Staining Techniques Identifying Structural Features of Bacteria Asdren Zajmi Management and Science University Shah Alam, Malaysia Frances Fischbach, Marshall B. Dunning II.2009.A Manual of Laboratory and Diagnostic Tests. Edt 9. Lippincott / Williams & Wilkins Staining Simple stains Differential stains – Gram stain – Acid-fast stain – Endospore stain Special stains – Capsule (Negative) stain – Flagellar stain – Fluorescent stains Staining for electron microscopy Staining Reaction Stains - salts composed of a positive and negative ion, one of which is colored (chromophore) Basic Dyes - chromophore is the positive ion – dye+ Cl- Acid Dyes - chromophore is the negative ion – Na+ dye- Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE Common Basic Dyes – crystal violet – methylene blue – safranin – basic fuchsin (magenta dye) Dyes become Stains In 1884 the Danish microbiologist Hans Christian Gram discovered that crystal violet irreversibly stains certain bacteria but can be washed from others. The dye has been widely used ever since for the Gram stain technique, which identifies bacteria as gram-positive (the stain is retained) or gram-negative (the stain is washed). The Gram stain, which divides most clinically significant bacteria into two main groups, is the first step in bacterial identification. Bacteria stained purple are Gram + - their cell walls have thick peptidoglycan and teichoic acid. Bacteria stained pink are Gram – their cell walls have thin peptidoglycan and lipopolysaccharides with no teichoic acid. The Gram stain has four steps: 1. crystal violet, the primary stain 2. iodine, which acts as a mordant by forming a crystal violet-iodine complex, then 3. alcohol, which decolorizes, followed by 4. safranin, the counterstain. Is this gram stain positive or negative? Is this gram stain positive or negative? Gram staining tests the bacterial cell wall's ability to retain crystal violet dye during solvent treatment. Safranin is added as a mordant to form the crystal violet/safranin complex in order to render the dye impossible to remove. Ethyl-alcohol solvent acts as a decolorizer and dissolves the lipid layer from gram-negative cells. This enhances leaching of the primary stain from the cells into the surrounding solvent. Ethyl-alcohol will dehydrate the thicker gram- positive cell walls, closing the pores as the cell wall shrinks. For this reason, the diffusion of the crystal violet- safranin staining is inhibited, so the bacteria remain stained. Gram Variability Some Gram-positive bacteria appear Gram- negative when they have reached a certain age, varying from a few hours to a few days. On the other hand, some Gram-negative bacteria may become Gram-positive in older cultures. For this reason it is strongly recommended to use very young cultures for the staining procedure, after growth has become just visible. Acid Fast stain Frances Fischbach, Marshall B. Dunning II.2009.A Manual of Laboratory and Diagnostic Tests. Edt 9. Lippincott / Williams & Wilkins Acid-fast cells contain a large amount of lipids and waxes in their cell walls – primarily mycolic acid Acid fast bacteria are usually members of the genus Mycobacterium or Nocardia – Therefore, this stain is important to identify Mycobacterium or Nocardia Disease associated with Mycobacterium or Nocardia Mycobacterium: – M. leprae – leprosy – M. tuberculosis – tuberculosis Nocardia: – N. brasiliensis- nocardiosis (lung disease) – N. asteroids – nocardiosis (lung disease) Advantages and Disadvantage of Acid Fast Bacteria Advantages: – Acid-fast cells contain a large amount of lipids and waxes in their cell walls, making them relatively impermeable and resistant to many disinfectants – Also enables resistance to desiccation, antibiotics, and other toxins Disadvantage: – Waxes delay nutrient uptake, so cells grow slower Ziehl-Neelsen Method is used to stain acid-fast bacteria Ziehl- Neelsen Procedure 1. Make a smear. Air Dry. Heat Fix. 2. Flood smear with Carbol Fuchsin stain – Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate the cell wall 3. Cover flooded smear with filter paper 4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed 5. Cool slide 6. Rinse with DI water 7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains 3% HCl and 95% ethanol – The waxy cell wall then prevents the stain from being removed by the acid alcohol (decolorizer) once it has penetrated the cell wall. The acid alcohol decolorizer will remove the stain from all other cells. Ziehl- Neelsen Procedure (continued) 8. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop by drop) until the red color stops streaming from the smear 9. Rinse with DI water 10. Add Loeffler’s Methylene Blue stain (counter stain). This stain adds blue color to non-acid fast cells!! Leave Loeffler’s Blue stain on smear for 1 minute 11. Rinse slide. Blot dry. 12. Use oil immersion objective to view. Blue=Non acid-fast bacteria Red= acid fast bacteria Endospore Stain Frances Fischbach, Marshall B. Dunning II.2009.A Manual of Laboratory and Diagnostic Tests. Edt 9. Lippincott / Williams & Wilkins These are very resistant structures made by only a few genera of bacteria. – Clostridium is an anaerobic organism that forms spores. Tetanus, botulism, gas gangrene and pseudomembranous colitis are diseases caused by different species in this genus. – Bacillus is a common aerobic genus whose species can form endospores. Anthrax and Bacillus cereus food poisoning are two diseases caused by members of this genus. Spores are extremely resistant structures, difficult to destroy with heat or other physical and chemical disinfecting agents. Endospore destruction is the standard for testing the operation of an autoclave. Dormant cell Produced when starved Resistant to adverse conditions – high temperatures – organic solvents contain calcium dipicolinate Spore • Peptidoglycan layer – cortex – between two membranes – less cross-linked – dehydrated muramic acid (lactam) The endospore exosporium coat outer membrane cortex Inner membrane Staining procedure – Prepare a smear of Bacillus megaterium, allow the smear to dry and then heat-fix. – Place the slide on the staining rack in the sink and flood the smear with malachite green stain. – Heat the stain to steaming by passing a lit bunsen burner over the smear. Don't overheat the stain! Once the steaming stops, pass the bunsen burner over the slide again. As the stain evaporates add more stain. Continue this procedure for 5-10 minutes. (Safety Note: Please remember that the acetone-alcohol decolorizer from the gram stain experiment is extremely flammable. Do not perform this flaming step while people are gram staining!) – Wash the smear gently and thoroughly with running water. – Counterstain with aqueous safranin for 1 minute. – Wash the slide with water, blot gently and allow the smear to air dry. – Observe under oil immersion and compare what you see with the illustrations in Leboffe and Pierce. http://www2.hawaii.edu/~johnb/micro/m140/syllabus/week/handouts/m140.6.1.html Capsule Stain Frances Fischbach, Marshall B. Dunning II.2009.A Manual of Laboratory and Diagnostic Tests. Edt 9. Lippincott / Williams & Wilkins Some bacteria excrete a viscous substance which coats the cell called a capsule A capsule is a slime layer that surrounds the bacterial cell Capsule Structure Some capsule layers are thick, while others are very thin Most capsules are made of polysaccharides, others are made of protein Capsule layers are water soluble Functions of a Capsule Provides an osmotic barrier for the cell Carbohydrate Reserve Adhering to surfaces (sticks to surfaces) Protection from phagocytosis (the slime layer is difficult to ingest or absorb by phagocytes) Capsule Staining Theory Capsule staining is a negative stain Most staining techniques are based on formation of an ionic bond between the stain and the recipient (cell structure) Because the capsules are non-ionic, dyes will not bind to the capsule – (so, capsules do not stain) Capsule Staining Theory (continued) Therefore, we visualize the capsule indirectly by staining the cell and the area outside the capsule This is called a negative stain, because the structure we want to visualize is not stained Capsule Stain Procedure 1. Make a smear in a drop of Congo red dye 2. Air dry – DO NOT HEAT FIX!! (we cannot heat fix capsule smears because the heat will denature the protein or melt the polysaccharide) – Also, because the cells are dried in the Congo red stain, shrinking caused by heat-fixing would cause an artificial white halo around those cells, causing a false impression of a capsule Capsule Stain Procedure (continued) 3. Flood the stain with Maneval’s stain, let stand for 1 minute 4. Drain and rinse with DI water 5. DO NOT BLOT DRY! (because if smears are not heat fixed, they are likely to wash off the slide) 6. Use oil immersion objective to view Flagellar Stain Frances Fischbach, Marshall B. Dunning II.2009.A Manual of Laboratory and Diagnostic Tests. Edt 9. Lippincott / Williams & Wilkins The flagella stain allows observation of bacterial flagella under the light microscope. Bacterial flagella are normally too thin to be seen under such conditions. The flagella stains employs a mordant to coat the flagella with stain until they are thick enough to be seen. These staining techniques are typically very difficult. Many bacteria are motile - accomplish this motility by means of flagella. Flagella can vary by number and location. – Monotrichous - the flagella is at the end of the cell; this placement is called polar. – Amphitrichous – the flagella at either end of the cell; – Lophotrichous multiple flagella located in a tuft at one end of the cell – Peritrichous – the flagella is all over the cell Monotrichous Lophotrichous Peritrichous Functions of Flagellum Protein, antigenic Locomotory strucutre Adherence factor Differentiation Staining procedures Flagella staining is the most difficult histochemical staining and usually requires some amount of practice. Also note that no heat- fixation is used when staining bacteria flagella! – Prepare a thoroughly cleaned and well dried microscope slide. – With forceps and a scalpel, cut out the colony in which you detected motile bacteria. – Gently touch the culture side of the agar piece to a clean microscope slide. – Allow the imprint to air-dry. Do not heat-fix! – Flood the dried bacteria film with pararosaniline (basic fuchsin) stain until a golden film with precipitate forms. This may take up to 15 min. – Rinse very gently with water – Air dry preparation, do not blot dry! – Cover dry preparation with immersion oil. Observe first with low power (20× objective) to locate a good view field. Add a drop of immersion oil and swing the 100× oil immersion objective lens into the oil. Use only fine focus to bring image into focus. Describe the cell form and number of flagella that you observe in your preparation. Prepare a drawing of your cells to document staining results in your lab report. Margaret E. Heimbrook, Wan Lan L. Wang, Gail Campbell. 1989. Journal of Clinical Microbiology. p.2612-2615. Margaret E. Heimbrook, Wan Lan L. Wang, Gail Campbell. 1989. Journal of Clinical Microbiology. p.2612-2615. References Frances Fischbach, Marshall B. Dunning II.2009.A Manual of Laboratory and Diagnostic Tests. Edt 9. Lippincott/Williams & Wilkins Margaret E. Heimbrook, Wan Lan L. Wang, Gail Campbell. 1989. Journal of Clinical Microbiology. p.2612-2615. Laboratory Exercise # 8: Other Staining Techniques (See the material provided in the CD) An Introduction to Cells and Procaryotic Cell Structure and Function (See the material provided in the CD) Microbiology Section 1 – Laboratory Week Three (See the material provided in the CD) Lab 7: Bacteria (See the material provided in the CD) Endospore Stain: Exercise 7 (See the material provided in the CD) http://www2.hawaii.edu/~johnb/micro/m140/syllabus/week/handouts/ m140.6.1.htm FALEMINDERIT!