Repeat Testing on Pipe "C" at Site #8 - March 19, 2011

4-Pole Creek at moderate level of 1.5 to 2', turbid water. Only Site #5 (under bridge) was tested along with Pipe "C", because at overall site #8, the site #1 (upstream control) has been nearly identical with the downstream site #5 (under bridge).  The object in this experiment was to continue to track the coliforms from Pipe "C" because the numbers seem to be inconsistent. On some days they are lower than the creek itself, and more recently, they are significantly higher. There is also the inconsistent appearance of suds. When these samples were collected (early in the day) there was abundant suds, similar to one of the previous test days.  We didn't have the camera when we took the samples, and the picture shown here was taken later in the day, and there were no suds at that time.  

                            # colonies                                   Coliforms/dl 
                      
Pipe "C"   1ml        4 (blue)                                         (400)

Pipe "C" 10ml     TNTC - blue dye                                N/A
                           destroyed by TNTC,
                           can't estimate

Site # 5     1ml        2 (blue)                                        (200)

Site # 5   10ml       55                                                  550

We can conclude that the Pipe C has more coliform cfu's than the stream itself on this date, but our numbers on the Pipe C, 1ml and Site # 5, 1ml have statistically inaccurate #'s of colonies (4 and 2 respectively).
The numbers of colonies on the 10ml analyses are much higher for Pipe C, but the blue dye was destroyed by there being too many colonies.  All we can say is that the coliform counts in Pipe C were much higher than in the creek itself.

March 14 Data - Continuation of Testing on Site #8

The March 14 samples represent "normal" conditions, where the creek was 1 to 2' deep, cloudy and occasionally misty, but no rain. The coliforms/dl are similar to the results we obtained during conditions
of moderate rain, whereas the post-flooding conditions of March 11 gave the highest numbers. 
The unusual data in this March 14 testing was the numbers from pipe "C", which exceeded sites #1 and #5, where previously the numbers at pipe C were lower than either #1 or #5. Also, when we sampled at this time, there was a lot of detergent-type foaming from pipe "C" (see picture).      

Continued Study of Site #8- Near Flood Stage - March 11,2011

Pipe C under water - gray effluent showing

Conditions: Depth was ~5', heavy rain, muddy water. Pipe C was under water and the effluent was visible - gray, fairly clear against the muddy creek water. My dad took all of the samples because of the dangerous flowing. Pipes A and B have been ignored from testing because we did not get any coliforms from them.

The coliform counts were higher than on the 9th, but the pattern was the same, since pipe C didn't contribute as many coliforms as the creek itself.                                                                                                           

Continued study of Site #8 using dry incubation and testing different weather situations

 The creek was fairly high on this 3/9/11 date, with 15hrs. of medium downpour before the sampling time.The water was turbid and 3 to 4 feet deep.

Site #1(upstream, above pipe C) was at 2400col./dl. Site #5 (downstream, under bridge) was about the same, 2200col./dl. Pipe C was low in coliforms (700/dl), but on a plate we left at room temp, before we had a chance to photograph it, the environmental bacteria had a chance to grow out, and were very high in their count. The coliforms were blue, and the env. bacteria were not, but it looks like pipe C carries drainage from the surface (along side 8th street, above the creek?), since that is where most of the env. bacteria are.

                                                                                                                                        

Methodology Problems

The first pic shows plates that have no colonies. We have no idea what happened, except that this has happened twice before, and gives us no data, after the time spent on collection, filtration, plating, and incubation. We suspect that the immersion of our plates in plastic bags, in water at 44.5C is not always working right. We acquired a dry incubator, designed specifically for coliforms, and automatically kept at 44.5C.  The last #8 site test was done in the dry incubator, and worked well (see second pic).

            In this pic, Alex shows how the plates are loaded into slots. The closet is then closed, and the temperature is accurately held at 44.5C. This dry incubator works very well.

Biochemical tests used for identification of Escherichia coli from other coliforms

Glucose Fermentation: The tube is red originally from the phenol red acid/base indicator. If the organism ferments glucose, acids are produced and the tube turns yellow. An inverted tube traps gas and shows that gas is produced as well as acids. All coliforms produce acid and gas on both glucose and the lactose  fermentation tube (second tube). The third tube is the citrate test. If the organism uses citrate, it grows. If it doesn't use it, it doesn't grow. E. coli is negative, but Citrobacter, Klebsiella, and Enterobacter are all +.
The 4th tube is the SIM test - 3 tests in one tube - The most important one here is the indole test. If the organism produces indole, it can be detected by adding Kovac's reagent, which turns red in the presence of indole. E. coli is positive, the others are negative. The phenylalanine test is the 5th tube. If the organism breaks down phenylalanine, then a compound is formed which turns green when you add ferric chloride solution. All of the coliforms are negative for this, but one of the non-coliform fecal bacteria - Proteus - is +.  The swab is for the oxidase test. All the coliforms are oxidase neg.
Since E. coli is the only coliform that is found only in feces, and is not naturally environmental, as the other coliforms occasionally are, it is important to check to see if E. coli is found in the testing site (Four Pole Creek) as well as the other coliforms.  This pic is from a colony from the preliminary testing site below the tennis court area. It proves that E. coli is there. Alex inoculated 4 colonies from a plate into sets of biochemical tests the same as in the picture. This pic shows E. coli, and the other 3 were indole +, showing that E. coli was in those colonies, but it appeared to be mixed with other coliforms, since the citrate was positive in all of the others.  All 4 of these tubes were inoculated by Alex.