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Name: Class: Date:

STANDARD QUALITATIVE ANALYSIS OF WATER

(Multiple Tube Technique and Most Probable Number Method)

OBJECTIVES

· To understand the principle of Multiple Tube Technique and Most Probable Number Method.

· To be able to interpret the presumptive test and the confirmatory test to detect the presence of coliform bacteria in water.

INTRODUCTION

A primary source of water pollution is fecal contamination. The presence of bacteria that are common inhabitants of gastrointestinal tract in water is an indication of fecal contamination. Presence of coliforms in water is not in itself a dangerous thing, but their presence is used as an indicator of fecal contamination which could result in contamination with true pathogens such as Salmonella. The purpose of this technique is to estimate the concentration (in bacteria/100mL) of coliforms in a water sample. The technique we will use consists of two parts: 1) a presumptive test that gives an estimated concentration of coliforms using the most probable number method and 2) a confirmatory test that more specifically indicates the presence of Escherichia coli.

PROTOCOL

Day 1

Presumptive Test

These tubes contain lactose, a pH indicator, peptones, and a Durham tube. A true positive result for presumptive coliforms is growth with the production of both acid and gas from lactose fermentation.

1). Label three of your single strength fermentation tubes ‘0.1’ and label the other three ‘1’. Label three one and a half strength fermentation tubes ‘10’. Also label with your name and lab section.

2) Add water from your sample to each of the tubes using a plastic pipette: 0.1 mL water to each of the ‘0.1’ tubes, 1 mL of water to each of the ‘1’ tubes and 10 mL of water to each of the ‘10’ tubes.

3) Place your tubes in the incubator.

Table 1: Most Probable Number calculations for Multiple Tube Method results.

# of tubes in each group giving a true positive reaction	MPN per 100 mL	# of tubes in each group giving a true positive reaction	MPN per 100 mL
10 mL	1 mL	0.1 mL		10 mL	1 mL	0.1 mL
0	0	0	< 3	3	0	0	23
0	0	1	3	3	0	1	39
0	1	0	3	3	0	2	64
1	0	0	4	3	1	0	43
1	0	1	7	3	1	1	75
1	1	0	7	3	1	2	120
1	1	1	11	3	2	0	93
1	2	0	11	3	2	1	150
2	0	0	9	3	2	2	210
2	0	1	14	3	3	0	240
2	1	0	15	3	3	1	460
2	1	1	20	3	3	2	1,100
2	2	0	21	3	3	3	≥2,400
2	2	1	28

Source: Standard Methods for the Examination of Water and Wastewater, 13th Ed. New York: American Public Health Association, 1971.

Day 2

Most Probable Number estimation and Confirmatory Test (2.5pts)

1). Record your results below: Results	Number of tubes in each group with a positive result
	10 mL	1 mL	0.1 mL
Growth
Acid
Gas
Positive for all three (true positive)

2). Based on your number of true positives in each group, look up the Most Probable Number per 100 mL (MPN per 100 mL) using the table on the previous page.

MPN of coliforms/100mL: ______________________

3). If you have a true positive result in any of your tubes, take a sample from the MOST DILUTE tube with a true positive result and streak for isolation on EMB agar (Confirmatory Test). If you do NOT have any true positive results, streak a sample from the pure E. coli culture onto EMB agar instead.

Day 3

Confirmatory Test Results (1pt)

1) Record the results from your EMB agar below. Record results from your own culture, record the results from a known Escherichia coli plate as well:

Source of inoculum	Growth	Color	Interpretation/Conclusion

Q1 Would your original water sample be safe to drink? Why or why not? Discuss both water samples used for this experiment at your table. (1.5)