Monitoring for Bacteriological Contamination with MicroFunnel™ Plus Filter Funnels 5.2.1

Monitoring for Bacteriological Contamination with MicroFunnel™ Plus Filter Funnels

Membrane filtration (MF) is widely accepted throughout the world for the analysis of microbial contamination in fluid samples. Using the MF technique, microorganisms are captured on a microporous membrane filter surface and grown into countable colonies by incubation of the filter with a suitable growth medium. Pall Life Sciences has designed the MicroFunnel Plus filter funnel (see Figure 5.6) to simplify this testing process. This filter funnel can be used in accordance with the MF procedures referenced in Standard Methods for the Examination of Water and Wastewater, 20th edition, and the U.S. Environmental Protection Agency’s Microbiological Methods for Monitoring the Environment, 600/8-78- 017. MicroFunnel filter funnels also meet the USP-24 requirements for products used for the MF technique.  

Figure 5.6

MicroFunnel Filter Funnels for Microbiology Testing

 

The MicroFunnel Plus filter funnel is supplied pre-assembled and sterilized by gamma irradiation to eliminate the potential for toxic residuals associated with EtO sterilization. The integral unit and membrane ensure high bacterial recoveries and no grid line inhibition. After capture of the microorganisms, the membrane is easily removed from the base via a unique squeeze separation process, without interfering with the membrane. This greatly simplifies transfer to the growth media-soaked absorbent pad incorporated into the cap. The full specifications of the MicroFunnel Plus system are summarized in Table 5.6. Three membrane types are available:

• GN-6 Metricel® membrane: a 0.45 μm mixed cellulose ester membrane ideal for use in most applications.
• Supor® membrane: a 0.2 μm polyethersulfone membrane ideal for its low binding characteristics and superior retention of small organisms such as Pseudomonas sp.
• Metricel Black membrane: a 0.45 μm modified polyethersulfone membrane ideal for yeast and mold analysis or providing good contrast for light-colored colonies.

Table 5.6

Specifications for MicroFunnel™ Plus Microbiology Testing Device

	Parameter
Specification	MicroFunnel Plus 100 mL	MicroFunnel Plus 300 mL
Membrane	0.45 μm GN-6 Metricel® mixed cellulose ester membrane 0.2 μm Supor® polyethersulfone membrane Black 0.45 μm Metricel modified polyethersulfone membrane
Device      Support Pad       Cylinder Base and Petri Dish Lid (LP)       Cover       Plug       Funnel Adapter	Cellulose Polypropylene Polysyrene Polyethylene Polyethylene (not autoclavable)
Effective Filtration Area	13.46 cm2
Dimensions      Height (with Lid Petri Dish)       Height with Cover       Diameter(with Lid Petri Dish) 6.40 cm (2.52 in.)       Diameter with Cover	8.05 cm (3.17 in.) 7.62 cm (3.00 in.) 6.40 cm (2.52 in.) 6.05 cm (2.38 in.)	NA 9.09 cm (3.58 in.) NA 8.84 cm (3.48 in.)
Capacities	100 mL	300 mL
Operating Vacuum	635 mm Hg (25 in. Hg)
Sterilization	Individually packed and sterilized by gamma irradiation

In this section, protocols will be described for use of MicroFunnel™ Plus disposable devices for testing aqueous samples for microbial contamination. Culture media ampoules are available for a range of bacterial, yeast, and fungal contaminants and are summarized in a product selection guide in Table 5.7.

Table 5.7

Application Selection Guide for MicroFunnel Plus

Test Method	Method Number*	Recommended Filter	Media Broth
Total Coliforms	9222	GN-6 Metricel®	MF-Endo
Fecal Coliforms, E. coli	9222	GN-6 Metricel	M-FC
Total Bacteria	9215; USP 61; 71	GN-6 Metricel, Supor	M-TGE; TSB-USP; HPC
Lactobacillus sp. detection	--	GN-6 Metricel	Orange Serum
Yeast and Mold	9610	GN-6 Metricel, Black Supor	M-Green YM
Fecal Streptococcus	9230	GN-6 Metricel	KF-Streptococcal
Pseudomonas sp.	9213	Supor	Pseudomonas

*Standard Methods for the Examination of Water and Wastewater, 19th ed. and U.S. Pharmacopoeia XXIV

Protocol for Monitoring for Bacteriological Contamination with MicroFunnel Plus Filter Funnels

A. Materials Required

1. MicroFunnel Plus pre-sterilized devices in 100 and 300 mL volume sizes
   1. 0.45 μm GN-6 Metricel membrane, gridded
   2. 0.2 μm Supor membrane
   3. Metricel Black modified polyethersulfone membrane
2. MicroFunnel Plus LP (PN 4810) with 0.45 μm GN-6 Metricel membrane, white, gridded, sterile with absorbent pad incorporated into the cap for the funnel, 50/pkg
3. Selective growth media ampoules (2 mL volume)
4. Sterile petri dishes and absorbent pad (PN 7245)
5. Sterile phosphate buffered saline (PBS)
6. Stainless steel forceps (PN 51147)
7. Filter funnel manifold, single place in aluminum (PN 15408)
8. Hand operated vacuum pump (PN 4041) and filtering flask with sidearm (PN 4040)
9. Source of vacuum [63.5 cm Hg (25 in. Hg)] fitted with an in-line VacuShield hydrophobic PTFE filter (PN 4402) 
10. Microbiological incubator

B. Operation of the MicroFunnel™ Plus Device to Collect Microorganisms and Growth Inside the Same Device

In this next section, handling of the above funnel system will require the use of aseptic techniques to avoid environmental contamination of the sample to be tested. This manual assumes that the operator is aware of basic sterile techniques which will not be covered in detail in the following protocols. There are several culture media ampoules available for a range of bacterial, yeast, and fungal contaminants summarized in a product selection guide in Table 5.7. It may be necessary to process several parallel samples in different culture media to cover bacterial, yeast, and fungal contaminants.

1. Carefully remove the vented lid under aseptic or clean conditions to protect the sterility of the MicroFunnel device.
2. Collect the sample and introduce into the open device with a sterile or clean measuring cylinder. Make a notation of the sample volume.
3. Snap the vented lid back in place and seal the device for transport to the microbiology lab for testing. Label device to identify the sample.
4. Before mounting on the filtration manifold (PN 15408), remove the plug or vent tab from the base of the device. Place on manifold and draw liquid sample through the membrane filter.
5. Wash the membrane surface with 2 x 25 mL of sterile PBS.
   Tip: This step may require more than 2 x 25 mL of sterile PBS, depending on whether the sample matrix is more complex than a water sample. It is important to remove components of the original sample that might inhibit rapid growth on the specific culture media. Organisms may be present in the sample and only growing very slowly.
6. Gently grasp the funnel and remove and discard the lid (see Figure 5.7).

   Figure 5.7
   

   Separation of the Funnel from the Base of the MicroFunnel Plus Filter Funnel
   
    

7. Release funnel cylinder from base by firmly squeezing the midpoint of the cylinder.
8. Culture the samples in the MicroFunnel filter funnel base by gently dispensing 2 mL of culture media over the surface sample and allowing it to be drawn into the pad. It may be necessary to momentarily apply vacuum just until the media is drawn through the membrane into the pad.
9. Replace the cover onto the base of the MicroFunnel™ unit to convert into a Petri dish and discard the cylinder.

C. Culture of the Microorganisms on the Filter in a Petri Plate

1. Soak adsorbent pad in sterile petri dish (PN 7245) with appropriate selective growth media.
2. Remove filter from the MicroFunnel base with sterilized forceps and carefully place on the absorbent pad in the petri dish.
3. Replace the cap on the petri dish, invert, and incubate at 37 ºC till colonies appear on the gridded membrane (see Figure 5.8).

Figure 5.8

Addition of Growth Medium to the Cap of the MicroFunnel Plus LP Device

 

D. Culture of the Microorganisms in the Cap of a MicroFunnel Plus LP

1. Dispense the contents of an ampoule of culture medium onto the absorbent pad in the Petri dish lid kit (supplied only with MicroFunnel LP, PN 4810).
2. Following the procedure described in Step 1.2.7 and remove the membrane filter from the base with forceps.
3. Place the membrane filter onto the broth-soaked absorbent pad in the petri dish lid kit. Cover, invert, and incubate.

E. Interpretation of the Results

1. After incubation for 24-48 hours (depending on media used), single isolated colonies of microorganism should be visible on the plates (see Figure 5.9).

   Figure 5.9
   

   Examples of Microorganisms Cultured on the Membranes in a MicroFunnel™ Plus Device
   
   

2. Count the number of colonies by placing on a light box to backlight the plates. Calculate the number of organisms per unit volume of sample.
   Tip: Duplicate samples should be run to allow for uneven distribution of the contaminating microorganism in the original sample.
3. If a large number of colonies are seen that are too dense to count, it may be necessary to repeat the study with an original sample diluted with sterile PBS to lower the number of organisms cultured on the filter.

Ordering Information for Monitoring for Bacteriological Contamination with MicroFunnel™ Plus Filter Funnels

Microfunnel Filter Funnels

Part Number	Description	Pkg
4807*	0.45 μm GN-6 Metricel® membrane, white, gridded, sterile, individually bagged, 100 mL capacity	50/pkg
4801	LP unit with 0.45 μm GN-6 Metricel membrane, white, gridded, sterile, individually bagged, Petri dish lid	50/pkg
4808*	0.45 μm Metricel Black membrane, gridded, sterile, individually bagged,100 mL capacity	50/pkg
4809	0.2 μm Supor® membrane, white, gridded, sterile, individually bagged, 100 mL capacity	50/pkg
4823	0.45 μm Supor membrane, white, gridded, sterile, individually bagged, 100 mL capacity	50/pkg
4813	0.2 μm Supor membrane, white, gridded, sterile, individually bagged, 300 mL capacity	20/pkg
4814	0.45 μm Supor membrane, white, gridded, sterile, individually bagged, 300 mL capacity	20/pkg

*For use with ambient temperature samples only.

Microbiological Media (2 mL Plastic Ampoules)

Part Number	Description	Pkg
4302	M-FC Broth with rosolic acid, Fecal Coliforms	50/pkg
4306	Pseudomonas Broth, Pseudomonas sp.	50/pkg
4307	Trypticase Soy Broth – USP, Total Bacteria	50/pkg
4352	HPC Media with TTC Indicator, Total Bacteria	50/pkg
68105	MF-Endo Broth, Total Coliforms	50/pkg
68106	M-TGE Broth, Total Bacteria	50/pkg
68107	M-Green YM Broth, yeast and mold	50/pkg
68108	KF-Streptococcal Broth, Fecal Streptococcus	50/pkg
68109	Orange Serum Broth, Lactobacillus sp.	50/pkg