ACTIVITY 2: Preparation of Wet, Fixed smear, and the gram staining method.

3 glass slides and coverslips

1 wire loop

1 alcohol lamp

2 clean toothpick

2 droppers, Marthylene blue, 0.85% NaCl and reagents for gram staining

Materials used in Activity 2

Crystal violet

Gram’s iodine

Ethyl Alcohol

Safranin

Reagents for gram staining used

purple

crystal violet

amber

Gram’s iodine

clear

Ethanol

red/ pink

safranin

Fixed Smear Method

Step 1: Teeth Scraping/Buccal Swab

Step 2: Place Sample on a Glass Slide

Step 3: Fix the sample Using Heat

Step 4: Staining with Methylene Blue

Step 5: Prepare slide for microscopy

Step 6: Examine the Slide Using the Microscope

Inside the cheek

Where should we rub the toothpick to get the buccal cells?

glass slide in a thin layer

After getting the buccal cells, we will spread it to the

alcohol lamp

what material will be using when heating the glass slide?

Quickly for 5 times to avoid overheating

how frequent we will pass the slide to the alcohol lamp.

covering it using the cover slip and adding immersion oil on top

After heating the glass slide, the next step will be

Scanner 4x (40x)

Low Power Objective 10x (100x)

High Power Objective 40x (400x)

what objectives will be using to examine the slide?

Wet Smear Method

Step 1: Teeth Scraping/Buccal Swab

Step 2: Place Sample on a Glass Slide

Step 3: Drop of 0.85 NACL (Sodium Chloride) solution

Step 4: Covering the sample

Step 5: Examine the Slide Using the Microscope

O.85% NaCl

What reagent used in the wet smear method

It uses heat

What is the unique steps in fixed smear method?

Gram Staining Method

Step 1: Teeth Scraping/Buccal Swab

Step 2: Place Sample on a Glass Slide

Step 3: Fix the sample Using Heat

Step 4: Staining (Crystal Violet)

Step 5: Staining ( Gram’s iodine)

Step 6: Staining ( Ethyl Alcohol)

Step 7: Staining ( Safranin)

Step 8: Prepare slide for microscopy

Step 9: Examine the Slide Using the Microscope 

Crystal Violet

What is the primary stain that color all cells with a purple hue

1 minute

How many minute to let it sit before wash and dry for crystal violet, gram’s iodine and safranin?

10-15 secs

time to let it sit before wash and dry for Gram’s iodine

Iodine

Acts as a mordant, forming a complex with the crystal violet dye within the bacterial cells

Ethyl Alcohol

decolorizes the smear, allowing differentiation between gram positive and gram negative based on their cell wall structure

Safranin

stains the decolonized gram negative bacteria and gram positive

pink

gram negative color

purple

gram positive color

Buccal cells under HPO

Before: Transparent

After: All positive and negative become purple

Crystal Violet (Primary stain) before and after color of bacteria

Before: Purple

After: remain purple

Gram’s iodine (mordant) before and after color of bacteria

Before: Purple

After: (+)- stay purple

(-) colorless

Ethyl Alcohol ( Decolorizer) before and after color of bacterias

Before: (+) still purple

(-) colorless

After: (+) remain purple

(-) pink/red

Safranin before and after color of bacterias

Squamous cell ae present

Motile bacterias are present

Wet smear result

- Squamous cells are present. More defined and clear image was seen under OIO.

- Non-motile bacteria were present.

Fixed smear result

Fixation process

is a process where the sample is fixed by agitating the slide over a heat for 5 times. This ensures that bacterias are killed.

Inclined

…, and slowly drop. If bubbles are present, slowly press down to eliminate bubbles.)

- Gram Positive Cells (Purple) were present

- Gram Negative Cells (Red) were present

- Non-motile Bacteria were present

Gram staining result

Hans Christian Gram

who first introduced gram staining in 1882, mainly to identify organisms causing pneumonia.

Gram Positive Organisms

retain the primary color and appear purple-brown (Squamous epithelial cells)

Gram Negative Organisms

do not take the primary color and appear as red (WBC and Macrophages)