absence of microorganisms
6 Aerobic plate count
6.3.1 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.
Total plate count
• Petrifilm™ Aerobic Count plates (3M Microbiology Products)
• Laboratory incubator set to 35 ± 1°C
• Laboratory incubator set to 32 ± 1°C (for milk and dairy products)
6.3.2 Procedure
a) Preparation of the samples and serial dilutions.
Follow the procedures described in Chapter 2.
b) Inoculation. Place the Petrifilm plates on a flat surface. Select three adequate dilutions of the sample for inoculation. Inoculate 1 ml of each dilution on separate Petrifilm™AC Total Count Plates (3M Company), lifting the top film and dispensing the inoculum at the center of the cir-cular area of the bottom film. Lower the top film covering the inoculum, place the spreader (which comes with the Petrifilm kits) with the recessed side down on the center of the plate and press gently to spread the inoculum evenly over the plate growth area.
c) Incubation. Wait one minute for the gel to solid-ify and incubate at 35 ± 1°C/48 ± 2h, in stacks of no more than 20 films, without inverting them.
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In the case of milk and dairy products, incubate at 32 ± 1°C/48 ± 3h.
d) Counting the colonies and calculating the results. The circular growth area of the Petri-film plates are about 20 cm2 in size and counting should be performed on the Petrifilms exhibiting 30 to 300 colonies (25 to 250 for dairy products).
Count all the red colonies, irrespective of size or color intensity. Calculate the number of CFU per gram or milliliter of sample by multiplying the number of colonies by the inverse of the dilution.
To determine the number of colonies on Petrifilm plates with more than 250 colonies, count the number of colonies in one or more representative 1 cm2 square area(s), calculate the average per cm2 and multiply by 20.
Detection limit: 1 CFU/ml for liquid samples or 10 CFU/g for solid samples.
6.4 Plate count method APHA 2001 for aerobic psychrotrophic bacteria in foods
Method of the American Public Health Association (APHA), as described in Chapter 13 of the 4th Edition of the Compendium of Methods for the Microbiological Examination of Foods (Cousin et al., 2001) and Chapter 8 of the 17th Edition of the Standard Methods for the Examination of Dairy Products (Frank and Yousef, 2004).
The Compendium recommends that the samples intended to be analyzed for the enumeration of aerobic psychrotrophics be analyzed within a 6h interval, counting from the moment the sample was collected.
Cold storage does not inhibit their multiplication and the generation time of several of these microorganism falls within this time interval. Freezing is not recom-mended for these samples, since it can cause injuries to or the death of several microorganisms. If freezing is considered indispensable, it must be taken into account when evaluating the results that part of the microbiota may have been lost.
Before starting activities, carefully read the guide-lines in Chapter 3, which deal with all details and care required for performing plate counts of microorgan-isms, from dilution selection to calculating the results.
The procedure described below does not present
these details, as they are supposed to be known to the analyst.
6.4.1 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.
Spread plate count
• Culture media: Plate Count Agar (PCA) which can be substituted for Trypticase Soy Agar (TSA) or Petrifilm™ Aerobic Count plates (3M Microbiology Products)
• Laboratory incubator set to 7 ± 1°C
• Laboratory incubator set to 17 ± 1°C (optional)
6.4.2 Procedure
A general flowchart for enumeration of aerobic psychro-trophic bacteria in foods using the plate count method APHA 2001 is shown in Figure 6.2.
Follow the same procedure as described for total aer-obic mesophilic counts, changing only the incubation condition(s).
Chapter 13 of the Compendium (Cousin et al., 2001) recommends plating on Petrifilm or spread plate, using Plate Count Agar (PCA) or Trypticase Soy Agar (TSA) as culture media. Pour plate is not recommended because these bacteria are heat-sensitive and may be affected by the hot or warm culture medium. Incubation can be done at 7 ± 1°C for 10 days or 17 ± 1°C for 16h, followed by 3 more days at 7 ± 1°C.
The Standard Methods for the Examination of Dairy Products (Frank and Yousef, 2004) recommends, for milk and dairy products, pour plate using PCA cooled to 45 ± 1°C, before pouring the culture medium over the inoculum. Incubate at 7 ± 1°C for 10 days.
6.5 References
AOAC International (2010) Rapid Methods Adopted as AOAC Offi-cial MethodsSM. [Online] Available from: http://www.aoac.org/
vmeth/oma_testkits.pdf [Accessed 26th April 2011).
Cousin, M.A., Jay, J.M. & Vasavada, P.C. (2001) Psychrotrophic micro-rganisms. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 13, pp. 159–166.
Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds) (2005) Standard Methods for the Examination of Water & Waste-water. 21st edition. Washington, American Public Health Asso-ciation (APHA), American Water Works AssoAsso-ciation (AWWA)
& Water Environment Federation (WEF).
Frank, J.F. & Yousef, A.E. (2004) Tests for groups of microrganisms.
In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products. 17th edition. Washington, Ameri-can Public Health Association. Chapter 8, pp. 227–248.
Hatcher, W.S., Parish, M.E., Weihe, J.L., Splittstoesser, D.F. &
Woodward, B.B. (2001) Fruit beverages. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Exami-nation of Foods. 4th edition. Washington, American Public Health Association. Chapter 58, pp. 565–568.
Horwitz, W. & Latimer, G.W. (eds) (2010) Official Methods of Anal-ysis of AOAC International. 18th edition, revision 3. Gaithersburg, Maryland, AOAC International.
Hunt, M.E. & Rice, E.W. (2005) Microbiological examination. In:
Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds).
Standard Methods for the Examination of Water & Wastewater.
21st edition. Washington, American Public Health Association (APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF). Part 9000, Section 9215, pp. 9.34–9.36.
Laird, D.T., Gambrel-Lenarz, S.A., Scher, F.M., Graham, T.E. &
Reddy, R. (2004) Microbiological count methods. In: Wehr, H.M. & Frank, J.F (eds). Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association. Chapter 6, pp. 153–186.
Lewis, D., Spomer, D., Smith, M. & Clark, W. (2004) Milk and milk products standards. In: Wehr, H.M. & Frank, J.F (eds). Stand-ard Methods for the Examination of Dairy Products. 17th edition.
Washington, American Public Health Association. Chapter 16, pp. 537–550.
Morton, R.D. (2001) Aerobic plate count. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Exami-nation of Foods. 4th edition. Washington, American Public Health Association. Chapter 6, pp. 63–67.
Wehr, H.M. & Frank, J.F (eds) (2004) Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association.
Homogenization 1ml 1ml
25g of sample +
225ml of Peptone Water (PW)
9ml PW
10-2 10-3
7±1 C/10 days or
17±1 C/16h followed by 7±1 C/3 days
o
o o
AEROBIC PSYCHROTROPHIC BACTERIA CFU/g
9ml PW 10-1
0.1ml
0.1ml 0.1ml
Plate Count Agar (PCA) or Tripticase Soy Agar (TSA)
(spread plate)
PCA or TSA PCA or TSA
Figure 6.2 Scheme of analysis for enumeration of aerobic psychrotrophic bacteria in foods using the plate count method APHA 2001 (Cousin et al., 2001).
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