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.

What you have done so far:

1. Isolated bacteria from your nose and skin using mannitol salt agar.
2. Took one colony from one of your plates (one that fermented mannitol if you had one).
   So you have one result:  + or – for mannitol fermentation
3. Grew it on blood agar.
   This is what you will use to determine what species you have isolated.

This week using the bacteria on your blood plate:

1. Determine if it exhitibs hemolysis (see your lab book).  Beta-hemolysis is indicative of Staphylococcus aureus (although not all strains of the species are B-hemolytic).
2. Do a gram stain. [Don't forget the control.]
3. See if it exhibits catalase activity.
   Catalase is an enzyme that breaks hydrogen peroxide down into oxygen and water.
   Mix a loop of your bacterium with a drop of hydrogen peroxide and see if it bubbles.
4. See if it exhibits oxidase activity.
   Oxidase is an enzyme that is part of the electron transport chain.
   For this test take a loop of bacteria and spread it on a part of an oxidase test card. [Four people can use one test card.]
   Look for a dark purple/blue color which is positive for oxidase activity.
5. See if it exhibits coagulase activity.
   Coagulase is an enzyme that coagulates blood forming a clot which protects the bacteria from neutrophils.
   Use a coagulase test kit and look for clumping of the blue latex.  [Follow the directions in the lab book or put out for you in lab.]

From your results you should be able to determine if your isolate is one of the following:

• Staphylococcus aureus
• S. epidermidis
• Micrococcus
• Not any of these three but some other undetermined species. (”Other”)

Test for Antimicrobial Drug Resistance

1. Dump a small amount of sterile Trypticase Soy Broth directly onto your blood plate.
2. Mix the broth with the bacteria using a sterile swab.
3. Take the swab and make a lawn of bacteria on the surface Trypticase Soy Agar plate, just as you did to test disinfectants.
4. Using aseptic technique place four different discs containing antibiotics on the surface of the plate.
   Record the names and concentrations of each of the four antibiotics.
5. Incubate at 37 degrees C.

For your return lab you will examine the plates you have made and

1. Measure the diameter of the zone of inhibition in mm.
2. Determine if your bacterium was resistant, susceptible, or intermediate to each of the 4 antibiotics by referring to the table of standards provided.

Report your results:

• In your return lab session you will be required to fill out a form that reports your results:  what you isolated and its resistance to antibiotics.

Write and turn in two papers:

• One paper on your findings in terms of your bacterium:  Isolation, Identification and Drug Resistance (50 points)
  • A paper copy before 4 PM on Tuesday November 24th.
  • Due online by 11:55 PM Tuesday November 24th.
• A second paper on the class results of the antibiotic portion of the study. (50 Points)
• Detailed instructions are located on Vancko Hall.

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

• View the comic in Vancko Hall.
• Work in teams of three students.
• Each student prepares a lawn of differenct bacteria on the surface of a plate of agar following the directions. The bacteria are:
  • E. coli, Staphylococcus aureus, Salmonella sp.
• Then follow the directions and test the same set of disinfectants on each plate.
• Plates are then incubated at 37 degrees C for 48 hours and the zone of inhibition is measured to determine which worked the best against which bacterium.

Exercise 15:  Identification of Gram Negative Bacteria

• Work in groups.  Each lab will have 6 groups because we are using 6 species of bacteria:
  • E. coli
  • Enterobacter aerogenes
  • Alcaligenes faecalis
  • Salmonella sp.
  • Proteus vulgaris
  • Pseudomonas aeruginosa
    
• One of the most important things is to GET ORGANIZED — pick a person to keep a check-list of media and to be sure that all tubes are inoculated.
  • Be sure that all tubes are labeled in such a way that if the person doing the labeling is absent for the return lab, other members of the group can identify the tubes as to bacterium and type of medium.
• Each group will get a known gram negative bacterial culture and an unknown gram negative bacterial culture.
  • BE SURE TO RECORD THE NUMBER OR LETTER OF THE UNKNOWN.
• Then you will inoculate TWO sets of microbiological media:  one set with the known and the other with the unknown.
• Your inoculated cultures will be incubated for 48 hours and then you will collect results for your known and unknown.
• Basically you are looking for products of reactions that indicate the bacteria were able to utilize the substrate, i.e. sugar or amino acid, given them in the medium.
  • Sometimes you just look at the tube for a color change; for some media you must add reagents.
• Results of the knowns will be shared so that you can try and figure out what your unknown was.
• Media will include the following:
  • OF glucose — agar deeps (one with air and one shielded from air)
  • Fermentation Broths with phenol red containing sugars:
    • Glucose
    • Lactose
    • Sucrose
  • MR-VP Medium — a broth
  • Simmons Citrate Agar slants
  • Urea Broth
  • Phenylalanine agar slants
  • SIM medium (semi-solid agar deeps)
  • Nitrate broth

For this exercise you will work in a team to determine the number of bacteria per gram of hamburger.  Half of the teams will have meat that has been left out at room temperature for a number of hours and the other half will have meat that has been continuously refrigerated.  We will compare the results of the two teams for the return lab.

We are using the pour plate technique to mix a known portion of the food with agar, letting it incubate, and counting the number of resulting colonies.  This will give you an estimate of the number of bacteria in a gram of the food.

Dilution

• Mixing a gram of hamburger with agar will result in a huge number of colonies — clearly more than you can count.  A plate with more than 300 colonies is considered (and reported as) Too Numerous to Count (or TNTC).
  • Note that a plate with less than 30 colonies is considered Too Few to Count (TFTC).
• Sooo the hamburger must be diluted and then the dilution is placed in the agar.
• You are going to doing three dilutions with the hamburger:
  • 20 in 180 or 20/200 = 1 to 10 (or 1/10 or 1:10 or 0.1)
  • then that will be diluted 1 in 99 = 1 to 100 (or 1/100 or 1:100 or 0.01)  [total dilution 1/10 and 1/100 = 1/1,000]
  • and that will be diluted 1 in 99 = 1 to 100 (or 1/100 or 1:100 or 0.01)  [total dilution 1/1000 and 1/100 = 1/100,000]
• We’ll go over this at the start of lab.

Pour Plate

• You will be placing known quantities of your dilutions in empty sterile petri dishes.
  • We are duplicating everything this week.  [Why is replication necessary and important?]
• Then mixing sterile melted agar with your dilution in the plate, allowing it to solidify, and then incubating it at 37 degrees C for 48 hours.

Determining the Number of Bacteria:

• For the return lab you will count the number of colonies on those plates that can be accurately counted:
• those containing between 30 and 300 colonies.
• Then you will calculate the number of bacteria per gram of meat by the following formula:
  • # bacteria/gram = # colonies x 1/mL plated x 1/dilution plated

COME TO LAB PREPARED BY CAREFULLY READING THE LAB AND THIS BRIEFING!!!

There is NO Lab Quiz this week.  HOWEVER — there is a set of questions on dilutions that is due on your return lab on Wednesday or Thursday!!!  NO LATE ASSIGNMENTS WILL BE ACCEPTED WITHOUT A DOCTOR’S EXCUSE!!!!!  It is posted in Vancko Hall.

The Setup

• You each get a tube with a mixed broth culture. Be sure to record your number.
• Each contains a mixture of 2 bacterial species which are visibly different either in shape, gram stain characteristics, or both.

Your Task (document everything with notes and drawings)

• Isolate each bacterial species by growing them on trypticase soy agar.
• Determine the following for each species:
  • Their colony characteristics.
  • Their shape and arrangement of cells.
  • Their gram stain characteristics.
  • Their motility.
• Document each of these characteristics with digital drawings or pictures.
• Write up your report and hand it in on time.
• You will have until Friday March 20 to work on your unknowns.
• Your report is due at the start of your lab session on Tuesday March 24th.

Day 1

• Isolate:  2 streak plates — one incubated at 37 degrees C, the other at 25 degrees C.
• Characterize from the broth cultures:
  • Motility — hanging drop or wet mount
  • Gram stain — prepare at least two slides for gram stains of you unknown.

Day 2

• Examine plates for isolated colonies (ideally with 2 different appearances).
  • Take a digital picture of your plate.
  • If you failed in your isolation attempt you may do one additional streak plate (that’s all, just one more).
  • If you do an additional plate it is your responsibility to incubate the plate and to remove it from the incubator.  Do not leave plates at 37 degrees C for more than 48 hours — they will dry out.  At least come in and put your plate in the refrigerator.
• Confirm isolation by doing a gram stain from each colony.
  • Don’t forget that a control (mixture of Gram + and Gram -) must be done with each and every gram stain.
• Negative stain if you didn’t do it on Day 1.

Due Dates etc.

• You have two weeks to complete your unknown project:
  • Both labs this week and one hour of the first lab next week.
  • You may come in on your own time to work on it until Friday October 30
• Your final paper (both digital and paper copies) is due before the start of your lab on Monday Nov. 2 or Tuesday Nov. 3rd (depending upon the date of your first lab of the week).
• The instructions for your paper are posted on the Lab Briefing Blog and there are sample papers posted in  lab.
  • NOTE:  do not go through the steps of the different stains and other procedures — just say that you did them and that you used aseptic technique throughout!

Even if you have completed all of your stains you still must attend lab.

Your instructor will check your drawings and your slides before you can leave lab.

1. You must save the reference slides that you made.
2. You might want to clean a few more slides in preparation for next week’s lab.  [I noticed that in my lab some of the negative stains tended to "puddle up" a bit too much which indicates that the slides were not clean enough.]

Next week it is Project 1:  Morphological Unknowns!   Be Prepared!

We use three staining techniques in General Microbiology:

1. Simple Stain — uses one stain and all bacteria are the color of the stain.  Is not a differential stain.
   • Preparation of the specimen is the same as for the Gram Stain except that a control is not necessary because this is not a differential stain.  [Any technique that is differential distinguishes between two or more types of bacteria.]
2. Gram Stain — uses four reagents and results in distinguishing between bacteria with two types of cell wall structures (gram positive and gram negative).  This is a differential staining technique.
   • The preparation is the same as for the simple stain except that a known gram positive and gram negative are used as controls.
   • The control:  the control is a mixture of Gram + and Gram – bacteria of two shapes.
     • We use Staphylococcus aureus which is a Gram + spherical-shaped (coccoid) bacterium and Escherichia coli [E. coli] which is a Gram - rod-shaped (bacillus) bacterium.
     • Since we use bacteria of different shapes we can mix the two controls and do not have to do a separate + and - control.
   • Cells that are Gram + are purple and those that are Gram - are red (or pink).
3. Negative Stain — uses one stain (Congo Red) which has a negative charge and does not adhere to the negatively charged cells.
   • The background is stained, not the bacterium.  Hence if you use a black stain it looks like a photographic negative.  We don’t use a black stain, we use Congo Red, which is red because it works much better than Nigrosin which is a black negative stain.
     • NO HEAT is applied in the preparation of this stain.  Therefore the cells appear more “natural” — more their normal size and shape.
     • However, this means that they may still be viable and must be treated as such.  All slides with negative stain are disposed of in disinfectant and will be sterilized and recycled for your use.
     • NOTE that for the simple and gram stain, since the specimens are killed by heat fixing they are not to be discarded in the disinfectant.  You may keep them in your slide boxes OR you may clean and reuse them.  If you are going to discard the slides do so in the glass discard barrel NOT in the buckets under the hood.

All stained slides are to be viewed at 1000x total magnification.  Lower magnifications are not sufficient to really see and describe the bacteria accurately.

Oil is applied directly to the dry stained slide.  No cover slip is used.

BE SURE TO READ THE COMIC BOOK ON STAINING — IT GOES THOUGH THE PROCEDURE IN DETAIL.

Practice is the key to successful staining technique.

In lab do these stains:

1. One simple stain
2. One negative stain
3. The following gram stains

• Do a gram stain from your thioglycollate broth from the soil lab. What do you expect to see here?
• Create reference slides (slides you will keep to refer to as you do your unknown) of the following:
  • Staphylococcus [gram + cocci in clusters]
  • Micrococcus luteus [gram + cocci in a sarcinate arrangement]
  • Streptococcus faecalis [gram + cocci in a string; note these sometimes look like slightly elongated cocci]
  • Bacillus subtilis or B. megaterium (whichever one we have out)
    • fairly large gram + rods; can form endospores
    • older cultures of Bacillus can sometimes be “gram variable” which means that some will look gram negative.
  • Eschericia coli (E. coli) [gram - rods]
  • Salmonella sp. [The "sp." means "species" -- I'm not sure what species we have.]  [gram - rods]

  Save your reference slides by dabbing off the oil and placing them in your blue slide box.

• Remember that for each and every gram stain you do, you must include a control mixture.
  • If you wish you can fit three samples on 1 slide:  one control and two other kinds of bacteria in each of two other circles.
  • Exercise 10 Isolation of Bacteria

Exercise 10 Isolation of Bacteria

In order to isolate bacteria you must spread the sample so thinnly that it separates individual cells which can then grow into visible colonies.   This is done in order to make a pure culture which can then be used for identification of the bacteiral species that is isolated.  [See Koch's postulates for the importance of this procedure.]

We use the “Streak Plate Technqiue”.

READ THE COMIC BOOK AS WELL AS YOUR LAB BOOK FOR THIS TECHNIQUE.

Each student will be doing two streak plates using a mixed culture that we provide:

1. One on a general-purpose medium:  Trypticase Soy Agar (TSA)
2. One on a selective medium  [Be sure you know what this means.]
   • Mannitol Salt Agar (MSA)*
   • Eosin Methyleneblue Agar (EMB)*
   • Phenylethylalcohol Agar (PEA)

• *These are also differential media.

These plates will be incubated for 48 hours at 37 degrees C and evaluated in the comeback lab period.

Aseptic technqiue is crutial in this process!  Review your aseptic technqiue.

Be sure to follow the directions exactly.

For what these media are used for and their ingredience see the lab book and the microbiological media information sheet in Vancko Hall.

Microbiological media comes in three forms:  broth (liquid), semisolid agar, and agar (solid)

• Trypicase soy broth and agar are the nutritionally the same; the latter has agar added (15 gm/l) to make it solid.
  • Remember that agar alone has no nutritional value for most bacteria; it is just a solid medium to which nutrients must be added to support bacterial growth.
  • You can find a listing of kinds of media that we use and their composition in the introductory module in Vancko Hall Lab.
• Broth is usually dispensed in tubes and it is what we use for our pure cultures of bacteria.
• Semisolid agar is usually dispensed in “deep” tubes — about 10ml per tube.  It is used for detecting motility.
• Agar may be dispenses in tubes as agar deeps or slants (allowed to cool at an angle so a slanted surface forms) or into plates.

You will take one of three available species of bacteria and aseptically transfer it to one tube of broth, one tube of semisolid agar, and one agar slant.  You are inoculating these sterile media.  The three species are:

• Staphylococcus aureus
• E. coli
• Proteus vulgaris

Aseptic Technique enables you to safely transfer bacteria from a culture to a sterile medium without contaminating the culture with another bacterium and without contaminating you or your work place.  It is second only to the use of the microscope in importance in microbiology.

Be sure to read the lab book AND the comic book on this laboratory exercise.  You may even find it helpful to print the comic.

Exercise 8 — Isolation of Soil Bacteria

The bacteria found in soil often form resistant structrures called endospores.  These resist drying and other hazards (like oxygen if it is an anerobic organism).

• In this exercise you will heat a soil sample to kill any vegetative (living) cells that are present and presumably leaving on the endospores.
• After heating you will inoculate a tube of specialized medium (thioglycollate medium) which promotes the growth of anaerobes.
• Next week you will stain your soil cultures.

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.]