1. Faculty of Medicine and Health Sciences
Microbiology Lab
Exp 3
Visualization of Bacterial Cells By Simple stains Wet Mount Smear Preparation Simple Staining By Acidic and Basic stains
First Semester

2. Visualization of Bacteria in Wet Mount
Bacterial cells are very small. They are about µm diameter and about 2 – 8 µm length. So it is not possible to see them by naked eye and they can only be seen using the microscope If your are enough skilful and upon using highest magnification, it is possible to see unstained bacterial cells by the light microscope. In this case , bacterial cells appear light-grayish (almost colorless)

3. This is how bacterial cells appear in urine sample obtained from a patient with UTI without staining at 1000 X magnification

4. Wet Mount Preparation:
Material needed:
Bacteria grown on plates
Distilled water and dropper
Glass slides
Cover slips
Loop
Bunsen Burner
Microscope
Oil
Lens paper and Xylene

5. Before you start, make sure of the following:
Have your lab coat on you and wear cloves if available
Have the door and the windows of the lab closed ( you do want to have an air current that may contaminate your culture)
Disinfect your bench with 70% alcohol
Allow your bench to dry, then light up the Bunsen burner

6. Procedure:
Place a drop of distilled water on the middle of the glass slide
Sterilize the loop by flaming, allow to cool
Uncover the plate and pick up a bacterial colony using the sterilized loop. ( Do not leave the plate uncovered)
Mix the picked bacterial colony with the water drop on the slide
Re-sterilize the loop by flaming, allow to cool and place it on the bench
Cover the drop water drop which has been mixed with bacteria using the cover slip (hold the cover slip at 45 degrees and the edge of the drop and then lay it on the drop so that the water spreads underneath the cover slip)
Examine the wet mount using the oil immersion lens
Note: It is possible to use bacteria grown in broth. In this case, dilute the booth culture and place a drop of the diluted broth on the slides and continue ( points 6 and 7)

7. Expected results: as seen in the following images
Note: After you finish, make sure you clean the lenses of your microscope with Xylene, turn off your microscope and place it in the cabinet
What can we learned from the visualization of unstained bacterial cells?
1- bacterial cell morphology
2- arrangement of bacterial cells

10. Bacterial Smear Preparation:
Preparation of bacterial smears on a glass slide is very useful in case you want to stain bacterial cells on a glass slide.
Staining bacterial cells placed on a glass slide implies that bacterial cells have to be adhered to the slide, other wise, they will be washed off upon washing the smear to remove excess stain.

11. Material needed:
Bacteria grown on plates
Distilled water and dropper
Glass slides
Loop
Bunsen Burner
Wooden forceps
Before you start, make sure of the following:
Have your lab coat on you and wear cloves if available
Have the door and the windows of the lab closed (you do not want to have an air current in the lab that may contaminate your culture)
Disinfect your bench with 70% alcohol
Allow your bench to dry, then light up the Bunsen burner

12. Place a very small drop of distilled water on the slide
Procedure:
Place a very small drop of distilled water on the slide
Flame the loop, cool, and touch it to the colony
Mix the picked up colony with the water drop
Use large circular motions to spread the drop to about the size of a HLAF SHEIKEL
Re-flame the loop
Hold the slide with the wooded forceps ( make sure the forceps do not get in contact with bacteria in the smear)
Heat fixation: is done by rapidly passing the slide (smear side up) through a flame several times. Use a slide holder and avoid contact with hot glass.
Label the slide with a graphite pencil in the ground glass section on the far left of the slide since the reagents used in staining (such as the decolorizer in the Gram stain) can remove your labels if you use pen or wax pencil)
Note:
If the bacterial smear is being made from a broth, simply place a drop of diluted broth on the slide and spread, do not use additional water.
Continue points 6 through 9

14. Heat fixation:
Heat fixation is an an important step that:
1- Kill bacterial cells (safer to handle)
2- Allow bacterial cells to adhere to the glass (not to be washed off by the stain the washing steps)
3- Preserve the morphology of bacterial cells
Note: In certain cases, heat fixation cannot be used ( Example: capsule staining). In this case, chemical fixation must be used instead.

15. Simple Staining Procedures:
1- By Using Basic Stains: In solutions, these stains generates positively charged ions that bind easily to the negatively charged cytoplasm of bacterial cells.
Examples of Basic Stains:
Crystal violet
Carbolfuchsin
Methylene blue
Safranin
Basic staining:
1- Improve visualization of bacterial cells under the microscope
2- You will able to determine the morphology and arrangement of bacterial cells
Note: Basic stains are simple stains and not differential stain. So, you cannot tell whether the stained bacterial cells are Gram positive or Gram-negative.

16. The color of the stained bacterial cells depend on the color of the used stain.
Examples: if you use
Crystal violet : the stained bacterial cells appear violet
Carbolfuchsin: the stained bacterial cells appear Fuchsia
Methylene blue: the stained bacterial cells appear blue
Safranin: the stained bacterial cells appear Pink to Red

17. Material needed:
Prepared Bacterial Smear
Basic stain
Water
Bibulous paper
Microscope
Oil
Lens paper and Xylene
Before you start, make sure of the following:
Have your lab coat on you and wear cloves if available
Disinfect you bench (allow your bench to dry if you used ethanol and then light up the Bunsen burner)

18. Procedure:
1. Prepare a bacterial smear.
2. Saturate the smear with a basic for approximately 1-3 minutes
3. Rinse the slide gently with water to remove excess stain
4. Carefully, blot-dry with you stained smear with bibulous paper
5. Clean the lenses of the Microscope with Xylene
6. Observe the stained smear under the microscope, using proper microscope technique (use oil immersion lens)

19. A Coccus Bacterium Stained by Methylene Blue (Basic Satin)

20. Oral Smear Stained by Methylene Blue
Notice the bacterial cells on epithelial cells

23. 2- By Using Acidic Stains (Negative Stain) Example: Nigrosin
Nigrosin is an acidic stain. This means this stain generates negatively charged ions in solution.  
Since the bacterial cell cytoplasm is negatively charged, the cells repels the stain.
This stain can be used to stain the surface of the glass slide (back ground), but not the bacterial cells.
By using this type of stains, the bacterial cells will not be stained and be seen as empty spots that take the morphology (the shape) against a dark background.
Negative stains are particularly useful for the determination of the morphology and arrangements of heat-sensitive bacteria
Note: Bacteria stained with this stain are usually heat sensitive so that heat fixation cannot be used.

24. Material needed:
Acidic stain (Nigrosin)
Glass slides
Bacteria
Microscope
Oil
Lens paper and Xylene
Before you start, make sure of the following:
Have your lab coat on you and wear cloves if available
Disinfect your bench ( Allow your bench to dry if you used Ethanol), then light up the Bunsen burner

25. Procedure:
1. Place a single drop of Nigrosin on a clean microscope slide, adjacent to the edge.
2. Using a flamed loop , remove some bacteria from your tube or plate and mix it into the drop of Nigrosin.
3. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism and spread the drop out into a film. This is done by contacting the drop of Nigrosin with the clean slide and using the capillary action of the dye/slide to spread the Nigrosin across the slide.
4. Allow the film to air dry. 
5. Observe the slide under the microscope, using proper microscope technique

27. Negative staining by using Congo Red:
Congo Red can be used for negative staining. However, unlike Nigrosin that does not stain bacterial cells because of its negatively-charged ions, Congo Red does not stain bacterial cells because it has a very large molecular weight, so that molecules of Congo Red cannot go through the cell wall of bacterial cells.
Procedure:
1. Place a single drop of Congo Red on a clean slide, adjacent to the edge.
2. Using a flamed loop , remove some bacteria from your tube or plate and mix it into the drop of Nigrosin.
3. Place the end of another clean slide at an angle to the end of the slide containing the organism and spread the drop out into a film. This is done by contacting the drop of Congo Red with the clean microscope slide and using the capillary action of the
dye/slide to spread the Congo Red across the slide.
4. Allow the film to air dry. 
5. Observe the slide under the microscope, using proper microscope technique