Staining techniques of Microorganisms

Includes methods of staining different types of bacteria and fungi.

1. STAINING TECHNIQUES of microorganisms KHADIJA SAEED

2. DEFINITION OF STAINING The use of selected dyes to color biological specimens TO ASSIST IN EXAMINATION AND IDENTIFICATION of: Cells Thin slices of tissues Microorganisms Why do we use staining? Because microbes are : colorless highly transparent

3. dyes BASIC DYES (+) = Basic dyes bind to negatively charged molecules such as nucleic acids The dyes can be : crystal violet safranin malachite green etc. ACIDIC DYES (-) = Acidic dyes bind to positively charged cell structures like some proteins . The dyes can be : Acid fuchsin Nigrosin etc. Neutral dyes = Neutral dyes are produced by combining basic and acid dyes. These dyes are: Eosinate of methylene blue  giesma stain

4. Types of staining Simple staining gram staining Spore staining Capsule staining Negative staining Acid fast staining Staining of fungi

5. 1) Simple staining Simple stain can be used as a quick and easy way to determine : Cell shape Size Arrangement of bacteria It is a very simple procedure as it requires a single solution of stain. The solutions that we can use are: methylene blue safranin crystal violet

6. PRINCIPLE Basic dies are used for simple staining. These stains are positively charged (+). The surface of most bacterial cells and cytoplasm is negatively charged (-). So the stain adheres quickly to the cell surface. After staining, morphology of bacteria can be examined .

7. PROCEDURE A bacterial smear is prepared, air dried and heat fixed The heat fixed smear is flooded with dye and allowed to react for 1-2 minutes. After adding dye, the smear is washed under running water It is air dried and observed under microscope.

8. OBSERVATION Methylene blue stain

9. 2) GRAM STAINING Gram staining is a common technique used to differentiate two large groups of bacteria based on their cell wall constituents. The gram stain procedure distinguishes between : Gram positive and gram negative groups by coloring these cells red or violet. THE DYE USED are: Crystal violet Safranin

10. PRINCIPLE Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls. Which retains the crystal violet these cells are stained with because of presence of teichoic acid. Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall . Which does not retain the crystal violet during the decoloring process due to absence of teichoic acid. Crystal violet has positive charge and teichoic acid has negative charge.

11. PROCEDURE Fix the smear of specimen either by heating or alcohol fixation. Cover the fixed smear with crystal violet stain for 30 seconds and then wash off with water. Cover the smear with iodine for 30 seconds and wash it off with water. Decolorize by pouring acetone iodine (5 seconds) and rapidly wash off. Now cover the slide with safranin for 30 seconds and wash it off with water. Allow the slide to air dry, observe under microscope.

12. PROCEDURE

13. observation

14. 3) Spore staining Spore staining is the type of staining to recognize the presence of endospore in bacterial vegetative cells. Endospores are structures that ensure the survival of a bacterium through periods of environmental stress. ENDOSPORE

15. principle Spore staining is used to distinguish between the vegetative cells and the endospores. The dyes used are: malachite green. Safranin. Schaeffer-Fulton method is used.

16. procedure Prepare a smear of the spore producing bacteria such as bacillus megaterium. Flood the smear with malachite green. Heat the slide but make sure the dye does not evaporate. Let the slide cool down and rinse with water. Flood the smear with safranin (30-60) seconds. Rinse with water, blot dry and observe under microscope.

17. observation

18. 4) Capsule staining Capsule stain is a type of differential stain which uses: Acidic and basic dyes To stain background & bacterial cells respectively So that presence of capsule is easily visualized. Capsule is synthesized in the cytoplasm and secreted to the outside of the cell where it surrounds the bacterium, attached to the cell wall.

19. principle Capsule stain is a type of differential stain which involves the use of two stains: India ink Crystal Violet Bacterial capsules are non-ionic , so neither acidic nor basic stains will adhere to their surfaces. Therefore, to visualize them We stain the background using an acidic stain. And stain the cell itself using a basic stain.

20. procedure Use a loop to mix a drop of water, a drop of India ink and a small amount of Streptococcus pneumoniae together at the end of a slide. Use another slide to spread the smear like a blood smear. Allow the smear to air dry. Don't heat fix! Flood the smear with crystal violet, 1 minute. Wash with water, blot, dry, observe.

21. observation

22. 5 ) Negative staining NEGATIVE STAINING IS A TECHNIQUE FOR CONTRASTING A THIN SPECIMEN WITH AN OPTICALLY OPAQUE FLUID. In this technique, the background is stained, the bacteria is not stained. The main purpose of Negative staining is to study the : morphological shape size arrangement of the bacteria cells that is difficult to stain. It can also be used to stain cells that are delicate to be heat-fixed.

23. principle Negative staining requires an acidic dye such as India ink or Nigrosin . The stain are negatively charged (-) . the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.

24. PROCEDURE Place a drop of nigrosin dye on a clean slide. Place small amount of culture with inoculating loop on top of the drop of nigrosin . Smear the dye + bacteria on the slide with another slide. Allow the smear to dry without heating. Observe under microscope.

25. OBSERVATION

26. 6) ACID-FAST STAINING IT IS THE DIFFERENTIAL STAINING TECHNIQUE WHOSE IS TO DIFFERENTIATE BACTERIA INTO ACID FAST GROUP AND NON-ACID FAST GROUPS. The dyes used are : carbol fuchsin methylene blue.

27. PRINCIPLE Acid-fast organisms like mycobacterium contain large amounts of : lipid substances within their cell walls called mycolic acids. Mycolic acids resist staining by ordinary methods such as a gram stain. These bacteria are termed "acid-fast" because they resist decolorization with acid alcohol.

28. PROCEDURE To a smear, add carbol fuchsin and heat for several minutes. Cool down the slide and wash with water. The smear is then treated with a decolorizing agent like acid alcohol, which removes the red stain from bacteria that are not acid fast. Wash the slide with water again and add methylene blue. Wash with slide with water and dry the slide. Observe under microscope.

29. OBSERVATION A = NON ACID-AST BACTERIA B = ACID-FAST BACTERIA

30. 7) STAINING OF FUNGI Staining of fungi is done by adding respective dye to the culture. The fungal cell wall absorbs this dye and fungi is stained. Dyes used for fungal staining are: Lactophenol cotton blue = It stains chitin in cell walls of fungi. Acid-Schiff stain = It stains polysaccharide in the cell wall of fungi. Fungi stain pink-red with blue nuclei. Gomori methenamine silver stain = Silver nitrate outlines fungi in black color due to ppt formation of silver on fungi cell wall.

31. principle FUNGAL CELLS HAVE: macroscopic structure microscopic structure. THE COMMONLY USED DYE IS: Lactophenol Cotton Blue Reagent : used for staining wet mounting of fungi. THE PHENOL OF THIS DYE: Kills organisms suspended in stain It deactivates lytic cellular enzymes so the cells do not lyse.

32. procedure Place a drop of 70% ethanol on a clean microscopic glass slide Remove a fragment of the fungi colony using an inoculating loop and place on ethanol. Add a drop of lactophenol cotton blue stain on the top of fungal colony. Apply a coverslip. Do not push down or tap the cover slip as this may dislodge the conidia from the conidiophores. Examine the preparation under microscope for the presence of characteristic mycelia and fruiting structures.

33. observation Aspergillus on LPCB Mount