Exercise 13:  Testing Disinfectants

• 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

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

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.

Start Morphological Unknowns! (Exercise 11)

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!

Continue Staining!

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!

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.  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.