1. Experiment one Examination of Bacteria

2. Visualizing Bacteria
Staining is required to properly visualize bacteria

3. Microscopy Bright Field Microscopy

4. Microscopy Bright Field Microscopy
Three different objective lenses are commonly used
10x: To scan the slide for specimens
40x: To view parasites, filamentous fungi
100x: To observe single cells
Total magnification= (objective lens magnification) X (ocular lens magnification)

5. Microscopy Bright Field Microscopy
Note how light scatters via refraction
Refractive index of air is lower than that of glass
Loss 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 microscopy
Little contrast exists between structures with similar transparency
Insufficient 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 mircoscopy
Internal features are more easily viewed

9. Microscopy Phase-Contrast Microscopy

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 targets
Digital merging with differential stains

13. Electron Microscopy
Uses magnetic coils to direct a beam of electrons through the specimen onto a screen
Uses a very short wavelength, thus magnification and resolution is improved
Samples 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 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

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 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

18. Examination Methods: Direct Examination
India Ink
Darkens the background rather than the cell
Useful in detecting Cryptococcus capsules
Capsule 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. choleriae
Gram -ve

22. Morphological Observation Of Bacterial Cells
Streptococcus pneumoniae

23. Morphological Observation Of Bacterial Cells
Salmonella typhi

24. Morphological Observation Of Bacterial Cells
Clostridium tetani
Club 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 stain
MATERIALS:
Simple stain
Gram stain
Acid-fast stain
Special stain
Spore stain
Capsule stain
Flagella stain
Metachromatic 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, safranin
Microscope slides

27. Gram stain PROCEDURE: Smear: size of a dime to form a thin film
Dry : air dry
Fix: through the warm air above the flame two or three times.

28. Process of Gram’s Stain
Crystal violet
(primary staining)
Washing
1min
Lugol’s iodine
(mordant staining)
Washing
1min
95%ethyl alcohol
(decolorization)
Fuchsion red
(counterstaining)
Washing
30s
Washing
30s
Blot dry with bibulous papers
Observation with the oil immersion lens
Results :Gram –positive blue color Gram-negative red color

29. Gram Staining
In 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 content
Lipid is dissolved by alcohol
The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan
Thus 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.