In this experiment, we aimed to test the common statement that Pine- O- Cleen kills 99.9 of E.Coli on bathroom surfaces .To test this myth we used sterile swabs, sterile physiological saline & sterile pipettes. Tile was used to represent bathroom surface and E .coli culture to represent unclean or contaminated surface. E coli culture was spread on the tile with sterile glass spreader evenly and then Pine-O-Cleen was sprayed on it. Therefore in this experiment, the independent variable is the length of time E coli was on the surface of the tile used and the dependent variable is the number of E coli forming units (cfu) or the Percentage of E.coli cells remaining on the Nutrient Agar Plate and Malt extract plate after being cleaned using Pine-O-Clean and controlled variables were length of time E.coli was left on tile, type of bacteria onto the tiles (E.coli), the surface area that bacterial growth is tested on & number of bacteria initially present on tiles

By using sterile pipettes, sterile glass spreader and using a known concentration of e .coli culture (1 ml) and pine- o-cleen (1 ml) on both of the tiles used, other variables were controlled such as incubation temperature & incubation time.

Null Hypothesis: There is no difference in number of E. coli cells surviving on bathroom surfaces with or without treated with Pine -O-Cleen.

The effect of pine o clean in removal of bacteria from a contaminated surface was tested in weeks 12 and 13. Basically we tested the effect of Pine-O-Cleen on E. coli cells on bathroom surfaces. The results of the experiment in week 12 identified that the number of cfu on each plate were too low to be accurate and dilutions did not had decreasing number of colonies and some plates had 0 colonies and even control had colonies in it , so calculations were not done that week. Results in week 13 we obtained approximated cfu were transferred from the bathroom tiles and after it was treated with pine -o- cleen. This was determined by counting cfu on pour plates of the undiluted solution from week 13.

Table 1: Pour plate counts of serial dilutions and total number of colony forming units (cfu) transferred from the bathroom tile contaminated with E.coli culture to a swab and the ones treated with pine -o -cleen.

* The values were accurate for wk 13 since they were in the range of 25-250 cfu/plate for 10-4, 10-5 dilutions and plates were having decreasing number of colonies as dilution factor was increasing.

Interpretation and conclusion:

As mentioned above, total number of bacteria transferred from the tile with E.coli which was contaminated and after it was treated with Pine O’ Cleen. Based on the results, it can be concluded that the pine O cleen has some effect on number of bacteria on the tile. Plates had decreasing number of colonies as dilution factor increased. There was roughly a 10 fold difference between successive dilutions. Although some bacteria survived the cleaning process, pine o cleen still kills germs on bathroom surfaces. Pine -O -Cleen possess antimicrobial substances and that surface bacteria die within minutes but it doesn’t kill 99.9 of E .coli cells.

In agreement with our results, previous studies have shown very little amount of pine o cleen is needed to kill a particular species of bacteria (Fidalgo, 2002). Other variables to consider is that length of time E.coli was left on tile, type of bacteria onto the tiles (E.coli), the surface area that bacterial growth is tested on & number of bacteria initially present on tiles.

Decontamination reagents used for flucloxacillin wipe sampling and degradation/stability tests included: isopropanol 50 and 70, ethanol 60 and 100, benzalkonium chloride 0.1 (Pine O Cleen), sodium hypochlorite 0.5 (Chloroclens), sodium hydroxide 0.03M and distilled water

Previous studies have shown that Pine O cleen is very effective against bacteria and only 0.001 of bacteria was left behind after using Pine-O- Cleen . ( Fidalgo,2002).

Based on our preliminary results, together with the results reported on other recent studies, which all suggest that pine -O-Cleen is effective against bacteria, we reject Null hypothesis since pine- o -cleen does have an effect on number of E.coli cells since in treatment we got 0 colonies.

Critical evaluation of experimental design

In week 12, we set out to evaluate the number of E. coli cells transferred from the surface of the bathroom tile with E .coli culture to a moistened swab. A dilution series was set up and spread plates of each dilution were performed. Unfortunately the number of CFU was too low to be accurate. Therefore the experimental design was modified in the second experiment in wk 13 to compensate for this problem.

Due to limitation of the number of plates and bottles, the experiment was designed such that test in wk 12 were duplicated. We used 10-4, 10-5, 10-6 instead of 10-5, 10-6, 10-7.This introduced the possibility of variables due to potential differences in techniques and in concentration of E coli culture use. Therefore, the experiment could be improved by performing the duplicates at the same time, choosing treatments that altered the minimum of confounding variables, incorporating as many effective controls as possible, keeping number of replicates as high as feasible,

Since total number of replicates available for the experiment were limited by resources, compromised between number of treatment and number of replicates per treatment, accounting for the time taken to apply treatments and then recording results, ensuring same number of replicates is present in each treatment, finding out the limitation on resources.

If more materials were ordered like more bottles, tiles, NA plates it would have been ideal to increase the number of replicates for each test in order to make the results more statistically relevant.

A recent study has demonstrated that the transfer of bacteria to food increased when cells were applied to the surface n the presence of growth media compared with dilution buffer (Moore et al, 2007). This additional variable could also be tested in future studies.