SOP: Detection and Enumeration of Motile Aeromonads (Aeromonas hydrophila Group) in Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP detects and enumerates motile aeromonads (the Aeromonas hydrophila group) in foods following the APHA Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 30, 4th Edition. Dilutions of the food are spread-plated on starch ampicillin (SA) agar, where starch hydrolysis is the differential trait (few food bacteria hydrolyse starch) and ampicillin suppresses the background microflora. After incubation at 28 °C, the plates are flooded with Lugol’s iodine and amylase-positive colonies surrounded by a clear zone of hydrolysed starch are counted. When low numbers or injured cells are expected, the sample is first enriched in alkaline peptone water or tryptic soy broth with ampicillin, with quantitation by the most probable number (MPN) procedure.

2. Apparatus required

• Stomacher laboratory blender and sterile stomacher bags
• Sterile pipettes
• Sterile bent glass rods, for spreading
• Sterile Petri dishes
• Incubator capable of maintaining 28 °C
• Water bath, for tempering agar
• Dissecting microscope or colony counter (optional)

3. Reagents

• A. Starch ampicillin (SA) agar: phenol red agar base — 31.0 g; soluble starch, reagent grade — 10.0 g; distilled water — 1.0 L
• To prepare: suspend the ingredients in distilled water and dissolve by heating while stirring; autoclave for 15 min at 121 °C; after sterilization and tempering, dissolve 10 mg of ampicillin in a very small quantity of sterile distilled water and add to 1 L of the medium before pouring into sterile Petri dishes
• B. Modified SA agar (named in the chapter; no formulation given): recommended for samples in which swarming of Proteus may occur; colonies show a light halo against a blue background and no iodine flood is needed
• C. Bile salts brilliant green starch (BBGS) agar: proteose peptone — 10.0 g; Lab-Lemco powder — 5.0 g; bile salts — 5.0 g; soluble starch — 10.0 g; agar — 15.0 g; brilliant green (0.05% w/v solution) — 1.0 mL; distilled water — 1.0 L
• To prepare: suspend the ingredients in distilled water and dissolve by heating while stirring; autoclave for 15 min at 121 °C; final pH should be 7.2; recommended for samples in which large numbers of Proteus species might be encountered
• D. Alkaline peptone water (APW), enrichment broth (named in the chapter; formulation not given in the media chapter)
• E. Tryptic soy broth + ampicillin (TSBA): tryptic soy broth (commercial) with 30 mg of ampicillin per litre of broth, enrichment broth
• F. Peptone (0.1%) water: peptone — 1.0 g; distilled water — 1.0 L
• To prepare: dissolve the peptone in distilled water; autoclave for 15 min at 121 °C
• G. Nutrient agar or tryptic soy agar (TSA), commercial, for verification
• H. DNase test agar, commercial, for verification
• I. Kaper’s medium: proteose peptone — 5.0 g; yeast extract — 3.0 g; tryptone — 10.0 g; L-ornithine·HCl — 5.0 g; mannitol — 1.0 g; inositol — 10.0 g; sodium thiosulfate — 0.4 g; ferric ammonium citrate — 0.5 g; bromcresol purple — 0.02 g; agar — 3.0 g; distilled water — 1.0 L
• To prepare: heat to dissolve all ingredients completely in distilled water and adjust the pH to 6.7; dispense the medium into tubes (5 mL per tube) and autoclave at 121 °C for 12 minutes; the single tube shows fermentation of mannitol and inositol, ornithine decarboxylase and deamination, indole production, motility, and hydrogen sulphide production from sodium thiosulfate and cysteine
• J. Lugol’s iodine solution, for flooding plates and the amylase spot test
• K. Vibriostatic agent O/129 discs, 10 µg and 150 µg, for the resistance test
• L. Oxidase test reagent: 1% N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride
• M. Gram stain reagents
• N. Kovac’s reagent, for the indole test
• O. 3% hydrogen peroxide solution, for the catalase test

4. Sample preparation

Motile aeromonads grow readily in fresh foods at 5 °C, so process the food samples as soon as possible after they arrive at the laboratory. They are somewhat sensitive to pH values below 5.5, so process acidic foods soon after arrival.

Aseptically weigh 25 g of the food into a stomacher bag
↓
Add 225 mL of sterile peptone (0.1%) water
↓
Blend for 2 to 3 min in a stomacher laboratory blender
↓
Prepare further dilutions in peptone water as needed

5. Procedure

5.1. Direct plating (quantitative)

Spread 0.1 mL portions of the dilutions onto the surface of SA agar plates, distributing evenly with sterile bent glass rods
↓
(For samples in which large numbers of Proteus species might be encountered, use BBGS agar; modified SA agar may be used where Proteus swarming is a problem)
↓
Incubate the plates at 28 °C overnight, for a maximum of 24 hours
↓
Pick any suspect colonies for verification BEFORE the next step, since the iodine is rapidly lethal to the cells
↓
Flood the SA and BBGS agar plates with 5 mL of Lugol’s iodine solution
↓
Count typical colonies (5 mm, yellow to honey-coloured) surrounded by a clear zone of hydrolysed starch against a black background as Aeromonas hydrophila group organisms (on modified SA agar, count colonies surrounded by a light halo against a blue background; no iodine flood is needed)
↓
On overcrowded plates, the growth may be scraped off with a sterile loop and the zones counted to estimate the number of colonies

5.2. Enrichment variant (low numbers or injured cells expected)

Inoculate an enrichment broth — alkaline peptone water or tryptic soy broth with ampicillin — with a 25 g sample of food or with dilutions of the original 1:10 slurry from the stomacher bag
↓
Incubate at 28 °C
↓
When quantitation is desired, incorporate either broth into an MPN procedure using various dilutions in a 3- or 5-tube scheme
↓
After 24 hours, streak portions of the broths onto SA agar and BBGS agar and observe the plates after 24 hours at 28 °C
↓
Score plates as positive or negative for typical A. hydrophila colonies and obtain the quantitative estimate from MPN tables

5.3. Verification of isolates as A. hydrophila group

Pick typical colonies from the SA agar plate and streak them onto nutrient agar, tryptic soy agar, or another suitable growth medium that does not contain carbohydrate; also streak a plate of DNase test agar
↓
Test the suspected colonies from the SA plate for amylase activity by placing a drop of Lugol’s iodine on each colony
↓
If the colonies are amylase-positive, incubate the plating media for 24 hours at 28 °C; before incubation, place a disk of O/129 in a heavy streak area so that zones of inhibition can be seen
↓
From the nutrient agar or TSA plate, check that a pure culture was obtained, prepare a Gram stain (short gram-negative rods), and perform the catalase test by adding a few drops of 3% hydrogen peroxide to an isolated colony (bubbles indicate a positive reaction)
↓
Read the O/129 resistance: Aeromonas species are resistant to both the 10 µg and the 150 µg disks; any zone of inhibition around the 150 µg disk means the isolate is sensitive to the vibriostatic agent and is not Aeromonas
↓
Test growth with and without NaCl in 1% tryptone broth for 24 to 48 hours at 35 °C: Aeromonas species grow with and without NaCl, whereas Vibrio species require NaCl for growth
↓
Optionally, inoculate Kaper’s single-tube medium for presumptive identification: observe fermentation of mannitol and inositol, ornithine decarboxylase and deamination, indole production (Kovac’s reagent), motility, and hydrogen sulphide production from sodium thiosulfate and cysteine

6. Calculation

CFU per g = N / (V × D)

Where,

• N = number of typical colonies counted on the plates
• V = volume of dilution plated, in mL (0.1 mL per plate)
• D = dilution of the plated dilution (for example, 10^−1)

For the enrichment variant, read the MPN from the tables using the pattern of positive and negative plates.

7. Precautions

• Process samples promptly; motile aeromonads can grow competitively in most foods held at 5 °C.
• Pick suspect colonies for verification before flooding plates with iodine — the iodine kills the cells rapidly.
• Do not incubate the primary plates longer than 24 hours at 28 °C before reading.
• The significance of A. hydrophila group organisms in foods is currently not known; they are readily isolated, often in high numbers, from a wide variety of fresh and processed foods and water supplies, and are recognised as pathogens in select groups of individuals such as immunocompromised persons, patients with underlying malignancies, young children, and travellers in developing countries.

8. References

• APHA, Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 30, 4th Edition.