Performing a Gram Stain on a broth culture of D. radiodurans

Gram Stain Basics: Procedure Summary

December 22nd, 2021

Introduction

Microorganisms are fascinating entities to the human mind, and this is due to their unique living styles and coping mechanisms. Some organisms are favorable to others because they possess advantageous traits that aid in their survival. Take bacterial cell walls as an example; some species of bacteria have a thick cell wall made of peptidoglycan (sugars and amino acids) with no outer membrane, and they are called gram positive because they lack that outer lipid membrane. This is advantageous in itself because a thicker cell wall makes such species less susceptible to lysis and osmotic stress, providing a protective shield against numerous environmental factors. On the other hand, other species of bacteria have a thin cell wall with an outer lipid membrane which makes them gram negative. The advantage of such species being gram negative is that they are more resistant to antibiotics and other drugs, making them resilient when they enter the human immune system. A gram stain is a differential stain that informs whether a bacterium is gram positive or gram negative, and this is useful when diagnosing and treating pathologies caused by pathogenic bacterial species. This laboratory experiment helps science students determine whether a sample of Deinococcus radiodurans grown in a controlled environment is gram positive or gram negative, and based on this knowledge, appropriate measures are taken when experimenting on this bacterium. Examples of experiments include inducing growth of D. radiodurans by exposing the bacterium to oxidative stress through chemicals, ultraviolet radiation, etc. It is hypothesized that the bacterium D. radiodurans has a gram positive cell wall, and if a gram stain were to manifest this trait, purple cells would be present under the microscope. 

Procedure

There are several steps to performing a gram stain. Beforehand, a clean microscope slide is labeled on the back with a circle drawn in the middle to indicate where the smear will be, the name of the bacterium and the laboratory student's initials on the side. Then, on the other side of the slide, the inside of the circle is smeared with three consequent loopsful of D. radiodurans broth culture using a sterilized inoculation loop. After letting the smear dry up a bit, the slide is heat fixed with the flame from a Bunsen Burner. Now the slide is ready for the gram stain. To do so, the following steps are performed in order:

• Step one includes loading a few drops (3-4) of the crystal violet dye inside the boundaries of the circle on top of the heat fixed smear, then a 60 seconds waiting period follows to ensure the dye binds to the cell wall of the bacterial cells. Afterwards, the slide held in a slanted manner and rinsed with deionized water (DI water) until the excess violet dye is completely off the slide.
• Step two involves loading a few drops of iodine inside the circle and waiting 60 seconds. Then the slide is rinsed off with DI water similar to step one. 
• In step three, ethyl alcohol is dripped onto the slide that is held in a slanted manner to wash off the dyes from the gram negative cell walls. This is essential in gram negative bacteria with a thinner cell wall (peptidoglycan layer) because it helps get rid of the crystal violet-iodine complex, making the differential stain possible. Alcohol does not completely wash off this complex from gram positive bacteria due to the thicker peptidoglycan layer which makes it harder for the dye to fall off. 
• Lastly, step four involves loading a safranin dye (red in color) on the smear spot and waiting 60 seconds for it to settle. This step is also essential because only gram negative bacteria will pick up the dye; the previous crystal violet-iodine dye complex has been washed off with alcohol in gram negative bacteria. In contrast, the gram positive bacteria still have the crystal violet-iodine complex bound to its cell wall, and because this dye is darker than the safranin dye, the safranin dye cannot be clearly seen in gram positive bacteria. After the 60 seconds wait time, the dye is rinsed off with DI water for one last time. Furthermore, the slide is dried up of any liquid with bibulous paper to be viewed under the microscope.

Results 

Because D. radiodurans is expected to be gram positive, the microscope image needs to show bluish purple bacterial cells. After a series of trials to find the best resolution by altering the microscope's lens and focus, the succeeding image was captured:

It is evident that this experiment confirms D. radiodurans is gram positive because the cells do appear to be violet and not red (as would be the case with gram negative bacteria). Knowing whether a bacterium is gram positive or negative is very important in medicine because it determines the type of bacteria responsible for infection and how to treat it. By knowing the type of bacterium, for example, it can be predicted whether it is aerobic or nonaerobic, resistant to certain drugs and antibiotics, or whether it possesses other virulence factors that aid in its spread and survival. In lab, knowing the type of bacterial cell wall helps determine the ways experiments can be conducted efficiently. For example, gram positive bacteria are harder to lyse because of their thicker cell walls, so cell lysis procedures should be assigned more time and more rigorous materials and devices. All in all, the gram stain is a differential stain very easily performed, yet extremely helpful information it does provide. 

References

York, J.J. (June 2021). Smear Preparation and Gram Stain Procedure. PPT slides for Lab 3: Gram Stain. 

Shawn Soares - helped me along the way and handled viewing the gram stain slide under the microscope.