PRACTICE PROBLEMS
Answers coming on Monday.

Just as if you were reading your book and learning lots of new things in the paragraphs surrounding the topics we have covered in the lab, so you will learn some new things in the questions below. / Many of you wonder what to read in order to study. I think the problem is that you don't know how to take notes in class. Write down the key words I say, or that are in the handouts. Then look those up and see in which context they are discussed in the book and lab manual. I notice that when I am talking, only a few of you are writing anything. People who already know it all, don't need to write down at least a few words - the key words. If you are a good note-taker, help those who don't seem to writing much. AND those who don't know what to study, get together with someone who does and learn how it is they know. Band together as a team or at least a group of teams. Remember, nurses-to-be, life on the "floor" are "story problems" and not multiple choice! You're going to have to work your way through problems.

Finally Mary Lou and Jimmy were assigned to work together! They were given two strains of bacteria (A & B). Both grew up to red colonies on MacConkey agar that contained lactose (just like you used in lab). ((What kind of bacteria were they using?)) However only Strain A would grow on a plate that contained streptomycin. It was thus contained a mutation in its ribosome gene that conferred streptomycin resistance (strR). They were told to subject "A" to UV light until only 10% of the cells were still alive, and then to make a streak plate (fig 3.3b) on MacConkey agar (with lactose) again. Mostly red colonies grew up, but sprinkled among them were some pale colonies, which presumably had mutations in the lactose-using gene (lac). Fearing these might be contaminants, they selected 20 of the pale colonies using sterile toothpicks, and put them as dots onto MacConkey agar that had glucose rather than lactose. All 20 gave rise to red colonies. ((What kind of bacteria are these likely to be?)) Next their directions said to mix these pale lac- bacteria with Strain B, allow them to sit quietly for an hour, and then streak plate them on MacConkey agar (with lactose AND with streptomycin). While mostly pale colonies grew up, there were now a sprinkling of red colonies that were obviously lac⁺ and strR. Explain in 5 words or less what happened to Strain A as it went from lac⁺ strR (original) to lac^- strR and then back to lac⁺ strR.
A bacterial cell that has flagella sprouting from all over its surface has what style of flagellation? (Hint: it is not polarly flagellated.)
The reason that dysentery and typhoid fever are not treated with penicillin is because....
When Mary Lou and her classmates separately made plate counts from a bottle of culture, Mary Lou got 88 colonies on her plate from tube #2 and 51 from tube #3, while her classmates got 52 colonies from their tubes #2 and 6 colonies on their lanes from their tubes #3. What two things could Mary Lou have done wrong? Or did she do it right? (Note: their "Army" process included 10-fold serial dilutions and transferring 10μL onto the plates.)
How many cfu/ml were in the bottle in the previous question?
Why should it be expected that it is so hard sterilizing the skin on your hands?
What is the function of a streak plate?
You treated some soil bacteria with alcohol and then with boiling, before making a streak plate on nutrient agar. The next day their were many colonies on the agar surface. Why should you not expect these colonies to grow on MacConkey agar?
Eighth-grader Eva Dickinson was invited by Jason to visit Prof. Bengston's lab and work with Mary Lou, Cheryl, Wilbie and Jimmy. They were preparing a strain of Citrobacter (another enteric) into being a vaccine against typhoid fever. Eva was told that Citrobacter freundii (Cf) had the same capsular antigen as did Salmonella typhi. But what is so nice about Citrobacter is that it is not pathogenic - hence future vaccine manufacturers don't need to worry about getting sick as they culture it in huge batches to make vaccine. Each student aseptically transferred some of the Cf into their culture flasks of nutrient broth to incubate overnight. ((What is the word for putting bacteria into something?)) The next morning it was discovered that Eva had not put her flask into the 37°c incubator but had left it on her benchtop at room temperature (about 16°c). Nevertheless, her flask was now cloudy and hence her bacteria had grown just as had everyone else's. So they set about boiling their bacteria to kill them and injecting mice with differing doses of each student's preparation - some mice getting none, others 10 μL, and other more and more. Two weeks later, all mice were "challenged" by placing drops of Salmonella typhimuriam (rodent typhoid with the same surface antigen as human typhoid) into their mouths. Three days later all those mice that had gotten no vaccine died, as did also all those that got 250μL or less of the vaccine - EXCEPT for Eva's mice which all lived even though getting as little as 10μL. Which source gave the stronger vaccine - those incubated at 37°c or at room temperature?
Being that Cheryl and Jimmy were the only college seniors who were taking advanced biochemistry, they moved the Cf vaccine project to their lab course. They grew Cf at room temperature, at 37°c and at 42°c, and then extracted the capsular materials from these three cultures. They then did some fancy analysis and discovered that the 42°c-culture produced no capsule at all (the bacteria were swimming around in their underwear!), the 37°c-culture contained cells wearing normal capsule, while the room temperature culture had cells that were wearing something that might be called very shaggy coats. Speaking more scientifically: hot bacteria couldn't link together the sugars to make capsule, the normal body-temperature bacteria made capsule chains that were medium in length, and the room temperature bacteria made capsule polymers that were very, very much longer chains. Which, then, is most important for making an effective vaccine - more of the stuff, or longer chains? (This expt was actually reported by Eva as an 8th grader at an international meeting, and is responsible for the saving of many additional lives each year around the world.) ((Mnemonic: the colder it is, the shaggier the coat the bacterium produces.))
Eva, Jason and Wilbie, went on with their experiment that Friday afternoon, and Mary Lou wanted to join them, but given her track record, they decided to send her out for donuts. When she returned, she found three very excited students. They had stuck their fingers and had mixed whole blood with tow milliliter of each of the three different Cf cultures that had been grown at the different temperatures. After 10 minutes, they made streak plates from these blood-cultures onto agar plates that contained citrate, tap water and agar. On Monday, they discovered that no bacteria grew up from the 42°c culture, a few grew up from the 37°c culture, and a great many grew up from the room temperature culture. What is this telling you?
When Cheryl and Jimmy heard about the "youngsters'" results in the previous question, they took drops of blood from Mary Lou's vaccinated mice, and added that to three new tubes containing the cultures of CF grown at different temperatures. Again, these were plated on "citrate agar." Two days later they found no colonies on any of the plates. Fearing that the plates were not made correctly - perhaps some toxin in them, they then put spots of stock Cf on them, and those spots did grow. So the plates were alright. So why didn't anything grow up from the mouse-blood culture mixes?
Regarding question #1: A little later, Mary Lou was wondering why the mixed culture had to sit quietly for a while. So she made a new mix of B with the mutant A, and then sadistically put the flask to sloshing on a shaker table. This time when the mix was plated on the MacConkey(lactose) plate containing streptomycin, only pale colonies grew up. What should you think the sloshing did?
Meanwhile Prof. Bengston was working evenings in his lab. He had a strain of Sal. typhimuriam, and had irradiated the culture with UV and discovered that some of the cells no longer gave a stain for the presence of fimbriae. When he put drops of this culture into the mouths of mice, the mice were still happily running around a week later, while, as you saw mice getting the original strain all died in a few days. Explain why these mice getting the mutant lived.
By this time you should be fully impressed (or is it depressed?) with the importance of the various parts of the bacterial envelope. Draw a diagram of the fully dressed Gram-negative bacterium.
Why do you use tap water in making up bacteriological media?
Name five methods of sterilizing various things.
What are the parameters of operation of an autoclave/pressure cooker? Temperature? Pressure? Length of Time?
What is the purpose of making a streak-plate?
Why is it important to rid the surface of agar of bubbles when the plate is to be used for plate-counts?
Physiological saline is 0.9%. How much NaCl (FW=58) should you weigh out to make 100 mL of physiological saline (aka normal saline = NS).
You may use tap water to dilute bacteria, but not to dilute blood. For blood you must use NS. Why?
Name five pathogenic species of bacteria, and five non-pathogenic bacteria. (See: http://www.science-projects.com/safemicrobes.htm)
Why are chloroplasts considered prokaryotic organisms rather than eukaryotes? (fig 5.12, also see 5.11a) WARNING! Be sure you know how to differentiate prokaryotic and eukaryotic organisms if you see them.
You are directed to add a compound that ends in "-ose" to a culture medium. Would that be a protein, sugar, fat, or what?
Know why MacConkey agar works. What's the function of each of the ingredients: lactose, peptone, neutral red, crystal violet, bile salts? (e.g.: the peptone is the major food ingredient. what is more, is that if it is consumed in large amounts because the cells cannot consume the lactose, then a lot of ammonia is given off and, as we all know, ammonia in water is ammonium hydroxide - a base, and thus the pH of the medium actually goes the "wrong" way for a positive test.)
What is a "selective" medium? (see text)
Scrabble word: what does the microbiological word "axenic" mean? (What does the non-microbiological word "syzygy" mean? And that is a great "hangman" word!)
Why is agar used and not gelatin for make gels in the petri plates?
Where is the only natural wheel found?
Name two foods for humans that are naturally antibacterial.