Laboratory Exercise 8

NEGATIVE, SIMPLE, AND GRAM STAINING

Unstained bacterial cells are nearly transparent when observed by light microcopy and
hence are difficult to see. However, most bacteria can be easily stained. Staining involves
spreading a drop of an aqueous suspension of cells on a glass slide, allowing it to dry in air,
fixing it with gentle heat, and then applying staining reagents. Dyes as stains are used to make
microorganisms more easily seen, to show certain cell structures, or to reveal their chemical
nature.

Objectives: At the end of the experiment, the student must be able to:
1. Prepare stained bacterial smears for microscopic examination;
2. Enumerate the advantages of staining microorganisms;
3. Explain the basic principles of staining;
4. Perform the different staining techniques and ;
5. Recognize different morphological characteristics.

A. Negative Staining

Materials:
microscope slides (with polished edges)
nigrosine solution or india ink
TSB broth cultures of S. aureus and B. megaterium
inoculating needle and loop
alcohol lamp

Procedure:
1. Clean the glass slide, remove grease or oil by washing with soap and water. After
cleaning, dry slides and place them on laboratory towels until ready for use. They may
also be stored or dipped in alcohol and polished clean (free of grease) with a tissue or
soft cloth.
2. Sterilize the slides by passing it over the flame at least five times. Place the slide on the
table with the flamed slide up. Remember to hold clean slides with their edges.
3. Label the slide with the name of the microorganism, the type of staining, your initials and
the date.
4. Place several loopfuls of nigrosin or india ink in the center of the microscopic slide. Drop
should not exceed 1/8” diameter and should be near one end of the slide.
5. Use a sterile inoculating loop to transfer the organisms to the liquid and mix the
organisms into the stain.
6. Move the spreader slide toward the drop of suspension until it contact the drop causing
the liquid to be spread along its spreading edge.
7. Once the spreader slide contacts the drop on the bottom of the slide, the suspension will
spread out along the spreading edge.
8. Push the spreading slide to the left dragging the suspension over the bottom slide. Air
dry and examine under the microscope.

B. Preparation of smear from a broth culture

Materials:
microscope slides, inoculating loops, alcohol lamp, TSB broth cultures of E. coli and B.
subtilis

Procedure:
1. Wash a slide with soap and water, removing all dirt and grease. Handle the clean slide
by its edges.
2. Label the slide with the name of the microorganism, the type of staining, your initials and
the date.
3. To provide a target on which to place the organisms, make a 1⁄2″ circle on the bottom
side of the slide, centrally located, with a marking pencil. Later on, when you become
more skilled, you may wish to omit the use of this “target circle.”
 4. Shake the culture vigorously and transfer two loopfuls of organisms to the center of the
slide over the target circle. Be sure to cool the loop completely before inserting it into a
medium. A loop that is too hot will spatter the medium and move bacteria into the air.
5. Spread the organisms over the area of the target circle.
6. Allow the slide to dry by normal evaporation of the water. Don’t apply heat.
7. After the smear has become completely dry, pass the slide over an alcohol lamp flame to
heat-kill the organisms and fix them to the slide. The purpose of fixation is to kill the
microorganisms, coagulate the protoplasm of the cell and cause it to adhere to the slide.
Heat fixed smears to prevent washing out when staining is already applied. Heat fixation
is performed by rapid passage of the air-dried smear (smeared portion) two to three
times over the flame. Don’t overdo this procedure as it might cause the disruption of the
bacterial cell membrane

C. Preparation of Smear from a Solid or Semi-solid culture

Materials: microscope slides, sterile saline solution, sterile distilled water (SDW) inoculating
loops, alcohol lamp, TSA plate cultures of E. coli and B. subtilis

Procedure:
1. Wash a slide with soap and water, removing all dirt and grease. Handle the clean slide
by its edges.
2. Label the slide with the name of the microorganism, the type of staining, your initials and
the date.
3. To provide a target on which to place the organisms, make a 1⁄2″ circle on the bottom
side of the slide, centrally located, with a marking pencil. Later on, when you become
more skilled, you may wish to omit the use of this “target circle.”
4. Put a drop of sterile saline solution (or SDW) to emulsify the culture using a sterile
inoculating loop or needle.
5. Only the tip of the sterile needle or loop should touch the culture to prevent transfer of
too many cells. Do not dig into the agar. Be sure to cool the loop completely before
inserting it into a medium. A loop that is too hot will spatter the medium and move
bacteria into the air.
6. Spread the organisms over the area of the target circle.
7. Allow the slide to dry by normal evaporation of the water. Don’t apply heat.
8. After the smear has become completely dry, pass the slide over an alcohol lamp flame to
heat-kill the organisms and fix them to the slide. The purpose of fixation is to kill the
microorganisms, coagulate the protoplasm of the cell and cause it to adhere to the slide.
Heat fixed smears to prevent washing out when staining is already applied. Heat fixation
is performed by rapid passage of the air-dried smear (smeared portion) two to three
times over the flame. Don’t overdo this procedure as it might cause the disruption of the
bacterial cell membrane

D. Simple Staining: In simple staining, the bacterial smear is stained by a single reagent.
Basic stains with positive charge chromogen are preferred because bacterial nucleic acids
and certain wall components carry a negative charge that strongly attracts and bind to the
cationic chromogen. The purpose of simple staining is to show the morphology and
arrangement of bacterial cells. The most commonly used basic stains are methylene blue,
crystal violet and carbol fuchsin

Materials: microscope slides, inoculating loops, alcohol lamp, methylene blue, wash bottle,
bibulous paper, slides with heat-fixed smear

Procedure:
1. Use the previously prepared smears, one from broth cultures and one from the plate
cultures.
2. Place the slides on a staining rack and flood them with methylene blue. Leave the stain
on for 1 minute.
3. Gently wash the smear with tap water to remove the excess stain. Hold the slide parallel
to the stream of water. In this way, you can reduce loss of organisms from the
preparation. Wash off the stains that got on the bottom of the slides.
4. Air dry the slide or blot it dry with bibulous.
5. Examine the slide under oil immersion objective.
 6. Draw microscopic observations.

E. Gram Stain
Differential staining requires the use of at least three chemical reagents that are applied
sequentially to a heat-fixed smear. The first reagent is called primary stain. Its function is to
impart its color to all cells. In order to establish a color contrast, decolorizing reagent is used.
Based on the composition of the cellular components, the decolorizer may or may not remove
the primary stain from the entire cell. The final reagent, the counterstain has a contrasting color
to that of the primary stain. If the primary stain is removed, the decolorized cellular components
will accept and assume the color of the counterstain. In this way, cell types or their structures
can be distinguished from each other on the basis of the stain that is retained.
The most important differential stain used in bacteriology is the Gram stain, named after Dr.
Christian Gram. It divides the bacteria into two major groups: gram positive and gram negative,
which makes it an essential tool for classification and differentiation of microorganisms. The
gram stain reaction is based on the difference in the chemical composition of bacterial cell walls.
Gram positive cells have thick peptidoglycan layer, whereas the peptidoglycan layer in gram
negative cells is much thinner and surrounded by outer lipid-containing layers.

Materials:
slides with heat-fixed smears, wash bottle of distilled water, gram-staining kit and wash bottle,
bibulous paper, slides with heat-fixed smear

Procedure:
1. Use the previously prepared heat-fixed smears, one from broth cultures and one from plate
cultures.
2. Place the slides on a staining rack and flood and cover the smear with crystal violet and let
stand for 20 seconds.
3. Briefly wash off the stain, using a wash bottle of distilled water. Drain off excess water.
4. Cover the smear with Gram’s iodine solution and let it stand for one minute.
5. Pour off the Gram’s iodine and tilt the slide and let 2-3 drops of decolorizer 95% ethanol run
over the slide. If the last drop is still purple, continue decolorizing, 2-3 drops at a time, until
the decolorizer runs clear. Do not over decolorize!
6. Stop action of the alcohol by rinsing the slide with water from wash bottle for a few seconds.
7. Cover the smear with safranin for 45 seconds.
8. Wash gently for a few seconds, blot dry with bibulous paper, and air-dry.
9. Examine the slide under oil immersion. Record the morphology, arrangement and gram
reaction of the bacteria. Draw microscopic observations.
Study Guide Questions:
Short but concise answers must be placed after every question. Copy each question first before
writing your answer. Maximum of 2 sentences.

1. What is the purpose of heat fixation? What happens when too much heat is applied?
2. Which is more effective in staining, acidic or basic dyes? Why?
3. Describe the advantages and disadvantages of negative staining.
4. Describe the advantages of differential staining procedures compared with simple staining
techniques.
5. Give the purpose of each of the following reagents in a differential staining procedure:
a. Primary stain
b. Counter stain
c. Decolorizing agent
d. Mordant
6. Compare the reactions of gram negative with gram positive bacteria in every step of the
staining process.