Performing a Gram Stain on a broth culture from possible contaminant of D. radiodurans

Gram Stain: Procedure Summary

March 11th, 2022

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 determining the morphology of the bacterium and when diagnosing and treating pathologies caused by pathogenic bacterial species. This laboratory experiment helps scientists determine whether a sample of a yellow contaminant grown in a controlled environment similar to that of Deinococcus radiodurans is gram positive or gram negative, and based on this knowledge, the shape, size, and type of this bacterium is determined. It is hypothesized that the yellow contaminant bacterium is from the genus Micrococcus and the species type luteus. In addition, it has a gram positive cell wall, and if a gram stain were to manifest this trait, purple cells would be present under the microscope. This hypothesis was established by Dr. Deutch while he examined the yellow circular colonies of the contaminant grown in TGY medium, which were then inoculated into TGY broth and grown over two days to perform the gram staining procedure. 

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 hypothesized 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 loopfuls of Micrococcus luteus 
(the predicted species name will be used to simplify it to the reader, but the species itself is still under investigation) 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 

First, an image of the agar plate containing the grown streaks and colonies of the contaminant is shown. Because Micrococcus luteus 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 images were captured:

It is evident that this experiment confirms Micrococcus luteus is gram positive because the cells do appear to be violet and not red (as would be the case with gram negative bacteria). The microscope images further strengthen the claim that the bacterium under investigation is in fact Micrococcus luteus because it has an almost identical morphology to Deinococcus radiodurans; the cells under the microscope appear to be cocci with two bellies, and they form grape-like clusters. Knowing whether a bacterium is gram positive or negative is very important in medicine because it determines the type of bacteria responsible for the contamination and how to eliminate it. Micrococcus luteus is also thought to have contaminated the growth of Deinococcus radiodurans as D. radiodurans was being spread onto an agar plate with TGY. This knowledge is acquired because the contaminant colonies on the agar plate used to create broth cultures yielded the same yellow contaminant colonies when it was plated again on a different plate with TGY. 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 and AJ Federico - helped with the process of viewing the gram stained slide under the microscope. 

Dr. Charles Deutch helped us predict the genus and species of the bacterium.