Environmental Sampling Detection of Microorganisms Preparation of Sampling Material

Laboratory procedure MFLP-41B
July 2006

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Health Protection Branch
Ottawa

Microbiological Methods Committee¹, Karen Catherwood², Patti Wilson³, and Walter Zandstra⁴

¹ Evaluation Division, BMH,
Food Directorate, PL: 2204A1
HPFB, Ottawa, Ontario, K1A 0L2

E-mail: Don_Warburton@hc-sc.gc.ca

² Canadian Food Inspection Agency
3155 Willingdon Green,
Burnaby, BC, V5G 4P2

E-mail: CatherwoodK@inspection.gc.ca

³ Food Laboratory
Canadian Food Inspection Agency
P.O. Box 1060,
1992 Agency Drive
Dartmouth, N.S. B2Y 3Z7

E-mail: wilsonpa@inspection.gc.ca

⁴ Microbiology Specialist
Canadian Food Inspection Agency
2301 Midland Avenue,
Scarborough, Ontario M1P 4R7

E-mail: zandstraw@inspection.gc.ca

1. APPLICATION

This method is applicable to the preparation of necessary material in order to take environmental samples in food processing plants in support of compliance activity relative to Section 7 of the Food and Drugs Act.

This revised method replaces MFLP-41B dated April 1998 and the Supplement to Method MFLP-41B dated September 1999.

2. DESCRIPTION

This procedure describes methods of preparation for material to be used when environmental sampling is done in a food plant environment for microbiological evaluation. For information on how to take a sample from a food contact surface in a food plant environment please refer to MFLP-41A.

3. PRINCIPLE

It is essential that the methods and materials used for environmental sampling be standardized since the presence of pathogens or high numbers of bacteria in a food plant establishment may be a sign that foods have been produced under poor sanitary conditions or that the plant houses microbial niches. Methods used will demonstrate the presence or estimate the number of viable microorganisms sampled on food contact surfaces. Samples obtained are then inoculated into or onto selective media specific to the type of microorganism(s) of interest. It is assumed that each viable microorganism will then multiply under specified conditions of incubation and give rise to visible growth which can be measured, counted and identified. This method is still considered semi-quantitative since varying proportions of the total number of viable cells may be recovered. However, identification of microorganisms isolated may provide valuable information. It is essential that microbiological analysis be initiated as soon as possible after sampling in order to avoid any loss due to die-off.

4. BEFORE SAMPLING

Before initiating environmental sampling, the person responsible for performing the environmental sampling must notify and consult with laboratory personnel.

5. MATERIALS AND SPECIAL EQUIPMENT

5.1 Sterile material may be prepared by laboratory personnel or purchased

1) Spoons (short- and long-handled), sterile

2) Forceps, sterile

3) Knives (Victorinox or equivalent), sterile

4) Plastic cups (8 oz., Becton Dickinson Labware, #4015, or equivalent), sterile

5) Swabs (cotton, calcium alginate, Dacron or Rayon)

6) Sponges (celluose or polyurethane). Commercially available with or without neutralizing buffer (Qualicum Scientific, Oxoid) or equivalent.

7) Swatches (J-cloths, gauze or cloths)

8) Jars, polypropylene or other unbreakable material (Nalgene or equivalent)

9) Whirlpak™ bags, sterile

10) Screw-cap tubes, polypropylene or other unbreakable material for swabs

11) Disposable overalls, head cover, overshoes, facial hair-cover (if sterile clothing is needed)

12) Prepackaged surgical gloves (wrist), sterile

13) RODAC™ plates (Falcon)

14) Petrifilm™ plates, various types

15) Neutralizing Buffer, commercially available (Difco, Qualicum Scientific, Oxoid) or equivalent

16) D/E Neutralizing Agar, commercially available (Difco, Qualicum Scientific, Oxoid) or equivalent.

17) Microbial Content Test Agar, commercially available (Difco, Qualicum Scientific, Oxoid) or equivalent.

18) Violet Red Bile Agar, commercially available (Difco, Oxoid) or equivalent.

19) Dichloran Rose-Bengal Chloramphenicol (DRBC) Agar Base, commercially available (Difco, Oxoid) or equivalent.

20) Baird Parker Agar Base, commercially available (Difco, Oxoid), or equivalent.

21) Letheen Broth/Agar, commercially available (Difco) or equivalent.

22) Transport medium, commercially available (Difco) or equivalent.

5.2 Surface Treatment for Chemical Germicides

If sampling is to be carried out on surfaces previously subjected to chemical germicide treatment, appropriate neutralizers should be incorporated into the medium. Neutralizing media are usually commercially available. Although efficacy of the neutralizers for agar contact sampling has not been demonstrated definitively, it has been found useful.

See Table 1 for a list of media, neutralizers, the compounds which are neutralized and the reference

6. PREPARATION OF BACTERIAL CARRIERS

It is recommended that each lot of bacterial carriers (sponges, swatches, swabs) be tested for inhibitory properties against the selected bacteria by using the method of Libras and Rose (8.1, Appendix 1) or another acceptable method. See Appendix 1 of this method.

6.1 Sponges

6.1.1 Put sponge(s) (approx. 4 cm X 8 cm) in a wide mouth Nalgene jar containing 10 to 15 mL of Neutralizing Buffer or any other buffered rinse solution which contains neutralizers to completely moisten each sponge. Sterilise jars at 121°C for 15 minutes.

Alternately, place pre-sterilized sponges and Neutralizing Buffer into sterile Whirlpak bags or jars.

Indicate the sterility of the sponges with autoclave tape. Mark each container with the preparation or expiry date.

Alternatively, individual packaged sponges, pre-moistened with neutralizing buffer, are commercially available.

6.2 Swatches (J-cloths, gauze and cloths)

6.2.1 Cut J-cloths 35 X 60 cm in half in order to obtain a cloth of 17.5 X 30 cm. Fold the swatches in such a way that they can be easily removed by inspectors. Five swatches can be put in a sampling jar with the last side folded on the top to facilitate removal while using forceps or gloves.

6.2.2 Add 200 mL of neutralizing buffer per sampling jar. Label each jar with the identity of the media, the date and the number of cloths in the container. Autoclave at 121°C for 15 min.

6.3 Swabs

6.3.1 Swabs of approximately 2 cm with the head firmly attach to an applicator stick 12 to 15 cm long may be used. Swabs made of calcium alginate fibres are soluble in aqueous solutions containing 1% sodium hexametaphosphate (or sodium glycerophosphate, or sodium citrate, or 1% of any mixture of these) allowing the release of the captured organisms. Pre-sterilized swabs in various transport media are commercially available.

6.3.2 For sterile dry swabs, prepare screw-capped plastic vials containing 20 mL of sterile Neutralizing Buffer or any other buffered rinse solution which contains neutralizers.

Also, prepare screw-capped tubes of sterile transport media to contain the swab used to sample the environmental surface.

6.4 Keep bacterial carriers refrigerated until picked up by the inspector. Ship bacterial carriers to inspectors with ice packs.

7. SAMPLING

There are two types of sampling: qualitative and quantitative. Qualitative sampling includes liquid media which promote the growth of the organism of concern. Quantitative sampling estimates the number of bacteria isolated from a determined sampling area using Petrifilm™ and RODAC™ plates.

7.1 Qualitative Sampling and Procedure

7.1.1 Bacterial carriers and controls must be refrigerated when received by the laboratory and analyzed as soon as possible.

7.1.2 Add media or supplements to media as appropriate, and incubate following the appropriate method for the microorganism(s) of concern.

7.1.3 Detection of Microorganisms; table 2 lists appropriate steps to be taken when samples are received at the laboratory after inspection. Usually qualitative environmental sampling is done when the presence of Listeria monocytogenes, Salmonella, or Staphylococcus aureus is suspected. Therefore, only those are listed in the table. Other determinations can also be performed depending upon recommendations made by laboratory personnel to the inspector, and is usually determined on a case by case basis.

7.2 Quantitative Sampling and Procedure

7.2.1 Perform quantitative sampling using RODAC (Replicate Organism Detection and Counting plates) or Petrifilm plates.

7.2.2 Preparation of RODAC™ plates

Fill the disposable plastic RODAC™ plates aseptically with 15.5 to 16.5 mL of the appropriate sterile media. The meniscus of the agar should rise above the rim of the plate to give a slightly convex surface in order to allow a good contact with the surface to be sampled. Prefilled plates with test medium are also commercially available.

Media for some methods are listed in Table 3 and the formulae can be found in the Compendium, Appendix G (8.2). When sanitizer residues are present on the contact surface, choose an appropriate neutralizer to add to the media from Table 1. Media with neutralizers are also commercially available.

7.2.3 Preparation of Petrifilm™

Follow the manufacturer's instructions. Request the technical bulletin on SURFACE SAMPLING PROCEDURES from the manufacturer,and pay particular attention to the recommended diluents when sanitiser residues may be present.

7.2.4 Detection of Microorganisms

Count all developing colonies. Spreading colonies should be counted as one but care should be taken to observe other distinct colonies intermingled in the growth around the plate periphery or along a hair line. These should also be counted as one colony, as should bi-coloured colonies and halo type spreaders. It is generally agreed that 200 colonies is the approximate maximum that can be counted on contact plates.

Colony counts may be recorded by:

1) Individual counts,
2) Number of viable particles per selected area,
3) Means and standard deviations.

8. REFERENCES

8.1 Libras, C. M. and Rose, B. E. 1989 . Antibacterial Properties of Retail Sponges. J. of Food Prot. 52(1):49-50.

8.2 Health Canada. 2006. Appendix G In Volumes 1, 2 and 3. Compendium of Analytical Methods. Website: http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php. 8.3 American Public Health Association. 2001. Compendium of Methods for the Microbiological Examination of Foods; Fourth Edition. F.P. Downes and K. Ito (eds.). American Public Health Association Inc., 1015 Fifteenth Street, Washington, D.C. 20005. 8.4 Difco Laboratories. 1998. Difco Manual, Dehydrated Culture Media and Reagents for Microbiology; Eleventh Edition. Difco Laboratories Inc., Detroit, Michigan 48232. 8.5 Health Canada. 2006. Appendix K In Volumes 1, 2 and 3. Compendium of Analytical Methods. Website: http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php.

Table 1: Neutralizers of sanitisers for media

Media	Neutralizers	Compounds Neutralized	Ref.
Bacto D/E Neutralizing Agar (Dey and Engley Agar)	Sodium thyoglycollate Sodium thiosulphate Sodium bisulfite Lecithin (soybean) Tween 80	Quaternary ammonium, phenols, iodine, chlorine, mercurials (Merthiolate), formaldehyde, gluteraldehyde	APHA (8.3) Difco (8.4)
0.1% peptone water	For surfaces containing fatty materials 0.5% Tergitol Anionic 7 0.5-1.0% Tween 80	Phenols	APHA (8.3)
Letheen Broth/Agar	Lecithin Tween 80	Quaternary Ammonium, phenols, formalin, hexachlorophene, ethanol	Difco (8.4)
Bacto Neutralizing Buffer	Monopotassium phosphate, Sodium thiosulfate, Aryl Sulfonate Complex	Chlorine and quaternary ammonium	Difco (8.4)
Bacto Microbial Content Test Agar	Lecithin Tween 80	Quaternary ammonium, phenols, formalin, hexachlorophene, ethanol	Difco (8.4)

Table 2: Methods for Qualitative Analyses

Determination	Receiving	Analysis
Listeria monocytogenes	Add LEB (or the appropriate broth) at 100 mL/unit (sponges) or 10mL/unit (swabs), and incubate at 30°C for 48 hours (or the temperature specified in the selected method).	Proceed as per method chosen from the Compendium Appendix K (8.5) Listeria species and Listeria monocytogenes.
Staphylococcus aureus	Incubate for 3 hours at 35°C in 50 mL of double-strength TSB. Add 100 mL of a solution of TSB single strength containing 20% salt (sponges) or 10mL/unit (swabs). Incubate at 35°C for 24 hours. (8.3)	Proceed as per method chosen from the Compendium Appendix K Staphylococcus aureus
Salmonella	Add Nutrient broth (or the appropriate broth) at 100 mL / unit (sponges) or 10mL/unit (swabs), and incubate at 35°C for 18-24 hours.	Proceed as per method chosen from the Compendium Appendix K Salmonella

Table 3: Methods for Quantitative Analyses

Determination	Petrifilm/Rodac	Media	Method (or equivalent)
Coliforms	RODAC™	Violet Red Bile (VRB)	See manufacturer's procedure
	Petrifilm™	Coliform Count (CC) plates	MFHPB-35
	Petrifilm™	High Sensitivity Coliform Plates	MFLP-85
Escherichia coli	Petrifilm™	E. coli count (EC) plates	MFHPB-34
Numération des colonies bactériennes aérobie	RODAC™	Microbial Content Test Agar (MCTA)	See manufacturer's procedure
	Petrifilm™	Aerobic Count (AC)	MFHPB-33
Yeasts and Moulds	RODAC™	DRBC Agar	See manufacturer's procedure
	Petrifilm™	Yeast and Mould (YM) Count Plates	MFHPB-32
Staphylococcus aureus	RODAC™	Baird Parker (BP)	See manufacturer's procedure
	Petrifilm™	Staph Express plates	MFLP-21
Other bacteria, as needed			See this Compendium for suitable methods

APPENDIX 1

VERIFICATION OF INHIBITORY PROPERTIES

Published studies by Libras and Rose (8.1) have indicated that some sponges sold at retail outlets for environmental sampling may contain antibacterial agents. In order to prevent problems caused by inhibition from the use of sponges or similar material, each lot of bacterial carriers (sponge, J-cloth, etc.) should be tested for inhibitory properties using one of the two methods presented below. Before initiating the inhibitory test, bacterial carriers should be moistened with neutralizing buffer, put in a Nalgene jar (or equivalent) and autoclaved for 30 minutes.

PREPARATION OF CONTROL CULTURES

Keep cultures on TSA at 4°C. For each test, each culture must be transferred into test tubes containing 9 mL of TSB and incubated at 35°C overnight. Sample 0.1 mL from the first tube of TSB and transfer it into a second tube containing 4 mL of TSB. Incubate the second tube at 35°C overnight. Adjust the optic density of cultures at 0.5-0.6 at 600 nm wavelength. Adjust cell density by either diluting with sterile TSB or centrifuging and resuspending the cell pellet in a smaller volume of sterile broth.

METHODS

From Libras and Rose (8.1) The following two methods are suggested:

1. Solid Media

1) After the optical density of each bacterial culture is adjusted, a sterile swab is dipped into the culture. Drain excess liquid from swab by pressing the tip against the inside of the tube just above the level of the broth. The plate is swabbed in three directions to obtain a uniform lawn of growth. Three plates should be prepared for each bacterial strain.

2) Aseptically place two to three sponges, ca. a 1 cm square piece of the sponge, per plate for each bacterial strain. Incubate plates upright at 35°C for 20-24 h.

3) Roughly measure the inhibition zones, in millimetres, around the edges of the piece of sponge.

2. Liquid Media

1) Sterilize Neutralizing buffer at 121°C for 30 minutes and inoculate (MUST be cooled or you will kill the organisms) with appropriate bacterial strain in order to obtain approximately 20 cells per millilitre. The material analyzed should be completely submerged. The material tested (previously moistened and sterilized) is immersed into that suspension, pressed against the inside of the tube just above the level of the broth, draining the excess liquid in order to give a good impregnation. Follow the same procedure for each trial in peptone water without bacteria as a negative control.

2) After being immersed into the bacterial suspension, the bacterial carrier (sponge, Q-tips, J-cloth, etc.) is put into the appropriate nutrient broth i.e. enrichment broth for Listeria (LEB) to isolate Listeria monocytogenes or Nutrient Broth (NB) to isolate S. aureus, E. coli and Salmonella. Repeat this procedure three times in 3 different jars for each bacteria and each type of bacterial carrier tested. For each trial, a sample of 5 mL of inoculated peptone water is put into a nutritive media as a positive control.

3) LEB is incubated at 30°C and NB at 35°C for 24 hours.

4) In order to check for the growth of L. monocytogenes, LEB is inoculated onto PALCAM agar and Oxford agar. NB is streaked on Baird-Parker (BP) agar to check for the growth of S. aureus or on MacConkey (MC) agar to check for the growth of E. coli and Salmonella.