Serial Dilution Technique for Counting Microbes

I.

Objectives

Correlate absorbance value for a bacterial suspension with an accurate bacterial count.

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Become proficient at dilutions. Become proficient at performing a standard plate count and determining bacterial counts in a sample.

II.

Theoretical Background Scientists use a number of different methods to determine the number of micro-organisms that are present in a given population. This can be accomplished by using the spectrophotometer to measure the optical density of the population, by directly counting the microorganisms using a haemocytometer, or by serial diluting the bacteria and plating the diluted bacteria on media that supports the growth of the micro-organisms. The latter method is somewhat more time consuming, but provides statistically accurate and repeatable results. This method is also the ideal method for enumerating microorganisms in a given population because it only identifies the living organisms in that population. Microbial counting is useful in the basic sciences and is used determine the number of bacteria present for physiological or biochemical studies. For example, if one knows the number of bacteria present in a culture then one can calculate the amount of protein or DNA that can be isolated from that population. Microbial enumeration is also routinely used in the areas of public health. Food or water microbiologists test food, milk or water for the numbers of microbial pathogens to determine if these products are safe for human consumption. We will be using serial dilutions, plating and counting of live bacteria to determine the number of bacteria in a given population. To this end we will make serial dilutions of a solution containing an unknown number of bacteria, plate these bacteria and determine the total number of bacteria in the original solution by counting the number of colony forming units and comparing them to the dilution factor. Each colony forming unit represents a bacterium that was present in the diluted sample. The numbers of colony forming units (CFUs) are divided by the product of the dilution factor and the volume of the plated diluted suspension to determine the number of bacteria per mL that were present in the original solution.

III.

Materials and Method o o o o 6 Test tubes 5 Agar Plate 2 Pipettes 2 Beakers o o o Aspirator Alcohol lamp Glass Slide

Method: *Label each test tube and Petri dish accordingly. 1. Make sample solution with 9ml of tap water and soil.

2. Take 1ml of sample solution and add 9ml of water to new test tube #1. 3. Take 1ml of solution from test tube #1 and add that to 9ml of water into test tube #2. 4. Take 1ml of solution from test tube #2 and add that to 9ml of water into test tube #3. 5. Take 1ml of solution from test tube #3 and add that to 9ml of water into test tube #4. 6. Take 1ml of solution from test tube #4 and add that to 9ml of water into test tube #5. 7. Vortex each dilution tube. 8. Streak out 0.5ml of solution from tubes #1, #2, #3, #4, #5 onto an 5separate agar plates. 9. Spread out solution on the plates using sterilized glass slides. 10. Cover and secure. Incubate. 11. Let the plates grow over night and then count the number of colonies on each plate.

IV.

Data and Results (Estimated count)

Petri Dish

No. of Colonies after 24 hours

Petri Dish 1 (x10) Petri Dish 2 (x102) Petri Dish 3 (x103) Petri Dish 4 (x104) Petri Dish 5 (x105)

120

90

49

41

34

V.

Discussion The results from experiment one did not show a perfect serial dilution, the colony numbers per plate were not exactly 1/10. The numbers of colonies per plate however did decrease with each dilution. By dilution number 5 there were only 34 colonies on the plate compared to the 41 of dilution #4 and the uncountable numbers found on the plates that represented dilutions 1,2,3, and the stock.

VI.

Conclusion Base on this results you can easily count the colony of microbes by doing a serial dilution.

GROUP 4
Memebers: y Neil Dolendo y Daphne Ganancial y Janine Villas y John Lorenzo y Monnel Ordinario y Geofil Pangantihon y Charlotte Abilo y Cesarina Padernal y Ciarra Rodriguez