MICROBIOLOGY LAB MANUAL
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.
3. evaluate a Gram stain reaction quality and troubleshoot
causes of Gram staining problems.
4. describe the Gram stain reaction, cell shape and arrangement of S. aureus, E. coli
and Bacillus sp.
5. interpret unknown slides for Gram stain reaction, cell shape and arrangement.
6. explain the information gathered from AFB, endospore, capsule, flagella and
inclusion body stains.
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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.
Dr Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
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 2134) concerning the various staining techniques. A tutorial is also available at
http://www.microbelibrary.org/images/mayberry/gramstain/GrmStain_files/frame.htm
Materials:
Three isolation plates from lab 2
original environmental broth culture original TSA plate
Stock cultures
S. epidermidis
slides
E. coli
transfer loops
Bacillus sp.
Gram stain reagents
Crystal violet
Decolorizer - Acetone-Ethanol
Safranin
Iodine
(*note - not acid alcohol)
Dr Janet Fulks
Bakersfield College
August 2010
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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.
 One smear will be made of each of the known stock cultures (S. epidermidis, E. coli, or
Bacillus sp.).
 One smear from your broth.
 And one from each of your pure culture plates sub cultured from the environmental
cultures (Ex. 2)
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.
 Preparation of the slide- Clean and dry the slide thoroughly to remove oils.
 Preparation of the smear – From the broth culture use the loop to spread one or two drops
of specimen in the center of the slide spreading it until it is approximately the size of a
nickel. When making a smear from solid media cultures, start by putting a very small drop of
water in the center of the slide and then mix a loop full of bacteria from the surface of solid
media in the water, spreading it out to the size of a nickel.
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Preparing the Smears
MICROBIOLOGY LAB MANUAL

Fixation - The point of fixation is to attach the organisms and cells to the slide without
disrupting them. In this class we will use an electric fixing tray that will dry and fix the smears
in one step. Slides must be completely dry and fixed before staining, or they will wash off.
Note - Smears made from a broth look shiny even when they are dry.
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, and then rinse with water.
9. Allow the smear to air dry or place it on the drying and fixation tray.
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.
Dr Janet Fulks
Bakersfield College
August 2010
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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.
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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.
MICROBIOLOGY LAB MANUAL
Exercise 4 – Staining Microbes Lab Report
Name_________________
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.
Specimen
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
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Subculture #3
Dr Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
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
Dr Janet Fulks
Bakersfield College
August 2010
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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. The smear was done correctly but some of the Gram positive rods were dead and
stained Gram negative.
e. No conclusions can be made, the inoculum was probably too thick and the entire
procedure needs to be repeated.
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g. Gram positive & negative rods that were decolorized but the safranin counterstain
was forgotten
MICROBIOLOGY LAB MANUAL
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
Feature
Gram Positive
Gram Negative
Gram stain color
Cell wall
Components
Penicillin
Sensitivity
Tetracycline
Sensitivity
Toxins
Exotoxins
Endotoxins
Tolerance to Drying
High
Low
Typical examples of
Bacteria
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. The colors are necessary so observe this online to write your
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observations.
Dr Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
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.
Dr Janet Fulks
Bakersfield College
August 2010
MICROBIOLOGY LAB MANUAL
Lab Exercise Grading Rubric
Expected Outcome
Unsatisfactory
did not meet
expectations
Developing- demonstrates
partial completion of
expectations
Questions and
drawings are
complete
Missing part of the
assignment
1-2 blank questions or
drawings
Correctly answered Questions contain
questions.
more than 6 errors
Questions contain 3-5
errors
Drawings
demonstrate
understanding.
Some are
missing(1-2), or
there are several (3
or more) errors
Following
directions
Tables contain
blank spaces or
inaccurate
descriptions such as
Staph epi is gram
neg.
Some errors(1-2) show
gram stain process is not
clearly understood
Accomplished
demonstrates achievement of performance
level
Questions and drawings are complete
Questions are answered correctly or with only
1 or 2 errors
Appropriate color and morphology in
drawings
Some errors in following Tables are completed with attention to detail
using vocabulary and drawings which are
directions such as
exact and descriptive
describing the gram stain
morphology instead of the
macroscopic morphology
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TOTAL POINTS:
Dr Janet Fulks
Bakersfield College
August 2010