microbiological testing of beverages.docx
TRANSCRIPT
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Microbiological Testing of Beverages, Drinking Water
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INTRODUCTION
The consumer’s steadily growing requirements for the quality and the longer shelf life of foods
and beverages must be met by the manufacturer.
uality assurance can’t be limited to ins!ection of the final !roduct alone, such as a bottled
beverage or a !re!ared food !roduct. "nstead, continuous ins!ection of incoming raw materials
and in#!rocess quality control tests must be !erformed throughout !roduction. Microbiological
and ase!tic testing !lay a significant role in such quality assurance.
"n the soft drink industry the microbiological and hygienic quality including the biological
stability of the !roducts are im!ortant criteria for their assessment. The reason$ %ust a few
microbes are often all it takes to s!oil large quantities of a beverage. <hough the e'!losive
technological develo!ment has reduced the risk of contamination by s!oiling microbes, the issue
of shelf life has taken on new dimensions as a result of the enormous !roduction out!ut
!ossibilities of today. uality control of bottling and filling, in terms of chemical and, above all,
biological stability, must be ada!ted to this develo!ment by state#of#the#art test methods.
The requirements for a !ractical microbiological test method are that it !ermits quantitative and
re!roducible detection of trace contamination and that it can be !erformed efficiently and
economically under routine conditions.
These requirements are fulfilled o!timally by the membrane filter method. The !rinci!le of this
method is based on the concentration of microorganisms from relatively large sam!les on thesurface of the membrane filter, and on culturing these microbes on a nutrient !ad or an agar
culture medium.
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THE MEMBRANE FILTER METHOD
Description
The Membrane (ilter Method & membrane filter of the a!!ro!riate !ore si)e is !laced in a filter
holder, and the sam!le is filtered. "n this !rocess microorganisms in the test sam!le are retained
on the filter surface by the screening action of the membrane filter.
*rowth inhibitors can be removed by flushing the membrane with sterile +al solution after
filtration. &fterwards, the membrane filter is !laced on a culture medium and incubated.
(or the Monitor M(#Methode the monitor is ready to use due to a !re#asembled membrane and
!ad inside. The nutrient media is added from the to! and sucked into the !ad by a short vacuum
-/ sec.0 &fter removal of the funnel the lid and the base fit to a !etri dish. +utrients and
metabolites are e'changed through the !ore system of the membrane filter. olonies, which have
develo!ed on the membrane filter surface during incubation, are counted and related to the
sam!le volume.
The Advantages:
1 2roofen accuracy om!ared with the direct method, considerably larger sam!le volumes
can be tested. This concentration effect increases the accuracy of microbial detection.
1 uantitative results
The visible colonies can be related directly to the sam!le volume.
1 Documentation
The membrane filter with colony growth can be filed as a !ermanent record of the test.
No Inhiitors
"nhibitors, such as essential oils or disinfectants, can be flushed from the membrane filter after
filtration.
!M" #$a%it&
3artorius 3tedim Biotech Membrane (ilters are manufactured under *M2 conditions, ensuring
consistently quality and high re!roducibility from batch to batch and within each batch.
The C$%t$re Media
Microorganisms can be detected by different methods. Methods involving culturing techniques
and the microsco!e are used to detect microbes, whereas biochemical and serological
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techniques are commonly a!!lied to differentiate among such organisms. (or detecting
microorganisms in cultures, liquid and solid culture media are em!loyed. Microorganisms are
concentrated by growth in or on these culture media. uantitative detection is only !ossible with
solid culture media because the individually develo!ing colonies can be evaluated and counted
on the surface.
The following culture media can be used for microbiological testing$
1 N$trient "ad 'ets
N$trient "ad 'ets de(inite%& opti)i*e the )e)rane (i%ter )ethod+
The& standardi*e )icroio%ogica% test proced$res, )a-ing the) )$ch )ore e((icient+
The si)p%i(& %aorator& .or-+
The& he%p to save ti)e and )one&+
1 &bsorbent !ads to be wetted with culture media. 1 ulture media with agar or gelatin as the
solidifying agent. The nutrient 2ad 3ets are described on the following !ages and certainly offer
the most convenient way to use the membrane filter method.
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NUTRIENT "AD 'ET'
3artorius 3tedim Biotech +utrient 2ad 3ets have been used successfully in the membrane filter
method for over 45 years. 2ractical and easy to handle, they reduce labor and sim!lify many
microbiological testing !rocedures.
+utrient !ads are sterile, dehydrated culture media. 6nce they are moistened with 7.517.8 ml of
sterile and deminerali)ed -or distilled0 water they are ready to use immediately.
The level of moisture is o!timal when an e'cess ring of water surrounding the !ad is visible.
A%% N$trient "ad 'et t&pes are s$pp%ied .ith the appropriate )e)rane (i%ters, .hich are
a%so presteri%i*ed and individ$a%%& pac-aged or dispenser/read& pac-aged on a and (or the
$se .ith the Microsart0 e+)otion dispenser+
The membrane filters tailored to meet the s!ecial requirements of microbial detection are
available with 9: mm or 85 mm diameters.
+utrient !ad sets -+230 are continuously enhanced as !art of our develo!ment !rogram to ada!t
our !roducts to changing a!!lication requirements. Besides the new +23 ty!es, we have also
u!dated our !ackaging design. The standard +23 bo' contains /55 sterile nutrient !ads, each of
which is individually inserted in a !etri dish and sterili)ed. Ten each of these !etri dishes are
sealed in an aluminum bag. This s!ecial !ackaging in bags !rotects the sensitive formula
constituents of the nutrient !ads during trans!ort and storage from fluctuations in humidity and
tem!erature. &s a result, it guarantees the high quality of our +23 throughout their entire shelf life of u! to 49 months.
Ho. to Use N$trient "ad 'ets
"t’s so easy to use +utrient 2ad 3ets$ +23 and go Before starting with the tests remove
everything that is not essentially needed for this work. arefully clean and disinfect your
working area. (or sim!le microbiological tests a laminar flow bo' is not needed. When used
un!rofessionally, a laminar flow bo' increases the risk of secondary contamination instead of !rotecting from it. & good !rotection against airborne contamination, however, is to work close
to the flame of a Bunsen burner. "nstruments like force!s should be !laced into a glass with
alcohol.
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Description and T&pica% !ro.th Eva%$ation Res$%ts
Caso N"'
Ty!e /95;7
3oybean#asein Digest medium for isolating microorganisms and for determining the total f<count. Dehydrated culture medium for cultivating microorganisms in !harmaceuticals,
cosmetics, raw materials, water -general quality0, waste water, foods and other !roducts.
Re(erences:
&2=& -dairy0, &2=& -food0, &2=& -water0, &6&, D&B, >* ?@A@7, >2, (D&, "D(, "36 ::59,
"36 @/??, "36 ?75@#/ /??5C, "36 ?75@#/ 455/C, 2, "36 ::59
Inc$ation Conditions:
I 8 days at 75178
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Eva%$ation and T&pica% Res$%ts:
2redominantly bacteria grow on this medium. Their colonies are of different si)e and color, most
of them are white or colorless.
Re)ar-s$ 3tressed and chlorine#tolerant bacteria are stimulated by this medium in combination
with lower incubation tem!eratures and longer incubation time.
'tandard TTC 2I )od+3 N"'
Ty!e /9588
Meat e'tract#!e!tone medium for determining the total f< count based on the J&2=& -water0K
and modified by the addition of TT.
Dehydrated culture medium for cultivating microorganisms in raw materials, water -general
quality0, waste water, beverages, beer, foods and other !roducts.Re(erences:
&2=& -water0, "36 ::59, LHB
Inc$ation Conditions:
8 days at 75178
Eva%$ation and T&pica% Res$%ts:
2redominantly bacteria grow on this medium. The ma%ority of their colonies are stained red by
TT reduction.
3ta!hylococcus aureus >scherichia coli Bacillus subtilis
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Tota% Co%on& Co$nt
'tandard N"'
Ty!e /95;9
Meat e'tract#!e!tone medium for determining the total f< count based on the J&2=&
-water0K. Dehydrated culture medium for cultivating microorganisms in raw materials, water
-general quality0, waste water, beverages, beer, foods and other !roducts.
Re(erences:
&2=& -water0, "36 ::59, LHB
Inc$ation Conditions:
8 days at 75178
Eva%$ation and T&pica% Res$%ts:
2redominantly bacteria grow on this medium. The mor!hology and color of their colonies vary.
Ty!e /95:;
Try!tone *lucose >'tract medium for isolating microorganisms and for determining the total
f< count. Dehydrated culture medium for cultivating microorganisms in raw materials, water
-general quality0, waste water, beverages, soft drinks, concentrates, foods and other !roducts.
Re(erences:
&2=& -dairy0, &2=& -food0, &2=& -water0,&2", "36 ::59
Inc$ation Conditions:
8 days at 75178
Eva%$ation and T&pica% Res$%ts:
6n this medium !redominantly colonies of bacteria grow that can have different si)e and colors.
4east E5tract N"'
Ty!e /95?5
(or the detection of the total count of aerobic heterotro!hic bacteria. Dehydrated culture medium
for cultivating microorganisms in water -general quality0 and other !roducts.
Re(erences:
>* ?@A@7, =M36, "36 ;444, "36 ::59,
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"36 @/??
Inc$ation Conditions:
99 N9 hours at 7; N4
;@ N9 hours at 44 N4
Eva%$ation and T&pica% Res$%ts:
2redominantly bacteria grow on this medium. The ma%ority of all colonies are colorless.
1+ E+ Co%i and Co%i(or)s, Enteroacteria CHROMOCULTOP N"'
Ty!e /95@:
(or the detection of total coliforms and >scherichia coli. Dehydrated culture medium for
cultivating microorganisms in raw materials, water -general quality0, waste water, beverages,
foods and other !roducts.
Re(erences:
"36 ::59, Qournal (ood 2rotection,
Ren=yg -%ournal of hygiene0
Inc$ation Conditions:
4514@ hours at 7; N4
Eva%$ation and T&pica% Res$%ts:>. coli develo!s dark#blue to violet colonies, other coliforms red to !ink colonies. 6ther gram#
negative colonies are colorless, a few with S#*lucuronidase activity are light blue to turquoise.
Femarks$ To confirm >. coli give one dro! of ovacs indole reagent on each dark blue colony.
herry red color after a few seconds is a !ositive reaction.
ECD N"'
Ty!e /95@4
3elective culture medium for detecting and identifying >scherichia coli. Bile salt inhibits the
accom!anying flora of microbes not living in the intestine. Dehydrated culture medium for
cultivating microorganisms in raw materials, water -general quality0, waste water, beverages,
foods and other !roducts.
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Re(erences:
&2=& -water0, D"+ /5//5, >* ?@A@7,
"36 ::59, "36 @/??, "36 ?75@#/ 455/C,
HMB*, scherichia coli based on the "36
?75@#/.
Endo N"'
Ty!e /9587
3elective medium for detecting and enumerating >. coli and coliform bacteria. Dehydrated
culture medium for cultivating microorganisms in raw materials, water -general quality0, natural
water, waste water, beverages, soft drinks, concentrates, fruit %uice, sugar, sugar !roducts, foods
and other !roducts.
Re(erences:
&2=& -dairy0, &2=& -food0, &2=& -water0,
D*=M, "36 ::59, "36 ?75@#/ /??5C, M+6,
. coli form red colonies with a metallic sheen and a red dot at the underside of the membrane.
6ther coliforms grow as dark to light red colonies without metallic sheen. olorless colonies of
lactosenegative bacteria are not counted.
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MacCon-e& N"'
Ty!e /95?:
(or the isolation and differentiation of coliform bacteria and other enterobacteriaceae.
Dehydrated culture medium for cultivating microorganisms in !harmaceuticals, cosmetics, raw
materials, water -general quality0, natural water, waste water, beverages, soft drinks,
concentrates, fruit %uice, foods and other !roducts.
Re(erences:&2=& -dairy0, &2=& -food0, &2=& -water0,
&6&, D&B, D"+ 7@9//, D*=M, >2, "36
::59, HMB*, M+6,
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Ty!e /95;@
(or the detection of >. coli and faecal coliform bacteria according to *eldreich et al. Dehydrated
culture medium for cultivating microorganisms in raw materials, water -general quality0, waste
water, beverages, foods and other !roducts.
Re(erences:
&2=& -food0, &2=& -water0, &6&, >2&,
(D&, "36 ::59, "36 ?75@#/ /??5C, . coli and coliform bacteria form blue colonies with a blue surrounding. This color is dark blue
at faecal coliforms with strong lactose fermentation and lighter blue for non#faecal coliformswith weaker lactose fermentation. Hactose#negative bacteria grow with different colors and are
not evaluated.
Re)ar-s$ =igher incubation tem!eratures largely su!!ress the nonfaecal coliforms.
Teepo% N"'
Ty!e /95;:
Hauryl 3ul!hate medium for the detection of >. coli and faecal coliform bacteria according to
Burman, +.2. -/?;:0. Dehydrated culture medium for cultivating microorganisms in water
-general quality0, waste water, beverages, foods and other !roducts.
Re(erences:
&(+6F, &2=& -water0, B3, (D&, "36 ::59,
"36 ?75@#/ /??5C, . coli and coliform bacteria form /14 mm diameter yellow colonies surrounded by a yellow
)one. +on#lactose fermenting bacteria develo! red or colorless colonies without yellow )one.
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Tergito% TTC N"'
Ty!e /958;
3elective and differential medium for the detection and enumeration of coliform bacteria and >.
coli according to 2ollard modified acc. to ha!man. Dehydrated culture medium for cultivatingmicroorganisms in raw materials, water -general quality0, waste water, beverages, foods and
other !roducts.
Re(erences:
&2=& -food0, >* ?@A@7, "36 ::59, "36 @/??,
"36 ?75@#/ /??5C, "36 ?75@#/ 455/C
Inc$ation Conditions:
/@149 hours at 7; N4
Eva%$ation and T&pica% Res$%ts:
Hactose#!ositive microorganisms form yellow#orange colonies with a yellow surrounding and
have a yellow dot under the membrane filter. &ccording to "36 ?75@#/ all colonies that show
yellow color under the membrane filter are counted as !ositive. Femarks$ Tergitol : inhibits
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*ram !ositive colonies and minimi)es the swarming of 2roteus. (urther differentiations of >.coli
and coliforms with 6'idase# and "ndol#Tests are required.
A*ide N"'
Ty!e /958/
(or the detection and enumeration of intestinal enterococci according to 3lanet) and Bartley.
Dehydrated culture medium for cultivating microorganisms in raw materials, water -general
quality0, natural water, waste water, beverages, foods and other !roducts.
Re(erences:
&2=& -food0, &2=& -water0, >* ?@A@7,
=M36, "36 ::59, "36 :@??#4, "36 @/??,
HMB*, M+6Inc$ation Conditions:
9519@ hours at 7; N4
Eva%$ation and T&pica% Res$%ts:
>nterococci form red, !ink or reddish brown colonies with a diameter of 5.814 mm. Femarks$
>nterococci are considered to be indicator organisms of faecal contamination. They are less
sensitive to chemical effects than are >. coli organisms and are therefore longer detectable, for
instance in waste water and in chlorinated water.
Bis)$th '$%(ite N"'
Ty!e /958:
3elective culture medium according to Wilson and Blair for isolating 3almonella ty!hi and other
salmonellae. Dehydrated culture medium for cultivating microorganisms in !harmaceuticals,
cosmetics, raw materials, water -general quality0, waste water, foods and other !roducts.
Re(erences:
&(+6F, &2=& -dairy0, &2=& -food0, &6&,
D*=M, (D&, =M36, "36 ;8:? /?@/C,
"36 ::59,
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Eva%$ation and T&pica% Res$%ts:
Most salmonellae form light colored colonies with brown to black centers surrounded by a black
)one with a metallic sheen -Jfish eyeK0. 3ome 3almonella s!ecies develo! uniformly dark brown
to black colonies which may lack the ty!ical )one. Femarks$ "f a very slight contamination with
salmonellae is sus!ected, !re!are a selective enrichment culture and subsequently streak the
sam!le with an inoculation loo! on a membrane filter that has been !laced on the !re#wetted
+23.
Cetri)ide N"'
Ty!e /95:8
(or the detection and enumeration of 2seudomonas aeruginosa according to Howbury.
Dehydrated culture medium for cultivating microorganisms in cosmetics, raw materials, water
-general quality0, waste water, foods and other !roducts.
Re(erences:
&2=& -water0, &6&, &3M, D"+ 7@9//,
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>* ?@A@7, (D&, "36 ::59, "36 @/??,
>+ /4:@5, >+ "36 /;4;;
Inc$ation Conditions:
9519@ hours at 7; N4
Eva%$ation and T&pica% Res$%ts:
2seudomonas aeruginosa forms blue, blue#green or yellow#green colonies with /14 mm diameter
and blue )ones. The colonies !roduce !yocyanin and fluorescein and show fluorescence in
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Inc$ation Conditions:
718 days at 75178
Eva%$ation and T&pica% Res$%ts:
6nly Jwild yeastsK -not belonging to the genus 3accharomyces0 which utili)e lysine as sole
source of nitrogen grow on this medium, they form white or cream colored colonies brewery
culture yeasts grow not at all or very !oorly.
Ma%t E5tract N"'
Ty!e /95@;
(or the isolation and enumeration of yeasts and molds. Dehydrated culture medium for
cultivating microorganisms in beverages, wine, soft drinks, concentrates, fruit %uice, foods andother !roducts.
Re(erences:
&2=& -food0, &6&, "(<
Inc$ation Conditions:
718 days at 45148 or at 75178
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de!ending on the target of the investigation
Eva%$ation and T&pica% Res$%ts:
Eeasts normally develo! smooth white, rarely colored colonies. Molds generally form velvety or
fluffy, cotton#like colonies that are white during the early growth !hase and later, after
conidios!ore formation, of various colors.
Re)ar-s: The low != of this medium su!!resses the growth of most bacteria. This medium is
available with two different ty!es of membrane filters.
'ao$ra$d N"'
Ty!e /95;?
(or the cultivation and enumeration of yeasts and molds. Dehydrated culture medium for
cultivating microorganisms in !harmaceuticals, cosmetics, raw materials, water -general quality0,waste water and other !roducts.
Re(erences:
&2=& -food0, &6&, >2, 2 V
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Eva%$ation and T&pica% Res$%ts:
Molds develo! velvety or fluffy whitish or greenish colonies which can get various colors after
conidios!ore !roduction. Eeasts have a smooth surface. &cid forming sugar fermenters are
whitish to yellow, non#acid formers are, by contrast, greenish to bluegreen.
Re)ar-s: The low != su!!resses the growth of most bacteria. This medium is available with
various ty!es of membrane filters$ 7 different !ore si)es and 4 different colors.