Part 8. Treating Natural Water Samples with Ozone
(Fall Semester, 2006 and Spring/Summer Semester, 2007)

Carter Lake, Iowa/Nebraska, November, 2006. 
    In the left figure below, we are bubbling the 0.25% ozone through a sample of Carter lake water.  After 15, 30, and 45 minutes, we stopped and removed about 3 mL water and placed it on a new agar plate.  We also placed about 3 mL of untreated water on an agar plate.  After one week in the refrigerator, the agar with the untreated water had grown all of the coliform bacteria shown in the next photograph.  Row 2: Left: another picture of the same non-treated sample, another week later and Row 2, Right: The ozone treated water was free of any colonies.

Missouri River at Omaha, Nebraska.
    We collected samples of Missouri River water at Omaha, Nebraska during the Spring of 2007.  The samples shown below are water samples just minutes after being collected from the  river.  The left sample was collected in February 2007 and the right sample was collected in March 2007.  The February sample was collected on a very cold  day and there was a considerable amount of ice floating in the river.  The water was quite clear with virtually no sediment present.  The turbid March sample was allowed to settle overnight. 

General procedure.  If the agar method is being used, the entire experiment can be done on less than 10 mL sample.  We also intended to use the Hach method which requires two 100 mL samples.  Thus, 110 mL samples of Missouri River water were treated with ozone (800 nmol/min) for 45 minutes as shown in the figure above.  Small samples of about 3.5 mL were removed after 0 min, 15 min, 30 min and 45 min and were added to agar plates.  The plates were covered and sealed and refrigerated for one week yielding the following results, shown below.  Upper left:  river water control (not treated with ozone); upper right: water sample after 15 minutes of ozone treatment did not kill the bacteria present (colonies appeared as pink clumps indicating coliform); lower left (30 minutes) and lower right (45 minutes).  In both the 30 and 45 minute exposures, all bacteria have been killed. 

 Upper left: untreated water (control)

29 March 2007
    Missouri River water was collected and refrigerated overnight, allowing it to settle.  On 30 March 2007, a 110 mL sample of the water was treated with ozone for 45 minutes.  Small samples (3 - 4 ml each) were removed after 15 min, 30 min, and 45 min and added to agar plates.  These were refrigerated for analysis one week later.  The remaining 100 mL was analyzed by the Hach coliform/e coli test (wich gives results in 24 hours)  A control (untreated, but settled Missouri River water from the same sample was analyzed by the Hach kit as well.  Results are shown below.  The plate on the left is of the treated water showing only one red colony of coliform  and zero colonies of E. coli (blue).  The plate on the right is of the untreated water showing 68 colonies of coliform (red) and 40 colonies of E. coli (blue).  The blue colonies were very patchy.

Laleh and Stephanie getting ready to collect a Missouri River water sample in March, 2007

August/September, 2007.  Water samples collected August 23, 2007 from Missouri River at Omaha, Nebraska and Carter Lake in Omaha.  On August 24th, approximately 15 mL of water from each location was treated with the ozone stream from our little generator.  Samples (3 g) were removed after 5 minutes, 15 minutes and 30 minutes and placed on Nutrient agar plates (Fisher Scientific S716931A).  After 40 hours of incubation at room temperature, the results shown above were observed.  In the top series (Missouri River) , all bacteria were killed within 5 minutes; only the untreated sample ("Missouri River t = 0 min") produced bacteria colonies which appear as a somewhat opaque yellow-brown discolored film across the surface.  The other three samples contained no colonies.  In the bottom series (Carter Lake), bacteria grew in the untreated sample and in the t = 5 minutes of ozone samle, but did not grow in the t =15 minutes or t = 30 minutes samples.  The white shadows in the 15 minute and 30 minute samples above are not bacteria and do not appear in the actual samples, only in the photograph.  They are possibly from shadows and reflections caused by the covers being in place. Here are the same plates with the lids off:

We repeated this experiment using time-lapsed photography.  The camera took one picture per hour for 4.5 days.  This time, the bacteria grew in clumped colonies in the untreated water (lower petri dish) and not at all in the sample treated with the ozone stream for 15 minutes (upper dish.) The sequence of photos were assembled into the following eighteen second movie in which the bacteria can be seen to grow at about 20,000 times faster than in the actual experiment (1 s = 6 hr.)  Colonies start appearing after 12 hours or so. (Some moisture condensation on the cover obscures part of the viewing area.)
View a movie of this process: