Exercise 4  - Staining Microbes

 Learning objectives

Following this exercise the student should be able to:
1. prepare a smear properly from broth or solid cultures.
2. list reagents, functions and steps of a Gram stain.
evaluate a Gram stain reaction quality and troubleshoot causes of Gram staining problems.
describe the Gram stain reaction, cell shape, and arrangement of S. aureus, E. coli and Bacillus sp.
interpret unknown slides for Gram stain reaction, cell shape and arrangement.
explain the information gathered from AFB, endospore, capsule, flagella and inclusion body stains.

Microbes are invisible to the naked eye and difficult to see and identify even when using a microscope. A simple stain visualizes the microorganisms, a differential stain displays the chemical differences in cellular structures, including the cell wall and cell membrane because the macromolecules within the structure bind to different components of the stain. An example of this differential staining is seen in staining used for blood smears.

Staining white blood cells with a differential stain displays the difference between the five white blood cell types; basophils, eosinophils, neutrophils, monocytes and lymphocytes. The intracellular granules of basophils stain dark blue because of their affinity to basic portion of the stain Basophil means basic loving. On the other hand, the eosinophil (acid loving) stains red as a result of the intracellular granule’s affinity to the acidic portion of the stain. Treatment of microbial diseases depends upon the correct identification of microorganisms and depends upon the ability to read and interpret stained smears. Bacterial cells are commonly stained with a differential stain called the Gram stain and protozoal cells with the Trichrome stain; this reveals the internal structural differences and aids in identification. Properly preparing slides for staining is important to ensure good results. Remember, you cannot see the material you are working with so you must develop good technique based upon principles.

Always start with clean slides using lens paper to clean them. Slides can be made from direct clinical material (a wound, sputum, knee fluid, the throat etc.), broth cultures, and from solid media cultures. The first principle is that some fluid is needed to emulsify the material if it is dry, however too much fluid may make the microbes hard to find. Slides from clinical cultures are usually placed directly on the slide without the addition of water, as are slides from broth cultures. Slides from solid media require water to emulsify and separate the individual bacterial cells for better observation, but a very single drop of water is usually adequate. Next the material must be attached to the slide so they don’t wash off with the staining process. The second principle involves fixing the slide using either a chemical fixative or heat. In this lab a heat-fixing tray will be used.

This lab will use the principles above to make and stain bacterial slides using a differential staining technique called the Gram stain. Initially 3 stock cultures (known types) of bacteria will be stained, and then the 3 isolated unknown microbes from the environmental cultures will be stained and examined. The environmental culture will contain a variety of bacteria and possibly some fungi. Bacterial cells can be observed for shape (rod, coccus, or spirillum) and arrangement (in chains, clusters, etc.). Arrangements of cells are best observed from clinical and broth cultures because the emulsification process disrupts the natural arrangement from colonies "picked" from solid media. Read the portion in the Atlas (pages 21-34) concerning the various staining techniques. A tutorial is also available at http://www.microbelibrary.org/images/mayberry/gramstain/GrmStain_files/frame.htm


Three isolation plates from lab 2

original environmental broth culture original TSA plate

Stock cultures - 1) S. epidermidis, 2) E. coli, 3) Bacillus sp.


transfer loops

Gram stain reagents

      Crystal violet
Decolorizer - Acetone-Ethanol (*note - not acid alcohol)

Preparing the Smears

1. Collect the broth and pure subcultures you made from Ex. 2. Observe the colony morphology. Ask the instructor to critique your isolation technique. This will be very important in later labs. Practice the isolation technique on a new plate if you need some more experience.

2. You will produce several smears in this lab.

It is important to label the slides accordingly; be sure you know which side is up.

3. Put on your gloves. There are 3 steps in preparing a smear for staining. Remember to use aseptic technique and flame the loop before and after each use.

Staining the Smears

In the beginning it is wise to make a single broth slide and a single solid medium slide and then stain and observe these before making the other slides. This allows you to alter your technique if the results are not optimal.

 4. Begin with the known culture smears (S. epidermidis, E. coli, or Bacillus sp.). Place the smear on the staining rack over the sink.


5. Cover the smear area with the crystal violet (gentian violet) stain and leave it for 15 seconds and then rinse the slide with a gentle stream of water.


6. Apply Gram's iodine covering the smear completely for 15 seconds and then rinse.


7. Using the Gram’s decolorizer, apply it a drop at a time to the smear area until no more color leaves the area. (This is the most crucial and difficult step in the procedure. Apply it evenly to the entire smear.) Quickly rinse with water to stop the decolorizing process.


8. Apply Safranin to the smear for 15 seconds, then rinse with water.


9. Allow the smear to air dry or place it on the drying and fixation tray.

(See flow chart) 

Observing and Evaluating the Smear

10. The slide should appear only lightly colored to the naked eye. A good slide is evenly stained and the bacteria are spread thinly enough that you can identify individual cells. The bacteria should not be in clumps as this will alter the amount of stain retained in that area.


11. Observe the slides under oil immersion. You will be able to see little more than color using the scanning or low power. Locate the bacteria on high power; do not use the coarse adjustment to do this. It is of no value to try and observe bacteria on high power. Always go to oil immersion for observation of these small organisms. *Between slides it is not necessary to go back to low and high power. Leave the oil objective at the same location and simply slip a new slide on the stage.


12. Observe the bacterial cells to determine their Gram reaction. These colors are hard to differentiate at first. The purple or violet coloring is Gram positive, the pink coloring is Gram negative. The Staphylococcus epidermidis and the Bacillus sp. should stain Gram positive. Record your observations concerning the Gram reaction, cell shape and arrangement on the lab report sheet. Have the instructor verify your conclusions. Repeat the procedure for the remaining cultures.


13. Clean up.

Thoroughly clean the microscope. Discard the used slides in the sharps container at the biohazard table. Place the cultures in the biohazard can.


14. Check out with the instructor and return your microscope to its original location. Clean your area, wipe down the desk and wash your hands before leaving.

Exercise  4 – Staining Microbes Lab Report



Lab Partners_________________

1. Have the instructor critique your isolation technique from the environmental sub-culture. Write down notes to improve your next isolation plate.








2. Describe the microscopic details (Gram reaction, cell shape and arrangement of the organisms) and the macroscopic details of the colony morphology.



Gram Reaction

Gram + or Gram -

Cell Shape

(cocci, rods, spirilla)

Cell Arrangement
(single, chains, clusters, tetrads, etc.)

Colony Morphology
Correlate the colony appearance with the microscopic appearance

(Use terms from lab 3)

Staphylococcus epidermidis





E. coli






Bacillus sp.






Subculture #1






Subculture #2






Subculture #3







3. Using appropriately colored pencils draw the following cells.

a. Gram positive rod in chains


        b. Several singular Gram negative rods


c. Gram negative coccobacilli


         d. Gram positive staphylococci, if the mordant (iodine) was not applied


          e. Gram negative diplococci, if the decolorizing step was forgotten


  f. Gram positive tetrads


  g. Gram positive & negative rods that were decolorized but the safranin (counterstain) was forgotten



4. During one lab period a student produced a smear that was very thick. The center of the smear looked like Gram positive rods and the periphery appeared to be Gram negative rods. What would you conclude about the gram reaction of these bacteria? Circle all that apply.

a.      The smear reveals the presence of both Gram positive and Gram-negative rods in the specimen.

b.      The smear reveals Gram positive rods that were uniformly over-decolorized.

c.      The smear reveals Gram negative rods that were under-decolorized in the center of the smear.

d.      No conclusions can be made, the inoculum was probably too thick and the entire procedure needs to be repeated.


5. Suppose you are viewing a clinical specimen smear made from a wound culture. You notice that there are Gram positive cocci in clusters and Gram negative rods along with a large number of WBC's. Circle the following assumptions that are consistent with the smear.

a. This is the typical smear of a sterile wound.

b. The presence of WBC's indicates probable infection.

            c. The fact that some bacteria stained GPC and some GNR indicates the stain was done incorrectly.

d. The presence of GPC and GNR would indicate good staining and a wound with a mixed infection.

            e. The bacteria will need to be isolated and identified separately in order to fully treat the infection.


6. Fill in the chart below

Differences Associated with Gram Stain Reaction




Gram Positive


Gram Negative


 Gram stain color






Cell wall








Are these organisms killed by Penicillin?







Are these organisms killed byTetracycline?







Primary type of toxins




Endotoxins and exotoxins


Tolerance to Drying






Typical examples of








7. Observe the clinical smears below. Draw arrows to and identify bacterial cells, red blood cells, and white blood cells. Identify whether they are prokaryote and eukaryote cells and describe the Gram stain reaction, cell shape and arrangement of the bacteria.









Expected Outcome

did not meet expectations

Developing- demonstrates partial completion of expectations

demonstrates achievement of performance level

Questions and drawings are complete

Missing part of the assignment

1-2 blank questions or drawings

Questions and drawings are complete


Correctly answered questions.

Questions contain more than 6 errors

Questions contain 3-5 errors

Questions are answered correctly or with only 1 or 2 errors


Drawings demonstrate understanding.

Some are missing(1-2), or there are several (3 or more) errors

Some errors(1-2) show gram stain process is not clearly understood

Appropriate color and morphology in drawings


Following directions

Tables contain blank spaces or inaccurate descriptions such as Staph epi is gram neg.

Some errors in following directions such as describing the gram stain morphology instead of the macroscopic morphology

Tables are completed with attention to detail using vocabulary and drawings which are exact and descriptive







Text Box: Lab Exercise Grading Rubric





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Bakersfield College | Kern Community College District | Dr. Janet Fulks
1801 Panorama Dr. - Bakersfield,

 Bakersfield College | Kern Community College District | Dr. Janet Fulks
1801 Panorama Dr. - Bakersfield, CA 93305 - (661)395-4381
Date last updated 09/12/2011
©Janet Fulks