selling the portfolio - thermo fisher safety/4...selling the portfolio . ... • confirmation –...

The world leader in serving science Proprietary & Confidential

Julie Elston Global Product Manager, Immunodiagnostics and Product Training Manager

Selling the Portfolio

2 Proprietary & Confidential

• Industrial Workflow • Food testing

• Media, DryBags, Precis • Confirmation – biochemical ID and latex agglutination

• Water Testing

• Media and confirmation

• Pharmaceutical • Environmental monitoring – TWIP and *NEW* Contact Plate Applicator

Agenda


• Extensive product range

• Our core business is manual microbiology

• We are moving forward with technological advances

• BUT...... ......we must protect our market share and we must protect our customer base

in preparation for future developments

• There are still many opportunities , especially cross-selling products from the customer’s workflow

Our portfolio


Industrial Workflow

Sample Collection

Fisher Scientific

Sample Enrichment

/Dilution

Sample Preparation/Purification

Detection Confirmation/ Identification Further ID


• Food Testing

• Water Testing

• Pharmaceutical testing

Industrial workflow


Sample Collection

Fisher Scientific

Sample Enrichment

/Dilution

Sample Preparation/Purification

Detection Confirmation/ Identification Further ID

Food Testing – laboratory workflow

Presenter

Presentation Notes

The Thermo Scientific microbiology portfolio includes an extensive range of products for the isolation, identification and enumeration of foodborne pathogens. These products range from culture media and diagnostic kits to quality control organisms. Our focus on providing quality products, on-time delivery and superior support is matched by our commitment to provide complete solutions that meet your testing needs.

http://www.uaz.edu.mx/histo/pathology/ed/ch_9b/c9b_staphaurex.jpg


Food Testing – What do people look for and why?

• Pathogens • To ensure that food is safe and meets legislation

• Toxins • Some organisms such as Salmonella produce dangerous toxins

• Indicator organisms • Counts, TVC, E. coli, coliforms, Enterobacteriaceae A change in expected number indicates that something in the process has

gone wrong or changed

• The presence of specific organisms may indicate the increased likelihood of certain pathogens being present (monitoring normal levels not just presence absence)

Listeria spp. monitoring could indicate L. monocytogenes levels and E. coli monitoring could indicate E. coli O157

• Spoilage organisms • Organisms that will cause food to go off or cause undesirable traits e.g. Yeast & Moulds


Microbiological Food Testing

1. Pre-enrichment

2. Selective Enrichment

3. Selective Plating

4.Confirmation Tests


Pre-enrichment

• Helps growth of target organisms that may be stressed e.g. drying, low pH and high temperature

• Helps grow sufficient numbers of bacteria to be detected on media

• No select agents (antibiotics) as this may harm the target organism

• Competitor organisms grow as well

1. Pre-enrichment


Selective enrichment

• Contains selective agents to kill non-target organisms • Increases growth of the target organism

2. Selective Enrichment


Selective Plating

• More selective agents included to inhibit the growth of non-target organisms that grew in selective enrichment

• Allows differentiation of target organism usually by • Colour of colonies • Clear zones

3. Selective

Plating


Confirmation of positives • Confirms that you have isolated your target

organism

• A wide selection of tests can be performed, according to target organism

• Physical tests • Gram stain, motility, shape

• Biochemical tests • Sugar metabolism • Oxidase • Catalase

• Immunological tests

• Agglutination of homologous antibody/antigen

4.Confirmation Tests


Salmonella Précis and Listeria Précis Workflows


2 days

Salmonella Precis

ENRICHMENT ONE Broth-Salmonella

PLATING Brilliance™ Salmonella

CONFIRMATION Oxoid Salmonella Latex

or ISO 6579 methods


Listeria Precis Workflow

2 days

ENRICH ONE Broth-Listeria

PLATE Brilliance™ Listeria

CONFIRM O.B.I.S. mono

or ISO 11290


Innovative format and workflow solution for efficient food pathogen testing

DryBags


Automated Salmonella Précis Preparation

Peristaltic Pump

ONE Broth Dry-Bag

Automated Diluter

Brilliance Salmonella

Salmonella Latex

15 – 30 mins 1 sample/min 24 hours/48

hours 15 mins

Sample Collection

Sample Enrichment

Sample Purification Detection Confirmation ID/Charact

erization

2 days


Automated Précis Preparation

Peristaltic Pump

ONE Broth Dry-Bag

Automated Diluter

Brilliance Listeria

O.B.I.S Listeria

15 – 30 mins 1 sample/min 24 hours/48

hours 15 mins

Sample Collection

Sample Enrichment

Sample Purification Detection Confirmation ID/Charact

erization

2 days


Food Microbiology Confirmation Methods


Customers

Food Laboratories… • World population is growing and consuming more, leading to an increase in the number of food microbiology tests carried out each year

• Higher demands and expectations as well as increased levels of regulation as governments attempt to ensure that manufacturers meet minimum safety levels for food

• QC laboratory manager is the key person in deciding which tests to run, where they are done, what methods are used and the frequency of testing

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/

Purification Detection Confirmation Identification/

Characterization


Biochemical Identification - What it is and how it works

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Thermo Scientific™ Remel™ Spot Tests

Single-substrate reagents for rapid identification of microorganisms • Skilled microbiologists can identify the majority of isolates seen routinely using

colony and Gram-stain morphology, and one-step biochemical spot tests • E. coli can be identified based on growth and lactose fermentation of MacConkey

Agar, indole positivity, oxidase negativity, and beta glucuronidase positivity (MUG) within one hour at low cost

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Rapid Spot Test range

Product # Product Name Organism ID

R21132 Microdase Disk Modified oxidase test for differentiation of staphylococci from micrococci

R21135 MUG Disk For presumptive identification of Escherichia coli by fluorogenic assay

R211667 Nitrocefin Disk Haemophilus spp., N. gonorrhoeae, M. catarrhalis, staphylococci, enterococci, and anaerobes

R21134 Novobiocin Disk For differentiation of Staphylococcus saprophyticus from other coagulase-negative staphylococci

R211357 PRO Disk w/Reagent For identification of yeast and anaerobic microorganisms primarily Candida albicans and Clostridium difficile

R211172 PYR Disk w/Reagent For presumptive identification of enterococci, Lancefield group A streptococci, and E. coli

R21138 PYR/Esculin Disk For presumptive identification of group A streptococci and enterococci

R21137 Pyridoxal Disk For demonstration of growth by nutritionally deficient streptococci

R21152 Urea-PDA Disk For demonstration of urease activity and phenylalanine deamination in Enterobacteriaceae

R21115 ALA Disk For demonstration of porphyrin synthesis in Haemophilus spp.

R261605 Beta Lactam Disk Detection of beta-lactamase production in Haemophilus spp., N. gonorrhoeae, and staphylococci

R21120 Beta Lysin Disk For detection of CAMP factor by group B streptococci

R21122 Bile Disk Screening test to separate Bacteroides fragilis from other gram-negative anaerobic bacilli

R21128 Caffeic Acid Disk For demonstration of phenol oxidase production by Cryptococcus neoformans

R21121 Catarrhalis Test Disk For rapid detection of butyrate esterase for presumptive identification of Moraxella catarrhalis

R211820 Gram-Sure Rapid test disk for differentiation of aerobic gram-positive and gram-negative rods or coccobacilli

R21085 Hippurate Disk Detect hydrolysis of sodium hippurate by group B streptococci, G. vaginalis, and C. jejuni

R21087 Indoxyl Acetate Disk For rapid identification and differentiation of Campylobacter, Wolinella, and Helicobacter

R21129 LAP Disk For the rapid detection of leucine aminopeptidase activity in streptococci and related genera

R30168501 LAP Disk w/Reagent Differentiation Aerococcus/Leuconostoc from Streptococcus/Enterococcus/Lactococcus/Pediococcus


Oxoid Biochemical Identification Systems

• OBIS PYR (ID0580M) - determination of PYRase activity in streptococci and Citrobacter • OBIS Mono (ID0600M) - differentiation of L. monocytogenes from other Listeria species • OBIS Campy (ID0800M) - differentiation of Campylobacteriaceae from other Gram negative

organisms • OBIS Salmonella ( ID0570M) - to differentiate Salmonella spp. from similar organisms • OBIS albicans (ID0700M) - differentiation of Candida albicans from other yeast species

• Position as quick, safe and simple, confirmatory tests • Results in 2 – 12 minutes from testing colony • Reduce the number of more expensive high level identifications • Quickly screen out negatives

Thermo Scientific™ O.B.I.S.™

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


O.B.I.S. Kit General Protocol

1. Carry out orientation tests such as catalase, oxidase and/or Gram stain 2. Smear organism into reaction area(s) of card 3. Moisten reaction area of card 4. Allow to react (time and temperature depend on test kit; 30 seconds – 1

hour) 5. Add developing solution to reaction area(s) 6. Wait for reaction to take place (20 seconds) 7. Look for colour reactions and record results 8. Determine if further identification is required

Refer to pack insert for exact methodology of each kit

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

Presenter

Presentation Notes

Detailed protocols to follow


• AOAC Official Method – first choice of system in food

• 15 biochemical tests detecting specific enzymes and/or metabolic end products

• Self-contained test system with reagent impregnated discs

• R38370 – Micro-ID Listeria ID System • 4 hour screen for L. monocytogenes

• Positive esculin and rhamnose disks

• 24 hour incubation for species identification

• R38145 – Micro-ID Identification System • 4 hour incubation • Enterobacteriaceae identifications

Thermo Scientific™ Remel™ MICRO-ID™ Identification System

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Thermo Scientific™ Remel™ MICRO-ID™ LISTERIA

• Test Gram-postive, oxidase-negative, catalase-positive colonies • Prepare organism suspension to McFarland No. 1 • Inoculate each well • Close lid and incubate at 35 – 37oC for 24 hours • After 4 hours, l. monocytogenes suspsected if esculin (E) and rhamnose (RHAM) are

positive • After 24 hours add reagent to VP disk • Rotate device to moisten disks. Read and record colour change

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


• Self-contained biochemical test system

• MB1128A – Microbact Listeria 12L • 11 carbohydrate utilization test and 1 rapid microhaemolysis test

• Identification in as little as 4 hours

• No prior subculturing on non-selective medium, can be used with chromogenic or selective agars

• MB1074A – Microbact GNB Kit (several configurations)

• 24 tests total available in strips of 12 tests which can be used independently or together

• MB1561A – Microbact Staphylococcal 12S • 12 tests based on sugar utilization and enzyme detection substrates

Thermo Scientific™ Oxoid™ Microbact™

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


• Test Gram-postive, oxidase-negative, catalase-positive colonies

• Prepare suspending medium • Suspend single isolated colony for 24 hr test • Prepare McFarland 0.5 Standard for 4 hour test

• Inoculate each well with 4 drops of suspension • Add 1 drop of haemolysin reagent to Well 12 • Replace lid and incubate at 35 – 37oC for 4 or 24 hours • Remove lid and read and record colour change

Thermo Scientific™ Oxoid™ Microbact™ Listeria 12L

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Thermo Scientific™ Remel™ RapID™ Systems

• When a confirmation must be made, waiting for an ID panel to do its job can be frustrating

• Traditionally, manual, confirmatory ID panels provide results in 24 – 48 hours because they utilize carbohydrate fermentation reactions that are dependent on organism growth

RapID works differently

• It uses enzyme technology to reduce the time-to-results to 4 hours • Based on microbial degradation of specific substrates • Combination of conventional tests and single-substrate chromogenic tests • The advantage - provide a definitive answer to bacterial ID faster

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


RapID Principle

• Ideal for stand-alone testing or for complementing automated systems when testing for anaerobes, yeast, Neisseria-Haemophilus, Enterobacteriaceae, staphylococci, streptococci, Corynebacteria, non-fermenters, and urinary tract isolates

• Reaction cavities contain dehydrated reactants and the tray allows the simultaneous inoculation of each cavity with a predetermined amount of inoculum

• A suspension of the test organism in RapID Inoculation Fluid is used as the inoculum which rehydrates and initiates test reactions

• After incubation of the panel, each test cavity is examined for reactivity by noting the development of colour

• The resulting pattern of positive and negative test scores is used as the basis for identification of the test isolate by comparison of test results to reactivity patterns stored in a database

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


RapID Range


Features & benefits

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

Presenter

Presentation Notes

The chapter focuses on the features and benefits of the RapID product range


• Simple biochemical tests help to address current lab issues • Best use of the laboratory’s resources • Assure quality test results • Reasonable cost

• Extensive range of single step biochemical tests

• Part of the end-to-end workflow solution

Spot Tests and O.B.I.S. Competitive Advantage

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


MICRO-ID™ Competitive Advantage

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

• AOAC Official Method • First choice identification method for customers

• 4 hour Enterobacteriaceae identification

• 4 hour L. monocytogenes screen • 24 hour species level identification for all Listeria species



Oxoid™ Microbact™ Competitive Advantage

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

• 4 hour incubation for Listeria species

• 24 hours for Gram negative bacteria and Staphylococcus species

• Simple, strip format

• Can be an alternative to Micro-ID products



RapID™ Systems Competitive Advantage

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Latex Agglutination - What it is and how it works

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Thermo Scientific™, Oxoid™ and Remel™ Latex Agglutination Kits

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

• For the detection or identification of micro-organisms using immunological technology

• Polystyrene latex particles are sensitised with either a specific antigen • Ready-to-use reagents • Simple, easy to perform procedures • Rapid results • Particle agglutination

• Red blood cell agglutination • Latex agglutination

• wet latex • dry latex

• FDA 510K approved • CE marked


Test Principles – Agglutination tests

The tests are always

• Rapid • Simple • Visual

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Test Principles - Latex Agglutination

• For the detection or identification of micro-organisms or specific antibodies using immunological technology

• The polystyrene latex particles are sensitised with either a specific immunoglobulin or specific antigen

Key

Bacteria (antigen)

Latex particle

Y (sensitised with antibody)


A positive latex agglutination reactions is indicated by the visual clumping of particles with clearing of the opaque background A negative latex agglutination reaction is indicated by a lack of clumping and the opaque background stays unchanged

Positive Negative

Agglutination Tests


Food Agglutination Kits (wet)

Name No. of tests Code • Wellcolex E. coli O157:H7 50 R30959501/R30959601 • Wellcolex Colour Salmonella 50/200 R30858301/R30858302 • Staphaurex 120/400 R30859901/R30859902 • Staphaurex Plus 150/450 R30950102/R30950201 • Campylobacter Test Kit 50 DR0155M • Staphylase 100 DR0595A • E.coli O157 Kit 100 DR0620M • Staphytect Plus 100/500 DR0850M/B • PBP2I 50 DR0900A • Salmonella Test Kit 100 DR1108A • Listeria Test Kit 100 DR1126A

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Food DrySpot Agglutination Kits

Name No. of tests Code • Staphytect Plus 120 DR0100M • E. coli O157 120 DR0120M

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization


Dryspot Latex Reagents

• Completely dry including Control Reagents • less handling • no dispensing errors • Less chance of contamination

• Room temperature storage

• fridge space

• Extended shelf life • 2 years

• Improved reproducibility


spotted Agglutinate with antigen Extract

5. 4.

Antibody absorbed to latex

Dry formulation spotted

Bacterial suspension or sera added

Agglutination observed

• Reagent Formulation

DrySpot Latex Reagents


Features & benefits

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

Presenter

Presentation Notes

The chapter focuses on the features and benefits of the RapID product range


Latex Agglutination Competitive Advantage

Sample Collection

Sample Enrichment

/Dilution

Sample Prep/


Characterization

• Large range which includes ‘’Gold Standard’’ kits

• Test and Control reagent eliminates reporting of non specific agglutination

• Wellcolex patented coloured latex technology

• DrySpot room temperature storage and extended shelf life

• One step in the end-to-end workflow solution offered by Thermo Scientific™


Water Testing Workflow


Water Testing – laboratory workflow

Presenter

Presentation Notes

Culture based methods have traditionally been used for detection of micro-organisms in food. After collection of the sample a pre-treatment or enrichment step is often needed to get the number of any target organism up to a detectable level so that it can be grown an visualized either in a broth or more usually in colony format on a plate. Over the last couple of decades new technologies have enabled the introduction of tests that provide a more convenient and or rapid solution by replacing the plating step with another means of detection such as immunological capture or molecular techniques. Some efforts to reduce the time of enrichment such as immuno-magnetic separation, centrifugation, and specially designed media have brought about some benefits but normally they only reduce the total time to result by a few hours and may add significant cost and hands-on time to the process. Confirmation and identification tests is another area where significant simplification has been introduced.


Legionnaire’s Disease

• Legionnaires’ disease, named after the outbreak in 1976 at the American Legion Convention in Philadelphia

• Caused by Legionella pneumophila and other Legionella species

• Characterised as an acute febrile respiratory illness ranging in severity from mild illness to fatal pneumonia

• The mortality rate can be as high as 25% in untreated immunosuppressed patients


• Outbreaks of Legionnaires' disease receive significant media attention, especially when a large number of people become ill or die

• Single infections often go unnoticed. The Centers for Disease Control and Prevention (CDC) estimate between 8,000 and 18,000 people are hospitalized with Legionnaires' disease in the US each year.

• The major reservoir of Legionella appears to be fresh water sites, air-conditioning units and various water plumbing fixtures

• The immediate consequences for the building owner or manager faced with liability claims and negative publicity can be devastating and extremely costly.

• Proactively managing the risk of Legionella bacteria in cooling towers and water systems is more cost effective than responding to an outbreak

Legionnaire’s Disease


Legionella

• Legionella pneumophila is the most common cause of Legionnaires’ disease.

• At present, 14 different serotypes exist of which Legionella pneumophila serogroup 1 accounts for 90% of cases

• Isolates derived from environmental and clinical samples may be cultured on standard non-selective or selective Legionella culture media.

• Legionella species on primary isolation have an absolute requirement for L-cysteine hydrochloride. Confirm that an isolate is a Legionella by showing that it cannot grow on any medium which does not contain L-cysteine hydrochloride.


• Legionella CYE Agar Base (CM0655) • Charcoal Yeast Extract Agar for the isolation of Legionellaceae when used with

Legionella BCYE Growth Supplement SR0110 (Edelstein BYCE Agar)

• This medium can be further supplemented with one of the following: • Legionella BMPA Selective Supplement (SR0111) • Legionella GVPC Selective Supplement (SR0152) • Legionella GVPN Selective Supplement (SR0215) • Legionella MWY Selective Supplement (SR0118)

• Additionally, a medium omitting L-cysteine may be prepared from Legionella CYE Agar Base (CM0655) and BYCE Growth Supplement (SR0175)

Legionella Culture Media


Oxoid Legionella Latex Test DR0800M • A fast and simple screening procedure for predominant pathogenic Legionella species and

serotypes

• The DR0800 (wet kit) kit contains: • DR0801 Legionella pneumophila serogroup 1 Test Reagent • DR0802 Legionella pneumophila serogroups 2–14 Test Reagent • DR0803 Legionella species Test Reagent • These 3 sub-kits are also available seperately

• The Legionella species reagent is reactive with the following species and serotypes: Legionella longbeachae 1 and 2; Legionella bozemanii 1 and 2; Legionella dumoffii; Legionella

gormanii; Legionella jordanis; Legionella micdadei; Legionella anisa

DrySpot format - same as the wet kit, only available as three separate kits: • DR0200 Legionella pneumophila serogroup 1 Test Reagent • DR0210 Legionella pneumophila serogroups 2–14 Test Reagent • DR0220 Legionella species Test Reagent


• Recommended for the enumeration of enterococci in water supplies to detect and enumerate enterococci by the technique of membrane filtration or direct plating

• The medium is very selective for enterococci • Plates are incubated at 35oC for 4 hours and then at 44-45oC for 44 hours • All red or maroon colonies may be accepted as presumptive enterococci

Traditional Culture Media - Slanetz & Bartley Medium

http://www.google.com.br/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwirqLin3K_LAhWjCpoKHax-D60QjRwIBw&url=http://www.solabia.fr/solabia/produitsDiagnostic.nsf/SW_PROD/94CF44F2F8052117C12574B100510379?opendocument&LG=EN&&bvm=bv.116274245,d.d24&psig=AFQjCNGQeV_ymDFxWsru7lvEKwKgH60LRA&ust=1457479454609550


• ONPG – weak colour intensity and freely soluble, therefore unsuitable for use in solid media

First-use of chromogens

ONPG -ve ONPG +ve

• Demonstration of β-galactosidase production on tryptone nutrient agar

with ONPG discs

Presenter

Presentation Notes

ONPG is used to detect the presence of β-galactosidase, an enzyme found in lactose-fermenting organisms like E. coli. β-Galactosidase is not lactose specific and can act on simple galactosides including the ONPG (o-nitrophenyl-β-D-galactopyranose) substrate. ONPG hydrolysis results in the release of galactose, and the yellow chromogenic compound (o-nitrophenol). The test substrate, ONPG, does not depend on an induced or constitutive permease enzyme to enter the cell, therefore reactions are rapid and occur within a 24-hour period even for late lactose fermenters As ONPG is insoluble and unsuitable for use in solid media ONPG discs are used to detect β-galactosidase for the confirmation of E. coli in The Microbiology of Drinking Water 2002, Part 4, Method B: The enumeration of coliforms and Escherichia coli by single membrane filtration technique. Presumptive colonies are streaked onto Tryptone Nutrient Agar and ONPG discs placed on agar surface.


E. coli

Chromophore component (e.g. indoxyl derivative such as 5-bromo-4-chloro-3-indoxyl)

Sugar component (e.g. galactose)

“X”-gal

Uptake

β-galactosidase

Chromogen

Background and Principles of Chromogenic Media

Presenter

Presentation Notes

Chromogen – “a chemical compound, itself without colour, that can be transformed into a coloured compound, or can react with another material to form a coloured compound”



NH

HO

5-bromo-4-chloro-3-indoxyl ('X')

Br

Cl• “X”-gal

Presenter

Presentation Notes

X-gal mimics lactose, and therefore may be hydrolysed by the β-galactosidase enzyme. Lactose fermenting organisms such as E. coli will hydrolyse X-gal as they posses the β-galactosidase enzyme It is this production of blue that is the key as it helps to identify and differentiate colonies on agar plates



NH

HO

Cl

6-chloro-3-indoxyl ('Rose')

Presenter

Presentation Notes

Rose gal works under the same mechanism as X-gal but instead of a blue colour it produces a pink colour, this demonstrates that different chromophores can have the exact same application but produce different colours whish enables dual chromophores to be used in a single Chromogenic agar further aiding in differentiation identification and


• E. coli • E. coli O157 • Coliforms • Enterococci • VRE • Genera that cause urinary tract

infections • Staph. aureus • MRSA

Chromogenic Media Exist For….

• Listeria spp and L. monocytogenes

• Bacillus cereus • Enterobacter sakazakii • Clostridium perfringens • Vibrio parahaemolyticus • Candida spp, C. albicans

and C. tropicalis

Presenter

Presentation Notes

Chromogenic media exist for a wide range of organisms as listed they can be applied to Clinical applications for resistance screening such as MRSA, Diagnostic and also have a place in Food and Water microbiology.


Culture Media Design

ß-galactosidase C8-esterase

α-glucosidase

ß-glucuronidase

ß-glucosidase

E.coli

Ent.sakazakii

Salmonella

Salmonella Shig. sonnei

Shig. sonnei

Shig. sonnei Shigella spp. Vibrio spp.

Pseudomonas spp.

Klebsiella spp. Enterobacter spp.

Citrobacter freundii

Citrobacter koseri

Sal. arizonae

Phenylalanine deaminase

Proteus spp. Providencia spp.

Hafnia alvei

Hafnia alvei

Vibrio vulnificus

P. vulgaris Enterobacter spp.

Serratia spp.

E.coli

Gram-negative organisms


1. Growth of target organism

2. Inhibition of non-target organisms

3. Differentiation of target organisms

Three Key Principles of Design


1. Growth of Target Organism Genus level or species level

• Careful selection of: • Amino acids and nitrogen nutrients • Peptones - acid or enzymatic digested protein rich extracts from Meat, Soya,

Casein & Pea or Yeast Extract Inclusion of specific vitamins and growth promoting factors

• Blood – e.g. Campylobacter • Serum • Vitamins Right energy source

• Carbohydrates – often glucose, but can be sucrose or other sugars Mineral salts and metals

• Common metals are Ca++,Mg++, Fe+++ • Trace metals can include Manganese, Zinc, Molybdenum and Copper

Key Principles of Design

Presenter

Presentation Notes

Understand growth requirements of target Organisms, what level do we need to get to, genus or spp Careful selection of ingredients that provide the optimum growth environment for our target


2. Inhibition of non-target organisms Antibiotics

• Often will inhibit G+ve or G-ve bacteria Chemical agents

• Inorganic salts - Bismuth, lithium, tellurite, tetrathionate Dyes

• Acriflavin, Brilliant green, crystal violet Surface active agents

• Bile, cetrimide, lauryl sulphate Others

• Dichloran, irgasan, Inhibigens


Presenter

Presentation Notes

Once we understand the growth requirements of the target we need to consider the organisms that could be present in our sample that we want to inhibit. This inhibition can be achieved by the use of........


3. Differentiation of Target Organisms Chromogens

• Single or dual combination

pH indicator Dyes

• Phenol red .e.g Brilliant Green agar

Morphology

Biochemical

• Hydrogen sulphide e.g. XLD

Zone/halo formation due to proteolytic action

• e.g. Baird-Parker or Brilliance Listeria Natural colour of organism e.g. Staph aureus, Pseudomonas


Presenter

Presentation Notes

How do we go about differentiating target organisms from uninhibited non-target organisms, as it is this that really makes Chromogenic media beneficial in terms of ease of use. This can be achieved in many ways.......


• Formulation contains:

• Peptones and Yeast Extract – growth components

• Lactose – utilised by E.coli and coliforms producing acid

• Sodium Lauryl Sulphate – inhibits Gram +ve bacteria

• Phenol Red – pH sensitive dye used to colour colonies

• 5-bromo-4-chloro-3-indolyl-b -D-glucuronide – chromogenic substrate

• Agar – Gelling agent

Membrane Lactose Glucuronide Agar (MLGA)

Presenter

Presentation Notes

MLGA which is an chromogenic agar specified in The Microbiology of Drinking Water 2002, Part 4, Method B: The enumeration of coliforms and Escherichia coli by single membrane filtration technique.. The 3 key principles of media design have been applied in its design.... The correct mix of peptone and yeast extract is included to support growth of target organisms which is E. coli and Coliforms Lactose is used as the energy source which is utilised by our target organisms Sodium Lauryl sulphate inhibits non target organisms Phenol red is used a colour indicator to help differentiate Chromogen BCIG is used for further differentiation


Differentiation of E.coli and coliforms is facilitated through two biochemical reactions:

1. Lactose fermentation by lowers the pH turning colonies yellow

2. Chromogenic substrate 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (BCIG) is cleaved by the enzyme glucuronidase and produces a blue chromophore which builds up in the cells.

Membrane Lactose Glucuronide Agar – How Does It Work?

Organism Lactose fermentation

Glucuronidase enzyme

Colony colour

E.coli Yes (yellow) Yes (blue) Green coliforms Yes (yellow) No Yellow Pseudomonas No No Pink Bacillus (Gram +ve) - - Inhibited

Presenter

Presentation Notes

MLGA – 2 detection systems pH and chromogen The energy source lactose is utilised by E. coli and Coliforms this lowers the pH and turns the colonies yellow allowing for identification of Coliforms The chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (BCIG) is cleaved by the enzyme glucuronidase and produces a blue chromophore which builds up in the bacterial cells. As Escherichia coli is able to utilise both lactose as an energy source and possesses glucuronidase enabling it to utilise BCIG e. coli appears as green colonies as both blue and yellow builds up within the cells.


• Extensive product range

• Our core business is manual microbiology

• We are moving forward with technological advances

• BUT...... ......we must protect our market share and we must protect our customer base

in preparation for future developments

• There are still many opportunities , especially cross-selling products from the customer’s workflow

Our portfolio


Any questions?

selling the portfolio - thermo fisher safety/4...selling the portfolio . ... • confirmation –...

Documents