PURPOSE

In this experiment we have examined water by looking at its microbiological content.Our aim was to determine the number and kinds of microorganisms in water sample by applying different test methods.

THEORY

The presence of bacteria and pathogenic (disease-causing) organisms is a concern when considering the safety of water. Pathogenic organisms can cause intestinal infections, dysentery, hepatitis, typhoid fever, cholera, and other illnesses.So,water should be safe enough to be consumed and used.There are four types of water:

· Spring or well water ( taken from well or spring and bottled with minimum treatment)

· Specifically prepared drinking water (mineral content is adjusted and controlled)

· Purified water (minerals are less than 10 mg/l)

· Flouridated water (fluoride is added to the water)

There are some microorganisms such as pseudomonas,flavobacterium and moraxella in water.Coliforms are rarely found in water.They are divided into 2 groups which are fecal and nonfecal coliforms.Non-Fecal coliforms can contaminate wıth water by airborne sources or product contact surface and they are available in soil,water.Fecal coliforms indicate sewage contamination.

There are some methods used to determine the microorganisms in water.These are:

· Heterotropic plate count

· Coliform count

· Aerobic plate count

· Fecal coliforms

· Mold and yeast count

· E.coli

The Heterotropic Plate Count (HPC) (microbiological) (35^o C, 48 hours) in drinking water should not exceed 500 colonies per ml. The HPC is a microbiological test used to determine the quality of the water in terms of its general bacterial content.  This test is used as a supplement to the routine analysis for coliform bacteria.  Heterotrophic Plate Counts can also be used to monitor disinfection efficiency at water treatment plants and as a measure of water quality deterioration in distribution lines (e.g. biofilm formation) and reservoirs.

Water supplies have to be constantly monitored for a variety of materials—-bacteria, nitrates, pesticides, metals, etc. In this lab, you will analyze water for bacteria, and, in particular, an indicator group of bacteria called the coliforms. They are indicators of other potentially harmful microorganisms in drinking water. They are all gram negative bacteria that ferment lactose, and are non-sporeforming.Coliform count can be divided into 2 methods

· MPN( most probable number method)

· Membrane fitler method

Membrane filter technique:  Filtering 100 ml of water through a millipore filter with holes smaller than the bacteria causes the bacteria to be trapped on top of the filter.  The filter pad is then placed on special coliform media which allows a colfirm count to be done.

Most probable number:  The water sample is diluted and inoculated into a variety of specialized media tubes.  The MPN is determined with the help of a standard chart, based on the number of tests that have turned positive.

The results are given as number of coliforms per 100 ml of water.   If coliforms are present, the lab will generally recommend that a second sample be analyzed. If the number of coliforms was over 30, a second sample is essentially useless.

MATERIALS and METHODS

We have used total plate count and coliform count tests to determine the amount of microorganisms in water sample

Materials

· test tubes

· plate count agar

· laurly sulphate tryprone broth

· alcohol

· iodine solution

· crystal violet

· pipettes

· microscope

· slides

· xylol

· distilled water

· petri

PROCEDURE

Total Plate Count

· Dilutions were prepared from none to 10^-5 by using water sample taken from alleben valley

· 0,2 ml of each dilutions was inoculated to PCA (plate count agar) petries

· İnnoculation was spreaded

· Then it was left for incubation at 37 °C for 24 hours

Coliform count

· Microscope slide was divided into two parts and metallic shine colonies observed in total plate count method were taken one side of slide

· Pink colonies were taken the other side of the slide and distilled water was added to each side of the slide

· Then the slide was dried in air

· Heat fixation was applied

· Crystal violet was added to each side of the slide

· İt was waited for 1 minute

· Washed with distilled water

· İodine solution was added and it was left for 1 minute

· Washed with water then alcohol was added (30 sec wait)

· Washed and safranin was added

· Slide was washed and dried in air

· İmmersion oil was added

· Slide was observed under 100* objective for bacterias

· Bacterias were examined according to their shapes,colors and G (-), G(+)

RESULTS and CALCULATIONS

Total Count

Groups	Non	10-1	10-2	10-3	10-4	10-5
1-3	TNTC	TNTC	270	54	6	0
2-4	TNTC	TNTC	168	290	64	0

Non = TNTC

10^-1= TNTC

10^-2   = (270+168)/2 *10²   =  10950

                      0,2

10^-3
= (290+54)/2 *10³    =
860.000

                   0,2

10^-4   = (64+6)/2 * 10⁴    =
1.750.000

                   0,2

10^-5
 = 0

Colıform Count Presumptive test

Groups	Non	10-1	10-2	10-3	10-4	10-5
1-3	3	3	3	3	3	2
2-4	3	3	3	3	3	2

3:3:2
= 24 (from MPN table)

Number
of microorganisms = 24 *10⁴ / 100 = 2400

Confirmed test

After incubation we observed metallic shine and pink colonies in petri

Completed test

 G(-)
                                           G(+)

_Rod
shaped
_ rod shape

_red
color
_ purple color

DISCUSSION

In this experiment we have examined microbial content of water by using different test methods.In coliform count test, we have observed high number of microorganisms in water sample.That was because that water sample was taken from a valley which contains so many microorganisms and it is not in safe conditions.At the result part for total count test, result of dilution 10^-2 was smaller than the result of dilution 10^-3.This is not possible because we diluted our water sample with distilled water so microbial content of 10^-2 should be greater than the microbial content of 10^-3.This mistake could be because of wrong determination or carelessness.In coliform count test we have used different chemicals such as iodine ,alcohol,crystal violet and saffranine.Some bacterias gives specific color when they are exposed to some chemicals.For example G(+) bacterias are purple when crystal violet is added but G(-) are purple in crystal violet,colorless in alcohol and red color in saffranine.This color change may be because of different absorptivities of membranes of these bacterias.They can absorb some of these chemicals so we can define them easily by looking at their shapes and colors then we can decide their types.