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6.1: The Gram Stain

HGTC BIO 225: Microbiology Lab Manual · LibreTexts

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    From LibreTexts, under CC BY 4.0, as the source states it; its equations are written in TeX, which the PDF shows typeset. This page at LibreTexts.

    Learning Objectives

    • Discuss the purpose of the Gram Stain as a differential stain.
    • Understand the structural differences of the cell wall in gram positive and gram negative bacteria. 
    • Prepare microscope slides for gram staining with bacterial species whose cell wall structure is unknown.
    • Determine if the bacteria are gram positive or gram negative. Describe cell morphology. 
     

    Differential Stains

    The gram stain is a differential staining technique used to determine if bacteria are gram positive or gram negative based on their cell wall structure. Developed in 1884 by the Danish scientist Hans Christian Gram, the gram stain is a key microbiology technique used in clinical diagnosis to determine proper antimicrobial treatments.

    Differential stains, like simple stains, are used to increase microscopy contrast. However, differential stains are far more complex and can be used to differentiate cellular structures, cell types, and bacterial species. Differential stains can even dye two cellular structures in one bacterial body. Typically, a differential stain will use more than one dye, use other chemical reagents such as mordants and de-colorizers, and involve heat fixation. In this course, you will complete three differential stains: the gram stain, the acid-fast stain, and the endospore stain. Each stain will be described in depth in their separate chapters, but below is a table to provide a brief comparison of these differential stains. 

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    Table 1. Differential stains assist with microbe identification as these stains focus on specific cell characteristics. The gram stain identifies peptidoglycan quantity, the acid-fast stain determines if a cell has mycolic acid, and the endospore stain helps identify if cells can produce dormant spores. 

     

    Gram Positive and Gram Negative Bacteria 

    Bacteria are separated into two main groups based on the composition of their cell wall: gram positive and gram negative. The cell wall of a gram positive bacterium has a thick layer of peptidoglycan, between 30 and 100nm thick. (OpenStax, 2024) Peptidoglycan is a unique bacterial carbohydrate that provides significant structural support and stability in bacteria. Peptidoglycan is also the target of the crystal violet primary stain during the gram stain procedure. Additionally, the gram positive cell wall contains teichoic acids whose function to further stabilize the peptidoglycan layer and bind surface proteins on host cells. The cell wall of gram negative bacteria has a very thin layer of peptidoglycan, less than 4nm thick. (OpenStax, 2024)  Since the peptidoglycan layer is so thin, gram negative bacteria are covered by an additional lipid bilayer called the outer membrane. This extra outer membrane contains lipoproteins that attach to the peptidoglycan layer, porin proteins for nutrient transportation, and lipopolysaccharides that aid in bacterial infection and toxin production. 

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    Figure 1. Illustration of the gram positive and gram negative cell wall make-up. Gram positive cell walls have thick layers of peptidoglycan and do not need an extra outer layer for protection or stability. Gram negative cell walls have very thin layers of peptidoglycan and, thus, have an extra protective layer called the outer membrane. 

     

    Gram Stain Procedure and Reagents 

    Prior to completing a gram stain, bacteria must be prepared as a bacterial smear and heat fixed. Heat fixation serves to adhere bacteria to the microscope slide, kill cells, and increase stain uptake as described in "4.1 Simple Stains".The gram stain then uses 2 stains and 2 reagents: Crystal Violet Stain, Safranin Stain, Gram's Iodine, and Gram's De-Colorizer (Ethyl Alcohol). Crystal Violet is the primary stain and will be added to the specimen first. Both gram negative and gram positive cells can absorb the crystal violet dye as it targets peptidoglycan. At this stage, all cells will be purple. Next, Gram's Iodine is added and acts as a mordant or "glue" and stabilizes the crystal violet stain. In gram positive cells, the iodine will bind to crystal violet forming a large molecular complex trapped by the thick layers of peptidoglycan. Gram negative cells can not "trap" the iodine-crystal violet complex as their peptidoglycan layer is too thin to contain it. After the application of iodine, the specimen is rinsed with a De-Colorizing agent, typically ethyl alcohol (ethanol). This wash cycle is the key step in cell wall differentiation. In gram negative cells, the outer membrane will be dissolved by the de-colorizing agent, exposing the thin peptidoglycan layer with the iodine-crystal violet complex. The de-colorizer will then "wash away" the dye complex, causing gram negative cells to lose the purple color and return to their natural, transparent appearance. Gram positive cells are only weakly affected by the de-colorizer and will retain the dye complex and, thus, cells remain purple. Transparent gram negative cells are not visible on the microscope. So, after the de-colorizer rinse, a counterstain called Safranin is applied. The colorless gram-negative cells will uptake the safranin dye and turn pink. Gram positive cell appearance will not be altered by safranin and gram positive cells will remain purple. 

    A table shows the Gram stain process. Each row consists of a column describing the step and a column describing the effect. Additional columns show drawings to support the description in the effects column. Step 1: Crystal Violet, the primary stain, is added to the specimen smear. This stains cells purple or blue. The Gram-positive and Gram-negative cells all look purple. Step 2: Iodine, the mordant, makes the dye less soluble so it adheres to cell walls. The cells remain purple or blue. The Gram-positive and Gram-negative cells all look purple. Step 3: Alcohol, the decolorizer, washes away stain from Gram-negative cell walls. Gram-positive cells remain purple or blue, Gram-negative cells are colorless. Step 4: Safranin, the counterstain, allows dye adherence to Gram-negative cells. Gram-positive cells remain a pruple or blue. Gram-negative cells appear pink or red.

    Table 2. This table describes the steps of a Gram Stain. Gram positive cells will absorb the primary purple stain and keep the stain tightly bound in their thick cell wall layer. Thus, gram positive cells should be purple through the entire gram stain prep. Gram negative cells will also absorb the primary stain, but will not be able to keep the dye in their cell wall because it is thin. Thus, gram negative cells should lose the primary stain, be clear/transparent for some steps of the gram stain, and finally be re-stained with the counter-stain making all gram negative cells pink. 

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    Figure 2. Potential results after completion of a gram stain. Samples containing only 1 bacterial species will either be all purple (gram positive) or all pink (gram negative). Mixed species samples may present both purple and pink cells depending on the cell wall make-up of the mixed cell types. 

     

    Potential Challenges

    Differential stains use multiple dyes, reagents, and steps which means there is more room for potential error during sample preparation. Gram stains work best with fresh cultures. Older cultures can have damaged cell walls, resulting in gram-variable results (pink and purple cells visible) or the incorrect gram reaction. Further, since the decolorization step is key to cellular differentiation, the amount of de-colorizer used significantly alters the gram reaction. Application of excessive de-colorizer can leach the crystal violet dye out of gram-positive cells, resulting in false gram negative results. Vice versa, failure to sufficiently rinse gram negative cells with de-colorizer may result in cells keeping the crystal violet dye and a false gram positive result. 

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    Figure 3. Since the gram stain is a differential stain with multiple steps, dyes, and reagents, the chance for error is increased. Improper heat fixation means the bacteria will be washed away in rinses cycles and there will be no specimen to view on the slide. Using too little de-colorizer means the primary stain, crystal violet, will not be fully washed out of gram negative cells and everything will be purple (a false gram positive result). Using too much de-colorizer will remove the primary stain from the gram positive cells which allows them to later absorb the counter-stain, safranin, and everything will be pink (a false gram negative result). 

    Other factors that can alter the success of your gram stain include:

    • Improper smear prep. Bacterial smears that are too thick won't react properly with reagents. 
      • Also, if you are not gentle with the inoculation loop, you risk disrupting the natural cell arrangement. 
    • Skipping the air drying and/or heat fixation step. Drying steps and heat applications are key to ensuring that bacterial cells are adhered to the slide and won't be washed away during rinse cycles. 
    • Time and reagent application. Stains and mordant application should be left on bacterial smears for 1 minute. Leaving the stains or mordant on a smear for too long or too short will alter the normal gram reaction. Cells may not have sufficient time to absorb the stain.
    • Wash Cycles. In between reagent application, wash cycles with DI water are used to clean the bacterial smear. Not washing the slide means stains and other reagents will not be properly removed before follow-up steps. This means different stains and the iodine will be mixed together to produce an incorrect gram result.
      • Excessive washing may also remove stains and even the bacteria resulting in slides with no specimen or an incorrect gram result.  
    Real-World Connections

    Bacterial Vaginosis 

    The human urinary tract and reproductive system comprise the urogenital system and are both exposed to external environments. This exposure makes the urogenital system prone to microbial imbalances and infections. A preliminary diagnostic tool for bacterial infections of the urogenital system is the gram stain.

    The gram stain can be used to diagnose vaginosis, inflammation of the vagina caused by bacterial overgrowth. Lactobacilli and Gardnerella vaginalis are both part of the vaginal normal microbiota, but in significantly different amounts. Lactobacilli cells are gram positive rods present in large quantities on the vaginal cell walls. These bacteria contribute to the acidic pH of the vagina which helps inhibit pathogenic growth. Normal, healthy vaginal secretions and samples should contain large populations of purple rods (Lactobacilli) when observed by microscope and gram stain.

    Gardnerella vaginalis is present in significantly smaller quantities than Lactobacilli. Overgrowth of G. vaginalis leads to bacterial build-up on the vaginal cell walls and cytotoxin production that targets red blood cells. These actions cause vaginal burning and itching for the patient.  Patients may develop G. vaginalis and vaginosis during unprotected sex, douching, or menstruation. (CDC, 2021). These activities can deplete normal Lactobacilli populations, allowing G. vaginalis to overgrow. G. vaginalis cells are coccus in shape and primarily gram negative (though gram variable results may occur). A patient sample containing high quantities of pink cocci and low quantities of purple bacilli is likely suffering from a vaginosis infection. This presumptive diagnosis can be used to help determine the severity of the infection and determine if antibiotic treatment is required.

     

     

    Attributions

    "Microbiology Textbook: Chapter 2,How We See The World" by OpenStax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Microbiology Textbook: Chapter 3, The Cell" by OpenStax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Microbiology Laboratory Manual: Labs, 1.10 Gram Stain" by Dr. Rosanna Hartline, West Hills College Lemoor, LibreTexts: Biology is licensed under CC BY-NC-SA 4.0

    "Microbiology Labs I: 17, Gram Stain" by Jackie Reynolds, Updated By: Delmar Larsen, LibreTexts: Biology , Dallas College, Richland Campus is licensed under CC BY-NC-SA 4.0

    "Microbiology Textbook: Chapter 4, Prokaryotic Diversity" by Openstax, Digital ISBN: 13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Microbiology Textbook: Chapter 23, Urogenital System Infections" by Openstax, Digital ISBN: 13: 978-1-947172-23-4 is licensed under CC BY 4.0

    "Sexually Transmitted Infections Treatment Guidelines, 2021: Bacterial Vaginosis " by Centers for Diseases Control and Prevention , Use of CDC Materials Statement is in the Public Domain

    "Microbiology Textbook: Chapter 9, Microbial Growth" by Openstax, Digital ISBN: 13: 978-1-947172-23-4 is licensed under CC BY 4.0

    Image Citations 

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    6.1: The Gram Stain is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by LibreTexts.