BACHELOR OF SCIENCE IN NURSING:

MICROBIOLOGY AND PARASITOLOGY LABORATORY

LABORATORY EXERCISE WEEK
SIMPLE STAINING AND
11
GRAM STAINING

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

• Learn and observe the proper procedures in preparing bacterial smears aseptically
• Explain the purpose of simple staining
• Classify bacteria according to their shape, size and arrangement
• Observe bacteria from simple staining under the compound light microscope
• Explain the different steps in Gram staining
• Understand the principle behind Gram staining
• Distinguish between Gram positive and Gram negative bacteria
• Understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on the
biochemical and structural differences of their cell walls

INSTRUCTIONS
• Study the principle of simple staining
• Watch the video tutorial on how to perform simple staining
• https://www.youtube.com/watch?v=2EykxVWGFGc
• Study the principle of Gram staining
• Watch the video tutorials on how to perform Gram Staining
• https://www.youtube.com/watch?v=FI0A3U1okXE
https://www.youtube.com/watch?v=sxa46xKfIOY
https://www.youtube.com/watch?v=2MaZyId3XXY
https://www.youtube.com/watch?time_continue=29&v=EdGnGKObzcI&feature=emb_logo
• Accomplish week 11 Lab activity
INTRODUCTION
Most staining procedures such as Gram staining, AFB staining and endospore staining begin with
preparation of bacterial smear. A bacterial smear is a thin layer of the specimen placed on the slide.
Bacterial smears from solid or broth media are performed to fix the bacteria to the slide so that the sample
will not be washed off during staining.

As in other microbiology laboratory activities, it is important to practice aseptic technique during the
preparation of a bacterial smear. These measures are done to prevent the contamination of the culture
and other materials in the laboratory, and to ensure the safety of the people who are performing the
procedure.

Some of these aseptic techniques include:

• Disinfecting the work area such as table tops before and after use
• Minimizing the time that cultures and growth media are open to the environment.
• Avoiding touching or breathing into the sterile culture media or the stock cultures.
• Sterilizing loops, needles, pipettes and other tools before and after they are used
• Refraining from placing tube caps on the table top.
• Holding the tube caps in the hands while inoculating
• Flaming the lip of test tube after the cap is removed

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.
In the 1800’s, Hans Christian Gram, a Danish bacteriologist, who was working with tissue samples from
the lungs of patients who had died from pneumonia, developed a technique for staining bacteria that is
still widely used today.

Gram staining is a differential staining method that involves the application of a series of dyes that leaves
some bacteria purple and others pink.
.
Bacteria that stain purple are considered Gram-positive, and those that stain pink, Gram-negative. The
specific stain reaction of a bacterium results from its cell wall composition. The difference in color of
bacteria after gram staining is due to the difference in the thickness of the peptidoglycan layer of their
cell walls.

Gram staining involves three processes: staining with a water-soluble dye called crystal violet,
decolorization, and counterstaining, usually with safranin.

PROCEDURE FOR PERFORMING GRAM STAINING

MATERIALS:
• glass slides
• toothpick
• microscope
• baby oil
• 95% ethyl alcohol
• distilled water / NSS
• inoculating loop
• Gram staining reagents:
o crystal violet
o Gram’s iodine
o acetone-alcohol or 95% alcohol
o safranin

PROCEDURE:

A. BACTERIAL SMEAR

• Obtain 2 clean glass slides

• Label your glass slides as follows:
o Slide #1 SS # (table #)
o Slide #2 GS # (table #)
• Sterilize your inoculating loop and add a small scoop of water on the glass slide
• Using a toothpick, scrape the inner lining of your cheeks gently. Avoid scraping
large chunks of cheek cells
• Spread the scrapings on a clean microscope slide.
 • Air dry the smear.
• While holding the slide at one end, quickly pass the smear over the flame of an
alcohol lamp two to three times.(heat fixation)

B. SIMPLE STAINING
• 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

C. GRAM STAINING
• Cover the smear with crystal violet and allow the stain to act for one minute.
Wash with water.
• Flood with Gram’s iodine for one minute. Wash with water.
• Decolorize with acetone-alcohol or 95% ethyl alcohol until visible stain has been
washed off. Wash with water.
• Counterstain the smear with safranin for 60 seconds. Wash with water.
• Blot dry and examine under oil immersion objective.

Engelkirk, P., Duben-Engelkirk, J., & Fader, R. (2018). Burton's microbiology for the health sciences.
LWW.

Gram stain | Microbiology lab. (n.d.). Lumen Learning – Simple Book

Production. https://courses.lumenlearning.com/suny-mcc-microbiologylab2/chapter/gram-stain/

The Gram stain: Its persistence and its quirks. (2013, February). Small Things
Considered. https://schaechter.asmblog.org/schaechter/2013/02/the-gram-stain-its-persistence-and-its-
quirks.html

Lab 6: Gram stain and capsule stain. (2020, July 14). Biology
LibreTexts. https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiol
ogy_Labs/Microbiology_Labs_II/Lab_06%3A_Gram_Stain_and_Capsule_Stain

Laboratory perspective of Gram staining and its significance in investigations of infectious diseases
Thairu Y, Nasir IA, Usman Y - sub-saharan Afr J Med. (2014, October 1). Sub-Saharan African Journal
of Medicine - Free full text articles from Sub-Saharan African Journal of
Medicine. https://www.ssajm.org/article.asp?issn=2384-
5147;year=2014;volume=1;issue=4;spage=168;epage=174;aulast=Thairu

Petersen. (2016). Laboratory exercises in microbiology: discovering the unseen world through hands-
on investigation. CUNY Academic
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Staining microscopic specimens | Microbiology. (n.d.). Lumen Learning – Simple Book

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 MICROBIOLOGY AND PARASITOLOGY LABORATORY
Week 11 Lab activity: SIMPLE STAINING AND GRAM STAINING

NAME:___________________________________ Date: ____________

Year and Section:___________________________

I. 1. Draw (with correct color and shape) and label completely what you saw under the
microscope.
A. Simple staining

B. Gram staining
II

List the steps of the Gram staining procedure and fill in the table.

STEP REAGENTS/DYES PURPOSE GRAM POSITIVE GRAM NEGATIVE

COLOR/REACTION COLOR/REACTION

III. Answer the following questions:

1. What is the importance of simple staining?

2. What are the advantages of gram staining over the simple staining technique?
3. What is the disadvantage of preparing thick smears for bacterial smears?

4. Give 3 purposes of fixation.

5. Differentiate the cell walls of gram positive and gram negative bacteria.

6. What is the importance of determining if a bacterium is Gram positive or Gram negative?