Question

A. Write three ways that you can use the factor that was used to dilute a sample

B. One of your fellow classmates got sick after drinking coca cola (with ice) from a soda fountain at a local fast food restaurant. He claims that toilet water is cleaner than the ice cubes that are dispensed from the machine at the restaurant.

1.What organisms do you think causes contamination of ice dispensers? ...toilet water?

2. How could you test the water and conclude with water is “cleaner”? Show your work by creating an appropriate dilution schemes. So one scheme for the ice water and one scheme for the toilet water.

Answer 1

Contamination:

As ice is made from water, the ice might be contaminated with water-borne pathogens. They include fecal coliforms, such as Escherichia, Enterobacter, Klebsiella, other bacteria, such as Vibrio, Shigella and Salmonella, Leptospira and Legionella. Toilet water is mixed with human gut microbiota, the main organism of which is E. coli. In fact, the presence of E. coli in a water sample is the indication of fecal contamination.

Parameters for water quality:

The quality of water depends primarily on the concentration of bacteria, lead, pesticides, nitrites/nitrates, chlorine, hardness, and the water's pH. Chlorine aids in disinfecting; nitrates leached from fertilizers are harmful to infants; calcium and magnesium (“hardness”) can cause scale buildup in pipes; and water with very high pH levels (acidic water) can corrode fixtures.

Procedure:

Step-1:

• The water sample taken was centrifuged and the supernatant was discard.

• Add 10 µL of the sample as minimal volume to 90 µL phosphate buffer or normal saline (sterile) in a micro centrigufe tube. Adding 10 µL makes the volume in centri-tube 100 µL. Now mix it thoroughly.

• This is needed to concentrate the bacteria in the sample. Label this tube as '10⁰'.

Step-2:

• Take 4 sterile micro-centrifuge tubes. Dispense 90 µL of buffer/saline into them.

• Take 10 µL from the sample in step 1 and mix with the second tube, mix. Label this 10^-1. This is dilution factor (=one-tenth diluted)

Step-3: Repeat the above step with the remaining three tubes and label them as 10^-2, 10^-3, and 10^-4.

Step-4: Spread them on sterile nutrient agar (or LB agar) plates separately.

Step-5: Count the number of colonies after growth.

Calculation:

(Number of colonies x Dilution factor) = number of colonies in 100µL of the concentrated sample in step 1.

Calculate the number of colonies for each dilution. Each colony is considered to be coming from a single bacterium, though this is not true always. Thus, it is expressed as colony forming units (CFUs) instead of number of cells.

Lesser the number of bacteria (CFUs), the cleaner is the sample.