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Lab 5: Differential Staining
Learning Objectives: Explain the principle of differential staining Apply differential staining to identify bacteria Differential stain uses two or more stains to specifically stain certain structures or cellular components which cannot be easily observed using simple stains. Differential staining principles are based upon the specific chemical nature and composition of cellular components and therefore, different structures are observed using different stains and staining procedures. Differential staining often becomes the basis of identification of the bacteria in clinical labs. In this lab, we will learn about four separate staining procedures. Gram Staining Endospore Staining Acid-fast Staining Capsule Staining (Special Staining) Exercise 1: Gram Staining Gram staining is used to identify cells based on the differences of the cell wall. While some bacteria may contain a thick layer of peptidoglycan that forms a thick rigid cell wall (Gram positive), other bacteria have a very thin layer of peptidoglycan forming a thin cell wall sandwiched between two cell membranes (Gram negative). The outer membrane is rich in lipopolysaccharides (LPS). Gram staining takes advantage of these differences in the cell wall of bacteria. Using two different stains that offer a lot of contrast, bacteria Bacteria with thin cell wall Bacteria with thick cell wall Primary Stain: Crystal Violet Mordant: Iodine
BIOS242 Lab 5 Name: containing a thick cell wall are stained purple. They are called Gram-positive cells. Bacteria containing a thin cell wall are stained pink and are called Gram- negative cells. In Gram staining, bacteria are first treated with a primary stain, crystal violet. Upon treatment with crystal violet, all cells are stained purple irrespective of presence of thick or thin cell wall. Stained bacteria are then treated with a mordant, iodine. Iodine helps crystalize crystal violet on the cell surface and therefore, cells retain crystal violet with higher affinity. After that, cells are rinsed with a decolorizing agent, a solution of acetone and alcohol. The decolorizing agent dissolves lipids and peptidoglycan from both gram positive and gram negative cell walls. However, the thicker peptidoglycan layer of the Gram positive bacteria helps to retain some of the crystal violet. This step is the most CRITICAL step in the staining process. Over decolorization can completely decolorize Gram positive bacteria as well. Under decolorization will make Gram negative bacteria appear as Gram positive. Lastly, bacteria are stained with a counterstain, safranin. Both Gram positive and Gram-negative cells will stain with safranin but the purple crystal violet will override the pink color in Gram positive cells. The Gram-negative cells, which are colorless cells after decolorization, will appear pink after using safranin. Materials: Broth cultures of S. epidermidis , E. coli, a mixed culture containing one Gram positive and one Gram negative bacteria; sterile loop, Gram staining kit, glass slides, Incinerator, DI water, marker, immersion oil, lens paper, bibulous paper, microscope Note to students: Wear gloves and use PPE before starting the lab work. Use aseptic technique to prevent contamination. Use only 1-2 drops of stain per slide. Avoid using excess. Method:
- Obtain glass slides and pure cultures. Write the name of the bacteria on the side of the glass slide.
- Using aseptic technique make a thin smear. Allow it to air dry and then heat fix it.
- Add a drop or two of crystal violet and allow staining for 1 minute.
- Gently wash off crystal violet using a few drops of DI water.
- Add a few drops of iodine solution to the slide and wait for 1 minute.
- Wash iodine solution using a few drops of water. Counterstain: Safranin Decolorizing agent: Acetone/Alcohol Gram positive cell Gram negative cell
Name: Nocardia waxy cell surface using carbolfuchsin. A mix of acid-alcohol is used to decolorize cells that do not have waxy cell surface and then counterstained with methylene blue. Non-acid-fast cells- stained blue
Name: Materials: Prepared slides of endospore, capsule and acid-fast staining; immersion oil, lens paper, microscope Method:
- Obtain the prepared slides.
- Observe slides using the 10X, and 40X objective lenses.
- Record observations made using 40X objective lens in the lab report. a. Check with instructor to determine if use of the oil immersion lens is necessary. If using the oil immersion lens, follow the steps outlined in Appendix A. b. Record observations in lab report.
- Complete the lab report.
Name: Additional observation/notes: Shape: cocci Color: pink Gram – negative or positive: Gram – negative Anaerobic or aerobic: Anaerobe bacterium
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Name: Mixed culture: S. Marcescens + E. Aerogenes Additional observation/notes: S. Marcescens has the cocci shape E. Aerogenes has the rod shape Shape: Rods and cocci Color: pink Gram – negative or positive: both gram – negative Anaerobic or aerobic: both are anaerobic
Name: Questions: Complete the following table by predicting colors of bacteria with thick and thin cell walls as they are processed through the steps of Gram staining. Steps of Gram Staining Bacteria containing thick peptidoglycan layer (cell wall) Bacteria containing LPS, thin cell wall Crystal violet treatment Purple Purple at first but then washes off Iodine Purple clear Decolorization Purple clear Safranin purple pink
- A fellow student showed you a Gram stained slide where cells containing thick cell walls were stained pink. What would you tell her about the staining procedure? Why? The cells turned pink because there was a contamination and also it could be that the dye rinsed off during the staining process. The staining procedure must be done correctly in order to yield good results.
- A fellow student showed you a Gram stained slide where cells containing LPS and a thin peptidoglycan layer were stained purple. What would you tell her about the staining procedure? Why? I would tell the student that without alcohol during the decolorizing step, the bacterial cell wall was not altered and that prevented the iodine and crystal violet from being rinsed off. Exercise 2: Differential Staining- Observation of prepared slides Draw the observation inside the circles provided. Label your image appropriately. Write the magnification at which the observation was made. Endospore staining:
Name: Acid-Fast Staining: Additional observation/notes: The shape is a coccus and this image is a gram negative stain because its purple.
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- Describe the differences between simple staining and differential staining principles. Differential staining is a procedure that allows people to visually distinguish between different types of bacterial cells and show that not all cell types stain equally. The simple stain can be used as a quick and easy way to see cell shape, size, and arrangement of bacteria.
- A student forgot to use heat during the endospore staining procedure. How will it impact the observation? Since the slide was not heat fixed, the impact that It will cause the smear to be washed off during the staining process.
- Why is the capsule stain called a negative staining method? The capsule stain is called a negative staining method because it stains the background and not the actual capsule or bacteria. Also it’s a gentle staining procedure.
- What is meant by the term “acid-fast”?
Name: Acid fast refers to the property of mycobacteria to retain carbol fushsin even in the presence of acid alcohol.
