Biotesting (Microbial Challenge) Method

A. Introduction 

DestructiveTo detect the presence of holes in hermetic packages by placing them in an agitated solution of fermentation bacteria in water for an extended period of time.> 10 μm pinhole, > 10 μm microchannelOff-line, > 1 week$1,000-5,000

B. Operation 

Microbial challenge tests, also called biotests, are based on the classical method developed by Bashfor, and have been designed to detect possible entry of microorganisms into sealed packages. A typical challenge test is conducted by immersing a filled and sealed package into a bacterial suspension, incubating the package for a given length of time, and quantifying the extent of microbial growth on the package contents. Alternatively, packages contained within a chamber may be sprayed with an aerosol mist containing microorganisms. The tested package often contains a nutritious medium that enhances the growth of microorganisms present, and allows growth of sublethally injured microbes which may not grow in the final product. A pH indicator may also be incorporated into the medium for visual confirmation of microbial growth. Challenge tests are performed using various nonpathogenic microorganisms such as Enterobacter aerogenes, Aerobacter aerogenes, and Pseudomonas aeruginosa.

The microbial populations used should be sufficiently high to ensure that all leakers are infected. Usually, populations greater than 108 of suspension are required. The results of biotests can vary widely depending upon factors such as the type (morphology) of microorganism used, the viscosity of package contents, the concentration of microbial suspension, the leak shape and size and the pressure or vacuum applied. Thus, comparison of results requires standardization of test conditions and parameters.

  • Water bath with temperature control and agitation
    1. Solution of Enterobacter aerogenes for foods, pH >5.0
    2. Solution of Lactobacillus cellobiosis for foods, pH <5.0
  • Sample packages
  • Apparatus to flex packages
  • Incubator
  1. Obtain representative samples. 
  2. Mix active bacteria in water at about 1.0 x 107/ml. 
  3. Immerse samples in mixture and agitate water bath and flex sample for 30 min. 
  4. Remove packages and rinse with chlorinated water. 
  5. Incubate samples for 2 weeks at 95-100oF. 
  6. Observe packages for swelling for 3 weeks.


A package swelled and/or the color/turbidity/viscosity of the product inside a package changed.


A package didn't swell and the color/turbidity/viscosity of the product inside a package remained the same.

False Positive

A package trapped too much air during a filling stage, and expanded during processing.

False Negative

Microorganisms didn't form enough gas. Air escaped during storage because of leak in a package.

C. Application 


DEFECTSFlexible PouchSemi-rigid and
Rigid plastic container
Plastic Can (Double-seam Metal End)Paperboard
Corner Dent   
Cut (Fracture)
Double Seam Defects*   
Foreign Matter Inclusion  
Label Foldover   
Leaker (Channel) 
Leaker (Corner)   
Leaker (Notch)   
Leaker (Perforation)   
Leaker (Pulltab)   
Leaker (Seal)   
Loose Flap or Ear   
Puncture (Pinhole)
Seal Defects (Blister)   
Seal Defects (Blocked)   
Seal Defects (Burnt)   
Seal Defects (Compressed)   
Seal Defects (Contaminated)  
Seal Defects (Convolution/Embossing)   
Seal Defects (Creep)   
Seal Defects (Crooked)   
Seal Defects (Incomplete)   
Seal Defects (Misaligned/Deformed)  
Seal Defects (Nonbonding/Weak)   
Seal Defects (Plastic Lumps)   
Seal Defects (Seal-width Variation)   
Seal Defects (Stringy)   
Seal Defects (Uneven Impression)   
Seal Defects (Uneven Juncture)   
Seal Defects (Wrinkle)  
Swell (Swollen Package)

D. Source 

  • ARNDT., G.W. JR. 1998. Chapter 22C Examination of Flexible and Semirigid Food Containers for Integrity. FDA Bacteriological Analytical Manual (8th Ed)
  • Chen, C., B. Harte, C. Lai, J. Pestka, and D. Henyon. 1991. Assessment of package integrity using a spray cabinet technique. J. Food Prot. 54(8):643-647.
  • Folinazzo, J. F., J. J. Kiloran and D. T. Maunder. 1968. Bio-test method for determining integrity of flexible packages of shelf-stable foods. Food. Technol. 22:615-618.
  • Keller, S.W., J.E. Marcy, B.A. Blakistone, G.H. Lacy, C.R. Hackney, and W.H.. Carter, Jr. 1996. Bioaerosol exposure method for package integrity testing. J. Parenter. Sci. Technol. 43:104-108 
  • Song, Y. and Hargraves, W.A. 1997. Post-process Contamination of Flexible Pouches Challenged by Immersion Biotest. Annual Report of the Collaborative Research Programs. Illinois Institute of Technology, National Center for Food Safety and Technology.