Staining Procedures PDF

STAINING (Smear Preparation, Simple Staining, Negative Staining, Gram Staining, Acid-Fast Staining, Capsular Staining, Endospore Staining, Flagella Staining ) FEROZ KHAN ( MPhil Scholar) UOB,Quetta  Sme...

STAINING (Smear Preparation, Simple Staining, Negative Staining, Gram Staining, Acid-Fast Staining, Capsular Staining, Endospore Staining, Flagella Staining ) FEROZ KHAN ( MPhil Scholar) UOB,Quetta  Smear Preparation The process of making a smear preparation is an important skill in the microbiology laboratory and is usually the first step in most staining procedures. The quality of the smear will directly affect the quality of the subsequent staining procedure. The smear preparation differs slightly depending on the specimen or culture  Supplies 1. Personal protective equipment 2. Sharps container 3. Biological waste container 4. Microscope slides with frosted-edge 5. Pencil or wax pencil 6. Sterile saline or water 7. Sterile pipettes 8. Loops or applicator sticks 9. Slide warmer, Bunsen burner, or methanol  Procedure of Simple Staining Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each: carbol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue, 1 to 2 minutes. Gently wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the stream of water; in this way you can reduce the loss of organisms from the preparation. Using bibulous paper, blot dry, but do not wipe the slide. Examine all stained slides under oil immersion Requirement of Negative Staining. Nigrosin,Microincnetator, Bunsen burner, inoculating loop, staining tray, glass slides, lens paper, and microscope  Procedure of Negative staining 1.Place of small drop of nigrosin close to one end of a clean side. 2. Place a side against the drop of suspended organisms at a 45 angle and allow the drop to spread along the edge of the applied slide, 3. Using aseptic technique place of loopful of inoculum from the bacterial culture in the drop of ingrowing and mix 4. Push the slide away from the drop of suspended organisms to forma thin smear. Air ,dry 5. Note. Do not heat fixed the slide 6. Examine the slide under the oil immersion.  Gram stain The Gram stain is a differential staining procedure used to categorize bacteria as Gram-positive or Gram-negative based on the chemical and physical properties of their cell walls. The bacteria are differentiated through a series of staining and decolorization steps. Gram-positive cells will stain purple and Gram-negative cells will stain red to pink.  Supplies and Reagents 1. Personal protective equipment 2. Slide rack 3. Timer 4. Absorbent paper, such as bibulous paper 5. Water (tap water or deionized) 6. Crystal violet 7. Gram’s iodine 8. Decolorizer 9. Safranin (or carbol fuchsin) 10. Bright field microscope with 100X objective 11. Immersion oil 12. Pencil  Instructions Place the prepared fixed smear on a slide rack then flood the slide with crystal violet. 2. Wait 15 seconds then rinse the slide with water. 3. Flood the slide with Gram’siodine. 4. After 15 seconds rinse the slide with water. 5. Apply the decolorizer to the slide. 6. Rinse the slide immediately with water. 7. Flood the slide with counterstain. 8. Wait 15 seconds then rinse the slide with water. 9. Blot the slide with absorbent paper. Be careful not to wipe the cells off the slide. 10. Allow the newly stained slide to air dry completely. 11. View the slide under oil using the oil immersion objective for a total magnification of 1000X. 12. Record results based on your laboratory’s criteria.  Preparation of microscope slide: ✓ Clean slide with a Kimwipe and alcohol to remove any fingerprints. ▪ Draw two circles with your Sharpie on the bottom of the slide. ▪ Using your inoculation loop, put two small drops of water in each circle. ▪ Using aseptic technique, remove a very small amount of bacteria from the culture tube. Make sure you flame the tube before and after you enter. Continue.. ▪ Smear the bacteria in the drop of water on your slide. You may go out of the perimeter of your circles! ▪ Let the slide air dry completely. ▪ Heat-fix the slide by running it through the flame 3-4 times with the ‘smear’ side up. Do not flame the side with the bacteria! ▪ Let the slide cool completely and you are ready to stain it.  Acid-fast Stain Procedure Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids.  Staining procedure: 1.Cover the smears with a piece of paper towel within the border of the slide. 2.Place the slide over a beaker of steaming water. Do not let the beaker boil dry 3.Flood the paper towel with carbolfuchsin and let the slide steam for 3-5 minutes. 4.Complete the rest of the procedure at the back sinks. 5.Remove the stained paper towel and discard it in the trash can, not in the sinks. 6.Gently wash out the slide with water to remove any pieces of a loose paper towel and tap dry. Continue.. Apply acid-alcohol for 15-30 seconds. Rinse off and tap dry. Counterstain with methylene blue for 1.5 minutes. Rinse with water and tap dry. Blot gently with bibulous paper. Dry the bottom of the slide before placing it on the stage of the microscope and view with the oil immersion lens.  Procedure of Capsule Stain 1.Prepare thin smears of bacterial culture on a microscope slide. 2.Allow the smear to only air-dry. Do not heat-fix as this will cause the capsule to shrink or be destroyed. 3.Apply 1% crystal violet and allow it to remain on the slide for 2 minutes. 4.With the slide over the proper waste container provided, gently wash off the crystal violet with 20% copper sulfate. Caution: Do not wash the copper sulfate and stain directly into the sink. 5.Blot the slide dry with bibulous paper. 6.Observe with the oil immersion lens  Principle of Endospore Staining 1. Take a clean grease free slide and make smear using sterile technique. 2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or towelling cut to fit the slide. 3. Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required. 4. Alternatively, the slide may be steamed over a container of boiling water. 5. Wash the slide in tap water. 6. Counterstain with 0.5% safranin for 30 seconds. Wash with tap water; blot dry. 7. Examine the slide under microscope for the presence of endospores. 8. Endospores are bright green and vegetative cells are brownish red to pink  Procedure of Flagella Staining ✓ Grow the organisms to be stained at room temperature on blood agar for 16 to 24 hours. ✓ Add a small drop of water to a microscope slide. ✓ Dip a sterile inoculating loop into sterile water ✓ Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water). ✓ Touch the loopful of motile cells to the drop of water on the slide. ✓ Cover the faintly turbid drop of water on the slide with a cover slip. A proper wet mount has barely enough liquid to fill the space under a cover slip. Small air spaces around the edge are preferable.  continue.. ✓ Examine the slide immediately under 40x for motile cells. ✓ If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes. ✓ Apply 2 drops of RYU flagella stain gently on the edge of the cover slip. The stain will flow by capillary action and mix with the cell suspension. ✓ After 5 to 10 minutes at room temperature, examine the cells for flagella. ✓ Cells with flagella may be observed at 100x.  Observe the slide and note the following: Presence or absence of flagella Number of flagella per cell Location of flagella per cell