D E T E C T I O N A N D E N U M E R A T I O N O F T H E M O S T

P R O B A B L E NUMBER (MPN) OF COLIFORM IN WATER

T.L.V. PEIRISGS/MSC/FOOD/3630/08
2008/2010
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4.0 Introduction
The MPN procedure involves a multiple tube fermentation technique where three or more
decimaldilutions of the sample are inoculated into tubes of broth medium and incubated at a
specifictemperature and for a specific time. The method is progressive; i.e., first determining the
presenceof coliforms in the tubes, then determining if these tubes also contain faecal coliforms,
and thenconfirming whether
E. coli
is present. Based on the number of tubes indicating the presence /absence of the three groups of
organisms, the most probable number present can be estimatedfrom a standard statistical MPN
table.This method has been shown to produce satisfactory results with naturally-contaminated
andartifically-contaminated water in sealed containers (including mineral and spring water) and
prepackaged ice.The presence of coliforms, faecal coliforms and aerogenic
E. coli
in water may be determined bymeans of the MPN procedure. Briefly, this method involves
serially diluting out the targetorganisms in the sample, in 5-replicate aliquots, to extinction .The
probable level of the targetorganisms is then statistically estimated from an MPN table.Gas
production is used as an indication of ability to ferment lactose from LST broth
(presumptivecoliform test); gas production from BGLB broth is considered confirmation of
coliform presence;gas production at 45
o
C from EC broth is used as confirmation of faecal coliform presence; andappearance of typical
nucleated, dark-centred colonies with or without metallic sheen when positive EC broths are
streaked onto L-EMB agar are indicative of
E. coli.
The typical colonies onL-EMB agar must be confirmed by further biochemical tests to prove the
presence of
E. coli.
Most probable number (MPN) test
Most probable number (MPN) test tubes of lactose containing Macconkey broth are
inoculatedwith the samples of interest (usually water) measuring 10 ml, 1 ml, and 0.1 ml.
During incubation,coliform organisms produce gas. Depending upon which tubes from which
water samples displayga s , an MPN t abl e i s cons ul t ed and a s t at i s t i cal r ange of
t he number of col i f or m bact er i a i s determined. The MPN test is very easy to
perform and interpret, but it does not determine theexact number of bacteria as the
standard plate count does.
4.1.1 Materials :
Beaker (1L), Pipettes (1ml), Pipettes (10 ml), Tubes containing Macconkey Broth (sterilized)
4.1.2 Procedure :
100 ml of tap water and pond water was measured. Two set of series consisting of 5 tubes
filledwith 10.0 ml of double strength Macconkey broth and another ten tubes (two sets) filled
with 10.0
2
ml of s i ngl e s t r engt h Macconkey br ot h, wi t h i nver t ed Dur ham t ubes was
pr epar ed. ( i t was ensured-that Durham tubes do not contain air bubbles.)5 t ubes wi t h
doubl e s t r engt h Macconke y br ot h was i nocul at ed wi t h 10. 0 ml al i quot s of
t hesample to be tested and another 5 tubes with single strength medium with 1.0 ml
aliquots of thesample and the other 5 tubes with 0.1 ml of the sample using sterile
pipettes. They were thenmixed well and kept at 37
0
C for 24
hours and observed the tubes at the end of 24
hours for acidand gas production. Negative tubes were re incubated for additional 24 hours.
Positive tubes wererecorded. These are considered as positive for presumptive coliforms and
estimate the microbialcontent using the MPN table."Note: For the first five tubes (tube
with double strength Macconkey broth), inoculum can be pr epar ed t hr ough a
s er i al di l ut i on as di r ect i nocul ums may cont ai n i mmeas ur abl e l oad of
microorganisms. This is applicable if the sample is contaminated water but not samples like
milk.
4.1.3 Results
T y p e o f
S a m p l e T a p
w a t e r P o n d
w a t e r Q u a n t i t y o f
w a t e r p u t u p i n e a c h t u b e 1 0
m l 1 m l 0 . 1 m l 1 0 m l 1 m l 0 . 1 m l
N u m b e r
o f t u b e
u s e d
5 5 5 5 5 5 Nu mb e r o f t u b e s
g i v i n g
p o s i t i v e reaction0
0 0
5 2
2
4.1.4 Conclusion
According to the MPN Table tap water has not been contaminated by coliforms and
pond water has been contaminated by micro organism. MPN of coliforms 100 ml of the pond
water 95.
MPN Table - for Coliforms / E.coli /100 ml water.
Quantity of water put up in eachtube1 0 m l 1 m l 0 . 1 m l
M o s t p r o b a b l e n u m b e r o f coliforms organisms in 100 mlof
the original water N u m b e r o f
t u b e s u s e d 5 5 5 Number
of tubes giving positive
0
005
0
002
0
120
0 *
2450
3
reaction 5
5
5555555555555555555552
2
2223333334444445555551
2
34501234501234501234570
95 *
120150175801101501752002501301702252753504252503505509001 6001 800
4.2 Conformation of coliform
Conformation of the results is necessary since positive presumptive tests may be the
result of organisms of non coliform origin that are not recognized as indic ators of
faecal pollution. Theconfirmed test requires that selective and differential media such as eosin
methylene blue (EMB)s t r e a k e d f r o m a p o s i t i v e l a c t o s e b r o t h t u b e
o b t a i n e d f r o m t h e p r e s u mp t i v e me d i a . E o s i n methylene blue contains the
dye methylene blue, which inhibits the growth of gram - positiveorganisms. In the
presence of an acid environment EMB forms a complex that precipitates out ont he col i f or m
col oni es pr oduci ng dar k cent er s and a gr een met al l i c s heen. Thi s r eact i on
i s characteristic for Eschirichia coli, the major indicator of faecal pollution.
4.2.1 Materials:
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EMB agar plates, Inoculating loops, lamps
4.2.2 Procedure :
Labeled EMB agar plates was inoculated by streaking Using lactose broth culture
which gave positive results in 24 hours and Incubated the plates in an inverted position for 24
hours at 37
0
CExamined the colony characters.
4.2.3 Results & Conclusion
T y p e o f
S a m p l e R e s
u l t s C o n c l
u s i o n Tape water no dark cerves and agreen
metallicsheenn o f a e c a l p o l l u t i o n i n t a p e water Pond Preducing dark centres
and a greenmetallic sheenTher e i s a f acal pol l ut i on i n pond water
4.3 Completed test :
The completed test is the final analysis of the sample. It is used to examine the coliform
coloniesthat papered on the EMB plates used in the confirmatory test. An isolated colony is
picked fromthe confirmatory test plate and inoculated into a tube of lactose broth and
streaked on a nutrient agar slant to perform Gram stain. The test tubes that shows acid and gas
in the lactose broth andthe presence of gram negative bacilli on microscopic examination after
inoculation and incubationare further confirmation of the presence of E.coli and they are
indicative of a positive completedtest.
4.3.1Result
When examined, slide which prepared from the EMB Culture plate (belongs to
sample of pondwater) by a microscope it contained pale to dark red coloured rod
shaped bacteria, when sporestain was done it got clared that these are non sporing
rods.Therefore this sample is bacteriologically unsatisfactory andit is contaminated by faecal
bacteria(E-Coli)
4.4 Discussion
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The test methods described in this Practical provide guidelines for detection and enumeration of
bact er i a of f ood and wat er or i gi n. Then we can pr event f ood bor ne
i nf ect i ons and di s eas es occurring more commonly in Sri Lanka.
Disadvantages of MPN Techniques
1. MPN procedure takes very long time for the confirmed test result.2. In MPN the results are
probability calculations and cannot be accurate.3. MPN requires more glass wares and media.4.
False positive results are of common occurrence.
Advantages of MPN Techniques
1. Interpretation of the results requires minimal experience and training as results can be got
bysimply observing for the presence of gas or no gas.2. Water samples with high turbidity can be
analyzed, since there is no apparent deleteriouseffect.3.Because of the dilutions used in the range
of 1:0 or 1:100, toxic substances present in thesample can be diluted out.4. MPN technique is the
effective method for analyzing samples such as muds, sludges, sedimentsetc
.
References :
0 1 . F o o d a n d Ag r i c u l t u r e o r g a n i z a t i o n o f t h e u n i t e d n a t i o n s 1 9 9 2
Ma n n a l o f f o o d q u a l i t y control 14/4 microbiological analysis FAO, Rome I
taly.0 2 . S r i L a n k a s t a n d a r d 6 1 4 P a r t 2 ( 1 9 8 3 )
s p e c i f i c a t i o n f o r p o t a b l e w a t e r ( P a r t 2 ) Bacteriological
Requirements.
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