Most Probable Number (MPN) & Biological Oxygen …cfcc.edu/blogs/srowe/files/2018/04/Most-Probable-Number...Most Probable Number (MPN) & Biological Oxygen Demand (BOD) Part 1: Presumptive

Most Probable Number (MPN) & Biological Oxygen Demand (BOD)

Part 1: Presumptive Coliform Test (MPN)

Introduction

This lab exercise will employ a commonly used multi-tube fermentation technique. The results from the test will be

used to estimate bacterial numbers in the sample water. This technique uses the principle of dilution to extinction to

estimate the number of bacteria in a sample. Decimal dilutions of the sample water will be added to replicate

fermentation tubes containing Lauryl Tryptose broth, a selective growth medium for coliforms. The cultures are

incubated and assessed by eye for gas production, bypassing tedious colony counting or expensive and tedious

microscopic counts. The degree of dilution at which an absence of gas production begins to appear indicates that the

solutions have been diluted so much that there are many subsamples in which no bacteria are present. It can

reasonably be assumed that if the technician continued to dilute the sample water, eventually a maximum dilution at

which growth can occur would be found. This maximum dilution would represent a volume containing a single

organism. The results of such an analysis are expressed in terms of the Most Probable Number (MPN). This represents

an estimate based on a statistical probability formula.

The major weakness of the MPN method is the need for large numbers of replicates at the appropriate dilutions to

narrow the confidence intervals. However, it is a very important method for counts when the appropriate order of

magnitude is unknown, which is the case for our water samples. Each team will process one water type doing three

basic dilution factors (undiluted, dilution of 10, dilution of 100), inoculating 5 fermentation tubes from each dilution for

a total of 15 fermentation tubes per team.

Materials

1 100mL graduated cylinder (sterile) 3 125mL Erlenmeyer flasks (sterile) 3 10+mL pipette (sterile) 1 pipette pump 1 sterile eyedropper Sterile Dilution Water 15 fermentation tubes in Styrofoam/cardboard container

Procedure

Making the Dilutions:

1. Acquire 70-90mL of raw sample water in a 125mL Erlenmeyer flask. Pour it directly into the flask from the supply jug; do NOT use your sterilized graduated cylinder. Label this with the assigned water type and mark it as the Undiluted (x1) solution.

2. Use a sterile 10+mL pipette and transfer 11mL of the above Undiluted (x1) solution to a second 125mL Erlenmeyer flask. Label the pipette used as your x1 pipette. Using the sterile graduated cylinder add 99mL of sterile dilution water to the flask. Swirl to mix and homogenize the solution. Mark it as your x10 dilution.

3. Use a different sterile 10+mL pipette and transfer 11mL of the above x10 dilution to a third 125mL Erlenmeyer flask. Label the pipette used as your x10 pipette. Using the sterile graduated cylinder add 99mL of sterile dilution water to the flask. Swirl to mix and homogenize the solution. Mark it as your x100 dilution

4. Label the remaining sterile 10+mL pipette as your x100 pipette.

Inoculating the Tubes:

1. Wash hands thoroughly with soap and water (see “Hand washing” procedures). 2. Obtain 15 fermentation tubes and LABEL the tops of the caps:

o First row of 5 tubes = ×1 o Second row of 5 tubes = ×10 o Third row of 5 tubes = ×100.

3. Use your x1 pipette and transfer 10mL of the Undiluted (x1) solution to each of the 5 tubes labeled ×1. o Remove the caps from the tubes one at a time and replace the cap on each tube immediately after

sample addition. 4. Invert the tubes 5-10 times to thoroughly mix the sample with the broth. 5. After the last inversion, make sure that the tube is upright and the durham tube is full of liquid (NO AIR

BUBBLES). 6. Use your x10 pipette and transfer 10mL of the x10 dilution to each of the 5 tubes labeled ×10. Remove the caps

from the tubes one at a time and replace the cap on each tube immediately after sample addition. 7. Invert the tubes 5-10 times to thoroughly mix the sample with the broth. 8. After the last inversion, make sure that the tube is upright and the durham tube is full of liquid (NO AIR

BUBBLES). 9. Use your x100 pipette and transfer 10mL of the x100 dilution to the last 5 of the broth tubes labeled ×100.

Remove the caps from the tubes one at a time and replace the cap on each tube immediately after sample addition.

10. Invert the tubes 5-10 times to thoroughly mix the sample with the broth. 11. After the last inversion, make sure that the tube is upright and the durham tube is full of liquid (NO AIR

BUBBLES). 12. Place the tubes in the incubator at a temperature of 35 ± 0.5° C. Please open and close the incubator door

quickly to allow the least amount of warm air to escape from the incubator. 13. After one hour, examine the durham tube for trapped air. If there is air in the durham tube, invert the

fermentation tube until the air escapes. This air is a result of heating the dissolved gases in your sample and is not due to the presence of bacteria.

14. Slightly loosen the caps on all 15 tubes. 15. Return the fermentation tubes to the incubator. Please open and close the incubator door quickly to allow the

least amount of warm air to escape from the incubator. 16. Allow 24 ± 4 hours for reaction time.

24 hours later…

1. Gently tap each fermentation tube and examine the durham tube for gas. a. If the broth appears cloudy and the durham tubes contain gas bubbles in at least some of the

fermentation tubes, coliform bacteria are present. If there is gas in any of the fifteen fermentation tubes, you do not need to re-examine them later. Record your results in your lab notebook.

b. If no gas is present in any of the fermentation tubes, return the tubes to the incubator and re-examine them again in 24 ± 2 hours. Record your results in your lab notebook.

2. Formation of gas in any amount constitutes a positive test, while the absence of gas at the end of the 48 hours constitutes a negative test.

Data

The following data table is included only as an example. You MUST record your data in your laboratory notebooks if you would like to receive credit for your work!

Note

The results that you obtain from this test are of “presumptive” quality. This literally means that your results are based

on statistical probability and presumption. In a laboratory environment, presumption is not always an acceptable

endpoint for your data. A Presumptive Coliform Test is only ideal because it allows a technician to quickly identify

sample water that may be contaminated, whereas a Confirmed Coliform Test is a little more time consuming. Imagine

running Confirmed Coliform Tests, which take 3-4 days, on samples that may all be negative for coliforms.

The Presumptive Coliform Test is typically run as the initial assay to identify which water samples most likely contain

coliforms, then a technician would follow-up with a Confirmed Coliform Test on the positive water samples. Most likely

your instructor will not have you carry out a Confirmed Coliform Test as it is quite similar to the Presumptive Coliform

Test.

Time & Date placed in incubator:

Time & Date checked for gas formation (24 ± 2 hrs later):

Second Time & Date checked for gas formation (if necessary):

Dilution Factor Presumptive Positive or Negative (+/-) Result

1 2 3 4 5

× 1

× 10

× 100

Results

Using the MPN index table below, it is possible to estimate the number of coliform organisms present in your sample

water by analyzing the positive and negative test results. The MPN index table was derived by HACH using a statistical

probability formula that you will not be responsible for knowing. This index is based on 95% confidence intervals.

Any series of decimal dilutions must contain both positive and negative tubes to be of any value. If all of the tubes are

positive, a greater degree of dilution was needed (if this is the case, be sure to record that in your Results section). If all

of the tubes are negative, the degree of dilution was too great (if this is the case, be sure to record that in your Results

section).

MPN Index Table

Number of Positive Reaction Tubes MPN Index per

100ml

Number of Positive Reaction Tubes MPN Index per

100ml × 1 × 10 × 100 × 1 × 10 × 100

0 0 0 0 1

0 0 1 2 0

0 1 0 0 0

< 2 2 2 4 2

4 4 4 4 5

2 3 3 4 0

1 0 1 0 0

26 27 33 34 23

1 1 1 1 2

0 1 1 2 0

1 0 1 0 0

4 4 6 6 4

5 5 5 5 5

0 0 1 1 1

1 2 0 1 2

30 40 30 50 60

2 2 2 2 2

0 1 1 2 3

1 0 1 0 0

7 7 9 9

12

5 5 5 5 5

2 2 2 3 3

0 1 2 0 1

50 70 90 80

110

3 3 3 3 3

0 0 1 1 2

0 1 0 1 0

8 11 11 14 14

5 5 5 5 5

3 3 4 4 4

2 3 0 1 2

140 170 130 170 220

3 4 4 4

2 0 0 1

1 0 1 0

17 13 17 17

5 5 5 5 5

4 4 5 5 5

3 4 0 1 2

280 350 240 300 500

4 4 4

1 1 2

1 2 0

21 26 22

5 5 5

5 5 5

3 4 5

900 1600

≥ 1600

* If using dilutions other than 1, 10, and 100, multiply the MPN index by the smallest dilution factor from the series used.

Part 2: 7-Day Biological Oxygen Demand (BOD7)

Introduction

Biological Oxygen Demand (BOD) – also known as Biochemical Oxygen Demand – is a quantitative expression of the

ability of microbes to deplete the oxygen content of a sample of water. This depletion takes place due to microbes

consuming organic matter in the water via aerobic respiration. Aerobic respiration uses oxygen as an electron acceptor,

and the organic material is consumed as a source of energy. In addition to microbe respiration, the organic matter in

the water sample can also be oxidized by trace chemicals in the sample. This can be measured using a Chemical Oxygen

Demand (COD) procedure that is not described in this lab procedure.

The following BOD procedure is a relatively simple procedure that employs detailed glassware cleaning, good water

collection techniques, and the proper use of a YSI multiparameter meter. The method outlined below requires water

samples be stored in the dark for 7 days between DO readings; the number of days necessary for storage will depend on

the water source that is being tested and may range from 5 to 20 days depending on lab protocol and/or known

microbiological factors. Historical convention dictates that BOD readings should be taken within 5 days which has led to

the universal scientific standard of the BOD5. You may be wondering what is significant about a five day wait period.

Why not three days, or ten days? The answer: Originally, the test was applied to wastewater treatment in England,

where five days is the maximum timeframe for river water to reach the sea. For reasons that will be discussed in lecture,

we will be using a 7-day BOD (or BOD7).

Materials

2 Glass BOD Bottles with ground glass stoppers

YSI with BOD probe

Dark incubator (room temperature)

Procedure

1. Obtain 2 BOD bottles with ground glass stoppers.

2. Properly clean and dry the BOD bottles and stoppers.

3. Carry the bottles to your water source: The CFCC floating dock where the R/V Martech ties up. You will be

collecting surface water from the end of this dock.

4. Rinse the each BOD bottle with site water at least two times.

5. Following the “Collecting Water Samples” instructions from your handout, collect your sample water.

6. Immediately carry the sample containers to the lab.

7. Using one of the YSI’s, ascertain the current Dissolved Oxygen (DO) level in your samples. Record these numbers

in your lab notebook. Note: You must test the DO within 30 minutes of collection for accurate results.

8. Return the stopper to your BOD bottles.

9. Properly LABEL your bottles: Sample number, sample source, date, time, and team members’ initials.

10. Place both bottles into a dark incubator at 20 ± 1°C (room temperature).

11. Incubate for 7 days ± 6 hours.

7 days later…

1. Using one of the YSI’s, ascertain the current Dissolved Oxygen (DO) level in your samples. Record these numbers

in your lab notebook.

2. Dump your sample water down the sink drain.

3. Thoroughly clean & rinse the BOD bottles and stoppers.

4. Place bottles on drying rack to air dry. Place stoppers in a drying tray or on a paper towel to air dry.

Data

The following data table is included only as an example. You MUST record your data in your laboratory notebooks if you would like to receive credit for your work!

Initial DO 7-Day DO

Date

Time

YSI Reading

(mg/L)

YSI Reading

(%)

Calculation

To calculate BOD:

1. Subtract the 7-Day DO from the Initial DO.

2. Record this number using mg/L or ppm as the units for BOD.

a. This number is the amount of oxygen that has been used, or demanded, by the microbes during the 7-

day incubation period.

b. Unpolluted natural waters should typically have a BOD of 5.0 mg/L or less.