Slides of university

1. PREPARATION FOR LIGHT
MICROSCOPE EXAMINATION

3. purpose
• The study of bacterial morphology includes
the study of the size, the shape, and the
arrangement of the bacterial cells, as well as
an examination of structures such as
endospores, granules, capsules, and flagella,
and other structures.

4. • Two general techniques are used to prepare
specimens for light microscope examination.
1-The Wet -Mount and Hanging Drop
Technique:suspend organisms in a liquid .
• The observation of unstained living organisms is
required for certain purposes:
1. To study the morphology of spiral bacteria.
2. To determine whether or not the bacteria are
motile.
3. To follow up the cell division.

6. 2- Staining technique:Dry,fix and stain smears.
- Help making cells more visible.
- Stains Acidic:stains positive cells.
Basic: stains negative cells.
Types of staining technique:
a-Simple staining..
-Use single stain.
-Shape & arrangement.
b-Differential staining..
- Use 2 counter stains.
- Separate in to groups.
e.g-Gram staining.
- Acid fast staining.
- See structures..( Capsule, Endospore, Flagella )

7. Before staining bacteria … Fixation is a MUST….
*Preparation of bacterial smear:
- Clean slide, label(with wax pencil or diamond pencil).
- Take a drop of water & touch by loop colony &mix it with
water then spread.
- Dry the smear (in air or heating gently).
- Fix the smear ( Pass through flame three times ).

9. Gram staining method
• Most important method (used for identification).
• Shows cell wall structure..
Gram -ve Gram +ve
-Thin peptidoglycan layer - Thick peptidoglycan layer
-Thick Lipopolysaccharide layer - Little Lps…

10. Gram staining procedure
1- Prepare bacterial smear (fresh young cells).
2- Primary stain (Crystal violet).
3- Wash with water (not too much…Why? ).
4- Mordant=Iodine… will intensify the reaction
between cell & dye ( CV-I complex).
5-Decolorizer=Alcohol-Aceton(Not too much).
6-Wash with water.
7- Counter stain (Safranin ).

13. Factors affecting Gram stain results:
1-Use actively growing cells. Old cells lose their
ability to hold the stain; they appear gram negative.
2- Avoid overheating the cells during heat fixation,
Excessive heat disrupts cell walls making gram-
positive bacteria appear gram-negative.
3- Adjust timing to your reagents.
4- Do not rinse too long. Water is a decolorizer.
5- Remember, gram positivity is a characteristic
that can be lost.