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Name: Parinnada
Status: Student
Grade:  9-12
Location: N/A
Country: United States
Date: March 2007


Question:
I am testing the bacteria content of reused water bottles. How would I go about measuring the bacteria content within the water bottle?



Replies:
One suggestion is using "growth plates". A "growth plate" is a sterile dish filled with a gel that promotes bacterial growth. The general procedure is to pour a carefully measured volume your water, mix into a gel called agar (kinda like jell-o, but for bacteria), let the mixture set, put it in a warm place overnight, then count the number of bacterial colonies that have grown. You will prepare several plates with different amounts of water (such as 0.1 ml, 1 ml, etc.).

There are lots of references online to give you specific instructions on how to do this. There are several steps, but don't be intimidated. It's no more difficult than making bread! However, be careful to make sure the recipe/instructions you use match your equipment (different plates have different instructions for use).
Here are some example references:

http://www.cfsan.fda.gov/~ebam/bam-3.html

http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/numbers.pdf

http://www.disknet.com/indiana_biolab/b038.htm

I haven't carefully reviewed these sites, so I can't endorse them, but I did a quick scan, and they look generally good. Also, another ask-a-scientist question addresses a similar topic:

http://www.newton.dep.anl.gov/askasci/bio99/bio99891.htm

Hope this info helps!

Burr


I would obtain some nutrient broth and fill the bottle with a measured amount, say-100ml, and close it. Then I would shake the bottle to get the bacteria into the broth. Let the broth incubate overnight. It should become cloudy with bacteria. Then you must do a serial dilution in order to count the bacteria. I would take 1 ml. of the broth and dilute it into 99 ml of sterile water, and shake to mix. Then, take 1 ml of that dilution and put it on a plate of nutrient agar (or another similar agar). This is a 1:100 dilution. Spread the bacteria over the surface of the plate.

Then take 1/10 of a ml. of the 1:100 dilution and put it on another plate and spread the bacteria. This will be a 1:1000 dilution. Next, take another 1 ml of the 1:100 dilution and put it a second 100 ml of sterile water. This will be a 1:10,000 dilution. Plate 1 ml of this on a 3rd nutrient agar plate, and then another 1/10 of the 1:10,000 dilution on a 4th nutrient agar plate.

This will be a 1:100,000 dilution. You can repeat this process for a 1:1,000,000 and a 1:10,000,000 dilution. Incubate these plates for 24-48 hours until you see growth. The plate that has between 30-300 colonies on it should be counted. Then you must multiply back by your dilution to determine the original number of bacteria in the bottle. For example, let's say your 1:10,000 plate has 52 colonies on it. 52x10,000 is 520,000, and you would report your answer as 5.2 x 10-5 bacteria/ml of water. This is because you counted 52 bacterial colonies on the plate you diluted 10,000 times, so there would have been actually 10,000 times more bacteria than that. If you diluted perfectly, then the 1:1000 plate should have 10x more, or 520 colonies, and your 1:1,000,000 plate should have about 5 colonies because it was diluted another 10x.

You can find the procedure for a plate count using serial dilution in most microbiology lab manuals.

vanhoeck


Using a sterile pipet, remove several drops of the bottle's water and place these drops on a nutrient agar plate. Then using a sterile spreader, spread these drops around the surface of the agar. Cover the plate and then incubate the plate in a warm room. For a negative control, spread sterile water on another plate, and for a positive control, dip your finger in a couple of mL of tap water and spread a couple of drops of this sample on a third plate. After a couple of days, you should see bacterial colonies on the third plate but not on the second plate. Depending on whether you see colonies on the first plate you can conclude whether or not there were bacteria in the bottle of water.

Ron Baker, Ph.D.



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