Microbiology

Aseptic Techniques

1. Upon entering the lab, wash hands and arms up to the elbow with soap.
2. Before and during experimentation wear rubber gloves, apron, and goggles at all times.
3. All glassware and material should be autoclaved for 30 minutes at 20 psi, 121 degrees Celsius.
4. Any open flask, container, or beaker should be covered with aluminum foil when placed in the autoclave.
5. Use 10 % bleach solution and wipe down the lab area and tabletop.
6. Transfer of any culture will be done with a mechanical pipette.
7. Place a biohazard sign in plain view for all to see. 

 

Preparing Hepa filter clean room

  1. Remove jewelry and wash hands prior to use.
  2. Wear a lab coat, rubber gloves, goggles and hair cap.
  3. Wipe down the clean room area with 10% bleach solution and soft cloth or paper towel
  4. Turn on the UV light and close the cabinet
  5. ***DO NOT TURN ON THE UV LIGHT WHEN SPECIMENS ARE IN THE WORKSTATION***
  6. Let stand with UV light on for 15 -30 minutes.

Working in the clean room

  1. Wear a lab coat, rubber gloves, goggles and hair cap.
  2. Lightly spray area with 10% bleach let dry for 20-30 seconds
  3. Before placing any item in the work station sterilized it with 10% bleach.
  4. Once you have equipment in the hood, do not put your arm over the top of the equipment.
  5. Lay all equipment sideways. Try not to place items behind each other.
  6. When all work is completed, close the guard. Turn off the blower.  
  7. Dispose of gloves and hair cap in a red biohazard bag and dispose of using Stericycle in the school's clinic
  8. Hang the lab coat and wash your hands upon leaving the lab.
  9. ***DO NOT TURN ON THE UV LIGHT WHEN SPECIMENS ARE IN THE WORKSTATION***

Preparing to use the Autoclave Sterilizer:

  1. Wear a lab coat, gloves,  and goggles
  2. Lubricate the edge of the lid with Vaseline (petroleum Jelly).
  3. Clean and Remove the large pot and grill.
  4. Clean the walls if needed.
  5. Add 2 inches of clean water to cover the heating element.
  6. Replace the grill and large Pot.
  7. Place items to be sterilized.
    1. Petri dishes should taped shut be placed in an autoclave bag.
    2. Glassware should be covered with aluminum foil.
    3. Instruments should be placed in a beaker and covered or wrapped in aluminum foil
  8. Place the cover. The metal tube goes into the slot on the side.
  9. Secure the lid by tightening the black knobs. Do not over tighten.
  10.  Set the heating level (black knob in front) to number 6.
  11. Turn the petcock valve down.
  12. Turn the unit on. 
  13. It will take approximately 20 minutes to reach required heat and pressure.
  14. Leave the Pressure Cooker on for 30 minutes, 121 degrees Celsius, at 20psi.
  15. When completed, turn the unit off and unplug
  16. Release the pressure (petcock switch up).
  17. Let cool and remove items.
  18. Dispose of all itmes in a red biohazard bag and dispose of using Biotech Solutions Inc (321) 345-0919  FLDOH #7639


Preparation of Nutrient Agar and Pouring Making Plates
1. Measure out 2.3 grams of nutrient agar dehydrated media.
2. Measure out 100 mL of distilled water in a graduated cylinder. Place in a 2 L Erlenmeyer flask.
3. Put flask with water onto hot plate and bring to a boil.
4. Once boiling, dissolve 2.3 grams of agar into the water. Boil for one minute to dissolve.
5. Pour a thin layer of agar on the bottom of each plate and cover immediately.
4. Let agar plate solidify. Liquid agar will solidify at about 42 º C.
5. After the medium agar has solidified, label and store upside down in the refrigerator.


Streaking Plates Adding Sterile Disk

  1. Wearing gloves goggles and aprons, sterilize work area with 10% bleach 
  2. Take out plates from refrigerator and allow to warm up to room temperature.
  3. Take out bacterial culture vial add 2ml of distilled water to the vial and shake.
  4. Insert a sterile inoculating loop into the stock bacterial culture solution.
  5. Using a new plate streak the bacterium onto plate being sure to cover the entire area.
  6. Plate can be streaked multiple times in a sweeping motion to ensure plate overage.
  7. Using a clean sterile disk and sterile tweezers insert the disk into the solution to be tested.
  8. Place the disk in the center of the agar plate with the bacterial lawn.
  9. Cover the plate and seal with tape.
  10. Incubate the plate for 24-48 at 37 degerees Celsius.
  11. Dispose of materials into a red biohazard bag and dispose of using Stericycle in the school's clinic

Cleaning up/Disoposal:
1. After each day of experimentation, clean the lab counter with 10% bleach.
2. Wash hands with 10% bleach.
3. Clean with 10% bleach all glassware and related items. 
4. Place taped Petri dishes and other disposable equipment will be autoclaved for 30 minutes at 20 psi, 121 degrees Celsius. Contact Strericycle for pick-up and disposed of in a red biohazard bag in the school's clinic.
5. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 121 degrees Celsius and stored for later use.

 

Pythium Mold Inoculation:
1. Obtain 2 slants of pythium culture.
2. Add 2 milliliters of saline solution to each tube.
3. Gentle scrape the slant to release the pythium into the saline solution. Culture the pythum on an agar plate as noted below.
4. Pour the remaining pythium solution into the first nutrient tank (pythium only)
5. Repeat this step with the other slant and pour that into the nutrient tank for the pythium and bacterial community.
6. After the nutrient tanks have been inoculated, wait three days and run a slant test to indicate the presence of pythium still exist.

Pythium Mold Culturing
1. On the underside of each petri plate label with each plate with a wax pencil.
2. Heat the agar with 10 V-8 juice in a microwave or water bath to 50º C. The V-8 juice has the nutrients needed for pytium growth.
3. Pour a thin layer of agar on the bottom of each plate and cover immediately.
4. Let agar plate solidify. Liquid agar will solidify at about 42 º C.
5. After the medium agar has solidified, streak the plate with the proper technique.
6. Once the plates have been streaked, invert and close the petri dish, tape the edges if you wish and incubate them at 25º C for 24-48 hours unless other wise noted.