Antibiotic Resistance and Gram Staining of Unknown Bacterial Samples

Microbiology Lab
July 1, 2013
Abstract:

In this final laboratory assignment, each student was given an agar plate. Each plate was swiped with a pure culture bacteria sample. Where the sample came from and what kind of bacteria was unknown to the students. We were asked to observe the colony morphology on our plates and to perform the antibiotic sensitivity/resistance test. The plates were put into a 37° incubator oven. The next day the plates were taken out of the oven and we calculated the zone of inhibition. We then performed a gram stain of our bacteria and analyzed it under a microscope. We were then able to assess what kind of bacteria we had and if it was gram positive or negative.
Introduction:
Simple staining (the use of a single stain) allows a microbiologist to observe the morphology (shape) and arrangement of bacteria. In order to classify bacteria into different groups a differential staining procedure must be done. A differential stain involves the use of two or more stains. Depending on the components of the bacterial cell wall or outer layers, the bacteria will either retain the primary stain or have the primary stain removed in a decolorizing step and then retain the secondary stain. The gram stain is the most common differential stain used in the microbiology laboratory to categorize bacteria. The primary stain is the cationic dye Crystal violet and the secondary stain is the cationic dye Safranin. Since both stains are cationic, they are both attracted to the negatively charged particles in the bacterial cell wall. So what causes some bacteria to retain the Crystal violet stain while others are decolorized by the alcohol and then pick up the Safranin stain? The current theory behind gram staining is that the crystal violet enters the thick layer of the peptidoglycan of the gram positive cell wall. Then the iodine acts as a mordant and binds to the crystal violet. This large complex is caught within the complex of the peptidoglycan and isn’t washed away by the alcohol, used as a decolorizer. In gram negative bacteria the crystal violet-iodine complex is mainly taken up by the lipids within the outer membrane. Lipids are soluble in alcohol so the stain is washed away with the alcohol decolorizer. Then the thin layer of peptidoglycan (found below the lipid layer) can be stained with the secondary stain, Safranin.
Materials and Methods: * Gloves * Prepared bacterial agar plate * Agar plate * Inoculating loop * Sterile swab * Antibiotics 1. Streptomycin 2. Ampicillin 3. Gentamycin 4. Vancomycin * 2 glass slides * Sharpie or wax pencils * Distilled water * Sterile water * Staining tray and rack * Bunsen burner * Crystal violet stain * Gram’s iodine * Ethyl alcohol * Safranin * Microscope * Oil * Bibulous paper 1. Take a clean slide 2. Draw a circle in the center of the slide 3. Label one end of the slide 4. Turn the slide over so that the unmarked side is up 5. With the inoculating loop, place a loop full of water in the ringer area of the slide. Using proper aseptic transfer techniques mix a small amount of bacteria from the pure culture plate in the water and spread it. Make sure the smear is light. 6. Allow the smears to air dry. You should be able to see a thin white film on each slide. If not, add another loop of water and more bacteria. 7. Heat-fix the smears by passing the slides rapidly through the Bunsen flame three times so that the smears will not wash off. 8. Stain each smear by the following procedures 9. Flood slide with crystal violet. Allow to stand for five minutes. 10. Wash off with tap water. 11. Flood with Gram’s iodine (a mordant). Leave for two minutes. 12. Wash off with tap water. 13. Decolorize with alcohol drop-wise until no more color washes off (10 seconds). Be careful not to over decolorize. 14. Wash off with tap water. 15. Apply Safranin (counterstain) for two minutes. 16. Wash off with tap water. 17. Drain and blot gently with bibulous paper. Air dry the slide thoroughly before examining under microscope. 18. Examine all slides under oil with the oil-immersion objective (1000X). 19. Record observations.

Results of Unknown Sample Plate #9: ColonyGroup | # of Colonies | Form | Elevation | Margin | Surface | Opacity | Pigmentation | 1 | Too many | Circular | Raised | Entire | Shiny | Opaque | Red on yellow | 2 | Too many | Circular | Raised | Entire | Shiny | Opaque | Yellow |

Antibiotic | Zone of Inhibition | Effective? | Steptomycin | 6 mm | Yes | Ampicillin | 0 mm | No | Gentamycin | 4 mm | Yes | Vancomycin | 0 mm | No |

Bacteria observation under oil-immersion objective: * Coccus * Purple * Gram positive

Discussion and Conclusions:
According to my observations my bacteria was gram positive and cocci shaped. It was resistant to Streptomycin and Gentamycin. It was circular, raised, shiny, opaque, red and yellow, with an entire margin. My bacteria were known as Staph Epidermidis.
This lab taught me about bacteria and antibiotics. It also taught me how to use a microscope.

Citation:
Bio2010 Lab Manual http://www.itc.csmd.edu/bio/kathleen/BIO2010LabManual/Laboratory%20Exercise%20VI%20Micro.pdf