5.1: Simple Stains

Learning Objectives

• Define "simple stain" and their purpose in microbiology.
  • Discuss the importance of "contrast" when using a microscope.
• Understand how simple stains work and physically stain structures in bacterial cells. 
• Discuss the importance of bacterial smears and heat fixation when preparing specimens for staining procedures. 
• Prepare microscope slides for simple staining with bacterial species whose cell shape and arrangement are unknown.
  • Describe cell morphology. 

 

Simple Stains

Bacteria are naturally colorless or transparent when observed by light microscopy. Transparent bacteria lack contrast and will blend in with the background of a slide making observation by microscope extremely difficult or impossible. In order to increase contrast, the ability to see a specimen clearly in relation to the color/darkness of the background, bacteria can be stained with chemical dyes of variable color. These dyes can either be simple stains, negative stains, or differential stains. All 3 stain types will be discussed in their corresponding lab manual chapter. 

 

Simple and differential stains are used for fixed specimen slides, sometimes called "heat prep" slides. The first step to creating a simple or differential stain is to prepare a bacterial smear. Bacterial smears are made by mixing a small amount of cells with sterile DI water on a microscope slide and allowing everything to air dry. It is important to use a small amount of bacteria to make a smear otherwise the staining reagents will not properly bind to cell structures and individual cells will be almost impossible to observe. Being gentle with the inoculation loop is also key when making a smear or you risk damaging the natural cell shape and arrangement of the bacterial species. Bacterial smears on fixed specimen slides are "fixed" or adhered to the slide through the application of heat or chemical treatments. In this course we will be fixing bacteria to our slides using heat treatments, a process called heat fixation. Heat fixation has 3 functions:

1. Kill the bacteria.
2. Adhere dead bacterial cells to the microscope slide.
3. Increase absorption of chemical stains into bacterial structures.

 

Heat fixed bacterial smears are then ready for simple staining. Staining procedures depend on the natural negative charge of bacteria. Bacteria have a negative charge from external and internal cell components like their cell wall, cell membrane, and nucleic acids. Simple stains are made using basic dyes that contain positive ion salts with color. These dyes are called positive stains based on their net positive charge. Positive stains are attracted to the negative charge of the bacterial cell body and the entire cell will be dyed the color of the ion salt. Common positive stains used for simple staining procedures are Methylene Blue and Crystal Violet, which will turn the entire cell body blue or purple, respectively. 

Figure 1. Bacteria have a natural negative charge that will attract the positive charge of the basic dye, causing the whole cell body to be stained one color. 

Simple stains are called "simple" for their straightforward procedure that requires few reagents. Simple stains use only one dye and will stain the entire cell body one color. For this reason, we can use simple stains to determine cell size, shape, and arrangement. However, simple stains can not be used to differentiate between different bacterial species. Since every cell present on a slide will be stained the same color, there is no cell differentiation (limiting the application of simple stains with mixed specimen samples). Differential stains are better for comparing differences in bacterial species (see "5.1 The Gram Stain"). 

Simple Stain: Methylene Blue	Negative Stain: Nigrosin Stain	Differential Stain: Gram Stain
E. coli stained with methylene blue (black arrows) and observed on a bladder epithelial cell.	C. neoformans yeast (red arrows) was stained with Nigrosin. The stain turns the background black while leaving the yeast cells clear/transparent.	Gram Stain mix of gram-negative E. coli (pink) and gram-positive S. aureus (purple).

Table 1. Simple stains are good for observing overall cell shape and arrangement (cell morphology). Negative stains also observe cell morphology, but these stains dye the background instead of the cell body. Differential stains can be used to identify differences in cell structures between bacterial species.

Bacterial Cell Morphology 

Using simple stains and light microscopy, bacterial cell shape and arrangement can be determined. This cell morphology data can subsequently be used to aid in an initial clinical diagnosis. Below is a table with the most common cell shapes seen in prokaryotes. 

Figure 2. In this course, students will be observing coccus (round) and bacillus (rod-shaped) cells. 

Figure 3. Coccus and bacillus shaped bacteria are able to connect to neighboring cells creating a distinct cell arrangement. Coccus cells form more arrangement patterns than bacillus cells as the sphere shape of the cell increases the surface area available for the cells to connect to each other. 

Real-World Connections

Staph Infections: A Common Occurrence in Hospitals

Unfortunately, staph infections are a commonality in the healthcare setting. The causative agent, Staphylococcus aureus, is typically a benign bacterium that is found on the skin and nose of about 30% of people (CDC, 2024). However, if given the opportunity, S. aureus can cause infections, specifically in susceptible and immunocompromised patients in hospitals. These infections typically result in skin conditions, like pimples or boils under the skin, but in more severe cases can result in sepsis (bloodstream infections) pneumonia, endocarditis (infection of the hearth valves), and bone infections. People at the most risk of these infections include those with chronic conditions, medical implants, weakened immune systems, in ICU, or recovering from surgery.

Observing the characteristics of microorganisms in clinical samples can help microbiologists narrow down the list of suspects that caused the infection. Each bacterial species has a specific shape and arrangement, this includes Staphylococcus aureus.  S. aureus is a cocci (sphere) shaped bacterium with a staphylo (clustered) arrangement, hence the genus name Staphylococcus. Other pathogenic bacteria, for example Vibrio Cholera, have their own specific arrangements as well. Vibrio cholerae, the causative agent of cholera, has a curved rod or vibrio shape. Observing the shape and arrangement from a clinical sample, could help identify the causative agent of an infection. However, these results are not definitive, as many bacteria share similar shapes and arrangements. Therefore, the shape and arrangement of a bacterium should not be the only evidence used when diagnosing a patient.

Figure 4. Every prokaryote has a specific shape and arrangement. For example Staphylococcus aureus (shown in the left image) is round (coccus) and arranges in clusters (staphylo). The image on the right depicts Vibrio cholerae, the causative agent of cholera. This bacterium has a curved rod, or vibrio shape. 

Attributions

"Microbiology Laboratory Manual: Labs, 1.9 Simple Stain" by Dr. Rosanna Hartline, West Hills College Lemoore, LibreTexts: Biology is licensed under CC BY-NC-SA 4.0

"General Microbiology Lab Manual: Labs 3, Simple, Negative, and Gram Stain" by Nazzy Pakpour and Sharon Horgan, LibreTexts: Biology, Coastal State University, Easy Bay is licensed under CC BY-SA

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

"MB352 General Microbiology Laboratory 2021: Staining Techniques 4.3" by Joan Petersen and Susan McLaughlin , LibreTexts: Biology , Queeensborough Community College is licensed under CC BY-NC-SA

"Staphylococcus aureus: Basics" by Center for Disease Control, CDC Materials Statement is in the Public Domain

"Cholera: About Cholera" by Center for Disease Control, CDC Materials Statement is in the Public Domain

Image Citations

Figure 1, Modified From:

• "Microbiology Laboratory Manual: Labs, 1.9 Simple Stain" by Dr. Rosanna Hartline, West Hills College Lemoore, LibreTexts: Biology is licensed under CC BY-NC-SA 4.0

Figure 2, "Microbiology Textbook: Chapter 3, The Cell" by OpenStax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0

Figure 3, Modified From:

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

Table 1, Modified From:

• "UPEC adhered to BEC" by Stefan Walkowski is licensed under CC BY-SA 4.0.
• "Microbiology Textbook: Chapter 2, How We See the Invisible World" by Openstax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
• "Gram stain 01.jpg" by Y tambe, WikiMedia Commons is licensed under CC BY-SA 3.

Figure 4, Modified From:

• "Staphylococcus aureus 01.jpg" by Janice Carr, Wikimedia Commons is in the Public Domain
• "Vibrio cholerae.jpg" by Tom Kirn, Ron Taylor, Louisa Howard, Wikimedia Commons is in the Public Domain

---