Pharmaceutical Manufacturing: Applied Biopharmaceutical 2 Experiment Protocol 9: Environmental Monitoring Purpose: Environmental Monitoring (EM) encompasses many different

aspects of sampling in the pharmaceutical manufacturing plant. These sampling included non-viable particulates monitoring such as dust and skin flakes to viable particulates surrounding the environment such as bioburden in process water (PW). In this lab exercise, 4 commonly used testing techniques will be performed. A) Spread Plate method B) RODAC contact (Replicate Organism Detection and Counting) agar plate C) Membrane filtration of processed water. D) Settling plate using both Tryptic Soy Agar (TSA) and Potato Dextrose Agar (PDA) Note: PDA is typically used to identify presence of mold and yeast and is incubated at a much lower temperature of 25-30C for 3-5days. Material: Prepared Tryptic Soy Agar (TSA) RODAC contact (TSA) Potato Dextrose Agar (PDA) Inoculating spreader Alcohol Alcohol and burner TOC vials Nalgene analytical Filter unit Water samples Forceps Procedure: A) Spread Plate Method: 1) Inoculating the plate by aseptically transfer 0.1mL water sample (collected by an operator in the manufacturing plant) onto center of a TSA plate using the Gilson pipette(P200) and STERILE pipette tips. 2) Inoculation in Spread Plate technique is done using a bent glass rod or plastic spreader. 3) The spreader MUST be sterilized by first dipping it into a 95% alcohol solution, allow it to drain for a 10-15sec then burn off the excess by passing it quickly through the alcohol burner flame. 4) Streak the spreader back and forth across the plate working up and down several times. (Unlike streaking for colony isolation, you want to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees and streak a third time) Do not sterilize the glass rod between plate turnings. 5) Cover the plate and wait several minutes before turning it upside down for incubation. This will allow the broth to soak into the plate so the bacteria won't drip onto the plate lid. 6) Incubate the plates inverted at 370C. After >24hr period, examine the plates for colony formation. The result is expressed in cfu/mL.

B) RODAC contact agar plate 1) Examine the plate to ensure it had not been contaminated. 2) Find a location in the lab to be sampled. Ensure to label on the back of the RODAC plate with the following information: location, time/date, sampler initials. 3) Remove the lid of the RODAC plate and gently press the exposed agar to the sampling surface. Hold it position for <1sec. and cover the plate with the lid. 4) Incubate the RODAC contact plate at 370C of >24hr. Examine the result for colony formation. C) Membrane filtration of PW and other water samples (Demonstration by Instructor) 1) Refer to the diagram below. Perform the EM on the samples provided. Record the sample number, time/date, sampler initials on the TSA plate.

Step #7 Place the Cellulose Nitrate membrane on the TSA surface aseptically and incubate at 37C of >24hr.

D) Settling plate sampling 1) This sampling involves the monitoring of the critical work zones such as adjacent to
doors, between HEPA's in clean rooms and in corners of production rooms.

2) In settling plate sampling, Petri dishes containing agar medium are opened at a pre-determined location and exposed for a given period of time. The airborne microorganisms, typically attached to larger particles, will deposit onto open culture plates by gravity. 3) The culture plates used are TSA and PDA (for mold and fungi). 4) Place the instruction sheet (provided by instructor) along with agar plates with the lids still on at the locations of your choosing. 5) Raise lid to expose the surface of the agar medium, rest the lid on the very edge of the plate so that the entire agar (Figure 1b) surface is completely exposed. Take care not to put fingers on plates. Avoid passing anything over the top of the exposed plates. 6) Leave plates exposed for the full work session. The exposure time should be recorded before sending the plates for incubation. 7) After exposure, replace lids of plates. Collect all exposed plates, and return to Room A239 for incubation. Figure 1b:

Post lab questions: 1) What type of agar media is used to look for mold in the environment? 2) If you are sampling for the bacterial flora of a technicians cleanroom garment, what type of EM method were you use? 3) Typically, what is the pore size of the nitrocellulose membrane in Membrane filtration?