Staining techniques. Heat fixing kills the organisms, makes them adhere to the slide, and permits them to accept the stain.

Transcription

1 Staining techniques Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope. In some cases, staining is unnecessary, for example when microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be placed directly on the slide and observed. A preparation such as this is called a wet mount. A wet mount can also be prepared by placing a drop of culture on a cover slip (a glass cover for a slide) and then inverting it over a hollowed out slide. This procedure is called the hanging drop. In preparation for staining, a small sample of microorganisms is placed on a slide and permitted to air dry. The smear is heat fixed by quickly passing it over a flame. Heat fixing kills the organisms, makes them adhere to the slide, and permits them to accept the stain. 1

2 (1) Simple staining techniques Simple = only one dye is used during the staining procedure Staining can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. Such a procedure is the simple positive staining. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged (acidic) dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving the cells clear and unstained. This technique is called the simple negative staining technique. 2

3 Simple positive staining procedure 1. Add one loopful of the sample (mixture of microorganisms) onto a glass slide. 2. Allow it to air-dry. 3. Heat-fix the specimen on the glass slide, unless the specimen is heat-fixed, the bacterial smear will wash away during the staining procedure. 4. Flood slide with crystal violet and wait 1 min. or safranin and wait 3-4min. 5. Wash the smear with tap water to remove the excess of stain. 6. Blot dray, than add cederwood oil (immersion oil) and examine under a microscope. Simple negative staining procedure 1. Place a small drop of nigrosin at the end of the slide. 2. Place a loopful of sample ( mixture of microorganisms) and mix with drop of nigrosin. 3. Using the edge of another slide, spread the drop out across the slide. 4. Allow to air dray. 5. Place one drop of immersion oil and and examine under a microscope. The results of simple staining procedures (look at the figure below): Simple positive staining: all bacteria are colored, Simple negative staining: background is dark, bacteria are without any color 3

4 (2) Differential staining techniques The differential staining techniques are based on the application of a set of several different dyes which react differently with different types of microorganism. Hence, they can be used to distinguish among them. An example is the Gram staining technique. This procedure separates bacteria into two groups: Gram positive bacteria and Gram negative bacteria. Crystal violet is first applied, followed by the mordant iodine, which fixes the stain. Then the slide is washed with alcohol, and the Gram positive bacteria retain the crystal violet iodine stain; however, the Gram negative bacteria lose the stain. The Gram negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil immersion lens, while Gram positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step. Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal violet-iodine complex that occurs during staining, while Gram-negative cells have only a thin layer of peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol, and Gramnegative cells do decolorize. This allows the Gram-negative cells to accept the counter stain safranin. Gram Stain Procedure 1. Place a drop of NaCl onto the glass slide. 2. Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony. 3. Gently mix the bacteria into the NaCl drop. 4. Let the bacterial sample air dry 5. Using slide holder, pass the dried slide through the flame of Bunsen burner 3 or 4 times, smear side facing up. 6. Apply primary stain: Flood slide with crystal violet stain. 7. Rinse: After 1 minute, rinse the slide with water. 8. Apply mordant: Flood the slide with iodine. 9. Rinse: After 1 minute, rinse the slide with water. 10. Apply decolorizer: Flood slide with acetone alcohol. 11. After 10 or 15 seconds, rinse the slide with water. (Do not leave the decolorizer on too long or it may remove stain from the Gram-positive cells as well.) 12. Apply secondary stain (counterstain): Flood slide with safrinin. 13. Rinse: After 1 minute, rinse the slide with water. 14. Dry on air 15. Place the stained smear on the microscope stage smear side up, apply oil directly to the smear and focus using the 100X objective. 4

5 Gram-positive bacteria appear blue or purple Gram-negative cells will appear pink to red Results of the Gram staining procedure: Examples of Gram-positive and Gram-negative bacteria: Gram-positive bacteria: Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphteriae Gram-negative bacteria: Neisseria meningitidis, Escherichia coli, Pseudomonas aeruginosa 5

6 Acid fast staining technique Another differential stain technique is the acid fast staining technique. This technique differentiates species of Mycobacterium from other bacteria. Because the cell wall is resistant to water-based stains, acid-fast organisms require a special staining technique. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a diluted acid alcohol solution. Mycobacterium species resist the effect of the acid alcohol and retain the carbolfuchsin stain (they appear bright red under the microscope). Other bacteria lose the stain and take on the subsequent methylene blue stain (they appear blue under the microscope). The Ziehl-Neelsen staining procedure with Kinyoun modification (acid fast technique) 1. Place a drop of NaCl onto the glass slide. 2. Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony. 3. Gently mix the bacteria into the NaCl drop. 4. Let the bacterial sample air dry 5. Using slide holder, pass the dried slide through the flame of Bunsen burner 3 or 4 times, smear side facing up. 6. Flood slides with Kinyoun carbolfuchsin for 5 minutes. 7. Rinse gently with water until the water flows off clear. 8. Flood slides with acid-alcohol (3% HCl in ethanol) for 3~5 seconds. 9. Rinse gently with water until the water flows off clear. 10. Flood slides with methylene blue for 1 minutes. 11. Rinse gently with water until the water flows off clear. 12. Allow slides to air dry before viewing. Results of the Ziehl-Neelsen staining procedure: acid fast bacteria appear bright red non-acid fast bacteria appear blue Examples of acid fast and non-acid fast bacteria: Acid fast bacteria: Mycobacterium tuberculosis, Mycobacterium avium Non-acid fast bacteria: Escherichia coli, Staphylococcus aureus, etc. 6

7 Other staining techniques are designed to identify various bacterial structures of importance. For instance, a special staining technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed. A special staining technique is also used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. At the end of the procedure, spores stain green and other cells stain red. Capsule staining Bacterial capsules play a role in adherence, protection (they inhibit ingestion and killing by phagocytes), securing nutrients, and cell-to-cell recognition. PRINCIPLE: Chemically, the capsular material is a polysaccharide, a glycoprotein or a polypeptide. Capsule staining is more difficult than other types of differential staining procedures because the capsular materials are water soluble and may be dislodged and removed with vigorous washing. Bacterial smears should not be heated, because the resulting cell shrinkage may create a clear zone around the organism, an artifact that can be mistaken for a capsule. The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two dyes, one acidic and one basic, are used to stain the background and the cell wall, respectively. Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with india ink or nigrosin or congo red. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet, methylene blue or carbolfuchsin, bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope. Procedure of the capsule staining 1. Using sterile technique, add a loopful of bacterial culture to tube with 1 ml NaCl. 2. Add one drop of carbolfuchsin into the tube and mix gently. 3. Heat the mixture under flame 1min. 4. Place a drop of mixture onto the glass slide. 5. Place a drop of nigrosin onto the same glass slide next to drop of mixture of bacteria and the dye. 6. Use the other slide to drag the nigrosin-cell mixture into a thin film along the first slide. 7. Allow to air dry for 5-7 minutes (do not heat fix). 8. Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells. 7

8 Results of the capsule staining procedure: Encapsulated bacteria: Clear halos (capsules) are observed around pink bacteria against dark background Bacteria without capsules: pink bacteria with no clear halos Examples of encapsulated and non-encapsulated bacteria: Encapsulated bacteria: Bacillus anthracis, Klebsiella pneumoniae, Streptococcus pneumoniae, Neisseria meningitidis, Clostridium perfringens; Non-encapsulated bacteria: Neisseria gonorrhoreae, etc. 8

9 Summary of the staining techniques: 9