Comparing Raw & Pasteurized Milk and Testing Surfaces for Microbial Contamination Hands-on Module Food Safety Scientist Curriculum © 2014 Board of Regents, South Dakota State University iGrow.org This project was supported by the USDA NIFA grant number

iGrow.org 3M™ Pertifilm™ Aerobic Count 3M™ Pertifilm™ E.Coli/coliform Count 2 Disposable Pipettes 3M™ Spreader 2 Tablespoons Raw Milk 2 Tablespoons Pasteurized Milk Materials For Comparing Pasteurized & Raw Milk © 2014 Board of Regents, South Dakota State University iGrow.org Aerobic Petrifilm™ Pasteurized Milk Raw Milk 3M™ Spreader Disposable Pipettes

iGrow.org Procedure © 2014 Board of Regents, South Dakota State University iGrow.org Sample SourceAerobic Petrifilm™E.coli/coliform Petrifilm Raw22 Pasteurized22 Control You do NOT need to do this. The teacher will incubate 1 Petrifilm™ of each for the entire class. There will be a total of 8 Petrifilm™ for the milk samples (4-Raw/4-Pasteurized) Label 4-Aeroboic count Petrifilms™ and 4- E.Coli/coliform Petrifilms™ with: Sample Source Your Initials Date

iGrow.org Procedure 1. Use a disposable pipette to collect 1 milliliter (mL) of Pasteurized milk from bottle 2. Peel back the top film on the Petrifilm™ plate and squeeze the collected milk in the middle of the surface 3. Gently drop the top film onto the sample to prevent air bubbles from forming. 4. Place the spreader on top of the Petrifilm™ with the flat side facing up; gently press the spreader to evenly distribute sample. Repeat the process as needed for the experimental design – refer to next slide. © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Procedure 5. Repeat steps 1-4 using the raw milk *Each group will have an overall total of 10 Petrifilm™ samples including the two controls prepared by the teacher 6. Carefully place all 10 Petrifilms™ inside the incubator at approximately 90ºF (32ºC) for 48 hours © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Materials for Testing Surfaces for Microbial Contamination 3M™ Quick Swabs 1 cm Swabbing Template 3M™ Pertifilm™ – Aerobic Count 3M™ Pertifilm™ E.Coli/coliform Count Spreader © 2014 Board of Regents, South Dakota State University iGrow.org Aerobic Petrifilm™ 3M™ Spreader 1 cm Swabbing Template 3M™ Quick Swabs

iGrow.org Procedure 3M™ Quick Swabs can collect samples from either wet surfaces or dry surfaces; a. Dry Swab Method for Wet Surface Examples: Sinks, facets, water fountains, toilets, inside the mouth, etc. b. Wet Swab Method for Dry Surface Examples: tables, cell phones, door knobs, book covers, locker doors, etc. © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Procedure The following swabs will be taken from wet and dry surfaces using a different swab for each sample There will a total of 10 Quick Swab samples taken: 4 Wet, 4 Dry, 2 Controls DO NOT FORGET TO LABEL ALL PETRIFILM & QUICK SWABS – Location swab was obtained, initials, date © 2014 Board of Regents, South Dakota State University iGrow.org Quick Swabs Aerobic Petrifilm™ E.Coli/ Coliform Petrilim™ Wet Method22 Dry Method22 Control11

iGrow.org Procedure: Wet Method 1. Break the reservoir on the 3M™ Quick Swab 2. Swish the tube to wet the cotton tip of the swab 3. Choose a DRY surface with the 1 cm swabbing template to collect bacteria sample. 4. Swab 1 cm area on the dry surface © 2014 Board of Regents, South Dakota State University iGrow.org Dry Surface Dry Surface: Back of Chair

iGrow.org Procedure: Wet Method 5. Place the swab back in its case 6. Close the case tightly and swirl it to suspend the bacteria throughout the solution 7. Make certain it is labeled correctly: Location swab was obtained, initials, date. 8. Repeat 1 through 7 (sampling three other DRY surfaces in a different location using a different swab for each) © 2014 Board of Regents, South Dakota State University iGrow.org Dry Surface (continued) Light Switch Computer Mouse Door Knob EXAMPLES

iGrow.org Procedure: Dry Method 1. Choose a WET surface and cover the surface with 1 cm swabbing template to collect a bacteria sample 2. DON’T break the reservoir, but remove the dry swab from the case 3. Swab a 1 cm area on the wet surface 4. Place the swab into the case and close tightly © 2014 Board of Regents, South Dakota State University iGrow.org Wet Surface Wet Surface: Sink

iGrow.org Procedure: Dry Method 5. Break the reservoir now and swish the swab to suspend the bacteria throughout the solution 6. Label the swab, if not already labeled 5. Repeat 1 through 6 (sampling three other WET surface in a different location using different swabs) © 2014 Board of Regents, South Dakota State University iGrow.org Wet Surface EXAMPLES Inside mouth Toilet Water Fountain

iGrow.org Procedure: Culturing the Quick Swab Samples Label 4 aerobic count petrifilm™ and 4 E.Coli/Coliform petrifilm™ (if not already completed) with date, sample source, and initials Label Control Slides for each petrifilm™. Fluid from quick swab poured onto each type of petrifilm. Do not do any swabs. © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Procedure: Culturing the Quick Swab Samples Follow the steps from 5 to 12 in milk test to prepare petrifilm plates. Substitute the liquid in the 3M Quick Swabs in place of milk. © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Incubate all samples for 48 hours before analyzing the results © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Interpreting Data After 48 hours, remove the Petrifilm from the incubator If the Petrifilm has a number of colonies that can be easily counted, count all the colonies directly Leave the top layer on the Petrifilm down © 2014 Board of Regents, South Dakota State University iGrow.org E.Coli Petrifilm™Aerobic Petrifilm™

iGrow.org Interpreting Data When the sample has too many colonies to count directly, estimate the number of colonies by: Picking a square in the middle of the sample area and count the colonies in this square area Multiple the number of colonies in this square area by 20, and that is approximately how many bacteria colonies there are on the petrifilm © 2014 Board of Regents, South Dakota State University iGrow.org

iGrow.org Interpreting Data Compare the number of colonies from different sample sources and interpreting the results of the experiment © 2014 Board of Regents, South Dakota State University iGrow.org Source CFUs (colony-forming units) on E. Coli Petrifilm CFUs (colony-forming units) on Aerobic Petrifilm (refer to lab report)