Enumeration and confirmation of indicator species

in water by Membarane filtration and Most

probable number

Bennet Cherukara
BI-209 101
300972119
Submited to:- Prof.Parimala Vidhya
Results:

McFarlands standard are used to standardize the approximate number of bacteria in a liquid

suspension and as per that, the concentration of Eschericia coli culture has been identiied as 1.8

x 109 and t he Enterococcus faecalis culture to be as 9.0x108 .The next was equalizing the cultures

into similar concentration and further diltution was made to recieve 60-80 cfu/filter over agar

plates.Then individually both the 5ml and 10ml filters were cultured on m-Endo agar and m-

enterococcus agar plates (Table 1.b).The positive and negative control resuls are marked in the

(Table 1.a).MPN technique was used to confirm the coliform count, the preseumptive test using

LSTB tubes showed gas production which is an indicated of presence of coliforms(Table 2.a).The

BHI agar was used to confirm the presence of enterococci The Gram staining from the LSTB

culture illustrated the prensence of gram negative rod shaped organisms and the colonies from

BHI under gram stain showed he presence of gram positive cocci in pairs and chains(Table

2.b).The confirmatory test BGLB showed turbidity and gas production for coliform(Table 3.a).

MF verification for enterocci was achieved after obtaining the negative results for catalase test, change

of media color to black on BEA plate, appearance of growth and turbidity on BHI broth + 6.5% NaCl and

BHI broth (Table 3.c). Subsequently, the differences between coliforms and fecal coliforms were

confirmed after inoculating the EMB plate, m-FC Agar plate, EC broth tube and negative control tubes

with both the species (Table 3.b).

1: Membarane filtration- follow up results after plating.

● Colony count observed

Table 1a. Growth obtained on the qualiy control plates of m-Endo and m- Entero agar.

Control type Endo agar Entero agar

Positive Control No growth 0.5-1mm,circuar red
colonies
Negative Control No growth No growth

Table1b. Growth of test organism obtained in m-endo and m-entero agar.

No # Agar media Filter 5ml Filter 10ml

1 m-Entero agar 5 cfu/ml 12 cfu/ml

2 m-Endo agar 9 cfu/ml 23 cfu/ml

● Colony morphology

Endo agar - Circular,reddish colonies

Entero agar - Green metalic sheen

Table 2a: Results of LSTB tubes which are inoculated with colnies from m Endo agar.

LSTB Tube No # Colour Turbidity Gas formation

Tube 1 Light pink Present Positive

Tube 2 Light pink Present Positive

Catalase test results:-

Catalase test of the colonies from BHI agar – Identified negative(No gas production)
Table2.b: Colony morphology and Gram staining of colony from BHI agar plate and LSTB tube

BHI agar LSTB tube

●Colony appearence : ●Media appearence

0.5-1mm beigh coloured circular small colonies Turbid with gas production

●Gram stain ●Gram stain

Gram positive cocci in pairs or in short chains. Gram negative rod shaped bacilli

Table:3a: confirmation of membrane fileterd coliforms in BGLB tubes and compared with negative
controls and expected results.

Tubes Results Negative controls

BGLB 1 Turbidity observed No gas production

Lactose fermentation observed
Coliform present

BGLB 2 Turbidity observed No gas production

Lactose fermentation observed
Coliform present

Table 3b: confirmation and verification of coliforms and feacal coliforms using m-FC agar plate and EC
broth. And compared with negative control.

No: # Plates or Escherichia coli Enterobacter aerogenes Negative control

tubes
1 EC-broth 1
2 EC-broth 2
3 EMB plate 1/2
4 EMB plate1/2
5 m- FC plate1/2
NA Turbidity and growth No growth observed
observed
Turbidity and growth NA No growth observed
observed
Green metalic sheen NA No growth observed
observed
NA 0.5-1.5mm Pink mucoid No growth observed
colonies observed
NA No growth observed
0.5–2mm blue colour
flat colonies
 6 m- FC plate1/2 NA 2-5mm flat White to No growth observed
creamish colonies
observed

BEA streak plates:

Table 3c: conformation of enterococci in BHI broth with salt and without salt and compared with
negative control.

BHI broth +6.5% NaCl salt BHI broth without salt Negative control

Turbidity observed, No turbidity and absence of No

White precipitate and growth growth
present

DISCUSSION:
There where many oraganism which was enumerated and identified in the water sample by

the membrane filtration method and MPN method .The bacterial cell culture were using the

McFarland standards and two different filters including(10 ml & 5 ml filter). These were

performed in m-Endo agar and m-Enterococcus agar. m-Endo agar is used to enumerate the

presence of coliforms and m-enterococcus agar is used for the isolate enterococci. For the

results obtained we got more amount of organisms on the 10 ml filtration on the both m-Endo

agar and for the m-Enterococcus agar (Gullickson, 2017).

While working with the controls, positive controls on m-Endo agar did not receive growth, this

can be due to the following reasons including using hot loop directly over the test organism,

errors in the media preparation, or improper incubation of plate.

The expected colony count was ranging from 60-80 colonies on m-Endo and m-Entero plates ,

but the colony count received was almost three times lesser than the expected, that might be
because of the following reasons such as, the errors occurred during dilution, cross

contamination with any kind of inhibitors (Grabow, 1990). Verification of coliforms resulted

in expected result observations. Demonstration of difference between coliform and fecal

coliform yields expected results. Other than m-Endo agar and m-Entero agar we can also use

LES Endo and PES agar the benefit of LES Endo is rich in nutrients and also it is a best selective

media for coliforms. Similarly, PSE agar is an alternative for for m-Entero agar in which it has

the esculin in it (Rose, 1975).

When we compare the methods MPN and membrane filtration we could understand about

MPN that it requires less materials, able to enumerate low counts, and also one of the best

method which we could use for E.coli enumeration but as cons we cannot confirm the organism

without plating, time consuming and chances of occurring false positives. In case of membrane

filtration we can use even concentrated large samples, it allows removal of bactericidal agents,

less preparation time, less turnaround time and moreover, it provides good isolation and

enumeration but the cons are costly equipments, chances of fouling effect (Thomas, 1956).

LSTB is a selective media for coliforms and the lactose present acts as the main supporting

which provides the gram negative enough nutrients and the gas produced from the

fermentation has been demonstrated by the help of durham tube by looking at the gas bubble

produced. In the BGLB Bile is an inhibitory to gram-positive microorganisms, while brilliant

green dye inhibits selected gram-negative bacilli. Lactose-fermenting organisms resistant to

these inhibitors are detected by the production of gas. Gas production is noted by the

appearance of bubbles in the durham tube (Difco Laboratories, 1998). The ingredients like
tryptose, yeast extract, glucose and lactose in m- enterococcus agar helps in the growth of the

organism not only it provides the nutrients but also it inhibits the growth of Gram negative

micro organisms. m-Endo agar main ingredients are tryptose, thiopeptone, casitone, yeast

extract which enables and promotes the growth of Gram negative organisms by providing the

essential growth nutrients (Rose, 1975).

The additional verification and confirmation of the enterococci was done through catalase test

in which the catalase was identified as negative. Observation and confirmation of enterococci in

BEA streak plate and its morphology confirms the presence of enterococci, the plate appeared to

be black in colour and the colonies observed as black in colour measuring 0.5-1mm pinpointed

grey coloured colonies and also the BHI broth +6.5% salt inhibits the growth of the other

organisms and it only promotes the growth of enterococci. In the BEA plate the enterococci

produced black coloured pinpointed colonies and moreover it also converted the media colour

to black because it was able to hydrolysis the bile esculin present in it (Grabow, 1990).

To sum up, the membrane filtration and MPN techniques are better ways for testing the

account of the organisms in water. From this experiment it is identified that the water tested

contains different species of organisms which reveals to be not potable to drink and it helps us

to understand the difference between coliforms and faecal coliforms. So, this is an effective and

simplest method to enumerate microbes from water.

REFERENCES:

Difco Laboratories (1998). Difco manual, 11th ed. Difco laboratories, Sparks, MD, USA

Gullicksonet. al. (2017) Pharmaceutical Microbiology Laboratory Manual. Centennial College: 8-21
Rose, R. E., E. E. Geldreich, and W. Litsky. (1975). Improved membrane filter method for fecal
coliform analysis. Appl. Microbiol. 29:532-536.

Thomas, H. A., R. L. Woodward, and D. W. Kabler. (1956). Use of molecular filter membranes for
water potability control. J. Am. Water Works Assoc. 48:1391-1402

W.O.K. Grabow (1990). Microbiology of drinking water treatment: Reclaimed waste water.
In: Drinking Water Microbiology — Progress and Recent Developments (Ed. McFeters).
Springer Verlag, New York, pp. 185–203.