Growth Patterns in Broth
- Control: clear
- Flocculent: clumps
- Uniform fine turbidity: even cloudiness.
- Pellicle: top surface membrane.
- Sediment: cells sink to bottom.
Why do we need to stain bacteria?
To improve contrast between the background and bacterial cells, making them easier to see under the microscope.
What charge do basic stains carry?
- Positive (+) charge
- Bond with negatively charged cell surface
Differential stains determine:
- Cell morphology (rod, coccus, spiral)
- Cell arrangement (single, pairs, chains, clusters)
- Cell Size
They can show structures like:
- Spores
- Flagella
- Capsules
- Cell wall differences (Gram + vs Gram -)
Chromogen
whole colored molecule
Chromophore
part that gives it color
Auxochrome
- changed portion of chromogen
- allows dye to bind to cells
What do we need to do to the smear slide of bacteria before we stain it?
Heat-fix
Why do we heat-fix bacteria?
- kills the organisms
- sticks to the slide
Types of stains:
- Crystal violet, purple/blue color, (30-60 seconds)
- Safranin, red/pink color, (60 seconds)
- Methylene blue, (30-60 seconds)
Simple Stain Procedure
- Prepare smear:
- Drop of water + add bacteria → spread → air dry.
- Heat-fix (flame pass 2-3x). - Cover smear with stain (30-60 sec).
- Rinse with DI water.
- Blot with bibulous paper.
- Observe at 100x oil immersion.
What does Gram staining tell us about bacteria?
The type of cell wall a bacteria has through the amount of peptidoglycan in its cell wall
Gram’s Staining uses two different stains:
- Crystal Violet (purple primary stain)
- Safranin (red counter stain)
Two types of cell walls with different amounts of peptidoglycan:
- Gram Positive: thick peptidoglycan, retains CV (crystal violet)
- Gram Negative: thin peptidoglycan + outer membrane, loses CV, takes safranin (red)
During the destaining and rinsing process, which bacteria type will retain the stain better?
Gram Positive
Gram staining uses a mordant called Gram’s iodine
- A mordant is used to enhance staining
- Gram’s iodine enhances crystal violet staining by creating a crystal violet-iodine complex
Gram Stain Steps
- Crystal Violet (60 sec) → rinse.
- Gram’s Iodine (60 sec) → rinse.
- Acts as mordant (enhances stain, forms CV-I complex). - Decolorizer (alcohol/acetone) for 5-10 sec, until runs clear → rinse.
- Removes lipids from Gram - outer membrane → makes porous. - Safranin (60 sec) → rinse, dry.
Gram Stain Results
- Gram Positive Bacteria: blue (dark purple)
- Gram Negative Bacteria: red (dark pink)
Problems with Gram Staining
- Over decolorize or leave alcohol on too long: Reddish Gram Positive cells
- Under decolorize or remove alcohol too soon: Purplish Gram Negative cells
- Inconsistency in heat-fixing the cells on slide
- Some Gram-Positive cells (e.g. Bacillus sp.) can lose their ability to retain crystal violet(within 24 hr)
