ANTIMICROBIAL CHEMICALS

Disinfectants, Antiseptics, and Spices

Many herbs and spices MAY have (some documented, some just hearsay) antimicrobial
activity: thyme, cinnamon, garlic, ginger, chamomile, oregano, sage, echinacea, wasabi,
etc. We are going to try some out in this lab: fresh or dried, leaf or powder. In addition, you
will be running various antimicrobial chemicals against your bacteria. To save on time, for the
identification of antibacterial spices, each group will extract a different plant tissue and prepare
enough herb/spice extract for each group. Each table will run 2 bacterial organisms with the 4
disinfectants and antiseptics along with the spice extract for their table plus 1 from another
table. Therefore, each table will be running 6 chemicals against 2 bacterial organisms.

OBJECTIVES:

Compare the antimicrobial capabilities of different antiseptic and disinfectant chemicals.

Identify which categories of chemicals are most effective.
Identify antimicrobial properties of some plants.

MATERIALS NEEDED: per table

broth cultures of Staphylococcus and E. coli

2 large nutrient agar deeps, liquified---in water bath
2 sterile petri dishes
sterile paper disks
an herb or spice (1 per table)
antiseptic and disinfectant chemicals (4 per table)
small beakers for antiseptics and disinfectants
forceps and beakers of ethanol
1ml pipettes + pi-pump
mortar and pestle
centrifuge tube

PROCEDURES:

Prior to the Lab

1. Pick a chemical that you have at home or work, or bring a chemical from the store. On a single piece of
paper (hand-written or typed):
the brand name of the antiseptic/disinfectant
the active chemical ingredient (not the inert chemicals such as glucose or water)
2. Remember to bring a fresh herb/spice (preferable) or a dried herb/spice (1 per table).

Preparing spice and herb extracts

1. Weight out 1 gram of spice or herb, and transfer to a mortar (grinding bowl).
2. Add 5 ml of water for the 1 gram of tissue.
3. Grind the tissue sections with the pestle until a thick homogenous suspension is formed.
 4. Label a centrifuge tube with the name of the tissue and a group identification. Transfer
as much of the liquid homogenate as you can into the centrifuge tube.
5. Centrifuge the homogenate for 5 minutes at 3000 x G to pellet cell debris.
6. Pour off the supernatant and use this spice extract for the following exercise.

Using The Spice/herb Extracts and Antimicrobial Chemicals

1. Inoculate 0.5ml of each species into tubes of liquified nutrient agar which has cooled
to 45oC. Mix well and pour into 2 sterile petri dishes, allowing them to harden. Each
table should have the 2 different bacteria in the 2 pour agar plates.
2. Your table can choose 4 antimicrobial chemicals plus 2 spices/herbs to test each
organism against. That will be 6 discs on each agar plate.
3. Pour a small amount of each of your antiseptics and disinfectants, plus 1-2 spice/herb
extracts into each of the beakers.
4. Using alcohol-flamed forceps, pick up a sterile paper disk and dip it halfway into one of
the antimicrobial solutions, then place the disk on the inoculated media.
5. Repeat the procedure with the other chemicals. Both organisms will be tested with the
same chemicals.
6. Incubate the plates at room temperature, 25 oC.
7. AFTER INCUBATION, measure the zone of inhibition for each chemical in
millimeters . If there is no zone around the disk, call it 0.
8. Record the results for the entire class in the table on this handout.

INTERPRETATION:

Record the results for the various chemicals and spices used in this exercise.
DIAMETER ZONE OF INHIBITION
Chemical (brand name) or Spice Active Ingredient (in mm)
Staph E. coli

QUESTIONS:

1. The space created around the chemical disc is called the ________________.

2. The most effective active ingredients found in the store-bought chemicals was:

Fall 2011 – Jackie Reynolds, Richland College, BIOL 2421