microbiological testing of beverages.docx

8/20/2019 Microbiological Testing of Beverages.docx

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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. &lthough 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


8 days at 75178


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


8 days at 75178


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


8 days at 75178


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 @/??


99 N9 hours at 7; N4

;@ N9 hours at 44 N4


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


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


/@149 hours at 7; N4


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:



>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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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;;




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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718 days at 75178


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&, "(<


718 days at 45148 or at 75178


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de!ending on the target of the investigation


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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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.

microbiological testing of beverages.docx

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