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MICROBIOLOGICAL EXAMINATION METHODS OF FOOD AND WATER
MICROBIOLOGICAL
EXAMINATION METHODS
OF FOOD AND WATER
A Laboratory Manual
S E
NEUSELY DA SILVA, MARTA HIROMI TANIWAKI,

VALÉRIA CHRISTINA AMSTALDEN JUNQUEIRA,
NELIANE FERRAZ DE ARRUDA SILVEIRA,
MARGARETE MIDORI OKAZAKI &
RENATO ABEILAR ROMEIRO GOMES
Institute of Food Technology – ITAL, Campinas, SP, Brazil
Originally published in Portuguese as: ‘Manual de Metodos de Análise Microbiológica de Alimentos e Água ©2010, Livraria Varela, Sao Paulo, Brazil
English edition ‘Microbiological Examination Methods of Food and Water: A Laboratory Manual’, CRC Press/Balkema, Taylor & Francis Group, an
informa business ©2013 Taylor & Francis Group, London, UK
Translation to English: Paul van Dender†
CRC Press/Balkema is an imprint of the Taylor & Francis Group, an informa business
© 2019 Taylor & Francis Group, London, UK
Typeset by Apex CoVantage, LLC
All rights reserved. No part of this publication or the information contained herein may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, by photocopying, recording or otherwise, without written prior permission from the publisher.
Although all care is taken to ensure integrity and the quality of this publication and the information herein, no responsibility is assumed by the
publishers nor the author for any damage to the property or persons as a result of operation or use of this publication and/or the information contained
herein.
Published by: CRC Press/Balkema

Schipholweg 107c, 2316 XC Leiden, The Netherlands
e-mail: Pub.NL@taylorandfrancis.com
www.crcpress.com – www.taylorandfrancis.com
Library of Congress Cataloging-in-Publication Data

Names: Silva, Neusely da., author.
Title: Microbiological examination methods of food and water : a laboratory manual / Neusely da Silva, Marta Hiromi Taniwaki,
Valeria Christina Amstalden Junqueira, Neliane Ferraz de Arruda Silveira, Margarete Midori Okazaki & Renato Abeilar
Romeiro Gomes.
Other titles: Manual de metodos de analise microbiologica de alimentos e agua. English
Description: Second edition. | Leiden, The Netherlands ; Boca Raton : CRC Press/Balkema, [2018] | Includes bibliographical
references and index.
Identifiers: LCCN 2018034220 (print) | LCCN 2018035352 (ebook) | ISBN 9781315165011 (ebook) |
ISBN 9781138091887 (hardcover : alk. paper) | ISBN 9781138057111 (pbk. : alk. paper)
Subjects: MESH: Bacteriological Techniques—methods | Food Microbiology—methods | Water Microbiology |
Laboratory Manuals
Classification: LCC QR115 (ebook) | LCC QR115 (print) | NLM QW 25.5.B2 | DDC 579/.16—dc23
LC record available at https://lccn.loc.gov/2018034220
ISBN: 978-1-138-05711-1 (Pbk)

ISBN: 978-1-138-09188-7 (Hbk)
ISBN: 978-1-315-16501-1 (eBook)
Table of contents
About the authors XXVII

Preface XXIX
List of tables XXXI
List of figures XXXV
1 Sampling, transport and storage of samples for analysis 1

Revision history 1
1.1 Introduction 1
1.1.1 Lot 1
1.1.2 Lot sample and sample unit 1
1.1.3 Lot sampling plans 2
1.1.4 Analytical unit 2
1.2 Collecting samples for analysis 2
1.2.1 Selection and preparation of containers for the sampling of foods contained in
non-individual packages 3
1.2.2 Procedures for the sampling of foods contained in non-individual packages 3
1.2.3 Sampling of foods involved in foodborne diseases 4
1.2.4 Sampling of water 4
1.3 Transportation and storage of samples until analysis 5
1.3.1 Foods with low water activity 5
1.3.2 Frozen foods 5
1.3.3 Refrigerated foods 5
1.3.4 Commercially sterile foods in sealed packages 6
1.3.5 Water samples 6
1.4 References 7
2 Preparation of samples for analysis 9

Revision history 9
2.1 Introduction 9
2.2 Homogenization of samples and withdrawal of the analytical unit 10
2.2.1 Procedure for homogenization and withdrawal of analytical units from
liquid products 11
2.2.2 Procedure for homogenization and withdrawal of analytical units from solid
or concentrated liquid products 11
2.2.3 Procedure for withdrawing the analytical unit using the surface swabbing technique 12
2.2.3.1 Swab sampling 12
2.2.3.2 Sponge swab sampling 13
2.2.4 Procedure for withdrawing the analytical unit using the surface washing technique 13
2.2.4.1 Procedure for washing poultry carcasses 13
2.2.4.2 Procedure for washing other foods 13
2.2.4.3 Procedure for washing packages 14
2.2.5 Keeping of counter-samples 14
2.3 Preparation of the first dilution of the analytical unit 14
VI Table of contents
2.3.1 Diluents for presence/absence tests 14

2.3.2 Diluents for tests requiring differentiated handling of the sample 14
2.3.3 Diluents for general quantification tests 14
2.3.4 How to prepare an initial 1:10 (10−1) dilution 15
2.3.5 How to prepare an initial dilution different from 1:10 16
2.3.6 Procedure for the preparation of the first dilution of liquid samples 16
2.3.7 Procedure for the preparation of the first dilution of solid or concentrated
liquid samples 16
2.3.8 Procedure for the preparation of the first dilution of samples obtained by surface
swabbing or surface washing 16
2.4 Serial decimal dilution of the sample 16
2.5 References 17
Annex 2.1 – Procedures for homogenizing the content and withdrawal of the analytical unit of
different types of foods 18
a) Powdered products 18
b) Pasty or ground products 18
c) Yogurts with fruit pieces 18
d) Cheeses 18
e) Very hard food products 18
f ) Pieces of solid foods 18
g) Eggs in the shell 18
h) Meat cuts for analysis of non-surface contamination 19
i) Bivalves 19
j) Gastropods 19
k) Whole and sliced cephalopods 19
l) Whole crustaceans such as crabs 19
m) Sea urchins 19
n) Whole fresh fish 19
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 19
a) Liquids with low levels of contamination 19
b) Fatty foods 20
c) Lump-forming powders 20
d) Thickeners or products containing natural antimicrobial compounds 20
e) Gelatin 20
f ) Acid products 20
g) Fine flours or meals, cereal grains, animal feed 21
h) Cocoa and chocolate 21
i) Egg white 21
j) Fermented products containing live microorganisms intended for the
quantification of the contaminating microflora (except probiotics) 21
k) Powdered dairy products (dried milk, dried sweet whey, dried acid whey,
dried buttermilk, lactose) 21
l) Butter 21
m) Frozen dairy products and ice cream 21
n) Cheeses 22
o) Fermented dairy products 22
p) Casein and caseinates 22
q) Rennet casein difficult to dissolve 22
Table of contents VII
r) Powdered milk-based infant foods 22

s) Custard, desserts and sweet cream (pH > 5) 23
t) Mollusks (bivalves and gastropods) and sea urchins 23
u) Sea cucumbers (Holothuroidea) and tunicates 23
3 Basic plate count techniques for enumeration of microorganisms 25

Revision history 25
3.1 Introduction 25
3.2 Pour plate technique 26
3.2.1 Material required for the analyses 26
3.2.2 Procedure 26
3.3 Spread plate technique 27
3.3.2 Procedure 28
3.4 Drop plate technique 29
3.4.2 Procedure 29
3.5 Membrane filtration 30
3.5.2 Procedure 30
3.6 Counting colonies and calculating results according to APHA 31
3.6.1 Pour plate calculations 31
3.6.1.1 Rules for calculating the pour plate results in the standard situation 32
3.6.1.2 Rules for calculating the pour plate results in unusual situations 33
3.6.1.3 Calculating the pour plates results for samples prepared by
the surface swabbing technique (swabs or sponges) 35
3.6.1.4 Calculating the pour plate results of samples prepared by the surface
washing technique 36
3.6.2 Spread plate calculations 36
3.6.3 Drop plate calculations 36
3.6.4 Membrane filtration calculations 36
3.7 (revised) Counting colonies and calculating results according to ISO 7218:2007/Amd.1:2013 37
3.7.1 (new) General requirements for the calculation of results 37
a) Number of Petri dishes per dilution 37
b) Maximum and minimum acceptable number of colonies on counting plates 37
c) Decimal dilution of the number of colonies 38
d) Acceptable variation between counts of the pair of plates of a duplicate 38
e) Presentation of the result 38
3.7.2 General rules for the calculation of results 38
3.7.3 Rules for calculation in unusual situations 41
3.8 References 43
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) 44
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) 45
4 Basic techniques for microbial enumeration by the most probable number (MPN) method 47
Revision history 47
4.1 Introduction 47
VIII Table of contents
4.2 Multiple dilution test 48

4.2.2 Procedure 49
4.3 Single dilution test 50
4.3.2 Procedure 50
4.4 Calculation of the results 51
4.4.1 Calculating the results of the multiple dilution test 51
4.4.1.1 Calculation using the MPN tables (for decimal dilutions) 51
4.4.1.2 Calculating using the Thomas formula (for non-decimal dilutions) 52
4.4.1.3 Calculating the results of the samples prepared by the surface swabbing
or surface washing techniques 52
4.4.2 Calculating the results of the single dilution test 53
4.4.2.1 Rules for calculations performed using Table MPN-3 53
4.4.2.2 Calculation for samples prepared by the surface swabbing or surface
washing techniques 53
4.5 References 53
Annex 4.1 – MPN tables 54
5 Basic techniques for the detection of the presence/absence of microorganisms 57

Revision history 57
5.1 Introduction 57
5.1.1 Enrichment 57
5.1.1.1 Pre-enrichment 58
5.1.1.2 Selective enrichment 58
5.1.2 Isolation in solid media (selective differential plating) 58
5.1.3 Confirmation 59
5.1.3.1 Catalase test 59
5.1.3.2 Citrate test 59
5.1.3.3 Amino acid decarboxylation tests 59
5.1.3.4 Phenylalanine deaminase test 59
5.1.3.5 Carbohydrate fermentation tests 59
5.1.3.6 Indole test 60
5.1.3.7 Malonate test 60
5.1.3.8 Oxidation/fermentation (O/F) test 60
5.1.3.9 Oxidase test 61
5.1.3.10 Nitrate reduction test 61
5.1.3.11 Urease test 61
5.1.3.12 Methyl red (MR) test 61
5.1.3.13 Voges-Proskauer (VP) test 62
5.2 Material required for the analyses 62
5.3 Procedure 62
5.3.1 Pre-enrichment 62
5.3.2 Selective enrichment 62
5.3.3 Differential plating 62
5.3.3.1 Streak plating technique for obtaining pure cultures 62
5.3.4 Selection of colonies and subculturing of cultures for confirmation 63
5.3.4.1 Technique for the subculturing of pure cultures starting from colonies
isolated from plates 63
Table of contents IX
5.3.5 Confirmation tests 63

5.3.5.1 Gram staining (Hucker’s method) 63
5.3.5.2 (new) KOH test for confirmation of doubtful Gram staining
(Gregersen, 1978) 64
5.3.5.3 Spore staining (Schaeffer-Fulton’s method) 64
5.3.5.4 Spore staining (Ashby’s method) 64
5.3.5.5 Wet mounts for direct (fresh) microscopic observation 64
5.4 References 64
6 Aerobic plate count 65

Revision history 65
6.1 Introduction 65
6.1.1 The importance and significance of the total aerobic mesophilic count 65
6.1.2 Definition of psychrotrophs 66
6.1.3 Comments on methods of analysis 67
6.2 (revised) Plate count method APHA 8:2015 for aerobic mesophilic bacteria in foods
and water 69
6.2.1 Material required for analysis 69
6.2.2 Procedure 70
6.2.2.1 Pour plate technique 70
6.2.2.2 Spread plate technique 71
6.2.2.3 Membrane filtration technique 71
6.3 (revised) Petrifilm™ AOAC official methods for aerobic mesophilic bacteria in foods 73
6.3.2 Procedure 73
6.4 (revised) Plate count method APHA 13.61:2015 for aerobic psychrotrophic bacteria
in foods 73
6.4.2 Procedure 74
6.5 (new) Plate count methods ISO 4833-1:2013 and ISO 4833-2:2013/Corr.1:2014 for
aerobic mesophilic bacteria in foods 75
6.5.2 Procedure 75
6.6 (new) Plate count method BAM/FDA:2001 for aerobic mesophilic bacteria in foods 77
6.6.2 Procedure 77
6.7 References 79
7 Yeasts and molds 81

Revision history 81
7.1 Introduction 81
7.1.1 Yeasts and molds in foods 81
7.1.2 Comments on methods of analysis for total yeast and mold counts 82
7.1.3 Psychrotrophic fungi 82
7.1.4 Heat-resistant molds 84
7.1.5 Preservative-resistant yeasts (PRY) 84
7.1.5.1 Zigosaccharomyces bailii 85
7.1.5.2 Zygosaccharomyces bisporus 85
7.1.5.3 Schizosaccharomyces pombe 85
X Table of contents
7.1.5.4 Candida krusei 85

7.1.5.5 Pichia membranaefaciens 86
7.1.6 Osmophilic yeasts 86
7.1.6.1 Zygosaccharomyces rouxii 86
7.1.7 Direct plating method ICFM for particulate foods 87
7.2.A Plate count method APHA 21:2015 for yeasts and molds in foods 87
7.2.A.1 Material required for analysis 87
7.2.A.2 Procedure 87
7.2.B (new) Plate count methods ISO 21527-1:2008 and ISO 21527-2:2008 for yeasts
and molds in foods 90
7.2.B.1 Material required for analysis 90
7.2.B.2 Procedure 90
7.2.C (new) Plate count method BAM/FDA:2001 for yeasts and molds in foods 92
7.2.C.1 Material required for analysis 92
7.2.C.2 Procedure 92
7.3 (revised) Plate count method APHA 13:2015 for psychrotrophic fungi in foods 93
7.3.2 Procedure 93
7.4 (revised) Plate count method APHA 22.4:2015 for heat-resistant molds in foods 95
7.4.2 Procedure 95
7.5 Pitt and Hocking (2009) methods for preservative-resistant yeasts in foods 97
7.5.2 Procedure 97
7.5.2.1 Presence/absence method 98
7.5.2.2 Direct plate count method 99
7.6 (revised) Membrane filtration or plate count methods APHA 17.3:2015 for osmophilic
yeasts in foods 99
7.6.2 Procedure 99
7.6.2.1 Membrane filtration method 99
7.6.2.2 Plate count method 100
7.7 (new) Direct plating method Hocking et al. (2006) for percentage of fungal infection
in particulate foods 100
7.7.2 Procedure 101
7.8 References 101
8 Enterobacteriaceae 103
Revision history 103
8.1 Introduction 103
8.1.1 Taxonomy 103
8.2 (revised) Plate count method APHA 9.62:2015 for Enterobacteriaceae in foods 105
8.2.2 Procedure 105
8.3 (revised) Presence/absence (P/A) or most probable number (MPN) method APHA 9.61:2015
for Enterobacteriaceae in foods 106
8.3.2 Procedure 106
Table of contents XI
8.4 (revised) AOAC official method 2003.1 (Petrifilm™) for Enterobacteriaceae in selected foods 108
8.4.2 Procedure 108
8.5 (new) Plate count method ISO 21528-2:2017 for Enterobacteriaceae in foods 109
8.5.2 Procedure 109
8.6 (new) Presence/absence (P/A) or most probable number (MPN) method ISO 21528-1:2017
for Enterobacteriaceae in foods 111
8.6.2 Procedure 111
8.7 References 113
9 Total and thermotolerant coliforms and Escherichia coli 115

9.1.1 Definition of total coliforms 115
9.1.2 Definition of thermotolerant coliforms 116
9.1.3 E. coli 116
9.1.4 Use as indicators 116
9.2 (revised) Most probable number (MPN) method APHA 9:2015 for total/thermotolerant
coliforms and E. coli in foods 119
9.2.2 Procedure 120
9.3 Most probable number (MPN) methods ISO 4831:2006 and ISO 7251:2005 for total
coliforms and presumptive E. coli in foods 123
9.3.2 Procedure 123
9.4 (revised) Most probable number (MPN) method APHA/AWWA/WEF:2012 for total
and thermotolerant coliforms and E. coli in water 126
9.4.2 Procedure 126
9.5 (new) Most probable number (MPN) method BAM/FDA:2017 for total/thermotolerant
coliforms and E. coli in foods 128
9.5.2 Procedure 129
9.6 (revised) Plate count method APHA:2015 for total coliforms in foods 130
9.6.2 Procedure 130
9.7 (new) Membrane filtration method ISO 9308-1:2014/Amd.1:2016 for total coliforms
and E. coli in water 132
9.7.2 Procedure 132
9.8 References 134
10 Staphylococcus aureus 135

10.1.1 Taxonomy 135
10.1.1.1 The genus Staphylococcus 135
XII Table of contents
10.1.1.2 The coagulase-positive staphylococci 136

10.1.1.3 Staphylococcus aureus 137
10.1.2 Epidemiology 137
10.1.2.1 Staphylococcus aureus enterotoxins 137
10.1.2.2 Staphylococcal food poisoning 138
10.2 (revised) Plate count method APHA 39.63:2015 for coagulase-positive staphylococci
and Staphylococcus aureus in foods 140
10.2.2 Procedure 140
10.3 (revised) Most probable number (MPN) method APHA39.62:2015 for coagulase-positive
staphylococci and Staphylococcus aureus in foods 143
10.4 (revised) Presence/absence method APHA 39.61:2015 for coagulase-positive staphylococci
and Staphylococcus aureus in foods 145
10.5 References 147
11 Bacillus cereus 149

11.1.1 Taxonomy 149
11.1.1.1 Bacillus cereus group 149
Bacillus anthracis 150
Bacillus thuringiensis 150
Bacillus mycoides 150
Bacillus pseudomycoides 151
Bacillus weihenstephanensis 151
Bacillus cytotoxicus 151
New species 151
11.1.1.2 Bacillus cereus 151
11.2 (revised) Plate count method APHA 31.61:2015 for Bacillus cereus in foods 153
11.3 (revised) Most probable number (MPN) method APHA 31.62:2015 for Bacillus cereus in foods 157
11.4 References 159
12 Clostridium perfringens 161

12.1.1 Taxonomy 161
12.1.2.1 Clostridium perfingens type A food poisoning 162
12.1.2.2 Clostridium perfringens type C necrotic enteritis 163
Table of contents XIII
12.2 (revised) Plate count method APHA 33.72:2015 for Clostridium perfringens in foods 164
12.3 (revised) Presence/absence method APHA 33.71:2015 for Clostridium perfringens
in foods 167
12.4 (new) Plate count method BAM/FDA:2001 for Clostridium perfringens in foods 169
12.5 (new) Plate count method ISO 7937:2004 for Clostridium perfringens in foods 171
12.6 (new) Membrane filtration method ISO 14189:2013 for Clostridium perfringens in water 173
12.7 References 175
13 Enterococci 177
13.1.1 Taxonomy 177
13.1.1.1 Enterococci 178
Description of the genus Enterococcus 178
13.1.1.2 Fecal streptococci 179
Description of the genus Streptococcus 180
13.1.1.3 Differentiation of enterococci from group bovis fecal streptococci 180
13.2 (revised) Plate count method APHA 10.5:2015 for enterococci and fecal streptococci
in foods 181
13.3 (revised) Most probable number (MPN) method APHA 10.2:2015 for enterococci and fecal
streptococci in foods 183
13.4 (revised) Membrane filtration method APHA/AWWA/WEF 9230C.3c:2012 for enterococci
and fecal streptococci in water 184
13.5 Membrane filtration method ISO 7899-2:2000 for intestinal enterococci in water 186
13.6 References 186
14 Lactic acid bacteria 189

14.1.1 Carnobacterium 189
14.1.2 Enterococcus 191
14.1.3 Fructobacillus 191
XIV Table of contents
14.1.4 Lactobacillus 192

14.1.5 Lactococcus 192
14.1.6 Leuconostoc 193
14.1.7 Oenococcus 193
14.1.8 Pediococcus 194
14.1.9 Streptococcus 194
14.1.10 Tetragenococcus 195
14.1.11 Weissella 195
14.2 (revised) Plate count method APHA 19.52:2015 for lactic acid bacteria in foods 198
14.3 (revised) MPN methods APHA 19.526:2015 and APHA 19.524:2015
for lactic acid bacteria in foods 200
14.3.2 Procedure using MRS broth 200
14.3.3 Procedure using Rogosa SL broth 202
14.4 (new) Plate count method ISO 15214:1998 for lactic acid bacteria in foods 202
14.5 References 205
15 Campylobacter 207
15.1.1 Taxonomy 207
15.1.1.1 Campylobacter 207
15.1.1.2 Thermotolerant Campylobacter 209
15.2 (revised) Presence/absence method ISO 10272-1:2017 for thermotolerant Campylobacter
in foods 210
15.3 (new) Plate count method ISO 10272-2:2017 for thermotolerant Campylobacter in foods 213
15.4 References 216
16 Cronobacter 217
16.1.1 Taxonomy 217
Cronobacter Iversen et al. (2008) 218
16.1.3 Codex Alimentarius microbiological criteria for Cronobacter spp. in powdered
infant formulae 219
16.2 (revised) Presence/absence method ISO 22964:2017 for Cronobacter in foods 220
Table of contents XV
16.3 (new) Presence/absence method BAM/FDA:2012 for Cronobacter in dehydrated powdered

infant formula 223
16.4 References 225
17 Pseudomonas 227
17.1.1 Taxonomy 227
17.1.1.1 Pseudomonas 227
Pseudomonas in treated water intended for human consumption 229
Pseudomonas in mineral water and natural water 230
Pseudomonas in foods 230
17.1.1.2 Shewanella 230
Shewanella putrefaciens (synonym Pseudomonas putrefaciens) 231
17.1.1.3 Janthinobacterium 231
Janthinobacterium lividum (synonym Pseudomonas mephitica) 232
17.1.1.4 Stenotrophomonas 232
Stenotrophomonas maltophilia (synonym Pseudomonas maltophilia) 232
17.2 (revised) MPN method APHA/AWWA/WEF 9213:2012 for Pseudomonas aeruginosa
in water 233
17.3 Membrane filtration method ISO 16266:2006 for Pseudomonas aeruginosa in water 234
17.4 Plate count method ISO 13720:2010 for presumptive Pseudomonas spp. in meat
and meat products 237
17.5 Plate count method ISO 11059:2009 for Pseudomonas spp. in milk and milk products 237
17.6 References 240
18 Listeria monocytogenes 243

18.1.1 Taxonomy 244
18.2 (revised) Presence/absence or MPN method BAM/FDA:2017 for Listeria monocytogenes in foods 248
18.2.2.1 Presence/absence test and MPN count 250
18.2.2.2 Direct plate count 253
18.3 (revised) Presence/absence method USDA/MLG:2017 for Listeria monocytogenes in foods 254
XVI Table of contents
18.4 (revised) Plate count method ISO 11290-2:2017 for Listeria spp. and Listeria
monocytogenes in foods 257
18.5 (revised) Presence/absence method ISO 11290-1:2017 for Listeria spp. and Listeria
monocytogenes in foods 260
18.6 References 262
19 Salmonella 265
19.1.1 Taxonomic classification of Salmonella 266
19.1.2 Serological classification of Salmonella 268
Somatic (“O”) antigens 268
Capsular (surface or envelope) antigens 268
Flagellar (“H”) antigens 269
The White-Kauffmann-Le Minor system 269
Salmonella serovar nomenclature 269
Serovars most commonly found 269
19.1.3 Biochemical characteristics of Salmonella 270
19.1.5 Comments on traditional methods used for the examination of Salmonella 272
19.1.6 Comments on alternative methods for the analysis of Salmonella 274
19.1.7 Composite samples for analysis 274
19.2 (revised) Presence/absence method ISO 6579-1:2017 for Salmonella in foods 274
19.3 (revised) Presence/absence method BAM/FDA:2018 for Salmonella in foods 282
19.4 (revised) Presence/absence method MLG/USDA:2017 for Salmonella in foods 292
19.5 References 296
20 Vibrio cholerae and Vibrio parahaemolyticus 299

20.1.1 Taxonomy 300
20.1.2.1 V. cholerae 305
20.1.2.2 V. parahaemolyticus 305
20.1.2.3 V. vulnificus 305
20.2.A Presence/absence method BAM/FDA:2004 for Vibrio cholerae in foods 307
Table of contents XVII
20.2.B (revised) Presence/absence and MPN methods APHA 40.61:2015 for Vibrio cholerae
in foods and water 309
20.3.A MPN method BAM/FDA:2004 for Vibrio parahaemolyticus in foods 313
20.3.B (revised) Presence/absence or MPN method APHA 40.62/40.63:2015 for Vibrio
parahaemolyticus and Vibrio vulnificus in foods 315
20.4 (revised) Presence/absence method ISO 21872-1:2017 for potentially enteropathogenic
Vibrio cholerae and Vibrio parahaemolyticus in foods 318
20.5 References 322
21 Yersinia enterocolitica 325

21.1.1 Taxonomy 325
21.2 Presence/absence method ISO 10273:2017 for pathogenic Yersinia enterocolitica
in foods 328
21.3 References 332
22 Bacterial spore count 333

22.1.1 The bacterial spore 333
22.1.1.1 Sequence of spore formation 333
22.1.1.2 Spore ultrastructure 334
22.1.1.3 Mechanisms of spore resistance 334
22.1.2 Taxonomy of spore-forming bacteria important in foods 335
22.1.2.1 Aeribacillus 335
22.1.2.2 Alicyclobacillus 335
Alicyclobacillus acidoterrestris 336
Alicyclobacillus acidocaldarius 336
Alicyclobacillus acidiphilus 336
Alicyclobacillus contaminans 336
Alicyclobacillus dauci 336
Alicyclobacillus fastidiosus 337
Alicyclobacillus herbarius 337
Alicyclobacillus pomorum 337
Alicyclobacillus sacchari 337
22.1.2.3 Aneurinibacillus 337
Aneurinibacillus thermoaerophilus 338
XVIII Table of contents
22.1.2.4 Anoxybacillus 338

Anoxybacillus contaminans 338
Anoxybacillus flavithermus 338
Anoxybacillus tepidamans 339
22.1.2.5 Bacillus 339
Bacillus coagulans 339
Bacillus smithii 340
Bacillus sporothermodurans 340
22.1.2.6 Brevibacillus 341
22.1.2.7 Clostridium 341
Clostridium botulinum 342
Proteolytic clostridia 343
Saccharolytic clostridia 343
Psychrophilic and psychrotrophic clostridia that cause the
spoilage of refrigerated vacuum-packed meats 344
22.1.2.8 Cohnella 344
22.1.2.9 Desulfotomaculum 345
Desulfotomaculum nigrificans 345
22.1.2.10 Fictibacillus 345
Fictibacillus gelatini 345
Fictibacillus nanhaiensis 346
22.1.2.11 Geobacillus 346
Geobacillus stearothermophilus 346
22.1.2.12 Hathewaya 347
22.1.2.13 Jeotgalibacillus 347
Jeotgalibacillus alimentarius 347
22.1.2.14 Lentibacillus 347
22.1.2.15 Lysinibacillus 348
22.1.2.16 Moorella 348
22.1.2.17 Oceanobacillus 349
22.1.2.18 Paenibacillus 349
22.1.2.19 Paraclostridium 349
22.1.2.20 Sporolactobacillus 350
22.1.2.21 Thermoanaerobacter 350
22.1.2.22 Thermoanaerobacterium 350
T. thermosaccharolyticum 350
22.1.2.23 Virgibacillus 351
22.2 (revised) Methods APHA 25:2015 and 26:2015 for spores of total and flat-sour
thermophilic aerobic sporeformers in foods 351
22.2.2 Procedure for the analysis of sugar 352
22.2.3 Procedure for the analysis of starch 352
22.2.4 Procedure for the analysis of whole tomatoes, tomato pulp, tomato puree
and concentrated milk 353
22.2.5 Procedure for the analysis of nonfat dry milk 353
22.2.6 Procedure for the analysis of milk cream 354
22.2.7 Procedure for the analysis of other foods and ingredients (general) 354
22.3 (revised) Methods APHA 27:2015 for spores of thermophilic anaerobic sporeformers
in foods 356
Table of contents XIX
22.3.2 Procedure for the analysis of sugar and powdered milk 356
22.3.3 Procedure for the analysis of starches and flours 357
22.3.4 Procedure for the analysis of cereals and alimentary pastes 357
22.3.5 Procedure for the analysis of fresh mushrooms 357
22.3.6 Procedure for the analysis of “in-process” products 357
22.4 (revised) Methods APHA 28:2015 for spores of sulfide spoilage anaerobic sporeformers
in foods 357
22.4.3 Procedure for the analysis of starch and flour 358
22.4.4 Procedure for the analysis of skim milk powder 358
22.4.5 Procedure for the analysis of soy protein isolates 358
22.5 (revised) Methods APHA 23:2015 for spores of mesophilic aerobic sporeformers in foods 359
22.5.2 Procedure for foods in general 359
22.5.3 Procedure for the analysis of milk and dairy products 361
22.5.4 Procedure for the analysis of water 361
22.6 (revised) Methods APHA 24:2015 for spores of mesophilic anaerobic sporeformers
in foods 361
22.6.3 Procedure for the analysis of starch, flours and other cereal products 362
22.6.4 Procedure for the analysis of dehydrated vegetables 363
22.6.5 Procedure for the analysis of seasonings and spices 363
22.6.6 Procedure for the analysis of egg powder, milk powder and other powdered
dairy products 363
22.6.7 Procedure for the analysis of fluid milk and cheeses 363
22.6.8 Other procedures for mesophilic anaerobic sporeformers 364
22.7 Methods IFU 12:2007 for Alicyclobacillus in foods 364
22.7.2 Procedure for the analysis of raw material 365
22.7.3 Procedure for analysis of the finished product 366
22.7.4 Interpretation and calculation of the results 366
22.8 References 367
23 Commercial sterility 373

23.1.1 Definition of commercial sterility 374
23.1.2 Classification of commercially sterile foods 374
23.1.3 Parameters for evaluating the heat resistance of microorganisms 374
23.1.3.1 Survival curve and decimal reduction time (D value) 374
23.1.3.2 Number of decimal reductions 376
23.1.3.3 Thermal destruction curve and temperature coefficient (z value) 376
23.1.4 D and z values of microorganisms of importance in foods 377
Vegetative cells 378
Heat-resistant mold spores 378
Bacterial spores 378
Strictly thermophilic aerobic spore-forming bacteria 378
Strictly thermophilic anaerobic spore-forming bacteria 378
XX Table of contents
Facultative thermophilic aerobic spore-forming bacteria 378

Mesophilic aerobic spore-forming bacteria 378
Mesophilic anaerobic spore-forming bacteria 379
23.1.5 Dimensioning heat treatments and thermal processing 379
23.1.5.1 Definition of the intensity of the thermal process 379
23.1.6 Microbial spoilage of canned foods 380
23.1.6.1 Underprocessing 380
23.1.6.2 Post process contamination (leakage) 380
23.1.6.3 Spoilage by strictly thermophiles 380
23.1.6.4 Microbial multiplication before heat treatment 381
23.1.6.5 Non-microbial causes of spoilage 381
23.1.7 Useful terms 381
23.2 (revised) Method APHA:2015 for commercial sterility or cause of spoilage of low-acid
canned foods 382
23.2.3 Interpretation of the results 386
23.3 (revised) Method APHA:2015 for commercial sterility for cause of spoilage of acid
canned foods 389
23.3.3 Interpretation of the results 394
23.4 References 396
24 Guidelines on preparation of culture media 399

24.1.1 Ingredients used in the formulation of culture media 399
24.1.1.1 (revised) Water for preparing media and reagents 399
24.1.1.2 Nutrient sources for culture media 400
Peptones 400
Meat extract, yeast extract and malt extract 401
Carbohydrates 402
Minerals and essential metals 402
24.1.1.3 Selective agents 402
Antibiotics 402
Bile and bile salts 402
Chemical compounds 402
24.1.1.4 Differential agents 402
pH indicators 403
Hydrogen sulfide (H2S) indicators 403
Other differential agents 403
24.1.1.5 Reducing agents 403
24.1.1.6 Buffering agents 403
24.1.1.7 Chromogenic and fluorogenic substrates 403
X-Glucuronide 404
MUG 404
ONPG 404
Salmon-Gal 404
X-Gal 404
Table of contents XXI
X-Glu 404
X-Alpha-glicoside 404
X-Phos-inositol 404
24.1.1.8 Agar 404
24.1.2 (revised) Types of culture media 404
Chemically defined medium 404
Chemically undefined medium or partially undefined medium 404
Chromogenic or fluorogenic medium 405
Liquid medium 405
Solid and semisolid medium 405
Transport medium 405
Preservation medium 405
Suspension medium (diluents) 405
Resuscitation medium 405
Pre-enrichment medium or enrichment medium 405
Selective enrichment medium 405
Non-selective enrichment medium 405
Isolation medium 405
Selective isolation medium 405
Non-selective isolation medium 405
Differential medium 405
Identification medium 406
Ready-to-use medium 406
Medium prepared from commercially dehydrated formulations 406
Medium prepared from individual components 406
24.2 Procedure for preparation of culture media 406
24.2.1 Storing supplies and ingredients for preparation of culture media 406
24.2.2 Weighing and rehydration 406
24.2.3 Dissolution and dispersion 407
24.2.4 Verification and adjustment of the pH before sterilization 407
24.2.5 Distribution 407
24.2.6 Sterilization by moist heat 407
24.2.7 Sterilization by filtration 408
24.2.8 Verification after sterilization 408
24.2.9 Preparation of supplements for culture media 409
24.2.10 Storage of sterilized media until the moment of use 409
24.2.10.1 Recommendations from ISO 11133:2014 409
24.2.10.2 (new) Recommendations from Standard Methods for the Examination
of Water and Wastewater (Hunt, 2012) 409
24.2.11 (revised) Preparation of the media at the moment of use 410
Melting of the agar in solid media 410
Addition of supplements to basal media 410
Distribution of solid media over plates 410
Drying of media in plates intended for surface plating 410
Deaeration of the media for anaerobic microorganisms 410
24.3 References 410
Annex 1. Preparation of media and reagents 411
Acetamide agar/broth 412
XXII Table of contents
Acetamide broth ISO 412

Acid phosphatase reagent 413
AE sporulation medium modified for Clostridium perfringens 413
Agar Listeria Ottaviani & Agosti (ALOA) 414
Agar plug (Agar 2%) 415
Agar plug with thioglycolate 415
Alcoholic solution of iodine 415
3:1 alcoholic solution of iodine 415
Alkaline peptone water (APW) 416
Alkaline saline peptone water (ASPW) 416
All purpose Tween (APT) agar/broth 416
All purpose Tween (APT) agar acidified 417
All purpose Tween (APT) agar sucrose BCP 417
All purpose Tween (APT) agar glucose 417
Ammonium iron (III) sulfate solution 417
Arginine glucose slants (AGS) 417
Asparagine broth 418
Bacillus acidoterrestris (BAT) agar/broth 418
Baird-Parker (BP) agar 418
Bile esculin agar 419
Bile esculin azide agar 419
Biosynth Chromogenic Medium (BCM®) Listeria monocytogenes (R&F Listeria monocytogenes) 420
Bismuth sulfite (BS) agar 420
Blood agar 421
Bolton Broth 421
Brain heart infusion (BHI) agar/broth 422
Brilliant green agar (BGA) 423
Brilliant green bile (BGB) broth 423
1 brilliant green solution 423
Brilliant green sulfa (BGS) agar 424
Brilliant green water 424
Bromcresol purple dextrose (BCP) broth 424
0.04 Bromothymol blue indicator 424
Buffered Listeria enrichment broth (BLEB) 425
Buffered peptone water (BPW) 425
Modifications 426
Buffered peptone water modified (mBPWp) 426
Butterfield’s phosphate buffer (PB) (phosphate dilution water) (Butterfield’s phosphate buffered
dilution water) 426
Butterfield’s phosphate buffer with 40% glucose 427
Carbohydrate fermentation medium 427
Cefsulodin irgasan novobiocin (CIN) agar 427
Cellobiose colistin (CC) agar 428
Cellulase solution 428
Cephalothin sodium fusidate cetrimide (CFC) agar 429
Christensen urea agar 429
CHROMagarTM Listeria 430
CHROMagarTM Vibrio 430
Citrate azide agar 430
Chromogenic coliform agar (CCA) 431
Table of contents XXIII
Columbia Blood Agar (CBA) 431

Congo red magnesium oxalate (CR-MOX) agar 432
Cooked meat medium (CMM) 432
Coomassie brilliant blue solution 432
Chromogenic Cronobacter isolation (CCI) agar 433
Cronobacter selective broth (CSB) 433
Decarboxylase broth Falkow 434
Decarboxylation medium 434
Dextrose tryptone agar (DTA), dextrose tryptone broth (DTB) 435
DFI chromogenic agar 435
Dichloran 18% glycerol (DG18) agar 435
Dichloran rose Bengal chloramphenicol (DRBC) agar 436
Diluent with α-amylase 436
Dilution water (see magnesium chloride phosphate buffer, PBMgCl) 436
Dipotassium hydrogen phosphate solution (K2HPO4) 436
Dipotassium hydrogen phosphate (K2HPO4) solution with antifoam agent 437
Double modified lysine iron agar (DMLIA) 437
E. coli (EC) broth 438
E. coli broth with 4-methylumbelliferyl-β-D-glucuronide (EC-MUG) 438
Elliker agar/broth 438
Enterobacteriaceae enrichment broth (EEB) 439
m-Enterococcus agar (Slanetz & Bartley Medium) 439
Ethanol 70% 439
Fermentation medium for Clostridium perfringens 440
Ferric chloride solution 10% 440
Formalinized physiological saline solution 440
Fraser broth 441
β-Galactosidase reagent (ONPG reagent) (o-nitrophenyl-β-d-galactopyranoside) 441
Glucose agar 442
α-Glucosidase enzymatic assay solution 442
Glycerol salt solution buffered 443
Gram-stain reagents (Hucker) 443
Gum tragacanth and gum arabic mixture 444
Half Fraser broth (demi-Fraser broth) 444
Halotolerance saline peptone water 445
Hektoen enteric (HE) agar 445
Horse blood overlay medium (HL) 446
m-HPC agar 446
Hydrochloric acid solution 446
3% hydrogen peroxide (H2O2) 446
Indole Kovac’s reagent (5% p-dimethylaminobenzaldehyde solution) 447
Indoxyl acetate discs (2.5 to 5.0 mg) 447
Irgasan ticarcillin potassium chlorate (ITC) broth 447
Iron milk medium modified 448
K agar 448
KF Streptococcus agar 449
KF Streptococcus broth 449
Kim-Goepfert (KG) agar 449
King’s B medium 450
Koser’s citrate broth 450
XXIV Table of contents
Lactose broth (LB) 451

Lactose broth supplemented with anionic Tergitol 7 or Triton X-100 451
Lactose broth supplemented with cellulase solution 451
Lactose broth supplemented with papain solution 451
Lactose gelatin medium 451
Lactose sulfite (LS) medium (LS) 452
Lauryl sulfate tryptose (LST) broth 452
Levine’s eosin-methylene blue (L-EMB) agar 453
Liver broth 453
Liver veal agar (LVA) 454
Lysine iron agar (LIA) 454
MacConkey (MAC) agar 454
MacFarland standards 455
Magnesium chloride phosphate (PBMgCl) buffer 455
Malonate broth 455
Malt acetic agar (MAA) 456
Malt extract agar (MEA) with antibiotics 456
Malt extract broth (MEB) 457
Malt extract yeast extract 40% glucose (MY40G) 457
Mannitol egg yolk polymyxin (MYP) agar 457
de Man Rogosa & Sharpe (MRS) agar/broth 458
m-Enterococcus agar (Slanetz & Bartley Medium) 458
Methyl red solution 459
Modified cellobiose polymyxin colistin (mCPC) agar 459
Modified charcoal cefoperazone deoxycholate (mCCDA) agar 460
Modified Oxford (MOX) agar 460
Modified semisolid Rappaport-Vassiliadis (MSRV) agar 461
Modified University of Vermont (UVM) broth 462
Morpholinepropanesulfonic acid-buffered Listeria enrichment broth (MOPS-BLEB) 463
Motility medium for Bacillus cereus 463
Motility nitrate medium 463
Motility test medium 464
Motility test medium ISO 464
MR-VP broth 465
Muller-Kauffmann tetrathionate novobiocin (MKTTn) broth 465
Nessler reagent 466
Ninhydrin solution (3.5% mass/volume) 466
Nitrate broth 466
Nitrate test reagents 467
Nitrate test reagents ISO 7937:2004 467
Nutrient agar (NA), nutrient broth (NB) 468
Nutrient agar with manganese (NAMn) 468
Nutrient agar with trypan blue 468
Nutrient broth with lysozyme 468
NWRI agar (HPCA) 468
Orange serum agar (OSA), orange serum broth (OSB) 468
Oxford agar (OXA) 469
Oxidase Kovac’s reagent (1% N,N,N,N-tetramethyl-p-phenilenediamine dihydrochoride
aqueous solution) 470
Oxidation fermentation (OF) glucose agar 470
Table of contents XXV
Oxoid Chromogenic Listeria Agar (OCLA) 470

Papain solution 5 471
PE-2 medium 471
Penicillin pimaricin agar (PPA) 471
Peptone sorbitol bile (PSB) broth 472
Peptone water (PW) 472
Phenol red carbohydrate broth 472
Phenylalanine (tryptophane) deaminase agar 473
Phosphate buffered saline (PBS) 473
Phosphate buffered solution according to ISO 6887-4:2017 474
Phosphate buffered solution according to ISO 6887-5:2010 474
Phosphate saline buffer 0.02M (pH 7.3 to 7.4) 474
Plate count agar (PCA) standard methods agar (SMA), (tryptone glucose yeast extract agar) 475
Plate count agar (PCA) supplemented with 0.1% soluble starch 475
Plate count agar (PCA) with chloramphenicol (100 mg/L) 475
Potassium cyanide broth (KCN) 475
0.5 Potassium hydroxide saline solution 476
Potato dextrose agar (PDA) acidified 476
Potato dextrose agar (PDA) with antibiotics 477
Preservative-resistant yeast (PRY) medium 477
Preston broth 478
Pseudomonas CN agar 478
Purple agar/broth for carbohydrate fermentation 479
Pyrazinamidase agar 479
R2A agar 480
Rapid’L.mono agar 480
Rappaport-Vassiliadis (R-10) broth 481
Rappaport-Vassiliadis (RV) medium 481
Rappaport-Vassiliadis soya (RVS) broth 482
Reconstituted nonfat dry milk 483
Reinforced clostridial medium (RCM) 483
Reinforced clostridial medium (RCM) with lactate 484
R&F Cronobacter Chromogenic Agar 484
R&F Listeria monocytogenes (see Biosynth Chromogenic Medium (BCM®) Listeria monocytogenes) 484
Ringer’s solution quarter strength 484
Rogosa SL agar/broth 485
Saline decarboxylase broth 485
Saline nutrient agar (SNA) 486
Saline peptone water (SPW) (peptone salt solution) 486
Saline tryptophan broth 486
Selenite cystine (SC) broth 487
Sheep blood agar 488
Simmons citrate agar 488
Sodium citrate solution (Na3C6H5O7·2H2O) 489
0.5 Sodium desoxycholate solution 489
Sodium dodecyl sulfate polymixin sucrose (SDS) agar 489
Sodium hippurate solution 490
Sodium hydroxide solutions 490
Sodium tripolyphosphate solution 490
Spore stain reagents (Ashby’s) 490
XXVI Table of contents
Sudan black B solution 0.3% in ethanol 70 491

Sulfide indole motility (SIM) medium 491
Sulfite agar 491
T1N1 agar and T1N1 broth 492
T1N0 and T1N3 broth 492
Tetrathionate (TT) broth 492
Tetrathionate broth Hajna and Damon (1956) (TTH) 493
Thermoacidurans agar (TAA) and thermoacidurans broth (TAB) 494
Thioglycollate medium (TGM) fluid 494
Thiosulfate citrate bile sucrose (TCBS) agar 494
Toluidine blue DNA agar 495
Triphenyltetrazolium chloride soya tryptone (TSAT) agar 495
Triple sugar iron (TSI) agar 496
Trypticase soy agar/broth (TSA/TSB) 496
Trypticase soy agar/broth with 0.6% yeast extract (TSA-YE or TSB-YE) 497
Trypticase soy broth (TSB) with 10% NaCl and 1% sodium pyruvate 497
Trypticase soy broth (TSB) with 20% NaCl 497
Trypticase soy broth (TSB) with polymyxin 497
Trypticase soy broth (TSB) with 0.5% potassium sulfite (K2SO3) 497
Trypticase soy broth (TSB) with 35 mg/L ferrous sulfate 497
Trypticase soy agar (TSA) with 5% sheep blood 497
Tryptone glucose extract (TGE) agar 497
Tryptone glucose yeast extract acetic (TGYA) agar 498
Tryptone glucose yeast extract acetic broth (TGYAB) 498
Tryptone (tryptophan) broth 498
Tryptose sulfite cycloserine (TSC) agar 499
Tween esterase test medium 500
Tyrosine agar 500
Universal pre-enrichment broth 500
Urea agar (Christensen) 501
Urea broth 501
Urea broth rapid 502
Vaspar 502
Violet red bile (VRB) agar 502
Violet red bile glucose (VRBG) agar 502
Voges-Proskauer (VP) broth modified for Bacillus 503
Voges-Proskauer (VP) test reagents (5% α-naphthol alcoholic solution, 40% potassium
hydroxide aqueous solution) 503
Xylose lysine desoxycholate agar (XLD) 504
Xylose lysine Tergitol 4 (XLT4) agar 504
Yeast extract starch glucose (YSG) agar/broth 504
Annex 2. Sampling plans and microbiological limits recommended by ICMSF for foods 507
Index 515
About the authors
Neusely da Silva is a scientific researcher at the Food Technology Institute

(ITAL), a government research agency of the State of São Paulo, Brazil. She
graduated in Food Engineering and has a PhD in Food Science from the State
University of Campinas (UNICAMP, Brazil). Director of the Microbiology
Reference Laboratory of the Food Technology Institute from 1995 to 2007,
she was responsible for the accreditation of the laboratory assays according to
ISO 17025. She is author of over 70 publications in the area of Food Micro-
biology and her major research areas are bacterial physiology and methods for
detection of bacteria responsible for food-borne diseases and bacteria respon-
sible for food spoilage. E-mail: neusely@ital.sp.gov.br.
Marta Hiromi Taniwaki, PhD, is a scientific researcher at the Food Technology

Institute (ITAL) at the Center of Quality and Food Science in Campinas, Brazil.
She graduated in Biology and has a PhD in Food Science and Technology from
the University of New South Wales, Australia She is author of over 100 publica-
tions in the area of Food Mycology, Mycotoxins and Food Microbiology. She
is member of the International Commission on Food Mycology (ICFM) since
1997; member of the Brazilian delegation at the Codex Contaminants in Food
(CCCF) since 2006; Member of the International Commission on Microbiologi-
cal Specifications for Foods (ICMSF) since 2010 and editorial board of Myco-
toxin Research since 2012. Her major research areas are: fungi and mycotoxins in
foods; biodiversity of toxigenic fungi in foods, fungal physiology and mycotoxin
production, polyphasic approach to biosystematics of Aspergillus species. E-mail:
marta@ital.sp.gov.br
Valéria Christina Amstalden Junqueira was a scientific researcher at the Food

Technology Institute (ITAL) at the Center of Quality and Food Science (CCQA)
in Campinas, SP, Brazil from 1988 to 2016 and Director of the Microbiology
Reference Laboratory at ITAL from 2011 to 2015. She graduated in Biology and
has PhD in Food Technology from the State University of Campinas (UNICAMP,
Brazil) in the area of hygiene and legislation of foods. She has been a member of
the Brazilian delegation at the Codex Committee on Food Hygiene (CCFH)
since 2002. Her major research activities are on the control of the microbiological
quality of food with an emphasis on anaerobic bacteria, spoilage microorganisms
of processed foods, microbiological quality of water and non-alcoholic beverages.
E-mail: valeriacaj@gmail.com.
XXVIII About the authors
Neliane Ferraz de Arruda Silveira is a scientific researcher at the Food Techno-

logy Institute (ITAL) at the Center of Quality and Food Science in Campinas,
Brazil. She is a biologist with PhD in Food Technology from the State University
of Campinas (UNICAMP, Brazil), in the area of hygiene and legislation of foods.
Her major research areas are the control of microbiological quality of food with
emphasis on fish and fish products, minimally processed vegetables, foods served
in collective meals, meat products and bacteriological quality of drinking water.
E-mail: neliane@ital.sp.gov.br
Margarete Midori Okazaki is a scientific researcher at the Food Technology

Institute (ITAL), at the Center of Quality and Food Science in Campinas, Brazil.
She graduated in Food Engineering and has a Master Science degree (Msc.) in
Food Technology from the State University of Campinas (UNICAMP, Brazil).
She is vice director of the Microbiology Reference Laboratory of the Food
Technology Institute and concentrates her activities on the control of the micro-
biological quality of food and water and on technical training focused on micro-
biological examination methods. E-mail: okazaki@ital.sp.gov.br
Renato Abeilar Romeiro Gomes is a scientific researcher at the Food Technology

Institute (ITAL), Campinas, Brazil. He graduated in Agricultural Engineer and
has a Master’s degree in Agricultural Engineering from the Federal University of
Viçosa, with MBA specialization. He is currently a researcher at the Dairy Tech-
nology Center of the Food Technology Institute. E-mail: rarg@ital.sp.gov.br.
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[785] ‘Subieron allá dos vigas rollizas para desde alli echarlas sobre las casas
reales y hundirlas.’ Sahagun, Hist. Conq., 30. Peter Martyr supposes the temple to
have been long held by the enemy, but this is contrary to what Cortés and
Sahagun say.
[786] Three hundred, says Gomara.
[787] For a full description of this pyramid see Native Races, ii. 579 et seq. Some
horses had been taken to clear the approaches, but they slipped on the smooth
pavement, and were sent back as unserviceable. Bernal Diaz, Hist. Verdad., 105.
[788] ‘Acometiola tres o quatro vezes, y otros tantos dias,’ is Gomara’s

interpretation, in order to fill up the time assumed by him. Hist. Mex., 156.
[789] Ojeda appears to be the sole authority upon which Herrera relies for these
two struggles. dec. ii. lib. x. cap. ix. Clavigero doubts them; yet there is nothing
unlikely in either attempt.
[790] It was related afterward that when the natives first sought to remove the
virgin image their hands clove powerlessly to it for some time, and left their marks
upon it. Oviedo, iii. 510. Montezuma, being told of this miracle, ordered the image
to be left in its place. Afterward, ‘pareciò, segun supimos, que el gran Monteçuma
tenia ó deuocion en ella, ó miedo, y la mandó guardar.’ Bernal Diaz, Hist. Verdad.,
104, 102. Others, as will be shown, suppose it to have been saved by its owner,
Villafuerte, perhaps when Cortés withdrew the troops from the temple, or to have
fled by its own miraculous power to the shrine at Remedios.
[791] ‘Comierõ de los caualleros Mexicanos muertos.’ Herrera, dec. ii. lib. x. cap.
ix.
[792] Cortés, Cartas, 130-1. According to Bernal Diaz the sally with the engines
was directed against the temple, which he appears to place at some distance. It
was held by 3000 or 4000 Indians, ‘all chiefs,’ and cost the Spaniards 46 lives,
every man being beside wounded. They returned hard pressed by the enemy. ‘Se
mostrò Cortes mui varõ, como siẽpre.’ Hist. Verdad., 103-4. ‘Murieron todos
quinientos Indios, como valientes.’ Gomara, Hist. Mex., 157. ‘En trecientos
caualleros que alli estauan no quedaron seys viuos.’ Herrera, loc. cit. This author
describes on a later occasion the capture of a tower attached to Montezuma’s own
palace, from which missiles fell with telling effect. Cortés goes with 200 men to
reduce it, and is hotly received; yet the Indians, relying upon the execution to be
made by some loose beams which are to be rolled down upon the assailants at a
favorable moment, allow the Spaniards to rush forward and gain the tower, putting
almost every occupant to the sword. This story is probably a version of the temple
fight.
CHAPTER XXV.
DEATH OF MONTEZUMA.
June, 1520.
A Living Death—The Old Imperial Party and the New Power—Aztec

Defiance—Perilous Position or the Spaniards—Disappointment to
Cortés—Another Sally—The Dying Monarch—He has no Desire to Live
—His Rejection of a New Faith—He will None of the Heaven of the
Spaniards—Commends his Children to Cortés—The Character of
Montezuma and of his Reign.
Long before this the Spaniards had learned that the power which
had arisen in Montezuma’s stead was of a different quality from that
lately wielded by the poor caged monarch, whose proud spirit they
had so blighted and brought low. No Quetzalcoatl or other
personage, fair or dark, heaven-descended or of import infernal,
might now interpose to prevent the killing and cooking of the
strangers. Cortés had thought that the late spoliation of idols would
fill the people with awe toward beings so superior to their gods. But
when he threatened that if they did not lay down their arms not a
man of them should remain alive, nor one stone be left on another
throughout all their city, they laughed at him, the priests abetting.
“How speak you so foolishly,” they said, “mortal as we now know you
to be, when for every Spanish life we are prepared to sacrifice, if
need be, twenty-five thousand of our own lives?” They had cut off
retreat at the causeways, so that the lake alone was open to exit,
and here they were prepared with fleets of canoes filled with resolute
men. Even should the Spaniards hold out against assault, hunger
and thirst must overcome them in the end. “The truth of this was too
evident,” observes Cortés, “for hunger alone would have soon killed
us.”
The imperial party, which had sunk to insignificance since the
elevation of Cuitlahuatzin to the leadership, and was now sustained
only by a few relatives of Montezuma, had no longer a voice in the
direction of affairs. Their efforts to make terms with the Spaniards
might have gained public approval, but the ambition of Cuitlahuatzin
stood in the way of any compromise. To release the strangers would
be to restore Montezuma, and he preferred to occupy the throne
himself. He was also covetous of military fame; and knowing the
desperate condition of the besieged, he hoped by their reduction to
add to his record of glorious achievements.[793]
The soldiers felt the peril of their position more than the general.
They had been cheered for a moment by victory, only to find how
barren it was; only to realize that many such triumphs would prove
their ruin. In order to counteract this growing despondency, Cortés
resolved on a night sally with half his force. The Indians being
unprepared for this, the party advanced with comparative impunity,
destroyed several barricades, and fired a large number of houses
along the Tlacopan road, where the roof assault had been so severe.
The warriors having finally gathered in sufficient force to render
retreat advisable, the Spaniards destroyed a number of buildings in
the vicinity of their quarters before entering, and thus secured
additional immunity.[794]
The present purpose of the Spaniards was to open an exit from
the city. At a council, called to consider the situation, it was admitted
that delay would only reduce their strength without corresponding
gain, and with the prospect of closing more effectually the gate
against them.[795] It was a great disappointment to Cortés thus to
abandon his hard-earned advantage. There were those who would
exult over his misfortunes, and never could he hope to win favor
from the king except by some brilliant success. But this he would yet
achieve, God willing, or perish in the attempt.
The engines were strengthened, and every preparation was
made to meet the rapidly accumulating difficulties. At dawn a large
force set out in the direction of the Tlacopan causeway to secure its
approaches.[796] The advance was made in the order of the day
previous, with guns and pioneers, and with cavalry in front and rear.
The late destruction of houses proved of no considerable advantage,
but the cannon being brought to play on the barricades, an opening
was soon made. The engines, with their fortified sides and covers,
proved more efficient than formerly in checking assaults from the
roofs. The soldiers accordingly advanced with firmer resolution, and
although the showers from the house-tops were still troublesome,
and resistance on the streets was as fierce as ever, yet one after
another the first four canals were captured. The nearest houses
were razed, and with the débris roadways were thrown across the
channels.
These operations were carried on in the face of a bitter
onslaught, and occupied the entire day. Evening being at hand the
crossings were left in charge of a strong guard, composed of the
freshest men, while the rest returned to the fort.
Montezuma the while lay a-dying, prostrate a-dying, not as
Vespasian would have an emperor die—standing; but with manhood,
and the aspirations of man, ay, even the regrets and remorse
incident to foiled endeavor, all crushed he was killed when the insults
of his people fell upon him; he scarcely heeded their darts and
stones.
It is not necessary always that breath shall cease before one can
be dead. From Ianthe’s spirit fell the shackles of sense, the body
being left with its animal life, but soulless. And though corporal life
was yet present in Montezuma, the soul was already free: the
accursed aliens had done their worst. When the might of sacred
sovereignty was extinguished, the remains were less than man,
though they walked, and talked, and wept.
Compared with his present condition, how dignified and happy
death would have been by the hands of his brother priests, before
the gods, in the eyes of the nation, on the sacred sacrificial stone!
Or, like that among the Massagetæ, told of by Herodotus, who
sacrificed and ate their old people, holding natural death a
misfortune—even this or any other stepping down and out would
have been preferable to thus dying like a silly hare in a trap!
He refused food and any attention to the wounds, which were far
from fatal. He tore off the bandages, threw from him all medicines,
and bared his body to disease, even as his soul had been long since
bared, and stretched out his hand to hasten the cold stony grasp of
death. What a farce was life, and honor, and majesty, all to end in
poverty and disgrace! Feeling the all-changing moment at hand, he
summoned Cortés; for despite his long maltreatment he entertained
a kind of affection for the monster, who might even yet prove to be
the demi-god of some far away incomprehensible world. Moreover,
the Spaniard’s intellect and arm were the stronger; he was his son-
in-law and probable successor; therefore, though his jailer, he would
speak with him. And when he came Montezuma said: “The end for
me approaches, Malinche; it is even here. You cannot harm me
further, nor help me if you would. I have given you all; you have
taken all—my liberty, my kingdom, my life, and that which is more to
me than kingdom, liberty, or life, the affection of my people, the love
of my counsellors and friends; and respect—respect of self, and that
sacred respect which, living or dead, is mine by inheritance, and by
virtue of my office. But I would not upbraid you; I pray only that my
ruin will benefit you; I beg of you care for my children, and I conjure
you to avenge me on my rebel subjects and their leaders.”[797]
Moved by the touching appeal, Cortés promised all that was
asked of him, while remonstrating with the monarch for rejecting food
and medicine. Montezuma then, in like manner, exhorted his nobles
who were prisoners with him, and was touched by their sorrow for
the sad state of the empire, and their manifestation of affection for
himself. Father Olmedo, who had never relaxed his efforts for the
captive’s conversion, now pressed to his aid the general. But in vain.
All else these beings maledict had taken from him; they should not
now rob him of his religion. His faith was as dear to him, as true, as
pure, as efficacious, as was theirs to them. Away with another’s
gods! Let each live and die by his own. He was high-priest, too, and
for him to prove recreant to the national faith would overshadow all
his former crimes combined. “What is this they would have of me?”
he groaned within himself. Then turning suddenly to Olmedo, he
asked, “Do Spaniards go to this heaven of yours?” “Assuredly,” was
the reply; “it was made for them, and is held by Christians, against all
others, as the reward of their pure belief and gentle deeds.” “It is
enough; I will none of it,” said Montezuma, who from that moment
would not listen to a word of Christian exhortation.[798] It was early in
the morning of the 30th of June,[799] three days after the trying
scene in the presence of his people, that the monarch breathed his
last. And even the Spaniards forgot for a moment their diabolisms,
and allowed their minds to dwell on the virtues of this magnificent
heathen, this mighty sovereign, their sweet-tempered prisoner, and
kind and generous host.[800]
Of a truth, despite his pusillanimity with regard to the Spaniards,
which was indeed little else than pardonable superstition, this man
was in many respects not unworthy the title of Great so freely
bestowed upon him. Montezuma was but forty-one[801] at the time of
his death, and had wielded the sceptre for nearly eighteen years with
wonderful success. Under him the Aztec empire acquired its widest
extent and greatest glory. While his armies by well directed
operations spread the terror of his name to distant provinces and
increased the national domain by fresh conquests, his subtle
intrigues secured advantages at home, and established the
supremacy of Mexico in the tripartite alliance. With a high regard for
the dignity of his throne, he caused the sovereign to be worshipped
almost like a god, and sustained the grandeur of his surroundings
with lavish expenditure. This severe and ostentatious pride kept him
above the reach of his people, and failing to understand their wants
or to sympathize with their condition, he ruled not by love, but by
fear. Thus it is that we find the native records dwell upon his fitful
cold-blooded cruelty and superstition, not as a tyrant, however, but
as an administrator of their own cruel yet revered rites. He was
reputed just, but this quality was to be found rather in the intention
than in the act. With all his pride he appears to have been most
affable and kind to those with whom he came in contact. The
Spaniards certainly found him so. In their later intercourse other
considerations may have ruled him, however, and with the cunning
and secrecy of his race he may have submitted to the inevitable
demands of circumstances.[802]
Surrounded by fawning ministers, whose existence depended on
his favor, he was encouraged in the extravagant habits of a
magnificent court, which promoted their schemes at the expense of a
tax-ridden people. The ambition to extend his fame and power
required the maintenance of immense armies, of numerous
garrisons, and of costly campaigns, which proved another drain on
the people. This was augmented in subjected provinces by the
extortions of imperial officers, who found means to prevent the cry of
the oppressed from reaching the throne. Perhaps the most terrible
infliction was the levy on the youth of both sexes for slaves, and for
sacrificial victims to appease the bloody appetite of Aztec gods—an
appetite which had increased in horror with the abject superstition of
this otherwise enlightened monarch. Enlightened he undoubtedly
was, for as high-priest he had become versed in the higher learning
of the priesthood. The study of mythology came naturally to him,
while astronomy and natural history were favorite subjects with the
lords of the lake peoples, the former connected with myths and
divinations, the latter illustrated by specimens from different regions,
and collected in the botanic and zoologic gardens of Mexico and
other cities. The studies of his youth had gained for him a well
merited respect from his priestly confrères, and the prudence and
sagacity which controlled the well stored mind commanded attention
in the council.[803] While yet a young man there seemed to develop
qualities which fitted him for the position of high-priest, also as
counsellor, to which his princely rank paved an easy way. Besides
this he had shown himself possessed of great courage, and had
established his fame as a general by many victories.
It was with this reputation, as zealous and learned priest,
prudent statesman, and brave soldier, that he ascended the throne in
1503, while only twenty-three years of age. It is in such terms that
his colleague Nezahualpilli in his coronation address refers to the
hopes entertained of the youthful ruler.[804] Though ever a devout
servant of the gods, the effeminate pleasures of the court weakened
the nerves and energy of the soldier, till his warlike ardor survived
only in a taste for military reviews and for the chase. The caution of
the general remained, but timidity saves few leaders from disaster.
Vanity and designing ministers overruled too often the dictates of
wisdom in the administration of affairs.[805] His path had been
prepared by able predecessors, and answered well for the policy of
aggrandizement which became the leading feature of his reign. In
this his natural liberality and talent for intrigue, fostered by priestly
training, served him well and procured blindly devoted instruments
for his plans. Thus, by fair means and foul, the empire was raised to
the pinnacle of its glory, but not being of a natural or healthy growth it
proved unstable, and crumbling under the strong commotion created
by the approach of Castilians, it revived only for a moment in the
present uprising like the mental illumination preceding death.
Montezuma could not have failed to recognize the insecurity of the
bonds which held it, and influenced by the predictions of its downfall
he readily fell beneath the spell of the superior intellects which were
to assume control. It was his misfortune to have lost the sanguine
energy of his youth, which might have enabled him to rise above the
weaknesses of himself and his age. Duty and honor were overcome
by superstition and absorbing love of power, of life, and he reaped
the natural fruit of puerile and misdirected efforts by losing both.
Resistance might not long have delayed the inevitable, but it would
at least have procured for him an end worthy of his grandeur.
Of his many wives may be named the princesses Teitlalco,
Acatlan, and Miahuaxochitl, of whom the first named appears to
have been the only legitimate consort.[806] By her he left a son,
Asupacaci, who fell during the noche triste, and a daughter,
Tecuichpo, baptized as Isabel, married consecutively to
Quauhtemotzin, the last Mexican sovereign, to visitador general
Alonso Grado, to Pedro Andrade Gallego, and to Juan Cano de
Saavedra. She had children by the latter two, from whom descend
the illustrious families of Andrade-Montezuma and Cano-
Montezuma.
By the Princess Acatlan were left two daughters, baptized as
María and Mariana. The latter alone left offspring, from whom
descends the Sotelo-Montezuma family. By the third wife came to
the emperor the son Tlacahuepantzin, known after baptism as Pedro
Yohualicahuacatzin Montezuma, whose descendants, the condes de
Montezuma y de Tula, intermarried with the noblest families of
Spain, and connected the name with the highest offices of state, and
with the title of grandee.[807]
FOOTNOTES
[793] In Manuscrit de 1528, Aubin Col., Cihuacohuatl and Tzihuacpopocatzin,
brothers of Montezuma, are named as the leaders of the opposition party,
connected also with the followers of Quetzalcoatl, who abhorred the cruel rites of
the Aztecs. They succeeded, it is said, in introducing provisions for the besieged.
Brasseur de Bourbourg, Hist. Nat. Civ., iv. 317-18. But this is doubtful.
[794] This is probably the sally which Herrera intrusts to Salcedo. dec. ii. lib. x.
cap. viii.
[795] ‘Por importunidad de muchos ... acordó de salir della.’ Carta del Ejército, in
Icazbalceta, Col. Doc., i. 429. ‘El ... capitan dilataba de cada dia la dicha salida,’
but the officers insisted that he should leave. Segunda Probanza de Lejalde, in Id.,
423.
[796] Clavigero directs the operation against the Iztapalapan road, which would
have been a useless manœuvre. Cortés is explicit enough on the point.
[797] In a privilegio in favor of the daughter Isabel, Cortés refers to this interview,
saying that three daughters were intrusted to him. No allusion is made to any son.
The affectionate terms in which he herein speaks of Montezuma must be due to
political reasons, and perhaps to a regard for the princesses. Panes, Vireyes, in
Monumentos Domin. Esp., MS., 67-8. Ramirez ridicules the idea of an appeal by
the emperor to Cortés, who was on unfriendly terms with him. Soc. Mex. Geog.,
Boletin, x. 359 et seq. But the effort of Montezuma to save the Spaniards shows
that intercourse had been reëstablished; and was not Cortés, as the husband of
one or more of his daughters, the proper person to protect their sisters?
[798] The question of his conversion has been much discussed. ‘No le pudo atraer
a que se bolviesse Cristiano,’ says Bernal Diaz, Hist. Verdad., 105; and Herrera is
even more explicit. dec. ii. lib. x. cap. x. Cortés and his followers, Martyr and
Oviedo, give no indications to the contrary. Ixtlilxochitl states, however, that he had
learned several prayers, and even begged for baptism, but that the rite was
deferred ‘por la pascua siguiente, que era de la resurreccion, y fue tan
desdichado, que nunca alcanzó tanto bien.’ Hist. Chich., 299. Yet he adds that it
has been said ‘que se bautizó y se llamó Don Juan.’ Relaciones, 457. According
to Gomara he asked for baptism in the beginning of lent, but it was determined to
postpone the rite till pentecost, for greater effect, and as more appropriate for so
high a personage. Owing to the troubles arising out of Narvaez’ arrival nothing
was done then, ‘y despues de herido oluidose con la prissa del pelear!’ Hist. Mex.,
154. Cortés had persuaded him, says Vetancurt, during the early days of his
imprisonment, to accept baptism, and he was taken to the temple for the purpose,
but at the last moment he excused himself on the ground that the Indians would
elect another lord and attack them all if he abandoned the faith. Teatro Mex., iii.
132-3. Father Duran, on the contrary, ever zealous for the natives, and particularly
for his hero, Montezuma, asserts that trustworthy men had vouched for his
baptism. Padre Olmedo had also expressed belief therein, although he had not
been present when it was administered. Hist. Ind., MS., ii. 445. The father’s
memory must have failed him with respect to Olmedo. Camargo has also been
assured of his baptism, with Cortés and Alvarado for sponsors. Hist. Tlax., 166.
Tezozomoc, who claims to have investigated the point, declares explicitly that the
rite was administered on his death-bed, when he received the name of Cárlos, and
that Cortés, Olid, and Alvarado were the sponsors. Recopilacion de verídicas
tradiciones, probando que el emperador Moteuhsoma recibió el santo sacramento
del bautismo. This author wrote at the close of the sixteenth century, and follows
traditions only. Bustamante, in modern times, has also reviewed the question, and
follows Tezozomoc implicitly. In support thereof he quotes a poem, by Captain
Angel Betancourt, wherein he refers to Montezuma as the ‘indio bautizado,’
introduces the vague utterance of Ixtlilxochitl, and even attempts to misconstrue a
certain expression of Cortés. Montezuma tells the latter to baptize his daughters,
and this Bustamante regards as proof that he himself desired the rite. He does not
suppose that the religion of the vicious Spaniards could have had great attractions
for him, but when about to die he accepted it, ‘as the drowning man does the
saving plank.’ Article in Chimalpain, Hist. Conq., i. 287-95. Still fuller is the review
of Ramirez, who follows Torquemada in pointing out the fact that neither Cortés
nor Alvarado ever referred to their supposed sponsorship, as they certainly would
have done in connection with so distinguished a personage had they felt
empowered. Soc. Mex. Geog., Boletin.
[799] Cortés’ chronology, as indicated in the Cartas, shows clearly that he left
Mexico during the night of June 30th, as will be demonstrated. He also implies
what Bernal Diaz and Herrera distinctly assert, that considerable fighting took
place on the Tlacopan road between the time the corpse was surrendered to the
Aztecs and the Spaniards returned to their quarters preparatory to evacuation.
Hence the death must have occurred early on that day. Herrera confirms Cortés’
testimony that he could not have died before the 30th, by saying, ‘en quatro dias
se murio.’ dec. ii. lib. x. cap. x. It is also generally admitted that he was not
wounded before the third day of the siege.
[800] ‘Cortes lloró por èl, y todos nuestros Capitanes, y soldados: è hombres huvo
entre nosotros ... que tan llorado fue, como si fuera nuestro padre.’ Bernal Diaz,
Hist. Verdad., 104. Cortés speaks highly of him, ‘y siempre tuvo mui buena
voluntad á los Españoles,’ but this is in the deed presented to his daughter. See
Privilegio, Monumentos Domin. Esp., MS., 66. In the Cartas he is referred to
merely as a captive who dies. After saying that he never consented to the death of
a Spaniard nor to injury against Cortés, Gomara adds: ‘Tambien ay quien lo
cõtrario diga.’ Hist. Mex., 154-5.
[801] Clavigero says 54, but Bernal Diaz, who was so much in his company, could
hardly have been mistaken, and the comparative youth of his children also
indicates that 41 is more correct.
[802] ‘Antes ni despues huvo en este mundo quien le igualase en magestad y

profanidad ... fue muy justiciero ... de condicion muy severo, aunque cuerdo y
gracioso.’ Ixtlilxochitl, Hist. Chich., 301. ‘Dizen los Indios que fue el mejor de su
linaje, y el mayor rey de Mexico.’ Gomara, Hist. Mex., 155. ‘Fu circospetto,
magnifico, liberale ... sua giustizia degenerava in crudeltà.’ Clavigero, Storia
Mess., iii. 132; Herrera, dec. ii. lib. x. cap. x.; Torquemada, i. 499-500.
[803] ‘Fue muy sabio, pues passaua por las cosas assi, o muy necio q̄ no las
sentia.’ Gomara, Hist. Mex., 155. ‘El hombre mas sábio de su siglo,’ is
Bustamante’s interpretation. He also discovers that Montezuma objected to
sacrifices! Chimalpain, Hist. Conq., 292-3.
[804] See Native Races, ii. 149-50.
[805] ‘Pareva aver cangiato di sesso, siccome dicevano i suoi sudditi.’ Clavigero,
Storia Mess., iii. 132.
[806] So Cano, the son-in-law, declares, Oviedo, iii. 549, and so Cortés intimates
in the privilegio to Isabel, whom he calls ‘su legitima heredera,’ especially
commended to him.
[807] Of the rest of the imperial wives and concubines nothing positive is known,
save that a number of them and their daughters were liberally bestowed, as a
mark of imperial favor, on prominent personages, including Spaniards. After the
conquest they sank into obscurity, although some of them still managed to
maintain a certain consideration among the natives, despite their poverty. Bernal
Diaz claims that he received one of the concubines; upon Olid was bestowed a
daughter, and upon Cortés two, it is said, one baptized as Ana, the other as Inés.
Two witnesses declare that Isabel also ‘cinco meses questava casada con ...
Gallego e que pario una fija y que hera del ... Cortés.’ Cortés, Residencia, ii. 242,
244; i. 63, 99, 221, 263. The three daughters confided to Cortés on their father’s
death-bed were not in the Spanish quarters at the time, at least not all of them, but
were found after the conquest and baptized. The eldest and legitimate, the
attractive Tecuichpo, was then the wife of the last and captive sovereign,
Quauhtemotzin, her cousin, who had married her chiefly with a view to strengthen
his hold on the throne, for she was too young for the married state. She was
baptized as Isabel, and her Indian husband having been executed, Cortés, on his
return from Honduras, gave her in marriage to the hidalgo Alonso Grado, of
Alcántara, who had succeeded Ávila as contador, and now held the position of
visitador general of New Spain. In consideration partly of Grado’s services and
partly of Isabel’s rank, the captain-general bestowed as dower, in the emperor’s
name, the town of Tacuba (Tlacopan), with the villages and farms subject to it,
together with the title of señora thereof. The deed, which recounts the services of
her father and the intrusting of his daughters to Cortés, is signed by him as
captain-general and governor of New Spain, and dated June 27, 1523. It is given,
among other books, in Monumentos Domin. Esp., MS., 65-8. Grado dying soon
after, without issue, she married Pedro Andrade Gallego, by whom she had one
son, Juan Andrade, the founder of the Andrade-Montezuma family. This branch
inherited the Villa Alta villages, in Oajaca, and other estates, which in 1745 were
bought up by the crown for a pension of 3000 pesos, continued by the Mexican
government in irregular payments. A member of this branch was the bishop of
Chiapas a few years later. Certificacion de las Mercedes, MS., 14-18. M. Fossey
describes a visit, in 1849, to the poverty-stricken yet proud descendants. Mexique,
497-500. The omission of Gallego’s middle name has led the critical Alaman,
among others, to assume that this family descends from Isabel’s fifth marriage
with Juan Andrade. Prescott’s Mex. (Mex. 1844), ii. 31. Nor is Prescott free from
error in connection with Montezuma’s descendants. The Andrade branch became
allied to the Condes de Miravalle, and a daughter of this house was the wife of
General Barragan, who became presidente interino of the republic, thus raising a
descendant of Montezuma once again to the supreme place in the country. The
Princess Isabel was married a fourth time, to Juan Cano de Saavedra, by whom
she had five children, the inheritors of the Tacuba estates, also exchanged for a
pension which was continued by the republic. Of the Princess Acatlan’s two
daughters, María and Mariana, the former left no issue. Mariana married the
conquistador Juan de Paz, bringing a dower of three towns, and after his death
she took for husband the conqueror Cristóbal de Valderrama. By him she had a
daughter, Leonor, who, marrying Diego Arias Sotelo, gave origin to the Sotelo-
Montezuma family. Fonseca, Hist. Hacienda, i. 464. This work, with its collection of
official papers and extracts, gives a mass of information about the imperial
descendants and estates. Prescott confounds the mother and daughter. Mex., ii.
351-2. Viceroy Mendoza, in a despatch to the emperor of December 10, 1537,
refers to the death, three weeks before, of Valderrama, and speaks of children by
the former husband, which are not admitted in Fonseca. Pacheco and Cárdenas,
Col. Doc., ii. 208. Cortés refers to three sons of Montezuma: the heir, who fell on
the causeway during the noche triste, and two surviving boys, ‘one said to be
insane, the other paralyzed.’ On leaving Mexico he took with him one son and two
daughters, his concubines probably, all of whom perished. Cartas, 135, 153.
Sahagun names two sons, who perished on that occasion. Hist. Conq. (ed. 1840),
122, 126. Ixtlilxochitl gives them different names. Hist. Chich., 302. Cano gives the
name Asupacaci to the heir, or only legitimate son, the brother of his wife Isabel,
and states that he was killed by Quauhtemotzin, who feared him as the only rival
to the throne. Oviedo, iii. 549. Brasseur de Bourbourg follows him, but prefers the
name of Cipocatzin for the young prince, while Axayoca is also applied. Cortés’
version is more likely to be correct, however. One of the surviving sons, ‘Signor di
Tenajoccan,’ Clavigero, Storia Mess., iii. 133, was baptized with the intervention of
his sponsor, Rodrigo de Paz, and died three years after the conquest, ‘y se enterrò
en la Capilla de San Joseph.’ Vetancvrt, Teatro Mex., pt. iii. 144. This author
assumes that the youth fled with the Spaniards from the capital and hid at
Tepotzotlan. The other prince, son of Miahuaxochitl, daughter of the lord of Tula,
and niece of Montezuma—baptized as María, says Vetancurt—received the name
of Don Pedro. He accompanied Cortés to Spain in 1528, it appears, at the age of
eighteen, and made repeated appeals to the emperor for a maintenance in
accordance with his rank. At first some trifling favors were granted, and he,
together with a cousin, was educated by the Franciscans in Madrid. Puga,
Cedulario, 85. President Fuenleal, of the audiencia, and other prominent persons
having added their recommendation, regular pensions and encomiendas were
bestowed, including the town of Tula, the seat of his maternal grandparents, upon
which was based the second title of Condes de Montezuma y de Tula, conferred
on his grandson. The line expired on the male side with the great great-grandson
of the emperor, whose daughter married Sarmiento de Valladares, duke of Atlixco,
and viceroy of New Spain, thus raising the name again to the highest position in
the country. Prescott, following Humboldt, Essai Pol., i. 191, 203, calls Valladares,
by mistake, a descendant of Montezuma. The cousin of the vice-queen married
Silva, the first marquis of Tenebron, whose descendants inherited the title and
estates from the other branch, and became grandees in 1765. Their pension
amounted at this time to 40,000 pesos, says Berni, Titulos de Castilla, which
represented in part the encomiendas withdrawn by the government. The republic
recognized this portion, as it had the pensions to the other branches. Shortly after
the independence of Mexico the holder of the title, Alonso Marcilla de Teruel
Montezuma, came over with the intention of asserting his claim to the throne of his
forefathers, but the prudent possessors of the power thought it best not to admit
him, and he passed on to New Orleans, there to put an end to his life some years
later. Prescott understands that the septuagenarian had been disappointed in love.
Mex., ii. 352. Several members of the Spanish nobility have intermarried with this
line, among them a branch of the Guzman family, whence the claim made for the
consort of Napoleon III. of having Montezuma’s blood in her veins. Gondra gives a
portrait of a member married into the Mendoza family. Prescott’s Mex. (ed. Mex.
1845), 219. One of the line, Padre Louis de Montezuma, wrote the Historia del
Emperador, which has been consulted by Alaman, Disert., i. app. ii. 158. Clavigero
gives a genealogic table in Storia Mess., iii. 235, and Carbajal, while plagiarizing
the statements and blunders of others, adds a few of his own. Hist. Mex., ii. 378-
88. In Fonseca, Hist. Hacienda, i. 455 et seq., are to be found several valuable
extracts concerning titles and estates; also in Reales Cédulas, MS., i. pt. i. 5, ii. 4
etc.; Certificacion de las Mercedes, MS.; Mex. Mem. Hacienda, 1848, 35-6;
Fuenleal, Carta, in Pacheco and Cárdenas, Col. Doc., xiii. 222. The family name
has been spelled in different ways, also by its possessors, as Motezuma,
Muteczuma, Moctezuma, Mocthecuzoma, Motecuhzuma, Moteuhzuma; but
Montezuma is the most common form.
The Historia de las Indias de Nueva-España y Islas de Tierra Firme, by Father
Diego Duran, is claimed by its author, in the introductory to chapter lxxiv., to be
devoted essentially to the life and rule of this monarch, ‘cuya vida é história yo
escribo.’ The preparation of the work was more directly prompted by a
compassion for the maltreated natives, whose champion he constituted himself, in
common with so many of the friars. This spirit led him naturally to color the
occurrences of the conquest; and a non-critical acceptance of whimsical legends
and statements in favor of his protégés tends further to reduce the value of the
work. His deep interest in the aborigines and their history may be explained by the
fact that he was born at Tezcuco, of a native mother. Franco wrongly calls him
Pedro, and Clavigero, Fernando. He professed as a Dominican at Mexico, in
1556, with missionary aspirations, no doubt, but a delicate constitution and
constant suffering confined him rather to the monastery, and directed his efforts to
researches and writing. Castellanos, Defensa, 28, attributes several works to him,
and Eguiara, Bib. Mex., 324, the compilation of the Dominican history of Dávila
Padilla, though not the style and form. Dávila also, ‘scrisse la Storia antica de’
Messicani, servendosi de’ materiali raccolti già da Ferdinando Duran Domenicano
da Tezcuco; ma questa opera non si trova.’ Clavigero, Storia Mess., i. 13. But this
may be a mistake. A similar rewriting would have greatly improved the Historia de
las Indias, which is exceedingly unpolished and slovenly, full of repetitions and bad
spelling, and showing great poverty of expression. On the other hand, it is relieved
by an admirable portrayal of character and knowledge of human nature, and by a
minute study of the effect of conversion on the natives. The work consists of three
tratados, the first in 78 chapters, giving the history of Mexico from its origin to the
conquest, terminating with the expedition to Honduras. This was completed in
1581, while the other two were finished two years before. The second tratado, in
23 chapters, treats of Mexican divinities and rites, and the third, in two, or more
properly nineteen, chapters, of calendar and festivals. Padre Duran died in 1588,
leaving the manuscripts to Juan Tovar, Dávila Padilla, Hist. Fvnd. Mex., 653, who
gave them to Acosta, then occupied in preparing his Natura Novi Orbis, and other
works. The contribution came most opportunely, and was used chiefly for his
account of Mexico, as he frankly admits, though giving the credit to Tovar, who
may have claimed the authorship. On the strength of this statement Clavigero,
with others, confirms the claim to the ‘nobilissimo Gesuita Messicano.’
Torquemada, i. 170-1, ii. 120, himself not spotless, takes advantage of the
confession to rail at Acosta for borrowed plumage, mutilated at that. The
manuscripts, now in the Biblioteca Nacional de Madrid, are written in double
columns and illustrated with numerous plates. Pinelo, Epitome, ii. 711, refers to
them as in two parts. A few copies have been taken, mine forming three volumes.
A set obtained by José Fernando Ramirez, one of Maximilian’s ministers, was
prepared by him for publication, but, owing to the death of the imperial patron, only
the first 68 chapters were issued at Mexico, 1867, in one volume, with notes and
considerable changes of the style. This mutilation, as some term it, may have
been a reason for the seizure of the whole edition, together with the separate
plates, by the republican government. Only a few copies escaped this fate, one of
which I succeeded in obtaining. Although independent issue was long withheld
from Duran, he has at least enjoyed the honor of being associated with one
possessed of far greater fame than he himself could ever hope to achieve.
The motives which impelled Joseph de Acosta to write on America were quite
pretentious. Among the many Spanish books on the New World, he says: ‘I have
not seene any other author which treates of the causes and reasons of these
novelties and wonders of nature, or that hath made any search thereof. Neither
have I read any booke which maketh mention of the histories of the antient
Indians, and naturall inhabitants.’ With a view to repair these omissions he issued
De Natvra Novi Orbis libri dvo, et de Promvlgatione Evangelii, apvd Barbaros, sive
de Procvranda Indorvm Salvte Libri sex. Salmanticœ, 1589. The first part, De
Natura, is a philosophic dissertation on physical features, on the probable
knowledge among the ancients of a western hemisphere, and on the origin of the
Indians. The second part, in six books, bearing a separate imprint under 1588,
though published only in connection with the previous two books, treats entirely of
the method and progress of Indian conversion. The Natura was translated into
Spanish, and incorporated, with some amendments, in the Historia Natvral y moral
de las Indias, Sevilla, 1590, dedicated to Infanta Isabel, which treats also of Indian
history and customs, and refers briefly to the conquest. The work achieved great
success, and was reproduced in numerous editions, in nearly every language,
though often without Acosta’s name, and in distorted form, as in De Bry and some
German versions. This may not be considered bad treatment by those who charge
Acosta with plagiarism, although he frankly admits following a number of authors,
among them ‘es vno Polo Ondegardo, a quien communmente sigo en las cosas
de el Piru: y en las materias de Mexico Ioan de Touar prebendado que fue de la
Iglesia de Mexico, y agora es religioso de nuestra Compañia de Iesvs. El qual por
orden del Virrey hizo dõ Martin Enriquez diligẽte, y copiosa aueriguaciõ de las
historias antiguas.’ See p. 396. There is no doubt that the interest and value of the
work are owing chiefly to the circumstance that the original authorities have
remained sealed, until lately at least; for, despite its pretentious aim, the pages are
marred by frequent indications of the then prevalent superstition and credulity. The
Procvrando Indorvm Salvte is more in consonance with the character of the Jesuit
missionary and scholastic.
Born at Medina del Campo about 1539, he had in his fourteenth year joined
the Society, to which four brothers already belonged. After studying and teaching
theology at Ocana, he proceeded in 1571 to Peru, where he became the second
provincial of his order. Returning to Spain seventeen years later—‘post annos in
Peruano regno exactos quindeciem, in Mexicano & Insularibus duos,’ says the
dedication of 1588 to Philip II., in De Natvra of 1589—he gained the favor of the
king, occupied the offices of visitador and superior, and died as rector at
Salamanca, February 15, 1600. Several other works, in print and manuscript,
chiefly theologic, are attributed to him—see Camus, 104-13—among them De la
criança de Cyro, dedicated to Filipe III. in 1592, which was also a borrowed text,
from Xenophon, and remained a manuscript in the Royal Library.
CHAPTER XXVI.
LA NOCHE TRISTE.
June 30, 1520.
The Captive-King Drama Carried too Far—Better had the Spaniards Taken
Montezuma’s Advice, and have Departed while Opportunity Offered—
Diplomatic Value of a Dead Body—Necessity for an Immediate
Evacuation of the City—Departure from the Fort—Midnight Silence—
The City Roused by a Woman’s Cry—The Fugitives Fiercely Attacked on
All Sides—More Horrors.
And now what must have been the feelings of the invaders, who,
like the ancient mariner, had killed the bird that made the breeze
blow! For assuredly they were responsible for the emperor’s death.
Indeed, the direct charge of murder against Cortés has not been
wanting, even among Spanish chroniclers; but this was owing greatly
to the effort of the general to extricate the army from its desperate
situation while the enemy was supposed to be distracted by grief and
engaged in solemn obsequies. We may be sure, however, that the
Spaniards did not kill Montezuma; that they did not even desire his
death; but regarded it at this juncture as the greatest misfortune
which could happen to them.[808] For in the vast evolvings of their
fast, unfathomable destiny, they were now all like sea-gulls poised in
mid-air while following a swiftly flying ship.
It is interesting to note the manœuvring on both sides over the
dead monarch, who having ministered so faithfully to his enemies
while living, must needs continue in the service after death. The
hostile chiefs were called and informed of the sad consequences of
their outrage on the emperor. The body would be sent to them, so
that they might accord it the last honors. The leaders replied curtly
that they had now a new chief, and cared no longer for Montezuma,
dead or alive. The corpse was nevertheless carefully arrayed in
fitting robes and given in charge of two prisoners, a priest and a
chief,[809] with instructions to carry it to the Mexican camp, and
explain the circumstances of the death and the grief of the
Spaniards. On appearing outside the fort a leader motioned them
back, and would probably have used force but for the priestly
character of the bearers, behind whom the gate had been closed. A
few moments later they disappeared from view. The disrespect
shown the living was not spared the dead. As the corpse was borne
through the streets jeers and insults fell from lips which formerly
kissed the ground on which the monarch had stood. Many declared
that a coward like Montezuma, who had brought so many
misfortunes on the country, was not worthy of even ordinary burial.
[810]The imperial party managed, however, to secure the body, and,
assisted by those to whom the royal blood and high priestly
character of the deceased outweighed other feelings, an honorable
though quiet cremation was accorded in the Celpalco, where
Sahagun intimates that the ashes remained.[811]
Shortly after the body had left the Spanish quarters Cortés sent
a fresh message to the Mexicans, believing that by this time the
presence of the august dead might have had its effect on them. He
pointed out the respect due to the remains of a sovereign, and
proposed a cessation of hostilities with that view, and till they had
elected a successor, one more worthy than the present leader, who
had driven them to rebel.[812] The chiefs replied that the Spaniards
need trouble themselves about nothing but their own safety. They
might come forth, they added tauntingly, to arrange a truce with their

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Microbiological examination methods

of food and water: a laboratory manual

Food Chemistry A Laboratory Manual 2nd Edition Miller

Microbiological Methods for Environment Food and

Laboratory manual [to accompany] Physical examination &

LooseLeaf Benson’s Microbiological Applications

A Laboratory Manual in Biophotonics 1st Edition Vadim

Scientific examination of documents : methods and

Molecular microbial diagnostic methods pathways to

Social Drivers In Food Technology Vivian-Lara Silva

NEUSELY DA SILVA, MARTA HIROMI TANIWAKI,

Translation to English: Paul van Dender†

© 2019 Taylor & Francis Group, London, UK

Typeset by Apex CoVantage, LLC

Published by: CRC Press/Balkema

Library of Congress Cataloging-in-Publication Data

ISBN: 978-1-138-05711-1 (Pbk)

About the authors XXVII

1 Sampling, transport and storage of samples for analysis 1

2 Preparation of samples for analysis 9

2.3.1 Diluents for presence/absence tests 14

r) Powdered milk-based infant foods 22

3 Basic plate count techniques for enumeration of microorganisms 25

4.2 Multiple dilution test 48

5 Basic techniques for the detection of the presence/absence of microorganisms 57

5.3.5 Confirmation tests 63

6 Aerobic plate count 65

7 Yeasts and molds 81

7.1.5.4 Candida krusei 85

9 Total and thermotolerant coliforms and Escherichia coli 115

10 Staphylococcus aureus 135

10.1.1.2 The coagulase-positive staphylococci 136

11 Bacillus cereus 149

12 Clostridium perfringens 161

14 Lactic acid bacteria 189

14.1.4 Lactobacillus 192

16.3 (new) Presence/absence method BAM/FDA:2012 for Cronobacter in dehydrated powdered

18 Listeria monocytogenes 243

20 Vibrio cholerae and Vibrio parahaemolyticus 299

21 Yersinia enterocolitica 325

22 Bacterial spore count 333

22.1.2.4 Anoxybacillus 338

23 Commercial sterility 373

Facultative thermophilic aerobic spore-forming bacteria 378

24 Guidelines on preparation of culture media 399

Acetamide broth ISO 412

Columbia Blood Agar (CBA) 431

Lactose broth (LB) 451

Oxoid Chromogenic Listeria Agar (OCLA) 470

Sudan black B solution 0.3% in ethanol 70 491

Neusely da Silva is a scientific researcher at the Food Technology Institute

Marta Hiromi Taniwaki, PhD, is a scientific researcher at the Food Technology

Valéria Christina Amstalden Junqueira was a scientific researcher at the Food

Neliane Ferraz de Arruda Silveira is a scientific researcher at the Food Techno-

Margarete Midori Okazaki is a scientific researcher at the Food Technology

Renato Abeilar Romeiro Gomes is a scientific researcher at the Food Technology

[786] Three hundred, says Gomara.

[788] ‘Acometiola tres o quatro vezes, y otros tantos dias,’ is Gomara’s

A Living Death—The Old Imperial Party and the New Power—Aztec

[802] ‘Antes ni despues huvo en este mundo quien le igualase en magestad y

[804] See Native Races, ii. 149-50.

June 30, 1520.

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