Gram Positive Bacterial Unknown for Microbiology
The information contained on this page is correct to my knowledge. It was all derived from my personal research and testing. Information from this site may be borrowed, but please site your references correctly. Incorrectly sited refrecnes constitute PLAGIARISM!!!!!! This is punishable by various laws and regulations.
James Arnold
Microbiology
December 2, 1999
Gram Positive Unknown
The gram positive unknown that was assigned to me was in solution with a gram negative unknown that also had to be identified. In order to identify them, the technique of streak for isolation had to be conducted in which the two bacteria were streaked on nutrient agar and then differentiated by their appearance. The two unknowns that I had were not easily distinguishable and considerable trouble in isolating them resulted. At one point, I only grew about 2 million cells of my gram negative unknown on a plate and about 200 cells of gram positive. From these statistics, I knew it would be impossible to isolate the individual bacteria. I figured out these statistics by performing a gram stain on the mixed culture to see if I had two bacteria growing. I did have two bacteria growing, but the gram negative was growing faster than the gram positive. After many attempts, Dr. Darden gave me the gram-positive bacteria and I had to continue with the project. Growing pure cultures of the gram negative was very easy because there were so many of them. The chances of picking one gram positive cell with all the other gram negative cells was very small and so a separate streak was done with the gram negative bacteria for their identification.
The gram-positive bacteria were gram stained using the method outlined in the lab manual on page 61. The procedure followed the steps:
1. The bacteria are smeared on a slide and allowed to dry.
2. The bacteria is heat fixed to the slide
3. Crystal violet pigment is added to the smear for 1 minute
4. Wash the pigment off with distilled water
5. Apply Gram’s Iodine for 1 minute
6. Wash iodine off with distilled water
7. Decolorize the smear with 95% ethyl alcohol drop by drop until it runs off clear
8. Wash off alcohol with distilled water
9. Counterstain with Safranin for 45 seconds
10. Wash off Safranin with distilled water
11. Blot dry with Bibulous paper
Following this procedure I found that my bacterial cells were both rod shaped and in chains. Their dark blue appearance on the slide made determining the shape and gramness of the bacteria rather easy. Following the flow chart that was given to us, I found that the next test that I had to conduct was a test for spores-formers. The test used was the spore stain also known as the Schaeffer-Fulton Method. This test is outlined on page 71 of the lab manual and consists of using the primary stain Malachite green to stain the cells. The cells are flooded with the primary stain for three minutes while on a hot plate. The heat is used to aid the primary stain to penetrate the impervious layers of the spore. Following this stain, both vegetative and spore cells are green. The next step is the decolorizing agent, which is distilled water. After heating the cells for three minutes, they are allowed to cool and then are decolorized by the distilled water. All the primary stain is removed from vegetative cells, but the spore will retain the stain. The final step is to counterstain the cells with Safranin. The Safranin will color the previously decolorized vegetative cells red so that they now contrast the green spore cells. After washing the Safranin off of the cells, they are examined under the microscope under oil immersion for spores. My unknown didn’t have spores and so that led me to the next test to determine if the bacteria were acid fast or not. At this point, I knew that the bacteria had to be either Mycobacterium or Lactobacillus in nature according to the flow chart. My guess at this point was Lactobacillus because I have a feeling that the acid-fast test would be negative.
The acid-fast test as stated on page 65 of the lab manual is a test that is designed to group bacteria into two groups, acid-fast and non-acid-fast. There are some bacteria such as the Mycobacterium that have a thick waxy capsule and cannot be stained with other staining methods, while other can be stained using simple stains and the Gram stain. This was the determining factor for the unknown bacteria. Was it a member of the genus Mycobacterium? The acid-fast stain also called the Ziehl-Neelsen Method uses three reagents. Carbol Fuchsin is the primary stain. Acid-Alcohol (3% HCl + 95% Ethanol) is the decolorizing agent. The counterstain is Methylene Blue. For this test, I used a 24-hour old culture of the bacteria as compared to a 72-hour culture for the spore stain. The test consists of applying Carbol Fuchsin to the bacterial smear for 5 minutes over heat to aid in penetration of the stain. After 5 minutes, the cells are decolorized by distilled water. The acid-alcohol is added drop by drop until it runs clear and then it is washed off using distilled water.
Finally the counterstain Methylene Blue is added for two minutes and then washed off with distilled water. The slide is then blotted dry using bibulous paper and then is viewed under oil immersion to determine if the bacteria are acid-fast. Non-acid-fast cells will undergo decolorization when the acid-alcohol is run over them and will become blue when the counterstain is added to them. Acid fast cells will retain the primary stain and remain red even after decolorization and counterstaining. The bacterial unknown was found to be non-acid-fast because it remained blue after the counterstaining. This means that the bacteria must be a member of the genus Lactobacillus. After looking in Bergey’s Manual, I found that my unknown bacteria were Lactobacillus casei.
The Lactobacillus genus is very useful to humans. It is very important in the production of fermented vegetables such as pickles and it is also used in producing such products as beer, wine, and sourdough breads. It has a commensal relationship with humans and animals in the digestive tract. Outside the human body this genus is found in animal feeds, manure, and milk because they dependent on lactose as a form of subsistence.
My Favorite Web Sites
Angelfire - Free Home Pages
My Main Page
Email: arnoldj@citadel.edu