Unit 3- Microscopy and Staining.pdf

Microscopy & Staining Objectives  Describe the major staining techniques used in a microbiology laboratory  Understand the principles behind major staining methods  Explain the principles of the Gram stain and the differences between the cell wall of Gram-positive and Gram-negative bacteria Revie...

Microscopy & Staining Objectives  Describe the major staining techniques used in a microbiology laboratory  Understand the principles behind major staining methods  Explain the principles of the Gram stain and the differences between the cell wall of Gram-positive and Gram-negative bacteria Review of the microscope  Microorganisms are invisible to the unaided eye,  Robert Hooke and Anton Van Leeuwenhoek were two of the first scientists to use microscopes.  By the late 1800s, the sophisticated light microscope was developed.  In the 1940’s the electron microscope was developed thus making the viruses and the smallest bacteria (for example chlamydia) visible. Microscope - units of measure  Microscopes permit extremely small objects to be seen.  Objects are measured in the metric system in micrometers and nanometers.  A micrometer (μm)   One millionth of a meter. A nanometer (nm)  One billionth of a meter. Microscope - Types 1. Light Microscope or Compound microscope 2. Dark‐field microscope 3. Phase‐contrast microscope 4. Fluorescent microscope 5. Electron Microscope 1. Transmission electron microscope (TEM) 2. Scanning electron microscope (SEM) Microscope - light Microscope  Compound microscope (common light microscope) contains two types of lenses that function to magnify an object.  Ocular - The lens closest to the eye.  Objective - The lens closest to the object.  Condenser - Apparatus located on the base of the microscope which condenses light rays to a strong beam.  Diaphragm - located on the condenser and controls the amount of light coming through it.  Both coarse and fine adjustments are found on the light microscope. Microscope - How does it work?  An object is magnified when light is projected through an opening in the stage, hits the object and then enters the objective. An image is created, and this image becomes an object for the ocular lens, which magnifies the image. Microscope - diagram Microscope - objective Lenses   Compound light microscopes often contain 3 – 5 objective lenses:  Scanning lens (4X),  Low‐power lens (10X),  High‐power lens (40 X),  Oil‐immersion lens (100 X). It also contains an ocular lens that magnifies 10 times. Stains Importance of Staining  Highlights the structures of microorganisms  Differentiate between different microorganisms. Types of staining techniques 1. Simple stains 2. Negative stains 3. Differential stains 4. Special stains Simple stains Simple stains  They produce color contrast but impart the same color to all the bacteria in the smear. Simple Stains  Usually cationic  The cell wall and cytoplasm of bacterial cells have a negative charge.  Flood the slide  Lacks detail on structure. Simple stains - examples  Crystal Violet  Methylene blue  Lactophenol cotton blue  Iodine Simple stains - crystal violet  Almost never used as a simple stain anymore  Used almost exclusively in Gram’s Stain Simple stains - methylene blue  Used to emphasize particular structures in the sample, shape, size and arrangement  In some cases, the methylene blue stain gives better results than the Gram stain. Simple stains - methylene blue  Corynebacterium diphtheriae   Y. pestis   typical bipolar "safety pin" appearance. Neisseria meningitidis and Haemophilus influenzae   bipolar staining, which gives it a "safety pin" appearance. Burkholderia pseudomallei   observation of metachromatic granules. In cerebrospinal fluid, better contrast between Gramnegative bacteria and the background Haemophilus ducreyi  observe the typical "bicycle chain" appearance. Simple stains - methylene blue  Respects cell structure better than Gram stain  Also used as a counterstain in several other stains procedures  Method of Moeller, Macchiavello, Stamp, Ziehl-Neelsen Simple stains - non-bacterial   Lactophenol cotton blue  Phenol – kill the organisms  lactic acid – preserves fungal structures  Cotton blue – stains the chitin found in the fungal cell walls. Lugol’s Iodine  differentiating parasitic cysts from host white blood cells Simple stains - lactophenol Simple stains - lactophenol Simple stains - lactophenol Simple stains - lactophenol Simple stains - lactophenol Simple stains - Lugol’s iodine  Parasites Negative stains Negative stains  The dye takes up the background leaving the organism visible Negative stains  India Ink  Nigrosin Negative Stain – India ink  acidic  anionic.  The colour of the background is changed by the dye causing the cells to stand out. Negative stains - India Ink Differential stains Differential stains - definition  Two stains are used which impart different colors to different bacteria or bacterial structures, which help in differentiating bacteria Differential stains  Allows observer to visually distinguish between different types of bacterial cells based on the physical properties of their cell walls. Differential stains - types 1. Gram 2. Acid Fast 1. Cold (kinyoun) 2. Heated (Ziehl-Neelsen) 3. Giemsa 4. Acridine orange 5. Albert stain for C. diptheriae (not discussed) Differential stains - Gram stain  The most widely used staining procedure in Microbiology.  Named after Hans Christian Gram, a Danish bacteriologist who originally devised it in 1882 (published 1884)  It is almost always the first test performed for the identification of bacteria. Differential stains - Gram stain  The stain includes the following steps:  Crystal violet - the primary stain  Iodine - the mordant  Decolorizer - made of acetone and alcohol  Safranin or Carbol Fuchsin - the counterstain Differential stains - gram stain counter stains   safranin  gram-positive cells appear blue to purple  gram-negative cells appear pink to red. fuchsin  gram-positive cells appear bright purple to purplishblack  gram-negative cells appear bright pink to fuchsia. Differential stains - Gram positive cell wall  Gram positive organisms take up the crystal violet  Fixed in the cell with the iodine mordant.  A crystal-violet/iodine complex forms which remains in the cell even after decolorizing. Why?  gram positive cell walls include a thick layer of peptidoglycans. (60-90%)  Decolorizing causes this thick cell wall to dehydrate and shrink, closing the pores in the cell wall, preventing the stain from exiting the cell. Differential stains - Gram positive cell wall Differential stains - Gram negative cell wall  Gram negative cells also take up crystal violet and iodine forming a crystal violet-iodine complex.  these cell walls do not retain this complex when decolorized. Why?  Gram negative cell wall -10-20% peptidoglycan layer (thin)   outer layer made up of lipids, polysaccharides, and proteins. When exposed to the decolorizer, the lipids in the cell wall dissolve  the crystal violet-iodine complex leaches out of the cells Differential stains - Gram negative cell wall Differential stains – Gram pos & neg cell wall Differential stains – Gram pos & neg reactions Differential stains – Gram pos examples Cocci Bacilli Differential stains – Gram negative organisms  Rods/Bacilli Cocci Differential stains - gram stain  Gram positive cocci / Gram negative bacilli/rods Differential stains - Applications of Gram stain Differential stains - Acid-fast  Used for those microorganisms which are not staining by simple or Gram staining method  particularly the member of genus Mycobacterium can only be visualized by acid-fast staining. Differential stains - Acid-fast types  Ziehl-Neelsen   Kinyoun   Hot Cold Fite (not covered in this talk) Differential stains - Acid-fast ZN  Carbol fuchsin    Application of heat  carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm.  Then after all cell appears red. Decolorized   solubilizes the lipoidal material present in the Mycobacterial cell wall 3% HCL in 95% alcohol Counterstain  methylene blue.  Only decolorized cells absorb the counter stain Differential stains - Acid-fast ZN  Acid fast cells are resistant to decolourisation   Large amount of lipoidal material in their cell wall prevents the penetration of decolorizing solution. The non-acid fast organism  lack the lipoidal material in their cell wall Differential stains - Acid-fast modified Kinyoun  The Kinyoun method can be modified as a weak acid fast stain   5% sulfuric acid instead of hydrochloric acid. The weak acid fast stain in addition to staining mycobacteria will stain organisms that are not able to maintain the carbol fuchsin after decolorizing with HCl  Nocardia spp.  Rhodococcus spp. Differential stains - Acid-fast Differential stains – ZN or Kinyoun M. tuberculosis Limitations?  Over 100 mycobacteria per milliliter of tissue are usually necessary before the organisms can be visualized by light microscopy Differential stains - Giemsa   contains a mixture of azure, methylene blue, and eosin dye  Azure and eosin are acidic dye that variably stains cytoplasm, granules, etc.  Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell. specific for the phosphate groups of DNA and attaches itself to where there are high amounts of adenine-thymine bonding. Differential stains - Giemsa applications Application in Parasitology - Plasmodium Differential stains - Giemsa applications Application in Parasitology – Toxoplasma gondii Differential stains - Giemsa applications Application in Parasitology – Histoplasma capsulatum Differential stains - Giemsa applications Application in Bacteriology – Yerinia pestis Differential stains - Giemsa applications Application in Bacteriology – Chalmydia inclusion bodies Differential stains - Giemsa Cell Components The color observed after staining Bacteria Pale or dark blue Chlamydia trachomatis inclusion bodies Blue-mauve to dark purple Borrelia spirochetes Mauve-purple Yersinina pestis coccobacilli Malaria parasite Blue with dark stained ends bipolar staining Malaria parasites have a red or pink nucleus and blue cytoplasm. P. vivax is seen, the Schüffner dots are seen as an even carpet of pink dots in the cytoplasm of red blood cells. If P. falciparum is observed, Maurer clefts will be seen as unevenly distributed, coarse bodies in the red cell cytoplasm. Differential stains - Acridine Orange  Cell permeable  Nucleic acid selective fluorescent dye   Emits green fluorescence when bound to dsDNA (at 520 )  Emits red fluorescence when bound to ssDNA or RNA (at 650 nm). Cationic dye  Enters acidic compartments such as lysosomes which, in low pH conditions, emits orange light. Differential stains - Acridine Orange  Used to confirm the presence of bacteria in positive blood cultures  Gram stain results are difficult to interpret  when the presence of bacteria is highly suspected, but none are detected using light microscopy  Enumerating the microbial load in a sample  Detection of cell wall-deficient bacteria (e.g., mycoplasmas) grown in cultures.  Used in a rapid screening tool for the detection of malaria. Differential stains - Acridine Orange Special stains Special Stains  Endospore  Capsule  Auramine-Rhodamine  Calcoflour White  Flagella stain  Cytoplasmic inclusion bodies Endospore stain - what is an endospore?  Metabolically inactive  highly resistant structures  produced by some bacteria  defensive strategy against unfavorable environmental conditions. Endospore stain  Schaeffer-Fulton’s method  Dorner’s Method Endospore stain - Schaeffer-Fulton’s method   a primary stain – malachite green  Forced into the spore by steaming the bacterial emulsion.  water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water. Counter stain – Safranin   counterstain any cells which have been decolorized. At the end of the staining process, vegetative cells will be pink, and endospores will be dark green. Endospore stain - Schaeffer-Fulton’s method Endospore stain - Dorner’s method  a primary stain – Carbol Fuchsin  Decolouriser – Acid Alcohol  Counter stain – Nigrosin   counterstain any cells which have been decolorized. At the end of the staining process, vegetative cells will be colourless, and endospores will be red and the background black. Endospore stain - Dorner method Endospore stain - endospore producing orgs  Clostridium perfringens  C. botulinum  C. tetani  Bacillus anthracis  Bacillus cereus Capsule stain - what is a capsule?  A gelatinous outer layer secreted by bacterial cell  Surrounds and adheres to the cell wall.  Composed of polysaccharides or polypeptides.  Notoriously poor at staining  Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. Capsule stain - positive stain techniques   Negative stain  Congo Red  India Ink  Nigrosin Counterstain  Crystal Violet  Methylene Blue Capsule stain - positive stain techniques Capsule stain - capsule producers  Streptococcus pneumoniae  Klebsiella pneumoniae  Haemophilus influenzae  Pseudomonas aeruginosa  Neisseria meningitidis  Cryptococcus neoformans Special stains - flagella stains  Most motile bacteria possess flagella  The shape, number, and position of flagella are important characteristics in differentiating genera and species identification. Special stains - flagella stains techniques  A wet-mount procedure (Ryu method)*  Dried-smear preparation (Leifson method) Special stains - flagella stains Impregnation  Bacterial cells and structures that are too thin to be seen under the light microscope are thickened by impregnation of silver salts on their surface to make them visible  Flagella  Spirochetes Special stains - flagella stains  Monotrichous   Amphitrichous   Single flagellum on both sides - Alkaligens faecalis Lophotrichous   Single polar flagellum - Vibrio cholera, Tufts of flagella at one or both sides - Spirillum Peritrichous  – Numerous falgella all over the bacterial body Salmonella Typhi, E. coli Special stains - flagella stains Special stains - flagella stains Flagellated organism Disease caused Escherichia coli Gastroenteritis Salmonella species Gastroenteritis Pseudomonas aeruginosa Wound infections Bacillus subtilis Septicaemia Bordatella pertussis Whooping cough Campylobacter jenuni Gastroenteritis Clostridium difficile Antibiotic associated diarrhoea Helicobacter pylori Gastric ulcers/cancer Proteus mirabilis UTIs Special stains - calcoflour white  Fluorescent stain that binds to cellulose and chitin which is found in the cell walls of yeast and moulds.  Exposure to the long-wavelength ultraviolet and short-wavelength visible light helps in colored observation.  can differentiate the hyphae from the collagen fibers and other artifacts unlike KOH wet mount Special stains - calcoflour white  A water-soluble, colorless dye and fluorescent whitener used in the textile and paper industry   The green coloration is due to barrier filters in the fluorescence microscope.   Fluoresces into green color when exposed to ultraviolet light Other elements present in the sample will fluoresce as reddish-orange. Evans blue is used as a counterstain  Diminishes the background fluorescence by using the blue light excitation (not UV) Special stains - calcoflour white applications  Visualise the hyphae, pseudohyphae, and yeast.   Chitin concentration is higher in the budding yeasts, calcofluor white stains the bud scars more intensely. Helps detect non-culturable fungus like Pneumocystis jirovecii. Special stains - calcoflour white applications   Helps observe non-fungal agents such as free-living amoebae  Acanthamoeba, Naegleria, and Balamuthia  larva of Dirofilaria. Helps in the rapid diagnosis of Acanthamoeba keratitis from corneal scrapings and keratectomy specimens. Live case Live from the lab  Ophthal  Male patient with persistent uveitis  No travel history  On steroids (oral & topical) Live from the lab Live from the lab Live from the lab References  https://microbiologie-clinique.com/Methylene-BlueStain.html  https://microbeonline.com/types-of-stainingtechniques-used-in-microbiology-and-theirapplications/  Wanger, A., Chavez, V., Huang, R., Wahed, A., Dasgupta, A., & Actor, J. K. (2017). Microbiology and molecular diagnosis in pathology: a comprehensive review for board preparation, certification and clinical practice.