Exercise 15

Posted on June 12th, 2009 in Microbiology lab by drstocksblog

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

Project 2:

Posted on June 12th, 2009 in Microbiology lab by drstocksblog

Exercise 16:  Staphylococcal Carrier Study

Staph Flow Chart

Overview:

This is the second big project of the semester and it counts a total of 100 points.  I suggest you do the paper in two parts:  I. Individual Results and II. Class Results.  Each is worth 100 points.  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.

Isolation:

  • Completed Thursday 06.10.2009:  Isolate bacteria from the nose and skin using the streak plate technique on mannitol salt agar incubated at 37 degrees C for 48 hours.
  • Plates were take from the incubator and refrigerated after 48 hours of incubation.
  • Tuesday 06.16.2009:  Grow isolate on Blood Agar
    • Did your isolate ferment mannitol?
      • If so take one of those colonies for this portion of the project.
      • If not, take any single colony from either plate.
    • Transfer one isolated colony from MSA to blood agar plate by placing the colony in 0.5 ml of sterile Trypticase Soy Broth (TSB) and then spreading it liberally on a plate of blood agar.
      • This will be incubated at 37 degrees C and you will use the growth to identify your isolate.

Identification Thursday 06.18.2009:

  • Perform the following tests to identify your isolate [Refer to detailed directions in your lab book]:
    • Examine blood plate for hemolysis.
    • Gram stain –don’t forget the control!
    • Oxidase Test
    • Catalase Test
    • Coagulase Test
  • After these are complete you should be able to tell what you think your isolate is.
  • Note that some isolates will not match enough of the criteria to make any identification.  In that case just say you identified something else or “other”.  You would have to conduct more tests to determine what it is and we don’t have time or the means for that.
  • Part I of your report is almost ready to complete.  It is the isolation and identification of your isolate.

Screen for Antimicrobial Drug Resistance Set up Thursday 06.18.2009 and complete Tuesday 06.23.2009

  • After you have completed your identification of your isolate follow the directions in the lab book and screen your isolate for drug resistance.
  • We are testing your isolate for resistance to
    • Penicillin
    • Streptomycin
    • Tetracycline
    • Tiple sulfa
  • These plates will be incubated for 24-48 hours and you will gather your results on Tusday June 23rd.

Report the results for your antibiogram for distribution to the class

  • Report your results.
  • These will be collected and class results will be distributed to you for your analysis.

Week 4: Bacteria in Food

Posted on June 8th, 2009 in Uncategorized by drstocksblog

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).

We will divide this up so that Mrs. Tarrant’s lab will do this first and then start their unknowns.  My lab will work on their unknowns and after Mrs. Tarrant’s lab is done with the food will do it.

Week 4 Unknowns

Posted on June 8th, 2009 in Microbiology lab, Unknown bacteria by drstocksblog

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 this week to work on your unknowns.
    • Any additional work you do must be done outside of the scheduled laboratory period and when there is a faculty or staff member on the floor.  Remember that since it is summer there is not always someone here.
  • Your report is due at the start of your lab on Thursday June 18th.

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.
    • Negative Stain

Day 2

  • Examine plates for isolated colonies (ideally with 2 different appearances).
    • 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 expect to come in on your own time to work on it.
  • Confirm isolation by doing a gram stain from each colony.
  • You should have isolated two bacteria with different microscopic morphology.  Their appearance should correspond to  what you saw on Day 1 in your broth culture.

Your Report (due June 18th)

  • I’ll post a grading rubric for it.  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 due at the same time as your hard copy.
  • 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!

Unknown Flow Chart

Week 3 Lab: Staining and Isolating

Posted on May 31st, 2009 in Uncategorized by drstocksblog

Exercise 9:  Staining Bacteria

We use three staining techniques in General Microbiology:

  1. Simple Stain — uses one stain and all bacteria are the color of the stain.  This 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.

Be sure to start at low power magnification (100x) and work your way up.
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. At least 3 gram stains — one of which must be of your soil isolation.
  2. One simple stain
  3. One negative stain

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.

Pictures and Video from Week 1

Posted on May 25th, 2009 in Uncategorized by drstocksblog

Some Pictures and Video From Lab

Here is a series of pictures with some questions.

Week 2: Exercises 5, 6, and 7 plus

Posted on May 22nd, 2009 in Microbiology lab by drstocksblog

Exercise 5 — Organisms in the Environment

This one is straight forward. You are to swab two surfaces:  one body surface and one environmental surface.  This is an individual exercise.

  • Then transfer the material to the surfaceof an agar plate and see what grows.
  • Before discarding each swab swish it in a tube of sterile broth and we’ll see what grows in that.
  • Read the more specific directions in your lab book.

Exercise 6 — Hand Washing  — Group Exercise

This is a group exercise; there should be 4-5 people per group.

  • Each group designs their experiment to evaluate the effectiveness of different methods of hand washing and then carries out the experiment.
  • Decide what you want to test. (Your objective)
  • Develop an hypothesis.
    • Remember that an hypothesis is a statement (not a question) of expected results.
    • It is specific to your experiment.
  • Each person should to exactly the same thing as every other person so that your experiment is replicated.
    • The easiest way to test different methods is to press a finger than has been washed on a portion of an agar plate.
    • Each plate can easily be divided into 4 sections.
    • One finger should be a control.
  • We have some items you can use:  various hand santitizers, antibacterial liquid soaps, regular liquid soaps, sterile surgical scrub brushes, etc.  You can bring your own thing to test.
  • Develop and carry out your experiment on Tuesday and get the results on Thursday.
    • Make a semi-quantitative assessment of your individual results.  For example, rate the growth from 0 (none) to 3 or 4 (a lot).  Then you can average your individual results and easily compare the different agents or methods.
  • Your report consists of a narrated PowerPoint presentation AND a one-page abstract which is a summary of your experiment and the results.  One report and one abstract per group.
    • There is a sample presentation on Vancko Hall along with some background information and guidelines and grading rubric for the project.
  • It will be due Thursday June 4th.

Exercise 7 — Media and Aseptic Technique

  • In this exercise you develop the essential skill of transferring bacteria from one medium to another without contaminating the pure culture, yourself, or your environment!
  • See the lab for details.
  • Read the comic on Vancko Hall too!

New Exercise — The Effectiveness of Disinfectants — Phenol Coefficients

  • This will be done on Thursday.  Incubations will be 48 hours and you’ll evaluate your results on the folowing Tuesday (the 2nd of June).
  • The exercise is available on Vancko Hall.  But you don’t need to print it.  We will have copies for you.
  • We’ll all see how this works because I haven’t done this one!

Micro Lab Briefings Blog

Posted on May 17th, 2009 in Microbiology lab, Microscopy by drstocksblog

Welcome to the General Microbiology Lab Briefings Blog for Summer 2009

This is a blog — basically a web page that I add to  and that you can link to via a RSS feed.  That just means that you can sign up so that you can get notification each time I update the blog.

What is this?

  • This is indended to give you a “heads up” on what is going on in lab before you have to be there.
  • So that you can be more organized and more prepared.
  • In summer lab meets for 4 hours twice a week.
  • That’s almost 3 regular semester weeks each week.  Yep, lots of stuff and not much time to cover it all.
  • I’ll usually post before Monday for that week’s lab.

What this is not.

  • This is not a substitute for reading the lab exercise.
  • Try to read the labs ahead of time.

This week in lab we’re covering Exercises 1, 2, 3, and 4 in the lab manual.

Lab Safety:

  • We’ll review this with you but be sure to read the lab safety information on Vancko Hall!
  • See also the Page “Always and Nevers for Microbiology Lab”

Exercise 1 Microscopy (Care and Use of the Microscope) should be completed on Tuesday

  • I have printed out the How to Use the Microscope comic and will hand it out in lab.
    • For future tutorial comics you are on your own because you’ll know ahead of time what’s coming up.
  • We will spend more time that usual going over the microscope and its use with you at the start of lab.
  • For this lab be sure to look at and draw the following:
    • Prepared slide of blood at 100x, 400x, and 1,000x total magnification.
    • A wet mount or hanging drop preparation of the hay infusion.
      • A hay infusion is a preparation of dry grass and water which has been allowed to sit around for at least a week.
      • It contains a great community of bacteria and protozoans.
      • Notice the relative size of each.
      • Do any of the bacteria exhibit motility?  If so, How do they move?
      • Can you see different bacterial shapes?
  • In the future you only need to draw the slides at one magnification (whatever is best for that specimen).
  • See the Page “Always and Nevers for Microbiology Lab” for do’s and don’ts of microscopy and lab in general.
  • You should draw and label everything you see in lab!  There is space in your lab book for drawings and your written descriptions.

Exercise 2:  Kingdom Protista (includes Algae and Protozoa)

Tuesday (prepared slides):

  • Protozoa  (view at the best magnification to adequately view it — usually 100x or 400x)
    • Amoeba
    • Paramecium
    • Trypanosoma – is a blood parasite so you will see red blood cells; view at 1,000x
    • Trichomonas
  • Algae
    • Volvox
    • Diatoms
  • Cyanobacteria
  • Oscillatoria - wet mount of live cultures (400x)

Thursday (mostly living material — make wet mounts)

  • Protozoa
    • Paramecium – apply some Protoslo solution to the slide to slow them down.
    • Amoeba – veiw as a demonstration on the microscope viewer in the front of lab.
    • You still have to draw it!
    • Vorticella – a sessile (non-motile) cilliate that looks like a wine goblet!
  • Algae — wet mounts
    • What you or your classmates or your instructors bring in!

Exercise 3:  Fungi (molds and yeasts)

Tuesday

  • Swab your tongue to see if you have yeast in your mouth.

Tuesday and/or Thursday

  • View the following:
    • Prepared slides of Rhizopus (at 100x), Penicillium (at 400x) , and Aspergillus (at 100x).
      • These three are on a single slide and are in the order in which they are listed on the label.
    • Agar plates of the same three genera of fungi.
    • Saccharomyces – make a wet mount and view at 400x; you may want to stain these for better viewing (see lab mannual)
    • Sourdough “Sponge” — wet mount at 400x
      • this is the starter culture that I maintain for my sourdough bread
      • In it you should see yeast, grains of flour, and rod-shaped bacteria
      • What is the function of the yeast?  … bacteria?

Exercise 4:  Helmenths (Parasitic Worms)

Tuesday and Thursday

View and draw the slides and plastic mounts of the parasites listed in your lab book with the following exceptions.

  • Since there is only one good slide of the liver fluke microscopic stages I have taken pictures of the metacercarium, miracidium, and cercarium as shown below.  [Note the diagram is of a blood fluke and the slides are actually of a liver fluke, Fasciolata hepatica.]

Fluke Cycle