COURSE
SCIE1046: Fundamental of Microbiology with Lab
- About the Lab
Learning Objectives:
- Describe the structure of the Gram-positive and Gram-negative bacteria.
- Appreciate theoretical and technical aspects of the Gram staining procedure.
- Know the most commonly made mistakes in Gram staining.
- Critically interpret the results of a Gram staining experiment using a light microscope.
Estimated Length: 45 to 55 minutes
MAKE THE CONNECTION
The background information in section 2 was adapted from the following Microbiology lecture course Tutorials:
1.2.4 Preparing Slides for Microscopy
1.2.5 Staining Methods
2.1.2 Gram-Negative Bacteria
2.1.3 Gram-Positive Bacteria
5.5.7 Infections of the Nervous System
- Background Information
The following background information will be helpful as you prepare for the simulation.
In this lab, you will be using principles of Gram staining to identify a pathogen found in a cerebrospinal fluid (CSF) sample. A CSF sample is typically acquired using a lumbar puncture, often referred to as a spinal tap. A lumbar puncture is performed by inserting a needle between the L3 and L4 or L4 and L5 vertebra and extracting somewhere between 1 to 15 ml of CSF.
The brain and spinal cord are both covered in protective membrane layers. These layers are referred to as meninges (meninx singular). There are three layers of meninges. The top layer directly attaches to the bone of the cranium and the bone of the vertebral foramen and is known as the dura mater. Dura mater is Greek for “tough mother”. It is a very strong thick white tissue that protects the structures of the central nervous system (CNS). The middle layer is the arachnoid mater. Arachnid is a class of joint-legged invertebrate animals that include spiders. The arachnoid mater gets its name because this tissue is lined with spider-web-like vessels that nourish and care for the brain and spinal cord. The arachnoid mater forms a space above the underlying meninx, the pia mater. This space is referred to as the subarachnoid space. CSF flows freely in this space, providing a protective cushion to CNS structures. The deepest meninx layer, the pia mater adheres directly to CNS structures and is very thin. Pia mater is Greek for “gentle mother” and is semi-permeable, allowing only select molecules to cross the membrane into the CNS tissues.
CSF flows through the subarachnoid space and drains through the ventricles in the brain. These ventricles are open spaces that allow for continuous circulation of CSF. As CSF flows in the subarachnoid space, it not only creates a protective cushion, it also picks up waste from the tissues.
The blood–brain barrier is a collection of cells that serve as a barrier between nervous system vessels and the CSF. Only pathogens with specific characteristics can cross the blood–brain barrier and reach the CSF. Pathogens can reach the CSF through four main methods: transcellular, paracellular, phagocyte piggyback, or non-hematogenous. You can learn more about these methods by reading section 5.5.7 in the microbiology lecture course.
2a. Gram Staining
The Gram stain procedure is a common and important type of differential staining. It classifies bacteria into one or two groups based on characteristics of their cell walls. This procedure was developed by Danish microbiologist Hans Christian Gram in 1884.
STEP BY STEP
- The first stain applied is called the primary stain. Crystal violet is added. This stains all of the cells purple.
- Next, Gram’s iodine is added as a mordant(a substance used to set or stabilize stains or dyes). Gram’s iodine complexes with the crystal violet to form a crystal violet–iodine clump that is harder to remove from thick layers of peptidoglycan (a carbohydrate) in cell walls.
- Next, the slide is rinsed with a decolorizing agentthat helps wash out the stain. Ethanol or an acetone/ethanol solution is most commonly used. Cells with thick peptidoglycan layers retain the original crystal violet stain much more easily during this step than cells with thin peptidoglycan layers.
- Finally, a secondary counterstain(a stain added after another stain to contrast with it in some way) is added. Safranin is most commonly used, and it stains the decolorized cells pink. The cells that have retained the crystal violet dye are affected much less noticeably.
Cells that retain crystal violet and remain purple are called gram-positive, and cells that lose crystal violet and become pink when dyed with safranin are called gram-negative. These differences reflect the differences in the peptidoglycan layer, but there are other important differences between gram-positive and gram-negative cells. You will learn more about these in the lessons about prokaryotic cells and the characteristics of taxonomic groups of bacteria.
IN CONTEXT
There are several considerations to remember when using the Gram staining procedure. First, it is important to use young, healthy colonies because older bacterial cells may have damaged cell walls that do not retain the primary stain as well as healthy cells do. Second, errors such as leaving the decolorizer on too long can affect the result. In some cases, most cells will appear gram-positive, while a few will to be gram-negative. This suggests damage to the individual cells or that the decolorizer was left on too long. The cells should still be classified as gram-positive if they are all the same species.
Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have implications with clinical relevance. Therefore, Gram staining is a valuable clinical tool, and knowing how to use it is an important skill.
EXAMPLE
Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. This can be helpful in selecting an appropriate antibiotic. You will learn more about the characteristics of gram-positive and gram-negative bacteria and about antibiotics in other lessons.
REFLECT
Most people have taken antibiotics at some point. Can you remember some of the antibiotics that you have been given? What do you think the doctor considered when deciding which antibiotics to use? Think about this, and pay attention the next time you or someone you know is given antibiotics.
| Gram Staining Process | ||||
| Gram Staining Steps | Cell Effects | Gram-Positive | Gram-Negative | |
| Step 1 Crystal violet The primary stain is added to the specimen smear. |
The cells are stained purple or blue. | |||
| Step 2 Iodine The mordant makes the dye less soluble, so it adheres to cell walls. |
The cells remain purple or blue. | |||