Bacteriology of Milk

Starting materials

Fresh pasteurized milk
Sterile cotton swabs
10 test tubes with about an inch of water in each one
8 nutrient agar petri plates
Directable flame source (e.g.: plumber's propane torch)
aluminum foil
marking pen to write on the petri plates

Methods

1 Before you continue, you must have 8 nutrient agar plates. Click to learn how to make beginner's nutrient agar plates.
And while you are sterilizing the agar for the plates, make up and sterilize a series of 10 test tubes with an inch of water in each one. Cover each tube with a little aluminum foil (don't use rubber stoppers!).
2 Carefully remove a sterile swab from its paper container. Thou shalt not touch the cotton end nor let it touch anything. Dip a sterile cotton end into some frest milk, and then jiggle it in the COOL water of the first tube in your series of ten tubes. (You have two extra tubes in case you drop one.) Discard the swab.
3 Obtain a new swab, and dip it into tube #1 and jiggle it in tube #2, and discard that swab. Continue using new swabs and transferring liquid from one tube to the next until you have done 8 tubes.
4 You have now made a "serial dilution" of the milk: each tube has less milk (and hence dairy bacteria) in it than the previous tube has.
5 Using the marking pen, number the bottoms of the plates from 1 to 8 using smallish numbers off near the edges.
6 Using a new swab, dip it in tube #1 and then rub it ALL OVER the surface of plate #1. The the same with the other tubes and plates always using a fresh swab each time.
7 Allow the plates to incubate at room temperature (or a little warmer) for several days. (Dairy bacteria grow rather slowly.) Daily, inspect the plates. When you see individual colonies on some of the plates, inspect the colonies themselves. Are they all alike (have similar colony morphology)? If so, they are probably all the same species of bacterium. If there are several colony morphologies present, you have at least that many different species of bacteria. What kinds of bacteria are common in cheeses, for example? Click this! Are the species few in number, or do they number in the hundreds?
8 CLEAN UP! Do not allow your plates to sit around and stink up the place! To dispose of the putrid, disgusting stuff, dribble a little household bleach directly onto the surfaces of the agar and tilt the plates to get the bleach to cover the whole surface. Instantaneous death to the microbes! Make sure that you splash the bleach onto your skin and clothes. Imagine the increasing pain as the bleach attacks you, and the racing stripes you will get on your clothes!
If you are using disposable plates, drain the excess bleach into a sink with running water, and trash the plates and their disgusting agar. If you are using non-disposable plates, allow the bleach to sit overnight, drain liquid into a sink, and then using a stick or spatula dig the agar out and flip into a trash can. Then wash the plates thoroughly.
9 Going back to steps #2 and #3, how could you make this protocol more precise? Afterall, how much liquid exactly does a swab hold? Suppose you used pipets instead of swabs. Suppose that your tubes contained 9 ml of water, and you transferred 1 ml of solution from tube to tube. Wouldn't each dilution be exactly 10-fold? Thus a 10-fold serial dilution series.
10 Only after you have cleaned up are you allowed to go further. Sit down with your colleagues (teacher and students) and do some real scientific thinking! Here are a few statements and questions to help you start. You have found lots of bacteria in milk (because you had to dilute it so much via the serial dilutions to get to isolated colonies). You have found that there are very few different species of bacteria in milk. Had you used a slurry of garden soil rather than milk, would you expect a greater variety of species? (Hint: yes!) If the milk you used contained so many bacteria of so few types, where did they come from? Do you think that they came in from contamination from the farm? (Hint: think dirt.) Or do you think that they came from the cow? Were they inside of the cow's utter? If so, what were they doing in there? Why didn't the cow have mastitis? How do you think that the cow acquired those bacteria in the first place? As you discuss those questions and possible answers, you will undoubtedly think of experiments to do. Ah, ha! The scientific method!!!

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1	Before you continue, you must have 8 nutrient agar plates.	Click to learn how to make beginner's nutrient agar plates.
1	And while you are sterilizing the agar for the plates, make up and sterilize a series of 10 test tubes with an inch of water in each one. Cover each tube with a little aluminum foil (don't use rubber stoppers!).
2	Carefully remove a sterile swab from its paper container. Thou shalt not touch the cotton end nor let it touch anything. Dip a sterile cotton end into some frest milk, and then jiggle it in the COOL water of the first tube in your series of ten tubes. (You have two extra tubes in case you drop one.) Discard the swab.
3	Obtain a new swab, and dip it into tube #1 and jiggle it in tube #2, and discard that swab. Continue using new swabs and transferring liquid from one tube to the next until you have done 8 tubes.
4	You have now made a "serial dilution" of the milk: each tube has less milk (and hence dairy bacteria) in it than the previous tube has.
5	Using the marking pen, number the bottoms of the plates from 1 to 8 using smallish numbers off near the edges.
6	Using a new swab, dip it in tube #1 and then rub it ALL OVER the surface of plate #1. The the same with the other tubes and plates always using a fresh swab each time.
7	Allow the plates to incubate at room temperature (or a little warmer) for several days. (Dairy bacteria grow rather slowly.) Daily, inspect the plates. When you see individual colonies on some of the plates, inspect the colonies themselves. Are they all alike (have similar colony morphology)? If so, they are probably all the same species of bacterium. If there are several colony morphologies present, you have at least that many different species of bacteria. What kinds of bacteria are common in cheeses, for example? Click this! Are the species few in number, or do they number in the hundreds?
8	CLEAN UP! Do not allow your plates to sit around and stink up the place! To dispose of the putrid, disgusting stuff, dribble a little household bleach directly onto the surfaces of the agar and tilt the plates to get the bleach to cover the whole surface. Instantaneous death to the microbes! Make sure that you splash the bleach onto your skin and clothes. Imagine the increasing pain as the bleach attacks you, and the racing stripes you will get on your clothes! If you are using disposable plates, drain the excess bleach into a sink with running water, and trash the plates and their disgusting agar. If you are using non-disposable plates, allow the bleach to sit overnight, drain liquid into a sink, and then using a stick or spatula dig the agar out and flip into a trash can. Then wash the plates thoroughly.
9	Going back to steps #2 and #3, how could you make this protocol more precise? Afterall, how much liquid exactly does a swab hold? Suppose you used pipets instead of swabs. Suppose that your tubes contained 9 ml of water, and you transferred 1 ml of solution from tube to tube. Wouldn't each dilution be exactly 10-fold? Thus a 10-fold serial dilution series.
10	Only after you have cleaned up are you allowed to go further. Sit down with your colleagues (teacher and students) and do some real scientific thinking! Here are a few statements and questions to help you start. You have found lots of bacteria in milk (because you had to dilute it so much via the serial dilutions to get to isolated colonies). You have found that there are very few different species of bacteria in milk. Had you used a slurry of garden soil rather than milk, would you expect a greater variety of species? (Hint: yes!) If the milk you used contained so many bacteria of so few types, where did they come from? Do you think that they came in from contamination from the farm? (Hint: think dirt.) Or do you think that they came from the cow? Were they inside of the cow's utter? If so, what were they doing in there? Why didn't the cow have mastitis? How do you think that the cow acquired those bacteria in the first place? As you discuss those questions and possible answers, you will undoubtedly think of experiments to do. Ah, ha! The scientific method!!!