Testing the efficacy of UV light in disinfecting bacteria

1. Objective

The objective of this laboratory work is to:

Test the UV lamps against cultured  E.Coli bacteria in water to check if they are effective in disinfecting the water.

2. Theory

2.1 Bacteria in Water

Bacteria are one of the major pathogens in water that can transmit diseases. Among them coliform bacterias are  a grouping of bacteria that includes many strains.These bacterias can live in soil, water, groundwater, as well as in the gastrointestinal tracts of animals. There are many which are harmless but also few which can cause diseases.Studies have shown that one can become immune to bacteria in their own water, but someone else not used to the water may suffer from diarrhea or other form of stomach bugs.^[1] Coliform bacteria are a group of bacteria consisting of various strains of bacteria. Escherichia coli are one of the types of coliform which are commonly found in the intestines of animals and humans, The presence of these bacteria in water therefore indicate presence of sewage and animal waste in water,this means possible presence of other pathogens that can feed off the waste in water and cause other diseases like cholera, dysentery and so on.^[2]

Figure.1 : Microbiological parameters determined by EU.^[3]( "EUR-Lex - 31998L0083 - EN - EUR-Lex - Europa.eu." 2015. 5 Apr. 2016 )

As per European Union ‘COUNCIL DIRECTIVE 98/83/EC of 3 November 1998 on the quality of water intended for human consumption’, the given sample of water should not contain any colony per 100 ml of sample taken.

2.2   Ultraviolet  light disinfection

The wavelength of Ultraviolet light  ( UV light) lie just below the visible light range. There are 3 subtypes of UV light. UV-A and UV-B are the radiations that are cause sunburns and their wavelength range from 100-280 nm while wavelength of UV-C range from 200-280 nm. These radiations are blocked by the Ozone layer in our atmosphere. UV lamps that are generally used to disinfect water also emit UV-C light, have wavelength peaking at 265 nm. 85% of total electricity is converted to Uv light while 15 % is consumed as heat. ^[4]

Ultraviolet disinfection has close relationship with dosage time therefore it is crucial that dosage time be considered along with the intensity of the light.

                                              (1)

 is the dosage of ultraviolet radiation [μW*s/cm2], I  is the fluence [μW*s/cm2], t is the exposure time[s]

The survival rate of microorganisms is dependent on the fact that some microorganisms require more or less irradiance than other species. It is also dependent on the dimensions of the UVIR. The survival fraction of a microbial population exposed to UV germicidal irradiation is an exponential function of dose:^[5]

                                       (2)

Where S  is the fraction of a microbial population that survives UVGI, k is a species dependent deactivation rate constant [cm2/μJ]

3. Apparatus and Equipments

• Bucket
• UV light
• 12 V Pump
• Inlet and outlet hose
• Incubator
• Laminar flow booth
• Autoclave
• Sterilized water
• Graduated measuring cylinder
• RAPID 'E.coli 2 Agar
• Peptone
• Beef extract
• Lab cultured E. coli bacteria
• Petri dish
• Test tubes ( 10 ml)
• Pipettes ( 100-1000 μl)
• Sodium chloride
• Erlenmeyer flask

4. Methodology

The experimental set is divided into four  main steps:

1. Sample preparation
2. Running the water through UV radiation  tube
3. Bacteria Count for before and after samples

4.1 Sample preparation

The first part of the sample preparation involved preparing the bacteria in broth instead of mixing the bacteria from the test tube directly to the water sample.

• Two 500 ml erlenmeyer flasks are taken to prepare broth (See appendix 1.) and inoculated with the E.coli bacteria sample and left in the mixer for 24 hours.
• After 24 hours in the shaker  the 100 ml  broth is then mixed into the 15 l  tap-water is filled in the  ‘Bucket 1’ and stirred vigorously for 5 minutes.
• 15 ml sample from the ‘Bucket 1’ is taken and marked as ‘1B’  
• Another identical bucket, ‘Bucket 2’ is taken with 15 l of tap water and 100 ml of the broth is then mixed to the water and mixed vigorously for 5 minutes.
• 15 ml sample from  ‘Bucket 2’ is taken and marked as ‘2B’

 4.2 UV treatment

The UV treatment system was turned on for 3 minutes with the pump running on full speed with tap water before the actual test was performed.

• The outflow rate when the valve was fully opened was measured to be 14 L /min and when partially opened was 11 L /min
• The pump is then immersed into the ‘Bucket 1’ and then run at full speed and valve fully opened, the outflow is collected in ‘Bucket 1 A’
• 15 ml sample from Bucket 1 A is marked as 1A
• Another outflow rate was measured to be 11 L/min when the valve was partially opened
• The pump is then immersed into the ‘Bucket 2’ and then run at full speed , the outflow is collected in Bucket 2A
• 15 ml of sample from Bucket 2A is marked as 2A

4.3 Bacteria Count

The bacteria is counted using the pour plate technique. 8 dilutions for each samples 1B, 2B, 1A, 2A and replicates for last 4 dilutions for all of the four samples.A standard zero sample dilution was also done.The agar plates were prepared and diluted samples were poured and incubated for 48 hours.( See appendix 2)

5. Results

The bacteria count from the plates are shown in the table below

Table 1: Bacteria count in the plates

Figure 2: Plates showing CFU formed in the Before samples

Calculations

Initially 0.5 ml of sample was diluted to 4.5ml sterilized saline water.

Dilution factor = 5/0.5 = 10

For sample 1B1

For  dilution, Final dilution factor =

 = =

For  dilution, final dilution factor =

 = =  

For sample 2B1

For  dilution, final dilution factor =

 = =

For  dilution, final dilution factor =

 = =

The number and colonies for  and  samples for both 1B1 and 2B1 were either too many or too erratic to count while the replicates were done for samples and no CFU were observed.

There were no CFU observed in any of the After samples for all dilutions.

6. Conclusion    

The results of the experiment quite clearly shows that the UV light is effective in disinfecting the water from the bacteria. Calculations in the before sample show that there was substantial number of  CFU of bacteria in the water but after the corresponding batch of the water was passed through the UV system at 14 L/min and 11 L/min, the water was disinfected. Although the test was done at only two flow rates the disinfection system should perform even better when the flow rate is decreased.

Appendix 1 : Broth Preparation

Appendix 2 : Plates Preparation