SOP: Detection and Enumeration of Pectinolytic and Pectolytic Microorganisms

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP detects and enumerates pectinolytic (pectin-degrading) and pectolytic (pectic acid/pectate-degrading) microorganisms in foods following the APHA Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 18, 4th Edition. Pectic substances are polymers of D-galacturonic acid in plant cell walls; pectin is substantially methyl-esterified, pectic acid is negligibly esterified, and polypectate (pectate) is pectic acid in the salt form. The organisms are grown on a gel medium containing the pectin or pectate substrate, and enzyme production is detected either by depressions in the gel around the colony where the substrate has been degraded, or by flooding the plate with a precipitant solution so that a clear zone appears around producer colonies while non-producers stay surrounded by opaque gel. A medium pH of 7.0 or above distinguishes pectate/pectin lyase producers (alkaline pH optima) from polygalacturonase producers (acidic optima). Colonies that cause depressions in the polypectate gel or that are surrounded by a clear zone after flooding are counted as pectinolytic.

2. Apparatus required

• Sterile Petri dishes
• Sterile pipettes
• Spreader or sterile bent glass rod
• Cork borer, for the well plate technique
• Incubator capable of maintaining 30 °C
• Long-wavelength UV light, for fluorescent pseudomonads
• Blender, for the polypectate gel medium
• Black background, for viewing hydrolysis zones

3. Reagents

• A. Crystal violet pectate (CVP) medium: boiling distilled water — 500.0 mL; crystal violet solution (0.075% w/v aqueous) — 1.0 mL; sodium hydroxide (1 N aqueous) — 4.5 mL; calcium chloride (fresh 10% w/v CaCl₂·H₂O aqueous solution) — 3.0 mL; agar — 2.0 g; sodium nitrate — 1.0 g; sodium polypectate — 9.0 g; sodium lauryl sulfate (10% w/v aqueous solution) — 0.5 mL
• To prepare: to the 500.0 mL of boiling distilled water, blending at very low speed in a preheated blender, add the crystal violet solution, the sodium hydroxide, the calcium chloride, the agar and the sodium nitrate ↓ blend at high speed for 15 seconds ↓ continue blending at low speed and slowly add the sodium polypectate ↓ pour the incomplete medium into a 2-L flask and add the sodium lauryl sulfate ↓ cap the flask with aluminum foil rather than a cotton plug and autoclave for 25 min at 121 °C ↓ avoid foaming and pour the plates as soon as possible, since the medium solidifies quickly and cannot be remelted ↓ allow the plates to dry for 48 hours at room temperature before use ↓ final pH 7.2
• Note: not all commercial polypectate preparations give suitable gelation in this medium
• B. MP-7 (Mineral Pectin 7) medium, for pectate lyase producers: pectin (citrus or apple) — 5.0 g; monopotassium phosphate — 4.0 g; disodium phosphate — 6.0 g; yeast extract — 1.0 g; ammonium sulfate — 2.0 g; agar — 15.0 g; mineral salt solution — 500.0 mL; distilled water to make — 1.0 L
• Mineral salt solutions (each salt dissolved in 200.0 mL of distilled water): ferrous sulfate — 0.2 g; magnesium sulfate — 40.0 g; calcium chloride — 0.2 g; boric acid — 0.002 g; manganese sulfate — 0.002 g; zinc sulfate — 0.014 g; cupric sulfate — 0.010 g; molybdenum trioxide — 0.002 g
• To prepare: mix the dry basal ingredients before placing in liquid for better dispersion, then add 500.0 mL of distilled water ↓ mix 1.0 mL of each of the eight salt solutions in 492.0 mL of distilled water to produce 500.0 mL of mineral solution ↓ add the mineral solution to the basal medium to bring the volume to 1.0 L ↓ adjust the pH to 7.2 ± 0.2 ↓ dissolve with gentle heating and sterilize at 121 °C for 15 min ↓ temper the medium to 48 °C and pour into sterile Petri plates
• C. MP-5 (Mineral Pectin 5) medium, for polygalacturonase producers
• To prepare: prepare 500.0 mL of double-strength MP-7 medium without agar and adjust the pH to 5 to 6 with 1 N hydrochloric acid ↓ prepare 500.0 mL of 3% agar solution and heat to dissolve ↓ sterilize both at 121 °C for 15 min and cool both to 48 °C ↓ mix the two sterile solutions and pour the plates immediately to prevent hydrolysis
• D. FPA (fluorescent pectolytic agar) medium, for fluorescent pectinolytic pseudomonads: proteose peptone No. 3 — 20.0 g; dipotassium phosphate — 1.5 g; magnesium sulfate heptahydrate (MgSO₄·7H₂O) — 0.73 g; pectin — 5.0 g; agar — 15.0 g; distilled water — 1.0 L
• Antibiotic supplement mixture: penicillin G — 75,000.0 IU; novobiocin — 45.0 mg; cycloheximide — 75.0 mg; ethanol — 1.0 mL
• To prepare: dissolve the basal ingredients in the distilled water by boiling ↓ adjust to pH 7.1 and sterilize at 121 °C for 15 min ↓ add the 1.0 mL of ethanol to the dry antibiotics and allow to stand 30 min ↓ dilute the antibiotic solution with 9 mL of sterile distilled water ↓ add 1.0 mL of the dilute solution to 100.0 mL of the sterile and cooled basal medium before pouring into sterile Petri plates
• E. Polypectate gel medium: sodium polypectate — 70.0 g; peptone — 5.0 g; dipotassium phosphate — 5.0 g; monopotassium phosphate — 1.0 g; calcium chloride dihydrate (CaCl₂·2H₂O) — 0.6 g; distilled water — 1.0 L
• To prepare: heat 500.0 mL of distilled water and place all the ingredients except the polypectate in a blender with the heated water to dissolve ↓ add the polypectate last, in small amounts per addition, with slow stirring to diminish occluded air ↓ adjust the pH to 7.0 and add the remainder of the distilled water ↓ sterilize by autoclaving at 121 °C for 15 min ↓ temper the medium to 48 °C and pour into sterile Petri plates
• F. Polysaccharide precipitant: hexadecyltrimethylammonium bromide — 1.0 g; water — 100.0 mL
• To prepare: dissolve the 1.0 g in 100.0 mL of water; the solution may be sterilized by autoclaving if desired
• G. Fungal pectin medium (for fungal pectate lyases): pectin — 10.0 g (1%); yeast extract — 2.0 g (0.2%); mineral salts; agar — 30.0 g (3%); distilled water — 1.0 L
• To prepare: dissolve the ingredients in the distilled water and adjust to pH 7.0 ↓ sterilize at 121 °C for 15 min and pour plates

4. Sample preparation

• Prepare a homogenous suspension and appropriate dilutions of the food.
• In food containing a large amount of glucose, dilute sufficiently to avoid catabolite repression of pectic enzyme synthesis.
• In high-sugar foods such as jams and jellies, dilute the sample with an appropriate buffer or isotonic solution to prevent osmotic shock to the cells.

5. Procedure

Common plating steps (all variants)

Place not more than 0.25-mL quantities of the appropriate dilutions on the surface of prepoured plates to obtain 20 to 30 colonies and to avoid a wet agar surface
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Distribute by the spread plate technique
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Incubate plates inoculated with plant materials at 30 °C for 48 hours; incubate plates inoculated with foods at the temperature at which the food is stored
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Note: some pectinolytic bacteria grow at 37 °C but do not produce pectate lyase until the temperature is 32 °C or below; E. carotovora subsp. atroseptica often will not grow above 28 °C

Variant 1 — Crystal violet pectate (CVP) medium

Plate the sample on the crystal violet pectate medium; the crystal violet makes the medium selective for gram-negative bacteria
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Incubate as above
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Detect pectolytic colonies by the formation of depressions in the pectate gel where the substrate has been enzymatically degraded

Variant 2 — MP-7 medium (pectate lyase producers)

Plate the sample on MP-7 medium, preferably by the spread plate technique
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Incubate as above until colonies have grown
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Pour the polysaccharide precipitant over the surface of the plate, taking care not to dislodge the colonies
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Zones of pectin hydrolysis appear quickly, usually within a few minutes; view them against a black background

Variant 3 — MP-5 medium (polygalacturonase producers)

Plate the sample on MP-5 medium and incubate as above
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Flood the plates with the polysaccharide precipitant as in Variant 2; colonies surrounded by a clear zone on the white background are polygalacturonase producers

Variant 4 — FPA medium (fluorescent pectinolytic pseudomonads)

Plate the sample on FPA medium by the spread plate technique and incubate as above
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Detect the fluorescent bacteria on the plates with long-wavelength UV light before adding the pectin precipitant
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Treat the plates with the polysaccharide precipitant as in Variant 2 and read the clear zones

Variant 5 — Polypectate gel medium

Plate the sample on the polypectate gel medium and incubate as above
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Count the colonies that cause depressions in the gel as pectinolytic

Variant 6 — Fungal pectin medium (fungal pectate lyases)

Plate the fungal sample on the fungal pectin medium and incubate for 3 to 5 days
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Flood the plates with the polysaccharide precipitant to precipitate the non-degraded pectin; clear zones occur around colonies that produce pectate lyase
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If the precipitant is not left in contact with the fungal cells for more than 5 minutes, viable colonies can be isolated from the flooded plates

Well plate technique for liquid samples
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Cut holes in the agar gel plates with a cork borer
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Fill the wells with the liquid sample, such as a culture filtrate
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Incubate and read the clear zones around the wells as above

6. Calculation and reporting

CFU per g or mL = N / (V × D)

Where,

• N = total number of pectinolytic colonies counted on all plates of the dilution
• V = total volume of diluted sample plated, in mL
• D = dilution of the plated dilution (for example, 10⁻¹)

A colony is counted as pectinolytic when it causes a depression in the polypectate gel or is surrounded by a clear zone after flooding the plate with the pectin precipitant.

7. Precautions

• Pectate lyase is not produced by some bacteria above 32 °C even when they grow at 37 °C; check the incubation temperature against the organism expected.
• In high-glucose foods, insufficient dilution causes catabolite repression of pectic enzyme synthesis and false-negative plates.
• Do not use hypotonic diluents for high-sugar foods; the resulting osmotic shock damages the cells.
• For the MP-7 and MP-5 media, the polysaccharide precipitant must be applied after the colonies have grown; dislodging the colonies during flooding makes the zones unreadable.

8. References

• APHA, Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 18, 4th Edition.