This lab consists of a series of handouts concerning an outbreak of diarrhea following a church supper in upstate New York.

• You will be forming a case definition and analyzing interviews with everyone who could be found after the outbreak.
• Your task is to find out what caused the outbreak and how it occurred in order to avoid future outbreaks.
• Bring your calculator if you have one; it will come in handy.
• Before lab be sure to read the Steps in an Epidemiological Investigation which is on Vancko Hall.
• Also be sure to listen to the online lecture in Vancko Hall — Lecture portion of the course.
  
• Also it will be helpful if you bring a calculator.

At the end of this lab you will have a take-home exercise which will be due Thursday.

In this lab exercise you will be examining the kinds of bacteria that occur in the mouth, GI tract, and Urinary System.

Oral Bacteria:

• For the mouth you will be doing a semi-quantitative examination of your oral bacteria in order to estimate the likelihood that you will get dental caries.
  • Dental caries are caused by the fermentation of sugars that you eat by the bacteria in your mouth.  These bacteria ferment the sugars forming organic acids which eat away at the enamel covering on your teeth!
• We’ll be using Snyder Test Agar which is an agar deep that will be melted and held in the water bath for you.  You will inoculate it with some of your saliva and check the results (for fermentation) at 24 and 48 hours.

Intestinal Bacteria

• We use dog feces for this lab.
• The bacteria in the intestine are anaerobic or facultative anaerobes.
  • Production of acid by fermentation makes the pH low so we’ll be trying to grow some bacteria on tomato juice agar.  We’ll incubate this under anaerobic conditions.  Specifically we’re trying to grow species of Lactobacillis.
• We’ll also be culturing fecal coliforms [You should already know that these are gram ___ organisms that ferment ____. ]  We’ll be growing them on MacConkey’s agar which is selective for gram ___ organisms and differential for those that ferment ___.  [Just like EMB which we've used before.]
• We’ll also be looking for a gram positive coccoid bacterium that is common in feces: Enterococcus faecalis.  We’ll be trying to grow this in a specialized broth medium:  SF Broth.  [SF stands for Streptococcus faecalis because E. faecalis used to be called S. faecalis.]

Bacteria in Urine

• In our fake urine we’re looking for fecal coliforms because it is fecal coliforms that most often cause urinary tract infections.  [In females the close proximity of the opening of the anus and urethra make urinary tract infections more common than in males.]
  • For this we’re using MacConkey’s agar as we did for the feces.
• You’ll also be doing a plate count of bacteria in the urine in order to determine if our “person” had a urinary tract infection.
  • You’ll do a dilution (actually two dilutions:  1/10 and 1/10 = total dilution of 1/100) and plate out 0.1 ml on the surface of a blood agar plate.
  • In this case since you are spreading the sample on the surface of the plate it is called the spread
    
  • plate technique (rather than the pour plate technique we have used before).

  
  
  

Project 2 — Exercise 16:  Staphylococcal Carrier Study

Overview:

This is the second big project of the semester and it counts a total of 100 points.  Normally I have you do one large (largish) paper for this project.  But this year I’m going to have you submit your results and your paper in sections which will be graded and when added together = 100 points.  At each step in the process you will have to submit your results.  Late submissions will result in points being deducted.

Objectives:

• to isolate and identify a species of Staphylococcus from your body.
• to screen that isolate for antimicrobial drug resistance.
• to evaluate the data from the class.

This Week:

• Isolate bacteria from the nose and skin using the streak plate technique on mannitol salt agar incubated at 37 degrees C.
• Transfer an isolated colony to blood agar in order to grow it up for tests to determine what species you have isolated.

Next Week:

• Identify your isolate.
• Screen for antimicrobial drug resistance.

Flow Chart for the Project

This exercise is pretty straight forward:

You will sample two environments — your body and any non-body environment.  For each:

• Swab the environment with a sterile swab; if the environmental surface is dry, moisten the swab with sterile water.
• Zig-zag the swab over the surface  of a trypticase soy agar plate.  [Don't forget to label the plate!]
• Then swish the swab in a tube of sterile broth.  [NOTE:  broth is just a liquid medium.]
  • What do you do with the swab?????
• Place the plate in the appropriate bin for incubation:
  • 37 degrees C for the body
  • 25 degrees C for room temperature
• Place both tubes of broth in a test tube rack which will be incubated at 37 degrees C.

Exercise 6 — Hand Washing Experiment

About the Scientific Method:

• All scientific investigations start out with observations.  These may include observations in nature or information from sources such as advertising claims and scientific reports and papers in refereed scientific  journals.
  • Do some background reading before you come to lab!!!!
• Such observations lead to questions that may be answered by good experimental design.  They lead first to objectives of the investigation overall.  For example, in this exercise your objective clearly is to determine the effectiveness of some method [you fill in the method] of hand cleansing.
• From this comes your hypothesis.  The hypothesis is based on observations.  It is a statement of the expected results of your experiment.  [It is not a question.]  It should be stated in a way that it can be affirmed or denied by the experiment you are designing.
• Your experimental design should follow from your hypothesis.
  • It should clearly test the hypothesis.  And must contain a control as well as one or more experimental variables.
  • For example an unwashed hand would be a control for most of your experiments.
  • If you were testing the effectiveness of water temperature you would have cold and hot water washing as your experimental variables.
• Your experiment must be replicated to insure that your results are dependable.
  • In this case each member of your group conduct exactly the same experiment.  That way each person is replicating the experiment.
• Your experimental results must be quantified in some way so that you can calculate a mean (average) of the replicated results!
  • The easiest way to do this is to use a scale of your design which will assign a numerical score (say 0 to 3) to the amount of growth on the agar plate.
• Your results will be “published” in a PowerPoint report!

Practicalities:

• Work in groups.  Four members is ideal.  Three is too few to get good replication and 5 is really too many.
  • Five will be allowed only with approval of your lab instructor and only when there is no other way to formulate groups.
• Each person gets one plate of agar.
• Test methods by lightly pressing a finger on the surface of the agar.
  • You can easily divide a plate into 4 sections:  one a control and one for each variation on a method.  [For example, time would be 1 minutes, 5 minutes, 10 minutes, 15 minutes.]
  • Remember that each person does exactly the same thing.
• Don’t overlap variables.  For example:  washing both hands with cold water, testing, then washing with hot water and testing is examining the effect of double washing not the differences between the two!
  • You can remedy this by using different fingers for different treatments OR by recontaminating your hands between treatments.
• All plates will be incubated at 37 degrees C for approximately 48 hours.
• Results will be collected and pictures taken of plates during the return lab.
  • Note the computer int he lab has a digital camera attached or you can use your own cameras.
• Completed reports will be  in one week — the date depending upon the date of Exam 1 to avoid having the exam and the reports due on the same day.
  • Reports will include PowerPoint with narration and a written abstract.  [Instructions on Vancko Hall.]

Be sure to view the sort on-line lecture on the fungi in Vancko Hall Laboratory.  This will be a short introduction to the group since I won’t be getting to it in lecture before you have lab.

You will be examining fungi that are unicellular (yeasts) as well as those that are filamentous (molds).

Filamentous Fungi — Three representative genera are Penicillium, Rhizopus, and Aspergillus.

• View and draw these as they appear in an agar plate of Sabauroud Dextrose Agar.  Do not open the petri dishes — spores will escape and contaminate everything for the rest of the semester!!!!!
• View and draw them from a prepared slide that contains all three genera.  They are located on the slide in the order they are listed on the label.  View:
  • Aspergillus and Rhizopus at 100x
  • Penicillium at 400x
• The spores you see are asexual spores — either conidiospores or sporangiospores depending upon the genus.

Yeast:

• View yeast of two genera — make wet mounts — Candida (opportunistic pathogen) and Saccharomyces (bread and brewing yeast).
• Make a wet mount of my sourdough “sponge”.
  • A sponge is a culture of yeast (and in this case bacteria).
  • You will see abundant yeast and small rod-shaped bacteria between the cells of yeast.  The yeast breakdown carbohydrates and produce alcohol and the bacteria (Lactobacillus ) produce acid from the alcohol.  Hence the sour taste of sourdough bread!
• Try to culture some yeast from your tongue.  Follow directions in your lab book.  We’ll incubate them at 37 degrees C and you’ll check them on Thursday and maybe into next week.

Exercise 4:  Helminths (Multicellular Parasites)

Be sure to view the online lecture on Helminths in Vancko Hall — lecture section.

You will be looking at and drawing the following:

Here is the table from Microbiology Perspectives by Wistreich.  [Note this book will be very handy this week so if you have it, bring it to lab.]

• Cestodes (tape worms)
  • Slides:
    • Scolex and immature proglottids
    • Gravid proglottids
  • Whole or part tapeworms in bioplastic
• Trematodes (flukes):
  • Slides:
    • Chinese liver fluke (Clinorchis senensis) [I took invertebrate zoology with my fiance and we were going to name our first son Clinorchis -- good thing we never had kids, huh?]
      • Whole mount (w.m.)
      • Ova (eggs)
  • Sheep liver fluke (Fasciola hepatica)
    • These you do not need to look at because they are in the pictures on the diagram above.  They include:
      • cercarium
      • metacercarium
      • miracidium
  • Blood fluke (Schistosoma japonicum)
    • male
    • female
    • eggs
  • Also various whole flukes in bioplastic or other preservative.
• Nematodes (roundworms)
  • Trichinella spiralis slides:
    • muscle section
    • isolated larvae