General Microbiology Lab Briefing

Tuesday May 11th:  Lab Quiz (50 Points)

When:  at the start of your lab period.

Where:  In room 226 Bush Hall — the Library Computer Lab.

What:

• Multiple choice (about 28)
• Some matching
• Lots of pictures
• Questions about
  • techniques
  • media
  • reagents
  • staining
  • dilutions
  • and the like

What is permitted:

• Any resource you want (except other people).
• That includes your lab book, any other books, online resources, etc.
• I suggest you open the Media file on Vancko Hall before you start the quiz — it opens in a new window.

What to study:

• I wouldn’t spend a lot of time studying.
• But I would be sure my lab book was well organized.
• I might even mark pages that contained kinds of media and their reactions.

If you’ve been in lab, been paying attention, doing good drawings,  and have answered questions in the lab book you should do fine.

You will not be able to view the online quizzes you have taken during this quiz.

You’ll have an hour to complete the quiz.

Epidemiological Investigation — investigation of a foodborne outbreak

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.
    • Complete Part I which is attached to a News Message in Vancko Hall (and linked below the Epidemiological Investigation document in Vancko Hall).
    • If you do not have Part I filled in when you arrive in lab on Tuesday you will have 5 points deducted from the assignment for Thursday!
      
• 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.

Continue Staphylococcal Carrier Study

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 exhibits 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.

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.

Before leaving lab on Thursday fill out a data sheet indicating what you have isolated, where you isolated it (nose or skin), and what reaction it had in the antibiotic resistance testing.  If you do not submit your results at this time you will receive a 10 point deduction from the project grade.

Write up your results and turn them in.  See Vancko Hall for directions.  The first portion of the project is due on Tuesday May 4th at the start of your lab section.  The second part of the project is due on Thursday May 6th at the start of lab.  For each part submit both a paper copy and a digital copy uploaded to Vancko Hall.

Exercise 16:  “Pee, Poop, and Spit” Lab  Exercise

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 14:  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:

• Tuesday: Isolate bacteria from the nose and skin using the streak plate technique on mannitol salt agar incubated at 37 degrees C.
• Thursday:
  
  • Observe (and describe) the growth on your mannitol salt agar plate.  Did it ferment that mannitol?  [If so, you likely have isolated S. aureus; if not you probably have isolated S. epidermidis.
    • Transfer an isolated colony to blood agar in order to grow it up for tests to determine what species you have isolated.  Use a colony that fermented mannitol if you have one.
    • Take a single isolated colony and carefully transfer it to a tube containing 0.5 ml of sterile saline or 0.5 ml of sterile Trypticase Soy Broth.
    • Then do a streak plate from the tube onto a blood agar plate.
    • Your blood agar plate will be incubated for 24-48 hours.

Next Week:

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

Flow Chart:

Exercise 13:  Testing Disinfectants

• View the comic in Vancko Hall.
• Work in teams of three students.
• Each student prepares a lawn of different 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

NOTE:  Just because your unknowns are due on Tuesday does not mean that you can come to lab unprepared!   Be sure to read this briefing!

Exercise 12:  Determining the Number of Bacteria in Food

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

Determining if Any Potential Pathogens are Present

• The pour plate technique only tells you the number of bacteria; it does not tell you if they are pathogens.
• You are going to do three streak plates with the first diluted sample on selective and differential media to see if potential pathogens are present:
  • EMB agar, Mannitol Salt Agar, and SS agar
  • Be sure you read about these kinds of media and know what they are used to isolate (and differentiate among).

On Tuesday the introduction to the lab will take about 30 minutes and if you are well-organized the remainder should take less than an hour.

There are dilution problems posted on Vancko Hall.  Do the problems! They are due at the beginning of your lab on Thursday. SHOW YOUR CALCULATIONS.

So far for your Unknowns:

• Streak Plates:  You should have made two streak plates on trypticase soy agar:  one was incubated at 25 degrees C (room temperature) and the other at 37 degrees C.  This is because one may grow better at one temperature than the other.  Also it may slow the growth of bacterial species that are motile and make it a bit easier to isolate them.
• Motility:  Ideally you have looked for motility by doing a hanging drop or wet mount of your unknown broth.
• Stains: You should have prepared two slides for gram stains.

This week:

• Gram stain your slides from last week and look at them if you already haven’t done so
  • Be sure to draw them, label your drawings, and describe what you see in words.  [This makes writing your report sooo much easier.]
  • You MAY if you wish take digital pictures through the ocular lenses of the microscope with your own digital camera.  If you choose to use these pictures be sure that they clearly illustrate the shape, color and arrangement of the bacteria.  In other words, it may be more clear to use a drawing that you scan to make digital than a digital picture.
    
  • I’ll leave this up to you — but you have been cautioned about having clear illustrations that show what you say they show.
• Observe Streak Plates (take digital pictures).  Be sure to describe the colony characteristics.
  • Ideally you will see two different kinds of colonies that are clearly isolated (separate) from other growth on the plate.  Hopefully these will correspond to what you saw when you gram stained from the broth.
  • In order to show that you have isolated the bacterial species you need to gram stain from the isolated colonies.
  • Be sure to get only bacteria from isolated colonies.
  • Don’t take too much — you don’t want your stain to be too thick!
  • If you have isolation that you can illustrate and describe you are pretty much finished.
• What do you do if you don’t have isolation?
  • Gram stain anyway to see what is growing.
  • Do another streak plate, after trying to figure out what happened, either from your broth or your growth on the plate.
• Do a negative stain — either from the plate or from your original broth. Be sure to draw it.

Write Up Your Report — due at the start of your lab period on Tuesday March 30th:

• A rubric is already posted in Vancko Hall.  Use the rubric as a check list to make sure you have done everything.
• Both a hard (paper) AND digital copy of your report must be turned in.
• Submit your digital paper as a single file (illustrations included) in Vancko Hall.  It is also due on March 30th by the start of your lab session.
• Some suggestions:
  • Don’t describe HOW TO do a gram stain or ASEPTIC TECHNIQUE — I assume you know how to do those things correctly.
  • DO include the kind of agar, incubated at what temperature for how long.  (Your plates from last week were incubated for 48 hours and then placed under refrigeration).  These are all things that may change from lab-to-lab or person-to-person.
  • Use descriptive titles for your illustrations like this:   Figure 1. Gram stain of unknown broth at 1000x.
  • Your illustrations should be referred to in the body of your text like this. (See Figure 1.)
  • Your illustrations may be embedded in the text but do not have to be.  They may all be placed at the end of the paper.  Either way you must clearly label your illustrations.
  • Be sure you describe your results thoroughly in words!  This is why we’ve been having you write descriptions of what you see in your lab book.  Don’t just have a picture of a gram stain describe it in words!
  • Be sure to follow the directions!

TUESDAY:  Exercise 10 — isolation of bacteria

Finish this exercise by examining your streak plates.

• Ideally you should see well isolated colonies at the end of the second were beginning of the third section on your plate of agar.
• on the TSA agar can you see more than one kind of colony?
• If you did not get good isolation talk to your instructor about what you might do to improve your technique.
• Be sure to look at and draw all three kinds of selective media.
• Review and be sure that you understand whether each of these kinda of agars are also differential. If they are differential be sure to know what they are differential for and how you interpret the results.

Assignment in lab on Tuesday:

• Prepare a gram stain from an isolated colony on any medium except eosinmethylene blue agar.  (why can’t you do an accurate Gram stain this kind of agar.
• On a piece of paper separate from your lab manual
  • draw and label the streak plate that you are using.
  • draw, label, and describe the results of your gram stain. (Don’t forget to draw your control!)
  • briefly tell if your Gram stain confirms (or not) that you have successfully isolated a species of bacteria.
• Hand in both your drawing, explanation, and your labeled slide before leaving the lab.
• Your effort is worth 10 points

THURSDAY:  begin Exercise 11 — Project one — Morphological Unknowns

In preparation be sure to read exercise 11 in your laboratory manual.

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 26th to work on your unknowns.
• Your report is due at the start of your lab session on Tuesday March 30th.

Pick a test tube containing your unknown bacteria.  It contains two different species which are visibly different with the gram stain.

• Be sure to record the number of your unknown on the paper provided as well as in your laboratory manual.

Today you should do the following:

• prepare to streak plates on trypticase soy agar plates.  One will be incubated at 37° C. and one at 25° C. (Some bacteria will grow better at one temperature than at the other.)
• prepare two slides from your unknown for gram staining.  (At least heat fix the slides and store them for staining later.)
  • Fresh cultures stain better — so it is best to prepare these slides for staining today!
• do a hanging drop or wet mount preparation to determine if your unknown is motile. You do not need to draw motility because it is hard to capture in a drawing.  You should, however, describe it.

Be sure to take good notes on what you have done.  You will need too refer to your notes and drawings when writing up your report which is worth 100 points!

After you are done for the day place your unknown culture in the designated rack; it will be refrigerated and available for you to work on next week.

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.
  • If you don’t get to stain or look at them, that’s OK.  But be sure to make the slides today while your cultures are fresh.

Exercises  7 and 8– left over from last week

Be sure to look at, describe, and draw your results from from exercise six, media and aseptic technique, and exercise seven, soil bacteria.since last week’s lab was canceled you can limit your results to tubes that you did. — Not all three species of bacteria.

• What color is the bacterial growth?
• Is your bacterium motile?
• Is the growth in the broth uniform or does it want at the bottom or float at the top? Does it appear mucoid?

Do not discard these tubes because you will use them to practice staining!

Gram stain your bacteria from the Thioglycollate medium. (See gram stain below.)

• What do you expect to see?
• That is, what kind of bacteria form endospores?

Exercise 9 Staining Bacteria

It is necessary to stain bacteria because they naturally have little color and are the color of the background when viewed with light microscopy.  We use three methods of bacteriological staining: simple stain, Gram stain, and negative stain.

Be sure to read this exercise ahead of time AND the comic book on staining.  Below are some things to note (not detailed directions).

1) Simple stain — one stain that stains all bacteria the same color.

• green (malachite green stain), blue (methylene blue stain), red (safranin stain)
• Preparation:  bacteria to be stained are placed on the slide, air dried on the slide warmer and then heat fixed.
• Heat fixing makes the bacteria stick to the slide and kills them so they are safe to handle.

2) Gram stain — a differential stain that distinguishes between gram positive (purple) and gram negative (red) cells.

• Preparation:  the same as the simple stain but since this is a differential stain a mixed control is always used.
• Mixed control:  Staphylococcus (gram + cocci — round bacteria) and E. coli (gram – bacilli or rod-shaped bacteria).
• The control and the unknown bacterium are always stained on ONE slide but in two different circles on the slide.
• Once the smears are prepared (dried and heat fixed) they are stained using 4 reagents:
  • Crystal violet (primary stain)
  • Gram’s iodine (mordant or fixative)
  • Ethyl alcohol (decolorizing agent)
  • Safranin (counter stain)

3) Negative Stain Technique — has nothing to do with gram stain characteristics.

• Bacteria is mixed with the stain which is congo red and the mixture is spread on the surface of a slide.
• It is then AIR DRIED — not heated in any way.  See the comic and your lab book for detailed directions.
• It can then be viewed.

ALL STAINED SLIDES MUST BE VIEWED AT 1,000x (UNDER OIL) TO BE ADEQUATELY SEEN!

No coverslips are used on stained slides.

Exercise 10 — Isolating Bacteria [We'll probably actually do this one on Thursday.]

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 Technique”.

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 at 37 degrees C and evaluated next Tuesday.

• They’ll be incubated for 24 hours and then removed from the incubator (by Mrs. Tarrants or myself) and stored refrigerated until lab on Tuesday.

Remember:

• Aseptic technique is crucial in this process!  Review your aseptic technique.
• Be sure to follow the directions exactly.
• For what these media are used for and their ingredients see the lab book and the microbiological media information sheet in Vancko Hall.

Exercise 7 –  Culture Media and Aseptic Technique

Culture media comes in three forms:  liquid, semisolid, and solid.

• liquid medium (or broth) has no added agar*.
• semisolid agar has a little agar; we’ll be using a semi-solid agar deep — which is just a tube of semisolid agar.
• solid medium has the most agar (15 gm/liter) and is the consistence of hard jello.  You’ll be using an agar slant which is a tube of agar medium that has been left at an angle when it was removed from the autoclave.  Hence, it has a slanted surface.
• Remember that agar* is a carbohydrate that has no nutritive value for bacteria (they do not have enzymes that will break it down).  Unlike gelatin it is solid at 37 degrees C.  Nutrients must be added to provide food for the bacteria.  The nutritive base for the three kinds of media that we are using is the same.  [Look up the ingredients of Trypticase Soy agar in Vancko Hall.]

For this exercise each person will inoculate three tubes of medium (broth, semisolid agar deep, and agar slant) with one of three species of bacteris:

• Staphylococcus aureus
• Escherichia coli
• Proteus vulgaris (we might have to use a substitute here; the last I heard the Proteus was not growing well.)

Aseptic Technique:

• To do the inoculating you will need to practice aseptic technique which prevents contamination of your cultures and yourself!  Be sure to read the comic book explanation as well as the lab book for how to use aseptic technique.
• Mrs. Tarrants and myself will take a short period of time in lab to show the technique but we expect you to have read over the material provided before lab!

Exercise 9 — Isolating bacteria from soil

Soil contains bacterial endospores.  These are resting stages that are able to resist drying and, in the case of obligate anaerobic bacteria, oxygen.

• What you are trying to do there is to kill the bacteria that do not form endospores that may be present in dirt and soil.  To do that you will heat a mixture of soil and water for a minimum of 30 minutes.
• This should leave only endospores in the dirt-water mixture.
• You will then inoculate a tube of thioglycolate medium with the mixture and incubate at 37 degrees C.
  • Read about this medium in your lab book.  (Know why we use it.)
• After the winter break you will gram stain your culture to see what grew!

There is a quiz at the end of this week.  It will be available Friday and due the Monday after  Winter Break!