SOP: Enumeration of Lipolytic Microorganisms in Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP enumerates lipolytic microorganisms in foods following the APHA Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 15, 4th Edition. Microbial lipases hydrolyze the ester bonds of triglycerides and release free fatty acids. The food dilutions are plated on a medium containing a fat substrate: either incorporated into a single layer of growth medium, or as a fat-containing base layer overlaid with nutrient agar (double layer method). Lipolytic colonies are detected by the zone of hydrolysis around the colony — a transparent zone on tributyrin agar, or a dark blue zone when Victoria Blue B is present with long chain fatty acid triglycerides. Only colonies surrounded by a lipolytic zone are counted, and the result is reported as the number of lipolytic microorganisms per millilitre or gram of the food.

2. Apparatus required

• Sterile Petri dishes
• Sterile pipettes
• Blender, for preparing the base layer medium
• Sterile bent glass rod, for spreading
• Glass columns, 2 to 3 cm in diameter x 10 to 15 cm in length, for purifying lipid substrates
• Incubator capable of maintaining 20 to 30 °C
• Stereoscopic microscope, for detecting weak zones of hydrolysis
• Laminar flow bacteriological safety hood, for drying base layer plates

3. Reagents

• A. Base layer agar with nutrient overlay agar
• Fat — 50.0 g
• Victoria Blue B (1:1500 aqueous solution) — 200.0 mL
• Agar — 15.0 g
• Distilled water — 800.0 mL
• To prepare: the Victoria Blue B solution is not necessary if tributyrin is used as the fat substrate; if it is not used, add 200.0 mL of distilled water to the base layer medium instead. Remove free fatty acids from the fat substrate as described below if needed. Sterilize the fat in the autoclave for 30 minutes at 121 °C. Sterilize the Victoria Blue B solution by membrane filtration. Dissolve the agar in 800.0 mL of distilled water and sterilize for 15 minutes at 121 °C. Cool the sterile ingredients to 50 °C and mix them together in a warm sterile blender for 1 minute. Pour 3.0 to 4.0 mL of the medium into the bottom of sterile Petri dishes. The plates may be used immediately or stored for 3 to 4 days in the refrigerator.
• B. Victoria Blue B (Colour Index 44045)
• To prepare the 1:1500 aqueous solution: dissolve Victoria Blue B in distilled water to 1 part in 1500
• If Victoria Blue B is not available, Victoria Blue without an alphabetic subdesignation can be used, although a deep blue zone of lipolysis may not result
• C. Fat substrates: reagent grade tributyrin; or corn, soybean, olive, or other liquid oils; or any fresh commercially available cooking oil; or solid fats such as lard, tallow, butter, or a solid vegetable shortening; or triolein or other defined synthetic glycerides. The fat concentration used is 5%; note, however, that some researchers have cautioned against the use of tributyrin in concentrations above 1%. Solid fats are melted before emulsification. The substrates should not contain antioxidants or other materials at concentrations that inhibit growth.
• Purification of tributyrin (if the substrate is known or suspected to contain substantial free fatty acids): dissolve 5 to 10 g of tributyrin per 100 mL of petroleum ether and pass the solution through a column of activated alumina. Collect the effluent and remove the petroleum ether from it under vacuum or by evaporation on a steam table under a nitrogen stream, resulting in purified triglyceride. A column of the size described below will remove the free fatty acids from 50 g of fat containing 2% to 3% free fatty acids.
• D. Petroleum ether, ACS specifications (boiling point 30 to 60 °C; the chapter prints “3° to 6°C”, evidently a misprint for the ACS 30–60 °C grade)
• E. Activated alumina, prewashed with petroleum ether, packed (25 g) into the glass columns described above
• F. Nutrient overlay agar: nutrient agar, standard methods agar, or tryptic soy agar, in that order of preference (used for the double layer method)
• G. Buffer, 10 to 40 mM, to provide a stable background pH in nutrient-free, agar-solidified lipid emulsion media used for the detection of free lipase
• H. Positive control organisms: Geotrichum candidum and Pseudomonas fragi; a positive result can also be generated by spotting a few microlitres of free fatty acid (for example oleic acid) onto a plate
• I. Negative control organisms: Proteus mirabilis or Escherichia coli JM101, which lack lipolytic activity

4. Sample preparation

• Prepare the sample and make dilutions as for the agar plate count.
• If high backgrounds of apparent lipolytic activity are observed in plates to which no samples have been added, free fatty acids are present in the lipid substrate: purify the substrate over activated alumina as described above.

5. Procedure

5a. Single layer method

Prepare pour plates using the appropriately diluted food samples, pouring the plates with 12 to 15 mL of the single layer medium containing the fat substrate
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Alternatively, spread or streak the samples on the surface of the solidified medium and incubate
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To detect free lipase only, spot aliquots of the samples of interest onto the test media or into wells cut into it (a nutrient-free, agar-solidified lipid emulsion, with 10 to 40 mM buffer for a stable background pH, may be used)
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Incubate the plates at 20 to 30 °C for 3 days if tributyrin is the fat substrate, or for 4 to 7 days for other fats; positive results may be apparent as early as one day after inoculation

Single layer plating with a fat-containing growth medium generally gives satisfactory counts and incorporates the organisms into the medium, which optimizes their enumeration.

5b. Double layer method

Pour the base agar layer as prepared above
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Prepare dilutions of the food product and spread 0.1 mL of each dilution on the surface of the base layer; allow the inoculum to dry (a few minutes)
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Pour 10 to 12 mL of the nutrient overlay agar over the base agar, suspending the cells in it by swirling the plates before the medium cools sufficiently to solidify
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Alternatively, pour both layers of agar and inoculate the plates by spreading or streaking the samples atop the solidified upper layer
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Incubate as in the single layer method above

The double layer method places a nutrient agar layer between the lipid-indicator dye and the sample. It increases the recovery of organisms whose growth is retarded by the indicator dye, the lipid substrate, or the products of lipolysis, and gives the best detection of weakly lipolytic bacteria. Dry the base layer plates before use in a laminar flow hood by partially opening the Petri dish lids for 10 to 15 minutes.

5c. Counting and reporting colonies

On tributyrin agar without Victoria Blue B, lipolytic microorganisms are detected by a transparent zone surrounding the colony on an opaque background
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On media containing long chain fatty acid triglycerides and Victoria Blue, a dark blue zone surrounds the lipolytic colonies against an opaque, light blue background (short chain, soluble fatty acids diffuse through the agar and give a weak blue color if Victoria Blue is present)
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Use a stereoscopic microscope to detect weak or indistinct zones if needed
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Count only the colonies surrounded by a lipolytic zone
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Report the result as the number of lipolytic microorganisms per mL (or g) of the food sample

5d. Controls

Run control plates for each batch of lipid substrate and dye
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Incubate Geotrichum candidum and Pseudomonas fragi for 3 to 7 days at 20 to 25 °C, and E. coli at 30 °C
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Interpretation: G. candidum hydrolyzes tributyrin very slowly, if at all, because its lipase isoenzymes preferentially hydrolyze ester bonds where the fatty acid contains a 9-10 cis double bond — it shows best lipolytic activity against oils such as olive, corn, and soybean oil. Weak zones of hydrolysis (not the dark blue of the typical Victoria Blue B reaction) for one or both of the known lipolytic organisms indicate inhibitory substances in the fats or oils. No zones of hydrolysis should be seen with P. mirabilis and E. coli.

6. Precautions

• The fat substrate is the critical reagent: high background activity in plates to which no sample was added is diagnostic of free fatty acids in the substrate — purify it before use.
• Indistinct zones sometimes occur because of weak lipolysis, the presence of free fatty acid in the lipid substrate, or the production of acid from sugar metabolism. Careful substrate preparation helps minimize weak, indistinct zones.
• The indicator dye, the lipid substrate, or the products of lipolysis can retard growth; use the double layer method when weakly lipolytic bacteria are expected or when carbohydrate must be added to obtain good growth.
• Tributyrin is convenient for the detection of lipolytic activity but is not fully specific for this class of enzymes.

7. References

• APHA, Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 15, 4th Edition.