BACHELOR OF SCIENCE IN NURSING:

MICROBIOLOGY AND PARASITOLOGY

LABORATORY
LABORATORY EXERCISE WEEK
6 8
SIMPLE STAINING

At the end of this exercise, each student must be able to:

o Explain the basic mechanism of simple staining
o Explain the purpose of simple staining
o Classify bacteria according to their shape, size and arrangement

INSTRUCTIONS
 Study the principle of simple staining
 Watch the video tutorial on how to perform simple staining
 https://www.youtube.com/watch?v=2EykxVWGFGc
 Accomplish Lab Report # 6

INTRODUCTION
Most cells and microorganisms lack color and contrast when viewed under the microscope. It is
therefore very difficult to observe important cellular structures and their distinguishing characteristics
without artificially treating specimens. Staining is done to give color to certain features of a specimen
before examining it under the microscope.
Simple staining is a procedure which utilizes only one kind of dye to color the bacterial cells. Basic dyes
such as methylene blue, safranin, or crystal violet are usually used. These positively charged basic
dyes readily adhere to the cell surface since most bacterial cells have negatively charged surface and
cytoplasm.

Simple staining allows one to observe the morphology such as cell size, shape and arrangement of the
bacterial cells. Morphology refers to “form” or shape of the cells. Cells of a given species tend to
arrange themselves in a typical pattern. Bacteria are usually classified according to the shape and
arrangement of their cells. Cocci have round shapes. Bacilli are rod-shaped bacteria. Spirilla are spiral
shaped bacteria. According to arrangement, bacteria may be classified as: strepto: arranged in chains,
diplo: small groups of two, tetra: groups of four, sarcina: groups of eight, staphylo: grape-like clusters,
palisades: parallel bacilli.

It must be noted that a properly prepared bacterial smear is essential to be able observe morphology
and arrangement of bacterial cells accurately.

PROCEDURE FOR PERFORMING SIMPLE STAINING

MATERIALS:
 glass slides
 bacterial cultures
 microscope
 cedarwood oil
 95% ethyl alcohol
 distilled water / NSS
 inoculating loop and needle
 stains: crystal violet, methylene blue, safranin

PROCEDURE:

 Prepare a smear from a bacterial culture on clean glass slide

o Place a drop of water or NSS on a clean glass slide by means of a loop
o Pick a small amount of a colony with a sterile loop and transfer this to the
drop on the slide.
o Spread to about 1 sq. cm. area and air dry.
o Fix the smear by passing the slide over a flame. Avoid excessive heating
since it will alter the composition of the bacterial cell.
 Place the slide on a staining rack and cover the smear with methylene blue,
safranin or crystal violet stain and allow to act for about 1-2 minutes.
 Remove the excess stain by washing gently in tap water.
 Blot dry the preparation with absorbent water. DO NOT RUB.
 Examine the stained smear under oil immersion objective
Engelkirk, P., Duben-Engelkirk, J., & Fader, R. (2018). Burton's microbiology for the health sciences. LWW.

OpenStax CNX. (2018, Mar 19). OpenStax Microbiology. Retrieved from http://cnx.org/contents/e42bd376-624b-4c0f-972f-
e0c57998e765@4.24

Petersen, J. a. (2016). Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation.
CUNY Academic Works. Retrieved from http://academicworks.cuny.edu/qb_oers/16

Staining microscopic specimens | Microbiology. (n.d.). Lumen Learning – Simple Book

Production. https://courses.lumenlearning.com/microbiology/chapter/staining-microscopic-specimens/

Fitzgerald, H. (2018) Unit 4: Streak Isolation from a Mixed Broth and Simple Stains
Creative Commons Attribution-NonCommercial 4.0 International License.
 MICROBIOLOGY LABORATORY
Lab Report # 6: SIMPLE STAINING

NAME: ARIANNE JANS E. MUNAR Date: 03-23-21 _

Year and Section:_ BSN-6

I. Perform the simple staining procedure using homemade resources. Show proof of
performance of the procedure by attaching pictures. Make sure you are included in the
picture.

II. Look for photomicrographs of different kinds, shapes and arrangements of

bacteria ( diplococci, streptococci, staphylococci, bacilli, vibrio etc. ) as seen under the
microscope using simple staining. Make sure to cite all your references.

III. Answer the following:

1. What is the importance of simple staining?
-The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells. The
most commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin.

2. Can you differentiate Gram positive and Gram negative bacteria using simple
staining procedure? Why?
 -Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive
and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain
during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained
by the safranin in the final staining process. The process involves three steps:

1. Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is
added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the
original crystal violet stain and iodine and is insoluble in water.
2. A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan
layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened
peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer
membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells
is unable to retain the crystal violet-iodine complex and the color is lost.
3. A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the
safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However,
the decolorized Gram negative cells are stained red.

3. Explain the principle behind simple staining

-Its principle is based on producing a marked contrast between the organism and its
surroundings by using basic stain. A basic dye consists of a positive chromophore,
which strongly attracts to the negative cell components and charged molecules like
nucleic acids and proteins.