PRACTICAL 4

Bacteriological Examination of Drinking Water Using the MPN Method OBJECTIVES

1. Determine the presence of coliform bacteria in a water sample using Most Probable Number (MPN) technique and membrane filter techniques. 2. To explain each steps (presumptive, confirmed and completed) in Most Probable Number (MPN) technique. 3. Student will learn about differential and selective

INTRODUCTION
Water is routinely tested to ensure that it is safe for drinking. A widely used indicator of the suitability of drinking water is coliform bacteria. Coliforms are gram-negative, non-endospore-forming rods that are facultative anaerobic and produce acid and gas from lactose within 48 hours at 35C. The key indicator organism in this group is E. coli, which is not normally present in soil and water, but present in large numbers in the intestine and feces. Therefore, the presence of E. coli is indicative of human or animal fecal waste. Water contaminated with fecal material, as determined by the presence of coliforms, is considered nonpotable, meaning unsuitable for drinking. Human fecal waste may also carry intestinal pathogens such as Salmonella typhi, Salmonella typhimurium, Vibrio cholera and Shigella sonnei, however, their presence is difficult to detect since they do not typically occur in a large number and do not survive long in soil and water. As a consequence, coliforms, especially E. coli, are used as the indicator of fecal contamination. The number of total coliform in water sample can be determined by a statistical estimation called the Most Probable Number (MPN) test. The MPN technique is a statistical method that is useful in determining low concentration of organisms (<100/ g or ml). In this method, sample will serially dilute so that the inoculant will sometimes but not always, contain

viable organisms. At each dilution, multiple volumes are transferred to 3, 5 and 10 tubes of liquid test medium. The tubes are incubated and the results are evaluated. Depending on the test performed, positives tubes may be identified by turbidity (growth) alone, or in combination with gas and acid production. After grading tubes positive (+) or negative (-), the initial (MPN/g (or ml) levels are determined with a MPN table (Fig. 4.1) MPN test involves a multiple series of Durham fermentation tubes and is divided into three parts: the presumptive, confirmed, and completed tests

The Most Probably Numbers (MPN) Tables.

Table 1. For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram and 95 percent confidence intervals. Pos. tubes MPN/g Conf. lim. Pos. tubes MPN/g Conf. lim. 0.1 0.0 0.001 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 0 0 1 1 2 3 0 0 0 1 1 2 2 3 0 0 0 1 1 1 0 1 0 1 0 0 0 1 2 0 1 0 1 0 0 1 2 0 1 2 <3.0 3.0 3.0 6.1 6.2 9.4 3.6 7.2 11 7.4 11 11 15 16 9.2 14 20 15 20 27 Low Hig 0.1 0.01 0.00 -0.15 1.2 1.2 3.6 0.17 1.3 3.6 1.3 3.6 3.6 4.5 4.5 1.4 3.6 4.5 3.7 4.5 8.7 9.5 11 18 18 38 18 18 38 20 38 42 42 42 38 42 42 42 42 94 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 3 3 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 0 1 2 0 1 0 1 2 0 1 2 3 0 1 2 3 0 1 2 3 21 28 35 29 36 23 38 64 43 75 120 160 93 150 210 290 240 460 1100 0.15 9.6 Low High 4.5 8.7 8.7 8.7 8.7 4.6 8.7 17 9 17 37 40 18 37 40 90 42 90 42 94 94 94 94 94 110 180 180 200 420 420 420 420 430 1,000 1,000 2,000 --

180 4,100

>1100 420

MATERIAL
1. Water sample (surface water, tab water, sewage water) 2. Media: i.
ii.

Lactose broth 9 x 10 ml broth with a durham tube per group Brilliant green lactose bile broth tube - 3 x 10 ml broth with a durham tube per group Eosin methylane blue (EMBA) plates 3 plates per group Nutrient agar slant 3 tube per group

iii. iv.

3. Chemical and reagent Gram-stain reagent 4. Equipment incubator, light microscope 5. Miscellaneous supplies
i.

Bunsen burner and inoculating loop Immersion oil Microscope slides Pipettes (10 ml, 1 ml, 0.1 ml) and pipette bulb Test tube racks

ii. iii. iv. v.

PROCEDURE
A.Presumptive test

1. Three series of five tubes each, or 15 tubes total, are inoculated

with a water sample. Each tube contains 10 ml of lactose broth/ Lauryl sulphate tryptose (LST) broth and a durham tube. 2. Each tube in the first series of five tubes receives 10 ml of sample; each tube in the second series of five tube receive 1 ml of sample; and each tube in the third series of five tubes receives 0.1 ml of sample. 3. After 24 hours incubation at 35C, tubes are examined for the presence of acid and gas, product of lactose fermentation. A positive tube, will turn yellow and has a gas bubble in the durham tube.

Negative tube, however, will unchanged colour (red) and has no gas bubble in the durham tube.
1. After 48 hours of incubation, negative tubes are examined again for

a delay positive reaction. All tubes after 48 hours are denoted as either (+) or (-), and a most probable number is assigned according to the MPN table. Record results of the presumptive test. 4. However, the presence of the positive tube is only considered as positive presumptive test that is, it presumes that coliforms are present. Thus, their presence must be confirmed in the next part, which is confirmed test.

B.Confirmed test

1. Using inoculating loop, from the tube that has the highest dilution of

water sample and shows gas production, transfer one loopful of culture to the brilliant green lactose bile broth tube. Incubate for 48 3 hours at 35C. The formation of gas at any time within 48 hours constitutes a positive confirmed rest. 2. Record results of the confirmed test.

C. Completed test

1. From the positive brilliant green lactose bile broth tube, streaked

onto eosin methylene blue (EMBA) agar and incubated for 24 hours at 35C.
2. This agar selects for and differentiates coliform bacteria. E. coli is

especially easy to differentiate since it produces a distinctive green, metallic sheen on this agar. The presence of colonies on EMBA agar with this characteristic is considered as a positive confirmed test that is, it confirms the presence of coliforms. However, their presence must be further substantiated by the completed test.
3. After 24 hours, colonies from EMBA with a green, metallic sheen are

transferred to a lactose broth tube and a nutrient agar slant.

4. If acid and gas are produced in the lactose broth tube within 24

hours and a Gram stain detect a Gram-negative rod, this is considered a positive completed test, meaning that the confirmation of coliform in water is complete (IMVIC test to characterise organism can also be carried out). The water is considered to contaminated with coliforms and unsafe to drink.

RESULTS, OBSERVATIONS AND DISCUSSIONS

A. Presumptive test
Sample added 10 ml 1.0 ml 0.1 ml Combination of positives = .. MPN index/100 ml = Presumptive test: positive or negative? .. Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Number of positive tubes

B.

Confirmed test

Number of tube of highest dilution streaked onto EMB plates Number of these plates with green, metallic-sheen colonies . Confirmed test: positives or negative? ..

C.

Completed test

Number of green, metallic-sheen colonies selected from EMB plates Number of these colonies that produced acid and gas from lactose and were Gram-negative rods

D.

Conclusion: water potable or nonpotable?

QUESTIONS
1. What are coliform? Why is their presence in drinking water routinely monitored?

2. What action should be taken if coliforms are detected in drinking water? 3. quantitative? Why is the MPN test qualitative rather than

4. What does a metallic green sheen indicate on an EMB plate? Pink to dark red colonies with a metallic surface sheen on LES Endo agar? 5. polluted water? What bacterial disease can be transmitted by

6. What does a positive presence-absence test indicate? A negative presence-absence test?

CONCLUSION

REFERENCES