Hello Everyone!

      Welcome to Week Three of Dylan and Devin Discovering Dirt! This past week Dylan and I made another leap and bound into our soil microbe research. The objective of the last step in our research was to determine if the soil microbe that we have specifically chosen is Gram-positive or Gram-negative. What are Gram-positive and Gram-negative cells you ask? Let me give you the DIRTY details.

      First, you need to know what a Gram stain is. A Gram stain is the method of taking bacteria and separating them into two distinct groups by using the physical and chemical properties of their cell walls to determine if peptidoglycan is present. Peptidoglycan is a polymer made of amino acids and sugars. These amino acids and sugar combine to form a net-like layer on the outside of the plasma membrane is the majority of bacteria, and this membrane is known as a cell wall. The amount of peptidoglycan in the cell wall is what differentiates Gram-positive from Gram-negative. 

This figure shows the difference in the amount of peptidoglycan present in a Gram-positive cell wall in comparison to a Gram-negative cell wall. It can be seen that the peptidoglycan layers in the cell wall of the Gram-positive bacteria are MUCH thicker than the single layer in the the Gram-negative bacteria.

Gram-positive Bacteria

      Now for the DIRTY details of what differentiates a Gram-positive and Gram-negative bacteria. Gram-positive bacteria are bacteria that give positive results in a Gram stain test. The bacteria absorb the crystal violet stain used in the test, which results in the purple appearance seen when looking at the bacteria through the microscope. This purple color is seen because the thick peptidoglycan layer in the bacterial cell wall has the ability to retain the crystal violet stain after it is washed away from the rest of the sample, in the decolorization stage of the test. 

Gram-negative Bacteria 

      Gram-negtive bacteria are unable to retain the crystal violet stain after the decolorization step. The alcohol used during decolorization degrades the outer membrane of gram-negative bacteria, which makes the cell wall more porous resulting in the inability to retain the crystal violet stain. The peptidoglycan layer of Gram-negative bacteria is much thinner and is in between an inner cell membrane and a bacterial outer membrane. This placement causes them to take up the counterstain (safranin or fuchsine), which causes the bacteria to appear pink.

The Gram staining process.

      So, now that you know the differences between Gram-positive and Gram negative bacteria and the process of a Gram stain, it's time to see how Dylan and I did in staining a bacteria for ourselves! Dylan and I went through the process of smearing our unknown bacteria onto a microscope slide. We then proceeded to flood the smear with crystal violet dye. Once the dye had been on the smear for a minute its was rinsed off with water, and then Gram's iodine was added to the smear to soak for one minute. After the Gram's iodine sat for one minute it was rinsed off gradually with 95% ethanol to remove any excess crystal violet dye on the smear. Once all excess dye was removed the Gram's safranin counterstain was added to the smear for 30 seconds and then rinsed off. It can be seen in the picture of the Gram stain process the Dylan and I stained our unknown bacteria (bottom slide: UK), a Gram-negative bacteria (middle slide: K. pneumonia), and a Gram-positive bacteria (top slide: B. megaterium).

     

     

Unknown bacteria slide through the
microscope lens.

      After the Gram staining process and after looking at our unknown slide in comparison to our Gram-positive and Gram-negative control slides under the microscope Dylan and I were unable to make a decision in full confidence as to which type of bacteria our unknown was. It can be seen in the picture to the left that out bacteria is purple in color, which lead us to come to the conclusion that our sample was Gram-positive but we were still uncertain. The uncertainty is due to the fact that the shapes of our bacteria aren't consistent throughout the entire smear. 

Coccus                                                         Bacillus                                                  Diplobacillus 

      The three types of bacterial shapes above are the bacterial shapes Dylan and I believe are seen in our unknown bacterial sample. After doing a little bit our research and consulting with our lab instructor Dylan and I arrived at the conclusion that our unknown sample was a type of bacteria known as endospore-producing bacteria. An endospore-producing bacteria is also known as sporulating bacteria. Sporulating bacteria are derived from the phylum Firmicute and consist mostly of Gram-positive bacteria . These endospore-forming bacteria belong primarily to the Bacillus and Clostridium genus. So, even though Dylan and I didn't get our prediction 100% accurate, we did come close in saying that the unknown was endospore-producing because endospore-producing bacteria are primarily Gram-positive. 

MacConkey agar plate including the unknown
bacteria, B. megaterium (+), K. pneumonia (-),
P. aurigenosa (-).

      Aside from looking at the color and the shape of the unknown bacteria through the microscope and comparing them to the Gram-positive and Gram-negative control bacteria, Dylan and I also set up a MacConkey agar in order to observe Gram-positive and Gram-negative growth. MacConkey agar is used for the isolation of Gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting Gram-negative bacteria. A compact version of the T-streak method was used on the MacConkey in order to test all four bacteria on one plate. When looking at the plate, it can be seen that the two Gram-negative controls both showed growth, but the Gram-positive control and the unknown bacteria had no growth. The fact that the B. megaterium and the unknown bacteria did not have any growth occur is just more evidence to prove that Dylan and I were correct in predicting that our unknown bacterial sample is Gram-positive. 

     To wrap up the blog Dylan and I have decided to do some critical thinking and answer the question, "How does the Gram status influence the treatment of bacterial infections?" Gram-positive bacteria have a much greater amount of peptidoglycan in their cell membrane, which means that they have a very thick outer layer of peptidoglycan around their cell wall. This thick outer layer, has the ability to absorb large amounts of foreign material. Gram-negative bacteria, on the other hand, have a very thin outer membrane. Even though the membrane of Gram-negative bacteria is very thin it is very difficult to penetrate. Because of their thin but strong cell membrane, gram-negative bacteria are often resistant to many types of antibiotics. The level of difficulty associated with penetrating the cell wall of a Gram-negative bacteria is one of the reasons that Gram-negative bacteria are harder to treat than Gram-positive bacteria.

      Another reason, and probably the most important reason, that Gram-negative bacteria are harder to treat than Gram-positive is their resistance to drugs. Dr. Brad Spellberg, an infectious-disease specialist at Harbor-U.C.L.A. Medical Center in Torrance, Calif., and the author of “Rising Plague,” a book about drug-resistant pathogens states that, “For Gram-positives we need better drugs; for Gram-negatives we need any drugs.” Dr. Azza Eleman also said, “You don’t really have much choice, if a person has a life-threatening infection, you have to take a risk of causing damage to the kidney.” This quote is in reference to the only two drugs really used for treating a Gram-negative bacterial infection; collision and polymyxin B. These drugs were established in the 1940's and have really not been put to much use since then because they have such deleterious effects on the kidney and cause serious nerve damage. Although these drugs have horrible side effects, like Dr. Eleman said, they have been very rarely used which means that the Gram-negative bacteria have not built up a resistance to them.

      So in conclusion, Gram-positive bacterial infections are much easier to treat than Gram-negative bacterial infections because even though they have a thicker peptidoglycan layer they do not possess an outer membrane. The lack of an outer membrane in Gram-positive bacteria allows their membranes to be penetrated much more easily than Gram-negative bacteria. This means that aside from very high drug resistance causing treatment difficulty, Gram-negative bacteria is also difficult to treat because it contains the outer membrane surrounding its thin peptidoglycan layer.

http://www.nytimes.com/2010/02/27/business/27germ.html?_r=0

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