How to perform a Gram Stain

Jasleen Singh

Table of Contents iii

Table of Contents Table of Contents ............................................................................................................................................. iii

Introduction ........................................................................................................................................................ 5

Terminology ....................................................................................................................................................... 7

Terms to be familiar with ............................................................................................................................. 7

Gram Staining ..................................................................................................................................................... 8

What is Gram Staining? ................................................................................................................................ 8

Gram-Positive ................................................................................................................................................ 9

Gram-Negative .............................................................................................................................................. 9

Bacterial Smearing ........................................................................................................................................... 10

Safety ................................................................................................................................................................. 11

Lab Safety ..................................................................................................................................................... 11

Equipment for the Smear Preparation .......................................................................................................... 12

Bunsen burner .............................................................................................................................................. 12

Inoculation loop ........................................................................................................................................... 12

Microscope slide .......................................................................................................................................... 12

Sterile water .................................................................................................................................................. 12

Broth culture ................................................................................................................................................ 12

Plate culture ................................................................................................................................................. 12

Equipment for the Gram Stain ....................................................................................................................... 13

Crystal Violet ............................................................................................................................................... 13

Gram’s Iodine .............................................................................................................................................. 13

95% Ethanol .................................................................................................................................................. 13

Safranin ......................................................................................................................................................... 13

Immersion oil ............................................................................................................................................... 13

Bibulous paper ............................................................................................................................................. 13

Lens Paper .................................................................................................................................................... 13

Steps to follow for Smear Preparation .......................................................................................................... 14

Smear Preparation from Plate culture ...................................................................................................... 15

Steps to follow for Gram Staining ................................................................................................................. 16

Gram Stain .................................................................................................................................................... 16

iv Table of Contents

Microscopy ........................................................................................................................................................ 17

Examining under the microscope ............................................................................................................. 17

Index .................................................................................................................................................................. 18

Citations ............................................................................................................................................................ 19

Introduction 5

Introduction Pre-med students have to take microbiology with its corresponding lab as a prerequisite to get into

the biology program at their university. During the first lecture, students are told that they will use

different staining procedures to view the organisms being discussed, but they aren’t told how to

properly stain. When students come into lab they are enthusiastic about being able to view the

different bacteria and organisms described to them in class. It is hard for students to conduct the

experiment correctly when they are having difficulty following the procedures given to them.

Students are required to differentiate bacterial species into two large groups, Gram-negative and

Gram-positive. It is difficult to differentiate species into these two groups when one doesn’t know

what a Gram Stain does and how to read the results of the stain. Students also need to be aware of

safety precautions. During the beginning of the lab, students are given a vague safety discussion, but

they need a more detailed discussion in which the precautions for the specific lab need to be

presented.

One of the best ways to observe cultured bacteria under a microscope is by staining it. Some bacteria

cells are negatively charged and some positively charged. Due to this difference in charges, different

stains have to be used to observe different bacteria under a microscope. This manual will teach you

how to properly conduct a Gram stain. It is written in chronological order to help you through the

process step by step.

Figure 1. Bacterial

Culture

Terminology 7

Terminology

Terms to be familiar with Broth culture-A bacterial culture in which the microorganism is suspended in a liquid

nutrient medium.

Bibulous Paper-Blotting paper

Crystal Violet- Primary stain (purple)

Gram’s Iodine- Mordant, added to enhance the crystal violet staining by forming a crystal

violet-iodine complex

Gram Stain- Method of differentiating bacterial species into two large groups, Gram-positive

and Gram-negative

Gram-negative cells- Cells that have a high concentration of lipids; dissolve in decolorizer

Gram-positive cells- Cells that are rich in mucopolysaccharides; resist decolorization

Inoculation loop- Used to transfer cultures of microorganisms

Peptidoglycan- A polymer consisting of sugars and amino acids that forms a mesh-like layer

outside the plasma membrane of bacteria, forming the cell wall

Plate culture- A bacterial culture that is grown on a Petri dish that contains agar plus

nutrients.

Safranin-Counter stain

95% Alcohol Solution-Decolorizer

8 Gram Staining

Gram Staining

What is Gram Staining? The method of Gram Staining was developed in 1884 by a Danish scientist Hans Christian Gram.

The technique serves to make bacteria more visible by providing a contrast between microorganisms

and their background.

This also allows differentiation between various morphologies. Gram Stain divides bacteria into

two groups, Gram-positive and Gram-negative, based on the ability of cells to retain the purple color

of crystal violet after decolorization with alcohol. This retention is based on structural differences

between the cell walls of the two groups(Beck, Hughes & O'Donovan, 2000).

Figure 2. Gram Stain of staphylococcus bacteria

Gram Staining 9

Gram-Positive The cell wall of Gram-positive bacteria consists of thick layers of peptidoglycan outside the cell

membrane. During the Gram Stain, an insoluble crystal violet-iodine complex is formed when a

mordant, Gram’s Iodine, is applied to the cells that have been stained with crystal violet. Once the

decolorizing agent alcohol is applied, the pores in the peptidoglycan layers of a Gram-positive cell

become dehydrated and close. This traps the crystal violet-iodine complex in the cells and gives

them a purple color.

Gram-Negative The cell wall of Gram-negative bacteria is more complex in structure. It consists of a thin layer of

peptidoglycan outside the cell membrane and an additional outer membrane layer (Beck, Hughes &

O'Donovan, 2000). When the decolorizing alcohol is applied to these cells, it easily penetrates the

outer membrane and thin peptidoglycan layer, allowing the dye-iodine complex to be easily

removed from the cell. The resulting cells are colorless. A counter stain, Safranin, is applied to give

the cells a red-pink color.

Figure 3. Gram Positive and Gram Negative cell wall structures.

10 Bacterial Smearing

Bacterial Smearing Before students can Gram Stain, they must prepare a bacterial smear. This includes fixing the

microorganisms of interest to the slide, so that they aren’t washed away during the procedure (Beck,

Hughes & O'Donovan, 2000). Bacterial smears can be made from either liquid or solid media. The

procedures for both are similar except the first few steps in which the microorganisms must be

retrieved from their specific medias. When using microorganisms from solid media, students must

first place a small amount of sterile water on their slides, and then add their culture. It is important

to know that only a small amount of culture must be used for the bacterial smear otherwise a thick

smear will be produced. Thick smears produce false results because they don’t stain evenly.

Students must also completely air-dry the slide before applying heat for a successful smear. Liquid

remains will produce steam during the heat fixing process, destroying the bacterial cells.

Figure 4. Bacterial Smears.

Safety 11

Safety

Lab Safety 1. Do not smoke, chew gum, eat food, or drink in the laboratory.

2. Leave hats, coats, backpacks on the coat and book rack.

3. Wipe down lab bench with disinfectant before and after each laboratory period to minimize

bacterial contamination.

4. When transferring cultures, always flame needles, loops, and mouths of test tubes before and

after each transfer.

5. Place biohazardous material in designated biohazard bag. Disposable and broken glass should

be placed in the designated broken glass box. All other trash is to be disposed of in trash cans.

6. If any culture container is broken or the culture spilled, immediately notify the instructor so that

the area can be decontaminated.

7. If you cut or scratch yourself, notify the instructor immediately.

8. Carry microscopes in the appropriate manner. The cord should be secured, both hands firmly

gripping the microscope, one under the base and the other on the arm.

9. Turn of all equipment and return supplies to their appropriate storage areas at the end of the lab.

12 Equipment for the Smear Preparation

Equipment for the Smear Preparation

Bunsen burner The Bunsen burner must be kept on throughout the period of experimentation. The burner is used to

sterilize equipment and to prevent contamination of the room and personnel with the

microorganism that is being used.

Inoculation loop An inoculation loop is a platinum wire forming a small loop at the tip. It is used to retrieve a culture

of microorganisms and transfer it.

Microscope slide A clean microscope slide must be used with every procedure in order to remove possibilities of

contamination. When a new microorganism is to be observed, a new slide has to be used. This also

prevents the microorganisms from being crossed.

Sterile water The microorganism is mixed with a couple of drops of sterile water placed on the clean microscope

slide.

Broth culture Culture of microorganisms grown overnight in a test tube containing liquid nutrient medium.

Plate culture Culture of microorganisms grown overnight on a Petri dish containing agar plus nutrients.

Equipment for the Gram Stain 13

Equipment for the Gram Stain

Crystal Violet A triarylmethane dye used as a method of classifying bacteria.

Gram’s Iodine Gram’s Iodine is a mordant that forms an insoluble crystal violet-iodine complex.

95% Ethanol A decolorizing agent.

Safranin A counter stain that colors all cell nuclei red.

Immersion oil Transparent oil used to increase the resolution of a microscope.

Bibulous paper Blotting paper

Lens Paper Cleans lenses and other delicate surfaces

14 Steps to follow for Smear Preparation

Steps to follow for Smear Preparation

Smear Preparation from Broth culture 1. Light the Bunsen burner.

2. Flame the inoculation loop.

3. Using the loop, retrieve one loop full of organism from the broth culture.

4. Spread the loop on the slide.

5. Place the slide near the Bunsen burner and allow it to air-dry completely.

6. Heat-fix organism to slide by passing it through the flame and then back again.

7. Let the slide to cool.

8. The slide is ready to be stained.

Figure 6. Bacterial smear from broth culture.

Steps to follow for Smear Preparation 15

Smear Preparation from Plate culture 1. Light the Bunsen burner.

2. Flame the inoculation loop.

3. Place one or two drops of water on the microscope slide using the inoculation loop.

4. Flame the inoculation loop again.

5. Retrieve small amount of organism from plate medium.

6. Transfer this to the slide and mix with water to form smear.

7. Place the slide near the Bunsen burner and allow it to air-dry completely.

8. Heat-fix organism to slide by passing it through the flame and then back again.

9. Leave the slide to cool.

10. The slide is ready to be stained.

Figure 7. Bacterial smear from plate culture.

16 Steps to follow for Gram Staining

Steps to follow for Gram Staining

Gram Stain 1. Prepare a bacterial smear (see pages 9 and 10).

2. Cover the smear with crystal violet and stain for 30 seconds to a minute.

3. Rinse briefly with sterile water.

4. Cover the smear with Gram’s Iodine.

5. Stain for 1 minute.

6. Decolorize by rinsing with 95% ethanol for 5-10 seconds; until the color stops coming off the

slide.

7. Immediately rinse with water.

8. Cover the smear with Safranin and stain for 30 seconds to 1 minute.

9. Rinse the slide with sterile water. Blot dry with bibulous paper.

Figure 8. Gram Stain

Microscopy 17

Microscopy

Examining under the microscope Examine the organism under oil-immersion.

1. Place the slide on the stage of the microscope.

2. View the organism under 10x and 40x magnification.

3. Place a drop of immersion oil onto the slide.

4. Change the magnification to 100x.

The Gram-positive bacteria will be purple, whereas the Gram-negative bacteria will be pink.

Figure 9. Gram Stain results.

18 Index

Index Bunsen burner, iii, 11, 13, 14

Crystal violet, iii, 7, 8, 9, 12, 15

Gram-negative, iii, 5, 7, 8, 9, 16

Gram-positive, iii, 5, 7, 8, 9, 16

Gram Stain, iii, 5, 7, 8, 9, 12, 15

Inoculation loop, iii, 7, 11, 13, 14

Iodine, iii, 7, 9, 12, 15

Microbiology, 5

Safranin, iii, 7, 9, 12, 15

Citations 19

Citations Beck, D. E., Hughes, L. E., & O'Donovan, G. (2000). Microorganisms A Laboratory Manual.

Figure 1. http://tal.ifas.ufl.edu/services_available.htm

Figure 2.http://hardinmd.lib.uiowa.edu/cdc/staph/sputum.html

Figure 3.http://water.me.vccs.edu/courses/env108/Lesson5_print.htm

Figure

4.http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205%20Labs/205%20Lab%20Lessonbuild

ers/Lab4SmearPrep/Lab4_print.html

Figure 5.http://biology.clc.uc.edu/fankhauser/labs/microbiology/Single_Colony_Isolation.html

Figure

6.http://202.116.160.98:8000/course/weishengwx/huanongen/shiyanjishu/jiandanransefa(y).asp

Figure

7.http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205%20Labs/205%20Lab%20Lessonbuild

ers/Lab4SmearPrep/Lab4_print.html

Figure 8.http://biology200.gsu.edu/houghton/4595%20'04/figures/lecture1/Gram-stain.jpg

Figure 9.http://labtestsonline.org/understanding/analytes/gram-stain/tab/test/