MIC 205 Microbiology Lecture 2: Microscopy and Staining PDF

MIC 205 - Microbiology Lecture 2: “Seeing is Believing” Ch. 4 Microscopy Ch.11 Shapes of Cells Lecturer: Patrick Daydif Office: UCENT 356 Phone: (602) 496-0599 Email: [email protected] Office Hours: Please refer to Canvas (or by appointment) The...

MIC 205 - Microbiology Lecture 2: “Seeing is Believing” Ch. 4 Microscopy Ch.11 Shapes of Cells Lecturer: Patrick Daydif Office: UCENT 356 Phone: (602) 496-0599 Email: [email protected] Office Hours: Please refer to Canvas (or by appointment) The best way to contact me and answer your questions is Face to Face. 1 3 Classes/Types of Microscopes Light microscopes (utilize visible and UV light) – Bright-field – Dark-field – Fluorescence – Confocal Electron microscopes (utilize a beam of electrons) – Transmission (TEM) – Scanning (SEM) Scanning probe microscopes (utilize physical means) – Atomic force (AFM) – Scanning tunneling Increase in resolution 2 2 Limits of Resolution 3 3 1  Microscopy Microscopes are standard tools for microbiologists in research and diagnostic laboratories Mechanics are based on the same principles that govern human vision General principles... – Wavelength of radiation (electromagnetic spectrum) – Refraction and magnification – Resolution and contrast 4 4 Electromagnetic Spectrum Wavelength scale shows the relationships between various forms of radiation. Shorter wavelengths correspond to greater resolving power. 5 5 Refraction and Magnification Refraction: bending of light (radiation) Magnification: perceived enlargement of the primary image Both principles depend greatly upon the medium (air, water, oil) through which the illuminating radiation travels 6 6 2  Resolution and Contrast Resolution – the capability of distinguishing between two separate but adjacent objects Contrast – difference in intensity (determine by color & Brightness) between two objects, or between an object and its background low optimal high MORE CONTRAST = LESS RESOLUTION 7 7 Staining Increases contrast and resolution by coloring specimens with non-fluorescent dyes Bacterial cells have a net negative charge, and therefore basic dyes are commonly used. Microbiological stains are usually salts composed of cation (+), and anion (-). – One is colored, known as a “chromophore.” – Acidic dyes (- charge) stain alkaline structures such as Indian ink – Basic dyes (+ charge) stain acidic structures such as crystal violet Typical preparation: a smear of microorganisms (thin film) air-dried to the slide and then fixed to the slide by heat or chemicals. 9 9 Bright-Field Microscopes – Compound May have one (monocular) or two (binocular) ocular lenses Most have a multiple lens system of objective lens (4x, 10x, 40x, 100x) Light rays pass through the specimen and into a series of lenses for magnification. – Specially-designed oil immersion lenses increase resolution because light refracts differently in a denser medium. – Total magnification = ocular mag. x objective mag. 10 3  Other Forms of Light Microscopes Dark-Field Fluorescence Confocal Specimen appears light against a dark A series of digital background Illuminate specimen images can be Increases contrast with high-intensity “stacked” on top of and enables light source (UV) each other observation in specimens containing to construct a more detail fluorescent molecules three-dimensional will illuminate. image!!! Organisms will appear colored against a black 11 background 11 Happy New Year Confocal Fluorescence A sampling of human dental plaque. The individual microbes were identified using a combination of fluorescent markers that label nucleotide sequences specific to each species of bacteria. 12 12 Electron Microscopes Use electrons instead of visible light to illuminate the sample (wavelength 0.01 nm to 0.001 nm) Short-wavelength electrons provide HUGE boosts in both resolving power and magnification Capable of magnifying objects 10,000 - 100,000 times!!! Provide detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms Electron micrographs almost always presented in black and white Two types... – Transmission electron microscopes (TEM) – Scanning electron microscopes (SEM) 13 13 4  Light vs. TEM & SEM Microscopes SEM TEM Light Microscope 400x Electron Microscope 14 14 Scanning Probe / AFM Incredibly sensitive microscopes allow researchers to see single molecules and large atomic clusters by a pointed probe (physical interaction). Magnification capabilities in the millions. DNA double helix circular DNA molecules 15 15 Staining Simple stains Differential stains – Gram Stain – Acid-Fast Stain – Endospore Stain Special stains – Negative (Capsule) Stain – Flagellar Stain – Fluorescent Stains Staining for Electron Microscopy 16 16 5  Simple Stains Only one dye is added to the sample. What is the charge of the bacterial cells? A. + (Positive) B. - (Negative) What type of dye should be used on this sample? A. + (Basic dye) B. - (Acidic dye) 17 17 Gram Stain (Know this Stain!) Four-step differential stain used most commonly in microbiological laboratories – Step 1: Crystal violet (purple dye) – Step 2: Iodine (mordant) – Step 3: Alcohol (decolorize) – Step 4: Safranin (red dye) Result: Gram-positive bacteria stain purple, while Gram- negative bacteria stain pink Differential stain sorts bacteria based on the composition (thickness) of the cell wall. 18 18 Morphology of Prokaryotic Cells Prokaryotic cells can be found in a variety of shapes... – Spherical (coccus, pl. cocci)* – Rodlike (bacillus, pl. bacilli)* – Bent rods (vibrio, pl. vibrii) – Corkscrew (spirillum, pl. spirilli)* – Variable (pleomorphic) **Three basic shapes** Cells of a defined shape usually are found in one of several arrangements that result from two aspects of bacterial cell division – Plane(s) in which cells divide – Separation of daughter cells 24 See page 322 24 6  Reproduction of a Prokaryotic Cell Cell replicates its DNA. Cell wall All prokaryotes Cytoplasmic Nucleoid membrane reproduce asexually Replicated DNA Most common The cytoplasmic membrane elongates, method is Binary separating DNA molecules. Fission Cross wall forms; membrane invaginates. Cross wall forms completely. Daughter cells may separate. 25 Snapping division “ OH SNAP!!” Variation of Binary fission occurs in some Gram-positive bacteria As the inner wall continues to grow. This creates tension on the outer wall. Increased tension will cause the outer wall to snap in one location. Daughter cells remain attached. 26 Arrangements of Cocci The formation of doublets, beaded strings, and clumps depends strictly on the cell division plane and whether the daughter cell separates. 27 27 7  Name the Cocci Arrangements Staphylococci Diplococci Staphylococcus aureus Neisseria gonorrhoeae Sarcina Kineococcus radiotolerans Tetrads Micrococcus luteus Streptococci Streptococcus pyogenes 28 28 Arrangements of Bacilli The formation of doublets, sausage ropes, and stacks of little smokies depends strictly on the plane of cell division and whether or not the daughter cell separates. 29 29 Name the Bacilli Arrangements Diplobacilli Single bacilli Bacillus cereus Escherichia coli Streptobacilli V-shaped and palisade Bacillus cereus Corynebacterium diphtheriae 30 30 8  In the News: Bacillus cereus https://news.yahoo.com/teen-ate-leftover-rice-noodles-155422253.html 31 31 Endospores Specialized dormancy structures produced by Gram- positive Bacillus and Clostridium species – Not reproductive structures! Each vegetative cell produces one endospore; each endospore germinates to form one vegetative cell Entire process constitutes a defensive strategy against hostile or unfavorable conditions – Extremely resistant to desiccation, heat, radiation, and lethal chemicals – Can remain viable for tens to thousands of years (e.g., amber) Provides unique challenges for the eradication of harmful members of these two genera – Serious concern to food processors, health care professionals, and governments 32 See page 74 32 Endospore Formation 1 5 2 6 3 7 4 8 33 33 9  Endospore Examples Medial endospores Terminal endospores Bacillus sp. Clostridium sp. 34 34 10