BIO 2247 MICROBIOLOGY LAB

ATENEO DE DAVAO UNIVERSITY

(No part of this material may be copied, reproduced, or disseminated in any form or by any means without
written permission from the Instructor and the Biology Department, Ateneo de Davao University)

ACTIVITY 3
STAINING METHODS FOR MICROORGANISMS

INTRODUCTION

Living bacteria are generally colorless and almost invisible because of their lack of contrast with the
water they may reside in. Thus, staining is necessary to make them readily visible to observe intracellular
structures and overall morphology. However, success at bacterial staining depends, first of all, on preparing a
suitable smear of the organisms. A properly prepared bacterial smear is one that withstands one or more
washings during staining without loss of organisms, is not too thick, and does not result in excessive distortion
due to cell shrinkage.

Dyes as stains are used to make microorganisms more easily seen, to show certain cell structures, or to
reveal their chemical nature. Basic stains are cationic; the chromogen portion exhibits a positive charge and,
therefore, has a strong affinity for the negatively charged constituents of the bacterial cell. On the other hand,
the acidic dyes have a negatively charged colored ion which binds more to the background.

There are several types of stains. A simple stain is sufficient to visualize gross morphology like size
and shape. Differential stains distinguish different groups of bacteria based on certain stained cell components.
An example of this is the Gram stain. In Gram staining, the Gram-positive bacteria are differentiated from the
Gram-negative ones by their thicker cell wall, which absorbs the primary stain and resists decolorization. The
other type is the structural stain which targets specific parts of the cell, allowing the visualization of that
particular component.

Bacteria are most often identified and classified according to their shapes. The basic shapes of bacteria
include coccus (spherical), bacilli (rods) and spiral (corkscrew or curved rod). As cocci-shaped bacteria, they
may arranged in singles (monococci), occur in pairs (diplococci), or groups of four (tetrads). Others occur in
chains (streptococci), in clusters (staphylococci), and in pockets of eight (sarcinae).

Rod-shaped bacteria (bacilli), on the other hand, may occur singly, in chains, or form palisades. Spiral shaped
bacteria are curved rods having a helicoidal or cork-screw shape. These include the comma-shaped (Vibrio),
cork-screw shape with rigid bodies (Spirilla), and flexible forms (Spirochetes).

I. OBJECTIVES: Present at least two objectives for this activity.

II. MATERIALS:

glass slides cover slips alcohol lamp inoculating loop& needle test tube rack microscope
markers masking tape sterile distilled water immersion oil 400ml beaker
Kimwipes test tube rack toothpick denatured alcohol 70%, 95% EtOH wash bottle
staining pan pencils erasers colored pens methylene blue stain carbol fuchsin
Nutrient Agar slant cultures (S. cerevisiae, B. subtilis, E. coli) Gram stain kit 3% KOH solution

II. PROCEDURE:

A. ASEPTIC TECHNIQUE
The general procedure for the aseptic technique is as follows:

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1. Work Area Disinfection. The work area is first treated with a disinfectant to kill any microorganisms that
may be present. This step destroys vegetative cells and viruses; endospores, however, are not destroyed in this
brief application of a disinfectant.

2. Flame Sterilization Process. Flame sterilization is a quick, simple method of killing microorganisms on an
inoculating loop or needle (Fig. 3.1 of the Act. 3 Supplement).

a. Inoculating loops and needles. The transfer of organisms will be performed with an inoculating
loop or needle. To sterilize the loop or needle before picking up the organisms, heat must be applied
with an alcohol lamp flame, rendering them glowing red-hot.

• Hold the inoculating loop or needle with your dominant hand, and flame-sterilize by passing it at
an angle through the flame of an alcohol lamp until the entire length of the wire becomes orange
from the heat. In this way all contaminants on the wire are incinerated.
• Allow the loop to cool for about 15-30 seconds before contacting the inoculum to avoid killing the
microorganisms.
• Make sure that the loop does not come in contact with anything. Never lay the loop down once it is
sterilized, or it may again become contaminated.
• Once cool, the loop or needle can be used for various culture manipulations.
• The loop should be twisted back and forth in the medium to disperse the organisms on the loop. If
an inoculating needle is used for stabbing a solid medium, the needle is inserted deep into the
medium.

b. Culture Tube Flaming. Before inserting the cooled loop or needle into a culture tube, remove the
cotton plug from the mouth of the tube by grasping the plug with the little finger of your non-dominant
hand. Flame the mouth of the culture tube, insert the loop into the broth culture or slant, and draw out
a loopful. Once the loopful of culture has been removed from the tube, the mouth of the culture tube
must be flamed again before returning the plug.

c. Petri Plate Sterilization. To inoculate a Petri plate, an inoculating loop is used for the transfer. Flame
the sides of the plate by rotating it with your fingers before opening the cover. When streaking the
surface of the medium, the cover should be held diagonally over the plate bottom to prevent air
contamination of the medium.

3. Final Disinfection. When all work is finished, the work area is treated with disinfectant to ensure that any
microorganisms deposited during any of the procedures are eliminated.

B. PREPARATION OF BACTERIAL SMEARS

1. Wash a slide with liquid detergent and water, removing all dirt and grease. Handle the clean slide
by its edges.
NOTE: A drop of water on a clean slide will spread out evenly. If the drop rounds up, clean the slide
again. The slide should be thoroughly cleaned and freed of grease.

2. Label one end of the slide properly by writing the group number and name of the organism with a marker
on the surface on which the smear will be made. Ensure that the label does not come into contact with staining
reagents.

3. To provide a target to place the organisms, make a circle on the bottom side of the slide, centrally located,
with a marker.

4. Flame-sterilize the loop and let it cool.

5. a. CULTURE TRANSFER FROM LIQUID MEDIA (Fig. 3.2 of the Act. 3 Supplement).

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 Shake the broth culture vigorously and transfer one to two loopfuls of cultures by spreading it evenly and
thinly to the center of the slide over the target circle. Be sure to flame the loop after it has touched the slide.

b. CULTURE TRANSFER FROM SOLID MEDIA (Fig. 3.3 and 3.4 of the Act. 3 Supplement).
Pick up a very small amount of surface growth in the slant or plate cultures, and mix it into the water on
the slide. Disperse the mixture over the area of the target circle. Be certain that the organisms have been
well emulsified in the liquid. Be sure to flame the inoculating needle before placing it aside.

6. Allow the slide to air dry, don’t apply heat. Make sure the smear has become completely dry before
undergoing a fixed smear.

7. Fix the smear with heat or alcohol. In heat-fixing, pass the slide, film-side up, over the flame of the alcohol
lamp 2-3 times. Do not heat directly the slide to prevent scorching of the specimen. For smears obtained
from milk samples, alcohol treatment is used, and the smear is covered with 95% ethyl alcohol for one
minute and then drained off. Milk has components that curdle during heating, resulting in a poor smear.

8. When working with stains, wear disposable gloves. Place the fixed smear on a staining pan or rack over a
sink or suitable receptacle. Handle the slide with a clothespin to avoid staining the hands since some stains
are carcinogenic. After each application of stain, tilt the slide to remove excess stain.

9. When protocol calls for washing the slide, avoid spraying water directly on the smear. Just allow water to
run down from the top of the slide.

10. Blot-dry the slide with a paper towel after the staining procedure. Do not rub so as not to dislodge the
smear.

C. Prepare the following smears: (Fig. 3.5 of the Act. 3 Supplement).

1. Saccharomyces cerevisiae (S. cerevisiae) for simple staining
Obtain a 24-48 hr. slant culture sample using a wire loop and spread it on a clean slide as a thin, even film
without water. Air-dry and fixed by heat.

2. Cheek scrapings for differential staining

With the toothpick's flat end, gently scrape the inner surface of your cheek and make a smear. Air dry and
fixed by heat.

3. Bacterial Cells for Gram staining

Deposit a loopful of bacterial suspensions of 18-24 hour B. subtilis and E. coli cultures on opposite ends of
the slide. Air dry and fixed by heat.

D. STAINING PROCEDURE

1. Simple Staining (S. cerevisiae cultures) (Fig. 3.6 of the Act. 3 Supplement).
Stain the smear with methylene blue for 3-5 minutes. Wash the stain off the slide with water for a few
seconds. Blot the slide dry with filter paper strips and cover with a cover slip. Observe the specimen under
the oil immersion lens.
Result: The cells will be stained blue.

2. Differential Staining (Cheek scrapings)

Stain with methylene blue for 5 seconds. Wash with water. Counterstain with carbol fuchsin for 3 seconds.
Wash with
water and air dry.

Results: The epithelial cells appear pink, while Diplococcus salivarius cells appear dark blue.

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3. Gram staining (B. subtilis and E. coli) (Fig. 3.7 of the Act. 3 Supplement).
a. Cover the smear with crystal violet and let stand for 1 minute.
b. Using a spray bottle, rinse thoroughly with distilled water. Drain off excess water.
Note: In all washing steps, do not spray water directly on the smear. With the slide tilted,
direct a stream of water from the top of the slide across the smear.

c. Cover the smear with Gram’s iodine solution and let it stand for 1 minute.
d. Rinse with distilled water thoroughly. Drain off excess water.
e. Decolorize with 95% ethanol for 15-30 sec. Do not decolorize for too long. Add the decolorizer drop
by drop until the crystal violet fails to wash from the slide. Rinse with water and remove the excess.
Note: This step is critical. Thick smears will require more time than thin ones.

f. Counterstain with safranin for about 45 seconds. Safranin preparations vary considerably in strength,
and different staining times may be required for each batch of stains.
Rinse with distilled water and drain off excess water. Blot dry with filter paper and air dry. Examine
the slide under the microscope. There is no need to place a coverslip on the stained smear.

Results: Gram-positive organisms stain blue to purple; gram-negative organisms stain

pink to red.

4. String Test using Potassium Hydroxide (3% KOH) (Fig. 3.8 of the Act. 3 Supplement).

The results from the Gram stain can be verified by performing the String Test:
a. Place a drop of 3% potassium hydroxide solution on a clean slide.
b. Take a loop of isolated discrete colony growth and emulsify in the solution.
c. Stir the suspension continuously for 60 seconds and slowly raise the loop from the cells.

Results: Gram-negative cells - Thick and stringy, forming long strands

Gram-negative cells will lyse in the KOH solution, releasing their DNA and causing the
liquid to be viscous. Often, “strings” of DNA adhere to the loop as it is raised from the slide.

Gram-positive cells - Suspension remains unaltered

Gram-positive cells do not undergo lysis in the KOH solution, so an increase in viscosity or
DNA strings will not be seen in these cells.

E. MICROSCOPIC EXAMINATION AND OBSERVATION

View all stained smears under OIO and record your observations in your worksheet. Draw some stained
microorganisms under each smear and identify their cell morphology (Fig. 1). Discard the used stained
slides in the disinfectant bucket (containing 95% EtOH) in the sink.

F. GUIDE QUESTIONS:

1. Provide 3 reasons why the aseptic technique is essential when handling microbial cultures in the laboratory.
2. Provide two examples of heat used during the inoculation of a tube culture.
3. For preparation of a smear on a slide, what is the purpose of heat fixation? What problems can arise when
the slide is heated in a flame?
4. How does the smear preparation of cells from a liquid medium differ from the preparation of cells from a
solid medium?
5. What is the purpose of simple staining?
6. Why are basic dyes more successful in staining bacteria than acidic dyes?

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Figure 1. Bacterial shapes and arrangements. (Cappuccino, J.G. and C.T. Walsh. 2019. Microbiology: A Laboratory
Manual. 12th ed. Pearson Education, Inc. New York, USA).

7. Name the reagent used and state the purpose of each of the following in the Gram stain:
a. mordant
b. primary stain
c. decolorizer
d. counterstain

8. Why must young cultures be used when doing a Gram stain?

9. How do gram-positive and gram-negative bacteria differ in cellular structure, and how does this contribute
to their differential staining properties?

References: _______________________

Sources:

Brown, A. and H. Smith. 2015. Benson’s Microbiological Applications: Laboratory Manual in General
Microbiology. 13th ed. McGraw- Hill Education. New York, USA. 481 pp.

Cappuccino, J.G. and C.T. Walsh. 2019. Microbiology: A Laboratory Manual. 12th ed. Pearson Education, Inc.
New York, USA. 561 pp.

Fernandez, W.L., I.F. Dalmacio, A.K. Raymundo, A.F. Zamora B.C. Mendoza. 2008. Laboratory Manual in
General Microbiology. 3rd ed. University of the Philippines Los Baños (UPLB). Microbiology Laboratory, IBS,
CAS, UPLB. 119 pp.

Public Health England- National Health Service (PHE-NHS). 2018. UK Standards for Microbiology
Investigations: Potassium Hydroxide Test. https://www.gov.uk/guidance/uk-standards-for-microbiology-
investigations-smi-quality-and-consistency-in-clinical-laboratories

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 BIO 2247 MICROBIOLOGY
ANSWER SHEET

ACTIVITY 3
STAINING METHODS FOR MICROORGANISMS

NAME: ___________________________________ Date Performed: ___________________

Group No. ________________________________ Date Submitted: ___________________

I. Objectives: (2 pts)

Complete the following drawings and table for the different staining methods. (15 pts.)
Sketch the cells observed under the microscope. Include the coloration and background.

Features Staining Methods

Simple Differential Gram
Microscopic
Observation

Organism

Cell
Morphology:
Shape

Arrangement
Cell color

Reaction

String Test

II. Compare the different types of stains in terms of the following: (10 pts.)

Type of Stain Characteristics Chemical dyes Color of Cells Limitations

used after Staining
Simple Stain

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 Differential stain

Acid fast stain

Negative stain

Spore Stain

Flagella Stain

III. Answer the guide questions: (16 pts.)

1. Provide 3 reasons why the aseptic technique is essential when handling microbial cultures in the laboratory.

2. Provide two examples of heat used during the inoculation of a tube culture.

3. For preparation of a smear on a slide, what is the purpose of heat fixation? What problems can arise when
the slide is heated in a flame?

4. How does the smear preparation of cells from a liquid medium differ from the preparation of cells from a
solid medium?

5. Why are basic dyes more successful in staining bacteria than acidic dyes?

6. Name the reagent used and state the purpose of each of the following in the Gram stain:
a. mordant

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 b. primary stain

c. decolorizer

d. counterstain

7. Why must young cultures be used when doing a Gram stain?

8. How do gram-positive and gram-negative bacteria differ in cellular structure, and how does this contribute
to their differential staining properties?

References: _______________________ (2 pts.)

Sources:

Brown, A. and H. Smith. 2015. Benson’s Microbiological Applications: Laboratory Manual in General
Microbiology.
13th ed. McGraw- Hill Education. New York, USA. 481 pp.

Cappuccino, J.G. and C.T. Walsh. 2019. Microbiology: A Laboratory Manual. 12th ed. Pearson Education, Inc.
New York, USA. 561 pp.

Fernandez, W.L., I.F. Dalmacio, A.K. Raymundo, A.F. Zamora B.C. Mendoza. 2008. Laboratory Manual in
General Microbiology. 3rd ed. University of the Philippines Los Baños (UPLB). Microbiology Laboratory, IBS,
CAS, UPLB. 119 pp.

Public Health England- National Health Service (PHE-NHS). 2018. UK Standards for Microbiology
Investigations: Potassium Hydroxide Test. https://www.gov.uk/guidance/uk-standards-for-microbiology-
investigations-smi-quality-and-consistency-in-clinical-laboratories