Microbiological examination methods of food and water : a laboratory manual

Cover
Half Title
Title
Copyright
Table of contents
About the authors
Preface
List of tables
List of figures
1 Sampling, transport and storage of samples for analysis
	Revision history
	1.1 Introduction
		1.1.1 Lot
		1.1.2 Lot sample and sample unit
		1.1.3 Lot sampling plans
		1.1.4 Analytical unit
	1.2 Collecting samples for analysis
		1.2.1 Selection and preparation of containers for the sampling of foods contained in non-individual packages
		1.2.2 Procedures for the sampling of foods contained in non-individual packages
		1.2.3 Sampling of foods involved in foodborne diseases
		1.2.4 Sampling of water
	1.3 Transportation and storage of samples until analysis
		1.3.1 Foods with low water activity
		1.3.2 Frozen foods
		1.3.3 Refrigerated foods
		1.3.4 Commercially sterile foods in sealed packages
		1.3.5 Water samples
	1.4 References
2 Preparation of samples for analysis
	Revision history
	2.1 Introduction
	2.2 Homogenization of samples and withdrawal of the analytical unit
		2.2.1 Procedure for homogenization and withdrawal of analytical units from liquid products
		2.2.2 Procedure for homogenization and withdrawal of analytical units from solid or concentrated liquid products
		2.2.3 Procedure for withdrawing the analytical unit using the surface swabbing technique
			2.2.3.1 Swab sampling
			2.2.3.2 Sponge swab sampling
		2.2.4 Procedure for withdrawing the analytical unit using the surface washing technique
			2.2.4.1 Procedure for washing poultry carcasses
			2.2.4.2 Procedure for washing other foods
			2.2.4.3 Procedure for washing packages
		2.2.5 Keeping of counter-samples
	2.3 Preparation of the first dilution of the analytical unit
		2.3.1 Diluents for presence/absence tests
		2.3.2 Diluents for tests requiring differentiated handling of the sample
		2.3.3 Diluents for general quantification tests
		2.3.4 How to prepare an initial 1:10 (10−1) dilution
		2.3.5 How to prepare an initial dilution different from 1:10
		2.3.6 Procedure for the preparation of the first dilution of liquid samples
		2.3.7 Procedure for the preparation of the first dilution of solid or concentrated liquid samples
		2.3.8 Procedure for the preparation of the first dilution of samples obtained by surface swabbing or surface washing
	2.4 Serial decimal dilution of the sample
	2.5 References
	Annex 2.1 – Procedures for homogenizing the content and withdrawal of the analytical unit of different types of foods
		a) Powdered products
		b) Pasty or ground products
		c) Yogurts with fruit pieces
		d) Cheeses
		e) Very hard food products
		f) Pieces of solid foods
		g) Eggs in the shell
		h) Meat cuts for analysis of non-surface contamination
		i) Bivalves
		j) Gastropods
		k) Whole and sliced cephalopods
		l) Whole crustaceans such as crabs
		m) Sea urchins
		n) Whole fresh fish
	Annex 2.2 – Special cases in which there are variations in the analytical unit and/or dilution and/or diluents recommended for the preparation of the first dilution of samples of different types of foods
		a) Liquids with low levels of contamination
		b) Fatty foods
		c) Lump-forming powders
		d) Thickeners or products containing natural antimicrobial compounds
		e) Gelatin
		f) Acid products
		g) Fine flours or meals, cereal grains, animal feed
		h) Cocoa and chocolate
		i) Egg white
		j) Fermented products containing live microorganisms intended for the quantification of the contaminating microflora (except probiotics)
		k) Powdered dairy products (dried milk, dried sweet whey, dried acid whey, dried buttermilk, lactose)
		l) Butter
		m) Frozen dairy products and ice cream
		n) Cheeses
		o) Fermented dairy products
		p) Casein and caseinates
		q) Rennet casein difficult to dissolve
		r) Powdered milk-based infant foods
		s) Custard, desserts and sweet cream (pH > 5)
		t) Mollusks (bivalves and gastropods) and sea urchins
		u) Sea cucumbers ( Holothuroidea ) and tunicates
3 Basic plate count techniques for enumeration of microorganisms
	Revision history
	3.1 Introduction
	3.2 Pour plate technique
		3.2.1 Material required for the analyses
		3.2.2 Procedure
	3.3 Spread plate technique
		3.3.1 Material required for the analyses
		3.3.2 Procedure
	3.4 Drop plate technique
		3.4.1 Material required for the analyses
		3.4.2 Procedure
	3.5 Membrane filtration
		3.5.1 Material required for the analyses
		3.5.2 Procedure
	3.6 Counting colonies and calculating results according to APHA
		3.6.1 Pour plate calculations
			3.6.1.1 Rules for calculating the pour plate results in the standard situation
			3.6.1.2 Rules for calculating the pour plate results in unusual situations
			3.6.1.3 Calculating the pour plates results for samples prepared by the surface swabbing technique (swabs or sponges)
			3.6.1.4 Calculating the pour plate results of samples prepared by the surface washing technique
		3.6.2 Spread plate calculations
		3.6.3 Drop plate calculations
		3.6.4 Membrane filtration calculations
	3.7 (revised) Counting colonies and calculating results according to ISO 7218:2007/ Amd .1:2013
		3.7.1 (new) General requirements for the calculation of results
			a) Number of Petri dishes per dilution
			b) Maximum and minimum acceptable number of colonies on counting plates
			c) Decimal dilution of the number of colonies
			d) Acceptable variation between counts of the pair of plates of a duplicate
			e) Presentation of the result
		3.7.2 General rules for the calculation of results
		3.7.3 Rules for calculation in unusual situations
	3.8 References
	Annex 3.1 – (new) Limits of agreement for sums of colony counts of two parallel Petri dishes or colony counts from one Petri dish per dilution step over two 10-fold dilution steps (with a probability of 99% per comparison) (ISO 14461-2:2005)
	Annex 3.2 – (new) Limits of agreement for colony counts of two parallel Petri dishes (with a probability of 99% per comparison) (ISO 14461-2:2005)
4 Basic techniques for microbial enumeration by the most probable number (MPN) method
	Revision history
	4.1 Introduction
	4.2 Multiple dilution test
		4.2.1 Material required for the analyses
		4.2.2 Procedure
	4.3 Single dilution test
		4.3.1 Material required for the analyses
		4.3.2 Procedure
	4.4 Calculation of the results
		4.4.1 Calculating the results of the multiple dilution test
			4.4.1.1 Calculation using the MPN tables (for decimal dilutions)
			4.4.1.2 Calculating using the Thomas formula (for non-decimal dilutions)
			4.4.1.3 Calculating the results of the samples prepared by the surface swabbing or surface washing techniques
		4.4.2 Calculating the results of the single dilution test
			4.4.2.1 Rules for calculations performed using Table MPN-3
			4.4.2.2 Calculation for samples prepared by the surface swabbing or surface washing techniques
	4.5 References
	Annex 4.1 – MPN tables
5 Basic techniques for the detection of the presence/absence of microorganisms
	Revision history
	5.1 Introduction
		5.1.1 Enrichment
			5.1.1.1 Pre-enrichment
			5.1.1.2 Selective enrichment
		5.1.2 Isolation in solid media (selective differential plating)
		5.1.3 Confirmation
			5.1.3.1 Catalase test
			5.1.3.2 Citrate test
			5.1.3.3 Amino acid decarboxylation tests
			5.1.3.4 Phenylalanine deaminase test
			5.1.3.5 Carbohydrate fermentation tests
			5.1.3.6 Indole test
			5.1.3.7 Malonate test
			5.1.3.8 Oxidation/fermentation (O/F) test
			5.1.3.9 Oxidase test
			5.1.3.10 Nitrate reduction test
			5.1.3.11 Urease test
			5.1.3.12 Methyl red (MR) test
			5.1.3.13 Voges-Proskauer (VP) test
	5.2 Material required for the analyses
	5.3 Procedure
		5.3.1 Pre-enrichment
		5.3.2 Selective enrichment
		5.3.3 Differential plating
			5.3.3.1 Streak plating technique for obtaining pure cultures
		5.3.4 Selection of colonies and subculturing of cultures for confirmation
			5.3.4.1 Technique for the subculturing of pure cultures starting from colonies isolated from plates
		5.3.5 Confirmation tests
			5.3.5.1 Gram staining (Hucker’s method)
			5.3.5.2 (new) KOH test for confirmation of doubtful Gram staining (Gregersen, 1978)
			5.3.5.3 Spore staining (Schaeffer-Fulton’s method)
			5.3.5.4 Spore staining (Ashby’s method)
			5.3.5.5 Wet mounts for direct (fresh) microscopic observation
	5.4 References
6 Aerobic plate count
	Revision history
	6.1 Introduction
		6.1.1 The importance and significance of the total aerobic mesophilic count
		6.1.2 Definition of psychrotrophs
		6.1.3 Comments on methods of analysis
	6.2 (revised) Plate count method APHA 8:2015 for aerobic mesophilic bacteria in foods and water
		6.2.1 Material required for analysis
		6.2.2 Procedure
			6.2.2.1 Pour plate technique
			6.2.2.2 Spread plate technique
			6.2.2.3 Membrane filtration technique
	6.3 (revised) Petrifilm™ AOAC official methods for aerobic mesophilic bacteria in foods
		6.3.1 Material required for analysis
		6.3.2 Procedure
	6.4 (revised) Plate count method APHA 13.61:2015 for aerobic psychrotrophic bacteria in foods
		6.4.1 Material required for analysis
		6.4.2 Procedure
	6.5 (new) Plate count methods ISO 4833-1:2013 and ISO 4833-2:2013/Corr.1:2014 for aerobic mesophilic bacteria in foods
		6.5.1 Material required for analysis
		6.5.2 Procedure
	6.6 (new) Plate count method BAM/FDA:2001 for aerobic mesophilic bacteria in foods
		6.6.1 Material required for analysis
		6.6.2 Procedure
	6.7 References
7 Yeasts and molds
	Revision history
	7.1 Introduction
		7.1.1 Yeasts and molds in foods
		7.1.2 Comments on methods of analysis for total yeast and mold counts
		7.1.3 Psychrotrophic fungi
		7.1.4 Heat-resistant molds
		7.1.5 Preservative-resistant yeasts (PRY)
			7.1.5.1 Zigosaccharomyces bailii
			7.1.5.2 Zygosaccharomyces bisporus
			7.1.5.3 Schizosaccharomyces pombe
			7.1.5.4 Candida krusei
			7.1.5.5 Pichia membranaefaciens
		7.1.6 Osmophilic yeasts
			7.1.6.1 Zygosaccharomyces rouxii
		7.1.7 Direct plating method ICFM for particulate foods
	7.2.A Plate count method APHA 21:2015 for yeasts and molds in foods
		7.2.A.1 Material required for analysis
		7.2.A.2 Procedure
	7.2.B (new) Plate count methods ISO 21527-1:2008 and ISO 21527-2:2008 for yeasts and molds in foods
		7.2.B.1 Material required for analysis
		7.2.B.2 Procedure
	7.2.C (new) Plate count method BAM/FDA:2001 for yeasts and molds in foods
		7.2.C.1 Material required for analysis
		7.2.C.2 Procedure
	7.3 (revised) Plate count method APHA 13:2015 for psychrotrophic fungi in foods
		7.3.1 Material required for analysis
		7.3.2 Procedure
	7.4 (revised) Plate count method APHA 22.4:2015 for heat-resistant molds in foods
		7.4.1 Material required for analysis
		7.4.2 Procedure
	7.5 Pitt and Hocking (2009) methods for preservative-resistant yeasts in foods
		7.5.1 Material required for analysis
		7.5.2 Procedure
			7.5.2.1 Presence/absence method
			7.5.2.2 Direct plate count method
	7.6 (revised) Membrane filtration or plate count methods APHA 17.3:2015 for osmophilicyeasts in foods
		7.6.1 Material required for analysis
		7.6.2 Procedure
			7.6.2.1 Membrane filtration method
			7.6.2.2 Plate count method
	7.7 (new) Direct plating method Hocking et al. (2006) for percentage of fungal infection in particulate foods
		7.7.1 Material required for analysis
		7.7.2 Procedure
	7.8 References
8 Enterobacteriaceae
	Revision history
	8.1 Introduction
		8.1.1 Taxonomy
		8.1.2 Comments on methods of analysis
	8.2 (revised) Plate count method APHA 9.62:2015 for Enterobacteriaceae in foods
		8.2.1 Material required for analysis
		8.2.2 Procedure
	8.3 (revised) Presence / absence (P/A) or most probable number ( MPN) method APHA 9.61:2015 for Enterobacteriaceae in foods
		8.3.1 Material required for analysis
		8.3.2 Procedure
	8.4 (revised) AOAC official method 2003.1 (Petrifilm™) for Enterobacteriaceae in selected foods
		8.4.1 Material required for analysis
		8.4.2 Procedure
	8.5 (new) Plate count method ISO 21528-2:2017 for Enterobacteriaceae in foods
		8.5.1 Material required for analysis
		8.5.2 Procedure
	8.6 (new) Presence/absence (P/A) or most probable number (MPN) method ISO 21528-1:2017 for Enterobacteriaceae in foods
		8.6.1 Material required for analysis
		8.6.2 Procedure
	8.7 References
9 Total and thermotolerant coliforms and Escherichia coli
	Revision history
	9.1 Introduction
		9.1.1 Definition of total coliforms
		9.1.2 Definition of thermotolerant coliforms
		9.1.3 E. coli
		9.1.4 Use as indicators
		9.1.5 Comments on methods of analysis
	9.2 (revised) Most probable number (MPN) method APHA 9:2015 for total/thermotolerant coliforms and E. coli in foods
		9.2.1 Material required for analysis
		9.2.2 Procedure
	9.3 Most probable number (MPN) methods ISO 4831:2006 and ISO 7251:2005 for total coliforms and presumptive E. coli in foods
		9.3.1 Material required for analysis
		9.3.2 Procedure
	9.4 (revised) Most probable number (MPN) method APHA/AWWA/WEF:2012 for total and thermotolerant coliforms and E. coli in water
		9.4.1 Material required for analysis
		9.4.2 Procedure
	9.5 (new) Most probable number (MPN) method BAM/FDA:2017 for total/thermotolerant coliforms and E. coli in foods
		9.5.1 Material required for analysis
		9.5.2 Procedure
	9.6 (revised) Plate count method APHA:2015 for total coliforms in foods
		9.6.1 Material required for analysis
		9.6.2 Procedure
	9.7 (new) Membrane filtration method ISO 9308-1:2014/Amd.1:2016 for total coliforms and E. coli in water
		9.7.1 Material required for analysis
		9.7.2 Procedure
	9.8 References
10 Staphylococcus aureus
	Revision history
	10.1 Introduction
		10.1.1 Taxonomy
			10.1.1.1 The genus Staphylococcus
			10.1.1.2 The coagulase-positive staphylococci
			10.1.1.3 Staphylococcus aureus
		10.1.2 Epidemiology
			10.1.2.1 Staphylococcus aureus enterotoxins
			10.1.2.2 Staphylococcal food poisoning
		10.1.3 Comments on methods of analysis
	10.2 (revised) Plate count method APHA 39.63:2015 for coagulase-positive staphylococci and Staphylococcus aureus in foods
		10.2.1 Material required for analysis
		10.2.2 Procedure
	10.3 (revised) Most probable number (MPN) method APHA39.62:2015 for coagulase-positive staphylococci and Staphylococcus aureus in foods
		10.3.1 Material required for analysis
		10.3.2 Procedure
	10.4 (revised) Presence/absence method APHA 39.61:2015 for coagulase-positive staphylococci and Staphylococcus aureus in foods
		10.4.1 Material required for analysis
		10.4.2 Procedure
	10.5 References
11 Bacillus cereus
	Revision history
	11.1 Introduction
		11.1.1 Taxonomy
			11.1.1.1 Bacillus cereus group
				Bacillus anthracis
				Bacillus thuringiensis
				Bacillus mycoides
				Bacillus pseudomycoides
				Bacillus weihenstephanensis
				Bacillus cytotoxicus
				New species
			11.1.1.2 Bacillus cereus
		11.1.2 Epidemiology
		11.1.3 Comments on methods of analysis
	11.2 (revised) Plate count method APHA 31.61:2015 for Bacillus cereus in foods
		11.2.1 Material required for analysis
		11.2.2 Procedure
	11.3 (revised) Most probable number (MPN) method APHA 31.62:2015 for Bacillus cereus in foods
		11.3.1 Material required for analysis
		11.3.2 Procedure
	11.4 References
12 Clostridium perfringens
	Revision history
	12.1 Introduction
		12.1.1 Taxonomy
		12.1.2 Epidemiology
			12.1.2.1 Clostridium perfingens type A food poisoning
			12.1.2.2 Clostridium perfringens type C necrotic enteritis
		12.1.3 Comments on methods of analysis
	12.2 (revised) Plate count method APHA 33.72:2015 for Clostridium perfringens in foods
		12.2.1 Material required for analysis
		12.2.2 Procedure
	12.3 (revised) Presence/absence method APHA 33.71:2015 for Clostridium perfringens in foods
		12.3.1 Material required for analysis
		12.3.2 Procedure
	12.4 (new) Plate count method BAM/FDA:2001 for Clostridium perfringens in foods
		12.4.1 Material required for analysis
		12.4.2 Procedure
	12.5 (new) Plate count method ISO 7937:2004 for Clostridium perfringens in foods
		12.5.1 Material required for analysis
		12.5.2 Procedure
	12.6 (new) Membrane filtration method ISO 14189:2013 for Clostridium perfringens in water
		12.6.1 Material required for analysis
		12.6.2 Procedure
	12.7 References
13 Enterococci
	Revision history
	13.1 Introduction
		13.1.1 Taxonomy
			13.1.1.1 Enterococci
				Description of the genus Enterococcus
			13.1.1.2 Fecal streptococci
				Description of the genus Streptococcus
			13.1.1.3 Differentiation of enterococci from group bovis fecal streptococci
		13.1.2 Comments on methods of analysis
	13.2 (revised) Plate count method APHA 10.5:2015 for enterococci and fecal streptococci in foods
		13.2.1 Material required for analysis
		13.2.2 Procedure
	13.3 (revised) Most probable number (MPN) method APHA 10.2:2015 for enterococci and fecal streptococci in foods
		13.3.1 Material required for analysis
		13.3.2 Procedure
	13.4 (revised) Membrane filtration method APHA/AWWA/WEF 9230C.3c:2012 for enterococci and fecal streptococci in water
		13.4.1 Material required for analysis
		13.4.2 Procedure
	13.5 Membrane filtration method ISO 7899-2:2000 for intestinal enterococci in water
		13.5.1 Material required for analysis
		13.5.2 Procedure
	13.6 References
14 Lactic acid bacteria
	Revision history
	14.1 Introduction
		14.1.1 Carnobacterium
		14.1.2 Enterococcus
		14.1.3 Fructobacillus
		14.1.4 Lactobacillus
		14.1.5 Lactococcus
		14.1.6 Leuconostoc
		14.1.7 Oenococcus
		14.1.8 Pediococcus
		14.1.9 Streptococcus
		14.1.10 Tetragenococcus
		14.1.11 Weissella
		14.1.12 Comments on methods of analysis
	14.2 (revised) Plate count method APHA 19.52:2015 for lactic acid bacteria in foods
		14.2.1 Material required for analysis
		14.2.2 Procedure
	14.3 (revised) MPN methods APHA 19.526:2015 and APHA 19.524:2015 for lactic acid bacteria in foods
		14.3.1 Material required for analysis
		14.3.2 Procedure using MRS broth
		14.3.3 Procedure using Rogosa SL broth
	14.4 (new) Plate count method ISO 15214:1998 for lactic acid bacteria in foods
		14.4.1 Material required for analysis
		14.4.2 Procedure
	14.5 References
15 Campylobacter
	Revision history
	15.1 Introduction
		15.1.1 Taxonomy
			15.1.1.1 Campylobacter
			15.1.1.2 Thermotolerant Campylobacter
		15.1.2 Epidemiology
	15.2 (revised) Presence/absence method ISO 10272-1:2017 for thermotolerant Campylobacter in foods
		15.2.1 Material required for analysis
		15.2.2 Procedure
	15.3 (new) Plate count method ISO 10272-2:2017 for thermotolerant Campylobacter in foods
		15.3.1 Material required for analysis
		15.3.2 Procedure
	15.4 References
16 Cronobacter
	Revision history
	16.1 Introduction
		16.1.1 Taxonomy
			Cronobacter Iversen et al. (2008)
		16.1.2 Epidemiology
		16.1.3 Codex Alimentarius microbiological criteria for Cronobacter spp. in powdered infant formulae
		16.1.4 Comments on methods of analysis
	16.2 (revised) Presence/absence method ISO 22964:2017 for Cronobacter in foods
		16.2.1 Material required for analysis
		16.2.2 Procedure
	16.3 (new) Presence/absence method BAM/FDA:2012 for Cronobacter in dehydrated powdered infant formula
		16.3.1 Material required for analysis
		16.3.2 Procedure
	16.4 References
17 Pseudomonas
	Revision history
	17.1 Introduction
		17.1.1 Taxonomy
			17.1.1.1 Pseudomonas
				Pseudomonas in treated water intended for human consumption
				Pseudomonas in mineral water and natural water
				Pseudomonas in foods
			17.1.1.2 Shewanella
				Shewanella putrefaciens (synonym Pseudomonas putrefaciens)
			17.1.1.3 Janthinobacterium
				Janthinobacterium lividum (synonym Pseudomonas mephitica)
			17.1.1.4 Stenotrophomonas
				Stenotrophomonas maltophilia (synonym Pseudomonas maltophilia)
	17.2 (revised) MPN method APHA/AWWA/WEF 9213:2012 for Pseudomonas aeruginosa in water
		17.2.1 Material required for analysis
		17.2.2 Procedure
	17.3 Membrane filtration method ISO 16266:2006 for Pseudomonas aeruginosa in water
		17.3.1 Material required for analysis
		17.3.2 Procedure
	17.4 Plate count method ISO 13720:2010 for presumptive Pseudomonas spp. in meat and meat products
		17.4.1 Material required for analysis
		17.4.2 Procedure
	17.5 Plate count method ISO 11059:2009 for Pseudomonas spp. in milk and milk products
		17.5.1 Material required for analysis
		17.5.2 Procedure
	17.6 References
18 Listeria monocytogenes
	Revision history
	18.1 Introduction
		18.1.1 Taxonomy
		18.1.2 Epidemiology
		18.1.3 Comments on methods of analysis
	18.2 (revised) Presence/absence or MPN method BAM/FDA:2017 for Listeria monocytogenes in foods
		18.2.1 Material required for analysis
		18.2.2 Procedure
			18.2.2.1 Presence/absence test and MPN count
			18.2.2.2 Direct plate count
	18.3 (revised) Presence/absence method USDA/MLG:2017 for Listeria monocytogenes in foods
		18.3.1 Material required for analysis
		18.3.2 Procedure
	18.4 (revised) Plate count method ISO 11290-2:2017 for Listeria spp. and Listeria monocytogenes in foods
		18.4.1 Material required for analysis
		18.4.2 Procedure
	18.5 (revised) Presence/absence method ISO 11290-1:2017 for Listeria spp. and Listeria monocytogenes in foods
		18.5.1 Material required for analysis
		18.5.2 Procedure
	18.6 References
19 Salmonella
	Revision history
	19.1 Introduction
		19.1.1 Taxonomic classification of Salmonella
		19.1.2 Serological classification of Salmonella
			Somatic (“O”) antigens
			Capsular (surface or envelope) antigens
			Flagellar (“H”) antigens
			The White-Kauffmann-Le Minor system
			Salmonella serovar nomenclature
			Serovars most commonly found
		19.1.3 Biochemical characteristics of Salmonella
		19.1.4 Epidemiology
		19.1.5 Comments on traditional methods used for the examination of Salmonella
		19.1.6 Comments on alternative methods for the analysis of Salmonella
		19.1.7 Composite samples for analysis
	19.2 (revised) Presence/absence method ISO 6579-1:2017 for Salmonella in foods
		19.2.1 Material required for analysis
		19.2.2 Procedure
	19.3 (revised) Presence/absence method BAM/FDA:2018 for Salmonella in foods
		19.3.1 Material required for analysis
		19.3.2 Procedure
	19.4 (revised) Presence/absence method MLG/USDA:2017 for Salmonella in foods
		19.4.1 Material required for analysis
		19.4.2 Procedure
	19.5 References
20 Vibrio cholerae and Vibrio parahaemolyticus
	Revision history
	20.1 Introduction
		20.1.1 Taxonomy
		20.1.2 Epidemiology
			20.1.2.1 V. cholerae
			20.1.2.2 V. parahaemolyticus
			20.1.2.3 V. vulnificus
		20.1.3 Comments on methods of analysis
	20.2.A Presence/absence method BAM/FDA:2004 for Vibrio cholerae in foods
		20.2.A.1 Material required for analysis
		20.2.A.2 Procedure
	20.2.B (revised) Presence/absence and MPN methods APHA 40.61:2015 for Vibrio cholerae in foods and water
		20.2.B.1 Material required for analysis
		20.2.B.2 Procedure
	20.3.A MPN method BAM/FDA:2004 for Vibrio parahaemolyticus in foods
		20.3.A.1 Material required for analysis
		20.3.A.2 Procedure
	20.3.B (revised) Presence/absence or MPN method APHA 40.62/40.63:2015 for Vibrio parahaemolyticus and Vibrio vulnificus in foods
		20.3.B.1 Material required for analysis
		20.3.B.2 Procedure
	20.4 (revised) Presence/absence method ISO 21872-1:2017 for potentially enteropathogenic Vibrio cholerae and Vibrio parahaemolyticus in foods
		20.4.1 Material required for analysis
		20.4.2 Procedure
	20.5 References
21 Yersinia enterocolitica
	Revision history
	21.1 Introduction
		21.1.1 Taxonomy
		21.1.2 Epidemiology
	21.2 Presence/absence method ISO 10273:2017 for pathogenic Yersinia enterocolitica in foods
		21.2.1 Material required for analysis
		21.2.2 Procedure
	21.3 References
22 Bacterial spore count
	Revision history
	22.1 Introduction
		22.1.1 The bacterial spore
			22.1.1.1 Sequence of spore formation
			22.1.1.2 Spore ultrastructure
			22.1.1.3 Mechanisms of spore resistance
		22.1.2 Taxonomy of spore-forming bacteria important in foods
			22.1.2.1 Aeribacillus
			22.1.2.2 Alicyclobacillus
				Alicyclobacillus acidoterrestris
				Alicyclobacillus acidocaldarius
				Alicyclobacillus acidiphilus
				Alicyclobacillus contaminans
				Alicyclobacillus dauci
				Alicyclobacillus fastidiosus
				Alicyclobacillus herbarius
				Alicyclobacillus pomorum
				Alicyclobacillus sacchari
			22.1.2.3 Aneurinibacillus
				Aneurinibacillus thermoaerophilus
			22.1.2.4 Anoxybacillus
				Anoxybacillus contaminans
				Anoxybacillus flavithermus
				Anoxybacillus tepidamans
			22.1.2.5 Bacillus
				Bacillus coagulans
				Bacillus smithii
				Bacillus sporothermodurans
			22.1.2.6 Brevibacillus
			22.1.2.7 Clostridium
				Clostridium botulinum
				Proteolytic clostridia
				Saccharolytic clostridia
				Psychrophilic and psychrotrophic clostridia that cause the spoilage of refrigerated vacuum-packed meats
			22.1.2.8 Cohnella
			22.1.2.9 Desulfotomaculum
				Desulfotomaculum nigrificans
			22.1.2.10 Fictibacillus
				Fictibacillus gelatini
				Fictibacillus nanhaiensis
			22.1.2.11 Geobacillus
				Geobacillus stearothermophilus
			22.1.2.12 Hathewaya
			22.1.2.13 Jeotgalibacillus
				Jeotgalibacillus alimentarius
			22.1.2.14 Lentibacillus
			22.1.2.15 Lysinibacillus
			22.1.2.16 Moorella
			22.1.2.17 Oceanobacillus
			22.1.2.18 Paenibacillus
			22.1.2.19 Paraclostridium
			22.1.2.20 Sporolactobacillus
			22.1.2.21 Thermoanaerobacter
			22.1.2.22 Thermoanaerobacterium
				T. thermosaccharolyticum
			22.1.2.23 Virgibacillus
	22.2 (revised) Methods APHA 25:2015 and 26:2015 for spores of total and flat-sour thermophilic aerobic sporeformers in foods
		22.2.1 Material required for analysis
		22.2.2 Procedure for the analysis of sugar
		22.2.3 Procedure for the analysis of starch
		22.2.4 Procedure for the analysis of whole tomatoes, tomato pulp, tomato puree and concentrated milk
		22.2.5 Procedure for the analysis of nonfat dry milk
		22.2.6 Procedure for the analysis of milk cream
		22.2.7 Procedure for the analysis of other foods and ingredients (general)
	22.3 (revised) Methods APHA 27:2015 for spores of thermophilic anaerobic sporeformers in foods
		22.3.1 Material required for analysis
		22.3.2 Procedure for the analysis of sugar and powdered milk
		22.3.3 Procedure for the analysis of starches and flours
		22.3.4 Procedure for the analysis of cereals and alimentary pastes
		22.3.5 Procedure for the analysis of fresh mushrooms
		22.3.6 Procedure for the analysis of “in-process” products
	22.4 (revised) Methods APHA 28:2015 for spores of sulfide spoilage anaerobic sporeformers in foods
		22.4.1 Material required for analysis
		22.4.2 Procedure for the analysis of sugar
		22.4.3 Procedure for the analysis of starch and flour
		22.4.4 Procedure for the analysis of skim milk powder
		22.4.5 Procedure for the analysis of soy protein isolates
	22.5 (revised) Methods APHA 23:2015 for spores of mesophilic aerobic sporeformers in foods
		22.5.1 Material required for analysis
		22.5.2 Procedure for foods in general
		22.5.3 Procedure for the analysis of milk and dairy products
		22.5.4 Procedure for the analysis of water
	22.6 (revised) Methods APHA 24:2015 for spores of mesophilic anaerobic sporeformers in foods
		22.6.1 Material required for analysis
		22.6.2 Procedure for the analysis of sugar
		22.6.3 Procedure for the analysis of starch, flours and other cereal products
		22.6.4 Procedure for the analysis of dehydrated vegetables
		22.6.5 Procedure for the analysis of seasonings and spices
		22.6.6 Procedure for the analysis of egg powder, milk powder and other powdered dairy products
		22.6.7 Procedure for the analysis of fluid milk and cheeses
		22.6.8 Other procedures for mesophilic anaerobic sporeformers
	22.7 Methods IFU 12:2007 for Alicyclobacillus in foods
		22.7.1 Material required for analysis
		22.7.2 Procedure for the analysis of raw material
		22.7.3 Procedure for analysis of the finished product
		22.7.4 Interpretation and calculation of the results
	22.8 References
23 Commercial sterility
	Revision history
	23.1 Introduction
		23.1.1 Definition of commercial sterility
		23.1.2 Classification of commercially sterile foods
		23.1.3 Parameters for evaluating the heat resistance of microorganisms
			23.1.3.1 Survival curve and decimal reduction time (D value)
			23.1.3.2 Number of decimal reductions
			23.1.3.3 Thermal destruction curve and temperature coefficient (z value)
		23.1.4 D and z values of microorganisms of importance in foods
			Vegetative cells
			Heat-resistant mold spores
			Bacterial spores
			Strictly thermophilic aerobic spore-forming bacteria
			Strictly thermophilic anaerobic spore-forming bacteria
			Facultative thermophilic aerobic spore-forming bacteria
			Mesophilic aerobic spore-forming bacteria
			Mesophilic anaerobic spore-forming bacteria
		23.1.5 Dimensioning heat treatments and thermal processing
			23.1.5.1 Definition of the intensity of the thermal process
		23.1.6 Microbial spoilage of canned foods
			23.1.6.1 Underprocessing
			23.1.6.2 Post process contamination (leakage)
			23.1.6.3 Spoilage by strictly thermophiles
			23.1.6.4 Microbial multiplication before heat treatment
			23.1.6.5 Non-microbial causes of spoilage
		23.1.7 Useful terms
	23.2 (revised) Method APHA:2015 for commercial sterility or cause of spoilage of low-acid canned foods
		23.2.1 Material required for analysis
		23.2.2 Procedure
		23.2.3 Interpretation of the results
	23.3 (revised) Method APHA:2015 for commercial sterility for cause of spoilage of acid canned foods
		23.3.1 Material required for analysis
		23.3.2 Procedure
		23.3.3 Interpretation of the results
	23.4 References
24 Guidelines on preparation of culture media
	Revision history
	24.1 Introduction
		24.1.1 Ingredients used in the formulation of culture media
			24.1.1.1 (revised) Water for preparing media and reagents
			24.1.1.2 Nutrient sources for culture media
				Peptones
				Meat extract, yeast extract and malt extract
				Carbohydrates
				Minerals and essential metals
			24.1.1.3 Selective agents
				Antibiotics
				Bile and bile salts
				Chemical compounds
			24.1.1.4 Differential agents
				pH indicators
				Hydrogen sulfide (H2S) indicators
				Other differential agents
			24.1.1.5 Reducing agents
			24.1.1.6 Buffering agents
			24.1.1.7 Chromogenic and fluorogenic substrates
				X-Glucuronide
				MUG
				ONPG
				Salmon-Gal
				X-Gal
				X-Glu
				X-Alpha-glicoside
				X-Phos-inositol
			24.1.1.8 Agar
		24.1.2 (revised) Types of culture media
			Chemically defined medium
			Chemically undefined medium or partially undefined medium
			Chromogenic or fluorogenic medium
			Liquid medium
			Solid and semisolid medium
			Transport medium
			Preservation medium
			Suspension medium (diluents)
			Resuscitation medium
			Pre-enrichment medium or enrichment medium
			Selective enrichment medium
			Non-selective enrichment medium
			Isolation medium
			Selective isolation medium
			Non-selective isolation medium
			Differential medium
			Identification medium
			Ready-to-use medium
			Medium prepared from commercially dehydrated formulations
			Medium prepared from individual components
	24.2 Procedure for preparation of culture media
		24.2.1 Storing supplies and ingredients for preparation of culture media
		24.2.2 Weighing and rehydration
		24.2.3 Dissolution and dispersion
		24.2.4 Verification and adjustment of the pH before sterilization
		24.2.5 Distribution
		24.2.6 Sterilization by moist heat
		24.2.7 Sterilization by filtration
		24.2.8 Verification after sterilization
		24.2.9 Preparation of supplements for culture media
		24.2.10 Storage of sterilized media until the moment of use
			24.2.10.1 Recommendations from ISO 11133:2014
			24.2.10.2 (new) Recommendations from Standard Methods for the Examination of Water and Wastewater (Hunt, 2012)
		24.2.11 (revised) Preparation of the media at the moment of use
			Melting of the agar in solid media
			Addition of supplements to basal media
			Distribution of solid media over plates
			Drying of media in plates intended for surface plating
			Deaeration of the media for anaerobic microorganisms
	24.3 References
Annex 1. Preparation of media and reagents
	Revision history
	Acetamide agar/broth
	Acetamide broth ISO
	Acid phosphatase reagent
	AE sporulation medium modified for Clostridium perfringens
	Agar Listeria Ottaviani & Agosti (ALOA)
	Agar plug (Agar 2%)
	Agar plug with thioglycolate
	Alcoholic solution of iodine
	3:1 alcoholic solution of iodine
	Alkaline peptone water (APW)
	Alkaline saline peptone water (ASPW)
	All purpose Tween (APT) agar/broth
	All purpose Tween (APT) agar acidified
	All purpose Tween (APT) agar sucrose BCP
	All purpose Tween (APT) agar glucose
	Ammonium iron (III) sulfate solution
	Arginine glucose slants (AGS)
	Asparagine broth
	Bacillus acidoterrestris (BAT) agar/broth
	Baird-Parker (BP) agar
	Bile esculin agar
	Bile esculin azide agar
	Biosynth Chromogenic Medium (BCM®) Listeria monocytogenes (R&F Listeria monocytogenes)
	Bismuth sulfite (BS) agar
	Blood agar
	Bolton Broth
	Brain heart infusion (BHI) agar/broth
	Brilliant green agar (BGA)
	Brilliant green bile (BGB) broth
	1% brilliant green solution
	Brilliant green sulfa (BGS) agar
	Brilliant green water
	Bromcresol purple dextrose (BCP) broth
	0.04% Bromothymol blue indicator
	Buffered Listeria enrichment broth (BLEB)
	Buffered peptone water (BPW)
	Modifications
	Buffered peptone water modified (mBPWp)
	Butterfield’s phosphate buffer (PB) (phosphate dilution water) (Butterfield’s phosphate buffered dilution water)
	Butterfield’s phosphate buffer with 40% glucose
	Carbohydrate fermentation medium
	Cefsulodin irgasan novobiocin (CIN) agar
	Cellobiose colistin (CC) agar
	Cellulase solution
	Cephalothin sodium fusidate cetrimide (CFC) agar
	Christensen urea agar
	CHROMagar™ Listeria
	CHROMagar™ Vibrio
	Citrate azide agar
	Chromogenic coliform agar (CCA)
	Columbia Blood Agar (CBA)
	Congo red magnesium oxalate (CR-MOX) agar
	Cooked meat medium (CMM)
	Coomassie brilliant blue solution
	Chromogenic Cronobacter isolation (CCI) agar
	Cronobacter selective broth (CSB)
	Decarboxylase broth Falkow
	Decarboxylation medium
	Dextrose tryptone agar (DTA), dextrose tryptone broth (DTB)
	DFI chromogenic agar
	Dichloran 18% glycerol (DG18) agar
	Dichloran rose Bengal chloramphenicol (DRBC) agar
	Diluent with α-amylase
	Dilution water (see magnesium chloride phosphate buffer, PBMgCl)
	Dipotassium hydrogen phosphate solution (K2HPO4)
	Dipotassium hydrogen phosphate (K2HPO4) solution with antifoam agent
	Double modified lysine iron agar (DMLIA)
	E. coli (EC) broth
	E. coli broth with 4-methylumbelliferyl-β-D-glucuronide (EC-MUG)
	Elliker agar/broth
	Enterobacteriaceae enrichment broth (EEB)
	m-Enterococcus agar (Slanetz & Bartley Medium)
	Ethanol 70%
	Fermentation medium for Clostridium perfringens
	Ferric chloride solution 10%
	Formalinized physiological saline solution
	Fraser broth
	β-Galactosidase reagent (ONPG reagent) (o-nitrophenyl-β-d-galactopyranoside)
	Glucose agar
	α-Glucosidase enzymatic assay solution
	Glycerol salt solution buffered
	Gram-stain reagents (Hucker)
	Gum tragacanth and gum arabic mixture
	Half Fraser broth (demi-Fraser broth)
	Halotolerance saline peptone water
	Hektoen enteric (HE) agar
	Horse blood overlay medium (HL)
	m-HPC agar
	Hydrochloric acid solution
	3% hydrogen peroxide (H2O2)
	Indole Kovac’s reagent (5% p-dimethylaminobenzaldehyde solution)
	Indoxyl acetate discs (2.5 to 5.0 mg)
	Irgasan ticarcillin potassium chlorate (ITC) broth
	Iron milk medium modified
	K agar
	KF Streptococcus agar
	KF Streptococcus broth
	Kim-Goepfert (KG) agar
	King’s B medium
	Koser’s citrate broth
	Lactose broth (LB)
	Lactose broth supplemented with anionic Tergitol 7 or Triton X-100
	Lactose broth supplemented with cellulase solution
	Lactose broth supplemented with papain solution
	Lactose gelatin medium
	Lactose sulfite (LS) medium (LS)
	Lauryl sulfate tryptose (LST) broth
	Levine’s eosin-methylene blue (L-EMB) agar
	Liver broth
	Liver veal agar (LVA)
	Lysine iron agar (LIA)
	MacConkey (MAC) agar
	MacFarland standards
	Magnesium chloride phosphate (PBMgCl) buffer
	Malonate broth
	Malt acetic agar (MAA)
	Malt extract agar (MEA) with antibiotics
	Malt extract broth (MEB)
	Malt extract yeast extract 40% glucose (MY40G)
	Mannitol egg yolk polymyxin (MYP) agar
	de Man Rogosa & Sharpe (MRS) agar/broth
	m-Enterococcus agar (Slanetz & Bartley Medium)
	Methyl red solution
	Modified cellobiose polymyxin colistin (mCPC) agar
	Modified charcoal cefoperazone deoxycholate (mCCDA) agar
	Modified Oxford (MOX) agar
	Modified semisolid Rappaport-Vassiliadis (MSRV) agar
	Modified University of Vermont (UVM) broth
	Morpholinepropanesulfonic acid-buffered Listeria enrichment broth (MOPS-BLEB)
	Motility medium for Bacillus cereus
	Motility nitrate medium
	Motility test medium
	Motility test medium ISO
	MR-VP broth
	Muller-Kauffmann tetrathionate novobiocin (MKTTn) broth
	Nessler reagent
	Ninhydrin solution (3.5% mass/volume)
	Nitrate broth
	Nitrate test reagents
	Nitrate test reagents ISO 7937:2004
	Nutrient agar (NA), nutrient broth (NB)
	Nutrient agar with manganese (NAMn)
	Nutrient agar with trypan blue
	Nutrient broth with lysozyme
	NWRI agar (HPCA)
	Orange serum agar (OSA), orange serum broth (OSB)
	Oxford agar (OXA)
	Oxidase Kovac’s reagent (1% N,N,N,N-tetramethyl-p-phenilenediamine dihydrochoride aqueous solution)
	Oxidation fermentation (OF) glucose agar
	Oxoid Chromogenic Listeria Agar (OCLA)
	Papain solution 5%
	PE-2 medium
	Penicillin pimaricin agar (PPA)
	Peptone sorbitol bile (PSB) broth
	Peptone water (PW)
	Phenol red carbohydrate broth
	Phenylalanine (tryptophane) deaminase agar
	Phosphate buffered saline (PBS)
	Phosphate buffered solution according to ISO 6887-4:2017
	Phosphate buffered solution according to ISO 6887-5:2010
	Phosphate saline buffer 0.02M (pH 7.3 to 7.4)
	Plate count agar (PCA) standard methods agar (SMA), (tryptone glucose yeast extract agar)
	Plate count agar (PCA) supplemented with 0.1% soluble starch
	Plate count agar (PCA) with chloramphenicol (100 mg/L)
	Potassium cyanide broth (KCN)
	0.5% Potassium hydroxide saline solution
	Potato dextrose agar (PDA) acidified
	Potato dextrose agar (PDA) with antibiotics
	Preservative-resistant yeast (PRY) medium
	Preston broth
	Pseudomonas CN agar
	Purple agar/broth for carbohydrate fermentation
	Pyrazinamidase agar
	R2A agar
	Rapid’L.mono agar
	Rappaport-Vassiliadis (R-10) broth
	Rappaport-Vassiliadis (RV) medium
	Rappaport-Vassiliadis soya (RVS) broth
	Reconstituted nonfat dry milk
	Reinforced clostridial medium (RCM)
	Reinforced clostridial medium (RCM) with lactate
	R&F Cronobacter Chromogenic Agar
	R&F Listeria monocytogenes (see Biosynth Chromogenic Medium (BCM®) Listeria monocytogenes)
	Ringer’s solution quarter strength
	Rogosa SL agar/broth
	Saline decarboxylase broth
	Saline nutrient agar (SNA)
	Saline peptone water (SPW) (peptone salt solution)
	Saline tryptophan broth
	Selenite cystine (SC) broth
	Sheep blood agar
	Simmons citrate agar
	Sodium citrate solution (Na3C6H5O7·2H2O)
	0.5% Sodium desoxycholate solution
	Sodium dodecyl sulfate polymixin sucrose (SDS) agar
	Sodium hippurate solution
	Sodium hydroxide solutions
	Sodium tripolyphosphate solution
	Spore stain reagents (Ashby’s)
	Sudan black B solution 0.3% in ethanol 70%
	Sulfide indole motility (SIM) medium
	Sulfite agar
	T1N1 agar and T1N1 broth
	T1N0 and T1N3 broth
	Tetrathionate (TT) broth
	Tetrathionate broth Hajna and Damon (1956) (TTH)
	Thermoacidurans agar (TAA) and thermoacidurans broth (TAB)
	Thioglycollate medium (TGM) fluid
	Thiosulfate citrate bile sucrose (TCBS) agar
	Toluidine blue DNA agar
	Triphenyltetrazolium chloride soya tryptone (TSAT) agar
	Triple sugar iron (TSI) agar
	Trypticase soy agar/broth (TSA/TSB)
	Trypticase soy agar/broth with 0.6% yeast extract (TSA-YE or TSB-YE)
	Trypticase soy broth (TSB) with 10% NaCl and 1% sodium pyruvate
	Trypticase soy broth (TSB) with 20% NaCl
	Trypticase soy broth (TSB) with polymyxin
	Trypticase soy broth (TSB) with 0.5% potassium sulfite (K2SO3)
	Trypticase soy broth (TSB) with 35 mg/L ferrous sulfate
	Trypticase soy agar (TSA) with 5% sheep blood
	Tryptone glucose extract (TGE) agar
	Tryptone glucose yeast extract acetic (TGYA) agar
	Tryptone glucose yeast extract acetic broth (TGYAB)
	Tryptone (tryptophan) broth
	Tryptose sulfite cycloserine (TSC) agar
	Tween esterase test medium
	Tyrosine agar
	Universal pre-enrichment broth
	Urea agar (Christensen)
	Urea broth
	Urea broth rapid
	Vaspar
	Violet red bile (VRB) agar
	Violet red bile glucose (VRBG) agar
	Voges-Proskauer (VP) broth modified for Bacillus
	Voges-Proskauer (VP) test reagents (5% α-naphthol alcoholic solution, 40% potassium hydroxide aqueous solution)
	Xylose lysine desoxycholate agar (XLD)
	Xylose lysine Tergitol 4 (XLT4) agar
	Yeast extract starch glucose (YSG) agar/broth
Annex 2. Sampling plans and microbiological limits recommended by ICMSF for foods
Index