Basic Procedures for Microbiology Projects.
READ CAREFULLY!! …. The following are GENERAL INSTRUCTIONS…they are NOT specific to
YOUR project. YOU! must MODIFY them to fit your needs with your SPECIFIC Information.
BEFORE you start writing….
KNOW your idea, what (IV) are you using on your bacteria.
KNOW the Genus species name of the bacteria you should be using;
KNOW the catalog number from Carolina Biological where we will order your bacteria.
KNOW the agar that you need to grow your bacteria culture, they do not all like Nutrient Agar.
SAMPLE CHOICES for BACTERIA and the agar they require :
Escherichia coli………………………….nutrient agar
Micrococcus luteus……………….…. nutrient agar
Pseudomonas fluorescens…….…. nutrient agar
Stapholococcus epidermidis…... nutrient agar
Streptococcus salivarius…….……tryptic soy agar w/ 5% dextrose starch agar
OKAY, now that you have your information, use the following outline to create your
experimental design/procedures:
1. New bottles of the (list IV’s…Scope, Listerine. Etc.) being tested will be purchased for
use in this experiment and will notbe opened until just before inoculation of the bacteria
cultures.
2. Goggles and gloves will be worn. Hands will be washed in warm water with antibacterial
soap.
3. All table surfaces being used for the experiment will be cleaned before abd after use
with a 50% bleach solution to sterilize the work surface and prevent contamination.
4. Five hundred (500) milliliters of __________ agar will be sterilized and used to fill 2030 sterile, disposable Petri dishes.
5. The dishes will be covered and the agar allowed to solidify.
6. After the agar has solidified the plates will be turned over and labeled on the bottom of
the dish to mark quadrants, the bacteria species being grown and the IV (OR the location
from which the bacteria will be collected.)
7. The ________ _________ bacteria, purchased from Carolina Biological Supplies
(catalog # ______), will be cultured in a sterile _______ broth solution. (OR Bacteria
will be collected on a sterile swab from ________name locations here___________)
8. Each Petri dish will be inoculated with the bacteria by the following method:
(ATTENTION: there are TWO methods listed here. CHOOSE the ONE that will be
appropriate for your procedure, and MODIFY it with your details. Do NOT just copy
without reading….that will make you look foolish.)
1. The tube containing the ___________ bacteria culture will be shaken gently to
ensure an even distribution of bacteria in the liquid. The mouth of the broth tube
containing the __________ bacteria culture will be held in a Bunsen burner flame
to kill foreign organisms that might be on the lip of the tube.
2. A sterile cotton swab will be inserted into the tube, avoiding the sides of the tube,
where it will be held for 5 seconds to collect a sample of the broth.
3. The Petri dish will be held agar side toward the floor to prevent airborne bacteria
from landing upon it.
4. The swab containing the bacteria culture will be gently swiped across the surface
of the agar in a horizontal direction, and then the plate will be turned 90 degrees
and swiped again to cover the whole surface evenly.
5. The Petri dish will be covered with a lid.
6. The swab will be disposed of in a 50% bleach solution.
After all dishes are inoculated with bacteria they will be treated with your independent variable
(IV).
1. Filter paper discs will be sterilized in an autoclave.
2. A pair of forceps will be heated in the flame of a Bunsen burner to decontaminate
them.
3. A disc will be picked up with the forceps and dipped into an umcontaminated sample of
the ________ (e.x. scope mouthwash) from a brand new bottle.
4. The lid of the agar plate will be lifted at a 45 degree angle to reduce airborne
contamination, and the disc will be gently placed at the center of a quadrant.
5. Steps 1-4 will be repeated for each sample disc added to each quadrant of each agar
plate.
6. The control plate will be treated with discs dipped into sterilized distilled water.
7. All the plates will be taped closed for incubation at ____ oC (enter the temp for your
bacteria, most are 37 oC )
8. The plates will be incubated for 24-48 hours.
9. After incubating the plates will observed and the zones of inhibition will be measured
in four directions.
10. The size of he zone of inhibition will indicate the ability of the (___IV____) to kill
the bacteria.
11. All surfaces used during the inoculation and measuring process will be cleaned with a
bleach solution immediately after the activity is finished.
12. All used Petri dishes will be treated with bleach and autoclaved before disposal in a
biohazard bag.
13. Hands will be washed and protective gloves worn will be disposed of in biohazard bags.
ALTERNATIVE PROCEDURE…collecting bacteria from outside sources….
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Hands will be washed and protective gloves worn.
A sterile swab will be used to collect bacteria samples from ___________
(bathroom handles, dog’s mouth, lettuce leaves,, subject’s mouth, etc. )
The lid of a sterile agar plate will be lifted at an angle to prevent airborne
contamination. The swab will be swiped on the agar in a predetermined zig-zag
pattern.
The lid will be closed and sealed with masking tape.
The used swab will be disposed of in a 50% bleach solution.
The plate will be incubated for 24-48 hours.
After incubation, the area of the plate showing the growth of bacteria will
calculated.
All surfaces used during the inoculation and measuring process will be cleaned
with a bleach solution immediately after the activity is finished.
Hands will be washed and protective gloves worn will be disposed of in biohazard
bags.
All used Petri dishes will be treated with bleach and autoclaved before disposal
in a biohazard bag.
REMEMBER !! For ALL projects you MUST include
1. The scientific name and source of the bacteria (catalog # where
appropriate).
2. The cleaning/sterile techniques for the surfaces used to prevent crosscontamination
3. All used Petri dishes will be treated with bleach and autoclaved before
disposal in a biohazard bag.
4. ISEF Forms 2 and 6 and 4 if humans are involved ( I will provide forms at
our info meeting) THEY MUST BE SIGNED !! before you begin your
project.