4 Microscopy Bright Field Microscopy Three different objective lenses are commonly used10x: To scan the slide for specimens40x: To view parasites, filamentous fungi100x: To observe single cellsTotal magnification= (objective lens magnification) X (ocular lens magnification)
5 Microscopy Bright Field Microscopy Note how light scatters via refractionRefractive index of air is lower than that of glassLoss of refracted light is minimized by applying mineral oil (same RI as glass)
6 Microscopy Dark Field Microscopy Creates contrast between the object and the surrounding field. Background is dark and the object is bright.An annular stop ring permits light coming from the outside of the beam.When light from the stop is deflected and deviated by the object can it be seen.Advantageous for viewing thin bacteria(Ie. Treponema pallidum)Disadvantage: internal structure is not as clearly visable compared to bright field
7 Microscopy Dark Field Microscopy Light Field vs Dark Field
8 Microscopy Phase-Contrast Microscopy Most of the detail of living cells is undetectable in bright field microscopyLittle contrast exists between structures with similar transparencyInsufficient natural pigmentation.Organelles show wide variation in refractive index (the tendency of the materials to bend light) providing an opportunity to distinguish them with phase contrast mircoscopyInternal features are more easily viewed
10 Microscopy Fluorescent Microscopy Organisms are stained with fluorescent dies (fluorochromes) and then viewed.Fluorescent microscopes emit a shorter wavelength of light than in bright field microscopy.The short wavelength excites the fluorochromes, and they fluoresce.Allows for easier low magnification scanning.Bright object occurs against a dark background.
11 Microscopy Fluorescent Microscopy Fluorescent vs Dark Field
12 Microscopy Fluorescent Microscopy Different fluorescent stains can bind to different targetsDigital merging with differential stains
13 Electron MicroscopyUses magnetic coils to direct a beam of electrons through the specimen onto a screenUses a very short wavelength, thus magnification and resolution is improvedSamples are stained/coated with metal ions to create contrast.
14 Electron Microscopy Transmission electron microscope Electrons pass through the specimen
15 Electron Microscopy Scanning Electron Microscope Electrons bounce off the surface of a specimen and create a 3D image.
16 How to use the oil immersion lens of microscopy The substage condenser to raise to the highest positionThe iris diaphragm fully openedTo adjust the light entering the lens with low-power lensThe oil immersion lens to be rotated into positionThe specimen to be put on the center of stageA drop of oil to be placed on the slide directly over the area to be viewed
17 Using the fine adjustment knob, to bring the specimen into sharp focus Up the stage with the coarse adjustment knob, to let oil lens into the oilLooking into the ocular lens and down the stage slowly with the coarse adjustment knob until the specimen comes into focusUsing the fine adjustment knob, to bring the specimen into sharp focusThe oil immersion lens should be cleaned with lens paper after experiment
18 Examination Methods: Direct Examination India InkDarkens the background rather than the cellUseful in detecting Cryptococcus capsulesCapsule excludes ink
19 Morphological Observation Of Bacterial Cells S. aureus
20 Morphological Observation Of Bacterial Cells E. coli
21 Morphological Observation Of Bacterial Cells V. choleriaeGram -ve
22 Morphological Observation Of Bacterial Cells Streptococcus pneumoniae
23 Morphological Observation Of Bacterial Cells Salmonella typhi
24 Morphological Observation Of Bacterial Cells Clostridium tetaniClub shape is due to endospore production at one termini of the cell.
25 Staining of Bacteria PURPOSE : To make bacteria more easily observable To acquaint you with Gram stainMATERIALS:Simple stainGram stainAcid-fast stainSpecial stainSpore stainCapsule stainFlagella stainMetachromatic granules stain.
26 Gram stain purpose: MATERIALS : differentiating bacteria Slant cultures of and Escherichia coli and S.aureus (18 to 24 hours old)Crystal violet, iodine solution, 95% alcohol, safraninMicroscope slides
27 Gram stain PROCEDURE: Smear: size of a dime to form a thin film Dry : air dryFix: through the warm air above the flame two or three times.
28 Process of Gram’s Stain Crystal violet(primary staining)Washing1minLugol’s iodine(mordant staining)Washing1min95%ethyl alcohol(decolorization)Fuchsion red(counterstaining)Washing30sWashing30sBlot dry with bibulous papersObservation with the oil immersion lensResults :Gram –positive blue color Gram-negative red color
29 Gram StainingIn gram-positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell.Gram +ve bacteria have low lipid contentLipid is dissolved by alcoholThe alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycanThus causing the cell wall to trap the crystal violet-iodine complex within the cell.
30 Gram Staining Gram-negative bacteria have higher lipid contents The alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached.The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the cell is decolorized.
31 Gram Staining: Common Errors There are several factors that could result in a gram-positive organism staining gram-negatively:The method and techniques used. Overheating during heat fixation, over decolorization with alcohol, and even too much washing with water between steps may result in gram-positive bacteria losing the crystal violet-iodine complex.The age of the culture. Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex.The organism itself. Some gram-positive bacteria are more able to retain the crystal violet-iodine complex than others.Therefore, one must use very precise techniques in gram staining and interpret the results with discretion.