What types of bacteria or eukaryotic microbes will not grow with the technique used in this lab? LAB Module NumberPart Number SPC using 3M Petrifilm and Microliter Pipettor Objectives Upon successful completion of this lab, the student should be able to: . properly use a microliter pipettor to deliver accurate volumes perform a SPC using 3M Petrifilm. Introduction 3M Petrifilm plates utilize a ready-made culture medium system that contains appropriate nutrients, a cold-water soluble gelling agent, and an indicator that facilitates colony enumeration Petrifilm Aerobic count plates are used for the enumeration of aerobic bacteria and a red indicator dye colors the colonies. Since there is no need to prepare or sterilize media, using 3M Petrifilm is a relatively quick way to carry out a SPC but dilutions of the food sample still need to be made Incubation parameters for 3M products are usually similar to those of standard methods. There are many different types of Petrifilm available depending upon which organisms are to be enumerated 3M has interpretation guides available in print or online which 3M Petrifilm plates utilize a ready-made culture medium system that contains appropriate nutrients, a cold water soluble gelling agent, and an indicator that facilitates colony enumeration. Petrifilm Aerobic count plates are used for the enumeration of aerobic bacteria and a red indicator dye colors the colonies. Since there is no need to prepare or sterilize media, using 3M Petrifilm is a relatively quick way to carry out a SPC but dilutions of the food sample still need to be made. Incubation parameters for 3M products are usually similar to those of standard methods. There are many different types of Petrifilm available depending upon which organisms are to be enumerated. 3M has interpretation guides available in print or online which contain detailed instructions for using their products. Often the pH of the food should be adjusted after the first dilution, for instance; Aerobic pH 6.6 – 72 E coli pH 6.5 – 7,5 Staph, aureus pH 6-8 Microliter pipettors are used to deliver small volumes of liquid, usually 1 ml or less Care must be taken to choose the appropriate Pipettor and tip. pipettors come in a range of different capacities and tips must be matched to the appropriate pipettor, Proper technique is critical to ensure accuracy and precision. Although features may differ slightly amongst different brands of pipettors, they share common functionalities and a general procedure for setting and Operating the traptrument follows Course Number Thumb knob Decrease Volume Increase Volume Lock Lover VOLUME ADJUSTMENT To select the desired volume, loosen the lock nut mechanism by tuming it counterclockwise. To reduce the volume, tum the thumb knob clockwise. Tuming the thumb knob counter-clockwise will increase the volume. (Fig. 1) Set the desired volume on the digital display to correspond to the arrow mark molded on the base of the window frame. The selected volume is fixed by turning the lock nut clockwise and can be confirmed on the digital display Tip Elector Knob Fig. 1 OPERATING INSTRUCTIONS 1. Attach a clean tip firmly to the instrument. 2. Before entry into the sample solution, depress the thund knob to the first stop. (see Fig.2) 3. Now immerse the tip approximately 3mm into the sample solution. (Step 1. fig. 2) 4 Smoontly retum the plunger knob to the release position allowing the sample to enter the tip (Step 2) Do not allow the knob to “snap” back to the release position. 5. Withdraw the tip from the sample solution Do not wipe the tip 6. Puce tip against the side wall of receiving vessel (Step 3) 7 Smoothly depress the plunger knob to the first stop (Stop 4). pause, then depress the knob to the second stop (Step 5) NOTE: Whion dispensing serum and other sous luids, it is necessary to pause about two seconds before moving to the secondary stoc B. With the knob stillheid in its lowest prosty withdraw the tip while sliding it against the wall or eceiving vesse utum the knoes to the route position. Do not allow the nice to retrie 2 1st stop and stop B 0 Step 2 Stap 3 Step 4 Step 5 Step 8 RELEASE POSMON SECONO STOP Chow Fig. 2 AIDS TO PRECISION AND ACCURACY Listed below are some techniques found to improve sampling precision PLEASE READ THIS SECTION CAREFULLY 1. Try to effect the same speed for both the intake and delivery of all samples. Smooth depression and release of the plunger knob will give the most consistent results, Never allow the plunger to “snap” back. Consistency of technique is a key to precision 2. Always depress the plunger knob to the proper stop before insertion of the tip into the solution. Depression of the plunger knob after insertion may cause the formation of an air bubble in the tip and result in a filing error 3. Try to insert the tip to approximately the same depth in the sample each time never going deeper than 3mm. Hold the instrumentas vertically as possible (10″ maximum from vertical 4. Whion sampling hot or cold material, the tip temperature should be equalized to that of the solution to prevent contraction or expansion of sample Materials and Methods 1. A fresh raw milk sample will be provided. 2. Using one 90 ml. blank and two 9 ml blanks, carry out a serial dilution to 10. It is important to thoroughly mix the dilution blanks, the instructor will demonstrate the use of the vortex mixer for test tubes. 3. Lift the top film of the Petrifilm plate and pipet 1 ml of sample onto the center of the bottom film. Release the top film and allow it to drop, do not roll top film down. With the ridge side down, place spreader on the top film over inoculum and gently apply pressure on spreader to distribute inoculum over the circular area. Lift spreader and wait one minute for the gel to form 4. Don’t forget to pipet 1 ml of sterile peptone into one Petrifilm plate as a negative control. Do not use a 9 ml blank that you will be using as part of your serial dilution for the negative control, the resulting volume change is too significant. 5 Incubate Petrifilm with clear side up in stacks of no more than twenty for 48 hours at 32°C and enumerate.Describe the process of photosynthesis. Also write an equation to show the process. What would you expect to happen if plants were not able to carry out photosynthesis?

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