What Is Gram Staining?

Identification of Unknown # 15

Abstract. One of the most fundamental differential staining techniques used in the study of bacteriology is gram staining. There are two main types of bacteria, gram negative and gram-positive. The purpose of this experiment was to perform a variety of tests to identify the bacteria contained in the unknown sample labeled number 15. The following are the tests that were used to identify the two different bacteria. The SIM test, which tests for sulfur reduction, indole production, and motility, MRVP which is a test used to distinguish different organisms within Enterobacteriaceae, the citrate test which is used to identify
the source of carbon and energy for an organism, and the catalase test which is used to differentiate Staphylococci from Streptococci. Differential media has the ability to differentiate organisms that are closely related by exhibiting different growth patterns and selective media only allows specific organisms to grow in the media and may possibly inhibit the growth of other organisms present. The following are the types of differential and selective media used in this experiment: MSA, Blood Agar, MacConkey agar, PEA, and EMB. After running these tests, it was deduced that the two unknown bacteria could be identified as Staphylococcus aureus and Klebsiella pneumonia.

Key words: Staphylococcus aureus. Klebsiella pneumonia. MSA. PEA. EMB. MRVP. Enterobacteriaceae. Staphylococci. Streptococci.

Introduction
Differential staining is a useful technique when studying different types of bacteria. This technique is typically a four-step process to help individualize specific structures and/or microorganisms. A primary stain, usually crystal violet, is used to give the cells a specific color, a mordant, usually iodine is insoluble which helps to bind to the primary stain, a decolorizing agent, typically ethyl alcohol, is used to provide a distinction in color by removing the stain from a slide or embracing it, and a counterstain, like safranin, provides a color that is contrary to the primary stain (Beveridge 1990). One of the most fundamental differential staining techniques used in the study of bacteriology is gram staining. There are two main types of bacteria, gram negative and gram-positive. Gram-positive bacteria, when using crystal violet, stain a purple color. This is due to the thick peptidoglycan layer’s resistance to the decolorizing agent. Gram-negative bacteria usually stain a red color as a result of the outer and lipid membrane dissolving along with the decolorizing agent paired with the safranin (Beveridge 1990).
The catalase test is used to differentiate Staphylococci, which is positive, from Streptococci, which is negative by facilitating the discovery of the enzyme catalase in the bacteria. If the catalase enzyme reacts with an added dosage of hydrogen peroxide by indicating bubbles, the microbe is considered to be positive. This test is also beneficial in distinguishing between aerobic and obligate anaerobic bacteria, as anaerobes are generally known to lack catalase, giving an indication of the identity of an unknown bacterium.
The coagulase test is a test that has the ability to determine the disease-producing capacity of a pathogen, which can be used to differentiate a variety of Staphylococcus species from Staphylococcus aureus (Choi 2014). Generally Staphylococcus aureus is coagulase positive and the other types of Staphylococci species are negative. The coagulase enzymes occur in a free coagulase form and a bound, otherwise known as clumping coagulase form where a slide test or a tube test can be performed. A slide test is specifically for bound coagulase, where positive results would indicate clumping. If results are negative, a tube test, which uses inoculated rabbit plasma, can be used to confirm, and where positive results would again be indicative of clumping and negative would remain a liquid. The coagulase reacts with prothrombin in the blood, which is what ultimately results in a blood clot (Ryan 2004).
The Sulfide-Indole-Motility test otherwise known as SIM uses a SIM medium containing a combination of differential mediums to test for sulfur reduction, indole production, and motility. The sulfur reduction test is often used in distinguishing organisms that pertain to the intestines. The presence of an indole indicates the enzyme tryptophanase, which is used to break down tryptophan into small components, one of these components being indole. The indole test uses Kovac’s reagent which will react with an indole if one is present in the bacteria and will produce a pink/red or red/purple ring along the top of the test tube. If no indole is present, no color change occurs. The motility test detects flagella by determining the cloudiness in the medium, meaning that the more turbid a medium is, the more indication that the bacteria are motile. A clear result would be indicative of non-motile bacteria.
IMVIC is a common testing method that applies Indole testing, methyl red, Voges-Proskauer, and citrate tests.
The indole test for this method is the same method as listed above in the SIM testing method. The methyl red and the Voges-Proskauer tests are used to distinguish different organisms within Enterobacteriaceae. This process is done by performing a mixed acid fermentation test by adding methyl red to an MR-VP medium and then observing their ability to convert acidic fermentation products to non acidic products by using the Voges-Proskauer test and the VP reagent (Choi 2014). A positive MR test has a noticeable red tinge due to the vast amounts of acid being produced. A positive VP test has immiscible layers of red and yellow, where negative results would indicate immiscible layers of a copper/brown color. Citrate is a derivative of citric acid. The citrate test identifies organisms, using the Simmons Citrate medium, that use citrate as their means of carbon and energy and can also differentiate different members of Enterobacteriaceae. A blue color or any kind of bacterial growth indicates a positive result and a green color indicates a negative result (Gallup
2010).
Differential and selective media are extremely vital when trying to isolate bacteria from a mixed culture of microbes. Differential media has the ability to differentiate organisms that are closely related by exhibiting different growth patterns along with other distinctive changes due to the specific dyes and chemicals contained within the media. Selective media only allows specific organisms to grow in the media and may possible inhibit the growth of other organisms present. The Mannitol Salt agar (MSA) has high concentrations of salt, which will inhibit the growth of a vast majority of bacteria excluding Staphylococci (Choi 2014). A yellow color in the plate would indicate the mannitol fermenting. Blood Agar is used to cultivate meticulous organisms and also helps to distinguish the different hemolytic properties. Gamma hemolysis is indicates that there is no lysis of red blood cells, Alpha hemolysis indicates incomplete lysis of red blood cells, and Beta hemolysis is indicative of lysis in red blood cells and complete loss of blood (Choi 2014). The MacConkey agar has a specificity for gram negative organisms due to its ability to inhibit the crystal violet staining on a gram positive specimen. The Phenylethyl alcohol Agar (PEA) has the ability to set apart a vast majority of gram positive cocci. The Eosin Methylene Blue Agar (EMB) has the ability to make a distinction between enteric lactose fermenters and nonfermentors and can also identify Escherichia Coli, where in this case the colonies would have a green color, and other bacteria would produce a pinkish color. A colorless growth of bacteria would indicate an enteric organism (Ryan 2004).

Methods and Materials
This study took place in the Science building at Texas A&M University-Commerce. An unknown broth sample labeled number 15 containing two different bacteria was provided for this experiment along with the necessary reagents, tools, and blank slides. The purpose of this experiment was to perform a variety of tests to identify the bacteria contained in the unknown sample. All of the following procedures were performed as noted in the lab manual provided by Choi (2014). A Trypticase Soy Agar (TSA) spread plate was provided to isolate the two bacteria contained within the sample. After incubating the spread plate and identifying the two different types of colonies within the plate, a differential test was done with the isolated colonies by smearing each bacterium on its own slide and performing the methods given in the manual (Table 1). Considering that gram-negative bacillus and gram-positive cocci were observed, the following list of tests was performed in order to determine the unknown sample according to the lab manual procedures (Choi 2014). The following list includes differential and selective media Table 1 lists the test, purpose, reagents, and results for each of the following.
1. Catalase Test
2. SIM Test
3. Methyl Red Test
4. Voges-Proskauer Test
5. Citrate Test
6. Mannitol Salt agar
7. Blood Agar
8. MacConkey agar
9. Phenylethyl alcohol Agar
10. Eosin Methylene Blue Agar

Results
After the first spread plate was done, two different types of colonies were noted for isolation. One colony type was very large, irregular, cloudy, and white, and this was concluded to be Klebsiella pneumonia and the other colony type was sparsely spread and limited to about 4 colonies throughout the edges of the plate that were noted to be small, spherical, and yellow. The small, yellow bacterium was noted to be Staphylococcus aureus. After further assessing these isolations with additional isolation tests, the colonies were confirmed to be different and in fact isolated based on size, shape, color, and quantity. Furthermore confirming the results, the gram staining indicated a gram-positive Staphylococci structure (Figure 11) and a gram-negative Bacillus structure (Figure 12). Additional tests were completed to further confirm the results as listed in Table 1 and a flowchart was constructed to indicate the procedures performed to identify both bacteria contained in the unknown sample number 15 (Figure 1).
Table 1. Results for Tests performed in no specific order to Identify Unknown Sample Number 15

TEST
PURPOSE
REAGENTS
OBSERVATION
RESULTS

Catalase Test

To differentiate between Staphylococcus (Catalase +) and Streptococcus (Catalase -)
As well as Clostridium (catalase -) and Bacillus (catalase +)
Hydrogen Peroxide
Bubbling Occurred in both bacteria
Catalase positive for both bacteria

SIM Test

To determine sulfur reduction, Indole production, and motility
Kovac’s added to SIM media
Turbidity did not noticeably increase. No color changes in both bacteria
Sulfur reduction and Indole was negative for both bacteria, Motility was inconclusive for both bacteria

Methyl Red Test
Used to distinguish different organisms within Enterobacteriaceae
Methyl Red
Slight pink color change
Weak positive for both bacteria

Citrate Test

To identify organisms that use citrate as their main source of carbon and energy
Citrate media
No color change for both bacteria
Negative for both bacteria

Voges-Proskauer Test
Used to distinguish different organisms within Enterobacteriaceae
Alpha-naphthol and potassium hydroxide
No reaction for both bacteria
Negative for both bacteria
Mannitol Salt agar

To indicate fermentation and inhibit growth of a variety of bacteria excluding Staphylococci
MSA media
No growth for either bacteria
Negative for both bacteria

MacConkey agar

To indicate pH where a pink/red is acidic indicating that it’s an organism that can ferment lactose
MacConkey media
No growth for either bacteria
Negative for both bacteria

Phenylethyl alcohol Agar

To select for gram positive Cocci
PEA Media
Slight growth for gram positive and no growth for gram negative
Weak positive for gram positive bacteria and negative for gram negative bacteria

Eosin Methylene Blue Agar

To isolate gram negative enteric pathogens/ select for E. Coli
EMB Media
Noticeable growth for both bacteria
Positive for both bacteria

Blood Agar

To determine hemolytic activity
Blood Media
Noticable growth and hemolytic activity for both bacteria
Gamma for gram positive bacteria and Beta for gram negative

.

Figure 1. Flow chart indicating the chronological order of the testing process and results determined to identify the species contained in unknown bacteria # 15

Discussion

The results obtained were thought to be true after the first gram staining test was done on each bacterium. This determination was made based on the fact that one bacteria was noted to be gram positive and one was noted to be gram negative as well as their vast difference in shape and growth pattern. After doing a second streak plate bacteria A, or what we now know as Staphylococcus aureus had very small round, almost uniform lines growing across the plate (Figure 2). Klebsiella pneumonia, bacteria B, had very large and irregularly spaced globs of colonies growing (Figure 3), which ultimately ended up being much more abundant in the second streak plate than the few that grew in the first. The first streak plate likely had issues growing due to human error in the experiment; possibly using the inoculating stick in the bacterium while it was still too hot, which would ultimately cause the bacteria to die before they reached the streak plate. After obtaining the gram positive and negative results, it was easy to determine which tests to use in order to deduce the type of bacteria that was contained within the unknown. A variety of differential media was used for both bacteria A and B in order to determine the appropriate nutrient requirements for the growth of each species. One of the most telling of the differential medias was the Blood Agar which was effective in deducing the hemolytic activity of the gram positive cocci (Figure 4). This part of the test confirmed that there was gamma activity and from this it could be deduced that the red blood cells were completely destroyed. The MSA tests were usuful in demonstrating the isolation of Staphylococci, this test was used to further confirm the results for the gram positive bacteria A (Figure 5). After getting results that were inconclusive for this test (Figure 1), there was a debate on whether or not a human error was made by possibly using a hot inoculum loop and killing the bacteria, or if maybe there was an issue with the media. It was noted while doing this spread plate that the media was exceptionally thin, which may not have allowed for the proper growth of the bacteria. The PEA experiment was done due to its ability to select for gram positive bacterium (Figure 7), however since these results were also inconclusive it was thought that human error was likely the cause of this. In future experiments, it would be wise to be sure to cool the inoculum loop thoroughly before grabbing any bacteria to spread on the plate so that all of the bacteria remain intact and are able to grow on the media. When choosing to do the MRVP test, weak positives were indicated which concluded that there was a slight acidic nature to the mixture, which helped to further conclude the bacteria that were within the unknown (Figure 8).
The gram negative bacteria B, was determined to have Beta hemolytic activity (Table 1).
The MacConkey test was then used to confirm these results, since it has the ability to select for gram negative bacteria (Figure 9). With negative results for this test, a variety of bacteria were able to be eliminated from the list of bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter aerogenes. The EMB test (Figure 10), helped to further eliminate more bacteria including E. Coli. This confirmed our results along with the negative results for the Indole test and the Sulfur reduction test (Figure 6). What ultimately led to our conclusion after comparing our results was the positive catalase test, which indicated that we in fact isolated the bacteria and got results that confirmed that one of our bacteria was in fact Staphylococcus aureus and one was not, and with the other tests eliminating a vast majority of the bacteria we were able to conclude that Klebsiella pneumonia was the other bacteria within the unknown (Table 1, Figure
1).
There were several issues that were faced while performing all of the tests in this experiment. The main issue that was encountered was trying to differentiate the colonies enough to isolate into new spread plates. This was done based on growth patterns, shape, size, and color, however this seemed to be a little more bias, which could have very well skewed the results obtained for this experiment. The majority of the tests performed confirmed the results, however there were some inconclusive results that may or may not have affected what the bacteria were perceived to be. Another issue faced was technique and possibly contamination. The biggest issue with technique was likely using a still hot inoculum loop to obtain the bacteria to spread, which may have been why some of our results were inconclusive. Other than these mishaps, the majority of the tests and results obtained led to the conclusion made on which bacteria were actually contained within the unknown.
Staphylococcus aureus is a spherical shaped gram-positive bacterium that is responsible for many diseases. This bacterium is most commonly found in the respiratory tract and on the skin, and although it may not always cause disease, it is the common cause of respiratory and skin infections and frequently associated with food poisoning (Lindsay 2008). This bacterium has also had its entire genome sequenced, which has contributed to many advances in research.
Klebsiella pneumoniae is a gram negative rod shaped bacterium that is non-motile and has the ability to ferment lactose. This bacterium can commonly be found in the mouth, epidermis, lungs and intestinal tract. This bacterium is most detrimental to the lungs due to its ability to cause inflammation, bleeding, and ultimately cell death (Ryan 2004). Klebsiella pneumoniae is commonly found in those with a very weak immune system, as it can work much easier and faster when the body is not fighting back. This bacterium is known to cause a variety of diseases with the most common being pneumonia and is typically resistant to antibiotics and can be contagious. When an antibiotic does not work for this disease, a surgical clearing usually must take place in order to remove all of the bacteria.

References
Beveridge TJ (March 1990)."Mechanism of gram variability in select bacteria". Journal of bacteriology 172 (3): 1609–20.

Choi, D. 2014. Applied Microbiology BSc 306 Lab Manual. Unpublished Manuscript

Gallup, Dave. "E. Coli: A "Model Organism" From Theodor Escherich 's Legacy." 8.3 (2010): n. page. Web. 19 Feb. 2014. http://www.emlab.com/s/sampling/env-report-03-2010.html

Lindsay, Jodi. Staphylococcus: Molecular Genetics. 1st edition. 1. London UK: Caister Academic Press, 2008. 1-71. eBook.

Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill.

Figure Legend

Fig.1. Flow chart indicating the order of the testing process and results, along with the though process of how the results were determined to identify the species contained in unknown bacteria # 15
Fig.2.Isolation of unknown bacteria A
Fig.3. Isolation of unknown bacteria B
Fig.4. Blood Agar plate of both bacteria A and B
Fig.5. MSA plate of both bacteria A and B
Fig.6. SIM including specific Indole tests of both bacteria A and B
Fig.7. PEA plate of both bacteria A and B
Fig.8. MR test of both bacteria A and B
Fig.9. MacConkey test of both bacteria A and B
Fig.10.EMB test of both bacteria A and B
Fig.11. Gram positive bacterium at 40x magnification
Fig.12. Gram negative bacterium at 40x magnification

Figure 2. Second isolation of bacterium A from the unknown sample number 15.

Figure 3. Second isolation of bacterium B from the unknown sample number 15.

Figure 4. Blood agar plate results with bacteria A on the right and B on the left

Figure 5. MSA plate results with both bacteria A (right) and B (left)

Figure 6. SIM test including individual indole test for both bacteria A and B

Figure 7. PEA test results for both bacteria A and B

Figure 8. Methyl Red test for bacteria A and B

Figure 9. MacConkey test for both bacteria A and B

Figure 10. EMB test for both bacteria A(right) and B (left)

Figure 11. Gram positive test for Staphylococcus aureus (Bacteria A)

Figure 12. Gram negative test for Klebsiella pneumoniae (Bacteria B)