30 Jun there are experiment you should read to answer the questions
Exercise 4: Gram Staining Except certain bacteria such as actinobacteria (mycoplasma) and chlamydia, all bacterial cell walls contain polymers of amino acids and carbohydrates, called as peptidoglycan. Peptidoglycan is consisted with the repeated units of N-acetylgalactosamine and N-acetyl muramic acid. The thickness of peptidoglycan layers is different between Gram-positive and –negative bacterial cell walls―Only one~a few layer(s) of peptidoglycan would be present in Gram-negative cell wall but peptidoglycan layers are much thicker in Gram-positive, usually around 10 layers. Gram staining takes this difference as an advantage. One chemical dye, crystal violet (Figure 4.1), is used to stain the peptidoglycan layer; after quick destaining procedure, Gram-negative cells cannot retain crystal violet because of thin peptidoglycan layers but Gram-positive does. As described in the previous chapter, most specimens require contrast. Thus, safranin is used as the counter staining dye in Gram-staining. Some bacteria such as Mycobacteria are not stained well with Gram staining, because of the presence of the waxy layer above the peptidoglycan. Figure 4.1 Chemical structure of crystal violet (left) and safranin (right). Figure 4.2 Gram staining of Staphylococcus aureus (strain; ATCC 25933) [purple] and Escherichia coli (strain; ATCC 11775) [pink] mixture. 16 Materials You do not need to prepare solution, however, remember the name of staining reagent and the whole procedure. Crystal violet solution (Hucker’s) solution A crystal violet 95% ethanol solution B ammonium oxalate distilled water Mix solutions A and B. 2.0g 20.0ml 0.8g 80.0ml Gram’s iodine solution iodine potassium iodide distilled water 1.0g 2.0g 300ml Safranin solution safranin O solution 95% ethanol distilled water 0.25ml 10.0ml 100.0ml 95% ethanol Slide glasses cover glasses transfer loops microscopes Gram-positive bacteria (Staphylococcus aureus, Bacillus substilus, Lactobacillus etc.) Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli etc.) Note: It is strongly preferred to use freshly prepared, actively growing bacterial culture for Gram staining. Procedure A 1. Gram staining is tricky. I strongly recommend doing the staining of known Gram-positive and negative cells on the same slide (Figure 4.3). Alternatively, it is good to mix obviously distinguishable Gram-positive bacteria in your sample (I used to use Bacillus s because cells are elongated rods (more than several mm) and thus you can easily distinguish from majority of other bacteria, unless your sample contain Bacillus. 2. Then prepare bacterial smear as we did in the previous chapter. 3. Place the slide on a staining chamber. 4. First staining; cover all smears with crystal violet solution. Incubate for 1 minute. 5. Gently rinse with water. 6. Cover with Gram’s iodine solution (mordant) and let it stand for 1 minute. 7. Repeat the procedure 5. 8. This step, de-staining, can determine your success. Be accurate (Do not de-stain too much). 9. Quickly move to rinse step; gently rinse with water to remove ethanol. 17 10. 11. 12. 13. Now counter staining; cover smears with safranin solution. Incubate for 45 seconds. Gently rinse with water. Remove excess water and mount a cover slip. Observe your sample with a microscope. You would need high magnification objective lens such as 40X Figure 4.3 How to place the control(s) on the same slide Note: having control(s) on the same slide is very important. 18 Figure 4.4 Gram staining procedure (by A. Rich) Following videos may be useful to understand the entire process; https://www.youtube.com/watch?v=FassELAQa1Y http://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::530::530::/sites/dl/free/0073525502/9 30300/Gram_Stain.swf::Gram%20Stain 19 Post-lab questions exercise 4 1. What is the advantage of Gram staining over simple staining methods? Explain. 2. Briefly explain the purpose of reagent used at each step; 3. Why is it important to have both Gram-positive and -negative bacteria on the same slide? 4. Draw Gram-positive and negative cell wall structures. 5. List three pathogenic Gram-positive and Gram-negative bacteria (and diseases caused by those bacteria). Refer the textbook. 1 Post-lab Questions: Exercise 2 1. Summarize the purpose, or provide the definition, of following microscope components. a. Coarse- and fine-adjustment knobs: b. Condenser: c. Magnification: d. Resolution: 2. Why is it important to carefully watch the distance between an objective lens and the specimen? How can the specimen get damaged by quickly changing the objective lens too quickly? 3. Provide the calculation of how to determine the total magnification. 4. List how to properly prepare your microscope for storage (i.e. is the stage left in the uppermost level or lower most, which objective lens should be selected?). 5. Sketch at least one cocci and one rod bacteria below with low and high power (magnification). Your sketches should state the following information: name of organism, cell shape and magnification. University of Bridgeport Katsuhiro Kita, Instructor of Record Patricia Clark, Laboratory Instructor Exercise 9 Biochemical Characterization I Introduction Catalase activity is very important to characterize unknown bacteria (especially Grampositive bacteria). In addition, this assay is so easy and quick (no need to incubate bacteria another day!) so that this procedure is even useful in clinical settings; catalase assay allows quick identification of Staphylococcus aureus by the combination with other selective media. On the other hand, oxidase is an enzyme Materials of Catalase TSA agar plates 3% hydrogen peroxide transfer loop Bunsen burners slide glasses Bacteria grown on slants or plates (Staphylococcus aureus, Micrococcus luteus and bacteria of your choice) Procedure A: Catalase test 1. Here, we try the slide method. This would be the easiest method. Have a clean (VERY IMPORTANT; dirty slide glasses might have something that could degrade hydrogen peroxide!) slide glass. Place a few drops of 3% hydrogen peroxide on a glass. 2. Suspend a small chunk of bacterial colony. Recommendation: use bacteria grown in broth in this test – sometimes the quantity of bacteria may not be sufficient. 3. If catalase activity is present, soon you can see the formation of air bubbles (oxygen) as shown in the picture below. Figure 9.1 Catalase test with the glass method (from Microbeonline.com). Materials for Oxidase TSA agar plates p-aminodimethylaniline oxalate solution p-aminodimethylaniline oxalate distilled water 0.5g 50.0ml You may need to warm up the solution to completely dissolve p-aminodimethylaniline oxalate. Transfer loops Bunsen burners Procedure B: Oxidase test 1. First, you need to grow organisms on agar plates. This setting may be done a week before the exercise. Alternatively, we can quickly test by taking a chunk of bacterial colony with a sterile cotton swab containing p-aminodimethylaniline oxalate solution. 2. Add one to a few drops of p-aminodimethylaniline oxalate solution on an agar plate with bacterial streaks on it. 3. Let the plate stand at room temperature for a minute; you might need to wait for a few minutes. 4. Record the color change. Figure 9.2 Haemophilus influenzae on chocolate agar showing oxidase positive (dark blue colonies). This simple combination allows relatively simple and reliable identification of H. influenzae, a human pathogen. Figure 9.3 (right) Paper-based quick assay for oxidase detection (Difco Laboratories). Take bacteria with a sterile cotton swab and rub on the assay kit. Only oxidase positive (2) bacteria change the color. Alternatively, you can dip p-aminodimethylaniline oxalate solution and take fresh bacteria from slants or agar plates. Post-lab questions: Exercise 9 1. Summarize your observation in catalase test. Bacterial species Staphylococcus aureus Streptococcus pyrogens Micrococcus luteus Bacillus cereus Pseudomonas aeruginosa Catalase activity (+ or -) 2. Summarize the results of oxidase test. Bacterial species Staphylococcus aureus Streptococcus pyrogens Micrococcus luteus Bacillus cereus Pseudomonas aeruginosa Oxidase activity (+ or -) 3. Is catalase an endoenzyme or exoenzyme? Briefly describe your thought and reason beyond your choice. 4. How does p-aminodimethylaniline oxalate change the color of the colony in the presence of oxidase? Search internet to find out the mechanism and briefly describe below. 5. What is the end-product (chemical; showing the purple color) of the oxidase reaction? How long should you wait the development of the purple color? (use the back of the paper if no room). 1 …
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