absence of microorganisms
6 Aerobic plate count
8.3.1 Material required for analysis Preparation of the samples and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer
• Dilution tubes containing 9 ml 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to check on special cases in which either the type or volume of diluent vary as a function of the sample to be examined.
Enrichment and plating
• Enterobacteriaceae Enrichment Broth (EEB)
• Petri plates containing Violet Red Bile Glucose (VRBG) Agar
• Laboratory incubator set to 35 ± 1°C
8.3.2 Procedure
A flowchart for the enumeration of Enterobacteriaceae in foods using the MPN method APHA 2001 is shown in Figure 8.2.
a) Preparation of the samples and serial dilutions.
Follow the procedures described in Chapter 2.
Note a.1) If there is no need to quantify, but only to determine the presence/absence in the sample, inoculation can be done directly on Enterobacteriaceae enrichment broth (EEB), followed by incubation.
Homogenination 1ml 1ml
25g of sample +
225ml of Peptone Water (PW)
9ml PW
10-2 10-3
35 1 C/18-24± o h
Enterobacteriaceae CFU/g
9ml PW 10-1
1ml 1ml 1ml
VRBG Violet Red Bile Glucose (VRBG) Agar VRBG
(pour plate with overlay)
Figure 8.1 Scheme of analysis for the enumeration of Enterobacteriaceae in foods using the plate count method APHA 2001 (Kornacki and Johnson, 2001).
Homogenization 1ml
25g of sample +
225ml of Peptone Water (PW)
9ml PW
10ml 1ml 1ml
35 1 C/20-24± o h
Tubes of EEB with growth
Streak a loopful
Violet Red Bile Glucose (VRBG) agar 35±1 C/20-24o h
Plates with typical colonies
10ml 10ml 1ml 1ml 1ml 1ml
Confirmed tubes
MPN/g ENTEROBACTERIACEAE Double strength
Enrichment Broth
Enterobacteriaceae (EEB)(10ml)
Single strength Enrichment Broth
Enterobacteriaceae (EEB)(10ml)
Single strength Enrichment Broth
Enterobacteriaceae (EEB)(10ml)
Figure 8.2 Scheme of analysis for the enumeration of Enterobacteriaceae in foods using the MPN method APHA 2001 (Kornacki and Johnson, 2001).
7007TS-DASILVA-Book.indb 86
7007TS-DASILVA-Book.indb 86 11/26/2012 11:15:23 AM11/26/2012 11:15:23 AM
b) Inoculation (presumptive test). Inoculate three 10 ml-aliquots of the first dilution (10−1) in three tubes containing 10 ml of double strength Entero-bacteriaceae Enrichment Broth (EEB), three 1 ml-aliquots of the 10−1 dilution in three tubes containing 10 ml of single strength EEB and three 1 ml-aliquots of the 10−2 dilution in three tubes containing 10 ml of single strength EEB.
c) Incubation. Incubate the EEB tubes at 35 ± 1°C/20–24 h and observe any growth. In case growth is observed, pass on to the subsequent items.
d) Confirmation of Enterobacteriaceae. From the culture of each tube exhibiting growth streak a loopful on a plate of Violet Red Bile Glucose Agar.
Incubate the plates at 35 ± 1°C/20–24 h. Observe whether there is any growth of typical Enterobacte-riaceae colonies (red purple, 0.5 mm or greater in diameter surrounded by a reddish halo indicating precipitation of the bile salts).
e) Calculating the results. Record the number of confirmed tubes and determine the MPN/g or MPN/ml as detailed in Chapter 4, using one of the MPN tables.
8.4 Petrifilm™ AOAC official method 2003.1 for Enterobacteriaceae in selected foods
Official method of the AOAC International, as described in the Revision 3 of the 18th Edition of the Official Methods of Analysis of AOAC International (Horwitz and Latimer, 2010). Applied to milk, cheddar cheese, flour, frozen prepared meals, frozen broccoli, and nut pieces.
Petrifilm™ (3M Company) is a modified version of the Colony Forming Units (CFU) plate count method, consisting of two sterile, dry rehydratable films impreg-nated with culture medium and cold-water-soluble gel-ling agents. Inoculation is done on the surface of the plating surface (bottom film), which, after inoculation is covered with the top film. The inoculum is spread evenly using a plastic spreader and applying gentle man-ual pressure. After solidification of the gel, the plates are incubated for the development of colonies. The cul-ture medium is Violet Red Bile Glucose (VRBG) Agar supplemented with 2,3,5-triphenyltetrazolium chloride (an indicator which, when reduced, gives the colonies a deep red color) and with an pH indicator (to detect fermentation with acid production, with or without the
production of gas). Typical Enterobacteriaceae colonies are red, surrounded by a yellow halo with or without gas bubbles.
8.4.1 Material required for analysis Preparation of the sample and serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer
• Dilution tubes containing 9 ml 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to check on special cases in which either the type or volume of diluent vary as a function of the sample to be examined.
Inoculation and incubation
• Petrifilm Enterobacteriaceae plates
• Laboratory incubator set to 35 ± 1°C
8.4.2 Procedure
a) Preparation of the samples and serial dilutions.
Follow the instructions described in Chapter 2, how-ever, do not use diluents containing citrate, bisul-phite or thiosulphate with Petrifilm plates as they can inhibit growth. In those cases in which these diluents are recommended in Chapter 2, replace by Butterfield’s Phosphate Buffer. After homogeni-zation, withdraw an aliquot of known volume and check the pH. If necessary, adjust the pH to the 6.5–
7.5 range with NaOH or HCl 1N, and determine and record the volume of acid or base consumed in the adjustment. Add to the sample a proportional volume of sterile NaOH or HCl solution.
b) Inoculation. Select three appropriate dilutions of the sample and inoculate 1 ml of each dilution on a Petrifilm Enterobacteriaceae plate. To that pur-pose, follow the manufacturer’s instructions, i.e.:
Place the plate on a flat, level surface, lift the top film and place the tip of the pipette perpendicular to the center of the bottom film. Dispense 1 ml on the plating surface and cover the liquid with the top film, taking care to avoid the formation of bubbles. Place the plastic spreader with the recessed side down on the center of the plate. Press gently on the center of the spreader to distribute
the sample evenly over the entire Petrifilm plate growth area. Do not drag the spreader over the plate surface, just press gently. Remove the spreader and leave the plate undisturbed for two to five min-utes, to permit the gel to form and until complete solidification.
Note b.1) To select dilutions, follow the instructions con-tained in Chapter 3, since Petrifilm plate count is based on the same principles as the traditional plate count method.
Note b.2) Some foods may interfere with visualization of colo-nies in first dilution, such as is the case with coffee, chocolate or dried herbs (very dark).
c) Incubation. Incubate the plates at 35 ± 1°C/
24 ± 2 h, with the clear side up in stacks of no more than 20 plates.
Note c.1) It may be necessary to humidify the incubator to prevent over-drying and dehydrating the plates.
Moisture loss, as indicated by weight loss, should not exceed 15% after incubation.
d) Counting the colonies and calculating the results.
Select plates with 15 to 100 colonies. Count only typical colonies, which can be of three types: red with gas bubbles and without yellow halo, red with yellow halo but without gas bubbles or red with yellow halo and with gas bubbles. Determine the number of colony-forming units CFU/g or ml by multiplying the number of typical colonies by the inverse of dilution (CFU/g or ml = No colonies/
dilution).
Note d.1) Do not count the colonies present in the foam barrier at the edge of the film, since these did not undergo the action of the selective agents. Do not enumerate artificial air bubbles.
Note d.2) The circular growth area of the Petrifilm plates is about 20 cm2 in size. On plates with more than 150 colonies, counts may be estimated by counting the number of colonies in one or more representa-tive 1-cm square areas and calculating the average per 1-cm square area. Next, multiply this number by 20 to obtain the total number of colonies per plate.
Note d.3) After the incubation time the plates may be stored frozen (at −15°C or lower temperatures) for count-ing at a later time.
8.5 References
Brenner, D.J. & Farmer III, J.J. (2005) Family I. Enterobacteriaceae.
In: Brenner, D.J., Krieg, N.R. & Staley, J.T. (eds). Bergey’s Man-ual of Systematic Bacteriology. Volume 2. 2nd edition. New York, Springer Science+Business Media Inc. pp. 587–607.
Davidson, P.M., Roth, L.A. & Gambrel-Lenarz, S.A. (2004) Colif-orm and other indicator bacteria. In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products.
17th edition. Washington, American Public Health Association.
Chapter 7, pp. 187–226.
Horwitz, W. & Latimer, G.W. (eds) (2010) Official Methods of Anal-ysis of AOAC International. 18th edition, revision 3. Gaithersburg, Maryland, AOAC International.
Kornacki, J.L. & Johnson, J.L. (2001) Enterobacteriaceae, coliforms, and Escherichia coli as quality and safety indicators. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbio-logical Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 8, pp. 69–82.
Wehr, H.M. & Frank, J.F (eds) (2004) Standard Methods for the Examination of Dairy Products. 17th edition. Washington, Ameri-can Public Health Association.
7007TS-DASILVA-Book.indb 88
7007TS-DASILVA-Book.indb 88 11/26/2012 11:15:23 AM11/26/2012 11:15:23 AM