Lab 6: Structural Diversity Among Cultured Isolates
Gram Reaction Confirmation by use of Selective/Differential Media
Background on Selective and Differential Media
Selective media helps select for growth of certain organisms in a mixed population by using a ingredient that inhibits the growth of other microorganisms, but not the desired species or group. Differential media does not select for any particular group by inhibiting or enhancing the growth of one group over competitors, but this type of medium is able to show a visible difference between or among groups of microorganisms. A medium for microbial growth can be a combination of selective and differential, depending on its ingredients and its use.
Activity: Performing a Spot Inoculation Technique on Selective/Differential Media to Assess Cell Wall Composition (Confirm Gram stain)
You will inoculate each of your isolates onto both eosin methylene blue (EMB) and phenylethyl alcohol (PEA) solid media. Consult Culture Media: Use of Selective & Differential media to confirm Gram stain to learn how these media are able to select for either Gram positive or Gram negative bacteria and, in the case of EMB, to differentiate lactose fermenting bacteria from non-lactose fermenters. You should test all your isolates on both media to confirm your Gram stain results. You will be expected to know how these media select and differentiate for cell wall characteristics and/or metabolic differences.
Use a marker to divide the bottom of 2 plates of each medium into 4 sections and organize a labeling system in your lab notebook and on the plate so you can easily identify where you placed each of your soil isolates: 2 isolates and a positive and negative control on one plate of each medium and another plate of each medium can be used for the rest of your isolates with or without controls. (Use E. coli and Staph as controls.) You will spot inoculate the middle of each quadrant by taking a tiny amount of growth and inoculating a single thin zig-zag line in the center of a section. Incubate your plates at room temp for several days until you are sure that any inhibition of growth is from selection and not from slow growing bacteria. Put the plates in the refrigerator before they get overgrown. You can analyze them in Lab 7. See the illustration below of how to inoculate a plate with 4 different cultures.
Fig: 6-1. Testing of multiple isolates in one plate can be accomplished by dividing a plate into 4 (OR MORE) sections. Be sure the inoculum is placed in the center of each section and that you check the plate for growth regularly.
Assessing Bacterial Morphology and Characteristic Arrangement and Cell Wall Composition by Gram Stain
Today you will once again make smear slides (explained here, in lab 4, and in Protocols as Smear Slide Preparation) and perform Gram stains (found here and in protocols as Stains: Gram Stain).
Activity: Preparing a bacterial smear slide
1. Use a graphite pencil to label on the far left side of a clean, glass slide the code ID or name of three of your isolates. If you have more than 3 isolates, make two slides with no more than 3 organisms/slide.On an additional clean, glass slide, label a control slide as SE (Staphylococcus epidermidis), EC (Escherichia coli), and EC/SE mix.
2. Prepare the "control" slide first by using your loop to apply three tiny drops of deionized water to different parts of this pre-labeled slide. Flame sterilize the loop, allow it to cool for a few seconds and touch the cooled loop to a single colony of the control culture plate of S. epidermidis. Pick up a VERY, VERY TINY bit of growth from a single bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony will give you plenty of bacteria. If you take a glob, the bacteria will be so thick, you will not get good stain results.
3. Place the loop with the bacterial growth into the drop of water. Use a circular motion with the loop to make a smooth suspension of the bacteria in the water. Stop when you have spread out the circle of emulsified bacteria as much as possible, while making sure to leave room to make two other smears on the slide. Reflame your loop and repeat the process of inoculating theE. coli control into both the middle drop of water and the drop on the far right. Mix the middle drop of E. coli emulsion as you did for the Staph smear. Flame your loop and cool it for a few seconds. Touch your loop again to a Staphylococcus colony (just a TINY bit) and inoculate the drop of water on the far right, to mix the Staph with the E. coli in the last smear. Spread the bacteria in the mixed smear evenly.
4.Now you are ready to make smears of your isolates. Using your best aseptic technique, flame sterilize your loop. Apply a very small loopful of deionized water (bottle on your bench) to the slide and then add a tiny amount of bacteria from a fresh culture growing on solid medium of one your isolates. Create the smear as you did for the controls moving from left to right and spacing the smears so that 3 isolates can fit on one slide. Repeat for the other isolates. Broths can also be used to make a smear. In this case, be sure to mix the broth, carefully vortex or tap the bottom of the tube. Remove the culture tube top (NOT placing it on the bench) and pass the lip of the culture tube rapidly through the flame. Dip your loop into the broth culture and place a small drop of broth culture of that isolate on the appropriate place on the labeled slide. (The labeling from top to bottom should refer to isolates placed left to right.) Mix the drop with your loop to disperse the broth over an area that will not touch the next culture but is large enough to allow your slide to dry quickly.
5. Allow the smears to air dry completely! Be sure all the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step .
6. Heat fix (to kill and attach organisms to the slide) by passing the slide (smear side up) quickly through a flame 3 times. Use a clothes pin or slide holder to avoid contact with hot glass.
An example of a multiple smear labeled slide:
The Gram Stain
The Gram staining procedure as it is done today, involves: a) primary staining of all cells with crystal violet, b) precipitating the primary stain dye within the cells with iodine (a mordant), c) removing the dye-iodine precipitate from some cells (the Gram-negative) with a decolorizer such as 95% ethanol, acetone, or n-propyl alcohol, and d) counter-staining of the decolorized cells with safranin. Organisms that retain the crystal violet primary dye are termed Gram-positive, while those which lose the primary stain and show the red safranin counter-stain are termed Gram-negative. This differentiation is not absolute, because it is based on the differences in the rate at which the primary dye is lost from the cells. If you over decolorize for too long or with too harsh a decolorizer, Gram-positive organisms will appear Gram-negative. Truly Gram-positive cells, such as Bacillus subtilis or Staphylococcus aureus, will not retain the primary dye if the iodine step is omitted. Criteria for a true Gram-positive state include the requirement of iodine following the crystal violet.
Since the term Gram positive or Gram negative actually refers to a type of cell wall, not all organisms that retain the primary dye of the Gram procedure are really Gram-positive, because they lack that particular cell wall composition. For example, Mycobacterium species have a different type of cell wall but they will take up and retain crystal violet if you use heat. In this case, the crystal violet will be resistant to harsh decolorization and be retained. However, a Mycobacterium type cell wall does not require the use of a mordant, like iodine, to precipitate the stain. True Gram-positive organisms do not retain the primary crystal violet without precipitation by a mordant. Using crystal violet with heat, harsh decolorization, and no mordant describes an acid-fast stain rather than a Gram stain. Mycobacteria have an acid-fast type cell wall; they are neither Gram-positive or Gram-negative, despite the fact that they will appear purple if you do a Gram-stain on them.
Activity: Preparing a Gram Stain
The Gram stain is a standard DIFFERENTIAL staining technique useful for the identification of culturable bacterial organisms and you will perform it now. Remember that a differential stain is one in which different bacteria are subjected to the same stain procedure but they respond differently and show a visible differentiation. In this case, that visible difference is pink vs purple color and it is based on cell wall composition differences.
Use the smear slides you have prepared of your isolates and the controls and follow the Gram Stain Protocol found below. This protocol can also be found in BISC209/F13: Stains in the protocol section of this wiki.
Gram Stain Procedure:
Here is a link to a good video on Gram staining 
To Gram stain the previously prepared heat fixed, bacterial smear slides, perform the staining protocol from start to finish on one slide at a time. You must be careful to apply the staining reagents liberally so all the smears are evenly and completely covered and you must be sure to expose each smear to each reagent for the same amount of time.
1. Place a heat-fixed bacterial smear slide on the staining tray. It is important that the slide is level during staining; therefore, you should use paper towels under the tray to level the slide. If you do this, it is much easier to ensure that your smears will be covered evenly with each reagent with as little waste of the dye as possible (a crucial aspect of proper staining results).
2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.)
3. Rinse the slide by lifting the slide at a 45 degree angle (using gloves or a clothes pin or slide holder) and use a squirt bottle to direct a very gentle stream of water slightly above the top smear. Rinse until the waste water coming off at the bottom is relatively clear. Drain off excess water by touching the edge of the slide to a paper towel.
4. Dispense just enough Gram's Iodine (mordant) to completely cover each smear. Let stand for 1 minute. Rinse thoroughly with a gentle stream of water as in Step 1.
5. Place the slide flat on the staining tray and dispense just enough decolorizing Reagent (80% isopropyl alcohol, 20% acetone) to completely cover each smear. Let stand for exactly 10 seconds and IMMEDIATELY rinse in a very gentle stream of water that is directed above the top smear as in step 3. This step is tricky as it is easy to over- or under-decolorize. .
6. Place the slide flat on the staining tray and dispense just enough Counterstain (0.6% safranin in 20% ethanol) to cover each smear. Let stand for 1 minute; rinse with water as in step 3.
7. Blot dry using the bibulous paper package found in your orange drawer. Do not tear out the pages, just insert your slide and pat it dry.
8. Clean up your area; rinse your staining tray in the sink and leave it to drain upside down on paper towels.
Use of the Compound Light Microscope
Activity: View your stained bacteria.
Refer to the directions for using your compound brightfield microscope BISC209/F13:_Microscopy found in the Protocols section. Today you will use only the 10x and 100x objectives. Remember also to read and follow the directions for care of this precision instrument (particularly on how to avoid getting immersion oil on any objective other than the 100x oil immersion lens). Be aware that there would be no field of microbiology if there weren't good, functioning microscopes to view this unseen world.
Assessing Isolates for Physical Characteristics by Special Stains
Today you will make smear slides (Protocol found at Smear Slide Preparation) and then perform a few special stains to look for endospores and capsules. You may also want to repeat some of your Gram stains if you got ambiguous results on a previous attempt or if your EMB and PEA growth patterns conflicted with an earlier staining result.
Directions for the Gram Stain, Schaeffer-Fulton Endospore stain and Capsule negative stain are found in the Protocols section of the wiki at
All Gram positive bacilli or any bacteria that showed a spore shaped, unstained area in the cells when Gram stained should be stained for endospores. In addition, any Gram positive isolates growing from your dried soil extract on Glyerol Yeast Extract Agar (GYEA) medium should be stained for endospores. There is no need to stain Gram negative isolates for endospores. Most of the spore forming bacteria are common soil organisms. Why would the capacity to form a highly protective, heat tolerant, dessication resistant, non-metabolic spore be useful to soil community microorganisms? Would this capacity give those members a competitive advantage to survive weather extremes? Would you expect a tropical greenhouse habitat to contain relatively fewer or more spore forming members than other habitats?
Detecting Capsules by Negative Stain
Highly mucoid (sticky and wet) colonies could be tested for the presence of a capsule using the capsule stain protocol if you have time. A capsule is a secreted, protective, adhesive, polysacchride envelope layer outside the other parts of the cell envelope. Some, but not all bacteria produce a capsule. It tends to be a virulence factor in bacteria that are human pathogens because it makes it more difficult for the immune system to recognize the bacterium with a capsule as a foreign invader.
Repeating Gram Stains
If your Gram stain results were ambiguous or not what you expected from the growth patterns you observed on PEA and EMB media, you should probably repeat those Gram stains. If there are PEA and EMB plates available, consider reinoculating your latest cultures onto these media. Perhaps your earlier cultures were not pure? If not, what effect might this have on your genomic data? What other implications might there be?
1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.
2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.
3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.
4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.
5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.
6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.
7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.
8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.
9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.
10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.
11. Take off your lab coat and store it in the blue cabinet with your microscope.
12. Wash your hands.
Assignment (20 points): Molecular Techniques Theory and Use. Instructions: Assignment: Understanding Molecular Techniques
To Do Before the Next Lab:
One to 3 days (depending on how fast your organisms grow) prior to Lab 7, please prepare a fresh both culture of each isolate into sterile nutrient broth.
On the same day you make the broths, subculture by isolation streaking EACH of your isolates onto nutrient agar. In Lab 7 these cultures will be used by your team for the interaction assay.