SOP: Microbiological Examination of Sugar
GIFSQ — Global Institute for Food Safety & Quality
Principle
Sugar for canning and bottling must conform to microbiological standards (the book cites the National Canners Association standards and the Standards of the American Bottlers of Carbonated Beverages): the total colony count is determined by the pour-plate technique on nutrient agar; yeasts and moulds are counted on an osmophilic medium; and the thermophilic bacterial spores important as spoilage agents in low-acid canned foods — flat-sour bacteria (Bacillus stearothermophilus), thermophilic anaerobes not producing H₂S (Clostridium thermosaccharolyticum) and sulphide-spoilage bacteria (Clostridium nigrificans) — are detected by their prescribed culture techniques after a heat-shock that destroys vegetative cells. (These organisms are not of health significance, but excessive numbers may survive commercial heat processes and cause spoilage.)
Apparatus required
- Sterile petri dishes
- Colony counter
- Humidified incubator (55°C)
- 250-ml sterile conical flask marked to indicate 100 ml
- Clean dry test tubes plugged with cotton wool
Reagents
a. Nutrient agar (total colony count): dissolve 3 g of beef extract, 5 g of peptone and 15 g of agar in water with heating, and make up to 1000 ml.
b. Osmophilic medium (yeast and mould): 15 g of dried malt extract, 0.78 g of peptone, 2.67 g of ammonium chloride, 2.37 g of glycerine and 20 g of powdered agar in 1000 ml of 45° Brix sucrose syrup containing 8–10% invert sugar.
c. Culture media for thermophilic spores — B. stearothermophilus: glucose tryptone agar; C. thermosaccharolyticum: liver broth; sulphide-spoilage bacteria: iron sulphite agar — prepared as given in Chapter 23 of the book.
d. Sterile distilled water.
Sample preparation — Sugar for thermophilic spore examination
Draw a 200 g sample from 5 separate bags and place in clean containers
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From each sample, place 20 g of sugar in a 250-ml sterile conical flask marked to indicate 100 ml; add sterile water to the 100 ml mark
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Heat rapidly to boiling point and boil for 5 min; replace the water lost by evaporation with sterile water
Procedure — Total colony count
Take 20 ml of the nutrient agar medium in clean dry test tubes, plug with cotton wool, sterilize at 15 psig for 30 min, and cool
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Weigh 20 g of sugar into 100 ml of sterile distilled water and dissolve with the help of a sterile pipette
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Pipette 1 ml each into three sterile petri dishes
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Melt the medium contained in the tubes, cool to 44–46°C, and add to the petri dishes aseptically (see the general instructions for microbiological examinations in Chapter 20 of the book)
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Incubate the plates at 32°C for 4 days and count the colonies using a colony counter; express the results as the total number of colonies per gram
Procedure — Yeast and mould
Take 20 ml of the osmophilic medium in test tubes, plug with cotton wool, and sterilize at 15 psig for 30 min
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Count the yeast and mould by the pour-plate technique given for the total colony count
Procedure — Flat-sour bacteria (Bacillus stearothermophilus)
Follow the procedure given in Chapter 23 of the book, using 2 ml of inoculum into each plate and 5 plates
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Incubate the plates at 55°C for 35–48 hr in a humidified incubator to prevent drying of the plates; count the number of colonies in each plate
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The total count from the 5 plates represents the number of spores in 2 g of the original sugar; multiply by 5 to express the results as the number of spores per 10 g of sugar
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Confirm typical colonies: individual colonies are 1–5 mm in diameter with a typical opaque central “spot”, surrounded by a yellow halo (an insignificant or missing halo may be due to the low-acid-producing type); if the plate is thickly seeded the entire plate may have a yellow tinge; typical subsurface colonies are compact and may appear as pin points; if the subsurface colonies show reasonable purity of the formed flora, assume they are formed by similar bacterial groups; to determine whether typical subsurface colonies are flat-sour organisms, streak from the colonies to agar plates to determine the surface characteristics
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If the plates are heavily seeded, dilute the original solution and repeat the procedure
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Report the results as the number of flat-sour spores per 10 g of sugar — the ISI procedure for counting and reporting is: (A) the combined count from 5 plates multiplied by 5 should not be more than 75; (B) take the average of the combined count from 5 sets of plates to obtain the average number of spores per 10 g of sugar, which should not be more than 50
Procedure — Thermophilic anaerobes not producing H₂S
Divide 20 ml of the boiled sugar solution equally among 6 liver broth tubes; stratify the liquid medium with plain nutrient agar; allow the agar to solidify; preheat to 55°C and incubate at that temperature for 72 hr
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Thermophilic anaerobes not producing H₂S are identified by splitting of the agar, presence of acid, and at times the formation of a cheesy odour; the method is useful as a qualitative test but cannot be used for quantitative purposes
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Report the results as the number of tubes positive or negative; according to the ISI specifications, all the tubes should be negative
Procedure — Sulphide-spoilage bacteria
Divide 20 ml of the sugar solution equally among 6 freshly exhausted tubes containing the medium and incubate at 55°C for 48 hr
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Sulphide-spoilage bacteria form characteristic blackened spherical areas; as H₂S reacts with iron, no gas formation is observed (some thermophilic anaerobes not producing H₂S generate relatively large amounts of hydrogen, which splits the agar and reduces sulphite — this causes general blackening of the medium, readily distinguished from the restricted blackened area caused by Cl. nigrificans)
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Report the results as the number of sulphide-spoilage spores per 10 g of sugar; according to the ISI specifications, sulphide spoilage shall not be detected in any of the 6 tubes
Precautions
- For the microbiological sampling of sugar, use equipment and containers sterilized at 15 psig for 30 min in an autoclave, or at 170°C for 1 hr in a hot air oven.
- Use a humidified incubator for the flat-sour plates to prevent drying.
- Follow the general instructions for microbiological examinations in Chapter 20 of the book.
Note: the book defers the full preparation of the thermophilic-spore media and the flat-sour culture technique to Chapter 23; this SOP documents the sugar-side preparation, media composition and procedures exactly as the book gives them in Chapter 8. The full Chapter 23 media procedures are documented in the Chapter 23 SOPs once converted.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.