absence of microorganisms
6 Aerobic plate count
7.6.1 Material required for analysis Membrane filter method
• Sterile distilled water or equivalent
• Malt Extract Yeast Extract 40% Glucose (MY40G)
• Membrane filters 0.80 μm pore sizeAgar
• Sterile filtration apparatus
• Vacuum pump
• Laboratory incubator set to 30±1°C Plate count method
• Butterfield’s Phosphate Buffer with 40% Glucose (w/w)
• Malt Extract Yeast Extract 40% Glucose (MY40G)
• Laboratory incubator set to 30±1°CAgar
7.6.2 Procedure
A general flowchart for the enumeration of osmophilic yeasts using the membrane filtration method or the plate count method APHA 2001 is shown in Figure 7.5.
The Compendium presents two procedures for the enumeration of osmophilic yeasts, one using the mem-brane filtration technique and the other using the direct plating technique.
a) Membrane filtration method: This method is recommended for filterable non-spoiled samples or for the rinsing water of processing lines, in which the population of osmophilic yeasts may be very low, but even so still be potentially capable of causing spoilage. It is important to consider that dilution of the sample with sterile distilled water, though still needed to make filtration possible, may subject
the osmophilic yeasts contained in the sample to osmotic shock. The resulting possible reduction in the number of CFU obtained must always be taken into account when interpreting the results.
Procedure: Weigh 25 g of the sample and add 25–50 ml of sterile, reagent grade water, homog-enizing well (in the specific case of samples of rins-ing water from processrins-ing lines there is no need for dilution). Filter through a filter membrane with a pore size of 0.80 μm, graph side facing up. Before the membrane dries, wash the walls of the cup of the filtration system with 100 ml sterile distilled water, filtering the whole volume of water to wash the membrane. Turn off the vacuum pump before the membrane starts to dry excessively. Transfer the membrane to a plate containing Malt Extract Yeast Extract 40% Glucose (MY40G) Agar, placing it onto the surface of the culture medium, graph side up. Incubate the plate at 30ºC for five to seven days.
Note a.1) It is recommended that liquid sugar samples be filtered preferably using metal filter holders, since porous filter holders are difficult to clean and sani-tize after filtering this kind of product.
b) Plate count method: This method is recommended for samples that are not suited for filtration, such as concentrated juices and fruit syrups. It is also the most indicated for samples with a high population of osmophilic yeasts, because it allows inoculation of dilutions.
Procedure: Weigh 25 g of the sample and add 225 ml of Butterfield’s Phosphate Buffer supple-mented with 40% glucose (weight/weight) (dilu-tion 10−1). In the case of analysis of sugar, prepare the diluent without the addition of glucose and add 40% of the sample itself (or an equivalent weight, in the case of liquid sugar) to the diluent, consider-ing this dilution as the undiluted sample. Prepare the subsequent dilutions, using the same diluent.
Inoculate 1 ml of the undiluted sample, 1 ml of the 10−1 dilution and 1 ml of the 10−2 dilution in sterile Petri dishes and add approximately 20 ml of Malt Extract Yeast Extract 40% Glucose (MY40G) Agar. Select higher dilutions in the case of spoiled samples, in which the expected counts are higher.
Incubate the plates at 30ºC for five to seven days and count the colonies with the aid of a magnifying glass of a colony counter. Only osmophilic yeasts will be able to develop in MY40G agar forming
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25g of sample +
225ml Butterfield’s Phosphate Buffer (PB) with 40% Glucose
MEMBRANE FILTRATION METHOD PLATE COUNT METHOD
Homogenization
1ml 1ml
9ml
PB 40% Glucose
10-2 10-3
OSMOPHILIC YEASTS CFU/g
9ml
PB 40% Glucose 10-1
1ml
1ml 1ml
Malt Extract Yeast Extrct 40% Glucose Agar (MY40G) (pour plate)
30 C/5-7 dayso MY40G
25ml of sample +
25 to 50ml of sterile reagent grade water
Membrane on MY40G 30 C/5-7 dayso
Filtration (0.8 m membrane filter)µ
MY40G
Figure 7.5 Scheme of analysis for the enumeration of osmophilic yeasts using the membrane filtration or the plate count method APHA 2001 (Baross, 2001).
very small colonies after this incubation period, since the growth rate of these microorganisms is low as a result of the water activity conditions of the medium.
7.7 References
AOAC International (2010) Rapid Methods Adopted as AOAC Official MethodsSM. [Online] Available from: http://www.aoac.
org/vmeth/oma_testkits.pdf [Accessed 26th April 2011).
Baross, J.A. (2001) Halophilic and osmophilic microorganisms.
In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 17, pp. 187–199.
Bayne, H.G. & Michener, H.D. (1979) Heat resistance of Byssochlamys ascospores. Applied and Environmental Microbiology, 37, 449–453.
Beuchat, L.R. & Cousin, M.A. (2001) Yeasts and molds. 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 20, pp. 209–215.
Beuchat, L.R. & Pitt, J.I. (2001) Detection and enumeration of heat resistant molds. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition.
Washington, American Public Health Association. Chapter 21, pp. 217–222.
Casella, M.L.A., Matasci, F & Schmidt-Lorenz, W. (1990) Influence of age, growth medium, and temperature on heat resistance of Byssochlamys nivea ascospores. Lebensmittel-Wissenschaft &
Technologie, 23, 404–411.
Cousin, M.A., Jay, J.M. & Vasavada, P.C. (2001) Psychrotrophic microrganisms. 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.
DiGiacomo, R. & Gallagher, P. (2001) Soft Drinks. 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 59, pp. 569–571.
Gray, R.J.H. & Pinkas, J.M. (2001) Gums and spices. 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 52, pp. 533–540.
Hocking, A.D., Pitt, J.I., Samson, R.A. & Thrane, U. (eds) (2006) Advances in Food Mycology. New York, Springer. pp. 49–67.
Horwitz, W. & Latimer, G.W. (eds) (2010) Official Methods of Analysis of AOAC International. 18th edition, revision 3.
Gaithersburg, Maryland, AOAC International.
King, A.D., Michener, H.D. & Ito, K.A. (1969) Control of Byssochlamys and related heat-resistant fungi in grape products.
Applied Microbiology, 18, 166–173.
Pitt, J.I. & Hocking, A.D. (eds) (2009) Fungi and Food Spoilage. 3rd edition. London, Springer.
Splittstoesser, D.F. & Splittstoesser, C.M. (1977) Ascospores of Byssochlamys fulva compared with those of heat resistant Aspergillus. Journal of Food Science, 42, 685–688.
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8
8.1 Introduction
Most of the guidelines contained in this chapter are taken from the American Public Health Association (APHA), as described in the Chapter 8 of the 4th Edi-tion of the Compendium of Methods for the Microbio-logical Examination of Foods (Kornacki and Johnson, 2001). When different from or complementary to those of the Compendium, they were completed with infor-mation and recommendations from the 17th Edition of Standard Methods for the Examination of Dairy Products (Wehr and Frank, 2004), specific to the microbiological examination of dairy products.
8.1.1 Taxonomy
The members of the Enterobacteriaceae family are Gram-negative bacteria in the shape of straight rods, non-sporeforming, facultative anaerobic and oxidase-negative (except for the Plesiomonas genus, which was recently transferred to this family). Enterobacteriaceae are chemoorganotrophs with both a respiratory and fer-mentative metabolism. The majority ferments glucose and other carbohydrates, producing acids and gases.
They are non-halophilic, produce catalase (except for Xenorhabdus and some strains of Shigella dysenteriae) and reduce nitrate to nitrite (except for Saccharobacter fermentatus and some strains of Erwinia and Yersinia) (Brenner and Farmer III, 2005).
Escherichia is the type genus of the family, which includes several other genera of importance in foods, such as Citrobacter, Edwardsiella, Enterobacter, Erwinia, Hafnia, Klebsiella, Morganella, Pantoea, Pectobacterium, Proteus, Salmonella, Serratia, Shigella and Yersinia. This family also includes the bacteria of the total coliform and thermotolerant coliform ( fecal coliforms) groups, discussed in a specific chapter. The number of genera
and species of the family has continually increased, from 12 genera and 36 species in 1974, to 20 genera and 76 species in 1984, 30 genera and 107 species in 1994 and 44 genera and 176 species in 2005, according to the 2nd Edition of Bergey’s Manual of Systematic Bacteriology (Brenner and Farmer III, 2005).
Enterobacteriaceae are widely distributed throughout nature and are found in the soil, water, plants, fruits, vegetables, meats, eggs, grains, animals, insects and in man. Several species are pathogenic to plants and ani-mals, causing significant economic loss in agriculture and the food industry. Erwinia and Pectobacterium, for example, seriously affect corn plants, potatoes, apples, sugarcane, pineapple and other vegetable products.
Yersinia ruckeri and several species of Edwardsiella cause disease in tropical fish, thereby directly affecting the fishery sector. Klebsiella e Citrobacter freundii are caus-ing agents of mastitis in cattle (Brenner and Farmer III, 2005).
Several Enterobacteriaceae are also pathogenic for man, posing a serious hazard to public health. Salmo-nella is the most important, with poultry, eggs, sheep and swine being the major vehicles for the transmis-sion of salmonellosis to man. Other pathogenic genera and species transmitted by foods are Shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis, Cronobacter (formerly Enterobacter sakazakii) and enteropathogenic strains of E. coli, including enterohaemorrhagic E. coli strains (EHEC) such as E. coli O157:H7 (Brenner and Farmer III, 2005).
Enterobacteriaceae are used as indicators of the sani-tary conditions of manufacturing processes since they are easily inactivated by sanitizing agents and capable of colonizing niches of processing plants where clean-ing and sanitation procedures were inappropriately per-formed (Kornachi and Johnson, 2001).
Although Enterobacteriaceae are mostly mesophilic in nature, psychrotrophic strains are not uncommon,
particularly within the genera Yersinia, Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia and Hafnia (Kornachi and Johnson, 2001).
8.1.2 Methods of analysis
The quantification of Enterobacteriaceae can be achieved by the standard plate count method, using Violet Red Bile Glucose (VRBG) Agar as culture medium. VRBG is a differential selective medium containing crystal vio-let and bile salts, which inhibit Gram-positive bacteria.
Fermentation of glucose results in acids, detected by the red color of the pH indicator neutral red, and by the formation of a zone of bile salt precipitation surround-ing the colonies.
For products with low counts, the Most Probable Number (MPN) technique is recommended, which is performed in two stages: The first consisting of selective enrichment in Enterobacteriaceae Enrichment Broth (EEB), and the second of isolation of typical colonies on VRBG Agar. The presence of bile salts and brilliant green inhibits most of the accompanying microbiota.
Furthermore, the high buffering capacity of the medium prevents the deleterious effect of pH reduction on the Enterobacteriaceae. This procedure can also be used as a simple presence/absence test, if quantification is not necessary or required.
Another method recommended in Chapter 8 of the Compendium (Kornacki and Johnson, 2001) and in Chapter 7 of the Standard Methods for the Examination of Dairy Products (Davidson et al., 2004) is the Petrifilm Enterobacteriaceae method of the 3M Company ( AOAC Official Method 2003.1), which follows the same prin-ciples as the VRBG plate count.
8.2 Plate count method APHA 2001 for Enterobacteriaceae in foods Method of the American Public Health Association (APHA), as described in Chapter 8 of the 4th Edi-tion of the Compendium of Methods for the Microbio-logical Examination of Foods (Kornacki and Johnson, 2001).
Before starting activities, carefully read the guide-lines in Chapter 3, which deals with all details and care required for performing plate counts of micro-organisms, 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.
8.2.1 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.
Enumeration (pour plate)
• Sterile, empty 20 × 100-mm Petri dishes
• Culture medium: Violet Red Bile Glucose (VRBG)
• Laboratory incubator set to 35 ± 1°CAgar
8.2.2 Procedure
A general flowchart for the enumeration of Enterobac-teriaceae in foods using the plate count method APHA 2001 is shown in Figure 8.1.
a) Preparation of the samples and serial dilutions.
Follow the procedures described in Chapter 2.
b) Inoculation. Select three appropriate dilutions of the sample and inoculate in Violet Red Bile Glu-cose (VRBG) Agar. Use the pour plate technique and, after complete solidification of the medium, cover the surface with a 5–8 ml thick layer of the same medium.
c) Incubation. Incubate the plates in an inverted position at 35 ± 1°C/18–24 h.
d) Counting of the colonies and calculating the results. Select plates with 15–150 colonies and count only the typical Enterobacteriaceae colonies on the VRBG medium: red-purple, 0.5 mm or greater in diameter, surrounded by a reddish halo characteristic of the precipitation of the bile salts.
On crowded plates the colonies may remain small and fail to reach 0.5 mm. Determine the number of CFU/g or ml by multiplying the number of typical colonies by the inverse of the dilution.
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8.3 Most probable number (MPN) method APHA 2001 for
Enterobacteriaceae in foods
Method of the American Public Health Association (APHA), as described in Chapter 8 of the 4th Edition of the Compendium of Methods for the Microbiological Examination of Foods (Kornacki and Johnson, 2001).
The enumeration of Enterobacteriaceae by the MPN method is indicated for samples with low counts, lower than the detection limit of the plate count method.
Before starting activities, read the guidelines in Chap-ter 4, which deals with all the details for MPN counts of microorganisms, from dilution selection to calculat-ing the results. The procedure described below does not present these details, as they are supposed to be known to the analyst.
8.3.1 Material required for analysis