Instructions and workbook for Microbiological Examination of Food laboratory

exercise s

MICROBIOLOGICAL
EXAMINATION OF
FOOD
INSTRUCTIONS AND WORKBOOK FOR MICROBIOLOGICAL
EXAMINATION OF FOOD LABORATORY EXERCISES

Barbara Jeršek

2017

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Instructions and workbook for Microbiological Examination of Food laboratory
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Barbara Jeršek
Instructions and workbook for Microbiological Examination of Food
laboratory exercises

Publisher: University of Ljubljana

Biotechnical Faculty
Department of Food Science

Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani

COBISS.SI-ID=290218240
ISBN 978-961-6908-10-8 (pdf)

Access: (URL): http/www.bf.uni-lj.si/knjiznice-odd-za-zivilstvo/ucbeniki-v-elektronski-obliki

Ljubljana, April 2017

All rights reserved. No part of this publication may be reproduced or used in any other way (graphic,
electronic or mechanical, including photocopying, recording or transfer in the database) without the
written permission of the copyright owner.

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TABLE OF CONTENTS

INTRODUCTION __________________________________________________________ 1
Preparation of food samples for microbiological examination___________________________ 1
Preparation of food basic solution _________________________________________________________ 1
Preparation of food dilutions _____________________________________________________________ 2
General procedure of microbiological examination of food _____________________________ 2
General procedure of microbiological examination of food when absence of specific microorganisms in a
specified mass (volume) of the food is required _______________________________________________ 2
General procedure of microbiological examination of food when the number of specific microorganisms in
food is evaluated _______________________________________________________________________ 3
TOTAL NUMBER OF LIVE MICROORGANISMS _____________________________ 4
Total number of live microorganisms_______________________________________________ 4
Total microbial count ____________________________________________________________ 5
Number of yeasts and molds ______________________________________________________ 5
DETERMINATION AND QUANTIFICATION OF INDICATOR BACTERIA ______ 6
AEROBIC SPORE-FORMING BACTERIA ________________________________________ 6
GENERAL ___________________________________________________________________________ 6
ISOLATION AND IDENTIFICATION_____________________________________________________ 6
ANAEROBIC SPORE-FORMING BACTERIA _____________________________________ 7
General ______________________________________________________________________________ 7
ISOLATION AND IDENTIFICATION_____________________________________________________ 7
Enterobacteriaceae, COLIFORMS, Escherichia coli __________________________________ 8
GENERAL ___________________________________________________________________________ 8
ISOLATION AND IDENTIFICATION_____________________________________________________ 9
DETERMINATION AND QUANTIFICATION OF PATHOGENIC AND TOXIN-
FORMING BACTERIA ____________________________________________________ 14
Salmonella ____________________________________________________________________ 14
GENERAL __________________________________________________________________________ 14
ISOLATION AND IDENTIFICATION OF Salmonella _______________________________________ 14
Proteus _______________________________________________________________________ 17
GENERAL __________________________________________________________________________ 17
ISOLATION AND IDENTIFICATION OF Proteus __________________________________________ 17
Listeria monocytogenes __________________________________________________________ 18
GENERAL __________________________________________________________________________ 18
ISOLATION AND IDENTIFICATION OF L. monocytogenes __________________________________ 18
COAGULASE-POSITIVE STAPHYLOCOCCI ____________________________________ 21
GENERAL __________________________________________________________________________ 21
ISOLATION AND IDENTIFICATION OF COAGULASE-POSITIVE STAPHYLOCOCCI __________ 21
Bacillus cereus _________________________________________________________________ 23
GENERAL __________________________________________________________________________ 23
ISOLATION AND IDENTIFICATION OF Bacillus cereus ____________________________________ 23
SULPHITE-REDUCING CLOSTRIDIA___________________________________________ 24
GENERAL __________________________________________________________________________ 24
ISOLATION AND IDENTIFICATION OF Clostridium perfringens _____________________________ 24
MICROBIOLOGICAL EXAMINATION OF DRINKING WATER _______________ 26

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GENERAL ___________________________________________________________________ 26
MICROBIOLOGICAL EXAMINATIONS _________________________________________ 26
Escherichia coli and coliforms (SIST EN ISO 9308-1) ________________________________________ 27
Enterococcus (SIST EN ISO 7899-2) ______________________________________________________ 28
Number of colonies (SIST EN ISO 6222) __________________________________________________ 28
Clostridium perfringens (mCP) __________________________________________________________ 28
WORKBOOK ____________________________________________________________ 29
1. EXERCISE: Total microbial count _________________________________________ 30
2. EXERCISE: Number of spore-forming bacteria ______________________________ 31
3. EXERCISE: Coliform bacteria ____________________________________________ 32
4. EXERCISE: Escherichia coli ______________________________________________ 33
5. EXERCISE: Salmonella __________________________________________________ 34
6. EXERCISE: Listeria monocytogenes ________________________________________ 36
7. EXERCISE: Coagulase-positive staphilococci ________________________________ 38
8. EXERCISE: Bacillus cereus _______________________________________________ 39
9. EXERCISE: Sulphite-reducing clostridia ___________________________________ 40
10. EXERCISE: Microbiological examination of water __________________________ 41
NOTES __________________________________________________________________ 42
REFERENCES ___________________________________________________________ 44
PHOTOS ________________________________________________________________ 45

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INTRODUCTION

A ccording to the Law on health suitability of foodstuffs and products and materials that
come into contact with food (ZZUZIS) (2000, 2002) and the Law on amendments and
supplements to certain laws in the field of health (ZdZPZ) (2004) are two of the eleven
requests which food must meet to be suitably wholesome and safe, directly related to
bacteriological testing of foods.
Foods are wholesome and safe when:
1. they do not contain microorganisms or parasites or their developing forms or secretions,
which may adversely affect human health;
9. their composition or the organoleptic properties (taste, smell, appearance) due to physical,
chemical, microbiological or other processes are amended so that are dedicated useless.

The instructions are therefore outlined the main principles of determining the most important
indicator and pathogenic microorganisms in food and microbiological testing of drinking water.

Preparation of food samples for microbiological examination

Before we begin microbiological examination, we need to determine status of the packaging,
information on food, which are written on the label or packaging of any deviation from the
characteristic properties of food. In laboratory notebook we write the date of achievement of the
sample, the sampling procedure, the subscriber's name and address and the nature of the food. If
foods are not investigated immediately, they must be stored in a refrigerator at a temperature
below 4 °C. They should be stored in such a way to avoid any change in the number and types of
microorganisms present (ISO 7218, 1996). Stable, pasteurized and similar products should be
examined as soon as possible, before the expiration date, but fresh and chilled products within 24
hours. When samples are frozen at -18 °C it should be noted on the report of the investigation;
spoiling products need to be examined soon as possible or within 48 hours.

Foods for microbiological examination are prepared aseptically to prevent contamination from
environment. In the case of packaged food, packaging is wiped with 70% ethanol and in cases
where the packaging material is glass or metal, it is flamed. Packaging should be opened
aseptically with a sterile accessories (for example, a can opener, knife, scissors) and food taken
with sterile fittings (for example: spoon, pipette).

Preparation of food basic solution

Basic solution is a basic 10-fold dilution of the food. It is prepared in order to provide uniform
and constant distribution of microorganisms in a test quantity of the product. First we weigh out
the investigated mass or volume of food in a sterile beaker or plastic bag, than nine times of the
amount of solvent is added, and the suspension is homogenized (ISO 6887-1: 1999 (E)). In the
absence of specific rules, the minimum quantity of suspect food is 10 g (ml). Therefore, 10 g (ml)
of well-mixed sample is aseptically weighed into a 90 ml of the solvent (or 20 g (ml) in 180 ml of
solvent). This gives the basic 10-fold dilution of (R 10-1). The sample is homogenized by kneading
device or stirrer with blades ("stomacher", "ultra-turax").

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Preparation of food dilutions

Next a 10-fold dilution is obtained, such that one volume of food basic solution (1 ml) is added
in a nine-fold volume of the solvent (9 ml). The dilution is repeated so many times to obtain the
appropriate dilution of the food by inoculating in/on the medium. By diluting the food reduces
the amount of food being investigated and the number of microorganisms in unit volume.

As the solvent, there can be used peptone salt, buffered peptone water (ISO 6887-1: (1999)),
phosphate buffer, or a saline solution (Downes and Ito, 2001). For foods with a high fat content,
such as butter, cream, cheeses, ice cream, instead of saline solution 2% solution of sodium citrate,
which is heated to 45 °C is used. For acidic foods, such as fruit juice, syrup, refreshments, first
we need to measure pH and then 100 ml of the sample is neutralized with 0.1 N KOH solution.
The basic solution is prepared from the neutralized sample.

Frozen food is thawed in a sterile container, which is then, together with the remission of the
fluid homogenized. In the case of carbonated beverages, for example beer, refreshing non-
alcoholic carbonated beverages, dispense 150 ml beverage in a sterile flask containing glass beads
and 5-10 minutes shake to expel carbon dioxide.

The time from preparation of stock solution to food investigation should not be longer than 45
minutes. Dilutions should be prepared in the first 30 minutes.

General procedure of microbiological examination of food

Microorganisms in food can be detected in selected weight or volume of food (for example,
require: Salmonella spp. negative in 25 g of chicken meat) or microorganisms can be quantitative
evaluated (for example, require: the total number of aerobic mesophilic bacteria must not be
greater than 103 cfu/g). Therefore, there are two general procedures of microbiological
examination.

General procedure of microbiological examination of food when absence of specific

microorganisms in a specified mass (volume) of the food is required

1. Enrichment: Enrichment is used when we expect a very low contamination, or when

investigating higher weight of food (for example, the investigation of Salmonella in 25 g or 50
g) or when we assume that the microbial cells are damaged. For enrichment non-selective or
selective liquid medium are used. The purpose of the enrichment is multiplication of
investigated microorganism to a concentration which is higher than the sensitivity of the
methods used for the isolation. The homogenized food sample is incubated in enrichment
medium for a certain time at a certain temperature.

2. Isolation: After incubation enrichment suspension is transferred with an inoculating loop on

appropriate selective medium, which are selected according to the type microbiological
examination. Culture media are incubated for a certain time at a certain temperature. After
incubation, growth of typical colonies is observed.

3. Conformation and identification: Typical colonies are further investigated with biochemical
and serological tests and on that way result of isolation are confirmed and strains are
identified.

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General procedure of microbiological examination of food when the number of specific

microorganisms in food is evaluated

1. Preparation of dilution: sample dilution is prepared from food basic solution, in which food
has already been diluted 10x (R 10-1). Dilution is done with a sterile saline solution, which is
divided into the tubes (9 ml). For example, 100x dilution of the food (R 10-2) means that 1 ml
of food basic solution is pipetted into 9 ml of sterile saline, 1000x dilution of the food (R 10-3)
means that 1 ml of 100x diluted sample is pipetted into 9 ml of sterile saline, .... By preparation
of dilutions, it is important that the sample is always mixed well and used fresh pipette tips are
used. How many dilutions are prepared depends on the number of microorganisms present in
food.

2. Isolation: Food dilutions are inoculated in/on appropriate liquid/solid medium. After
incubation of isolating medium for a certain time at a certain temperature the results are
quantified. Examples:
• When solid medium is used for isolation, 0.1 ml of appropriate diluted food sample is
spread onto solid medium with sterile glass rod.
• When we do not use in advance prepared solid medium, we inoculate 1 ml of appropriate
diluted food sample in sterile Petri dish. Solid medium should be dissolved, cooled to 45
o
C and added (15 ml) in Petri dish with already inoculated food sample. The sample
should be mixed with media immediately as "3x up-down, 3x left-right".
• When for isolation liquid medium in tubes is used (most probable number, MPN)
appropriate diluted food sample is inoculated in liquid sterile media. The number of
dilutions and used tubes is dependent on the MPN method.

3. Quantification: After incubation of solid isolation medium, characteristic colonies are

counted in these Petri dishes where countable numbers of colonies are grown (in general,
countable plate for bacteria is 15 – 300 colonies, countable plate for fungi is 15 – 150
colonies). To calculate the number of microorganisms in 1 g or 1 ml of food sample (cfu/g or
cfu/ml), average number of colonies and the dilution of food sample should be considered.
Examples:

• If we have countable plates at a single dilution of food, the number of microorganisms is

calculated according to the following formula:
np
N=
R
N number of microorganisms in food (cfu/ml, cfu/g)
np average number of colonies
R dilution of food sample by which colonies are counted

• If we have countable plates at many dilutions of food, the number of microorganisms is

calculated according to the following formula:

N=
∑C
(n1 + 0,1 ∗ n2 ) ∗ R
N number of microorganisms in food (cfu/ml, cfu/g)
Σc sum of all colonies on countable plates
n1 number of plates at the first dilution of food sample
n2 number of plates at the second dilution of food sample
R the first dilution of food sample by which colonies are counted

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• In the case of using liquid media, appropriate MPN table are used to determine the most
probable number of microorganisms.

4. Conformation and identification: Typical colonies are further investigated with biochemical
and serological tests and on that way result of isolation are confirmed and strains are
identified.

TOTAL NUMBER OF LIVE MICROORGANISMS

T
he identification scheme is not the classification scheme. The identification scheme for a
group of microorganisms can be made only after the group of microorganisms is
classified, which means that, according to certain properties different from the other
groups.
In general, the properties of the microorganisms selected for the identification scheme, should be
easily determined. Likewise, it should be as little or as much of these properties or as it is
necessary to identify the microorganisms.

Total number of live microorganisms

Total number of live microorganisms capable of reproducing is determined by counting the
colonies on the plates (Plate count) or in cases where low number of microorganisms is expected
most probable number of microorganisms (MPN) is used. By microbiological investigations of
food the choice of the appropriate medium and conditions for the incubation are extremely
important because foods contain a variety of microorganisms (Harrigan, 1998).

In food can be microorganisms that are strict anaerobes or may be microorganisms which are
facultative anaerobes; some can grow at 37oC and not at 20°C, while others grow at 20°C and not
at 37oC. Also, if the food is examined in the four conditions of incubation aerobically at 20°C and
at 37oC, anaerobically at 20°C and 37oC, it is to determine the total number of living
microorganisms, because we do not know what proportion of the microbial population is able to
grow and multiply by two or more conditions of incubation.

We have similar problem by choice of medium. Plate count and MPN are two methods to
estimate the number of microorganisms as a proportion of the microbial population that can be
multiplied in a given conditions of incubation including our selection of growth medium. The
growth of colonies or turbidity of the liquid medium is measurement of microbial reproduction
in the conditions of incubation and not in situations that are typical for the product. This is
particularly important in processed foods that contain damaged or inhibited microorganisms.
When a great number of microorganisms is obtained by direct counting of cells under a
microscope and a small number of microorganisms is obtained by counting the colonies on the
plates, this does not necessary mean that the majority of microscopic determined microorganisms
are dead, but that the microorganisms are just incapable of growth in a given situation incubation.

In general, more complex composition of medium higher number of different microorganisms

will be grown on/in the medium. For example, on plates with the PC (plate count) agar that
contain glucose more colonies are grown than on plates with NA (nutrient agar)

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Determination of the number of viable microorganisms at incubation temperature of 25 - 30°C

may be an indicator of hygiene in food plants and incubation at temperature of 30 - 37oC may be
indicator of potential health risks.

Total microbial count

The number of bacteria, yeast and moulds in food should be therefore determined by prescribed
procedure. According to ISO 4833 (1991) the number of microorganisms in food is determined
as follows: in two sterile Petri dish 1 ml of the liquid food or 1 ml of stock solution or proper
dilution of the food is pipetted. Than 15 ml of PC medium is added in each Petri dish. PC agar
should be previously dissolved and cooled to 45°C. The inoculum is gently mixing with medium.
When the medium is solidified Petri dishes should be inverted and incubated at 30°C for 72
hours.

Calculation of the number microorganisms:

• Plates with on which 15 to 300 colonies were grown:

Count the number of colonies on the plates, on which 15 to 300 colonies were grown. Consider
two successive food dilutions. The number of microorganisms in 1 g or 1 ml of the food (N) is
calculated using the following equation:

N=
∑C
(n1 + 0,1 ∗ n2 ) ∗ R
N number of microorganisms in food (cfu/ml, cfu/g) expressed as number between 1.0 in
9.9 x 10x
ΣC vsota kolonij, ki so zrasle na vseh ploščah
n1 sum of all colonies on countable plates
n1 number of plates at the first dilution of food sample
n2 number of plates at the second dilution of food sample
R the first dilution of food sample by which colonies are counted

• Plates with less than 15 colonies:

If on two plates which were inoculated with sample less than 15 colonies were grown, calculate
the arithmetic mean of the colonies and the number of microorganisms in 1 g or 1 ml of the food
(NE) is estimated, using the following equation:

N E = m ∗ R −1
m arithmetic mean of the colonies
R dilution

• No colonies on plates:
If on plates on which we had inoculated samples no colonies grow we give the result as:
- Less than 1 microorganism in 1 ml (g) of food (N<1 cfu/ml(g)), when we inoculated liquid
food without preparing dilutions
- Less than 10 microorganism in 1 ml (g) of food (N<10 cfu/ml(g)), when we inoculated 10-1
dilution of food.

Number of yeasts and molds

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Number of yeasts and molds in a food is determined according to ISO 7954 (1987) so that in two
sterile Petri dish, pipette 1 ml of the liquid food or 1 ml of basic food solution. Then, make a 10-
fold dilutions and from each dilution in 1 ml is pipetted in sterile Petri dish. In Petri dishes 15 ml
of YEDC (Yeast extract-Dextrose-chloramphenicol-agar) is added, which was previously heated
and cooled to 45°C, and the inoculum carefully mixing. When the medium is solidified, invert the
Petri dishes and incubate them at 25°C for 5 days. Colonies are counted after three, four and five
days of incubation. Consider plates, which have 15 - 150 colonies and calculate the number of
yeasts and molds, as for determining the number of microorganisms in the food. Instead YECD
also OGY (Oxytetracycline glucose yeast extract agar), DRBC (dichloran rose bengal
chloramphenicol agar) can be used (Downes and Ito, 2001). If according to the morphological
characteristics of the colonies can not be determined whether they are colonies of fungi or
bacteria colonies, made from the examined colonies microscopic slide and with respect to
micromorphological properties determine the type of the microorganism.

DETERMINATION AND QUANTIFICATION OF INDICATOR

BACTERIA

AEROBIC SPORE-FORMING BACTERIA

GENERAL

For foods that have been heated (pasteurization), subsequent contamination can be determined
from proportion of aerobic spore-forming bacteria - Bacillus - according to the total number of
bacteria in food. If there is no subsequent contamination of food, the total number of bacteria
and the number of aerobic spore-forming bacteria should be identical (within experimental
error).

The first group of foods include foods which depending on their characteristics do not allow
spore germination, such as dried foods, foods with low aw and thermally processed foods, which
are immediately frozen after heat treatment. For these foods the same number of bacteria and
spore-forming bacteria means that food has not been recontaminated and that food has been
appropriate heated.

The second group of foods include foods which depending on their characteristics, allow
germination of bacterial spores, such as pasteurized milk - these foods need to be
microbiologically examined after filling and cooling, otherwise bacterial spore may germinate.

ISOLATION AND IDENTIFICATION

We prepare basic food solution (R = 10-1) and 10-for dilutions. In empty sterile Petri dish
pipette 1 ml of each dilution. Then add in the Petri dish 15 ml of PC (Plate count agar) which
was previously heated and cooled to 45 °C, and mix the inoculum gently. When the medium is
solidified, invert the Petri dishes and incubate them at 30 ° C for 2 days. After incubation count
the colonies and calculate the number of aerobic bacteria in food.

In parallel with the determination of aerobic bacteria we can determine the number of aerobic
spore-forming bacteria by pipetting 2x 10 ml of basic food solution into sterile tubes and heat
them in a water bath at 81 °C. In one tube add thermometer to control temperature of heating –
heat tubes until temperature in control tube is 80 oC, and then heat another 1 min. Then cool the
basic food solution and prepare further dillutions. In empty sterile Petri dish pipette 1 ml of each
dilution. Then add in the Petri dish 15 ml of PC (Plate count agar) which was previously heated

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and cooled to 45 °C, and mix the inoculum gently. When the medium is solidified, invert the
Petri dishes and incubate them at 30 ° C for 2 days. After incubation count the colonies and
calculate the number of aerobic spore-forming bacteria in food (Harrigan, 1998).

The number of aerobic spore-forming bacteria in a 1 g / 1 ml of the food can also be determined
by placing 50 ml/g of the food in 450 ml of physiological saline and homogenization of sample
in stomacher for 2 min. Three bottles with agar TGE (Tryptone glucose extract agar) or PC (100
ml to 500 ml bottles) heat and cool to 45 °C in a water bath. Add in the first bottle 10 ml, in the
second 1 ml and a third 0.1 ml of basic food solution. Then put the bottles with samples into a
water bath at 80 °C for 30 minutes. Then cool the bottles with the samples to 45 °C and poure
each sample into five sterile Petri dishes. Incubation of plates is at 35 °C for 48 hours. Then
count all colonies. The number of spore-forming bacteria (cfu/g) from the first bottle is equal to
the number of colonies grown on all five plates with sample from the first bottle. The number of
spore-forming bacteria (cfu/g) in the second bottle is equal to the number of grown colonies on
all five plates and multiplied by 10, the number of spore-forming bacteria (cfu/g) in the third
bottle is equal to the number of grown colonies on all five plates and multiplied by 100. Thus, we
can determine from 1 to 150,000 cfu/g (Downes and Ito, 2001).

ANAEROBIC SPORE-FORMING BACTERIA

General

Mesophilic anaerobic spore-forming bacteria belong to Clostridium genus. Mesophilic anaerobic

spore-forming bacteria that may be in foods are Gram-positive, catalase-negative rods of various
sizes with sub-terminal to central endospore. Although it is considered that Clostridia are strict
anaerobes, some strains can grow even in the presence of oxygen (Downes and Ito, 2001).

Anaerobic bacteria are widely distributed in nature, especially in soil and are therefore a frequent
contaminant of fresh vegetables and spices. Some species of anaerobes are also normally present
in the gastrointestinal tract and the excreta of animals and may therefore be contaminants in milk
and meat. Some anaerobes are also characteristic of fish, shellfish and aquatic environment.

As many mesophilic anaerobic spore-forming bacteria are highly resistant to heat, they can grow
in an atmosphere without oxygen in a wide temperature range in which canned food is produced
and stored, and because it can grow in other food products (e.g. cured meat in cooked foods, in
foods stored in the refrigerator, in foods that are packaged in a modified atmosphere or vacuum),
the representatives of these bacteria may be important food - spoilage microorganisms as they
can be proteolytic or saccharolytic.

ISOLATION AND IDENTIFICATION

If anaerobic mesophilic spore-forming bacteria is quantitated, prepare a basic food solution (BS)
(R = 10-1), which is heated in the case that we are looking for proteolytic bacteria at 80 °C for 10-
13 minutes, in the case of more sensitive spores at 60 °C for 15-30 minutes and in the case that
we are looking for more heat-resistant spores BS is heated at 71 °C for 10 minutes. Then prepare
a 10x- dilutions of the food. As a method for quantification MPN or plate count or colony count
in test tubes with semisolid agar can be used used.

To determine the number of mesophilic anaerobic spore-forming bacteria by a method of

counting colonies on plates add into empty sterile Petri dishes 1 ml of appropriate food dilution.

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Then add in each Petri dish 15 ml of TPGY medium (Tripticase peptone glucose yeast extract
agar), AC (Acetate differential agar) or TP (tripticase peptone agar), which was previously heated
and quickly cooled to 45 °C, and the inoculum is carefully mixing with added medium. When the
medium is solidified, pour a thin layer of thioglycollate medium and incubate plates in an
anaerobic jar at 30 °C to 35 °C for 2-5 days. Count the colonies and calculate the number
anaerobic mesophilic spore-forming bacteria in food (Downes and Ito, 2001). The drawbacks of
this method are considerable differences between replicates, they can form gas bubbles that
damage the solid substrate and thereby prevent accurate counting colonies. Interpretation of the
results must be cautious, because in addition to clostridia some strains of facultative anaerobic
spore-forming bacteria can grow.

Enterobacteriaceae, COLIFORMS, Escherichia coli

GENERAL

Raw food or food having not thermally treated food additives often contain coliform bacteria and
Escherichia coli. In food, these bacteria in certain circumstances may also replicate and therefore
there is no necessary link between their number in food and the initial level of contamination.
The presence of E. coli and other Enterobacteriaceae in food can be an indicator for the presence
of intestinal bacteria, while the absence Enterobacteriaceae does not mean that the product does
not contain any pathogenic intestinal bacteria. For example, raw milk, meat or eggs may contain
Salmonella, while not containing E. coli.

E. coli are generally less resistant to environmental factors as Salmonella. Thus, in those cases
where E. coli are of faecal origin true picture of fecal contamination of food is given only if food
is examined immediately after contamination. Because sampling and microbiological testing of
food are not carried out immediately after contamination of the food, whereas the growth
depends on the properties of food and its storage conditions, we have the following options:

• E. coli can be killed in a foodstuff. It is therefore incorrect to assume, in the absence of E.

coli, that also no other pathogenic enterobacteria.
• E. coli remain relatively constant concentration in the food. Even in this case is not correct to
assume that the same act other pathogenic enterobacteria.
• E. coli growing in the food and their concentration increases. In this case, we must bear in
mind that the situation in the food which are favorable for the bacteria E. coli, probably
favourable for other Enterobacteriaceae and other bacteria.

Although a high concentration of E. coli in the food does not necessarily mean the recent strong
or faecal contamination, such information should be taken as an indicator that indicates a
possible risk from the Enterobacteriaceae (Downes and Ito, 2001). Given the difference in
resistance of E. coli and Salmonella, it was suggested that the total number of Enterobacteriaceae
could be better indicator of poor sanitation (Downes and Ito, 2001)

Definitions (Harrigan, 1998):

1. Enterobacteriaceae: the family of bacteria (Citrobacter, Enterobacter, Erwinia, Escherichia,
Hafnia, Klebsiella, Kluyvera, Proteus, Salmonella, Serratia, Shigella, Yersinia), which grow in the
presence of bile salts and form acid from glucose (VRB agar with 1% the addition of
glucose).
2. Bacteria coli-aerogenes: Bacteria that grow in the presence of bile salts, or other
equivalent selective additives and form acid and gas from lactose at incubation at 30 °C.
3. Coliform bacteria: Bacteria that grow in the presence of bile salts, or other equivalent
selective additives and form acid and gas from lactose at incubation at 35 °C or 37 °C.

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4. Fecal coliform bacteria: Bacteria that grow in the presence of bile salts, or other
equivalent selective additives and form acid and gas from lactose during the incubation at
44 to 45.5 °C.
5. Escherichia coli: are those which, in addition to the above features, have a positive result in
the methyl red, negative result in Voges-Proskauer and do not use citrate as the sole
carbon source. Indole positive E. coli are termed E.coli type 1, which is assumed to
originate from the intestine as the natural environment.

The presence of any of said groups of bacteria in the heat-treated food product is an indicator of
one or more of the the following statements:
• Initial concentration of bacteria was so high that they are not destroyed by heat treatment
• Conditions of the heat treatment of foods were favorable for the development of
surviving bacteria
• Recontamination of heat-treated foods

Escherichia coli belong to Enterobacteriaceae. They are Gram-negative, facultative anaerobic rods.
Glucose and other carbohydrates normally decompose the acids and gas, grow well in the
presence of bile salts. The optimum temperature for growth is 37 °C (usually between 35 and 40°
C), some pathogenic strains can also grow below 7 °C and above 46 °C. These strains to survive
in foods stored at refrigerator temperatures (3 -7° C), as well as food, frozen at -20 °C. Usually
grow at a pH value between 4.4 (depending on the type of acid present) and 9.

E. coli are in the digestive tract of humans and warm-blooded animals, as well as in the ground
water, and elsewhere. They represent approximately 1% of the human intestinal microflora. Their
presence in foodstuffs indicates the possibility that food is contaminated with faecal matter. That
indicates the possible presence of other pathogenic microorganisms and low hygiene level of
food production. Strains of E. coli, which can be present in foods and of causing disease, are
divided into five groups:
• enteropathogenic strains of E. coli (EPEC): they do not normally synthesize enterotoxins,
but can cause diarrhoea;
• enterotoxigenic strains of E. coli (ETEC): they synthesize heat-labile enterotoxins, which
contain A and B substances (LTA and LTB) and heat-stable enterotoxins (ST-I and ST-
II);
• enteroinvasive strains of E. coli (EIEC): they do not synthesize enterotoxins, pathogenesis
is similar to that of shigellosis;
• enterohaemorrhagic strains of E. coli (EHEC) (representative is E. coli 0157: H7): they
synthesize two toxins (SLT-I and SLT-II);
• facultative enteropathogenic strains of E. coli (FEEC).

ISOLATION AND IDENTIFICATION

TOTAL NUMBER OF Enterobacteriaceae

Total number of Enterobacteriaceae is determined by samples by which we expect low level of
contamination, with following procedure (ISO 8523, 1991):

• non-selective enrichment: 1g of food or 1 ml of BS (basic food solution) is inoculated in 9

ml BPW liquid medium (buffered peptone water), incubation at 35 °C or 37 °C for 16-20
hours

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• selective enrichment: 1 ml of pre-enrichment suspension is inoculated in 10 ml EEB

(Enterobacteriaceae enrichment medium that contains bile) liquid medium, incubation at 35
°C or 37 °C for 18-24 hours
• isolation with loop on VRBG (violet red bile agar with glucose), incubation at 35 °C or 37
°C for 24 hours
• Typical colonies on VRBG are dark red with a dark red glow. Five randomly selected
colonies re-inoculate on NA (nutrient agar) agar plates and incubate at 35 °C or 37 °C for 24
hours. In case that there are no typical colonies re-inoculate 5 beige colonies by the same
procedure.
• With the colonies on NA non-selective medium are confirmed with:
• oxidase test: Enterobacteriaceae are oxidase-negative (!! Do not use nickel - chromium
loop but platinum-iridium loop or plastic loop!)
• Fermentation of glucose: Enterobacteriaceae ferment glucose

The total number of Enterobacteriaceae is determined for samples in which is the expected 10 to
100 cfu/g or more bacteria after preparation of decimal dilutions of the sample and they are
inoculated (1 ml) in an empty Petri dish and mixed with (15 ml) melted, and cooled to 45 ° C
VRBG. When the medium is solidified, spread (5 ml) VRBG on the top to prevent the growth of
colonies on the surface. The plates are incubated at 35 °C oz.37 °C. In some cases, also
incubation at 30 °C is performed. Confirmation is performed the same way as described above.

TOTAL NUMBER OF Enterobacteriaceae, NUMBER OF COLIFORM

BACTERIA, NUMBER OF FECAL COLIFORM BACTERIA AND
NUMBER OF Escherichia coli DETERMINED WITH MPN METHOD

Total number of Enterobacteriaceae:

According to ISO 21528-1 (2004) standard for determination of the total number of
Enterobacteriaceae we prepare decimal dilutions of food sample and aseptically inoculate:
• 3x 10 ml of undiluted food sample, when food in liquid; 3x 10ml BS, when food is not
liquid, in 10 ml BBGBG medium with double concentration (buffered glucose brilliant green
bile broth),
• 3x 1ml of undiluted food sample, when food in liquid; 3x 1ml BS, when food is not liquid,
in 10 ml BBGBG medium with normal concentration,
• 3x 1ml of diluted food sample 10-1, when food is liquid; 3x 1ml of diluted food sample 10-2,
when food is not liquid, in 10 ml BBGBG medium with normal concentration.

Nine of these tubes are incubated at 35 °C or 37 oC for 24 hours. When Durham tube are added
in BBGBG medium and if we add an indicator positive results are determined in relation to the
formation of gas in Durham tube and formation of acid that is seen according to change of
indicator colour.. From each positive tube with BBGBG confirmation need to be done by re-
inoculating 3 loops of the suspension onto a VRBG solid agar plates, which are incubated at 35
°C or 37 oC for 24 hours. Typical colonies are dark red with a dark red glow. Five randomly
selected colonies re-inoculate on NA agar plates, and incubate them at 35 °C or 37 oC for 24
hours. In case that there is no typical colonies re-inoculate 5 beige colonies under the same
procedure.
• With the colonies on NA non-selective medium are confirmed with:
• Oxidase test: Enterobacteriaceae are oxidase-negative (!! Do not use nickel - chromium
loop but platinum-iridium loop or plastic loop!)
• Fermentation of glucose: Enterobacteriaceae ferment glucose

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Calculation of MPN Enterobacteriaceae (most probable number of Enterobacteriaceae):

• Count positive results by each food sample dilution
• From MPN table determine index of MPN according to the number of positive results
• In case food was liquid the numer of bacteria is obtained by dividing index of MPN with 10;
in case food was not liquid index of MPN is equal to the number of bacteria in 1 g of food.

Number of coliform bacteria:

According to ISO 4831 (1991) standard for determination of the number of coliform bacteria we
prepare decimal dilutions of food sample and aseptically inoculate:
• 3x 10 ml of undiluted food sample, when food in liquid; 3x 10ml BS, when food is not
liquid, in 10 ml LSTB (lauryl sulphate tryptose broth) medium with double concentration,
• 3x 1ml of undiluted food sample, when food in liquid; 3x 1ml BS, when food is not liquid,
in 10 ml LSTB medium with normal concentration,
• 3x 1ml of diluted food sample 10-1, when food is liquid; 3x 1ml of diluted food sample 10-2,
when food is not liquid, in 10 ml LSTB medium with normal concentration.

Nine of these tubes are incubated at 35 °C or 37 oC for 24 hours. Positive result is gas in Durham
tube. If there is no positive result, incubation of tubes with LSTB is prolonged for another 24
hours. Conformation of coliform bacteria is done after positive result in LSTB with re-
inoculation 1 loop of suspension into BGLBB (brilliant green lactose bile broth) which is then
incubated at 30 °C or 35 oC for 24-48 hours. Positive result is gas in Durham tube and yellow
medium.

Calculation of MPN of coliform bacteria:

• Count positive results in LSTB by each food sample dilution
• From MPN table determine index of MPN according to the number of positive results
• In case food was liquid the numer of bacteria is obtained by dividing index of MPN with 10;
in case food was not liquid index of MPN is equal to the number of bacteria in 1 g of food.

Number of fecal coliform bacteria:

The procedure is the same as described for the number of coliform bacteria except that LSTB
medium is incubated at 44 oC.

Number of Escherichia coli:

According to ISO 7251 (1993) standard for determination of the numer of Escherichia coli we
prepare decimal dilutions of food sample and aseptically inoculate:
• 3x 10 ml of undiluted food sample, when food in liquid; 3x 10ml BS, when food is not
liquid, in 10 ml LSB (lauryl sulphate broth) medium with double concentration,
• 3x 1ml of undiluted food sample, when food in liquid; 3x 1ml BS, when food is not liquid,
in 10 ml LSB medium with normal concentration,
• 3x 1ml of diluted food sample 10-1, when food is liquid; 3x 1ml of diluted food sample 10-2,
when food is not liquid, in 10 ml LSB medium with normal concentration.

Nine of these tubes are incubated at 37 oC for 24 hours. Positive result is gas in Durham tube. If
there is no positive result, incubation of tubes with LSB is prolonged for another 24 hours.
Conformation of E. coli is done after positive result in LSB or after turbidity of LSB with re-
inoculation 1 loop of suspension into EC broth which is then incubated at 44 oC for 24-48 hours.
Positive result is gas in Durham tube. From each tube with a positive result re-inoculate a loop
into tube with peptone water containing tryptophan. Incubate tubes for 48 hours at 44 °C. After
incubation, add Kovacsev reagent and the red colour indicates the formation of indole, which is
characteristic of E. coli.

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Calculation of MPN of Escherichia coli:

• Count positive results in LSB by each food sample dilution
• From MPN table determine index of MPN according to the number of positive results
• In case food was liquid the numer of bacteria is obtained by dividing index of MPN with 10;
in case food was not liquid index of MPN is equal to the number of bacteria in 1 g of food.

NUMBER OF COLIFORM BACTERIA DETERMINED WITH PLATE

COUNT METHOD
The number of coliform bacteria may be determined for the samples in which the expected 10 to 100
cfu/g or more bacteria so that first we prepare decimal dilutions of the sample and inoculate them (1
ml) in an empty Petri dishes. Food dilutions are mixed with VRBL (purple red bile agar with lactose)
medium (15 ml) , which was previously melted and cooled to 45 °C (ISO 21528-2, 2004). When the
medium has solidified, 5 ml of VRBL is spread on the top to prevent the growth of colonies on the
surface. Plates are incubated at 35 °C oz.37 °C. In some cases, also incubation at 30 °C is performed.
Typical colonies of coliforms are large (0.5 cm or more), dark red and have a zone of precipitated bile
salts. To determine the number of coliforms VRBL agar plate with up to 150 colonies is taken into
account.

NUMBER OF Escherichia coli DETERMINED WITH PLATE COUNT

METHOD
Number of E. coli is determined in food samples where we expect 10 – 100 cfu/g or more
bacteria so that first we prepare decimal dilutions of the sample and inoculate them (1 ml) in an
empty Petri dishes. Food dilutions are mixed with VRBL medium as selective medium for
coliform bacteria as fermentation of lactose is expected. Bile salts and crystal violet inhibit the
growth of Gram-positive bacteria. Indicator for changes pH are crystal violet and neutral red.
Plates are incubated at 44 °C for 24-48 hours. E. coli forms colonies with a dark red purple red
centre - due to the precipitation of bile salts at a low pH, 2 - 3 mm in diameter. Also other
coliform bacteria grow on that medium (such as Enterobacter aerogenes that form dark red colonies).
Proteus mirabilis form colourless colonies

Sample or decimal dilutions are inoculated also on EMB (eosin methylene blue lactose) selective
medium. Ingredients of medium allow separation of genera Escherichia and Enterobacter (ferment
lactose and sucrose) from other Enterobacteriaceae. Incubation of plates is at 44 °C for 24-48
hours. E.coli form purple colony with a darker centre and a greenish metallic shine. By
fermentation pH value due to the acid is dropped, at low pH values cause the formation of an
amide bond between eosin and methylene blue, which is reflected in the metal shine of colonies.
Enterobacter aerogenes: red colonies with a dark centre, around the transparent zone. The
degradation of sugars is smaller and thus a little acid is formed, degradation is mainly to acetoin,
with no a large drop in pH. Salmonella and Shigella form colourless or pink colonies (not ferment
lactose and sucrose).

E. coli is confirmed and identified by IMViC test and microscopic slide preparation according to
Gram. Typical colonies from a selective agar plates are re-inoculated first on non-selective plating
medium and after 24 hours of incubation at 37 °C colonies are re-inoculated in appropriate media
for the IMViC test.

IMViC test:
IMViC test consists of 4 tests:

1. Indole: Typical colony from a selective medium or colony from non-selective medium is
inoculated in peptone water containing the tryptophan - amino acid with indole ring. After 24-

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hours incubation at 37 °C, evidence of indole formation is obtained by Kovac reagent. In the
case of a positive reaction, which is characteristic for E. coli bright red indole ring is formed.

Principle: Tryptophan is grafted by tryptophanases that are enzymes synthesized by certain

bacteria, and the water, and result is formation of indole and serine. Indole reacts with an
aldehyde group of the p-methylaminobenzaldehide (component of Kovac reagent) and forms of
a red coloured complex.

2., 3. Methil Red (MR) and Voges Proskauer (VP): Typical colony from a selective medium
or colony from non-selective medium is inoculated in the MR-VP liquid medium with glucose
where the conversion of glucose to acid(s) (MR) and to acetoin (VP) is observed after incubation
at 37 °C for 24-48 hours. Pour half of the contents of the first tube into the second tube. Add in
the first tube methyl red as an indicator. In the case of acid(s) formation, medium will be red (E.
coli), otherwise the colour is orange (Enterobacter aerogenes).

Principle of MR: Degradation of glucose is via pyruvate to acids (lactic, acetic, ...). The pH
value drops significantly below 4.4 and the colour of added methyl red indicator turns into red.
At a pH 6.0 the indicator is yellow. If there are small amounts of acids, the pH is reduced, but
does not fall below 4.4 and orange colour indicates a negative result.

In the second tube acetoin is confirmed with 5% solution of α-naphthol in ethanol (5 g in 100 ml
of 96% ethanol) and 40% KOH aqueous solution with crystals of creatinine. Mix suspension well
and if there is after 15 minutes formation of a red colour (Enterobacter aerogenes), the result is
positive. If the sample contains E. coli, the red colour is not formed.

Principle of Voges-Proskauer: Degradation of glucose is via pyruvate to acetoin with

formation of a small quantities of acids. Acetoin is after addition of KOH and under the
influence of oxygen from the air converted into diacetyl. This forms a red complex in the
presence of α-naphthol.

4. Using citrate as the sole carbon source (test according to Simmons): Bacteria that are
able to use Na-citrate as a sole carbon source, can use nitrogen of the ammonium phosphate (the
only source of nitrogen). Result is seen as change of pH in alkaline (indicator is bromothymol
blue). A positive result is considered when bacteria grow and colour of the medium is changed in
blue (alkaline reaction).
On the slant medium with citrate inoculate bacteria with a loop. After incubation at 37 °C for 24
hours in the case of the growth of bacteria and when colour of the medium changes from green
to blue green the result is considered as positive. E. coli do not grow, Enterobacter aerogenes grow.

Microscopy:
Microscope slide according to Gram is prepared from colony on non-selective media

DETERMINATION OF Escherichia coli AFTER ENRICHMENT

Brilliant green lactose bile broth (BGLBB) is a selective medium for the isolation of coliform
bacteria, based on their ability to ferment lactose. Bile salts and brilliant green inhibit growth of
Gram-positive bacteria. Particular volume or weight of food in which we are looking for E. coli is
weighed in 9x volume of BGLBB and icubated at 44 °C for 24-48 hours. Change in color of the
medium from green to yellow and gas Durham tube indicate the possible presence of E. coli.

Then mix enrichment suspension and reinoculate it with onto VRBL plate. Incubate plates for at
44 °C24-48 hours and continue the procedure the same as described for plate count of E. coli.

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DETERMINATION AND QUANTIFICATION OF PATHOGENIC

AND TOXIN-FORMING BACTERIA

Salmonella

GENERAL

Salmonella belong to Enterobacteriaceae and are Gram-negative, facultative anaerobic,

sporeforming rods with the respiratory and fermentative-type metabolism. Glucose decompose
to acid and gas, do not ferment lactose (with some exceptions as only some serotypes ferment
lactose). All salmonella serotypes fall into two species: Salmonella bongori (less than 10 serotypes
and are very rare) and Salmonella choleraesuis (over 2,500 serotypes). Division into serotypes is
based on the Kauffmann - White scheme: O antigens (somatic), H antigens (flagela) and K
antigens (capsular). Epidemiologically Salmonella spp. are divided into three groups:
• Infect only humans: S. Typhi and S. Paratyphi A and S. Paratyphi C; typhoid and
paratyphoid fever are the most serious diseases, which are caused by salmonella with the
highest mortality rate.
• Serotypes adapted to the host: S. Gallinarum - poultry, S. Dublin - cattle, S. equi-Abortus
- horses, S. Abortus-Ovis - sheep, S. Coleraesuis - pigs; man can be infected by food, and
some serotypes are pathogenic to humans and animals.
• Unadapted serotypes: This group includes most serotypes present in food and are
pathogenic to humans and animals.

The primary source of Salmonella is the intestinal tract of humans and many animals (pets, reptiles,
birds and occasionally insects). Sometimes they are also present in other parts of the body.
Organisms excrete salmonela through faeces, and from there can be transmitted through a variety
of vectors into the water and food. Salmonella is a common source of animal feed (bone and fish
meal). The presence in food: cocoa, coconut flour (dried food), salad dressings, eggs, mayonnaise,
meat (especially poultry) and products such as milk (uncoocked - S. Dublin, pointing to poor
hygiene at milking). The most common serotype in food is S. Typhimurium, but in the given
period was S. Enteritidis.

Growth conditions:
The optimum pH for growth is between 6.6 and 8.2, pH values from 4.0 to 9.0 and above are not
suitable for salmonela growth. Minimum pH for growth is dependent on the type of acid that is
used for lowering the pH. Aeration has positive influence on the growth at a low pH. The
optimum temperature for growth is 37 °C, below about 7 °C and above 45 °C salmonella can not
reproduce. NaCl and nitrate inhibit growth, so curing with 9% NaCl has bactericidal effect,
nitrate has the highest inhibitory activity at low pH.

ISOLATION AND IDENTIFICATION OF Salmonella

ENRICHMENT
For determination of Salmonella in accordance with ISO 6579 (2002) two enrichments are used.
The first non-selective growth medium is BPW (buffered peptone water), and then selective two
enrichments as RVS broth (Rappaport-Vassiliadis broth with soya) and MKTTn broth (Muller
Kaufmann tetrationatni broth with novobiocinom). After the earlier ISO 6579 (1993) standard
selective liquid culture media SC (selenite cystine) and RV (Rappaport- Vassiliadis) were used.

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The investigation is done by weight a specific mass of the foodstuff in which we are looking for
Salmonella (25 g or 50 g or ml) into a 9x-volume of BPW non-selective enrichment medium (225
ml and 450 ml), homogenize and incubate at 37 °C for 18 hours. After incubation, the
suspension is homogenized and:
• 0.1 ml of suspension is transferred in SSR selective enrichment medium (10 ml) and incubated
at 41.5 °C for 24 hours
• 1 ml is transferred to MKTTn the second selective enrichment medium (10 ml) and incubated
at 37 °C for 24 hours.

ISOLATION
After incubation enrichment medium is mixed well and isolation with loop on selective agar
plates is done:
• according to ISO 6579 (1993) they are BGA (brilliant green agar), SS (Salmonella-Shigella
agar) and BS (bismuth sulphite agar)
• according to ISO 6579 (2002) they are XLD (xylose lysine deoxycholate agar) and other
medium of choice.
Agar plates are incubated at 37 °C for 24 hours.

Salmonella-Shigela (SS) medium:

In SS medium sodium citrate, gallic acid and brilliant green have an inhibitory effect on Gram-
positive bacteria and some of the non-pathogenic enterobacteria. The medium contains lactose,
an indicator of changes in the pH value is neutral red. Salmonella and other bacteria that do not
use lactose, form a cloudy, clear, colorless colonies. Isolation of Salmonella is based on their ability
to form of H2S. The origin of the sulfur in the culture medium is Na-thiosulfate. Indicator for
formation of H2S the iron citrate
Characteristics of colonies on SS:
Salmonella: transparent, slimy with or without dark black center, similar colonies also form some
species of Proteus; black center of colonies is due to the formation of iron sulphide;
Shigella: transparent or slightly pink colonies, that can be irregular in shape;
Enterobacteriaceae that ferment lactose: pink to reddish.

Bismuth sulphite (BS):

Isolation of Salmonella is based on their ability to form H2S. Source of sulfur are protein
components, an indicator of the formation of H2S is ferric sulfate. Growth of E. coli and certain
other coliform bacteria is inhibited due to the addition of bismuth sulfite, ammonium citrate
and brilliant green.
Characteristics of colonies on BS:
Salmonella: black, with metallic luster, there is the zone of the black-colored agar;
Proteus: brown to brownish black;
E. coli: growth is inhibited.

Brilliant green agar (BZA):

Isolation of bacteria is based on their ability to ferment lactose and sucrose, decomposition of
protein. pH indicators are brilliant green and phenol red. Brilliant green inhibit the growth of
non-pathogenic enterobacteria and species of the genus Shigella.
Characteristics of colonies on BZA:
Salmonella and certain other species which do not ferment lactose: red, purple, similarly is colored
the medium around the colonies (S. Typhi and S. Paratyphi do not grow);
Proteus: pinkish, slimy, 2-3 mm in diameter, the medium around the colonies are red because of a
basic reaction; release of ammonium compounds resulting from the degradation of proteins;
E. coli: yellow to yellow-green, like the color the medium around the colonies that is due to the
fermentation of lactose to fall pH; brilliant green turn into yellow color.

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Xylose lysine deoxycholate (XLD) medium:

Isolation of the bacteria is based on their ability to ferment lactose, sucrose, and xylose, on
decomposition of proteins or amino acid lysine. The indicator for changes of pH is phenol red,
the source of sulfur in the medium is Na-thiosulfate, an indicator of the formation of H2S the
iron citrate.
Characteristics of colonies on XLD:
Salmonella: because they do not use lactose and sucrose they do not form acid however they
decarboxilate lysine, which results in an alkaline reaction and the phenol red, becomes red:
colonies are therefore dark red, and in the case that they form of H2S they have a black center.
Shigella: dark red colonies
Proteus: sone strains can also form red colonies or red colonies with black centers

CONFORMATION
Colonies typical for Salmonella (up to 5 typical colonies) are re-inoculated from selective media on
non-selective media (NA) and plates are incubated at 37 °C for 24 hours. Then biochemical
(triple sugar, hydrolysis of urea, decarboxylation of lysine, β-galactosidase reactions, VP,
formation of indole) and serological confirmations (agglutination serum for salmonela) are
performed (ISO 6579, 2002).

For shortened biochemical identification do the following tests:

• Kligler double sugar: inoculate colony as stitch in, and on surface of solid slanted medium
• Urea: inoculate colony on surface of solid slanted medium
• KCN: inoculate colony in liqid media and mix
• Phenylalanine: inoculate colony on surface of solid slanted medium
• IMViC (description is by E. coli)
The tests are incubated at 37 ° C for 24 hours and then the results are read.

Kligler double sugar:

This is a selective medium that allows identification of species of the family Enterobacteriaceae
based on utilization of glucose (in the medium is 0.1%) and lactose (in the medium is 1%), gas
formation and the formation of hydrogen sulphide. Na-thiosulfate is the source of sulfur, Fe-
sulphate indicator of H2S formation.

Typical growth and reaction after 24 to 48 hours at 37 °C:

S. typhi: yellow bottom, red slanted surface, no gas formation;
Salmonella: yellow bottom, red slanted surface, due to the formation of H2S medium turns black,
the formation of gas;
Shigela spp.: yellow bottom, red slanted surface, no gas and H2S formation;
Proteus: yellow bottom, red slanted surface, gas formation (medium is cracked), some strains form
H2S;
E. coli: yellow bottom, yellow slanted surface, no gas and H2S formation.

The results of other tests for Salmonella:

• Urea: -
• KCN -
• Phenylalanine -
• IMViC - + - +
Microscopy:
Microscope slide according to Gram is prepared from colony on non-selective media

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Proteus

GENERAL

Proteus belong to Enterobacteriaceae and they are Gram-negative, facultative anaerobic, catalase-
positive rods. The optimum temperature for growth is 37 °C. Proteus are present in the digestive
tract of humans and warm-blooded animals, soil, water, and on the plants. The presence in foods:
meat and meat products, vegetables, eggs, raw milk, pre-packaged food stored at room
temperatures. They synthesize proteolytic enzymes and decarboxilate histidine in histamine.

ISOLATION AND IDENTIFICATION OF Proteus

ENRICHMENT
If we are analysing Proteus in greater mass or volume of food, we have to weight this amount in
9x volume of non-selective enrichment medium such as nutrient broth, the suspension is
homogenized and then incubated at 37 °C for 24 hours.

ISOLATION
If we perform isolation of Proteus after enrichment, we make isolation with loop from enrichment
medium on selective media. If we perform isolation of Proteus from food sample, basic food
solution and appropriate dilutions should be prepared. On selective medium 0.1 ml of BS or
appropriate dilution should be inoculated.
Use the following selective media: BZA; SS, BS in XLD and incubate plates at 37 °C for 24
hours.

CONFORMATION
Colonies typical for Proteus re-inoculate from selective media on non-selective plates and incubate
them at 37 °C for 24 hours. Then make a biochemical confirmation with the following tests:
• Kligler double sugar: inoculate colony as stitch in, and on surface of solid slanted medium
• Urea: inoculate colony on surface of solid slanted medium
• KCN: inoculate colony in liqid media and mix
• Phenylalanine: inoculate colony on surface of solid slanted medium
• IMViC (description is by E. coli)
The tests are incubated at 37 ° C for 24 hours and then the results are read.

• Kligler double sugar: yellow bottom, red slanted surface, gas formation (medium is cracked),
some strains form H2S;
• Urea: +;
• KCN: +;
• Phenylalanine: +;
• IMViC: different results according to Proteusspecies.

Microscopy:
Microscope slide according to Gram is prepared from colony on non-selective media

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Listeria monocytogenes

GENERAL

L. monocytogenes are Gram-positive, non-spore-forming rods (1 - 2 µm long, 0.5 µm wide),

sometimes apaer as coci or diplococi. After 3 to 5 days of incubation they are long rods (6 - 20
µm) and they are often Gram-negative. Bakterije vrste L. monocytogenes grow over a wide
temperature range of 3 °C to 45 °C, the optimum is between 30 °C and 37 °C. They are
reproducing in aerobic and microaerophilic conditions at pH values up to 9.6. The growth of
listeria is stopped or greatly inhibited under anaerobic conditions and at a pH of less than 5.6.

L. monocytogenes are widespread in nature. They have been isolated from various sources, for
example from decomposing vegetation, soil, animal slurries, waste water, feed silos, water.
Approximately 5-10% of people have L. monocytogenes in the digestive tract. The result of listeria
ubiquitous nature is their frequent presence in the human food chain. From all non-spore-
forming bacteria listeria are the most resistant to different environmental conditions. The
presence of L. monocytogenes in many foods is mainly due to cross-contamination of finished
products. Since listeria can grow at low temperatures of food cooling and as they are relatively
resistant to higher concentrations of salt, the food is a favorable environment for their growth.
Most often L. monocytogenes are found in the following foods: milk, soft cheeses, ice cream, and a
pre-chilled foods, raw meat, ready-to eat poultry products, pâté, raw vegetables, salads, fish, fish
products, sandwiches and rice.

In Listeria genus are for human and animal three pathogenic species: L. monocytogenes, L. ivanovii
and L. seeligeri. Examples of the infection with L. seeligeri are rare in both animals and in humans.
There have also been rare cases of human infection with L. ivanovii. Most of listeriosis in humans
are caused by L. monocytogenes. In general, L. ivanovii are less pathogenic than L. monocytogenes.

The main source of human infection is eating of food contaminated with L. monocytogenes.
Information on the individual cases and outbreaks of listeriosis has shown that the incidence of
listeriosis is very low, despite the fact that L. monocytogenes is often encountered in a variety of
foods. The fact is that the human listeriosis is rare disease, but the mortality rate is very high,
between 20 and 50%. In humans, listeriosis is opportunistic infections. The disease most often
occurs in those people who are already suffering from another disease, in people older than 65
years, pregnant women, unborn children or infants. It is very dangerous for patients with
leukemia or other distortions, for those who are being treated with corticosteroids or radiation
for people with AIDS, for alcoholics, people with diabetes and people with prosthetic heart
valves. Clinical symptoms of listeriosis are very different. Depending on the prevailing clinical
symptoms vary in nine different forms of listeriosis.

ISOLATION AND IDENTIFICATION OF L. monocytogenes

ENRICHMENT
Primary enrichment

The bacteria of the genus Listeria may be in a low number in food, whilst the total number of
bacteria is very high. Hence they need enrichment. Since it is important to determine also the
presence of the damaged cells of L. monocytogenes, primarily food is enriched in the half-selective
enrichment medium with half the concentration of the selective ingredients Half Fraser
medium (HF) (ISO 11290-1, 1996). The culture medium contains half of concentrations of
acryflavine and nalidixic acid.

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The investigation is done by weight a specific mass of the foodstuff in which we are looking for
L. monocytogenes (i.e. 10 g or ml) into a 9x-volume of HF (90 ml), homogenize and incubate at 30
°C for 24 hours.

Secundary enrichment

Tenth ml of primary enrichment medium after incubation is transferred into 10 ml of Fraser (F)
that is liquid selective enrichment medium, which has a total concentration of selective
ingredients. Suspension is incubated at 37 °C for 48 hours.

ISOLATION
For isolation we use two selective media, Oxford and PALCAM on which primary and secondary
enrichment is inoculated with the loop. Incubation of Oxford plates at 30 °C is used when the
food is not heavily contaminated with other bacteria, otherwise, at 35 °C or 37 °C from 24 to 48
hours. PALCAM plates are incubated microaerophic or aerobic at 35 °C or 37 °C 24 - 48 hours.
Following changes of standard (ISO 11290-1, 1996 / DAM 1: 2004), selective media are ALOA
and the other medium is of choice.

Colonies typical for Listeria:

Oxford: After 24 hours, colonies of Listeria are small with a diameter of 1 mm, gray, with black
zone; after 48 hours, they become darker, may have a green glow, with diameter to 2 mm, a black
zone and the middle of the hollow.

PALCAM: When tha plates were incubated in microaerophilic conditions, plates need to be on
the air 1 hour before reading. After 24 hours of incubation, Listeria colonies are small (diameter
1.5 to 2 mm), gray-green or olive green with a black zone, sometimes with a black center. After
48 hours, colonies are green with black zone.

ALOA: Listeria colonies are small (diameter up to 2 mm), turquoise blue; L. monocytogenes and L.
ivanovii have around colonies transparent to whitish zone.

IDENTIFICATION

Identification of Listeria :
For identification up to 5 typical colonies are taken from each plate with selective medium and
re-inoculate them on TSAYE (Triptic soy agar with yeast extract) non-selective medium. Plates
are incubated at 37 °C for 24 hours. After 18 to 24 hours of incubation typical colonies for
Listeria is colorless and convex, 1 to 2 mm. From these colonies we perform folowing tests:

• Catalase: One colony is mixed on a microscope slide in a drop of hydrogen peroxide. If

bubbles appear immediately, it is a positive reaction.
• Preparation of microscope slide according to Gram: listeria are Gram-positive rods.
• Motility: Re-inoculate Colony from TSYEA plate in TSYEB liquid medium and incubate 8 to
24 hours or until the medium is not turbid at 25 °C. Mobility viewed on a microscopic slide.
As an alternative, the motility medium can be used for observation of motility in which a
typical colony from TSYEA plate is re-inoculated by needle. After 48 hours incubation at 25
°C growth around the injection site is observed.

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Identification of L. monocytogenes :

• Hemolysis: colony from TSYEA plate is re-inoculated on blood agar medium with sheep
blood. For negative control L. innocua is used. After 24 hours of incubation at 35 °C or 37 °C
L. monocytogenes form colonies with transparent zone, which means β-hemolysis
• Using of rhamnose and xylose: re-inoculate listeria colony in a liquid medium containing
rhamnose and xylose and incubate media at 35 °C or 37 °C up to 5 days. L. monocytogenes grow
in the medium with rhamnose and not in medium containing xylose.
• CAMP test: On blood agar inoculate in two parallel lines Staphylococcus aureus and Rhodococcus
equi, between perpendicularly re-inoculate listeria colony. After 18 to 24 hours of incubation at
35 oC ali 37 oC is increased hemolysis zone at Staphylococcus aureus, but not at Rhodococcus equi
when tested listeria is L. monocytogenes.

Final confirmation of L. monocytogenes

Bacteria, which are identified as L. monocytogenes is sent to the reference laboratory for serological
typing.

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COAGULASE-POSITIVE STAPHYLOCOCCI

GENERAL

Staphylococci are Gram-positive, sporeforming cocci that occur individually, in pairs, in the form
of clusters. They are facultative anaerobic, catalase-positive bacteria. Representatives of this genus
synthesize a number of enterotoxins that cause gastroenteritis. Furthermore they can synthesize
coagulase, DNase (thermostable, thermolabile), lecitinase, and hemolysins.

In humans, the primary sources of staphylococci are nasal cavity, ulcerated sores, sore throat;
they can be on skin, eyes, gastrointestinal tract. From here, staphylococci can pass into the air,
dust particles, clothes and other places from where they can be transfered to foods. Staphylococci
are the possible causes of mastitis, and milk is such a source of infection. In foodstuffs of animal
origin and in other heat-treated foods staphylococci are often present in at least a low number.
The most frequently occurring species in food is Staphylococcus aureus. Most strains of S. aureus
synthesize coagulase, thermostable DN-ase, enterotoxins, hemolysin. The optimum temperature
for growth of S. aureus is between 30 and 37 °C (35 and 40 °C), but they can grow in a
temperature range between 7 °C and 48 °C, however synthesise enterotoxins is between 10 °C
and 46 °C (optimal temperature for the synthesis of them is between 40 °C and 45 °C). S. aureus
grow well in the presence of NaCl (between 7 and 10%, some strains also at 20%). The
maximum salt concentration at which S. aureus grow depend on pH, aw, temperature, and Eh. The
optimum pH for growth is between 6 and 7. The minimum value of aw is 0.86, while the other
ideal conditions is somewhat lower.

The synthesis of enterotoxins: enterotoxins are chemically simple proteins. Thermally are more
stable than cells of S. aureus. The maximum synthesis of enterotoxins usually occurs in the
optimal conditions for growth. The size of the population to form enough enterotoxins that
cause gastroenteritis (1 ng/g of food) is usually in excess of 105 cells of S. aureus/g of food.
Staphylococcal toxin can be a heat-stable and even after 30 min of heating at 100 oC does not
lose its activity. Therefore, absence of S. aureus does not mean that the food has no toxin.

We determine Staphylococcus aureus in foods, because:

• We can confirm that these bacteria cause infection.
• We can determine if the food or food ingredient is potential source of enterotoxigenic
staphylococci.
• We can search for subsequent food contamination, which is usually due to human
handling of food, for example, after heat treatment, or the result of inadequate sanitation
in an environment where foods are stored. This subsequent contamination is especially
dangerous because food after heat treatment contains no more (or at least much less)
competitive microorganisms and thus staphylococci have much better conditions for
growth in the food

Foods that are most commonly associated with poisonings with staphylococcal toxins are meat
(beef, pork, poultry) and meat products (sausages, salami, hot dogs), lettuce (containing meat,
poultry, potatoes), creams filled confectionery and dairy products (cheese). As often as
staphylococci normally present on raw foods (especially foods of animal origin), it is even more
important separation and strict separation of work and the way raw and already processed foods
(Downes and Ito, 2001)

ISOLATION AND IDENTIFICATION OF COAGULASE-POSITIVE STAPHYLOCOCCI

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ENRICHMENT
If we determine S. aureus in a greater amount of sample, or if we are looking for these bacteria in
foods in which they are present in very low concentration (typically refers to the product after the
heat treatment or other treatment), we use less selective enrichment of the food in salted broth
(TSB with 20% NaCl) that is the strengthening of staphylococci. If we determine S. aureus in raw
foods or unprocessed foods even where we expect a high concentration of other microorganisms
in food, we use Giolitti Cantoni selective enrichment broth (Downes and Ito 2001; Harrigan,
1998). A certain amount of sample is weighed into a no-9x volume of enrichment broth,
homogenized and the suspension is incubated at 35 °C or 37 °C for 24 hours.

ISOLATION
If we determine S. aureus after enrichment, we re-inoculate enrichment medium on selective
medium. If we determine S. aureus in small quantities of foods we need to prepare basic food
solution and appropriate dilutions. On selective medium is inoculated 0.1 ml of BS or appropriate
dilution. According to SIST EN ISO 6888-2 (1999) standard for the isolation of coagulase-
positive staphylococci Baird - Parker medium is used, which is incubated at 35 °C or 37 °C for
24-48 hours.

Baird - Parker medium (BP):

It is a selective medium for isolation of coagulase-positive staphylococci from food. It contains
K-telurite, glycine, and lithium chloride, which inhibit the growth of other bacteria, pyruvate that
promotes the growth of staphylococci, and 20% of egg yolk.

Typical growth of colonies:

S. aureus: Round, black (due to reduction telurite), smooth, shiny, convex colonies 2-3 mm in
diameter (when it is medium number of colonies), usually with a narrow white (mat) edge, the
medium around the colonies is transparent. Due to the action of different enzymes on egg yolk
lipoprotein, formation of different soluble products is observed - opaque, transparent zone
around the colonies.
S. epidermidis: produce black colonies, medium around colonies is rarely transparent. Some types
of streptococci, corynebacteria and micrococci also form black colonies, but without the
formation of clear zones around the colonies.

IDENTIFICATION
Colonies typical for staphylococci is confirmed by the following tests:
Typical colonies are re-inoculated in BHI (Brain heart infusion broth) non-selective liquid
medium, homogenized and incubated at 37 °C for 24 hours..

Coagulation of blood plasma:

In sterile tube with 0.3 ml of blood plasma (rabbit’s plasma) add 0.1 ml culture from BHI, mix
and incubate at 37 °C. Coagulation of the blood plasma is observed after 4, 6 and 24 hours.
Coagulase-catalysed conversion of fibrinogen to fibrin and reaction is positive when it is more
than ¾ of the initial volume in tube as coagulate. So are determined coagulase-positive
staphylococci.

Blood medium (KA): Typical colonies from BP re-inoculate on KA and incubate at 37 oC for
24 hours. S.aureus form yellow-white colonies with β-hemolysis..

Microscopy:
Microscope slide according to Gram is prepared from colony on non-selective media.

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Bacillus cereus
GENERAL

They are Gram-positive, sporogenic, aerobic or facultative anaerobic rods. Typically, they are
present in soil, water, dust particles, on plants. Minimum temperature for growth is around 5 °C,
maximum is around 50 ° C. They synthesize lecitinase, proteolytic enzymes, heat-labile and heat-
stable enterotoxins. Heat-labile enterotoxin (inactivation 30 min at 56 °C for) is synthesized by
cells during the exponential growth phase. Emetic enterotoxin is thermally stable and cells
synthesize it in stationary growth phase. The presence of B. cereus in lower concentrations in many
foods is normal, whereas these bacteria are normally present in environment. Often are B. cereus
in foods such as rice, potatoes, pasta, meat, vegetable dishes, milk, puddings, soups. The hazard is
when food contaminated with B. cereus is left for a long time at room temperature. Food
poisoning usually occurs when the concentration of B. cereus is up to 107 cfu/g.

ISOLATION AND IDENTIFICATION OF Bacillus cereus

ISOLATION
For the quantitative determination of B. cereus basic food solution and appropriate dilutions are
prepared. 0.1 ml BS or appropriate dilutions is inoculated on selective medium in replicates.
According to ISO 7932 (2004) Bacillus cereus (BC) medium is used for the isolation and plates
are incubated at 30 °C for 18-24 hours

Bacillus cereus medium (BC):

BC medium contains mannitol, polymyxin B, indicator phenol red or bromothymol blue and egg
yolk. Polymyxin B inhibits growth of other bacteria, bromothymol blue indicator for utilization
of mannitol, lecitinase activity is determined with egg yolk.
Typical colonies of B. cereus:
Large blue green colonies (because it does not ferment mannitol (alkali reaction))with a diameter
of about 5 mm, and a serrated edge, colonies are surrounded by a highly opaque zone of the
medium, which is the same color as colonies, which indicates the precipitation of hydrolysed
lecithin and the formation of insoluble diacylglycerols.

IDENTIFICATION
Kolonije značilne za bakterije vrst Bacillus cereus potrdimo z naslednjimi testi: Cepimo tekoče
gojišče MR-VP, tekoče gojišče za redukcijo nitrata in krvni agar za ugotavljanje hemolize. Gojišča
inkubiramo 24 ur pri 30 oC.

Methil-red test (MR) – formation of acid(s) from glucose:

Mix MR-VP medium after incubation and ½ of volume pour into new tube. In the first tube
acid(s) are identified with addition of metil red.
Voges-Proskaver test (VP) – formation of acetoine from glucose:
In the second tube with MR-VP medium add α-naphtol, KOH solution and creatinine, mix well
and observe formation of red colour within one hour..
Reduction of nitrate:
In medium we add 0.2 to 0.5 ml nitrate reagent and in 15 min the medium is red because of
reduction of nitrate to nitrite.
Blood medium:
For B. cereus is tyoical that forms big grey colonies with z α-hemolysis.
Microscopy:
Microscope slides according to Gram and according to po Schaeffer-Fultonu are prepared.

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SULPHITE-REDUCING CLOSTRIDIA

GENERAL

Sulphite-reducing Clostridia are Gram-positive, spore-forming, anaerobic rods. The cells appear
individually, in pairs,or short chains. Spores are central or subterminale. Cells are able to reduce
Na-sulphite (sulfur source) to hydrogen sulphide. This combines with the iron ions (a component
of the medium) in the iron sulfide, which is black (an indicator of the presence of sulphite-
reducing clostridia).

Clostridium perfringens are widespread in nature. They are present in soil, water, dust, digestive tract
of humans and animals. They are mesophilic bacteria, the optimum temperature for growth is
between 37 °C and 45 °C, the minimum around 20 °C and a maximum of about 50 °C. They
grow in range of pH between 5.0 and 8.5. In the presence of more than 5% NaCl, their growth is
inhibited.

C. perfringens strains are devided on the basis of enterotoxins synthesis into five groups of A, B, C,
D and E. Strain of C. perfringens that synthesizes A enterotoxin is usually present in food,
sometimes also strain that synthesizes C enterotoxin. Enterotoxin synthesized by strains of type
A is sensitive to heat (inactivation after 10 minutes of heating at 60 °C). Cells normally synthesize
enteroxin at the same time as they form spores. C. perfringens usually do not form spores in foods,
but then when they get into the digestive tract. Therefore, in fooda are anylysing vegetative forms
and spore, as these bacteria in food can multiply and after consumption they can cause infection
or poisoning.

C. perfringens is often in meat and meat products, poultry, commercially prepared frozen foods,
fruits and vegetables, spices, fish, shellfish, dehydrated soups. Foods that were prepared a day or
more prior to consumption thzs represent a great risk. When it comes to poisoning, the food has
more than 100 - 1000 cfu/g of C. perfringens.

ISOLATION AND IDENTIFICATION OF Clostridium perfringens

ISOLATION

For the quantitative determination of vegetative cells and spores of C. perfringens basic food
solution and appropriate dilutions are prepared. Then 0.1 ml BS in replicates or appropriate
dilution is inoculated on selective medium or 1 ml of MR and appropriate dilution is inoculated
in sterile Petri dish and mixing with melted and at 47oC cooled selective media.
When determining spores of C. perfringens basic food solution is heated for 20 min at 75 °C
(Downes and Ito, 2001), then dilutions are prepared and inoculated as by determining the
vegetative cells and spores.

According to the ISO 7937 (1997) standard for the isolation SICA (egg yolk free tryptose sulphite
cycloserine agar iron citrate) medium is used, which is incubated in anaerobic conditions at 35 °C
or 37 °C for 24 hours. For C. perfringens typical colonies are black (due to reduction sulfite and
elimination of iron sulphide). Since the cycloserine is in the medium, it inhibits growth of
enterococci that can be present in certain food products in higher concentrations, and can
overgrow colonies of clostridia. Black colonies on SICA form olso other sulphite-reducing
Clostridia such as C. bifermentans, C. botulinum, C. paraperfringens, C. sardines and C. sporogens.

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In addition to SICA medium many other selective and differential media are used such as
neomycin blood agar (NKA), sulphite polymyxin sulfadiazine agar (SPS), tryptone-sulfite
neomycin agar (TSN), iron sulphite agar (ISA) and others (Downes and Ito, 2001). Selectivity of
media is achieved by the addition of various antibiotics, most of the media also contains iron, and
sulfite, as Clostridia reduce sulphite to sulphide, and the iron form iron sulfide, which gives the
typical black colonies clostridia.

Growth of Clostridia colonies on SPS medium after 24 hours of anaerobic incubation at 35° C
or 37 °C:
C. perfringens black colonies
C. sporogens black colonies
There maight be also good growth of Streptococcus faecalis in Staphylococcus aureus.

Growth of colonies in ISA medium after 3 to 5 days of anaerobic incubation at 35 °C or 37 °C:

All sulphite-reducing clostridia form black colonies as reducing Na-sulphite to form hydrogen
sulfide, which in turn binds to the iron ions (Fe-sulphide is black). Anaerobic spore-forming
bacteria which do not reduce the Na-sulphite form white colonies.

IDENTIFICATION

Up to 5 typical colonies are transfered with loop in tioglycollate medium, homogenized and
incubated in a water bath at 44 °C for 4 hours or at 35 °C or 37 °C for 12-18 hours.

After incubation:

• Prepare microscope slide according to Gram: C. perfringens are Gram-positive rods,

microscope slide according to Shaffer-Fulton is not prepared as clostridia in that medium
do not form spores.
• By needle colony is re-inoculated in a semi-solid medium for motility determination and
for reduction of nitrate. Media are incubated at 35 °C or 37 °C for 24 hours. C. perfringens
are not motile, red or red-orange color means they reduce nitrate to nitrite.
• By needle colony isre-inoculated in a medium with lactose and gelatin, media are
incubated at 35 °C or 37 °C for 24 to 44 hours. C. perfringens use lactose (gas and acid; the
color changes from red to yellow) and liquefy gelatin.
• Aliquotes of 0.15 ml of thioglycollate medium are reinoculated in medium with raffinose
and salicine and incubated at 35° C or 37 °C for 24 hours. C. perfringens can ferment
salicine, from raffinose form the acid without gas.

Directly from a selective agar typical colony can be transfered into two blood media plates, which
are incubated at 35 °C or 37 °C for 24 hours, one plate aerobically and the second anaerobically.
The finding of Gram-positive rods with spores or without, hemolysis on KA plates that were
incubated anaerobically, and the absence of growth on the KA plates that were incubated
aerobically is considered as alleged positive result for the presence of sulphite-reducing clostridia.

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MICROBIOLOGICAL EXAMINATION OF DRINKING WATER

GENERAL
Drinking water is according to Slovenian regulations (Uradni list RS 19/2004, 35/2004, 26/2006,
92/2006, 25/2009, 74/2015):
1. Water in its original state or after preparation, intended for drinking, cooking, food
preparation or other domestic purposes, regardless of its origin and regardless of whether it is
supplied from a distribution network system for potable water cisterns or as prepackaged
water
2. All water used for the production and marketing of food

Drinking water is healthy suitably when:

1. does not contain microorganisms, parasites and their forms in number, which may pose a
risk to human health;
2. does not contain any substances in concentrations which alone or in combination with other
substances can pose a threat to human health;
3. complies with the requirements for microbiological and chemical parameters, which are
specified by above stated regulations.

Compliance with the limit values of the parameters (hereinafter referred to as compliance) is
compliance with the requirements for the limit values for the parameters listed in Annex I, which,
if necessary, can be supplemented with additional parameters and their limits.

MICROBIOLOGICAL EXAMINATIONS

Microbiological parameters:

General requirements for drinking water:

Parameter Limit value Method
Escherichia coli 0 / 100 ml SIST EN ISO 9308-1
Enterococcus 0 / 100 ml SIST EN ISO 7899-2

Requirements for the water intended for packaging:

Parameter Limit value Method
Escherichia coli 0 / 250 ml SIST EN ISO 9308-1
Enterococcus 0 / 250 ml SIST EN ISO 7899-2
Pseudomonas aeruginosa 0 / 250 ml
Number of colonies at 37 °C 100 / ml SIST EN ISO 6222
Number of colonies at 22 °C 20 / ml SIST EN ISO 6222

Indicator parametrs:
Parameter Limit value Method
Coliforms 0 / 100 ml SIST EN ISO 9308-1
Clostridium perfringens (including 0 / 100 ml mCP
spores) *
Number of colonies at 37 °C 100 / ml SIST EN ISO 6222
Number of colonies at 22 °C Without unusual change SIST EN ISO 6222

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Parameters of regular testing:

Parameter Limit value Method

Escherichia coli 0 / 100 ml SIST EN ISO 9308-1
Coliforms 0 / 100 ml SIST EN ISO 9308-1
Pseudomonas aeruginosa* 0 / 250 ml SIST EN ISO 12780
Clostridium perfringens* 0 / 100 ml mCP
Enterococcus 0 / 100 ml SIST EN ISO 7899-2
Number of colonies at 37 °C Less then 100 / ml SIST EN ISO 6222
Number of colonies at 22 °C Without unusual change SIST EN ISO 6222
* Only in the case that the water is intended for the packing
** Spores only in the event that the water is surface origin or only influenced by

Sampling of drinking water

With burner burn off water tap, let water run for 2-3 minutes and then sample 500 ml in a sterile
bottle.

Sample preparation
The water sample should be mixed well. Dilution: depending on the expected contamination of
the sample make decimal dilutions with physiological saline (ISO 8199).

Escherichia coli and coliforms (SIST EN ISO 9308-1)

1. Coliform bacteria: are those bacteria that form colonies aerobically at 35 °C or 37 °C on a

selective or differential media with lactose with formation of acid (and aldehyde) in 24 hours.
• Filtration of 100 ml water through a filter (2r = 47 ali 50 mm) with pore size of 0,45 µm
• Filter is placed on a selected one or more isolation media: Lactose TCC agar with tergitol,
lactoze agar with Tergitol 7,
• Aerobic incubation at 35 oC ali 37 oC
• Reading of suspected coliform colonies
• Conformation: Each colony (or a representative number) is re-inoculated to lactose
peptone water (LAP) and incubated hours at 37 °C for 48, formation of gas confirm
coliforms

2. Thermotolerant coliforms: are those coliforms that form colonies aerobically at 44 °C or 44.5
°C on a selective or differential media with lactose with formation of acid (and aldehyde) in 24
hours.

Presumptive Escherichia coli: are thermotolerant coliforms that form gas from lactose (and
manitole) and indole from tryptophane at 44 oC in 24 hours.
• Filtration of 100 ml water * through a filter (2r = 47 ali 50 mm) with pore size of 0,45 µm
• Filter is placed on a selected one or more isolation media: Lactose TCC agar with tergitol,
lactoze agar with Tergitol 7
• Aerobic incubation at 44 oC
• Reading of suspected thermotolerant coliform colonies
• Confirmation thermotolerant coliform and E. coli: Each colony (or a representative
number) from filter incubated at 44 °C reinoculate in lactose peptone water (LAP) and
triptone water and incubate them at 44 °C for 24 hours, the formation of gas in lactose
peptone water is conformation of thermotolerant coliform bacteria in water; in triptone

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water 0.2 ml- 0.3 ml of the Kovac reagent is added and formation of a red color on the
surface is proof of indole that is typical for E. coli. For E. coli conformation when it is
required (Aeromonas) carry out oxidase test (needto be negative)

Calculation:

C=
( A × N × VS × F )
(B × Vt )
C confirmed number of colonies in100 ml
A actual number of of confirmed colonies
B number of colonies re-inoculated for confirmation
N number of typical colonies on the filter
Vt a test volume of water
VS a reference volume water – 100 ml
F dilution factor

Enterococcus (SIST EN ISO 7899-2)

100 ml of water is filtered through a sterile membrane filter with a pore size of 0.45 µm. The
filter is placed on m-Enterococcus selective medium acc. Slanetz & Bartley and incubated at 37
°C for 48h.
Count all pink, red or brownish colonies which are shiny and convex. Filter is placed on the
Aesculin agar (preheated at 44 ° C) at 44 ° C for 2 h and then colonies with black zone are
counted.

Number of colonies (SIST EN ISO 6222)

1 ml of water is inoculated in sterile Petri dish (4x), poured with nutritient agar medium; two
plates are incubated at 22 °C for 72 hours and two plates at 37 °C for 48 hours.

Clostridium perfringens (mCP)

100 ml of water is filtered through a sterile membrane filter with a pore size of 0,2 µm. The filter
is placed on mCP medium and incubated at 44 °C for 21h.
The suspected colonies - yellow opaque is inspected under hood after they are subjected from 20
s to 30s with the vapors of 25% ammonium hydroxide (poison!) and if they turn pink colonies
are C. perfringens.

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WORKBOOK

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1. EXERCISE: Total microbial count

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of the total count of bacteria and the number of yeast and mold

RESULTS:

Native microscopic slide: Coloured microscopic slide:

Sample: colony from OGY Sample: colony from NA

Magnification: Magnification:
Cells: Cells:

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2. EXERCISE: Number of spore-forming bacteria

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of number of aerobic spore-forming bacteria and anaerobic spore-
forming bacteria

RESULTS:

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3. EXERCISE: Coliform bacteria

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of coliform bacteria

RESULTS:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

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4. EXERCISE: Escherichia coli

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of Escherichia coli

RESULTS:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

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5. EXERCISE: Salmonella
PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of Salmonella

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RESULTS:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

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6. EXERCISE: Listeria monocytogenes

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of L. monocytogenes

RESULTS:

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Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

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7. EXERCISE: Coagulase-positive staphilococci

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of coagulase-positive staphylococci

RESULTS:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

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8. EXERCISE: Bacillus cereus

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of Bacillus cereus

RESULTS:

Microscope slide according to Gram:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

Microscope slide according to Schaeffer-Fulton:

Bacteria:
Magnification:
Cells shape:
Cells formation:
Gr+ / Gr-:

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9. EXERCISE: Sulphite-reducing clostridia

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Determination of spore and vegetative cells of C. perfringens

Figure 2: Determination of spore of C. perfringens

RESULTS:

Microscope slide according to Gram: Microscope slide according toSchaeffer-Fulton :

Sample: Sample:
Magnification: Magnification:
Cells shape: Spores:
Cells formation:
Gr+ / Gr-:

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10. EXERCISE: Microbiological examination of water

PURPOSE:

MATERIALS:

METHODS:
Figure 1: Microbiological examination of drinking water

RESULTS:

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NOTES

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SAFETY REQUIREMENTS
• In microbiology laboratory always wear protective gowns.

• In microbiology laboratory smoking, eating and drinking is not allowed.

• On the work-bench is only material that you need to carry out an investigation.

• Windows and doors must be closed during the experimental work due to the risk of
contamination.

• Always use aseptic technique.

• Gas burners at the beginning and at the end of the work open and close assistant or technician.

• When working near the gas burner be careful.

• If you come to direct contact with microorganisms, tell the assistant or technician.

• To avoid contamination of the environment, place of contamination must first be disinfected,

washed with water and wipe with a paper towel.

• Contaminated glassware and plastic tips for automatic pipettes always place in the prepared basket
or container with disinfectants.

• Waste should never be placed in trash as it is contaminated with microorganisms.

• Microorganisms can not be out of the microbiological laboratory.

• Work with microscopes with care; make sure that the microscop and cleaned.

• Microscopic slides can not be throwing in the trash, but in prepared dish with disinfectants.

• After finishing work clean and disinfect work desk.

• After finishing work, disinfect hands, wash them thoroughly with soap and water and dry with a
paper towel.

• Wuth dangerous and toxic substances, work in a fume hood.

The results of the observations should be written in the workbook.

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REFERENCES
Downes F. P., Ito K. 2001. Compendium of methods for microbiological examination of foods. 4th edition.
Washington, APHA, 659 str.
Harrigan W. F. 1998. Laboratory methods in food microbiology. 3rd edition. London, Academic Press Limited, 532
str.
International standard. ISO 7954. 1987. Microbiology - General guidance for the enumeration of yeast and moulds -
Colony count technique at 25oC. 1st ed. Geneve: International Organization for Standardization, 3 str.
International standard. ISO 4831. 1991. Microbiology - General guidance for enumeration of coliform - most
probable number technique. Geneve : International Organization for Standardization, 11 str.
International standard. ISO 4833. 1991. Microbiology - General guidance for the enumeration of microorganisms -
Colony count technique at 30 oC. 2nd ed. Geneve: International Organization for Standardization, 4 str.
International standard. ISO 6579. 1993. Microbiology - General guidance on methods for the detection of
Salmonella. 3rd ed. Geneve: International Organization for Standardization, 1993. - IV, 16 str.
International standard. ISO 7251. 1993. Microbiology - General guidance for enumeration of presumptive
Escherichia coli - most probable number technique. 2nd ed. Geneve: International Organization for
Standardization, 7 str.
International standard. ISO 7218. 1996. Microbiology of food and animal feeding stuffs - General rules for
microbiological examination. - 2nd ed., Geneve: International Organization for Standardization, 43 str.
International standard. ISO 11290-1. 1996. Microbiology of food and animal feeding stuffs - Horizontal method for
the detection and enumeration of Listeria monocytogenes. Part 1, Detection method. 1st ed. Geneve: International
Organization for Standardization, 16 str.
International standard. ISO 7937. 1997. Microbiology of food and animal feeding stuffs - Horizontal method for
enumeration of Clostridium perfringens : colony-count technique. - 2nd ed. - Geneve : International Organization
for Standardization, 10 str.
International standard. ISO 6887-1. 1999. Microbiology of food and animal feeding stuffs - Preparation of test
samples, initial suspension and decimal dilutions for microbiological examination. Part 1, Genearal rules for the
preparation of the initial suspension and decimal dilutions. - 1st ed. - Geneve: International Organization for
Standardization, 5 str.
International standard. ISO 6579. 2002. Microbiology of food and animal feeding stuffs - Horizontal method for the
detection of Salmonella spp. 4th ed. Geneve: International Organization for Standardization, 27 str.
International standard. ISO 11290-1. 2004. Microbiology of food and animal feeding stuffs - Horizontal method for
the detection and enumeration of Listeria monocytogenes. Part 1, Detection method. Amendment 1, Modification
of the isolation media and the haemolysis test, and inclusion of precision data. - Geneve: International
Organization for Standardization, 13 str.
International standard. ISO 7932. 2004. Microbiology of food and animal feeding stuffs - Horizontal method for
enumeration of presumptive Bacillus cereus : colony-count technique at 30 oC. 3rd ed. Geneve: International
Organization for Standardization, 8 str.
International standard. ISO 21528-1. 2004. Microbiology of food and animal feeding stuffs - Horizontal methods for
the detection and enumeration of Enterobacteriaceae. Part 1, Detection and enumeration by MPN technique with
pre-enrichment. 1st ed. Geneva: International Organization for Standardization, 12 str.
International standard. ISO 21528-2. 2004. Microbiology of food and animal feeding stuffs - Horizontal methods for
the detection and enumeration of Enterobacteriaceae. Part 2, Colony-count method. 1st ed. Geneva : International
Organization for Standardization, 10 str.
Praktikum mikrobiološke analize: skripta in delovni zvezek za študente IV. letnika živilstva. 2000. Uredila Barbara
Jeršek. Ljubljana: Biotehniška fakulteta, Oddelek za živilstvo, Katedra za živilsko mikrobiologijo, 32 str.
Pravilnik o pitni vodi. Uradni list RS 19/2004, 35/2004
Slovenski standard SIST EN ISO 6888-2.1999. Mikrobiologija živil in krmil. Horizontalna metoda štetje koagulazno
pozitivnih stafilikokov (Staphylococcus aureus in drugih vrst). 2.del, Tehnika uporabe agarja z zajčjo plazmo iz
fibrinogenov (ISO 6888-2:1999). 1. izd., 7 str.
Trkov, M. Praktikum mikrobiološke analize. Interno gradivo. Ljubljana, Biotehniška fakulteta, Oddelek za živilstvo,
Katedra za živilsko mikrobiologijo, 1997, loč. pag.
Zakon o zdravstveni ustreznosti živil in izdelkov ter snovi, ki prihajajo v stik z živili (ZZUIZS). Uradni list RS
52/2000; 42/2002
Zakon o spremembah in dopolnitvah določenih zakonov na področju zdravja (ZdZPZ). Uradni list RS 47/2004
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063335.htm

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Instructions and workbook for Microbiological Examination of Food laboratory
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PHOTOS

Salmonella on SS medium Proteus on SS medium

Salmonella on XLD medium Bacillus cereus BC medium

Staphylococcu aureus on BA medium Staphylococcu aureus on BP medium

Listeria on ALOA medium Listeria on Rapid L. mono medium

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Instructions and workbook for Microbiological Examination of Food laboratory
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Listeria on PALCAM medium Moulds on OGY medium

Aerobic plate count on NA medium Bacillus cereus on KA medium

E. coli on EMB medium E. coli on Tergitol medium

Listeria on Oxford medium Listeria on ALOA medium

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