SAMPLE PREPARATION - jornades.uab.catjornades.uab.cat/workshopmrama/sites/jornades.uab.cat.workshopmr...broth preparation 1-2 Test for Salmonella ... 1.1. Sample preparation and plating

SAMPLE PREPARATION

Buffered

peptone

water

Decimal

dilutions

Dilution 1/10

(25 g / 225 ml)

Petrifilm

yeast and mould

Dilucup

Vitroids

2 x

TEMPO

Eddy Jet 2

DryPlates

Soleris

Petrifilm Aqua

Colilert

Enterolert

Pseudalert

Quanti-Tray

NEO-GRID

Milliflex Quantum

LENTICULE Discs

easySpiral Dilute

SimPlate

AUTOMATED COLONY COUNT

Flash & Go

Chromogenic media

plating

Buffered

peptone

water

Fraser 1/2

VIP Gold

Listeria

Singlepath

L’mono

Reveal 2.0 Salmonella Singlepath

Campylobacter

Selective enrichment

broth preparation

1-2 Test for

Salmonella

Scan 1200

ENVIRONMENTAL CONTROL

BioFinder

CONTAM

SWAB

InSite

ESC Swab

Gluten flow through AllerFlow gluten

Contact slides

Sampler Sampl’air Sampler MicroBio

Count-Tact

plates RODAC plates

Quick Swab

HydraSponge

Sam

plin

g a

nd

co

un

ting

(co

lon

ies)

Luminometer

Clean-Trace NG

Luminometer

MVP ICON

Luminometer

AccuPoint Advanced

Bio

lum

inis

cen

ce (A

TP

)

Reveal 3-D

Clean Test AllerSnap FLASH

Co

lorim

etry

(pro

tein

s)

Co

lorim

etry

(bacte

ria)

Imm

un

olo

gy (p

rote

ins)

Oth

er

Petrifilm rapid

coliform count plate

Coliforms / Escherichia coli

CHROMOGENIC CULTURE MEDIA

chromID Coli RAPID’E.coli 2

Petrifilm select

E. coli

DryPlates EC

Brilliance

chromogenic

coliform

CCA coliform

cromogenic

ASAP

Salmonella spp.

Chromatic SALMONELLA

RAPID’Salmonella Brilliance Salmonella

IRIS Salmonella BBL CHROMagar

Salmonella

BBL CHROMagar O157

Petrifilm E. coli /

coliform count plate

Chromocult coliform

DIAGNOSTIC TESTS AND KITS

RapID ONE

Salmonella spp.

Agglutination tests

API

Microgen

latex Oxoid

latex test

Enterobacteria

EnteroPluri-Test

CHROMOGENIC CULTURE MEDIA

Staphylococcus aureus

Baird-Parker RPF BBL CHROMagar

Staph aureus

Campylobacter spp.

Brilliance CampyCount CASA

ALOA RAPID’L.mono COMPASS Listeria Brilliance Listeria

Listeria monocytogenes

DIAGNOSTIC TESTS AND KITS

Listeria monocytogenes

Microgen ID

Microbact Listeria 12 L

O·B·I·S· Rhamnose test

Enzyme confirmation cards

Different microorganisms

ID membranes

MYCOTOXIN DETECTION

Reveal Q+ and Accuscan GOLD reader

BBL Crystal ID Gram +

Ochratoxin-A rapid test

Workshop MRAMA: Laboratory sessions

Universitat Autònoma de Barcelona

1

Overview of the methods employed

1. Wednesday

1.1. Sample preparation and plating automated procedures

- Dilutor DiluFlow Pro:

DiluFlow gravimetric dilutors enable the automatic dilution of the sample with perfect reliability.

- Pulsifier:

The Pulsifier is used for dislodging microorganisms from foods without excessively breaking the food structure.

The Pulsifier has an oval metal ring that can house a plastic bag with sample and diluents. When the instrument

is activated, the ring will vibrate vigorously for a predetermined time (around 30-60s). During this time,

microorganisms on the food surface or in the food will be dislodged into the diluents with the minimum

destruction of the food.

Fung and colleagues in 1988 evaluated the Pulsifier against the Stomacher with 96 different food items

(included beef, pork, fish, shrimp, a variety of vegetables, cereal, etc) and found that the systems gave

essentially the same viable cell count in the food but the “Pulsified” samples were much clearer than the

“Stomached” samples and they had less debris.

- BagMixer SW:



2

- Dilucup-Dilushaker:

The Dilucup is delivered in blisters of rows of either 3 or 6 containers, each filled with 9 ml diluting media. It is

easy to separate the number of Dilucups needed. The Dilucups are placed on the tray of the Dilushaker. The

Dilucups are opened by tearing off the film covering the holes in the lids. When the Dilushaker is turned on,

the liquid in all Dilucups rotates. A sample of 1 ml is added to the first cup and is instantly mixed with the

media. After changing the pipette tip, 1 ml of sample is withdrawn from the first cup and added to the second.

With the Dilushaker operating the process is repeated until the required dilution factor is achieved.

- Spiral plater Eddy Jet 2:



3

- EasySpiral Dilute:

EasySpiral Dilute enables serial dilutions up to 10-5 with increased accuracy. It is a very user-friendly tool: one

button touch for the routine serial dilutions and automatic plating. It then enables automatic standardized

plating of 30 to 1 trillion countable CFU/mL on 1 single Petri dish.

1.2. Rapid viable cell count methodologies

- Vitroids:

Vitroids are discs that contain viable microorganisms in a certified quantity. Consisting of pure cultures of

bacteria or fungi in a solid water soluble matrix, they are stable for at least one year and are in a viable state

with a shelf life of 1-3 years.

The preparation can be performed in most solid and liquid medium or rehydration buffer can also be used.

Discs can be rehydrated in as little as 100 µL buffer, or in larger volumes, e.g. 100 mL medium. It is also possible

to add the disc to a cooled molten medium used for pour plate techniques. The rehydration process takes

approximately 10 minutes. On solid media, the disc forms a droplet that can be spread with a sterile loop. In

liquid media, the disc dissolves very quickly.

- Lenticule discs:

LENTICULE discs contain viable microorganisms in a certified and narrow defined quantity (ISO/IEC 17025),

produced under reproducible conditions (ISO Guide 34). The bacterial and fungal strains are prepared directly

from strains selected from PHE’s National Collection of Type Cultures (NCTC) and National Collection of

Pathogenic Fungi (NCPF). The discs consist of bacteria or fungi in a solid water soluble matrix. Microorganisms

in this form are stable for at least one year and are in a viable stage (no lag phase or recovery time). Each batch

is provided with a comprehensive certificate of analysis that specifies the mean number of colony forming

units (cfu), an expanded uncertainty about the mean value, details about the method used to determine the

product data and the number of passages (subcultures) from the original strain.



4

- Petrifilm and Petrifilm Aqua (plates and reader):

The 3M Petrifilm plate is an all-in-one plating system. They are heavily used in many microbiology-related

industries and fields to culture various micro-organisms and are meant to be a more efficient method for

detection and enumeration compared to conventional plating techniques.

3M Petrifilm™ Aerobic plate count

3M Petrifilm™ Aqua yeasts and moulds

The 3M Petrifilm Aqua AQYM Plate is a sample-ready culture medium system which contains nutrients

supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator that facilitates yeast and

mold enumeration. 3M Petrifilm Aqua AQYM Plates are used for the enumeration of yeast and mold in bottled

water industry.



5

3M Petrifilm™ EC/Coliform plate counts

Petrifilm E. coli/Coliform Count (EC) plates contain Violet Red Bile (VRB) nutrients, a cold-water-soluble gelling

agent, an indicator of glucuronidase activity, and an indicator that facilitates colony enumeration. Most E. coli

(about 97%) produce beta-glucuronidase which produces a blue precipitate associated with the colony. The

top film traps gas produced by the lactose fermenting coliforms and E. coli. About 95% of E. coli produce gas,

indicated by blue to red-blue colonies associated with entrapped gas on the Petrifilm EC plate (within

approximately one colony diameter).

- DryPlates:

It is a microbial detection that does not require media preparation. That is why it has the advantages of a

dehydrated media (long expiration term) and prepared medias (sterile, ready for immediate use, timer-saver).

It cold self-diffuses the sample ml, thus avoiding the need for handles, and eliminating the agar’s temperature

cooling critical point to avoid burning microorganisms and increasing by 10 the plates detection limit.

- Colilert-18 and Quanti-Tray:

Colilert-18 is a commercially available enzyme-substrate liquid-broth medium that allows the simultaneous

detection of total coliforms and E. coli. It is available in the most-probable number (MPN) or the

presence/absence (PA) format. The MPN method is facilitated by use of a specially designed disposable

incubation tray called the Quanti-Tray.



6

Two enzyme substrates are included in Colilert—a chromogen that reacts with the enzyme found in total

coliforms (galactosidase), and a fluorogen that reacts with an enzyme found in E. coli (glucuronidase). After

18 or 24 hours incubation at 35ºC, a total- coliform-positive reaction turns the medium yellow; an E. coli-

positive reaction causes the medium to fluoresce under a long-wave ultraviolet light (366 nm).

- Pseudalert:

The Pseudalert test detects the presence of Pseudomonas aeruginosa in bottled, pool, and spa water samples.

The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa

through the hydrolysis of a substrate present in the Pseudalert reagent. P. aeruginosa cells rapidly grow and

reproduce using the rich supply of amino acids, vitamins, and other nutrients present in the Pseudalert

reagent. Actively growing strains of P. aeruginosa have an enzyme that cleaves the substrate to produce a

blue fluorescence under UV light. Pseudalert detects P. aeruginosa at 1 cfu in either 100 mL or 250 mL samples

within 24 hours for non-carbonated water samples and within 26 hours for carbonated samples.



7

- Enterolert-E:

Enterolert-E detects enterococci, such as E. faecium and E. faecalis, in fresh and marine water. It is based on

IDEXX’s patented Defined Substrate Technology* (DST*). When enterococci utilize their ß-glucosidase enzyme

to metabolize Enterolert-E’s nutrient-indicator, 4-methyl-umbelliferyl ß-D-glucoside, the sample fluoresces.

Enterolert-E detects enterococci at 1cfu per 100 mL sample within 24 hours.

- NEO-GRID (hydrophobic grid membrane):

NEO-GRID tests are based on the concept of hydrophobic grid membrane filtration. The filter membrane is

embossed with hydrophobic ink to produce a grid containing 1600 individual squares. The ink’s hydrophobic

nature contains the growth of an organism isolated during the filtration process within the square of capture.

Samples are first diluted in a sterile buffer. The NEO-GRID filtration unit is then placed onto a manifold

connected to a vacuum source. Using the vacuum source, a portion of the diluted sample is filtered through

the filter membrane. The membrane is then transferred to an agar plate prepared with a medium formulated

to promote/differentiate the growth of the target organism. Following incubation, the plate’s squares

containing presumptively positive colonies are counted to yield a most probable number (MPN) result.

- Milliflex Quantum:

The Milliflex® Quantum system is a rapid fluorescent-based

technology designed for fast quantitative detection of

microorganisms over a broad range of filterable matrices. This

non-destructive method also allows you to identify any

detected microorganisms using the current ID methodology.



8

- SimPlate:

SimPlate Total Plate Count utilizes proprietary Binary Detection Technology to provide quantitative results for

total aerobic bacteria from food and environmental samples in just 24 hours. Proprietary chromogenic media

and patented plating device with isolation wells combine to produce easily counted results and eliminate

problems associated with food particle interference or liquefying of gels caused by certain bacterial enzymes.

- Soleris:

The Soleris system is a rapid optical system for the detection of microbial contamination based on an

innovative application of classic microbiology familiar to microbiologists. The optical assay measures microbial

growth by monitoring pH and other biochemical reactions that generate a colour change as microorganisms

in the broth grow and metabolize. The results are displayed by color-coded monitoring with an alert on

samples out of specification.

The technology is based on monitoring changes in the chemical characteristics of microbial liquid growth

medium in which the target microorganisms grow and are detected by optically sensitive reagents. The

reagents change their spectral patterns as the metabolic process takes place. These changes are detected

photometrically by an optical instrument and monitored at predetermined time intervals. The key to the

technology is the monitoring of these changes in a semifluid zone of the patented organism-specific vial. This

zone is separated from the liquid medium, thereby eliminating the masking of the optical pathway by the

sample matrix, or microbial turbidity. Various dyes, which are indicators of metabolic activity such as pH,

redox, and enzymatic and CO2 production, can be utilized in the system.



9

- Colony counters Flash & Go:

It is an automatic colony counter for pour plates and any type of spiral spreading. Complete instrument with

centering holder for different Petri dishes size. There is a software to count colonies in any media.

- Scan 1200:

2. Thursday

2.1. Environmental control procedures

- Count-Tact and RODAC plates:

- Hycheck and other contact slides:

Hycheck are hygiene contact slides used to assess the microbiological contamination

of surfaces or fluids. They are double sided. Feature a hinged paddle that bends for

easy sampling.



10

- Quick Swab:

It is a ready-to-use environmental swab system to detect microbial contamination. For Dry Sampling:

1) Remove swab from tube and swab targeted area. Place swab back into tube and bring to the lab.

2) Bend red snap valve to transfer all the broth into the tube.

3) Shake or vortex tube vigorously for 10 seconds to release bacteria from swab.

4) Pour contents onto, for example, a Petrifilm Plate.

- ESC swab:

ESC (Easy Surface Checking) swab range includes a new generation of sampling devices for

microbiological examination of surfaces, especially recommended for food and pharmaceutical

industries.

- Samplers Microbio and Sampl’air:

- MonitorMark:

3M MonitorMark Time/Temperature Indicators monitor product

temperature exposure through the entire supply chain. They provide

a non-reversible record of temperature exposure that is accurate and

easy to interpret.



11

2.2. Description and preparation of chromogenic culture media

- CASA:

This medium allows a high selective enumeration of Campylobacter spp. On the selective chromogenic CASA

agar, there is a strong inhibition of growth of the competitive bacteria from the intestinal flora

and Campylobacter colonies appear red and are easily detected.

- ASAP:

It is a chromogenic culture medium for Salmonella detection from food and environmental samples. The

principle of the ASAP medium is based on the detection of C8-esterase activity, which is found in

all Salmonella species. C8-esterase activity of Salmonella is visualized by the pink to purple coloring of their

colonies (specific cleavage of the substrate).

• Pink to purple colonies � C8-esterase detection Salmonella spp.

• Blue to blue-green colonies � Beta-glucosidase detection � Expl: Klebsiella, Enterobacter.

• Violet blue colonies � C8-esterase & beta-glucosidase detection � Expl: Serratia

- ALOA:



12

ALOA agar is a pre-prepared, selective and differential medium for the isolation of Listeria spp. from food

samples and for the presumptive identification of L. monocytogenes. To minimize the growth of contaminating

organisms, lithium chloride and a balanced antimicrobial and antifungal mixture is employed. The

incorporation of the chromogenic substrate X-glucoside for the detection of beta-glucosidase demonstrates

the presence of Listeria spp., whilst the detection of a specific phospholipase C enzyme produced by

pathogenic Listeria spp. including L. monocytogenes is also achieved. Listeria spp. grow on this medium

producing blue - green colonies, with pathogenic species (L. monocytogenes and L. ivanovii) producing similar

coloured colonies surrounded by a characteristic opaque halo after 24 hours incubation at 37ºC. Non Listeria

spp. produce white colonies.

- chromID Coli:

Detection and enumeration of E. coli at 44°C and simultaneous enumeration of E. coli and other coliforms at

37°C, in food products. The chromID Coli medium contains 2 chromogenic substrates, which enable the

simultaneous detection of coliforms and identification of E. coli, without the use of additional reagents.

• β-GAL (+) : presence of coliforms other than E. coli � blue colonies (β-galactosidase).

• β-GUR (+) : presence of E. coli � rose colonies (β-glucuronidase).

- Baird-Parker RPF agar:

Staphylococcus aureus is a Gram-positive coccus capable of producing enterotoxin which can induce food

poisoning. The organisms may be present in small numbers in many foods, and, if allowed to multiply

unchecked, may produce highly heat resistant enterotoxins. The ability of S. aureus to produce lecithinase and

lipase has been recognised for many years, and the detection of these enzymes in egg yolk media has become

a widely used procedure for the identification of this organism. Its ability to produce coagulase using a similar

basal formulation enables confirmatory diagnosis with the incorporation of rabbit plasma into the base

medium.



13

This medium is a modification of Baird-Parker Medium and is recommended for the selective isolation,

enumeration and confirmation of S. aureus from food and other specimens. The reduction in potassium

tellurite concentration in RPF Agar results in S. aureus strains forming white, grey or black colonies, which are

surrounded by an opaque halo of precipitation, i.e. the coagulase reaction.

- Brilliance CampyCount agar:

Campylobacter is a leading cause of enteric disease in most developed countries. The organism is endemic in

many poultry populations; 98% of food-borne infections are caused by C. jejuni and C. coli. In recent years,

there have been numerous improvements in animal husbandry and carcasses processing that have reduced

the prevalence of Campylobacter in poultry. However, it is unfeasible that the complete elimination of

Campylobacter can be brought about in the near future. However, to reduce human infection, it is generally

accepted that further reduction in the levels of Campylobacter on the fowl is a more feasible goal. In order to

bring this about, a shift in industry standards from a presence/absence testing to enumeration needs to occur.

Brilliance CampyCount Agar is a transparent medium which makes identification of C. jejuni and C. coli

significantly easier than on traditional charcoal or blood containing agars. It contains an indicator that, when

metabolised by the target organisms, changes colour. As it builds up in the cells it turns colonies dark red,

making all C. jejuni and C. coli colonies readily identifiable. The components of Brilliance CampyCount Agar

have been carefully designed to maximise growth of C. jejuni and C. coli while inhibiting non-target organisms.

This defined formulation means the medium can be used to accurately enumerate the loading of C. jejuni and

C. coli on poultry carcasses and related samples.

- Brilliance Salmonella agar (Oxoid Salmonella Precis method):

An Inhibigen compound is comprised of two components, combined together by a bond that can only be

cleaved by a specific enzyme. When bound together, the inhibitor compound is not toxic and therefore can

exist in a medium without harming microorganisms. Once inside the cell, the bond will be cleaved if the target

enzyme is present. When the bond is cleaved, the inhibitor molecule is released and disrupts cell wall

synthesis, causing death of the organism. As cells die and lyse, free inhibitor is released but cannot be taken



14

up by other cells, resulting in targeted inhibition. The Inhibigen in Brilliance Salmonella Agar targets E. coli.

Novobiocin and cefsulodin, presented as a freeze-dried supplement, are added to the medium to inhibit the

growth of other competing flora such as Proteus spp. and Pseudomonas spp.

Differentiation of Salmonella from the other organisms that grow on Brilliance Salmonella Agar is achieved

through the inclusion of two chromogens that also target specific enzymes: caprylate esterase and ß-

glucosidase. Caprylate esterase is an enzyme present in all samonellae as well as some species

of Klebsiella, Enterobacter and Proteus. Organisms possessing caprylate esterase cleave the chromogen to

release an insoluble purple chromophore. As the cells grow, the chromophore builds up and produces a

purple-coloured colony. Some Enterobacteriaceae, including Klebsiella and Enterobacter but not Salmonella,

possess ß-glucosidase. If these organisms grow, they will form blue or dark blue colonies, even if they are

esterase positive, which make them easy to differentiate from purple Salmonella colonies.

- Brilliance Listeria agar (Oxoid Listeria Precis method):

Brilliance Listeria Agar uses the chromogen X-glucoside for presumptive identification of Listeria spp. This

chromogen is cleaved by ß-glucosidase which is common to all Listeria species. Other organisms that possess

this enzyme, such as enterococci, are inhibited by the selective agents within the medium; lithium chloride,

polymyxin B and nalidixic acid, whilst amphotericin inhibits the growth of any yeasts and moulds present in

the sample. Listeria monocytogenes and pathogenic Listeria ivanovii are then further differentiated by their

ability to produce the phospholipase enzyme, lecithinase. This enzyme hydrolyses the lecithin in the medium,

producing an opaque white halo around the colony. The medium is designed to identify Listeria spp. based on

their utilisation of a chromogenic substrate. However, in this modification, the pathogenic Listeria spp. are

then further differentiated by the detection of lecithinase (phosphotidylcholine phospholipase C (PCPLC)

activity, rather than phosphotidylinositol phospholipase C (PIPLC) activity. Both enzymes, PCPLC and PIPLC,

are associated with virulence in Listeria spp., and, therefore, the presence of either enzyme is a useful

indicator of pathogenicity.



15

- Brilliance E.coli/coliform agar:

Brilliance E.coli/coliform agar is a differential agar used for the presumptive identification of Escherichia

coli and coliforms from food and environmental samples. The agar base uses two enzyme substrates to

differentiate between E. coli and other coliforms. One chromogenic substrate is cleaved by the enzyme

glucuronidase, which is specific for E. coli and produced by approximately 97% of strains. The second

chromogenic substrate is cleaved by galactosidase, an enzyme produced by the majority of coliforms. This

results in purple E. coli colonies, as they are able to cleave both chromogenic substrates, and pink coliform

colonies, as they are only able to cleave the galactosidase chromogen.

- RAPID’Salmonella:

The principle of RAPID’Salmonella chromogenic medium relies on the demonstration of two enzymatic

activities. Salmonella spp. takes the form of readily identified typical magenta colonies (detection of C8

esterase). Counter selection based on β-D-Glucosidase is used to reveal other bacteria with a different color.

As expected in the regulations, RAPID’Salmonella permits detection of motile and non-motile Salmonella, as

well as lactose-positive Salmonella, Salmonella Typhi and Salmonella Paratyphi.

- RAPID’E.coli 2:

RAPID’E.coli 2 is a selective chromogenic medium used for direct enumeration without confirmation, of

coliforms including Escherichia coli. The principle of the medium relies on simultaneous detection of two

enzymatic activities: β-D-Glucuronidase (GLUC) and β-D-Galactosidase (GAL). Coliforms (GAL+/GLUC-) form

blue to green colonies, E. coli (GAL+/GLUC+) forms violet to pink colonies.



16

- RAPID’L.mono:

The RAPID’L.mono chromogenic medium specifically detects the phospholipase of Listeria monocytogenes and

its inability to metabolize xylose. After 24 hours of incubation, L. monocytogenes forms characteristic blue

colonies without a yellow halo.

- Rhamnose test:



17

- IRIS Salmonella agar:

IRIS Salmonella Agar shows a high specificity for the detection of Salmonellae including atypical species and

serovars, which is a source of confusion on other medium. Indeed, the detection of Salmonella Typhi and

Paratyphi, lactose-positive Salmonellae (Salmonella Senftenberg and subspecies S. arizonae and S. diarizonae),

saccharose-positive strains are ensured. The media allows the detection of non-motile serovars (S. Pullorum

and S. Gallinarum) or monophasic strains. IRIS Salmonella Agar allows also the detection of strains which show

a light or absence of esterasic activity on other medium (Salmonella Bongori, Salmonella Dublin and Atento,

certain strains of S. houtenae and S. diarizonae subspecies). The selective agents permit the inhibition of Gram-

positive and some Gram-negative bacteria. IRIS Salmonella Agar may be used in the standard methods for the

detection of Salmonellae as second isolation medium.

- COMPASS Listeria agar:

COMPASS Listeria Agar allows the detection of blue colonies surrounded by an opaque halo, typical of Listeria

monocytogenes.

The peptones and growth factors (yeast extract, sodium pyruvate and magnesium sulfate) favor the excellent

growth of Listeria monocytogenes. Yeast extract is also a source of vitamin B complex. Sodium chloride

maintains the osmotic equilibrium of the media. Listeria hydrolyzes the 5-bromo-4-chloro-3-indolyl-β-D-

glucopyranoside (or X-β-glucoside). The resulting product is subjected to an oxidative dimerization that forms

a blue precipitate in the center of the colonies. Phosphatidyl-inositol is used as a substrate for the detection

of phospholipase C of Listeria monocytogenes. When it is degraded, an opaque precipitate is formed around

the colonies. Secondary microflora is inhibited by the association of lithium chloride and a judicious mixture

of selective agents that include several antibiotics and an antifungal agent.



18

- BBL CHROMagar Salmonella:

Specially selected peptones supply the nutrients. Gram-positive organisms are generally inhibited as a result

of the selective medium base. The addition of an antifungal agent prevents the growth of Candida species and

other antimicrobial agents are used to inhibit the growth of gram-negative, non-glucose fermenting bacteria

and Proteus species, which could potentially overgrow Salmonella colonies. A chromogenic mixture is included

in the medium. Due to metabolic differences in the presence of selected chromogens, colonies of Salmonella

species appear mauve (rose to purple) in colour, whereas undesired bacteria are either inhibited, or produce

blue-green or colourless colonies.

- BBL CHROMagar O157:

CHROMagar O157 is intended for the isolation, differentiation and presumptive identification of E. coli

O157:H7. Due to the chromogenic substrates in the medium, colonies of E. coli O157:H7 produce a mauve

color, thus allowing presumptive identification from the primary isolation plate and differentiation from other

organisms. Gram-negative organisms, other than E. coli O157:H7, will either be inhibited or produce colorless,

blue, green, blue-green (aqua) or natural color colonies.

- BBL CHROMagar Staph aureus:



19

S. aureus is a well-documented pathogen. It is responsible for infections ranging from superficial to systemic.

Due to the prevalence of this organism and its clinical implications, detection is of utmost importance.

Staphylococcal food poisoning caused by S. aureus is one of the most common types of foodborne illness

worldwide. Its detection and enumeration help provide information about the potential health hazard of food,

as well as being an indicator of poor hygiene. It is also recommended that this organism can be used as an

indicator of water quality.

BBL CHROMagar Staph aureus is intended for the isolation, enumeration and identification of S. aureus based

on the formation of mauve-colored colonies. The addition of chromogenic substrates to the medium facilitates

the differentiation of S. aureus from other organisms. The addition of selective agents inhibits the growth of

gram-negative organisms, yeast and some gram-positive cocci. The chromogen mix consists of artificial

substrates (chromogens), which release an insoluble colored compound when hydrolyzed by specific enzymes.

This facilitates the detection and differentiation of S. aureus from other organisms. S. aureus utilizes one of

the chromogenic substrates, producing mauve-colored colonies. The growth of mauve-colored colonies at 24

h is considered positive for S. aureus on BBL CHROMagar Staph aureus. Bacteria other than S. aureus may

utilize other chromogenic substrates resulting in blue, blue-green, or if no chromogenic substrates are utilized,

natural colored colonies.

- Chromatic SALMONELLA:

Proteose Peptone and meat extract provide amino acids and proteins. Yeast extract is a source of amino acids

and vitamins of group B. Sodium chloride maintains the osmotic balance of the medium. The special

chromogenic mix allows differentiation of Salmonella spp., including S. typhi, from other coliform and non-

coliform bacteria, on the basis of the colour and the morphology of the colonies. Beside the chromogenic mix

inhibits Gram-positive microorganisms. Tween 20 increases microbial growth.

Salmonella typhimurium and other Salmonella species will appear as light mauve to mauve – colored colonies.

Citrobacter and other coliforms will appear as light blue- green to blue- green colored colonies. Some

organisms that do not hydrolyze any of the chromgenic compounds may appear as colorless colonies. Final

identification must be performed by biochemical and/or serological tests.



20

- CCA chromogenic agar:

CCA Chromogenic Agar is a culture medium for the enumeration of Escherichia coli and coliform bacteria after

membrane filtration stage. The CCA is based on enzymatic reactions that give colour to the colonies of target

organisms for simultaneous detection of coliforms and E. coli. This medium is suitable for samples with low

microbial load as drinking water, swimming pools, disinfected waters and water treatment plants at the end

of the treatment.

Count ß-galactosidase positive colonies and ß-glucuronidase negative colonies (all colonies coloured from

salmon-rose to red) as coliform bacteria different from E. coli. Count ß-galactosidase positive colonies and ß-

glucuronidase positive colonies (all colonies coloured from deep blue to violet) as E. coli. Total coliform count

is obtained by the addition of the salmon-rose to red colonies plus the deep blue to violet colonies.

- Chromocult coliform agar:

It allows the simultaneous detection, enumeration and confirmation of

coliforms and E. coli in water within 24 hours. Chromocult® Coliform Agar comes

in a granulated format and is the only chromogenic culture medium which was

used for validation to prepare the new ISO 9308-1 (2014) version.

- ID membranes:

These membranes are for economical and rapid identification and confirmation of microorganisms in water,

food, environmental and clinical samples. They find their application in various sectors in food and dairy

industry, water industry, etc.

The membranes contain chromogenic substrates such as ONPG, X-Gal, or X-Glu and other substrates and

indicators which serve as the basis for the differentiation by color. The target organisms are characterized by

enzyme systems that metabolize the substrates and initiate the color change. The colors can then be visually



21

detected on the membranes and show the enzymatic activity of the microorganisms which is a helpful tool for

the identification of genus and species.

2.3. Miniaturization

- TEMPO EC (enumeration by miniaturized MPN)

The TEMPO instrument is the food industry’s first automated quality indicator testing system for the

enumeration of quality indicator organisms in food and environmental samples.

TEMPO is based on the Most Probable Number (MPN) method. The principle of the MPN method is to allow

any microorganism present in samples to grow in suitable conditions in tubes, using a minimum of three

dilutions and three tubes per dilution.

BioMérieux has automated and miniaturized this method, which was previously time-consuming and subject

to errors due to the numerous steps involved.

2.4. Preparation of diagnostic kits

- API (kits and reader):

The API (Appereils et Procedes d’Identification) microbial identification test system consists of an array of

around 20 e.g. API 20E. Each of the mini-test tubes contains dehydrated media and reagents that are designed

to detect the presence or, absence of selected biochemical pathways production, in the microbe of concern.

To perform an API test, pure microbial growth e.g. a colony from an agar plate, is mixed with sterile saline

solution to form a microbial inoculum of the correct density; this is manually dispensed into each of the mini-

test tubes using a sterile pipette. Some of the mini-test tubes are filled completely, while others are filled

partially and overlaid with sterile mineral oil to provide anaerobic conditions. After inoculation, the strip is

placed in a plastic incubation box along with some water, to prevent moisture loss from the mini-test tubes

during incubation. After incubation at an appropriate temperature e.g. 37°C, for an appropriate length of time

e.g.18 to 24 hours, growth and reagent utilisation is assessed by either by colorimetric, fluorescent or turbidity

means - sometimes this requires the addition of extra reagents. For example, one drop of Kovac’s reagent has



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to be added to develop the indole (IND) reaction; one drop of TDA reagent (10% ferric chloride) to develop

the tryptophan deaminase (TDA) reaction; one drop of VP1 (40% potassium hydroxide) followed by one drop

of VP2 (6% alpha naphthol) for the Voges Proskaueur reaction; and one drop of oxidase reagent for the

cytochrome oxidase (OX) reaction.

The reactions of each test tube are recorded with the aid of the manufacturer’s interpretation.

- BBL Crystal ID (kits and readers):

The BBL Crystal ID kits feature an easy-to-use identification method using fluorogenic and chromogenic

reagents. Various BBL Crystal Kits are offered to identify several gram-negative, gram-positive, and anaerobic

organisms. BBL Crystal ID Kits provide one-step convenience, easy inoculation, and a comprehensive list of

organisms that can be identified. Following an incubation period, the sample can be manually read on the

Crystal panel viewer to obtain a profile number.

- RapID ONE:

The RapID ONE System is a qualitative micromethod employing conventional and chromogenic susbtrates for

the identification of medically important Enterobacteriaceae and other selected oxidase-negative, gram-

negative bacilli isolated from food, water, human clinical specimens, etc.

A clear plastic tray contains reagent impregnated wells, allowing simultaneous inoculation of each cavity with

a predetermined volume of inoculum. A suspension of test organism in RapidID inoculation fluid is used as the

inoculums which rehydrates and initiates test reactions. After incubation, each cavity is examined for reactivity

by noting the development of a color. In some cases, reagents must be added to the cavities to provide a color

change. The resulting pattern of positive and negative scores is used as the basis for identification of the test

isolate by comparison of the test results to reactivity patterns stored in a database or through the use of a

computer-generated code compendium (ERIC).



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- O·B·I·S· Salmonella:

The O.B.I.S. Salmonella Test offers a rapid screening method to distinguish Salmonella spp. from those

organisms exhibiting similar colonial appearance on common selective Salmonella media. This reduces the

need for a full biochemical identification of suspect colonies.

Identification of Salmonella relies on primary isolation of the organism on selective enteric media. However

there are several other genera among the Enterobacteriaceae also capable of growth on such media and which

can have similar colony morphology to salmonellae. The lack of PYRase and NPA activity in Salmonella spp.

can be used to differentiate them from Citrobacter spp. Which possess PYRase activity 1,2,3 and Proteus,

Morganella and Providencia spp. Which have NPA activity 4.

Oxoid has developed a new system for PYRase testing 5, using a non-carcinogenic substrate in response to

health concerns associated with the use of b -naphthylamide (a potent carcinogen) 6. The PYRase area on the

O.B.I.S. salmonella Test Card is impregnated with L-pyroglutamic acid 7-amino-4-methylcoumarin (7AMC) 5.

Dimethylaminocinnamaldehyde is used as a colour development reagent. The enzymatic hydrolysis of the

substrate produces a purple colour on addition of the O.B.I.S. PYR Developing Solution 5.

The NPA area on the O.B.I.S. Salmonella Test Card is impregnated with nitrophenylalanine. Deaminatin of the

reagent is shown by an orange-brown colour when the O.B.I.S. NPA Developing Soloution (0.25M sodium

hydroxide) is added.

- Microbact:

The Microbact range offers a selection of self-contained, biochemical-based tests for the identification of key

pathogenic bacteria, including Gram-negative bacteria, Listeria species and Staphylococcal species.

- Microgen ID:

It is a standardized bacterial identification system. Presented in convenient microwell formats, these systems

provide flexible identification platforms which in turn provide savings in cost, time and key laboratory space

(incubation and storage).



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- EnteroPluri-Test:

EnteroPluri-Test makes possible the identification of the Enterobacteriaceae and other gram negative, oxidase

negative bacteria isolated from non-clinical samples. The identification is based on biochemical tests

performed on culture media containing specific substrates. The combination of positive and negative reactions

allows building up a code number that permits to identify bacteria by using the Codebook.

- Enzyme confirmation cards:

These tests utilize a specific substrate which, when hydrolyzed by a specific enzyme of the target organism

(during peptide hydrolysis), produces a blue/white fluorescence, or purple/blue color upon the addition of

Reagent B (color developer) when applicable.



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3. Friday

3.1. Preparation and reading of other environmental control procedures

3.1.1. ATP-bioluminescence

- Luminometers Clean-Trace NG:

3M Clean-Trace NG Luminometer NG3 is a simple, quick and reliable hand held diagnostic tool to verify

cleaning effectiveness and capture, store and manage data for later analysis.

Residues remaining on surfaces indicate poor cleaning and provide nutrients for spoilage or pathogenic

bacteria to thrive, which could lead to product contamination. With the unique swab-click-read format and

simple color change technology, this 3M test is very easy to use and implement in a busy production

environment. The results are semi-quantitative with four possible colors. The faster the test turns purple the

higher the level of contamination on a surface. The test detects protein and other reducing agents, considered

as a superior hygiene indicator to glucose as more difficult to remove from a surface.

- AccuPoint:

The AccuPoint Advanced ATP Hygiene Monitoring System is a handheld device that accurately detects ATP

from surfaces and rinse water samples. The three colour-coded samplers with liquid-stable chemistry are

unrivalled in their accurate recovery of ATP from surfaces and rinse waters. Unlike traditional swabs utilised

by other manufacturers, our samplers cover a larger surface area to extract ATP more consistently.



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- MVP ICON:

The MVP ICON is the first system to provide true HACCP management capabilities. Featuring advanced photon

counting sensor (PCS) technology, the MVP ICON provides superior accuracy, sensitivity, and reproducibility

for ATP, pH, temperature, conductivity, and concentration. The innovative design is sleek, lightweight and

features an easy to use touch-screen interface.

3.1.2. Colorimetry

- InSite Salmonella:

InSite Salmonella is an easy-to-use, self-contained,

environmental Salmonella test. Each device contains a liquid

medium formulated with growth enhancers and chromogenic

compounds selective for Salmonella species. Simply swab the test

area and incubate! A change in color after 24-48 hours of

incubation is considered presumptive positive for Salmonella

species.

- AllerSnap:

AllerSnap is an easy-to-use, quick and affordable test for verifying allergens have been removed

from surfaces. By detecting all protein residues, AllerSnap screens for a broad range of allergens.

This saves time and eliminates additional costs associated with running specific allergen tests.

- Contam Swab:

- FLASH:

FLASH is a total protein visual test that rapidly detects protein residues left on food contact surfaces after

cleaning. Protein is a difficult food residue to remove. Most allergens are proteins, so quick verification of



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surface hygiene helps minimize the risk of cross-contamination to allergen-free products. Using FLASH

regularly also helps reduce the opportunity for biological contamination.

- Clean Test:

The 3M Clean-Trace is based on the measurement of levels of ATP present on an environmental surface. A 3M

Clean-Trace ATP Surface Test is used to swab a selected test point. The test is then activated and the swab is

brought into contact with the test enzyme solution (luciferin-luciferase). The enzyme reacts with any ATP

residue present on the swab bud. A product of this reaction is the generation of light by the enzyme solution.

The Clean-Trace ATP Surface Test is then placed in the 3M Clean-Trace NGi Luminometer. This measures the

light generated by the enzyme solution and produces a result expressed in Relative Light Units (RLUs). The

greater the level of ATP present on the swab, the greater the amount of light generated by the test and

consequently, the higher the RLU level produced. The test can be performed in less than 30 seconds, providing

a real-time result that indicates the cleanliness of the surface tested. This provides an opportunity to take any

corrective action required such as re-cleaning and re-testing the surface.



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3.1.3. Others

- BioFinder

It reacts to detect the group of microorganisms attached to surfaces, called Biofilms. It immediately reveals

contaminated areas by simple visual inspection. It can be applied to the most commonly used surfaces in the

food industry, such as stainless steel, polypropylene and epoxy-coated surfaces. Biofinder has advantages over

other methods, with time and cost reduction being the most significant ones. It simplifies monitoring surface

hygiene of industrial processes. Its formula and packaging type make it possible to treat large areas. It doesn’t

stain or leave residues on surfaces due to its high water solubility which aids in rinsing. Thanks to its simple

application and response type, technical staff is not required for handling. It is not considered hazardous to

the environment, according to current legislation.

3.2. Preparation and reading of immunological detection methods

3.2.1. Confirmation by latex agglutination

- Microgen latex:

Microgen latex is a latex slide agglutination test for the confirmatory identification of presumptive pathogenic

colonies from selective agar plates.

- Oxoid latex test:



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A loop full of material is taken from the top of a positive indicator tube, for example, Salmonella Rapid Test

and it is mixed with a drop of the latex test reagent. If agglutination of the latex test reagent occurs, then

Salmonella is present in the material. Similarly, presumptive Salmonella colonies can be taken from an agar

plate, mixed with the latex test reagent and agglutination will occur if Salmonella is present.

3.2.2. Immunoprecipitation

- 1-2 Test for Salmonella:

The 1-2 Test is highly sensitive and specific. It uses a unique combination of a built-in selective enrichment,

the immunodiffusion principle, and a proprietary preparation of antibodies to detect Salmonella.

3.2.3. Lateral immunomigration

- Singlepath:

- VIP Gold:

VIP Gold is a single-step visual immunoassay for the detection of pathogens in food and environmental

samples. Each device contains a proprietary reagent system, which forms a visually apparent antigen-

antibody-chromogen complex if the pathogen is present.



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- Reveal 2.0:

Reveal 2.0 for Salmonella is a simple yet sophisticated lateral flow technology that is easy to perform with

minimal sample touch time. This robust lateral flow technology lends itself to multiple food matrices and is

scalable to any operation—from low volumes to a theoretically unlimited throughput. Reveal 2.0 for

Salmonella continues the tradition of convenient unitized-irradiated media which eliminates need for

autoclaving. The simple assay procedure produces clear results in 15 minutes following enrichment.

- Reveal Q+ for mycotoxins:

Reveal Q+ for mycotoxins, a range of simple tests to detect precise amounts of mycotoxins in grain and feed

samples. Due to the known severe threat to human and animal health, more than 100 countries have

established regulatory limits for mycotoxins in commodities intended as human food or animal feed. The

Reveal Q+ product line adds to Neogen’s unrivalled range of simple and accurate lateral flow, microwell and

immunoaffinity column mycotoxin testing options.

For example: Aflatoxin is considered by many to be the most potent, naturally-occurring carcinogen known. It

has been linked to a variety of health problems in both humans and animals. Aflatoxin is a by-product of mould

growth in a wide range of commodities. Two moulds that are major producers of aflatoxin are Aspergillus and

A. parasiticus.

- Reveal 3D for allergens:



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Neogen Reveal 3-D food allergen kits are easy-to-use and interpret strip tests that screen samples for the

detection of trace levels of specific food allergens in 10 minutes or less. The unique Reveal 3-D tests allow for

rapid screening for the presence of low levels of allergen in CIP rinses and environments swabs virtually

anywhere. The Reveal 3-D allergen tests utilize a 3-line readout: a control line confirms the method has been

performed successfully and two further lines differentiate low & high levels of detection.

- Ocratoxina-A rapid test:

Aerobic plate count DryPlates TC 2 Soleris 16SimPlate 12 Milliflex Quantum TSA 11

Enterobacteria API 20E 3RapID ONE 17EnteroPluri-Test 13

E. coli /coliforms Petrifilm rapid coliform count plate 1 Enzyme confirmation cards 11 TEMPO EC (E. coli ) 3Petrifilm select E. coli 1 Differential Coli-E. coli ID Membrane 11 Colilert 14Petrifilm E. coli / coliform count Plate 1DryPlates EC 2RAPID'E.coli 2 5chromID Coli 3Brilliance chromogenic coliform agar 17Chromocult Coliform Agar 11CCA coliforms chromogenic agar 15BBL CHROMagar O157 10

Salmonella spp. ONE Broth Salmonella 17 Brilliance Salmonella 17 Enzyme confirmation cards 11 Oxoid Salmonella latex test 17RAPID'Salmonella capsules 5 RAPID'Salmonella 5 Microgen Salmonella latex 6(for buffered peptone water) ASAP 3 Reveal 2.0 16Salmonella enrichment 4 IRIS Salmonella 4 1-2 Test for Salmonella 12 + IRIS supplement Chromatic SALMONELLA 13

BBL CHROMagar Salmonella 10L. monocytogenes ONE Broth Listeria 17 Brilliance Listeria 17 Microbact Listeria 17 VIP Gold Listeria 12

ALOA 3 Microgen Listeria ID 6 Singlepath L'mono 11RAPID'L.mono 5 O·B·I·S· Listeria 17COMPASS Listeria 4 Rhamnose test (RAPID'L.mono confirmation) 5

S. aureus Baird-Parker RPF 3 Enzyme confirmation cards 11BBL CHROMagar Staph aureus 10 ID Dnase membrane 11

BBL Crystal ID Gram + 10P. aeruginosa Pseudalert 14Campylobacter Brilliance CampyCount 17 O·B·I·S· Campy 17 Singlepath Campylobacter 11

CASA 3 3M España 1

Yeasts and moulds Petrifilm yeast and mould 1 Laboratorios MICROKIT 2Mycotoxins Ochratoxin A rapid test 21 bioMérieux España 3

Reveal Q+ 16 Bioser/Biokar Diagnostics 4Water Petrifilm Aqua 1 Bioser/Bio-Rad Laboratories 5Others NEO-GRID 16 Bioser/Microgen Bioproducts 6

Bioser/Otros 7

ITRAM HIGIENE 8

IUL 9Vitroids 11 DiluFlow Pro 18 Count-Tact plates and applicator 3 Luminometer Clean-Trace NG: 1 CONTAM SWAB 13 BD Diagnostic Systems 10

· E. coli Dilucup-Dilushaker 7 MacConkey agar RODAC plates 13 · Clean-Trace water - free ATP InSite 19 Merck/Sigma-Aldrich Química 11

· S. enterica BagMixer SW 18 Contact slide 1 13 · Clean-Trace water - total ATP MicroPlanet Laboratorios/BioControl Systems 12

· S. aureus Pulsifier 6 Sampler Sampl'air 3 · Clean-Trace surface - ATP FLASH 12 MicroPlanet Laboratorios/Liofilchem 13LENTICULE discs 11 Stomacher 20 Sampler MicroBio 7 Luminometer MVP ICON 12 Clean Test 13 IDEXX Laboratorios 14

· L. monocytogenes easySpiral Dilute 18 TSA lecithine/polysorbate RODAC plates 10 Luminometer AccuPoint Advanced 16 AllerSnap 19 PanReac AppliChem 15

Spiral plater Eddy Jet 2 9 HyCheck contact slide 10 · AccuPoint ATP water sampler Nirco/Neogen Europe 16

Colony counter Flash & Go 9 HydraSponge 1 · AccuPoint ATP acces sampler Reveal 3-D Total Milk Test 16 Thermo Fisher Scientific 17Colony counter Scan 1200 18 Quick Swab 1 · AccuPoint ATP surface sampler AllerFlow gluten 19 Interscience 18AccusScan Gold 16 ESC Swab 13 Gluten flow through 21 BC Aplicaciones Analíticas/Hygiena 19

Seward 20BioFinder 8 BioSystems 21

Selective broths

Reference strains Sample prepartion, automationCount (colonies)

Chromogenic culture media

Environmental control

Others

Other rapid count methodologies and/or miniaturizationDiagnostic kits

Colorimetry (bacteria)Environmental control

Immunology (proteins)

Environmental control

Immunological methods

Bioluminiscence (ATP)

Colorimetry (proteins)

Documents

SAMPLE PREPARATION - jornades.uab.catjornades.uab.cat/workshopmrama/sites/jornades.uab.cat.workshopmr...broth preparation 1-2 Test for Salmonella ... 1.1. Sample preparation and plating