Experiment oneExperiment one

Examination of BacteriaExamination of Bacteria

Visualizing BacteriaVisualizing Bacteria

• Staining is required to properly Staining is required to properly visualize bacteriavisualize bacteria

Microscopy Microscopy Bright Field MicroscopyBright Field Microscopy

Microscopy Microscopy Bright Field MicroscopyBright Field Microscopy• Three different objective lenses are Three different objective lenses are

commonly usedcommonly used

• 10x: To scan the slide for specimens10x: To scan the slide for specimens

• 40x: To view parasites, filamentous fungi40x: To view parasites, filamentous fungi

• 100x: To observe single cells100x: To observe single cells

• Total magnification= (objective lens Total magnification= (objective lens magnification) X (ocular lens magnification) X (ocular lens magnification)magnification)

Microscopy Microscopy Bright Field Microscopy Bright Field Microscopy

Note how light scatters Note how light scatters via refractionvia refraction

Refractive index of air Refractive index of air is lower than that of is lower than that of glassglass

Loss of refracted light Loss of refracted light is minimized by is minimized by applying mineral oil applying mineral oil (same RI as glass)(same RI as glass)

Microscopy Microscopy Dark Field MicroscopyDark Field Microscopy• Creates contrast between the object and the Creates contrast between the object and the

surrounding field. Background is dark and the surrounding field. Background is dark and the object is bright. object is bright.

• An annular stop ring permits light coming from An annular stop ring permits light coming from the outside of the beam. the outside of the beam.

• When light from the stop is deflected and When light from the stop is deflected and deviated by the object can it be seen. deviated by the object can it be seen.

• Advantageous for viewing thin bacteria Advantageous for viewing thin bacteria (Ie. (Ie. Treponema pallidum)Treponema pallidum)

• Disadvantage: internal structure is not as clearly Disadvantage: internal structure is not as clearly visable compared to bright fieldvisable compared to bright field

Microscopy Microscopy Dark Field MicroscopyDark Field Microscopy

Light Field vs Dark FieldLight Field vs Dark Field

Microscopy Microscopy Phase-Contrast MicroscopyPhase-Contrast Microscopy• Most of the detail of living cells is Most of the detail of living cells is

undetectable in bright field microscopyundetectable in bright field microscopy– Little contrast exists between structures with Little contrast exists between structures with

similar transparencysimilar transparency– Insufficient natural pigmentation. Insufficient natural pigmentation.

• Organelles show wide variation in Organelles show wide variation in refractive index (the tendency of the refractive index (the tendency of the materials to bend light) providing an materials to bend light) providing an opportunity to distinguish them with phase opportunity to distinguish them with phase contrast mircoscopycontrast mircoscopy

• Internal features are more easily viewedInternal features are more easily viewed

Microscopy Microscopy Phase-Contrast MicroscopyPhase-Contrast Microscopy

Microscopy Microscopy Fluorescent MicroscopyFluorescent Microscopy• Organisms are stained with fluorescent Organisms are stained with fluorescent

dies (fluorochromes) and then viewed.dies (fluorochromes) and then viewed.• Fluorescent microscopes emit a shorter Fluorescent microscopes emit a shorter

wavelength of light than in bright field wavelength of light than in bright field microscopy.microscopy.

• The short wavelength excites the The short wavelength excites the fluorochromes, and they fluoresce.fluorochromes, and they fluoresce.

• Allows for easier low magnification Allows for easier low magnification scanning.scanning.

• Bright object occurs against a dark Bright object occurs against a dark background.background.

Microscopy Microscopy Fluorescent MicroscopyFluorescent Microscopy

Fluorescent vs Dark FieldFluorescent vs Dark Field

Microscopy Microscopy Fluorescent MicroscopyFluorescent Microscopy

• Different fluorescent Different fluorescent stains can bind to stains can bind to different targets different targets

• Digital merging with Digital merging with differential stainsdifferential stains

Electron MicroscopyElectron Microscopy

• Uses magnetic coils to direct a beam Uses magnetic coils to direct a beam of electrons through the specimen of electrons through the specimen onto a screenonto a screen

• Uses a very short wavelength, thus Uses a very short wavelength, thus magnification and resolution is magnification and resolution is improvedimproved

• Samples are stained/coated with Samples are stained/coated with metal ions to create contrast.metal ions to create contrast.

Electron MicroscopyElectron Microscopy

• Transmission electron microscopeTransmission electron microscope– Electrons pass through the specimenElectrons pass through the specimen

Electron MicroscopyElectron Microscopy

• Scanning Electron MicroscopeScanning Electron Microscope– Electrons bounce off the surface of a Electrons bounce off the surface of a

specimen and create a 3D image.specimen and create a 3D image.

How to use the oil immersion lens of microscopy

The substage condenser to raise to the highest position

The iris diaphragm fully opened

To adjust the light entering the lens with low-power lens

The oil immersion lens to be rotated into position

The specimen to be put on the center of stage

A drop of oil to be placed on the slide directly over the area to be viewed

Up the stage with the coarse adjustment knob, to let oil lens into the oil

Looking into the ocular lens and down the stage slowly with the coarse adjustment knob until the

specimen comes into focus

Using the fine adjustment knob, to bring the specimen into sharp focus

The oil immersion lens should be cleaned with lens paper after experiment

Examination Methods:Examination Methods:Direct ExaminationDirect ExaminationIndia InkIndia Ink

• Darkens the background rather Darkens the background rather than the cellthan the cell

• Useful in detecting Useful in detecting Cryptococcus Cryptococcus capsulescapsules– Capsule excludes inkCapsule excludes ink

Morphological Observation Of Morphological Observation Of Bacterial CellsBacterial Cells

• S. aureusS. aureus

Morphological Observation Of Morphological Observation Of Bacterial CellsBacterial Cells

• E. coliE. coli

Morphological Observation Of Morphological Observation Of Bacterial CellsBacterial Cells

• V. choleriaeV. choleriae

• Gram -veGram -ve

Morphological Observation Of Morphological Observation Of Bacterial CellsBacterial Cells

• Streptococcus pneumoniae Streptococcus pneumoniae

Morphological Observation Of Morphological Observation Of Bacterial CellsBacterial Cells

• Salmonella typhiSalmonella typhi

Morphological Observation Of Morphological Observation Of Bacterial CellsBacterial Cells

• Clostridium tetaniClostridium tetani

• Club shape is due to endospore Club shape is due to endospore production at one termini of the cell.production at one termini of the cell.

Staining of BacteriaStaining of Bacteria • PURPOSE :PURPOSE : To make bacteria more easily observable To make bacteria more easily observable To acquaint you with Gram stainTo acquaint you with Gram stain• MATERIALS:MATERIALS:

– Simple stainSimple stain– Gram stainGram stain – Acid-fast stainAcid-fast stain– Special stainSpecial stain

• Spore stain Spore stain • Capsule stain Capsule stain • Flagella stain Flagella stain • Metachromatic granules stain. Metachromatic granules stain.

Gram stainGram stain

• purpose: purpose: differentiating bacteriadifferentiating bacteria

• MATERIALS :MATERIALS :– Slant cultures of and Escherichia coli Slant cultures of and Escherichia coli

and S.aureus (18 to 24 hours old)and S.aureus (18 to 24 hours old)– Crystal violet, iodine solution, 95% Crystal violet, iodine solution, 95%

alcohol, safranin alcohol, safranin – Microscope slides Microscope slides

Gram stainGram stain

• PROCEDURE:PROCEDURE:– Smear: Smear: size of a dime to size of a dime to

form a thin filmform a thin film– Dry : Dry : air dryair dry– Fix: Fix: through the warm air through the warm air

above the flame two or above the flame two or three times.three times.

Crystal violet (primary staining)

Lugol’s iodine(mordant staining)

95%ethyl alcohol(decolorization)

Fuchsion red(counterstaining

)

Washing

1min

Washing

30s

Washing 1min

Blot dry with bibulous papers Observation with the oil immersion lensResults :Gram –positive blue color Gram-negative red color

Washing 30s

Process of Gram’s Stain

Gram StainingGram Staining

• In gram-positive bacteria, the crystal violet In gram-positive bacteria, the crystal violet and iodine combine to form a larger and iodine combine to form a larger molecule that precipitates out within the molecule that precipitates out within the cell. cell.

• Gram +ve bacteria have low lipid contentGram +ve bacteria have low lipid content• Lipid is dissolved by alcoholLipid is dissolved by alcohol• The alcohol/acetone mixture then causes The alcohol/acetone mixture then causes

dehydration of the multilayered dehydration of the multilayered peptidoglycanpeptidoglycan– Thus causing the cell wall to trap the crystal Thus causing the cell wall to trap the crystal

violet-iodine complex within the cell. violet-iodine complex within the cell.

Gram StainingGram Staining

• Gram-negative bacteria have higher lipid Gram-negative bacteria have higher lipid contents contents

• The alcohol/acetone mixture, being a lipid The alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of solvent, dissolves the outer membrane of the cell wall and may also damage the the cell wall and may also damage the cytoplasmic membrane to which the cytoplasmic membrane to which the peptidoglycan is attached. peptidoglycan is attached.

• The single thin layer of peptidoglycan is The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine unable to retain the crystal violet-iodine complex and the cell is decolorized. complex and the cell is decolorized.

Gram Staining: Common Gram Staining: Common ErrorsErrors• There are several factors that could result in a There are several factors that could result in a

gram-positive organism staining gram-negatively: gram-positive organism staining gram-negatively: – The method and techniques used. Overheating during heat The method and techniques used. Overheating during heat

fixation, over decolorization with alcohol, and even too fixation, over decolorization with alcohol, and even too much washing with water between steps may result in much washing with water between steps may result in gram-positive bacteria losing the crystal violet-iodine gram-positive bacteria losing the crystal violet-iodine complex. complex.

– The age of the culture. Cultures more than 24 hours old The age of the culture. Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine may lose their ability to retain the crystal violet-iodine complex. complex.

– The organism itself. Some gram-positive bacteria are more The organism itself. Some gram-positive bacteria are more able to retain the crystal violet-iodine complex than others. able to retain the crystal violet-iodine complex than others.

• Therefore, one must use very precise techniques in Therefore, one must use very precise techniques in gram staining and interpret the results with gram staining and interpret the results with discretion.discretion.