Testing Of One New Drain Site and Retest Of Three Others On Four Pole Creek

Site Descriptions and Conclusions:

Repeat Testing at beginning of Four-Pole (Country) vs End (After Southside Drainage)

 

         Number of Blue Colonies                    Coliforms/dl

 Beginning Sample #1                        19                                                1900                                                                                               

 Beginning Sample #2                        20                                                2000                                                                                                 

 End Sample #1                                  39                                                3900                                                                                                                             

 End Sample 2                                    36                                                3600                                                                                                           

The date we have here is just the opposite of what we saw the first time we sampled from these same sites.  During the first series, we saw slightly higher coliform counts in the beginning, after country systems and before south side huntington. This second time, we saw higher coliform counts after the south side and lower counts after the country systems vs. after south side.  Another difference is that there were significantly higher coliform counts when counting the sum of both before and after in this last experiment. This is probably because the creek was higher, following heavy rain the day before, which seems to lead to a "catching" of leachate from septic tanks in the country and possibly broken sewer lines on the south side.  We will need to repeat these sites once more and try to arrive at some   conclusions from the data.                                                                                                                       

Study of Fecal Levels from Upstream 4-Pole (septic tanks) vs. Southside Huntington

The purpose of this study was to determine fecal coliform levels in 4-Pole after the stream has gone through
rural areas which have septic tank sewage treatment (or no treatment) and then measure the same thing as the stream flows through Ritter Park, which drains areas on the south side of Huntington, in which sewage treatment is in place. We took two replicate samples from 4-Pole across from the foot of HHS hill. This represented the rural systems. We then took two replicate samples from the stream at the end of Ritter Park, after it had flowed through the south side. We need to find out: 1. Do the rural systems leak greater or lesser levels of fecal coliforms than the area served by the city system.  2. Are the fecal coliform levels we have gotten previously (thousands/dl) in the south side city area the result of drain pipes containing sewage leakage into 4-Pole or just the accumulation from the rural systems upstream plus sewage leakage from the south side.

Results:

             Samples                   # Blue Colonies                Fecal Coliforms/deciliter
                                                    
                                           0.1ml    1ml     5ml
            
             B1                            0          7        36                          720

             B2                            1          7        27                          540

             E1                            0          2        14                          280

             E2                            0          1        22                          440

Where B1 and B2 are replicate samples from a site (Below HHS) representing the effect of rural coliform leakage, and E1 and E2, which were taken after the creek has flowed through the south side of Huntington.

Conclusions From Data Obtained from Area 8

   The earlier work was done for practice in getting familiar with techniques, and that site was below the tennis courts (see maps representing 2 different days of anayses). Site 8 was considered as an actual testing site.
   Site 8 consists of ~80 meters of 4-Pole Creek including 3 drain pipes, beginning below the Rose Garden and ending at the 8th street bridge. Since my original objective was to test for and quantify fecal contamination as a health issue, I was interested in the fecal levels in the creek itself and in the effluent flowing into the creek from drain pipes. I was also interested in any impact from drain pipe effluent on coliform levels downstream. I was told that the EPA has an interest in impacts such as this also. From the creek itself, I got coliform levels in the thousands per deciliter. The levels from samples taken upsteam from all the pipes were about the same as those below the pipes (samples taken under the bridge), so we concluded that the pipe C, the only one of the pipes that had coliforms, did not cause downstream levels of coliforms to be any higher than site #1 (upstream), and therefore had a low level of negative impact. I have shown proof via biochemical tests (see photos of these tests in the blog) that both in the creek and in effluent from pipe C, that Escherichia coli was present among the coliforms counted on my plates. This proves that the coliforms I detected are fecal since Escherichia coli is found only in feces, and not in the environment.  As I have mentioned earlier, I used Fmc medium in all of the testing, because it selects out gram negative bacteria only, and only lactose fermenters will be blue on this medium, and those will be the coliforms which I counted.

Drain pipes:

   We labeled the 3 drain pipes A, B, and C (see map of site 8).  We got 0 coliforms from both A or B from 2 different test times. We didn't test them further because they did not present a health problem. We noticed 2 things about pipes A and B, though. One is that they drained briefly when it rained, and stopped draining soon after the rain stopped. We theorized that they drained the surface water from 8th street and not any underground sources, because of the short duration of effluent. Also, when I accidentally left the sampling plates at ambient instead of 44.5C, we noticed very high levels of environmental bacteria, which are found mostly in the top levels of soil, and deeper soils such as deeper drainage systems have few or no bacteria from the soil itself.
   Pipe C was the most important focus of my testing. It ran all the time, ranging from about 1 or 2 liters/min. to something over 10 liters/min. It was hard to determine because I wasn't allowed to sample during flood stage because of the danger of falling in and being carried away. Even during normal levels, I had to crawl down to the outlet of Pie C as you can see in the photo for April 5 on the blog site.
   We found 2 important things about Pipe C. One was that the effluent from it contained coliforms in significant levels. We tested it again and again under different weather conditions, because sometimes we got coliform levels lower than he creek itself, and occasionally we got higher levels. Also, sometimes we saw a lot of detergent foam (white), but not every day, more like every few days. We theorized that either pipe C drained surface water and the detergent was from someone washing his car, or pipe C represents underground storm drainage and then picks up effluent from broken sewer lines.  This latter theory is more likely since it contains significant levels of coliforms, and it drains constantly, instead of just when it rains.
   In terms of the health hazard issue, wherever coliforms are found there is the danger of the spread of fecal-oral diseases, as I mentioned early in this blog. There are several bacterial, parasitic, and viral diseases spread this way, and they are all serious and sometimes fatal, but I have found during this work that fecal contamination is almost everywhere. So, where do you draw the line as to what represents a dangerous situation vs. a tolerable one.  I have learned also during this work that the "lines" that are drawn vary a lot. Drinking water shall contain 0 coliforms to be safe. Public swimming areas (non-chlorinated) are temporarily shut down when possibly 300 cfu's coliforms per deciliter are found, or maybe 100/dl, depending on health dept. recommendations, or state regulations vs. federal regs. such as state parks vs. national parks. No one knows all the 'right" answers on this issue.

Next Site:

   Since I have one third of the semester left to work, I would like to do another project on Four Pole.  I see a couple of possibilities:

1.  A large drain empties into 4-Pole a few feet upstream from the bridge to the tennis court area. It always has a strong flow, and it would be interesting to see if it contributes fecal coliforms, and if so at what level. Also what impact would it have as the stream continues through the park.
2.  Measure influence of totality of drains from country homes a half mile or more upstream to see what fecal levels occur there vs the levels as the stream travels through the park and picks up fecal contamination as we have seen from Pipe C at area 8.  
      

Last Sampling of Location 8, Site #5 (Under 8th Street Bridge During Flood Stage)

These pictures were taken on 4/16/2011, when the creek was at it's highest since this study began. The things of interest are: 
1. The effluent in pipes A and B is very slight in the case of A, and no effluent at all from pipe B, in spite of the fact that the rain had just stopped a couple of hours ago. We concluded that the drainage from those pipes was from surface run-off, especially since the environmental bacteria were so high, indicating that the drainage is through surface soil, which has much higher levels of bacteria. There were no coliforms from the effluent from these pipes, when we tested them a few weeks ago, therefore we haven't paid much attention to pipes A and B. 

2. We also tested the water from the creek itself once more to see if it still contains somewhat high coliform counts. The photo of the Fmc plate with 0.1ml sample is shown above with only 3 colonies, which to get deciliters would be multiplied by 1000 to give 3000 cfu's coliforms/dl. The low number makes it an estimate, but it is similar to the levels we have gotten before for flood like conditions, apparantly from the high creek levels catching leaky sewage pipes and failed septic tanks out in the country.

   We see no reason to test further at Site 8, since we have repeated our testing under different weather conditions and have consistent data on repeats of these different conditions. We have shown that fecal contamination is present in the creek itself, it is higher during high creek levels, and Pipe C apparently picks up fecal contamination, possibly by catching sewage from broken sewer lines, further up the hill from the creek, and empties it into the creek. The impact is not very great though, since coliform levels downstream from the Pipe C are close in number to numbers at oursite #1, which is upstream from all the pipes. .  

Proof of Escherichia coli presence from Pipe "C"

This is me sampling from Pipe "C"

 No detergent at the time I sampled

Early in my blog I tested 4 of the colonies (blue colonies on Fmc) in biochemical tests which determine what species of coliform you have in the samples. These samples were taken from the creek itself (not Pipe "C"). One of the colonies was definitely E.c. and probably the other 3 had E.c. present but they were contaminated with other coliforms, but the test still shows that these coliforms are from fecal material in the creek, since E.c. was among them, and that species isn't found anywhere else in nature except feces. I had to do the same thing with Pipe "C" effluent, so I passed 1.0ml, and 10ml volumes of my sample, collected on Sunday, April 3, through membrane filters, and placed the filters on Fmc plates, which were incubated at 44.5 C. 10ml gave TNTC, 1ml gave too many also, and the blue dye was broken down, but there were some isolated colonies at the edge of the plate, which I inoculated one of them into Lactose Fermentation (first Tube), Sulfide, Indole, and Motility (second tube), and Citrate (third tube). I had to add Kovacs reagent to the SIM tube. The results below show + lactose, + indole, and negative citrate - showing that the colony was Escherichia coli:

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.  

Photos of Maps of Four-Pole Creek

Getting Water Samples from Four-Pole Creek

My dad and I collected water samples from various locations at Four-Pole Creek. We did this to try to determine if bacteria was coming out of certain sewage pipes and if it had the potential of spreading fecal-oral diseases.

Trial Run on Examination of water in Four-Pole Creek for fecal contamination.

Purpose – Study impact of fecal contamination on Four-Pole Creek because of

potential fecal-oral spread diseases.

1. Method selected - MFc - membrane filtration method with incubation at 44.50C to select only mammalian coliforms vs. environmental.

2. Site selected – See map – approx. 50 m below bridge to tennis center.
3. Collected samples in sterile specimen cups with screw-capped lids.
4. Lab – Micro. Cons. - Passed 1ml, 10ml, and 25ml volumes of the sample through a membrane bacterial filter, which was placed on a pad in a small petri dish

soaked with MFc broth. This was incubated in a water bath at exactly 44.50C for 24hrs.

         Results – See photo of plates.  Counting could not be done on a colony counter because the plates were opaque. Instead, pictures of each plate were taken, and the colonies were counted on those.  

Tuesday's Samples

           

 25ml plate:  TNTC (Too High to Count)

            10ml plate: 157

              1ml plate: 16

25ml plate:  TNTC (Too High to Count)

            10ml plate: 144

              1ml plate: 16

            Confirmation that coliforms were fecal in origin:  Test for presence of Escherichia coli,which is found only in feces, and is not found in the environment except in feces.

            Method was to perform glucose fermentation, lactose fermentation, citrate utilization, sulfide production, motility, indole production, phenylalanine deaminase production, and oxidase production.  See photo of those tests, with the results:

Gram Stain Procedure

This was a gram stain procedure I did at Marshall with my dad around the dates February 4-7. He showed me how and what to do. It was interesting. Now I know how to do a gram stain procedure.


Here's what we did and the results:


Performed Gram stain on one G+ organism (Staphylococcus)

and one G- organism (E. coli).  Purpose - identification

1. Prepared smear using aseptic technique.
2. Air dried, heat fixed (gently).
3. Crystal Violet (10 sec.)
4. Iodine (1 min.)
5. Decolorizer (15sec,)
6. Safranin (1 min.)

Results: Staphylococcus appeared as purple spherical clumps, which was expected. The Escherichia coli was a faint pink bacillus, which was also expected.

Streak Isolation Technique

In Mr. McKee's lab I separated bacterial culture into isolated colonies, using a transfer loop and doing a sweeping motion across a plate of trypticase-soy agar, followed by incubation.


Though I don't remember the exact date, we did this I believe on the first week of February.

First Post

I started doing microbiological research about a week ago and will continue doing so until the end of the school semester and possibly into the summer. So far I have learned two procedures for identifying bacteria. I will post details and pictures of them soon. I will also be deciding exactly what research project I will be taking on very soon.

I am excited for being able to do research as well as the end results I might achieve. I've been wanting to do things like this since I was a little kid and I already find it very interesting.

Hopefully this research will be an adventure that myself, those involved, and those reading this blog will find exciting and rewarding!