Lab Exercise: Antiseptics and Disinfectants- Evaluation using filter paper
method
OBJECTIVES
1. Compare the antimicrobial capabilities of different antiseptic and disinfectant
chemicals.
2. Compare effectiveness of antiseptics with different type of bacteria.
3. Utilize aseptic techniques.
INTRODUCTION
Both of antiseptics and disinfectants are substances are
agents that kill, or at least control the growth of, microbes.
Antiseptics are agents that are used on living tissue.
Examples include alcohol or iodine. Disinfectants are used
on non-living things such as floors, counter tops, dishes, etc.
They are usually stronger and are too toxic to be used on
living tissue. A common disinfectant is bleach or Cavicide in
our lab. They have various ways of killing organisms such as
interfering with the way they make their cell walls so they
blow up, or prevent them from reproducing somehow. In this lab we will compare the
effectiveness of several disinfectants and antiseptics with each other on different
bacteria species. See page 102 in the Alexander atlas for more information on this
procedure, as well as photographs.
LAB EXERCISES
Class supplies
Antiseptics and
disinfectants
Sterile filter paper
disks
Forceps
Team supplies
Culture of Escherichia coli
Culture of Pseudomonas
aeruginosa
Culture of Bacillus cereus
Culture of Staphylococcus
epidermidis
Pipet and pipet tips
Metal spreader, turntable,
and alcohol
Individual supplies
2 nutrient agar plates
Protocol:
Day 1
1. Assign one test organism to each member of your team. Label both of your plates with
the species you are using.
2. Divide each plate into four quadrants, and label each quadrant with one of the
antiseptics or disinfectants (you will test a total of 8 chemicals).
3. Transfer 100 µl (0.1ml) of your test organism to each plate and top spread.
4. Using alcohol-flamed forceps, pick up a sterile paper disk and dip it halfway into a
disinfectant or antiseptic or choice, then place the disk on the appropriately labeled
quadrant of the inoculated media. The chemical will wick up into the paper disk. No need
to push in, the liquid should adhere to the agar.
5. Repeat the procedure with the other chemicals, using 4 chemicals per plate.
6. Incubate the plates at the appropriate temperature
Day 2
1. Measure the diameter of the zone of inhibition for each chemical in millimeters. If
there is no zone around the disk, call it 0.
2. Record the results in the table below.
DATA AND OBSERVATIONS
1. Record the results for all members of your team.
Chemical
Diameter of Zone of Inhibition (in mm)
B. cereus
S. epidermidis
E. coli
P. aeruginosa
2. Use the data above to answer the following questions:
a. The most effective agent I used was ________________________.
b. The least effective agent I used was _________________________.
c. The organism I used was __________________.
3. Which chemicals were most effective against each of the bacteria we used?
Escherichia coli______________________
Pseudomonas aeruginosa______________________
Bacillus cereus_____________________________
Staphylococcus epidermidis_____________________________
4. Is there a difference with gram + and gram - bacteria? Give examples with species and
chemicals.
DISCUSSION
1. Differentiate between antiseptic and disinfectant. Give examples of each.
2. What factors influence the size of the zone of inhibition produced by a chemical?
3. How might the physical differences between gram + and gram – bacteria contribute to
differences in chemical resistances?
Optional add on lab:
Use of spices as antiseptics and disinfectants.
Preparing spice or plant extracts
1. Obtain a mortar and pestle, as well as one of the plant tissues available.
2. Weight out .5 gram of spice (cinnamon, wasabi, jalapeño, cayenne).
3. Transfer spice to a mortar and pestle.
4. Add 2.5 ml of distilled water to the spice.
5. Grind the spice with the pestle until a thick homogenous suspension is formed.
6. Label a centrifuge tube with the name of the spice. Transfer as much of the ground
solution as you can into the centrifuge tube.
7. Centrifuge the homogenate for 5 minutes at 3000 x G to pellet cell debris.
8. Pour off the supernatant and use this spice extract for the above exercise.
DISCUSSION
1. Spices may help preserve foods. What spices do you think would be effective antiseptics.