Presence/absence method APHA 2001 and BAM/FDA 2004 for Vibrio cholerae in foods

Method of the Food and Drug Administration (FDA), as described in the May/2004 revision of the Bacteriological Analytical Manual Online (Kaysner and DePaola Jr, 2004) and of the American Public Health Association (APHA), as described in the 4^th Ed. of the Compendium of Methods for the Micro-biological Examination of Foods (Kaysner and De Paola Jr., 2001).

1. Material required for analysis

Isolation

•  Alkaline Peptone Water (APW)

•  Thiosulfate Citrate Bile Sucrose (TCBS) Agar

•  Modified Cellobiose Polymyxin Colistin (mCPC) Agar or Cellobiose Colistin (CC) Agar (optional)

•  Laboratory incubator set to 35 ± 2°C

•  Laboratory incubator set to 39–40°C (if available)

•  Water bath set to 42 ± 0.2°C (for oysters samples)

Screening

•  Arginine Glucose Slants (AGS)

•  T₁N₁ Agar (tubes)

•  T₁N₀ Broth (same formulation of T₁N₁ without agar and without NaCl) (tubes)

•  T₁N₃ Broth (same formulation of T₁N₁ without agar and NaCl concentration adjusted to 3%) (tubes)

•  0.5% Sodium Desoxycholate Solution (for string test)

•  0.85% Saline Solution

•  Oxidase Kovacs Reagent

•  V. cholerae polyvalent 01 and O139 antisera

•  Inaba and Ogawa antisera

Confirmation by biochemical tests

•  Kit API 20E or equivalent

Biotypes El Tor and Classical differentiation (optional)

•  Sheep Blood Agar

•  Polymyxin B discs 50 U

2. Procedure

A general flowchart for detection of Vibrio cholerae in foods using the presence/absence methods APHA 2001 and BAM/FDA 2004 is shown in Figure.1.

Safety precautions recommended by method BAM/FDA 2004: Perform the tests in properly equipped laboratories, under the control of a skilled microbiologist. Take care in the disposal of all contaminated material.

BAM/FDA 2004 recommendations for the samples storage: Samples intended for vibrios detection or count should be kept at 7°C to 10°C and analyzed as soon as possible. Direct contact with ice should be avoided. If frozen storage is required, a temperature of −80°C is recommended.

a)  Enrichment and selective differential plating:

Homogenize 25 g of sample with 225 ml of  Alkaline Peptone Water (APW). Incubate the flasks at 35 ± 2°C/6–8 h.

    After 6–8 h of incubation, inoculate a loopful from the surface pellicle of APW onto a  Thiosulfate Citrate Bile Sucrose (TCBS) Agar plate and incubate the TSBC plate at 35 ± 2°C/18–24 h. A plate of  Modified Cellobiose Polymyxin Colistin (mCPC) Agar or  Cellobiose Colistin (CC) Agar may also be included. If used, incubate the mCPC or CC plates at 39–40°C/18–24 h (preferably) or 35–37°C/18–24 h if an incubator set to 39–40°C is not available.

    For samples processed by heating, freezing or drying re-incubate the APW overnight and repeat the plating on TCBS (and mCPC or CC, if used).

    For raw oysters, include a second flask with 25 g of product plus 2475 ml of APW. Incubate the APW at 42 ± 0.2°C/18–21 h in a water bath and proceed with the plating on TCBS (and mCPC or CC, if used) from this flask too.

 Note a.1)   If the enumeration is desired, use the most prob-able number (MPN) technique: Homogenize 25 g of sample with 225 ml of Phosphate Buffered Saline (PBS). From this first dilution (10⁻¹) prepare decimal serial dilutions. Select three appropriate dilutions and inoculate three 1 ml portions of each dilution onto three tubes with 10 ml of APW. From this point of the procedure continue the analysis separately for each APW tube. For oysters include a second series of APW tubes for the incubation at 42°C.

b) Screening:  Select for screening three or more typical colonies from each plate. Typical colonies of  V. cholerae on TCBS are large (2 to 3 mm) and yellow (sucrose positive). On mCPC or CC the typical colonies are green to purple.

    Transfer the typical colonies to T₁N₁ agar slants. If selected from crowded plates purify the culture by streaking on  T₁N₁ Agar plates. Incubate T₁N₁ slants or plates at 35 ± 2°C/overnight and proceed with the screening tests below (from T₁N₁ plates use a single isolated colony for the tests).

Figure.1   Scheme of analysis for detection of Vibrio cholerae in foods using the presence/absence methods APHA 2001 and BAM/FDA 2004 (Kaysner and DePaola Jr, 2001, Kaysner and DePaola Jr, 2004).

b.1)  Growth on  Arginine Glucose Slant (AGS): From the T₁N₁ cultures inoculate tubes of AGS by streaking the slant and stabbing the butt. Incubate the AGS tubes at 35  ± 2°C/overnight (with the caps slightly loosened).

The typical characteristics of V. cholerae (and V. mimicus) on AGS are purple (alkaline) slant and yellow (acid) butt (arginine not hydrolyzed) without gas or H₂S.

b.2)   Salt requirement test: From the T₁N₁ cultures inoculate tubes of T₁N₀ and T₁N₃ broths. Incubate the tubes at 35 ± 2°C/over-night. V. cholerae does not require NaCl and grows in T₁N₀ (without NaCl).

b.3)  String test: From the T₁N₁ cultures emulsify a loopful in a drop of 0.5% Sodium Desoxycholate Solution. Examine within 60s for cells lyse (loss of turbidity) which allows the DNA to string when a loopful is lifted (up to 2–3 cm) from the slide. V. cholerae is positive.

b.4)  Oxidase test: Using a platinum/iridium loop or glass rod take a portion of the T₁N₁ growth and streak it onto a filter paper moistened with the Oxidase Kovacs Reagent. The appearance of dark purple color within 10s indicates a positive reaction. V. cholerae (and V. mimicus) are oxidase positive.

b.5)   Serologic agglutination tests: In three sections (about 1  × 2 cm) marked with a wax pencil on the inside of a glass Petri dish or on a glass slide add one drop of 0.85% Saline Solution. From the T₁N1 cultures emulsify a loopful in each drop and observe for auto-agglutination. If auto-agglutination does not occur, add one drop of polyvalent V. cholerae O1 antiserum to one section and one drop of anti-O139 antiserum to another section. Tilt the mixture in back-and-forth motion for one minute and observe for agglutination against a dark background. If the test is positive, repeat separately with Ogawa and Inaba antisera. The Hikojima serotype reacts with both antisera.

c)  Confirmation by biochemical tests: Use the API 20E (BioMérieux) diagnostic strip or an equivalent biochemical kit for the identification and confirmation of the isolates. Follow the manufacturer’s instructions.

d)  Differentiation of El Tor and Classical biotypes (optional)

d.1) Beta- hemolysis: From the T₁N₁ cultures inoculate Sheep Blood Agar plates and incubate the plates at 35 ± 2°C/18–24 h. Exam-ine for beta-hemolysis, indicated by a clear zone around the growth of the culture. El Tor strains are  β-hemolytic and Classical strains are not hemolytic.

d.2) Polymyxin-B  sensitivity: From the T₁N₁ cultures inoculate T₁N₁ agar plates and place a 50 unit disc of polymyxin-B on the surface of each plate. incubate the plates inverted at 35 ± 2°C/overnight. Classical strains are sensitive to polymyxin-B, showing an inhibition zone >12 mm. El Tor strains are resistant.

e) Enterotoxigenity:  For these tests it is preferable to send the cultures to a specialist or reference laboratory.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .

Kaysner, C.A & DePaola Jr., A. (2001)  Vibrio. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 40, pp. 405–420.

Kaysner, C.A & DePaola Jr., A. (2004) Vibrio. In: FDA (ed.) Bacteriological Analytical Manual, Chapter 5. [Online] Silver Spring, Food and Drug Administration. Available from:  http://www. fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070830.htm  [Accessed 3rd November 2011].