microbial water quality...instructions, and incubate for your group's predetermined time....

Water Quality, 05/2013 1

Laboratory for Environmental Pathogens Research Department of Environmental Sciences

University of Toledo

Microbial water quality Background: Pathogenic microorganisms that occur in polluted water include protozoa, bacteria and enteric viruses. Monitoring the microbial quality of water (drinking water or surface water) is necessary to control the presence of these organisms. The most commonly reported disease caused by microbes in water is gastrointestinal illness, resulting in diarrhea. The World Health Organization says that every year more than 3.4 million people die as a result of water related diseases, making it the leading cause of disease and death around the world. Most of the victims are young children, the vast majority of whom die of illnesses caused by organisms that thrive in water sources contaminated by raw sewage. Although gastrointestinal illness is usually non-life threatening in normal healthy adults, the risk of death increases among vulnerable groups such as infants, the elderly and immunosuppressed individuals. Many pathogens that exist in water can cause human illness (Table 1). Since it is impractical (due to expense) and technically unfeasible to monitor for all pathogens in water, the microbiological quality of water is often evaluated based on the density of indicator microorganisms, including total coliforms and Escherichia coli. Although this system is not perfect, it is an accepted means to determine if water quality is impaired. Coliform bacteria occur naturally in the environment and are not generally harmful themselves. However, their presence suggests (indicates) that other types of disease-causing organisms may exist in the drinking water. Because they can be easily detected and quantified, coliform bacteria have been selected as an indicator for the bacterial quality of drinking water in many nations. The presence of these bacteria in drinking water may signify that a source well is defective, or that there might be problems with the water treatment, or the water distribution system. The maximum acceptable density of coliforms in drinking water has been set at no organisms detectable 100 ml-1.


T ab le 1. Important waterbo rne patho gens that impa c t d rinking and surfa c e wa ters. F ro m N RD C .o rg


E. coli is a type of coliform bacteria commonly found in the intestines of humans and warm-blooded animals. Most strains of E. coli do not cause illness in healthy humans and are actually beneficial to the host, as they aid in digestion and preclude the presence of potentially harmful bacteria. Some strains, however, cause gastrointestinal distress in humans, including E. coli strain O157:H7, which produces a powerful toxin that can cause severe illness. The presence of E. coli in water is a strong indication of recent sewage or animal waste contamination. Furthermore, sewage is known to contain many other types of disease-causing organisms. Therefore, health organizations across the world have selected E. coli as an indicator for the bacteriological quality of drinking water. The maximum acceptable concentration for E. coli for drinking water has been set at no organisms detectable 100 ml-1, while the threshold above which primary contact with recreational water is discouraged is 235 cfu E. coli 100 ml-1.

Dilution plating and membrane filtration are simple techniques used to estimate the number of bacteria in an environmental sample. In Dilution plating, serial dilution involves repeatedly mixing a known mass or volume of source sample or culture with sterile liquid, usually a low-molarity buffer. In such a scheme, 1 ml of sample added to 9 ml of buffer yields a 10-fold dilution (1:10, or 10-1); 1 ml of the 10-fold dilution added to another 9 ml of buffer yields a 100-fold dilution (1:100, or 10-2), etc. When fixed volumes of this dilution series (e.g. 100 µl of each of the dilutions) are spread onto a solid growth medium and incubated, different numbers of colonies will be obtained. By noting the number of colonies, the volume of inoculant added, the dilution and the mass or volume of original sample, one can calculate the number of microorganisms in the original sample. In samples that exhibit very low bacteria densities, concentration of the sample is necessary, which is performed using membrane filtration. Here, up to liter volumes of water are filtered through membranes that contain pore sizes small enough (0.1 – 0.45 µm) to trap bacteria on the membrane. The membranes are then placed directly onto growth media and incubated. Colonies that form on the membrane are then counted and used to calculate the density of bacteria in the original sample.


In this lab exercise you will use two media types to estimate the number of bacteria in two water samples; a tap-water sample collected from a university drinking fountain and a freshwater sample collected from the Ottawa River. You will determine the density of (i) heterotrophic bacteria by culturing the sample on blood agar (Figure 1), and (ii) E. coli and coliforms by culturing the sample on a medium called Rapid E. coli 2 agar (Figure 2). Additionally, you will test the effectiveness of a simple water purification system (Aqua-Pure Tablets) in removing potentially pathogenic organisms from natural waters. Materials (per group): Twelve, 15 ml Falcon tubes containing 9 ml of sterile, 10 mM phosphate buffer

(pH 7) Three, labeled, 100 mm Petri dishes of Nutrient Agar Five, labeled, 47 mm Petri dishes of Rapid E. coli 2 Agar 2000 ml of river water (1 x 1000ml, and 2 x 500 ml in separate containers for

water treatment) Bunsen burner Cell spreaders 200 and 1000 ml pipettes with tips Three test funnels, each containing a 0.45 µm diameter pore size

polycarbonate membrane filter with absorbent pad

F igure 1. H etero tro phic bac teria exhib it d iverse mo rpho log ies on genera l med ia , suc h as b loo d aga r.

F igure 2. E . c o li appear as purp le c o lonies on Rap id E . c o li 2 agar, while c o lifo rms appear b lue.


Vacuum manifold (one manifold to be shared by the groups) Sterile forceps One beaker containing ~50 ml ethanol for flame sterilization One PotableAqua tablet (treats 500 ml of water) One timer Kim Wipes Marker Gloves EtOH to treat the bench top

• Wear gloves throughout the entire protocol. • Do not cross-contaminate your samples OR THE SOLUTIONS. Be aware of your

pipette tip. • Work clean, either on fresh blue bench paper, in the hood, or on an ethanol-

treated bench top.

A. Treating water with PotableAqua tablets

1. Each PotableAqua tablet contains 20 mg of tetraglycine hydroperiodide, which releases 8 ppm of titratable iodine. The tablets require 30 minutes on incubation to purify water. We would like to determine the impact of shortening that time on the microbial quality of the water. Therefore each group will incubate water samples for a different amount of time, ranging from 0 minutes (every group's enumeration of an untreated sample), to 30 minutes. Each group will be assigned an incubation time (5, 10, 15, 20, 30 minutes).

2. Add a single tablet to each of two containers of 500 ml of surface

water. Shake to disperse the tablet according the manufacturers instructions, and incubate for your group's predetermined time. Prepare for Step 3 by organizing your materials as described in Section D, below.

3. Paying careful attention to your time, proceed with enumeration

of E. coli/coliforms and heterotrophic bacteria according to the Section D, below.

B. Enumerating heterotrophic bacteria in river water.


1. Fill each of six - 15 ml falcon tubes with 9 ml of sterile buffer and

label each tube (not the cap) to reflect a ten-fold dilution of the previous tube (i.e. tube 1= 10-1, tube 2 = 10-2, etc.). This might be done for you already. Check with the instructor or TA.

2. Shake the river water container to thoroughly mix the contents.

Using a 1 ml pipette, transfer 1 ml of water sample to the first dilution tube (10-1) (Figure 3). Briefly vortex the 10-1 tube to mix.

3. Transfer 1 ml of the suspension from the 10-1 dilution tube to the

second dilution tube (10-2). Briefly vortex the 10-1 tube to mix.

4. Repeat steps 2 - 3 until the original sample is diluted to 10-6.

C. Plating the diluted samples Samples from three consecutive dilutions should be plated in duplicate to ensure that countable plates (between 30 – 300 colonies) will be generated.

1:10 1 :100 1 :1000 1 :10,000 1 :100,000

F igure 3. S etting up a seria l d ilution series.


Samples will be plated beginning with the lowest dilution (fewest cells). Therefore, the same pipette tip can be used for each of the three inoculated plates. NOTE: Normally, three replications are generated in an experiment like this. We are having each group do one replication, and we will use each of the five groups' single result as a replication to determine a class average. If you need to put the pipette down between pipettings, place the pipette on the bench surface with the tip extended over the edge of the bench top to avoid touching the tip to any surface. If the tip contacts any surface, replace it with a new one.

1. Transfer 100 µl of the 10-6 dilution to the surface of a labeled Nutrient Agar plate.

2. Sterilize a glass spreader by soaking its spreading surface in

ethanol and pass it through a flame one time. The alcohol will kill any bacteria on the glass surface and the flame will burn-off the alcohol. Alternatively, use a sterile, disposable spreader.

3. After the flame has extinguished, remove the lid from the Petri

dish. Touch the spreader to the agar surface to cool the spreader (if it was flamed). Do not touch the hot spreader to the plated cell suspension or you will kill some of the cells. Using a back-and-forth motion, spread the inoculant over the surface of the agar. Replace the lid of the Petri dish.

4. Repeat Steps 1 – 3 for the 10-5 and 10-4 dilutions.

D. Concentrating water samples (e.g., E. coli/coliform analysis for river water and treated river water)

Water samples in which the organisms of interest will be in low density require a concentration step, rather than dilution, prior to plating. The following steps outline a filtration procedure that traps bacteria on a membrane, thereby concentrating them from a large volume of water. The membrane is then incubated atop agar media and bacteria density is estimated in the same manner as above.

1. Sterilize forceps by soaking them in ethanol and passing through

a flame. The alcohol will kill any bacteria on the glass surface and


the flame will burn-off the alcohol. With sterile forceps, remove a filter membrane (0.45 µm) and absorbent pad from plastic wrapping and place it onto the base of a Millipore disposable filtration unit. Be sure to place the pad-side down with the filter facing up. Snap the filter cup onto the base. This might be done for you already.

2. Place the filtration unit onto a vacuum manifold (Figure 4). Close

all of the valves (horizontal position) that control flow to vacuum ports.

3. Place an appropriate volume of sample into the cup. Depending

on the number of bacteria expected in your samples, a few mls to ten of liters could be filtered. See Table 2 for guidance.

SAMPLE ACTION (1 each) River water

E. coli Filter 10 ml Filter 100 ml Filter 500 ml

Heterotrophic bacteria Plate 10-6 dilution Plate 10-5 dilution Plate 10-4 dilution

River water + Potable Aqua tablet

E. coli Filter 500 ml

Heterotrophic bacteria Filter 500 ml

4. Hook up the tubing from the vacuum pump to the manifold via the

back-flow preventer. Turn the pump on. Open all of the valves that control flow to vacuum ports that have filtration units attached. The water will be pulled through the filter membrane and cells present in the water will be trapped on the membrane.

T ab le 2. D ilutio n pa rameters and appro ximate vo lumes o f wa ter samples to be c onc entrated fo r hetero tro phic ba c teria and c o lifo rm/E . c o li enumera tio n.


5. Close the valves after each water sample has completely passed

through each membrane to increase the vacuum suction to the remaining samples. Turn off the pump when each of the water samples has passed through the membranes.

6. Open the valves to release any remaining suction. Remove the

filtration units from the vacuum manifold and remove the filtration cup (unsnap it) from that base without disturbing the membrane.

7. With sterile forceps, carefully separate the membrane from the

pad/base and place (bacteria-side “up”) into a Petri dish containing the proper media (Rapid E. coli 2 Agar for E. coli/coliform samples, and Nutrient Agar for the heterotrophic bacteria samples).

8. Replace the lid of the dish and incubate samples and go to

section “C” above for incubation and counting instructions.

E. Incubation and counting

1. Incubate the Nutrient Agar plates upside-down at 30o C for 24-48 hours.

F igure 4. T he va c uum manifo ld with three filter units a tta c hed . A va c uum pump pulls so lutions thro ugh the filters and c o llec ts the filtra te in a bac kflow preventer made fro m an E rlenmeyer flask.


Incubate the Rapid E. coli 2 Agar plates upside-down at 37o C for 24-48 hours.

2. After the incubation is complete, identify the dilution that resulted

in the growth of between 30 – 300 bacterial colonies (eg. column one, row two in Figure 5). Count the colonies and write the colony number on the bottom of the plate (not on the lid).

On Nutrient Agar, bacteria colonies will appear mostly as small, beige, variously-shaped colonies, while on Rapid E. coli 2 Agar, E. coli and coliforms will appear as round, purple and blue colonies, respectively, each about 2-3 mm in diameter.

3. Determine the average number of colonies on the two plates. 4. Using the following formulae, determine the number of bacteria in

the original sample based on the average number colonies determined in Step 3.

Each group is responsible for providing their data to all other groups so that a class average can be calculated for each of the following enumerations:

F igure 5. Results o f a d ilution p la ting series showing the log reduc tio n in c e ll number that o c c urs as a result o f the 10-fo ld d ilutio n o f the o rig ina l water samp le p rio r to p lating .


Heterotrophic bacteria density in the river sample. Heterotrophic bacteria density in the treated water sample. E. coli density in the river water sample. E. coli density in the treated water sample. Coliform density in the river water sample. Coliform density in the treated water sample. For samples that were diluted:

Number of colonies ml-1 of undiluted water

= !"# ! !"#!"#!

, where

AVE is the average number of colonies on the counted plates; DIL is the dilution factor for the Petri dishes that were counted; VOL1 is the inoculated volume (0.1 ml)

For samples that were concentrated:

Number of colonies ml-1 of unconcentrated water

= !"# ! !"#!

!"#!, where

AVE is the average number of colonies on the counted plates; CONC is the concentration factor for the Petri dishes that were counted (i.e., 10-2, 10-4, etc.); VOL1 is the inoculated volume (0.1 ml)

microbial water quality...instructions, and incubate for your group's predetermined time....

Documents