2016 - IFM - CLASSIC - METHODS (Mikrobiologi Method)

Methods for food-
borne pathogens
detection and
enumeration
Sabina Purkrtová
Methods in microbiology
Analyte – the measured component of a sample
Analytes in microbiology
microorganisms as microbial cells
component of microorganisms – e.g. DNA, proteins
products of microorganisms – e.g. enterotoxins of S. aureus
presence or absence of the amount of the analyte

analyte measured either directly or
in a certain amount of sample indirectly in a certain amount of
sample
qualitative method quantitative method
Amount: according to the legislative Amount: according to the legislative

in EU usually 25 g (or occasionally 10 g) in EU usually 10 g
Types of methods
1. International, regional or national standards
• contain sufficient and concise information on how to perform

We're ISO, the
the tests and/or calibrations International
Organization for
• do not need to be supplemented or rewritten as Standardization. We
develop and publish
international procedures (if these standards are written in
International Standards.
a way that they can be used as published by the operating
staff in a lab)
• optional steps in the methods or additional details shall be

documented
• ISO, International Dairy Foods Association (IDFA), FDA (Food

and Drug Administration), AIJN (European Fruit Juice
Association
2. Laboratory-developed methods
3. Non-standard methods
Microbiological analysis procedures
CONVENTIONAL CELL CULTIVATION
• relatively easy to use, but time (requires several days), labour (lots of procedural steps)
and material consuming
• many of them are recognised as approved for ISO and they are gold standard
procedures
• Colony count method (CCM)
• pour plate techniques
• spread plate techniques
• Membrane filtration
• Most Probable Number (MPN)
RAPID METHODS
• immunonological method (based on antigen/antibody-binding)
• based on molecular biological method (based on PCR)
• others (ATP Photometry, Direct Epifluorescent Filter Techniques (DEFT), Electrical
impedance method, Flow cytometry, etc.)
Phylogenetic tree of life
Γ-Proteobacteria:
Enterobacteriaceae
ε-Proteobacteria:
Campylobacter sp.
http://www.sciencedirect.com/science/article/pii/S000527280600140X
Cultivation methods
Cultivation methods - classical microbiology methods
Microorganisms are studied not as single cells, but like a
mass population of daughter cells arisen from one single
mother cell by multiplication
http://www.blackmould.me.uk/images/FungalLifeC
ycle.gif
Asexual life cycle of toxic black

mould
Yeast: budding (most

https://www.kullabs.com/uploads/Notes/PXlu4LSHCNgjaCpM.jpg
yeasts), binary fission
(Schizosaccharomyces
sp.)
http://leavingbio.net/BACTERIA%20Page_files/image011.jpg
6
Bacteria: binary fission https://kollokvium.files.wordpress.com/2014/07/saccharo.png
Cultivation methods
Cultivation methods - classical microbiology methods
Microorganisms are studied not as single cells, but like a mass population of daughter cells
arisen from one single mother cell by multiplication
Example: Growth of bacteria in the logarithmic

phase (generation time: 0.5 h)
Log10 (number of cells)
Hours
http://www.cs.montana.edu/webworks/projects/
stevesbook/artifacts/images/chapter_002/section
002/typical_growth_curve_002.jpg
7
Cultivation methods
This population („mass of cells“) is visible on solid media as a colony
Growth of a colony
from one mother
cell (teoretically)
Video:
https://www.youtube.c
om/watch?v=zrx7Xg0g
kQ4
http://ibis.inrialpes.fr/rubrique51.html
a well-isolated colony grown from only one mother cell ~ ideal state, but it can be theoretic (some cells in clumps etc.)
the number of colonies = number of colony forming units (CFU) (not number of „cells“)
Those populations are visible to grow in Those populations are able to give the
liquid media (broth) detectable signal in biochemical testing
8
http://accounts.smccd.edu/case/biol240/images/nutrientbroth2.jpg
Cultivation methods
The ability of microorganism to survive and to multiplicate („to grow“) under given
envirommental conditions depends on the compatibility of these conditions and the
physiological and biochemical featrues of the microorganism
Physical factors Chemical factors

• temperature* • Nutrition requirements*
• pH* • Source of carbon
• osmotic pressure* • Source of nitrogen
• atmosphere* • Source of energy
• pressure • Other
• UV radiation (UV, γ...) • Antimicrobial and
(bactericidal/bacteriostatic) and
other chemical compounds*
Biological factors Physiological state of cells („fitness“)

• The influence of other • cultivable cells
microorganisms (synergism -helping • cells in good state
grow or survive , competition, • cells demaged or suppressed by the
antagonism, symbiosis, food processing, conditions…
mutualism…) • uncultivable cells
*...Factors widely used in food microbiology cultivation methods 9
Cultivation methods
http://image.slidesharecdn.com/microbiologyunit2-3-150710082502-lva1-app6892/95/microbiology-
unit-23-bacteria-36-638.jpg?cb=1436518851
Cultivation methods
obligate anaerobes:
oxygen is toxic for them
https://o.quizlet.com/CXi.qp97gMpOFIXSYF0skA.png
Cultivation methods
Bacteria Tmin (°C) Topt (°C) Tmax (°C)
Listeria monocytogenes 1 30-37 45
Vibrio marinus 4 15 30
Pseudomonas maltophilia 4 35 41
Thiobacillus novellus 5 25-30 42
Staphylococcus aureus 10 30-37 45
Escherichia coli 10 37 45 Cultivation temperature for yeasts and
Clostridium kluyveri 19 35 37 moulds: 25 °C.
Streptococcus pyogenes 20 37 40
Streptococcus
25 37 42
pneumoniae
Bacillus flavothermus 30 60 72
Thermus aquaticus 40 70-72 79
Archaebacteria Tmin (°C) Topt (°C) Tmax (°C)
Methanococcus
60 85 90
jannaschii
Sulfolobus acidocaldarius 70 75-85 90
Pyrobacterium brockii 80 102-105 115
12
Cultivation methods
Teplotní rozsah růstu 27
kvasinek. Optimální růstová
teplota je označena
kolečkem.
S., Saccharomyces
Sc, S. cerevisiae;
Sp, S. paradoxus;
Smik, S. mikatae;
Sarb, S.arboricolus;
Scar, S.cariocanus;
Su, S. bayanus var. uvarum;
Sk, S. kudriavzevii;
Km, Kluyveromyces
marxianus;
Pf, Pichia fermentans;
Td,Torulaspora delbrueckii;
Cs, Candida stellata;
Hu, Hanseniaspora uvarum.
Salvadó Z et al. Appl. Environ. Microbiol. 2011;77:2292-2302

Cultivation methods
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Cultivation methods
http://image.slidesharecdn.com/microbiologicalprinciples-110819155057-phpapp01/95/food-
microbiological-principles-fst-241-32-728.jpg?cb=1313769603
Cultivation methods
http://image.slidesharecdn.com/factors
affectingthegrowthandsurvivalofmicro-
organismsinfoods-150306050511-
conversion-gate01/95/factors-affecting-
the-growth-and-survival-of-micro-
organisms-in-foods-30-
638.jpg?cb=1425640055
http://aqualab.decagon.com.br/assets/Article-Graphics/Key-concepts-in-water-activity-Table-1.JPG
Nutrition requirement
http://www.easynotecards.com/uploads/929/65/_4a88b4c5_140c98ff4b1__8000_00008247.png
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Nutrition requirement
http://www.oxfordscholarship.com/doc/10.1093/acprof:oso/978
0199586936.001.0001/acprof_9780199586936_graphic_012.gif
Most bacteria important in clinical and food microbiology, all yeasts and moulds
belong to chemoheteroorganotrophes: the source of carbon and energy and redox
compound are organic compounds. 18
Culture media - purpose
Liquid medium:
the cell absorbs by the whole
surface
Semisolid:
To see motility
Solid (can be converted to

liquid): with agar, single
colonies
Solid (cannot be liquified):

carrot, potato etc. – good for
moulds
http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/stomat/ptn/Microbiology,%2
0virology%20and%20immunology/2/Lesson_03_The%20main%20methods%20and%20principles%20of% 19
20pure%20cultures%20isolation.files/image052.jpg
Culture media - composition
Chemically defined media
the exact chemical composition is known
prepared from pure chemicals to contain all the growth requirement of the microorganism as source of
carbon, energy, nitrogen, osmotic compounds, for auxotrophes vitamins and trace compounds etc. (they
are not able to synthesize certain substances required for its growth and metabolism, it can be expansive)
http://classes.midlandstech.edu/carterp/courses/bio225/chap06/06-T03_MediumFastid.jpg
http://classes.midlandstech.edu/carterp/courses/bio225/chap06/06-T02_MediumChemo.jpg
Carbon and energy source (equal): glucose Fastidious bacterium: demanding for
Nitrogen source: ammonium phosphate nutrition
E. coli – easy to be cultivated 20
Culture media - composition
Complex media
Composed mainly from different natural materials and some simple pure chemicals
Due to natural materials the exact chemical composition is not known and varies slightly from
batch to batch of used natural materials and its preparation
Products of hydrolysis of different source
proteins (meat, soya, gelatine, caseine, vegetable
tissue...) by proteolytic enzymes (as pepsin,
trypsin, papain, pankreatin) =
peptones/tryptones from...., protein
hydrolysates = main source of nitrogen (also of
carbon and energy, but this utilisation runs
slower, some tryptones also source of vitamins)
Simple saccharides (as glucose, lactose,
maltose...) – source of energy and and carbon
Other growth factors: sheep/horse blood,
http://images.slideplayer.com/20/5940717/slides/slide_8.jpg NADH+
Mineral salts:
Meat extract, yeast extract: NaCl – osmotic pressure ( 0,85-0,90 % NaCl
Source of vitamins (yeast extract saline solution), phosphates, sulphates, Ca2+,
particularly rich in B vitamins and other Mg2+, Fe2+,3+, Mn2+ and traces compound –
organic growth factors improving MO growth in some level
(source of energy, carbon, minor source of Buffering salts
nitrogen) for maintaing the stable pH during the
cultivation (soluble phosphates, acetates....)21
Culture media - consistency
Solidified by agar or gelatine
• By agar in 1-3 % (polymerisation at 37-45 °C) - solid media – enabling to grow in single
well-isolated colonies
• By agar 0.2-0.5 % - semisolid media – detection of motility (bacteria can move)
• By gelatine – biochemical testing for liquidifying of gelatine in some bacterial species
(e.g. Clostridium perfringens)
agar (agar-agar) – complex polysaccharides (agarose – 70 % + agaropektin – 30 %), derived
from the cell wall of a marine alga (Floridae a Gelidium), only few microorganism can degrade,
so it remains solid, in water it liquifies around at 95 °C, by cooling (~40 °C) solidifies and
polymerises
gelatine – produced by hydrolysis of colagen tissue (from gristle, chord, mixture of partially
degradated peptides and proteins)
STRUCTURE
In the natural state, agar occurs as structural carbohydrate in the cell walls of
agarophytes algae, probably existing in the form of its calcium salt or a mixture of
calcium and magnesium salts. It is a complex mixture of polysaccharides composed
of two major fractions - agarose, a neutral polymer, and agaropectin, a charged,
sulfated polymer.
Agarose, the gelling fraction, is a neutral linear molecule essentially free of sulfates,
consisting of chains of repeating alternate units of β-1,3-linked- D-galactose and α-
1,4-linked 3,6-anhydro-L-galactose. Agaropectin, the non gelling fraction, is a
sulfated polysaccharide (3% to 10% sulfate), composed of agarose and varying
percentages of ester sulfate, D-glucuronic acid, and small amounts of pyruvic acid.
The proportion of these two polymers varies according to the species of seaweed.
Agarose normally represents at least two-thirds of the natural agar-agar.
http://www.agargel.com.br/agar-tec-en.html
http://www1.lsbu.ac.uk/water/images/gelatin.gif
22
Culture media
Ready-to-use
Dehydrated
mixture prepared
by the producer
Mixed from single parts

23
Culture media - purpose
http://classes.midlandstech.edu/carterp/courses/bio225/chap06/06-T05_CultureMedia.jpg
24
Culture media – general purpose
General (universal) culture media
• applicable for the growth of all those microorganisms, for which the nutrition
composition is suitable (no selective additive)
• the restriction of well-grown microorganisms may be partly done by the
cultivation temperature
E.g.
Plate Count Agar, Nutrient agar, Tryptic soy agar
Brain heart infusion broth, Tryptic soy broth, Buffered peptone water
Enriched media
• More rich in nutrient to support the growth of a wide variety of organisms,
including some of the more fastidious ones
E.g.
Columbia agar with 5 % of sheep blood – very nutrient, also for more fastidious bacteria
Blood agar is an enriched medium in which nutritionally rich whole blood supplements the
basic nutrients.
Chocolate agar is enriched with heat-treated blood (40–45 °C), which turns brown and gives
the medium the color for which it is named.
25
Nutrient Agar
Universal culture media for cultivating less fastidious
microorganisms.
Typical Composition (g/litre)
Peptone from meat 5.0; meat extract 3.0; agar-
agar 12.0.
pH: 7.0 ± 0.2 at 25°C.
26
Plate Count Agar (Casein-peptone Dextrose
Yeast Agar)
This medium does not contain any inhibitors or indicators;
it is mainly used to determine the total microbial content in milk,
dairy products, water and other materials.
Peptone from casein 5.0; yeast extract 2.5; D(+)glucose 1.0;
agar-agar 14.0.
pH: 7.0 ± 0.2 at 25°C.
27
Tryptic Soy Agar (TSA)
Universal culture media free from inhibitors and indicators for a wide
spectrum of applications.

Peptone from casein 15.0; peptone from soymeal 5.0; sodium chloride 5.0; agar-
agar 15.0.
pH: 7.3 ± 0.2 at 25°C.
Tryptic Soy Broth (TSB) CASO Broth (Casein-
peptone Soymeal-peptone Broth)
Universal culture media free from inhibitors and indicators for a wide spectrum of
applications
Typical Composition (g/litre) Peptone from casein 17.0; peptone from soymeal 3.0;
D(+)glucose monohydrate 2.5; sodium chloride 5.0; di-potassium hydrogen
phosphate 2.5
pH: 7.3 ± 0.2 at 25 °C.

Buffered Peptone Water (BPW)
For the preliminary, non-selective enrichment of bacteria, particularly pathogenic
Enterobacteriaceae, from foodstuffs and other materials.
Peptone from casein 10.0; sodium chloride 5.0; disodium hydrogen phosphate dodecahydrate 9.0;
potassium dihydrogen phosphate 1.5.
Mode of Action
The broth is rich in nutrients and produces high resuscitation rates for subletally injured bacteria and
intense growth. The phosphate buffer system prevents bacterial damage due to changes in the pH of
the medium.
pH: 7.0 ± 0.2 at 25 °C.

Brain Heart Infusion Broth
For the cultivation of various fastidious pathogenic microorganisms.
Nutrient substrate (brain extract, heart extract and peptones) 27.5; D(+)glucose 2.0; sodium
chloride 5.0; disodium hydrogen phosphate 2.5.
pH: 7.4 ± 0.2 at 25 °C
Columbia Agar
This superior, complete medium proposed by ELLNER et al. (1966) can be used for
the cultivation of even fastidious microorganisms and also as a base for the
preparation of various special culture media. (universal + differential when used )

Peptrone from casein 10.0; peptone from meat 5.0; heart extract 3.0; extract from yeast
5.0; starch 1.0; sodium chloride 5.0; agar-agar 13.0. pH: 7.3 ± 0.2 at 25°C.
Preparation of blood agar: Mix 5 ml blood homogeneously with 95 ml sterile
culture medium base. Pour plates. (usually Columbia 5 % sheep blood agar)
(universal + differential when used )
Preparation of gentamicin blood agar: Mix 100 ml defibrinated sheep blood and
0.11 ml gentamicin solution homogeneously with 900 ml sterile culture medium
base. Pour plates.
Preparation of boiled agar: Add 10 ml blood to 90 ml sterile culture medium base.
Heat the mixture in a water bath for about 10 minutes to 80°C swirling all the time
until the medium becomes chocolate brown in colour, pour plates.
Preparation of lactose milk egg-yolk agar: Dissolve 42 g dehydrated culture
medium, 12 g lactose, 1 g agar-agar in 1 litre demineralized water. Mix in 33 ml/litre
of a 0.1 % aqueous solution of neutral red, adjust the pH to 7.0 and autoclave (15
min at 121°C). Cool to 45-50°C, add approximately 35 ml egg-yolk emulsion/litre and
10 g dried milk/litre and mix homogeneously. Pour plates.
http://image.slidesharecdn.co
m/copyofrevision2014-
140501141600-
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RBC – red blood cell

http://image.slidesharecdn.com/strpyosequelae-100123053606-phpapp02/95/str-pyo-sequelae-6-728.jpg?cb=1435204288
Columbia Agar with
5 % sheep blood
Escherichia coli
http://www.eolabs.com/columbia-agar-sheep-blood.html
Streptococcus pyogenes
Culture media – selective/differential
Selective culture media
• Suppression of unwanted microorganisms/Encouraging target (desired) microorganisms
• Selective compounds:
• Surface active compounds: bile, bile salts, sodium laureth sulfate, tergitol
• Antibiotics: by choice to select the target microorganism
• Inorganic salts: lithium chloride (against G- and enterococci), tetrathionate (against
G+ and coliform bacteria), sodium azide (against G-)
• Dyes: acridine, triphenyl dyes, crystal violet, briliant green, malachite green
• Enrichment: Similar to selective media but designed to increase numbers of desired
microbes to detectable levels
Differential culture media
• Differentiation of colonies of target (desired) microorgnisms from others based on some
biochemical reaction
• E.g. fermentation of glucose, lactose / indicated by acidobasic indicators;
• E. g. degradation of chromogenic compound….
Selective-differential culture media

• Suppression of unwanted microorganisms
• Encouraging target (desired) microorganisms and their differentiation
35
Acidobazic indicator
http://image.slidesharecdn.com/lab17-141201085153-conversion-gate01/95/-10-
638.jpg?cb=1417424219
Chemical compounds
changing colour
http://mslavenda.com/images/bromocresol_purple_chart.jpg
according to the pH
(by accepting or
http://www.dlt.ncssm.edu/tiger/diagrams/acid-base/WeakAcidTitration-
releasing H+ ) General.gif
http://mslavenda.com/images/bromothymol_chart.jpg
http://2.bp.blogspot.com/-XaGKyJzCBPk/UgS8FU96L3I/AAAAAAAAAt8/7B6bSjxLjjI/s1600/lol.PNG
Composition
Ingredients Gms / Litre
Peptones (meat and casein) 3.000
Pancreatic digest of gelatin 17.000
Sodium chloride 5.000
Bile salts 1.500
Crystal violet 0.001
Lactose monohydrate 10.000
Neutral red 0.030
Agar 13.500
pH after sterilization( at 25°C) 7.1±0.2
Essential nutrients, vitamins

and nitrogenous factors:
pancreatic digest of gelatin
and peptones (meat and casein)
Osmotic balance: sodium
chloride
Selective action
MacConkey Agar crystal violet and bile salts –
recommended for selective isolation of Escherichia coli. It inhibitory to most species of
is also recommended for selective isolation and gram-positive bacteria (e.g.
differentiation of lactose fermenting and lactose non S. aureus – no growth).
fermenting enteric bacteria = Selective and differential Gramnegative, specially enteric
medium for cultivation of coliform organisms bacteria, grow well.
Lactose fermenting strains
MacConkey Agar
(coliform bacteria)
growth as red or pink and may be
surrounded by a zone of acid
precipitated bile.
Lactose non-fermenting strains,
(Shigella, Salmonella, Proteus...)
are colourless and transparent and
typically do
not alter appearance of the
medium.
Yersinia enterocolitica may
appear as small, non-lactose
fermenting colonies after
incubation at room temperature.
Composition Ingredients Gms / Litre Differential action (by the ability to ferment lactose):
Peptones (meat and casein) 3.000
Pancreatic digest of gelatin 17.000 Lactose monohydrate (fermentable source of carbohydrat
Sodium chloride 5.000 + Neutral red – acidobasic indicator
Bile salts 1.500 Fermentation of lactose – acid production – absorped by
Crystal violet 0.001
Lactose monohydrate 10.000
neutral red – when pH falls below 6.8 colour change to red
Neutral red 0.030 http://2.bp.blogspot.com/-
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Agar 13.500 AAAAAAAAAt8/7B6bSjxLjjI/s
1600/lol.PNG
pH after sterilization( at 25°C) 7.1±0.2

Chromogenic culture medium Coliform bacteria
(MPN method)
• One or more chromogenic substrates
• For detection of specific enzyme, which are
able to cleave out the chromogenic substrate,
that a designed chromophore (responsible for
the colour change) is released https://encrypted-
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CHROMOGENIC MEDIUM (Colilert): Coliforms use β-galactosidase

to metabolize ONPG and change it from colorless to yellow.
Fluorogenic culture medium
• One or more fluorogenic substrates
• For detection of specific enzyme, which are able to cleave out the
fluorogenic substrate, that a designed fluorophore (responsible for
the UV fluorescence) is released
FLUOROGENIC MEDIUM (Colilert):

E. coli use β-glucuronidase to
metabolize MUG and create
fluorescence (UV 360 nm). Since
most non-coliforms do not have
these enzymes, they are unable to E. coli (MPN
grow and interfere method)
http://ga.water.usgs.gov/projects/bacteria/pictures/summaryanalysisecoli.jpg
Preparation of culture media (1/4)
• water quality
• distilled water
•conductivity max. 25 µS cm-1 (25 °C), but preferred < 5 µS cm-1
• microbial contamination max. 103 cfu/ml, but max. 102 cfu/ml preferred
(regularly verification, ISO 6222, incubation at 22 °C for 68 ± 4h)
Microbiological and chemical requirements of laboratory grade water
Benson, T. S., Dutko, T., Ilnicki, L., Lankford, M., Pentz, C., Salinsky, J., Teirab, B. and Ziemer, W., 1998. Equipment
calibration, maintenance, and performance verification. Revision 1, 7/11/00. In: USDA/FSIS Microbiology Laboratory,
Guidebook 3rd Edition/1998.chapter 36 revison 1, 6/8/00 pp 18
• weighing
• balance with max. error 1 %
• adding the designed volume of water (not to add up to the final volume)
•mixing or solubilization
• agar media to leave few minutes to soak
• dispersing by repeating or continous stirring (e.g. (sterile) magnetic stirrer)
• if neccessary, followed by heating to dissolving – to avoid overheating
•bottle for preparation

• for preparation use bottle with volume 20% upper than volume of
prepared culture medium to avoid running over
•pH
• pH is designed for the final prepared media and reagents at 25 °C
•pH adjustment to do before sterilisation in order to obtain the desired
pH (±0.2 usually) in the final prepared medium
• In the case of agar media, take a small volume and allow it to cool down to 25°C
and carefully introduce a pH electrode into cooled agar and measure.
•Adjustement of pH
• c(NaOH) = 1 mol/l (~40 g/l)
• c(HCl) = 1 mol/l (~36.5 g/l)
• When adjusting of pH to be done after sterilisation, to use sterilised solutions
•pH meter http://1.bp.blogspot.com/-

4AtMuBrl0e8/VVcaZBdAxII/AAAAAAAAAc4/gNmaSs6I0wY/s640/phmetre
-Hanna-HI-99161.jpg
•stick pH meter
• sterilization
• autoclave: moist heat (steam)
• used for the sterilization of heat-stabile culture media and aqueous
solutions and the destruction of discarded cultures
• air must first be removed in order to achieve the 121 °C ± 3 °C for 15 mins
(for max. 1 l) necessary for successful sterilization.
• the temperature 121 °C to destroy bacillus spores.
•boiling in water bath
• some media with heat-labile compounds
• e.g. selective media for Enterobacteriaceae, the present inhibitors inhibits
the growth of G+ microflora)
•membrane filters (0.2 µm pores)
• usually employed for heat-sensitive substances, e.g. vitamin solutions;
• the filters are heat-sterilised before use
• to check the correct kind of membrane filter (e.g. not binding proteins or
antibitiocs) and moist before using
• the medium to be sterilised on the same day when prepared
Training of Shigella spp. and Yersinia enterocolitica detection, Prague, 19-23 October 2015
• supplements or enrichments
• safety rules to work with and discard – it can be toxic substances
(e.g. antibiotics, dyes)
•when powdered, to be dissolved according to the manufacturer´s
recommendation
• e.g. antibiotics in sterile water or mixture of EtOH and sterile water
•to be careful about storage
•antibiotics solution looses its activity by a longer storage even at 5-8 °C
• if not allowed, it should be consumed on the day of preparation
• or if possible, to freeze (but not to refreeze after defreezing)
• but the change in activity due to freezing must be determined by
the producer or the consumer
•adding into medium
• for termolabile supplement into medium cooled at 50 °C or below
• the supplement must be minimally at the room temperature (to
avoid gelidifying of agar etc.)
• to carefully and precisely mix
• Preparation of Petri agar plates
• after sterilisation/boiling to cool down to 47-50 °C in a regulated
water bath (the time depends on the volume, kind of medium,
number of bottles)
•to add correctly supplements and enrichments
•to pour out medium as soon as possible, maximally within 4 hours
•not to let solidify and remelt again !
•to pour out in sterile Petri dishes
• the height of agar minimally 3 mm
• for Petri dishes with average 90 mm ~18-20 ml of medium
• the higher volume (and the height) of agar is possible for
plates
• to be stored
• to be cultivated more than 72 h
• to be cultivated at the cultivation temperature higher than 40 °C
Selectivity
Culture media - selectivity
• what is the ability of the selective medium to Target microorganism
= what we want to grow on the agar medium/in the broth
supress the growth of the non-target and what we want to detect – distiguish from other
microorganisms ? microorganisms
• what is the ability of target microorganism to grow Non-target microorganism
= what we want to be inhibited and not grow or grow on the
preferably in the presence of non-target agar medium/in the broth or what we want not to give the
microorganism in the tested selective medium ? same reaction as the target
Non-selective medium
Different species or their strains have different
ability to grow
- some species or strains have the higher
0.2 ml growth rate than the others
- the growth rate depends on the
temperature, pH, present substances for
nutrition, present inhibitors...
- some species can inhibit the others – by
True amount of production of antimicrobial compounds, by
cells in a changing the physical properties of the
sample medium
(0.2 ml)
Selective medium
Selectivity (SF)
for demonstration that a medium suppresses the growth of a non-target microorganism
Culture media - productivity
selective media = selective pressure = tested media
• chemicals – antibiotics, dyes etc. to selectively inhibit some groups of
microorganisms
• by cultivation conditions (temperature etc.)
non-selective media = without selective pressure = reference media
Non-selective medium (reference)
Productivity
• what is the efficiency of the target
microorganism to grow in the tested
0.2 ml medium
• how many % of inoculated target
microorganisms is able to grow (in
comparison of reference media)
• selective pressure inhibits partly also
True amount of the target microorganisms (damaged
cells in a cells mainly), also for them it is usually
sample not very easy to accept the conditions
(0.2 ml)
Selective medium
Culture media - specifity
Specifity
• does the target microorganism grow specifically ? – to give the expected appearance
of colonies (colour, surface....) ?
- The specifity is used on some biochemical property = presence of enzyme
- But different strains of the same species can behave differently !!!
- No presence of tested enzyme...
- The enzyme has the lower activity – it works slowly...
The target
microorganism
gives the expected
reaction
Horizontal method for the enumeration of
microorganisms
Horizontal method = by using Petri dishes
SAMPLE PROCESSING
• resuspension in diluent - decimal dilution - spreading, pourring/inoculation
(MPN)
• filtration
ISOLATION ON SELECTIVE MEDIA
SUB-ISOLATION OF PRESUMPTIVE COLONIES (non-selective agar)
BACTERIA GROWING ON SELECTIVE MEDIA ARE NOT APPROPRIATE FOR

BIOCHEMICAL TESTING – SELECTIVE MEDIA DECREASED „FITNESS“ OF
BACTERIA AND INFLUENCES THEIR PRESENT BIOCHEMICAL BEHAVIOR (NOT
PROPERTIES), WHICH CAN INTERFER WITH THE EXACTNESS AND RELIABILITY
OF TESTING
CONFIRMATION/IDENTIFICATION
Horizontal method for the enumeration of
microorganisms
SAMPLE Sample: usually 10 g of solid or 10 ml of liquid food/feed
Diluent: e.g. saline solution with peptone, peptone water etc.
• solid food/feed: 10 g + 90 ml of appropriate diluent
DECIMAL • liquid food/feed: 10 ml + 90 ml of appropriate diluent
DILUTIONS = 1. dilution, to be done in a sterile special bag, sample to
be resuspended or mixed well
= further dilutions: in tubes (1 ml of suspension + 9
ISOLATION ml of diluent)
CULTIVATION
COLONY COUNTING
1:100 1:1000 1:10000 1:100000 1:1000000
1:10
SUB-ISOLATION OF CULTIVATION +
PRESUMPTIVE CONFIRMATORY RESULT
COLONIES TESTING
Horizontal method for the enumeration of microorganisms
Inoculation of some dilutions (chosen to cover the given limit)
SAMPLE • by spreading suspension (usually 0.1 or 0.2 ml) onto
medium in Petri dishes
• by pourring over suspension (usually 1 ml) in an empty
DECIMAL Petri dish by melted medium
DILUTIONS
ISOLATION
CULTIVATION 1:100 1:1000 1:10000 1:100000 1:1000000
COLONY COUNTING
http://www.studyblue.com/notes/note/n/lab-practical-exam-review-1/deck/6074578
SUB-ISOLATION OF (upraveno)
CULTIVATION +
PRESUMPTIVE
CONFIRMATORY RESULT
COLONIES
TESTS
Plating method
pourring over gives better results
for facultative anaerobes than
spreading
spreading used for aerobic

microorganisms
http://department.monm.edu/chemistry/chem
Streaking istry330/fall2004/abickert/images/streak.gif
http://classes.midlandstech.com/carterp/Courses/bio225/chap06/Microbi
al%20Growth%20ss5.htm
Horizontal method for the enumeration of microorganisms
SAMPLE Cultivation: time and temperature depends on the

microorganisms
Colony counting: limit of colony forming unit on a plate:
DECIMAL 10-300 for counting all colonies, 10-150 for counting
DILUTIONS presumptive colonies
Confirmation: choice of up 5 presumptive/typical/atypical
colonies per a plate to be confirmed by biochemical testing (see
ISOLATION specifity/selectivity)
Result: A.B x 10c CFU/g, ml (e.g. 5.2 x 104 CFU/g, ml)
CULTIVATION
COLONY COUNTING
http://www.studyblue.com/notes/note/n/lab-practical-exam-review-1/deck/6074578
(ADAPTED S.P.)
SUB-ISOLATION OF CULTIVATION +
PRESUMPTIVE CONFIRMATORY RESULT
COLONIES TESTS
Colony counting
N (colonies/g sample) = If n1 = n2=2 then C
C
V *2 , 2*d
If n1 = n2=1 then C
V ( n1  0 ,1n2 ).d V *1,1*d
• C… the number of all counted colonies on plates of two dilutions,

following each other, when at least one plate is more colonies then 10 (the
highest amount is 300 for total colonies/150 for typical and/or atypical
colonies or according to used ISO method for analysis)
• V…. the volume of the sample spread on the plate (ml) (if 1 ml is spread
over 3 plates, it is taken as 1 plate with V=1 ml)
• d…..the coeficient of the first dilution taken for calculation (10-x)
• n1 …the number of counted plates of the first dilution taken for calculation
• n2….the number of counted plates of the second dilution taken for
calculation
Colony counting
ISO 7218:2007 Microbiology of food and animal feeding stuffs – General
requirements and guidance for microbiological examinations (if not
determined by the used method differently)
1. Typical cases
- Each dilution plated on one Petri dish (if not asked different, but plating
two plates is widely asked and highly recommended)
- Maximally 300 colonies per one P. dish (but typical and/or presumptive
colonies maximally 150 per one P. dish)
a) Counting (all colonies) - max. 300 colonies per one P.dish
- at least one dish with minimally 10 colonies

C… all counted colonies on plates of two dilutions (one
C
N  V *1,1*d per each)
V…. the volume of the inoculum
d…..the coeficient relative to the first dilution taken for
calculation= 10-x
Colony counting
b) Counting for confirmation- max. 150 (typical and/or atypical colonies per
one P.dish)
a  *C
b C… presumptive colonies
A A…..number of presumptive colonies taken for
a
confirmation (typically A=5)
b….number of typical colonies positively confirmed
a….the number of confirmed colonies at the plate – then
V ( n1  0 ,1n2 ).d used for the calculation according to a)
Example of application:
coagulase-positive staphylococci- typical and atypical colonies can be present
b
a   cc 
c bnc
 cnc
Ac Anc
Ac is the number of typical colonies submitted to the coagulase test
Anc is the number of atypical colonies submitted to the coagulase test
bc is the number of typical colonies which have been shown to be coagulase-positive
bnc is the number of atypical colonies which have been shown to be coagulase-positive
cc is total number of typical colonies seen on plate
cnc is total number of atypical colonies seen on plate
Colony counting
c) Low numbers for the first used dilution !!!!! (d1)
• (4-10 colonies) On the plate with the first used dilution is less than 10 colonies, but
minimally 4 colonies:
According to the Poisson distribution for the 50 % acceptable of relative trueness - the
lower limit is 4 colonies at one plate and it is calculated by using the formulae before
(ISO/TR 13843:2000)
• (1-3 colonies) On the plate with the first used dilution is 1-3 colonies:
„Microorganisms are present, but in the number lower than 4/(V*d) per g or per ml.“
• 0 colonies
„less than 1/(V*d) microorganisms per ml or per g”
(note: if two parallel plates are used (each per 0.1 or 1 ml according to the method,
V should be considered in the formula „less than….“ twice times (as 0.2 or 2 ml) – because
the examined volume with no colonies is the volume spread over these two parallel plates)
d) More than upper limit for the last used dilution (d2)
“more than 300/(V*d2) or more than 150*(b/A)/(V*d2) microorganisms per ml or per g (if
confirmation is done)”
Counting all colonies
Colony counting
10 g + 90 ml PW , V= 1 ml
C
N  V *( n1  0,1n2 )*d
Dilution, V=1 ml 10-1 10-2
Colonies <300 >10

Maximally 300 colonies per one P. dish, at least
one dish with minimally 10 colonies
Colonies <300 <10
Colonies 255 38 Colonies 255 5

255  38 255  5
N  2.7 *103 CFU / g N  2.4 *103 CFU / g
1* (1  1* 0.1) * 0.1 1* (1  1* 0.1) * 0.1
Colonies >300 10-300 Colonies & 55
Only this used for N 

55

55
 5.5 *103 CFU / g
calculation 1* (1  0 * 0.1) * 0.01 1*1* 0.01
If using one general formula is
N  V 
C 55
*n*d 
confusing, the simpler formula is 1*1*0.01 5.5 *103 CFU / g
Enterobacteriaceae and coliform
bacteria
Family Enterobacteriaceae
found in soil, water, plants and animals and their gastrointestinal tracts
Gram-negative, straight rods, some of which motile
Most grow well at 37 °C, some better at 25 - 30°C,
facultatively anaerobic, fermenting glucose,
oxidase-negative and catalase-positive (except Shigella dysenteriae type 1),
resistant to bile salts
Proteus sp., Hafnia sp., Salmonella sp., Shigella sp., Pantoea sp., Yersinia sp., etc.
coliform bacteria = subgroup of Enterobacteriaceae
similar to E. coli = coliform
may be found also as normal intestinal flora of humans and/or animals,considered
opportunistic pathogens - indicator for faecal contamination in water or bad
hygiene/insufiecient storage conditions in food
Escherichia sp., esp. Escherichia coli; Enterobacter sp., Klebsiella sp., Citrobacter sp.
fermenting lactose (enzyme β-D-galactosidase)
Escherichia coli β-D-glucuronidase (app. 97 % of strains), indole positive
Enterobacteriaceae, coliform bacteria and E. coli
Biochemical properties used for isolation on selective media and
confirmation
Family Enterobacteriaceae
Gram-negative, resistant to bile salts VRBD Agar
fermenting glucose, oxidase-negative,
cultivation temperature: 37 °C
coliform bacteria VRBL agar

all features of Enterobacteriaceae MacConkey agar, ENDO agar,
+ fermenting lactose (enzyme β-D-galactosidase) Chromocult Coliform agar
Escherichia coli
all features of Enterobacteriaceae and coliforms
+ enzyme β-D-glucuronidase
(app. 97 % of strains, not present e.g. in E. coli
O157) TBX
indole positive
VRBD (Violet Red Bile Dextrose) Agar
Selective agar for the isolation and enumeration of all
Enterobacteriaceae species in foodstufs
Peptone from gelatine 7.0; yeast extract 3.0; sodium chloride 5.0;
D(+)glucose 10.0; bile salt mixture 1.5; neutral
red 0.03; crystal violet 0.002; agar-agar 13.0.
Selective activity
Crystal violet and bile salts inhibit the accompanying bacterial flora.
Differential activity
Degradation of glucose is accompanied by
production of acid, which is indicated by a colour change to red and
Escherichia coli ATCC 8739 by zones of precipitated bile acids surrounding
the colonies.
All Enterobacteriaceae are detected as they all degrade glucose to
acid. The culture medium is not,however, absolutely specific for these
organisms as some other accompanying bacteria (e.g. Aeromonas)
also show these reactions.
Shigella flexneri ATCC 29903

Enterobacteriaceae
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
Sample 10 g sample 1 ml (-1. dilution) 1 ml (-(X+1). dilution Sample

(if liquid) + 90 ml BPW + 9 ml BPW …. + 9 ml BPW preparation and 1. DAY
0. dilution - 1. dilution - 2. dilution - X. dilution decimal dilution
Pourring over of 1 ml by VRBD

Isolation on
selective agar
VRBD VRBD VRBD VRBD VRBD VRBD VRBD VRBD
Cultivation: 37 °C, 24 h±2 h

Confirmation of 2. DAY
typical/atypical
Choice of typical (or atypical) colonies to be confirmed – up to 5 colonies, if colonies
present from each plate used for calculation • Inoculation on
Inoculation on nutrient agar, 37˚C, 24±3 h non-selective agar
3. DAY
Biochemical test: test for oxidase, glucose fermentation under anaerobic condition – • Confirmatory tests
glucose agar (37 °C, 24 h)
Evaluation of biochemical tests Result 4. DAY

Calculation of CFU/g, ml
***1 ml of tested suspension pourred over (up to 15 minutes after suspension preparation) by VRBD (at 44 °C – 47 °C)
twice – semi-anaerobic conditions – at first by 10 ml, after solidification by other 15 ml
Oxidase test
Oxidase
•abbreviated general name for some kind of cytochrome c oxidases (cytochrome
oxidase or indophenol oxidase), enzymes involved in aerobic respiratory chain,
when oxygen is used as the final receptor for hydrogen.
•Oxidases are usually present only in aerobic organisms.
•Presence or absence of oxidase(s) is an important feature of the bacterial
metabolism, used mainly in the identification process of gram-negative bacteria.
Oxidase positive (OXI +) Aeromonas sp. Pseudomonas sp. Dark-blue to violet

Oxidase negative (OXI -) Enterobacteriaceae Bacillus sp. Colourless
Detection:
Reaction of the culture with redox indicator as
e.g N,N-dimethyl-p-phenylenediamine
(DMPD): dark-blue when oxidized and
colorless when reduced.
Different test performances are possible.
VIOLET RED BILE AGAR WITH LACTOSE
(VRBL)
Selective medium for the detection and enumeration of
coliforms in dairy products, water and food
FORMULA IN g/l
Lactose 10.00 Bile Salts 1.50
Gelatin Peptone 7.00 Neutral Red 0.03
Sodium Chloride 5.00 Crystal Violet 0.002
Yeast Extract 3.00 Bacteriological Agar 15.00
Final pH 7.4 ± 0.2 at 25ºC
Escherichia coli ATCC 11775 Selective action

Bile salts and crystal violet inhibit Gram-positive bacteria, bile salts
partially inhibits non-intestinal Gram-negative bacteria
Differential action
Lactose fermentation detected by neutral red as a pH indicator.
Lactose fermenters form red colonies with red-purple halos.
Occasionally the cocci of the intestinal tract can develop as
small, punctiform red colonies.
Appearance of colonies Microorganisms
red colonies with or without red-purple Coliform bacteria
halos of precipiation
small, punctiform red colonies Occasionally the cocci of the intestinal
tract
Escherichia coli Salmonella gallinarum colourless Lactose-negative Enterobacteriaceae
ATCC 25922 NCTC 9240 and others
Endo Agar
used to confirm the detection and enumeration of coliform bacteria following presumptive test of
drinking water, from milk, dairy products and food.
Composition Selective activity:
Ingredients Gms / Litre sodium sulphite and basic fuchsin inhibits partially Gram positive
bacteria
Peptic digest of animal tissue 10.000
Lactose 10.000
Dipotassium phosphate 3.500
Sodium sulphite 2.500
Basic fuchsin 0.500
Agar 15.000
Final pH ( at 25°C) 7.5±0.2
Shigella flexneri ATCC

12022
Differential activity:
Coliforms – lactose-fermenter - pink colonies
lactose non-fermenters - colourless colonies on the medium.
E. coli – this reaction is very pronounced as the fuchsin crystallizes,
exhibiting a permanent greenish metallic
luster (fuchsin luster) to the colonies.
67
Endo
Agar
ChromoCult®Coliform Agar
Selective agar for the simultaneous detection of total coliforms and
E. coli in drinking water and processed food samples
Peptone 3.0; sodium chloride 5.0; sodium dihydrogen phosphate 2.2; di-sodium
hydrogen phosphate 2.7; sodium pyruvate 1.0; tryptophan 1.0; agar-agar 10.0;
Sorbitol 1.0; Tergitol® 7 0.15; 6-chloro-3-indoxyl-beta-Dgalactopyranoside 0.2; 5-
bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid 0.1; isopropyl-beta-
Dthiogalactopyranoside 0.1. pH: 6.8 ± 0.2 at 25°C.
Coliform identification
Salmon-GAL – cleaved by β-D-galactosidase - characteristic
for coliforms - a salmon to red color of the colonies.
E. coli identification
Escherichia coli ATCC 11775 X-glucuronide – cleaved by β-D-glucuronidase - characteristic
for E. coli. E. coli cleaves also Salmon-GAL so the colonies
take on a dark-blue to violet color.
Citrobacter freundii ATCC 8090

Escherichia coli
ISO 16649-2:2001 Microbiology of food and animal feeding stuffs -- Horizontal method for the
enumeration of beta-glucuronidase-positive Escherichia coli -- Part 2: Colony-count technique at 44
degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide
Pourring over of 1 ml of TBX agar*

Isolation on
selective agar
TBX TBX TBX TBX TBX TBX TBX TBX
Cultivation: 44 °C, 18-24 h

Confirmation of
typical/atypical
colonies
ChromoCult ® TBX ( • Inoculation on
non-selective agar
Tryptone Bile X-
glucuronide ) Agar
• Confirmatory tests
Evaluation of typical colonies – the used biochemical reaction as taken to be Result 2. DAY
so specific, that only colonies of E. coli are blue-green – no confirmatory step evaluation
*about 15 ml temperated to 44-47 °C

ChromoCult ® TBX ( Tryptone Bile X-glucuronide ) Agar
Selective-differential chromogenic agar for the detection and enumeration
of Escherichia coli in foodstuffs, animal feed and water.
Typical composition ( g / Litre )
Peptone 20.0; bile salts No. 3 1.5; X- β - D - glucuronide 0.075;
agar-agar 10.0.
Selective activity:
Growth of accompanying Gram-positive flora is largely inhibited by the use of
bile salts and the high incubation temperature of 44 °C (also Gram-negative).
Differential activity
X-β-D-glucuronide (5-bromo-4-chloro-3- indolyl-β-D -Glucuronide) is cleaved
by the β-D-glucuronidase (this enzyme differentiates most E.coli ssp. from
other coliforms) to split the bond between the chromophore 5-bromo-4 -
Escherichia coli ATCC 25922 chloro -3-indolyle and the β-D-glucuronide. E.coli colonies -blue-green.
Appearance Microorganisms
blue-green E. coli β-D-glucuronidase positive (96 – 97 % of strains)
colourless β-D-glucuronidase-negative E.coli strains (3 - 4 %) form
colourless colonies, e. g. E.coli 0157, or they cannot grow at
elevated temperature of 44 °C, e.g. E. coli 0157: H7.
Escherichia coli DSMZ 502

Coagulase-positive staphylococci
enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) -- Part 1:
Technique using Baird-Parker agar medium
Spreading of 0.1 ml onto Baird-Parker agar (BP)*

Isolation on
selective agar
BP BP BP BP BP BP BP BP
Cultivation: 37 °C, 24 h/48 h (colony counting after both time)

Confirmation of 2./3.DAY
typical/atypical
Choice of typical (or atypical) colonies to be confirmed – up to 5 colonies, if colonies
present from each plate used for calculation • Inoculation on
Inoculation in BHI and blood agar, 37˚C, 24±3 h non-selective
agar
3./4. DAY
Biochemical test: plasma-coagulase test (incubation of culture grown in BHI
• Confirmatory
with rabbit plasma) – 37 °C , 24 h; detection of haemolysis
tests
Evaluation of biochemical tests Result 4./5. DAY

***instead of 0.1 ml on one Petri dishes (average 90 mm) also 1 ml on three Petri dishes by 90 mm, when low count
expected, Czech Republic ČR – ČSN EN ISO 6888-1:1999 – national version – spreading of 0.2 ml
BAIRD-PARKER AGAR (BP)
Peptone from casein 10.0; meat extract 5.0; yeast extract 1.0;
sodium pyruvate 10.0; glycine 12.0; lithium chloride
5.0; agar-agar 15.0.
Also to be added (supplement):
Egg-yolk tellurite emulsion 50 ml; if required, sulphamethazine
0.05 g/l.
pH: 6.8 ± 0.2 at 25°C
Nutrient: peptone, meat extract, yeast extract
Selective stimulation of the growth of staphylococci
(resuscitation partial): pyruvate and glycine
Inhibition of accompanying microflora: lithium chloride and Staphylococcus aureus ATCC 25923
tellurite
Differential mode for Staphylococcus colonies show two
characteristic features when grown in this opaque medium
(opaque, because of its egg-yolk content)
a. characteristic zones and rings are formed as a result of
lipolysis and proteolysis,
b. reduction of tellurite to tellurium produces a black
colouration.
The egg-yolk reaction and tellurite reduction are usually
found to occur together with a positive coagulase reaction
and can thus serve as an index for the latter (coagulase
positive staphylococci – S. aureus and other species)
http://www.bacteriainphotos.com/photo%20gallery/staphylococcus%20aureus%20baird%20parker.jpg
BAIRD-PARKER AGAR (BP)
Staphylococcus aureus ATCC 25923

enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) -- Part 1:
Technique using Baird-Parker agar medium
Confirmation of 2./3.DAY
Choice of typical (or atypical) colonies to be confirmed – up to 5 colonies, if typical/atypical
present from each plate used for calculation colonies
Inoculation in BHI and blood agar, 37˚C, 24±3 h • Inoculation on
non-selective agar
Biochemical test: plasma-coagulase test (incubation of culture grown in BHI 3./4. DAY
with rabbit plasma) – 37 °C , 24 h; detection of haemolysis • Confirmatory tests
Typical colonies: black or grey, convex, shiny, 1-5 mm in average, with the clearance zone wide by 2-5 mm,
after at least 24 h incubation there can be an opaque ring
Atypical colonies: grey, without the clearance zone, or black with narrow white edge or without, the
clearance zone is missing or badly visible. The atypical colonies are mainly formed by coagulase-positive
strains of staphylococci from dairy products, seafood or harslet
β- Plasma-
haemolysis coagulase
S. aureus + +
S. epidermidis - -
Coagulase test : coagulase is an enzyme with the ability to coagulate plasma
It differentiates S. aures from the other species, with less or without impact on
human health and food safety (enterotoxins production) as (S. epidermidis, S.
saprophyticus), other coagulase-positive staphylococci (S. schleiferi, S. intermedius)
are not so common, coagulase production usually correlates with the pathogenity
Staphylococcus aureus forms two types of coagulase
the free coagulase is an extracellular enzyme
the bound coagulase on the surface of the cell wall
Slide test: detect only bound
coagulase
Dense culture mixed in saline
solution mixed with EDTA-rabbit
plasma = „clumping“, a screening test
(false-positive reactions and Tube test
http://www.mgm.ufl.edu/~gulig/mmid/mmid-lab/images/coag-ex.jpg
autoagglutination can occur) • Grown liquid culture (grown in BHI) mixed with
rehydrated EDTA-rabbit plasma (for 4, 18 and 24 h)
• Detection of free and bound coagulase
• Free coagulase reacts with plasmatic factor to
produce staphylotrombin, which catalyzes changes
of fibrinogen to fibrin (clumps, sluts or completely
congelation in a tube – positive when ¾
http://faculty.mc3.edu/jearl/ML/coagulase.jpg
congelates)
Total count of microorganisms
ISO 4833-1:2013 Microbiology of the food chain -- Horizontal method for the enumeration of
microorganisms - Part 1: Colony count at 30 degrees C by the pour plate technique

Pourring over of 1 ml of PCA agar*

Isolation on non-
selective agar
PCA PCA PCA PCA PCA PCA PCA PCA
Cultivation: 30 °C, 72 h±3 h

Confirmation of
typical/atypical
colonies
• Inoculation on
non-selective agar
Colony counting of all grown colonies
Result
Calculation of CFU/g, ml 4. DAY
evaluation
*1 ml of tested suspension to pour over with 12-15 ml of melted PCA (44-47 °C)
Yeasts and moulds
enumeration of yeasts and moulds
Part 1: Colony count technique in products with water activity greater than 0,95 (DRBC to be used)
Part 2: Colony count technique in products with water activity less than or equal to 0,95 (DG18 to be used)

(if liquid) + 90 ml SP + 9 ml SP …. + 9 ml SP preparation and 1. DAY
Spreading of 0.1 ml onto DRBC/DG18 agar*

DRBC/
DG18 Isolation on
selective agar
DRBC/ DRBC/ DRBC/ DRBC/ DRBC/ DRBC/ DRBC/
DG18 DG18 DG18 DG18 DG18 DG18 DG18
Confirmation of
Cultivation: 25 °C, 3-5 days (by the bottom down – typical/atypical
colonies
not in the usual reverse position)
• Inoculation on
non-selective agar
Colony counting of all grown colonies Result
Calculation of CFU/g, ml evaluation 3.-6.
DAY
* Diluent – peptone water with 0.1 % peptone, for low counts also 3 plates by 0.3 ml each
Cultivation in an open plastic bag to avoid the dissemination of spores from plates .
Selective agar for the
Dichloran Rose Bengal enumeration of food spoiling
Chloramphenicol (DRBC) Agar yeasts and moulds.
Peptone 5.0; glucose 10.0; potassium dihydrogen phosphate 1.0;
dichloran 0.002; magnesium sulfate 0.5; Rose Bengal 0.025;
chloramphenicol 0.1; agar-agar 15.0. pH: 5.6 ± 0.2 at 25°C.
Low pH- inhibition of most bacteria
Chloramphenicol - broad-spectrum antibiotic
inhibitory to a wide range of Gram-negative and
Gram-positive bacteria.
Rose Bengal
• to inhibit bacterial growth and restricts the
Saccharomyces cerevisiae ATCC 9763 growth of rapidly growing molds
• incorporated in the cells of yeasts and molds,
turning these colonies pink.
Dichloran - to inhibit the rapid spreading of
mucoraceous fungi and restricts colony sizes of
other genera, easing the colony count.
Magnesium Sulfate provides trace elements.
Glucose, peptone - nutrition
Mucor racemosus ATCC 42647

Dichloran Rose Bengal Selective agar for the
enumeration of food spoiling
Chloramphenicol (DRBC) Agar yeasts and moulds.
Mucor racemosus ATCC 42647 Saccharomyces cerevisiae ATCC 9763

Dichloran Glycerol (DG18) Agar
Selective agar with low water activity (aw) for the
enumeration and isolation of xerophilic moulds in dried
and semi-dried foods as well as a general purpose
medium for counting yeast and moulds in foodstuffs.
Peptone 5.0; glucose 10.0; potassium dihydrogen phosphate 1.0;
dichloran 0.002; magnesium sulfate 0.5; chloramphenicol 0.1; agar-agar
15.0. pH:5.6 ± 0.2 at 25°C.
Preparation
Suspend 31.6 g in 1 litre of demin water and heat to boiling until
completely dissolved. Add 175 ml of glycerol p.a. to the medium, mix
Wallemia sebi and autoclave at 121°C for 15 min. Cool to approx. 50°C, mix well
and pour plates.
By reducing the water activity from approx. 0.99 to 0.95 with

18 % (w/w) glycerol and addition of chloramphenicol growth
of bacteria is prevented.
Low pH- inhibition of most bacteria
Chloramphenicol - broad-spectrum antibiotic inhibitory to a
wide range of Gram-negative and Gram-positive bacteria.
Magnesium Sulfate provides trace elements.
Glucose, peptone - nutrition
Eurotium rubrum
Dichloran Glycerol (DG18) Agar
YGC Agar (Yeast Extract Glucose Chloramphenicol
Agar
Selective agar for isolating and counting yeasts and
moulds in milk and milk products.

Yeast extract 5.0; D(+)glucose 20.0;
chloramphenicol 0.1;
agar-agar 14.9. pH: 6.6 ± 0.2 at 25°C.
Saccharomyces cerevisiae ATCC 9080 Chloramphenicol to suppress

accompanying bacterial flora (unlike
other antibiotics e.g. oxytetracycline,
it has the advantage of being
fully autoclavable)
Yeast extract, glucose - nutrion
Aspergillus niger ATCC 16404

YGC Agar (Yeast Extract Glucose
Chloramphenicol Agar
Saccharomyces cerevisiae ATCC 9080
Aspergillus niger ATCC 16404

Sabouraud Dextrose Agar
Sabouraud Dextrose Agar is used for the cultivation of yeasts, moulds and
aciduric bacteria.
Composition Ingredients Gms / Litre
Dextrose 40.000, Mycological, peptone 10.000, Agar 15.000
Final pH ( at 25°C) 5.6±0.2
Mycological peptone provides nitrogenous compounds.
Dextrose provides an energy source.
High dextrose concentration and low pH favours fungal growth and inhibits contaminating
bacteria from test samples.
Malt Extract Agar Base(w/ Mycological Peptone)

Composition Ingredients Gms / Litre
Malt extract 30.000, Mycological peptone 5.000, Agar 15.000
Final pH ( at 25°C) 5.4±0.2
Malt extract - an acidic environment and nutrients favourable for growth and
metabolism of yeasts and moulds.
Mycological peptone rapidly gives a luxuriant growth with typical morphology and
pigmentation. For mycological count, it is advisable to adjust the reaction of medium
more acidic with addition of 10% lactic acid.
Antibiotics may be added as sterile solutions to the molten medium in order to suppress
bacterial growth.
3M Petrifilm™ Count Plates - principle
an all-in-one plating
system made by the Food
Safety Division of the 3M
Corporation
cost-effectiveness,
simplicity, convenience,
and ease of use
3M Petrifilm™ Count Plates - principle
3M Petrifilm™ Count Plates:
Coating technology
1. Top Film
Plastic Film coated with
adhesive, indicator and
cold water soluble gel.
2. Bottom Film
Plastic coated paper printed
with a grid, adhesive
standard methods
nutrients, cold water
soluble gel.
3M Petrifilm™ Coliform Count Plates
Using the same typical biochemical features of
coliform bacteria as in ISO 4832:2006
=bacteria resistant to crystal violet and bile
salts, fermenting lactose to acid and gas
The dehydrated medium is based on VRBL agar

principles
• crystal violet inhibits G+ bacteria
• bile salts inhibit most G- bacteria
• fermentation of lactose to acid –
decreased pH is detected by acidobazic
indicator neutral red, which changes in
some range of pH its colour to red
Enumeration of coliforms (total or •but fermentation of lactose to gas is
thermotolerant). detected for all grown colonies
Reading facilitated by an indicator, which immediately by trapping gas bubbles in
colours colonies the gel
red. Upper film traps the gas produced
by lactose fermentation.
MEMBRANE FILTRATION
MEMBRANE FILTRATION
• used for capturing bacteria from larger volume
of sample
• filter is then tranferred onto a plate to let
them grow
• It also can be used for sterilisation of liquids
Pore size Particles that pass through them

in (µm)
Erythrocytes, yeast cells, bacteria, viruses,
10
http://microbiologyon- molecules
line.blogspot.cz/2009/08/filtration.html
5 Yeast cells, bacteria, viruses, molecules
3 Some yeast cells, bacteria, viruses, molecules
1.2 Most bacteria, viruses, molecules
0.45 A few bacteria, viruses, molecules
0.22 Viruses, molecules
0.10 Medium-sized to Small Viruses, molecules
0.05 Small viruses, molecules
http://www.flickriver.com/photos/artsyscience/2345558461/ 0.025 Only the very smallest viruses, molecules
Ultra-filter Small molecules
MEMBRANE FILTRATION
MEMBRANE FILTRATION
• used for capturing bacteria from larger volume
of sample
• filter is then tranferred onto a plate to let
them grow
• It also can be used for sterilisation of liquids
http://microbiologyon-
line.blogspot.cz/2009/08/filtration.html
• filtrable samples - (dilution in order

to obtain countable amount of
colonies) – filtration=capturing
bacteria on a membrane filter
• nonfiltrable samples – diluting –
pourring over or spreading on
according to the tested
microorganisms properties
Enumeration of Escherichia coli and coliform bacteria in water
ISO 9308-1:2014 Water quality -- Enumeration of Escherichia coli
sample and coliform bacteria -- Part 1: Membrane filtration method for
waters with low bacterial background flora
day
Membrane filtration (filter 0,45μm) → 100 ml
1. Tranfer of filter onto TTC agar
Cultivation 36±2 °C, 24 - 48 h
2. Lactose + colonies Lactose - colonies
Isolation on TSA → 36±2 °C, 21±3 h
3. OXI test IND → 44±0,5 °C, 21±3 hod

-
OXI- IND+
4.
Coliform bacteria Escherichia coli
Pitná voda - normy - Vyhl. č.252/2004 91
Lactose TTC Agar with Tergitol® 7
Selective differential medium for the detection and enumeration of
E. coli und coliform bacteria in water using the membrane filtration
method.
Lactose 20.0; peptone 10.0; yeast extract 6.0; meat extract 5.0;
bromothymol blue 0.05; Tergitol®7 0.1; agar-agar 12.7. Additive: TTC
0.025. pH: 7.2 ± 0.2 at 25°C.
Selectivity action : sodium heptadecylsulfate (Tergitol®7) and

2,3,5-Triphenyltetrazoliumchloride (TTC) to inhibit most Gram-
Citrobacter freundii positive bacteria.
ATCC 8090 from above Differential action
Degradation of lactose to acid is indicated by the pH indicator
bromothymol blue, which changes the colour of the medium
under the membrane to yellow.
The reduction of TTC by lactose-negative bacteria - dark red
colonies. Lactosepositive E. coli and coliform bacteria reduce
TTC weakly - yellow-orange colonies.
Escherichia coli
ATCC 25922 from below
Lactose TTC Agar with Tergitol® 7
Real samples – well water
• TTC (accompanying microflora– brown-violet CFU)
93
Lactose
TTC Agar
with
Tergitol® 7
TTC – from From below

above
Real sample
94
MPN
MPN - Most Probable Number - The end point dilution method
preparation a series of dilutions from a sample
Presumption: in the liquid medium
the distribution of cells is uniform,
cells do not cluster together and
that there is detectable growth
even if only one viable microbial
cell is present in the tube/well.
estimation of bacterial count is based by

using statistical tables
the number of positive tubes/wells

(showing characteristic microbial growth:
inoculation of each change in optical density, shifting of pH,
incubation
dilution into liquid media changing of redox values and subsequently
using 3-5 parallels colour, etc.)
MPN – coliform bacteria/ E. coli
MPN - Most Probable Number - The end point dilution method
Lauryl Sulfate Broth
Nutrients: tryptose, lactose, L-tryptophan +
phosphate bufferring system
Inhibition: lauryl sulfate (G+, non-coliform G-)
positive tubes for coliform bacteria =

gas formation due to lactose
fermentation to gas in Durham tubes
positive tubes for E. coli = gas
formation in Durham tubes and
cleaving of fluorogenic substrate (MUG)
- fluorescence (and positive indol
reaction – confimatory test)
ISO 7251:2005-MPN principle
Estimation of MPN values
1) Calculation by formulae – see IS0 7218:2007 10.5.6.1
2) Software programs
3) Using MPN tables
Table B.5 ISO 7218:2007
MPN index for 3 following dilutions with 3 tubes starting from 1 g in a tube – (43 possibilities)
- if x g in the first tube used for calculation – to multiply the result by 1/x
(for 10 g = to multiply by 0.1, for 0.1 g = to multiply by 10)-Tables are for 1 g in the first tube
Table B.7 ISO 7218:2007/ Table A.1 ISO 7251:2005(E) (more detailed)
MPN index for 3 following dilutions with 5 tubes starting from 1 g in a tube (63 possibilities)
Number of Index Category Limits of confidence Category:
positive results MPN (95%) If the analysis results are
Lower Upper exact, then 95 %
limit limit combinations should be
2 0 0 0.92 1 0.15 3.5 the category 1; 1.4% the
category 2; 0.9 % the
2 0 1 1.4 2 0.4 3.5 category 3 and 0.1 % the
2 0 2 2.0 0 0.5 3.8 category 0.
2 2 1 2.8 3 0.9 9.4
ISO 7251:2005-MPN principle
Using MPN tables – more than three dilutions for 3 tubes
Sa Liquid sample 1 ml 10-1 ml 10-2 ml 10-3 ml MPN
mp 10 ml
le
Other 10-1 g 10-2 g 10-3 g 10-4 g L. sample O.sampl.
samples 1 g (ml-1 ) (g -1 )
1 3 3 2 1 0 1.1x101 1.1x102
Lower limit 0.2x101 0.2x102
Upper limit 4.0x101 4.0x102
Number of positive Index Category Limits of
1 – better category
results MPN confidence (95%)
3-3-2:
Lower Upper higher total
Table B.5 ISO 7218:2007 limit limit number
3 3 2 110 1 20 400 of the positive
tubes
3 2 1 1.4 2 0.4 3.5
2 1 0 1.5 1 0.4 3.8
Horizontal method for the detection of
microorganisms
SAMPLE PROCESSING + PRE-ENRICHMENT + ENRICHMENT
• Sample amount according to EU legislative usually 25 g (or 10 g)
• resuspension in pre-enrichment non-selective or selective medium (X g of
sample + 9X ml of enrichment medium) – cultivation – in order to allow
stressed cells to start to grow
• Transfer the enriched suspension into selective enrichment medium (usually
0.1-1 ml into 10 ml volume) – in order to perform selective enrichment of
target microorganism
ISOLATION ON SELECTIVE MEDIA (usually 2 kinds of agar by different mode of action)
SUB-ISOLATION OF PRESUMPTIVE COLONIES (non-selective agar)

BACTERIA GROWING ON SELECTIVE MEDIA ARE NOT APPROPRIATE FOR
BIOCHEMICAL TESTING – SELECTIVE MEDIA DECREASED „FITNESS“ OF
BACTERIA AND INFLUENCES THEIR PRESENT BIOCHEMICAL BEHAVIOR (NOT
PROPERTIES), WHICH CAN INTERFER WITH THE EXACTNESS AND RELIABILITY OF
TESTING
CONFIRMATION/IDENTIFICATION
Detection of Salmonella spp. according to
ISO 6579:2002
25 g of sample + 225 ml of buffered peptone water; homogenization
ISO 6579:2002 Microbiology of Pre-enrichment

Incubation at 37±1°C for 18±2 h
food and animal feeding stuffs
-- Horizontal method for the 1. day
detection of Salmonella spp.
1 ml of homogenate to 10 ml MKTTn 0.1 ml of homogenate to 10 ml RVS Selective

incubation at 37±1°C for 24±3 h incubation at 41.5°C for 24±3 h enrichment
2. day
Plating out by 10 µ on two selective media and incubation at 37±°C for 24± 3h : Isolation on
•Xylose Lysine – Desoxycholate Agar (XLD) selective agar
plates
•Any other selective medium (BGA,HE, BS, SS, DC, chromogenic media…)
3. day
Isolation of a charasteristic colony on Nutrient Agar and incubation at 37±°C for 24± 3h
Confirmation
Confirmation 4.-6. day

ISO 6579:2002: Pre-enrichment
Pre-enrichment
• Why?
• in dried, processed foods are often present in low numbers and in
debilitated condition
• for the repair of cell damage, dilute toxic or inhibitory substances
NON-SELECTIVE PREENRICHMENT= RESUSCITATION BEFORE

TRANSFER INTO SELECTIVE MEDIA
• What is important?
• sampling
• higher amount of sample – BWP 37 ± 1°C
• pH
• sour food – minimal pH 4.5
Buffered Peptone Water (BPW)
For the preliminary, non-selective enrichment of bacteria, particularly pathogenic
Enterobacteriaceae, from foodstuffs and other materials.
Peptone from casein 10.0; sodium chloride 5.0; disodium hydrogen phosphate dodecahydrate 9.0;
potassium dihydrogen phosphate 1.5.
Mode of Action
The broth is rich in nutrients and produces high resuscitation rates for subletally injured bacteria and
intense growth. The phosphate buffer system prevents bacterial damage due to changes in the pH of
the medium.
pH: 7.0 ± 0.2 at 25 °C.

ISO 6579:2002: Selective enrichment
Selective enrichment
• Why?
• to favor a higher ratio of Salmonella to non- Salmonella
• allow Salmonella to multiplicate to level detectable after plating: need 103 -
104 CFU Salmonella /ml
• MKTTn (37±1°C for 24±3 h): Muller-Kauffmann Tetrathionate-
Novobiocin Broth
• growth of coliform and other enteric bacteria (tetrathionate)
• primarily Gram-positive bacteria (brillant green, novobiocin)
• the accompanying bacteria (bile salts)
• RVS (41.5°C for 24±3 h): Rappaport-VASSILIADIS Salmonella
Enrichment Broth
• high osmotic pressure (magnesium chloride concentration)
• low pH (pH = 5.2)
• malachite green (106 mg/L)
• minimal nutritional requirements (peptone: 5 g/L)
• selenite
Rappaport-Vassiliadis Salmonella Enrichment
Broth
Peptone from soymeal 4.5; magnesium chloride
hexahydrate 29.0; sodium chloride 8.0; dipotassium
hydrogen phosphate 0.4; potassium dihydrogen
phosphate 0.6; malachite-green 0.036.
pH: 5.2 ± 0.2 at 25°C
cultivation: 41˚C, 24±3 h
dipotassium hydrogen phosphate +
potassium dihydrogen phosphate =
phosphate buffering system
http://www.kisanbio.com/shopimages/kisanbiotech/0390100000492.jpg
Selectivity action:
Selective conditions for preferably growth of Salmonella spp. favor especially other
Enterobacteriaceae – Salmonella spp. are more resistant
• malachite green
• higher osmotic pressure by adding magnesium chloride
• low pH 5.2 ± 0.2
• lower nutrition (peptone from soymeal)
Rappaport-VASSILIADIS Salmonella Enrichment Broth is not suitable for selective

enrichment of S. typhi and S. paratyphi A.
ISO 6579:2002: Isolation on selective agar
plates
Isolation on selective agar plates
• non-lactose fermenting and produce hydrogen sulfide
• XLD
• second selective medium
• Hektoen Enteric Agar
• Deoxycholate citrate agar
• Salmonella-Shigella agar
• Brillant Green Agar
• Bismuth Sulfite Agar
• Chromogenic Agar: Rambach agar (Chromagar Co.); SMID agar
(bioMérieux); CSE Agar (PPR Diagnostics); Compass Salmonella
agar (Biokar Diagnostics).; Salmonella Chromogenic agar
(Oxoid); Chromagar Salmonella (Chromagar Co.); ABC medium
(Lab M)
ISO 6579:2002: Isolation on selective agar
plates
• XLD (37±°C for 24± 3h)
Xylose-lysine-deoxycholate citrate; lactose + sucrose: phenol red,

sodium thiosulfate and ferric ammonium citrate
• little or no production of hydrogen sulphide: S. Typhi, S. Senftenberg,

S. Pullorum
• slowly produce of lactose: S. Choleraesuis, S. Arizona
• poor growth of S. Typhimurium
• Citrobacter, Proteus, Pseudomonas

XLD (Xylose LysineDeoxycholate) Agar
Yeast extract 3.0; sodium chloride 5.0; D(+)xylose 3.75; lactose 7.5; sucrose
7.5; L(+)lysine 5.0; sodium deoxycholate 1.0; sodium thiosulfate 6.8;
ammonium iron(III) citrate 0.8; phenol red 0.08; agar-agar 14.5.
pH: 7.4 ± 0.2 at 25 °C
Degradation of xylose, lactose and sucrose to acid = phenol red to change its
colour to yellow.
Production of hydrogen sulfide is indicated by thiosulfate and iron(III) salt - to
form a precipitate of black iron sulfide in the colonies.
Decarboxylation of lysine to cadaverine - a purple colouration around the
Salmonella enteritidis NCTC 5188 colonies due to an increase in pH.
Klebsiella pneumoniae ATCC 13883

Salmonella – BG agar
Peptone from meat 10.0; meat extract 5.0; yeast extract 3.0;
disodium hydrogen phosphate 1.0; sodium dihydrogen phosphate
0.6; lactose 10.0; sucrose 10.0; phenol red 0.09; brilliant green
0.0047; agar-agar 12.0.pH: 6.9 ± 0.2 at 25 °C.
Selectivity action:
Brilliant green – inhibition of G+ bacteria, Salmonella Typhi
and Shigella
By addition of 0.2 % deoxycholate sodium – inhibition of
Proteus colonies to swarm
Salmonella enteritidis NCTC 5188 Fermentation of lactose – phenol red changes colour to
yellow from red (in alkaline)
Appearance of Colonies Microorganisms

Lactose- and sucrose-negative:
Red, surrounded by a Salmonella, Proteus (no
bright red swarming), Pseudomonas
zone (small, crenate colonies) and
others
Lactose- or sucrose-positive:
Yellow, surrounded by a
E. coli, Enterobacter, possibly
yellow zone
Citrobacter, Klebsiella and others.
Salmonella typhimurium ATCC 14028
ISO 6579:2002: Confirmation
• Isolation of charasteristic colony onto non-selective media – Nutrient agar (37±°C for
24± 3h)
• Confirmation:
• Biochemical (37±°C for 24± 3h)
• TSI
• Urea agar (Christensen)
• L-lysine decarboxylase
https://classconnection.s3.amazonaws.com/74/flashcards/1275074/jpg/biochemical_test_re
• β-galactosidase (ONPG) sults-_shigella1332821047536.jpg
• VP test
• Indole test
• Motile (semi-solid nutrient agar)
• Serological (37±°C for 24± 3h)
• O antigens (somatic or cell wall)
• H antigens (flagellar)
• Vi antigens (polysaccharide virulence)
• Phase I
http://www.keydiagnostics.com.au/images/stories/virtuemart
• Phase II /product/microgen-salmonella-latex-kit.jpg
Confirmation of genus Salmonella
Strains Salmonella spp.
S. Typhi S. Parathypi A S. Parathypi B S. Parathypi C Ostatní kmeny
reaction % reaction % reaction % reaction % reaction %
TSI: glucose (acid + 100 + 100 + + + 100

formation)
TSI: glucose (gas - 0 + 100 + + + 92
formation)
TSI: lactose (acid - 2 - 100 - - - 1
formation)
TSI: succrose (acid - 0 - 0 - - - 1
formation)
TSI: hydrogen + 97 - 10 + + + + 92
sulfide
Urea splitting - - 0 - - - - 1
Lysine + - 0 + + + + 95
decarboxylation
β-galactosidace - - 0 - - - - 2
formation
Voges-Proskauer - - 0 - - - - 0
reaction
Indole production - - 0 - - - - 1
Biochemical confirmation of Salmonella spp.
• TSI Triple Sugar Iron Agar (use a straight wire to stab the butt and streak the agar surface)
- to determine whether organisms can ferment glucose, sucrose and/or lactose with or without
production of gas and the ability of the organism to produce hydrogen sulphide from thiosulphate in
an acid environment is also tested.
inoculation in the butt by stab
inoculation on the slant on the
Formula / Liter
Enzymatic Digest of Casein .. 5 g
surface
Enzymatic Digest of Animal • Fermentation of dextrose
Tissue.... 5 g (glucose) to acid
Yeast Enriched Peptone ..... 10 g • anaerobically - yellow butt
Dextrose ...... 1 g • aerobically leads to products,
Lactose ..... 10 g which are later oxidised
Sucrose ..... 10 g
Ferric Ammonium Citrate ... 0.2 g
by air and pH remains alkaline (red)
Sodium Chloride ...... 5 g • Fermentation of glucose to gas
Sodium Thiosulfate ............ 0.3 g (anaerobically - in the stab)
Phenol Red ....... 0.025 g Cracks, splits, or bubbles in medium
Agar ....... 13.5 g indicate gas production
Results Final pH: 7.3 ± 0.2 at 25 °C • Production of hydrogen sulfide
An alkaline slant-acid butt (H2S) – reaction with ferrous
(red/yellow) - fermentation of sulphate and sodium thiosulphate -
dextrose only. a black precipitate in butt
An acid slant-acid butt • Lactose and/or succrose
(yellow/yellow) - fermentation of utilisation to acid
dextrose, lactose and/or sucrose. Acid production – yellow butt and
An alkaline slant-alkaline butt slant
(red/red) - dextrose or lactose
not fermented (non-fermenter).
Bacterium fermenting lactose
Phenol red – alkaline = red,
and/or succrose are able to
acid = yellow
ferment glucose
• TSI Triple Sugar Iron Agar (use a straight wire to stab the butt and streak the agar surface)
- to determine whether organisms can ferment glucose, sucrose and/or lactose with or without
production of gas and the ability of the organism to produce hydrogen sulphide from thiosulphate in
an acid environment is also tested.
Urea agar (Christensen) (use a 1 μl loop full)
- to determine whether an organism can split urea into ammonia and
carbon dioxide by the action of the enzyme urease.
Phenol red: Composition Gms / Litre

Medium is acidic - yellow Peptic digest of animal tissue
1.000
(pH 6.8±0.2) Dextrose 1.000
Alkaline reaction by urea Sodium chloride 5.000
hydrolysis - pink colour Disodium phosphate 1.200
Monopotassium phosphate 0.800
Nutrition: Phenol red 0.012
Peptic digest of animal tissues, dextrose Agar 15.000
Sodium chloride - osmotic equilibrium Final pH ( at 25°C) 6.8±0.2
http://image.slidesharecdn.com/lab17-141201085153-conversion-
gate01/95/-10-638.jpg?cb=1417424219 Phosphates - buffering of the medium.
35-37 °C
http://image.slidesharecdn.com/helicobacterpylori-copy-110516201901-
phpapp01/95/campylobacter-jejunihelicobacter-pylori-39-728.jpg?cb=1305577391
• Indole test (use a 1 μl loop full)
- to determine whether an organism can split indole from tryptophan.
FORMULA IN g/l
Indole Kovacs Reagent:
Tryptone 10.00
p-Dimethylaminobenzaldehyde 50.0 gm
L-Tryptophan 1.00
Sodium Chloride 5.00 Hydrochloric Acid, 37% 250.0 ml
Final pH 7.5 ± 0.1 at 25ºC Amyl Alcohol 750.0 ml
Inoculation below the surface
indole production from tryptophan Production of indole is detected

by Kovac´s reagent
http://www.microbiologyinfo.com/indole-test-principle-reagents-procedure-result-
interpretation-and-limitations/
• L-lysine decarboxylase (use a 1 μl loop full)
- to determine whether an organism can decarboxylate an amino acid (lysine) leading to formation of an
amine. After inoculation both tubes are overlayed with sterile paraffin oil.
Two steps detection:
1. step
Dextrose is fermented – H+ is released
Acidic conditions
• bromocresol purple changes colour to yellow
• in acidic conditions lysine decarboxylase is
activated
2. step
L-lysine is decarboxylated and OH- released
Alkaline conditions
http://fce-study.netdna-
ssl.com/images/upload- • bromocresol purple changes colour back to
Lysine Decarboxylase Broth flashcards/1029530/23326
Typical Composition (g/litre) 21_m.jpg purple
Peptic digest of animal tissue 5.000;
Yeast extract 3.000;Dextrose 1.000 (glucose prepared from starch, chemically
equivalent to glucose); L-Lysine hydrochloride 5.000; Bromocresol purple 0.020
Final pH ( at 25 °C) 6.8±0.2
Inoculate below the surface, cover by sterile paraffin oil
+ CO2 + OH-
• β-galactosidase (ONPG) (use a 1 μl loop full)
- to determine whether an organism has the enzyme ß-galactosidase by using the compound o-
nitrophenyl-beta-D-galactopyranoside (ONPG).
- this test is used to differentiate between lactose-negative and lactose-delayed organisms.
- ONPG is hydrolysed by the enzyme to o-nitrophenol, which is yellow, and galactose.
Lactose fermenting bacteria (ONPG Late lactose fermentering bacteria
positive): Permease – not present
two enzymes delayed in the production of acid from lactose
Permease – transferring of the lactose because of sluggish permease activity
molecule into the bacterial cell β-galactosidase – present
β-galactosidase - cleaving the galactoside - give a positive ONPG test
bond, producing glucose and galactose - Citrobacter spp., Arizona spp,
inducible enzyme - made ONLY in the
presence of the lactose substrate Non lactose fermenter (ONPG
Coliform bacteria as E.coli, Negative):
Klebsiella spp., Enterobacter spp. Permease – not present
β-galactosidase – not present
O-Nitrophenyl-β-D-galactopyranoside (ONPG) is Salmonella spp; Shigella spp; Proteus spp;
Providencia spp. and Morganella spp.
structurally similar to lactose (i.e. ONPG is an analog of
lactose), except that orthonitrophenyl has been
substituted for glucose. ONPG is colorless compound: O-
nitrophenol is yellow, visual evidence of hydrolysis.
hydrolysis
β-galactosidase (ONPG)
Procedure Results and Interpretations
Culture The rate of hydrolysis of ONPG
Bacteria grown in medium containing lactose (to induce the to o-nitrophenol may be rapid
production of the galactosidase enzyme) , such as Kligler for some organisms; producing
iron agar (KIA) or Triple sugar Iron (TSI) agar, (Note: β- a visible yellow color reaction
galactosidase enzyme (inducible enzyme) is made ONLY within 5 to 10 minutes.
in the presence of the lactose substrate). Most tests are positive within 1
Inoculation hour; however, reactions should
1.A loopful of bacterial growth is emulsified in 0.05mL of not be interpreted as negative
physiologic saline to produce a heavy suspension before 24 hours of incubation.
2.One drop of toluene is added to the suspension and The yellow color is usually
vigorously mixed for a few seconds to release the enzyme distinct and indicates that the
for bacterial cells. organism has produced o-
3.An equal quantity of buffered ONPG solution is added to nitrophenol from the ONPG
the suspension. substrate through the action of
4.The mixture is placed in a 37 oC water bath β-galactosidase
5.When Using ONPG Tablets
6.A loopful of bacterial suspension is added directly to the
ONPG substrate resulting from adding 1mL of distilled
water to a tablet in a test tube.
7.This suspension is also placed in a 37oC water bath
• Voges–Proskauer (VP) test (use a 1 μl loop full)
- to determine whether an organism can produce acetylmethylcarbinol
(acetoin) from fermentation of glucose. If present, acetylmethyl carbinol is
converted to diacetyl in the presence of ∝- naphthol (to be added first), strong
alkali (40% KOH), and atmospheric oxygen. The diacetyl and quanidine-
containing compounds found in the peptones of the broth then condense to
form a pinkish red polymer.
Positive Reaction:
A pink-red color at the surface
Examples: Viridans group streptococci
(except Streptococcus vestibularis), Listeria,
Procedure of Voges–Proskauer (VP) Test Enterobacter, Klebsiella, Serratia marcescens,
1.Prior to inoculation, allow medium to Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc.
Negative Reaction:
equilibrate to room temperature. A lack of a pink-red color
2.Using organisms taken from an 18-24 Examples: Streptococcus mitis, Citrobacter sp.,
hour pure culture, lightly inoculate the Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio
furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio
medium. parahaemolyticus etc.
3.Incubate aerobically at 37 °C, 24 hours. A copper color should be considered negative. A
rust color is a weak positive reaction.
4.Following 24 hours of incubation, aliquot Quality Control of Voges–Proskauer (VP) Test
2 ml of the broth to a clean test tube. MRVP broth (pH 6.9)
VP positive: Enterobacter aerogenes (ATCC13048)
VP negative: Escherichia coli (ATCC25922) Ingredients per liter of
5.Re-incubate the remaining broth for an
deionized water:
additional 24 hours. buffered peptone= 7.0 gm
6.Add 6 drops of 5% alpha-naphthol, and glucose= 5.0 gm
mix well to aerate. dipotassium phosphate= 5.0 gm
Voges-Proskauer Reagent
7.Add 2 drops of 40% potassium hydroxide,
A: Barritt’s reagent A Voges-Proskauer Reagent
and mix well to aerate.
B: Barritt’s reagent B
8.Observe for a pink-red color at the surface Alpha-Naphthol, 5% 50 gm
within 30 min. Shake the tube vigorously Potassium Hydroxide 400 gm
1000
during the 30-min period. Absolute Ethanol
ml Deionized Water 1000 ml
• Commercional delivered kits
• API 20E - Identification system for Enterobacteriaceae and other non-fastidious Gram-
negative rods (BioMerieux)
- a plastic strip holding 20 mini-tests with dehydrates substrates
http://www.retroscope.eu/wordpress/wpcontent/uploads/2010/10/SalmonellaAPI20E.jpg
After incubation in a humidity chamber for 18-24 hours at 37°C, the color reactions are read
(some with the aid of added reagents), and the reactions (plus the oxidase reaction done
separately) are converted to a seven-digit code which is called the Analytical Profile Index,
from which name the initials "API" are derived. The code can be fed into the manufacturer's
database via touch-tone telephone, and the computerized voice gives back the identification,
usually as genus and species. An on-line database can also be accessed for the identification.
Serological confirmation of Salmonella spp.
• Serological confirmation: slide agglutination
• O antigens
• H antigens
• Vi antigens
• Phase I
• Phase II
https://classconnection.s3.amazonaws.com/
664/flashcards/1813664/jpg/gram_neg_anti
• Elimination of auto-agglutinable strains gens1358634331589.jpg
• Place one drop of saline onto a clean glass slide. Disperse in this drop part of
the colony to be tested or a colony from a pure culture, so as to obtain a
homogenous and turbid suspension.
• Rock the slide gently for 30 60 seconds. Observe the result against a dark
background, preferably with the aid of a magnifying glass.
• If the bacteria have clumped together into more or less distinct units, the strain
is considered auto-agglutinable, and the detection of antigens will be
impossible.
• In practice, auto - agglutinating strains of Salmonella are rare; it is more
economical to perform poly O, H and Vi serology first.
Serological confirmation of Salmonella spp.
• Examination for O antigen
• Using one pure colony, recognised as non-autoagglutinable, proceed as above, using
one drop of the anti O serum instead of saline solution.
• If agglutination occurs, the reaction is considered positive for the presence of that
antigen.
•Examination for H antigens

• Inoculate a Semi-Solid Nutrient Agar (SSNA) slope with a pure nonautoagglutinable
colony from the XLDA or BGA plate. Incubate at 37± 1°C for 24± 3 hours.
• Use this culture for examination for H antigens, proceeding as above, but using one
drop of the anti H serum instead of saline solution.
• If agglutination occurs, the reaction is considered positive for the presence of H
antigen.
•Examination for Vi antigen

• Use this culture for examination for Vi antigens, proceeding as above, but using one
drop of the anti Vi serum instead of saline solution.
Interpretation of serological confirmation
A B
Slide Agglutination
Sample A is a positive reaction and
sample B is a negative reaction.
http://www.ssi.dk/~/media/Admin/Diagnostica%20
Downloads/Downloads%20UK/Brochures/BrochureS
almonella%20antisera%20ver%202%20high1411201 Salmonella on swarm agar
1145552.ashx
Thank you for your attention
Sabina.Purkrtova@vscht.cz

2016 - IFM - CLASSIC - METHODS (Mikrobiologi Method)

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2016 - IFM - CLASSIC - METHODS (Mikrobiologi Method)

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Methods for food-

microorganisms as microbial cells

component of microorganisms – e.g. DNA, proteins

products of microorganisms – e.g. enterotoxins of S. aureus

presence or absence of the amount of the analyte

Amount: according to the legislative Amount: according to the legislative

• contain sufficient and concise information on how to perform

• optional steps in the methods or additional details shall be

• ISO, International Dairy Foods Association (IDFA), FDA (Food

Asexual life cycle of toxic black

Yeast: budding (most

Example: Growth of bacteria in the logarithmic

Log10 (number of cells)

Physical factors Chemical factors

Biological factors Physiological state of cells („fitness“)

Salvadó Z et al. Appl. Environ. Microbiol. 2011;77:2292-2302

Solid (can be converted to

Solid (cannot be liquified):

Mixed from single parts

pH: 7.0 ± 0.2 at 25°C.

Typical Composition (g/litre)

pH: 7.3 ± 0.2 at 25 °C.

pH: 7.0 ± 0.2 at 25 °C.

Typical Composition (g/litre)

RBC – red blood cell

Selective-differential culture media

Essential nutrients, vitamins

pH after sterilization( at 25°C) 7.1±0.2

CHROMOGENIC MEDIUM (Colilert): Coliforms use β-galactosidase

FLUOROGENIC MEDIUM (Colilert):

Microbiological and chemical requirements of laboratory grade water

•bottle for preparation

•pH meter http://1.bp.blogspot.com/-

• the medium to be sterilised on the same day when prepared

SUB-ISOLATION OF PRESUMPTIVE COLONIES (non-selective agar)

BACTERIA GROWING ON SELECTIVE MEDIA ARE NOT APPROPRIATE FOR

CULTIVATION 1:100 1:1000 1:10000 1:100000 1:1000000

spreading used for aerobic

SAMPLE Cultivation: time and temperature depends on the

• C… the number of all counted colonies on plates of two dilutions,

Colonies <300 >10

Colonies 255 38 Colonies 255 5

Colonies >300 10-300 Colonies & 55

Only this used for N 

coliform bacteria VRBL agar

Shigella flexneri ATCC 29903

Sample 10 g sample 1 ml (-1. dilution) 1 ml (-(X+1). dilution Sample

Pourring over of 1 ml by VRBD

Cultivation: 37 °C, 24 h±2 h

Evaluation of biochemical tests Result 4. DAY

Oxidase positive (OXI +) Aeromonas sp. Pseudomonas sp. Dark-blue to violet

Escherichia coli ATCC 11775 Selective action

Shigella flexneri ATCC

Citrobacter freundii ATCC 8090

Pourring over of 1 ml of TBX agar*

Cultivation: 44 °C, 18-24 h

*about 15 ml temperated to 44-47 °C

Escherichia coli DSMZ 502

Spreading of 0.1 ml onto Baird-Parker agar (BP)*

Cultivation: 37 °C, 24 h/48 h (colony counting after both time)

Evaluation of biochemical tests Result 4./5. DAY

Staphylococcus aureus ATCC 25923

Sample 10 g sample 1 ml (-1. dilution) 1 ml (-(X+1). dilution Sample