottle:
4. 4 Gently swirl the bottle a final time to mix the solution:
5. 5 Pour the agar solution into the bottom half of four Petri dishes:
6. 6 Place the lids onto the dishes slightly offcenter to create a small space to allow condensation to evaporate and allow the:
7. 7 Select two surfaces from which to collect microorganisms Examples of possible sources include shoes a table teeth:
8. 8 Put on a pair of gloves and begin collecting your samples by rubbing a cotton swab on the first surface you selected Be:
9. 9 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 contam:
10. 10Carefully streak the cotton swab onto the gelled medium agar Ensure that you do not press too hard when streaking the:
11. 11 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:
12. 12Use a permanent marker to label the bottom the Petri dish with the source of the sample eg if you collect your sample:
13. 13Repeat steps 7 11 for one additional surface using a new sterile swab and a new agar plate:
14. 14Let the plates incubate upsidedown in a warm location for three days or until visible colony growth has formed:
15. 15Use the wax pencil to label the end of each slide with the sample source and a future treatment You will need two slides:
16. 16Use the wax pencil to make a circle in the center of the four remaining microscope slides:
17. 17Use a pipette to transfer one drop of sterile saline into each circle 4 drops total:
18. 18Find two welldeveloped and isolated bacterial colonies on the Petri dish for the two different sample sources:
19. 19Sterilize your inoculation loop with your candle:
20. 20Use your sterile inoculating loop to transfer a small amount of one of the bacte:
21. 21Repeat Steps 18 20 for three more colonies using the remaining three slides:
22. 22Light the candle Pick up one slide with the forceps and fix the sample by:
23. 23You will use the Simple slide for the rest of this procedure Place the Negative:
24. 24Over a sink or disposable plastic container place several drops of crystal violet:
25. 25Gently rinse the slide with deionized water for 30 seconds:
26. 26Use one half piece of bibulous paper to blot the excess water from the slide Take caution not to disturb the sample:
27. 27If you have a microscope available observe the stained slide under increasing magnification and record what you see at:
28. 28Place your slide in a disposable plastic container and pour the 10 bleach solution over the surface until the sample is:
29. 29Wrap the slide in Parafilm and dispose of it in the trash:

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