SOP: Determination of Bacterial Permeability of Plastic Films

GIFSQ — Global Institute for Food Safety & Quality

Principle

Pouches made of the plastic films under test are filled with a suitable bacterial growth medium, sealed under atmospheric pressure, and immersed in a liquid bath that is highly contaminated with bacteria. If the medium in the pouches remains sterile for a time equivalent to the expected shelf-life of the product, the pouch materials are considered impermeable to the microorganisms in the bath.

Apparatus required

• Pouch-making materials (films under test)
• Liquid bath with contaminated medium
• Petri dishes (pour plate examination)
• Anaerobic incubation facility

Reagents

a. Suitable bacterial growth medium (such as nutrient broth).

b. Contaminated liquid medium (bacterial slurry) for the bath.

Procedure — Preparation of pouches

Make pouches of practical size from the materials to be tested — normal pouch size gives a normal distribution of seal surface, stresses, creases, pinholes, etc., as found in practice

↓

Fill the pouches to the normal fill volume with the growth medium, and seal them under atmospheric pressure; retain some air in the pouch to support the growth of aerobic and facultative organisms, should they enter the pouch

↓

Test each pouch with a slight hand pressure after the final seal is made — pouches with faulty seals or other structural defects can thereby be detected

↓

Sterilize the pouches by radiation or other cold sterilization method (the book’s pouches were sterilized with 4.5 megarads of cobalt-60 gamma radiation; only retortable pouches may be heat sterilized)

Procedure — Exposure to contaminated medium

Immerse the pouches in the contaminated medium; adjust the height of the pouches until the surfaces of the sterile medium and the contaminated medium are at about the same level

↓

Store the system under the time and temperature conditions likely to be encountered in actual storage

↓

Remove the pouches from the contaminated medium and incubate them at a temperature and for a time suitable for the type of bacteria in the medium

Procedure — Examination of pouches

After incubation, examine all pouches for visual evidence of microbial growth (turbidity, gas formation, etc.); separate the pouches into three groups: (i) pouches showing growth; (ii) pouches showing possible growth; (iii) pouches showing no growth; and investigate as below:

First group: Discard the contents and test the pouches for structural defects such as sealing defects and pinholes. If defects are found, repeat the test and examine. If the pouches are again found defective, tentatively consider that the material is permeable to bacteria.

Second group: Aseptically sample the contents of each pouch and determine the presence of bacteria by the pour plate method (see the bacteriological examination chapter). For each pouch, pour four plates; incubate two plates at a suitable temperature; pour a second layer of agar into the remaining two plates and incubate them under anaerobic conditions at an appropriate temperature. When the optimum growth temperatures are not known, incubate over a range of temperatures using correspondingly more Petri dishes — this reveals both aerobes and anaerobes present. After all pouches are sampled, discard the contents and save the pouches. If any of the plates show growth, inspect the corresponding pouches as in the first group. If the plates show no growth, consider that the corresponding pouches were not permeable to the microorganisms in the contaminated slurry.

Third group: Check the contents for growth as in the second group (the pouches in the third group can be presumed impermeable to the microbes in the slurry if the pouch contents show no contamination). Inoculate each pouch with a small amount of the contaminated slurry and incubate them. This step checks whether the growth capability of the medium was impaired by the irradiation or other sterilization method used.

Notes

• Investigations cited in the book indicate that nylon-11 and polyethylene-coated polyester were 100% impermeable to bacteria; saran-coated nylon-11, polypropylene and polyester averaged more than 95%.
• The reliability of plastic pouches for bacterial impermeability depends, in addition to the material used, on the sealing equipment, sealing techniques, etc. In general, ordinary plastic films more than 0.5 mil thick are not permeable to bacteria.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Ronsivalli, L.J., J.B. Bernstein and B.L. Tinker, Food Technol., 20, 1074 (1966) — cited in the book. (The book prints the author name as “Bernsteins”; carried as Bernstein.)