The Ubiquity and Hardiness of Bacteria

    For the first part of this lab exercise, you will be sampling various parts of your environment to see whether or not you can find a place that is germ-free. The second part will see your subjecting a culture known to contain bacteria to a variety of conditions that might or might not be bactericidal.

Read through the following first to determine how many agar-filled petri plates you will need to make and what other supplies you should ask for. Only near the end of the semester will Dr. V do a lot of the routine activities for you - and then only if you have demonstrated skill at doing it yourself.

1. Explanation of Beginning-Beginner's methods

   

2. Ubiquity. Not including the inside of the autoclave, or oven, or the top of a burner, sample three items in your environment that you think might be rather deplete in bacteria. For this you will need one sterile nutrient agar plate, and some sterile swabs. Divide the plate's bottom into three approximately equal, longitudinal sections using your marker on the OUTSIDE of the bottom. Writing small near the end of each lane, indicate the source. On the cover of the plate write your initials. After swabbing, use three short bits of Scotch tape to keep plate closed.

3. Hardiness ONE. In this exercise you will be testing various hand-washing agents as to their disinfectant abilities. In a sense you will see that this exercise bridges "ubiquity" and "hardiness." Don't forget to make a control! (What would that be?) To save on expensive swabs, you will be concerned about treating one of your fingertips, and then you will lightly touch that fingertip to the surface of the agar. Suppose you can think of a control plus five candidate treatments. How will you divide up one nutrient agar plate to accommodate the six "printings?"

       After you have printed a trial, you should re-dirty your finger and then do the next "cleaning" step. That way you have standardized your method. What cleaning agents are available? Let's see: plain tap water; non-antibacterial soap and water; shampoo and water; antibacterial soap and water; salt water; 50% alcohol in water; 95% alcohol in water; what else? (Not recommended: immersing your finger in boiling water for 5 minutes; or holding it in a flame for 15 seconds.)

4. Hardiness TWO. In this procedure you will subject your standard "dirty water" to various concentrations of some agent. You will then take 10 microliters of that treated water and spread it out as a lane on a nutrient agar plate. Each colony that grows up will be derived from a single living cell in the original treated water. For example, your silty water might be heated for five minutes at intervals of 20 degrees. You would expect that the more sensitive bacteria would be killed early on, while the more "refractory" ones would survive at higher temperatures. Similarly the "alcohol group" would adjust the concentration of their silty water to various ethanol concentrations using 100% ethanol. Other agents might be NaCl and soap.

       In this exercise, you will learn some mathematics of dilutions. For example, if 120 colonies grew up from the 10 μL, how many bacteria were there per mL of the original bacterial suspension. Let's see: there are 1,000 μL in one mL. Thus there are a hundred 10 μL in a mL. So-, there must be 100 x 120 = 12,000 bacteria/mL in the original.

       In Project TWO, you will need to be skilled in counting bacteria! As they say in the Caribbean: "Dunt warry; Be hoppy!" Even 6th graders can do it!

       ((Psst! One of the things Dr. V suggests to his students once they have gained proficiency in bacterial enumeration is that they get a full-flow urine sample and test it. If it contains more than 10,000 bacteria per mL, they ought to see a urologist as they have some level of urinary infection. Normal people usually have a few hundred per mL; and those with "UTI" are well over 10,000/mL.))

       Nomenclature: Mentioned above was that one invisible LIVE bacterium in the original suspension of silty water will give rise to one easily-visible colony on an agar surface. Thus one live bacteria is said to be "one colony-forming unit" or one "cfu." Let's see if you can answer this tricky question: how many cfu does one dead bacterium equal? Under a microscope, can you tell a live bacterium from and dead one? Now do you see the value of "plate counts?"

5. The First Tally of Supplies. How many petri plates do you need to make? If each plate will contain 25 mL, how much nutrient agar must you make? How much nutrient agar powder must you add to that volume of water? (Hint: read the label on the nutrient agar powder bottle!) Why is the use of tap water better than using distilled water for making bacteriological growth media?

6. The Second Tally of Supplies. Add to the above tally that which you will need to make to assess the self-sterilizing abilities of your coatings. So go to that page.

7. Once you have made all your plates and finished the required exercises,
   1. If you are in one of Wednesday's labs, put them in the incubator. You will inspect them the next evening during a lecture break period.

   2. If you are in Thursday afternoon's lab, make sure that your plates are Scotch-taped closed and secure in a ziplok bag. Take them home to incubate. Observe them on Friday and Saturday. Record what you see has grown, not grown, how many colonies there are. Return the plates to the ziplok bag and return them scuzzy and overgrown to lab the next week, where we will sterilize them before trashing them.

          BEST OF ALL, see if you can use your digital camera to photograph your results! Trick: set plates on black felt outside preferably in the sun. Array labels next to plates. Remove covers and take close-up photos WITHOUT flash.