SCHOOL OF HEALTH AND NATURAL SCIENCES

LABORATORY ACTIVITY NO. 8

STAINING TECHNIQUE IN MICROBIOLOGY
(SIMPLE STAINING)

Name/s: ________________________________________ Date: ______________

Course and Year: ______________________________ Code: _____________

INTRODUCTION
Individual bacterial cells can be seen under the binocular microscope. It requires however
skillful preparation of smear of bacterial specimen on the microscope slides. In this activity, you will
practice preparing good smears in order to facilitate better microscopic examination of bacterial
morphology.

Meanwhile, you will use a simple staining technique on your smears. In simple staining, only
one type of dye will be used. So, all the bacterial parts will be stained evenly and no special part will be
distinguished. While other techniques like differential and selective staining would reveal some
anatomical parts of the bacterial cell and at the same time also show the bacterial morphology, the
simple staining is better used in studying the morphology of bacteria because it is easier and faster to
perform.

Most bacteria come in one of three shapes: rod, sphere, or spiral. Rod-shaped bacteria are called
bacilli. Spherical bacteria are called cocci, and spiral or corkscrew-shaped bacteria are called spirilla.
Some bacteria come in more complex shapes. A hairlike form of spiral bacteria is called spirochete.
Streptococci and staphylococci are well-known disease-causing bacteria among the cocci.

OBJECTIVES
1. To be able to prepare wet mount of bacterial specimen.
2. To perform the simple staining technique on bacterial specimens.
3. To observe the different bacterial morphology under the binocular microscope.
4. To illustrate the structure/appearance of an individual bacterium.

MATERIALS AND EQUIPMENT

Bacterial cultures Inoculating loop Zonrox
Binocular Microscope Tissue Paper Glass Slides
Coverslip Petri dish Dropper
Methylene blue dye Face Mask Laboratory gown
Alcohol lamp Sterile distilled water Inoculating loop
Rubbing alcohol (75%) Clean Cover Slip

PROCEDURE
Take out the bacterial cultures that you have prepared in the previous activity. Prepare a very
thin smear of bacterial specimen from the different types of colonies that you have identified in the
previous activity. You may need to observe a demonstration from your instructor on the preparation of
a good smear for microscopic study.

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A. Smear Preparation from Solid Media

1. Prepare your rubbing alcohol, glass slide, alcohol lamp, inoculating loop, and sterile distilled
water.
2. Before using the glass slide be sure that it is free from dirt or oil. Clean it with soap or alcohol
then blot dry with tissue paper.
3. Place a drop of sterile/distilled water in the middle of the glass slide.
4. Sterilize the inoculation loop by passing it through the flame until it becomes red hot.
5. After the inoculating loop cools down, scoop a loopful of bacteria from your grown colonies
from the incubated Petri dishes.
6. Spread the bacteria in your glass slide by dipping the inoculating loop on the drop of
sterile/distilled water. Create a thin film of bacteria by smearing it in a circular motion.

7. Heat-kill and fix bacterial smear on the glass slides passing it through the flame three times. DO
NOT BOIL. Then air dry.

B. Staining
1. Pour a drop of methylene blue on your smear and let it stand for one (1) minute.
2. Pass your bacterial smear on the glass slide over a flame three times. Do this procedure as swift
as you can so as not to boil your stained smear, otherwise the specimens will be destroyed.
Quickly passing the slide over the flame would fix the stain on the specimen.
3. Wash the blue stain under a flowing tap water or by using a dropper.
4. Dry the stained smear by gently blotting with soft tissue paper.

C. Observation
1. Put a clean cover slip over your stained smear.
2. Examine the stained smear under the LPO, High Powe Objectives.
3. You must see bacterial cells. Should you see only clumps of stains and/or other dirt under the
microscope, it would mean that you have not prepared a good smear. As such, you may need to
prepare another smear until you distinctly see the bacterial cells.
4. Document your specimens by drawing/illustrating the bacterial morphology that you see under
the microscope.
5. Use the blank tables provided in this activity sheet to illustrate the bacterial morphology that
you saw under the microscope.

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ILLUSTRATION
1. Draw the bacterial cells that you see under LPO and HPO. Describe the morphology and
information.

Type of colony where the specimen was smeared from:

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LPO HPO

Magnification: Magnification:
Description: Description:

Type of colony where the specimen was smeared from:

_______________________________________________

LPO HPO

Magnification: Magnification:
Description: Description:

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DISCUSSION

1. What are the different morphological structures that you saw under the microscope?
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2. Are the bacterial morphologies that you have observed in your smears of bacterial specimens
from the various types of colonies different from or similar to each other?
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3. Based on your answer in number 2, why do you think so?
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4. What information about bacteria can be observed using only simple stain/s?
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5. What are the advantages of using stain/s in microbiology?
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GENERALIZATION
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NAME CONTRIBUTION/PARTICIPATION PERSONAL PROTECTIVE

EQUIPMENT

Date Submitted: _______________________ Score: __________________

Instructor: ____________________________

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