This laboratory session deals with the isolation of pure cultures of microorganisms and the methods used to examine their microscopic morphology. The isolation of pure colonies of microorganisms is essential to the performance of any microbiological procedure. The most commonly employed technique for obtaining pure isolates is the streaking of culture material onto agar plates. This mechanical means of obtaining pure isolates is helped by using special media.


Tryptic Soy Agar (TSA): a general purpose, non-selective, non-differential, supportive medium that supports growth of all microorganisms that do not require special nutrients.

Blood Agar Plate (BAP): a good general purpose, enriched, differential medium that supports the growth of most of the clinically important microorganisms. This medium allows some differentiation of bacterial species based on their ability to lyse the red blood cells contained in the agar.

MacConkey agar (MAC): a selective, differential medium designed to allow growth of only Gram-negative microorganisms, specifically the gram-negative enteric bacilli. This medium allows differentiation of these organisms by their ability or inability to ferment lactose.

Phenyl Ethyl Alcohol Blood agar (PEAB): a selective medium for gram-positive bacteria that retards or completely inhibits the growth of gram-negative organisms. The addition of blood also makes it differential, similar to BAP.

Specimen material is streaked on the surface of these media to yield isolated colonies. Culture specimens may contain many different microorganisms. It is essential for the microbiologist to separate these organisms into distinct, pure colonies before any identification of the organisms can be done.

When plates are streaked for isolation, the organisms present in the inoculum are streaked over the surface of the agar so that individual organisms are dispersed across the plate. Thus each organism is a sufficient distance from all other organisms to allow it to multiply and yield a pure colony of itself. It is assumed that pure colonies result from the division of a single, isolated bacterium.


Aseptic technique refers to procedures by which cultures may be manipulated without infecting the worker or contaminating the cultures or the laboratory environment.

The flaming of lips of tubes and flasks must ALWAYS be done whenever culture liquid is to be poured from a container (e.g., pouring plates). Flaming should be routinely done when caps are removed from tubes during transfer of cultures. The purpose of flaming is not to sterilize, but to warm the tube and create warm air convection currents up and away from the opening. This "umbrella" of warm, rising air will help to prevent the entrance of dust particles upon which contaminating bacteria reside.

Petri dish lids prevent dust from falling directly onto plates but allow diffusion of air around the edges. There are no direct air currents into the plate, and to enter, dust particles would have to rise vertically more than a centimeter. This does not often occur because of the density of the particles. Whenever the lid is removed, it should be held over the plate as a shield. Do not place the lid on the bench top. Do not leave plates uncovered. Do not walk around the room with an open plate.

When working with cultures in testtubes, work as rapidly as is consistent with careful technique. Keep the tubes open a minimum amount of time. While the tubes are open, hold them at a 45 degree angle so that dust cannot fall into the open tube. Hold the tubes away from your face while transferring.

Testtubes are handled in the following manner:

Label all cultures with the name or number of the organism, and your name.

Always clean the work area thoroughly with disinfectant before leaving the laboratory! The last step before leaving the lab is to wash your hands with Septisol.


The Gram stain is the most useful stain in microbiology. It allows one to learn both the stain reaction and morphology of bacteria. Almost all decisions regarding procedures used to identify an organism, are made based upon the results of a Gram stain.

The procedure consists of staining with crystal violet (a purple dye), mordanting with iodine, decolorizing with acetone-alcohol, and counter-staining with safranin (a red dye). Bacteria are divided into two groups by this procedure. The stain reaction correlates with the type of cell wall makeup. Those bacteria that are not decolorized retain the purple color of the crystal violet, and are GRAM- POSITIVE. Those decolorized by the acetone-alcohol are stained red by the safranin counter-stain and are GRAM-NEGATIVE.

The distinction is not always perfect; dead or dying bacteria stain red whatever their cell wall makeup. Young cultures (18-24 hrs old) should be used for staining. Some bacteria are Gram variable under any condition, but this small group does not significantly detract from the usefulness of this technique.

Preparation of Smears: For any stain to be successful, the smear must be properly prepared. If the smear is too thick, individual cell morphology is obscured; if too thin, the cells will be lost on the vast field of the slide. If properly done, the smear should dry almost immediately, leaving a faint opacity. Many of the difficulties encountered (especially in the decolorization step) are due to improperly prepared smears.



Emulsify your sample taken from one colony with a sterile loop or needle in a very small drop of saline on a clean glass slide. Spread it out to leave a thin film.

  1. Allow smear to air dry. (Heating destroys the morphology of the cells.)
  2. Flame quickly to fix the smear to the slide.
  3. Place slide on wire rack, over the staining tray.
  4. Flood the smear with CRYSTAL VIOLET; let stand 15 seconds.
  5. Wash stain off with a gentle flow of water; drain.
  6. Flood smear with GRAM'S IODINE; let stand 15 seconds.
  7. Wash stain off with a gentle flow of water; drain.
  8. Decolorize with ACETONE-ALCOHOL until the solvent flows colorlessly from the slide. Use less solvent and time for thin films or for smears taken from liquid cultures.
  9. Wash off quickly with a gentle flow of water; drain.
  10. Flood smear with SAFRANIN; let stand 15 seconds.
  11. Wash stain off with a gentle flow of water; drain.
  12. Blot smear with bibulous paper (NOT LENS PAPER) and let dry before viewing.

Single Colony Isolation (Quadrant Method) Note: The Single Colony Isolation diagram shows widely spaced streak lines. This is only to illustrate the pattern of the streak lines. The streak lines on a properly streaked plate are very close together, almost touching.

  1. Sterilize the loop to redness and cool by stabbing the agar at the edge, Diagramor inserting the loop into the broth.
  2. Collect one or more colonies (or a loopful of broth) on the loop.
  3. Lift one edge of the plate cover while the plate rests on the bench.
  4. Place the inoculum on the agar at the top of the plate.
  5. Use close, parallel strokes to cross back and forth over 1/4th -1/3rd of the plate (a).
  6. Close the plate, turn the plate 1/4 turn counterclockwise and sterilize the loop.
  7. Cool the loop and streak over the old strokes about 6-8 times, Diagramending by extending the close parallel strokes into the middle of the plate (b).
  8. Close the plate, turn the plate 1/4 turn counterclockwise and sterilize the loop.
  9. Cool the loop and streak over the old strokes about 3-4 times, again ending by extending the close parallel strokes into the middle of the plate (c).
  10. Close the plate, turn the plate 1/4 turn counterclockwise and sterilize the loop.
  11. Cool the loop and streak over the old strokes 2-3 times and finish by extending the close parallel strokes into the middle of the plate (d).



Materials supplied: work in pairs) Procedure:
  1. Each pair of students will make smears and do a Gram stain of each microorganism on BAP. Each student will be responsible for knowing the Gram reaction and colonial characteristics of each organism presented.
  2. Each student will be given his/her own unknown mixture tube. Record the number on the Report Sheet. Prepare a Gram stain of the culture and inoculate each of the following media by the Quadrant Method to obtain single colony isolation. Retain your slides until the next lab period.
  3. Mark all petri dishes on the BOTTOM with your name and the name or number of the organism. Place the petri dishes in the 37C incubator in an inverted position (i.e., with the agar part of the plate up).


  4. Observe the TSA plate. Do Gram stains on 2 colonies that have distinctly different colony morphologies. Compare these slides with those completed during the previous lab, to note the effect of age on stain retention and to rate the consistency of your staining technique.
  5. Do Gram stains on the colonies that appear on the selective media. Match up by Gram reaction, the colonies appearing on the selective media with the colony type that appears on the TSA plate. Are the results consistent with what you know about these selective media?
  6. Coordinate the results of the Gram stains from the selective media so that they match the results obtained from the TSA plate Record your data and turn in the Report Sheet before the end of class.

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