Lab 10

Introduction to Staining

Slide Boxes
- Write your name with a sharpie on the tape on top of one of the slide boxes at your table.
- This box is for storing your prepared slides throughout the semester.
- Note that slides must be heat-fixed.

Slide Cleaning Process
- Use a mild abrasive like Comet to clean the slides, then rinse and wipe dry with tissues (wipes from the drawer).
- Pass cleaned slides through a flame twice to eliminate any oils or lint.
- Importance of Cleaning:
- Lint fibers appear massive under a microscope.
- Smears will not adhere to slides that have not been properly cleaned.
- Multiple smears can be placed on a slide (except for negative stains).
- Every staining procedure, except negative staining, requires this cleaning process prior to making a heat-fixed smear.

Purpose of Staining
- Staining is vital for visualizing samples as most bacterial cells are transparent.
- It is used to determine morphology (shape) and arrangement of the cells.
- Example Arrangements:
- Cocci arranged in clusters: Staphylococcus aureus
- Cocci arranged in tetrads: Neisseria sicca
- Bacilli arranged in chains: Bacillus megaterium

Chemical Stains
- Contrast is obtained through chemical stains with a charged, chromogenic component.
- Simple Stains:
- Involves only one dye.
- Charge of Bacterial Cell Walls:
- Cell walls are slightly negatively charged.
- The electric charge of the dye determines which parts are visible based on dye color.

Basic Stains
- Positively charged.
- Attracts to the negatively charged cell wall, resulting in the cell being colored.
Acidic Stains
- Negatively charged.
- Repelled by the cell wall; everything except the cell is colored.

Types of Staining Techniques
- Simple Staining:
- Uses one dye with a rinsing step, allowing visualization of morphology and arrangement.
- Differential Staining:
- Utilizes two or more dyes, includes a decolorizing step, and requires multiple rinsing steps to differentiate between bacterial species based on characteristics.
- Heat-fixing Requirement:
- Most staining procedures require heat-fixing, except for negative staining.

Characteristics of Negative Staining
- Based on the observation of color patterns in slides.
- The dye must be negatively charged, as the dye repels from the cell, leaving the cell colorless while the background is stained.
- The method leads to observing naturally occurring morphology and arrangement, particularly useful for spirochetes like Treponema.

Negative Staining Procedure
1. Prepare two negative stains per student (1 with negative mix, 1 with yogurt).
2. Clean 2 slides for stained samples and 2 additional slides as spreaders.
3. Place one drop of “negative mix” (Bacilli, Cocci, and Yeast cells) on a cleaned slide.
4. Add one drop of nigrosin to the drop of negative mix.
5. Use the spreader slide to spread the mixture across the cleaned slide, allowing it to dry completely.
6. Repeat the process for the second slide using yogurt.
7. View slides under an oil immersion lens and record data.
8. Dispose of slides in provided disinfectant if finished viewing.

General Steps
1. Wearing gloves and eye protection, place a small drop of water at one end of a clean slide using an inoculating loop.
2. Add the bacteria aseptically to the water drop. Avoid over-inoculating; if transferring too much, switch to an inoculating needle.
3. Allow the smear to air dry, ensuring it appears slightly cloudy.
4. Pass the smear through an upper flame two or three times using a slide holder for heat-fixing. Be careful to avoid overheating.
5. Cool the slide and proceed with the staining protocol.

Reasons for Heat-Fixing
- Adheres cells to the slide, preventing them from washing off.
- Kills living bacteria, reducing potential contamination during rinsing.
- Coagulates cytoplasmic proteins, enhancing visibility of cells.
- Disruption of Morphology:
- Disruption can cause confusion in identifying bacterial species based on arrangement.

Steps for Simple Staining
1. Start with a heat-fixed emulsion (can include more than one organism).
2. Place the slide over a staining tray and cover the smear(s) with stain, letting excess drop into the tray.
3. Hold the slide at an angle and rinse gently with distilled water into the staining tray, disposing of the stain as per lab practices.
4. Blot dry gently with bibulous paper or towels without rubbing. Observe under oil immersion when dry.

Observation Under Oil Immersion
- Record the following details:
- Organism Name
- Dye Used
- Appearance of each organism with sketches or photographs noting color, morphology, and arrangement.