Lab 11 - Gram Staining Study Notes
Preparation for Gram Staining
Clean 4 microscope slides in preparation for gram staining.
Use mild abrasive (e.g., Comet) for cleaning.
Rinse slides and wipe them dry using tissues from the box in your drawer.
Pass the cleaned slides through a flame 2 times to remove any oils or lint.
Lab Exercise 5: Gram Staining
Overview of Gram Staining
Gram staining is a laboratory procedure used for the classification of bacteria into two groups based on their cell wall structure and composition.
Differential Stains versus Simple Stains
Simple Stains:
Only one dye is added, allowed to sit, and then rinsed.
All cells are stained the same color, hence no differentiation between species with similar morphology can be made.
Differential Stains:
Used to differentiate between different bacterial species.
Involves a multi-step procedure that includes:
Primary dye
Secondary dye
Decolorizing agent
Mordant
The combination of two dyes and a decolorizer leads to some cells retaining the primary dye while others take on the secondary dye's color, allowing differentiation.
Ex. 5 – Gram Staining
Hans Christian Gram:
Discovered the gram stain technique by accident while seeking a method to differentiate human cells from bacterial cells in biopsy samples.
Noted that human cells are gram negative as they lack a cell wall.
Observed that bacterial cells turn purple or red/pink upon gram staining, consistently, allowing for differentiation between bacteria.
Dyes used in this procedure:
Crystal Violet (primary dye)
Safranin (secondary dye)
Gram positive cells appear brighter and more distinct than gram negatives, aiding in easier identification.
Gram Stains
Components of the Gram Staining Technique:
Crystal violet: Primary dye.
Safranin: Secondary dye.
Iodine: Acts as a mordant that locks the primary dye into the cell.
Ethyl Alcohol (Ethanol): Decolorizer used to wash away excess primary stain allowing secondary dye to color some cells.
Gram Positive vs. Gram Negative Cell Wall Anatomy
Gram Positive Cells:
Composed mainly of peptidoglycan.
Have a very thick wall made of multiple layers with only 1 plasma membrane.
Gram Negative Cells:
Have a thin layer of peptidoglycan.
Possess 2 plasma membranes (outer and inner).
Feature a lipopolysaccharide (LPS) layer outside the outer membrane.
Detailed Cell Wall Anatomy for Gram Positive and Negative Cells
Gram Positive:
Contains:
Peptidoglycan
Teichoic acid
Surface protein
Lipoteichoic acid
Gram Negative:
Contains:
Peptidoglycan
Outer membrane (with LPS)
Receptor proteins
Porin proteins
Periplasmic space
Cytoplasmic membrane
Cytoplasm
Cells are transparent prior to staining.
Post-Staining Observations After Primary Dye and Mordant Application
Gram Positive Cells:
As long as the crystal violet-iodine complex is retained inside the cell, the cells will appear purple.
Gram Negative Cells:
The complex will be removed during decolorization.
Effect of Decolorization with Ethyl Alcohol
Gram Positive Cells:
Remain purple upon decolorization due to the thick peptidoglycan layer retaining the dye.
Gram Negative Cells:
Crystal violet is washed away after decolorization; after adding safranin, they appear red or pink.
Staining Procedure Steps
Begin with up to three heat-fixed emulsions on one slide.
Place the slide on a rack over a staining tray.
Cover smear(s) with crystal violet stain for 1 minute, ensuring excess stain is caught in the tray.
Rinse gently with distilled water into the staining tray.
Cover smear(s) with Gram iodine stain for 1 minute, capturing runoff.
Rinse with distilled water.
Decolorize with Gram decolorizer by allowing it to trickle down until runoff is clear, immediately rinsing with diluted water.
Counterstain with safranin for 2 minutes, washing away excess before drying.
Blot gently and view under oil immersion microscope.
Common Challenges with Gram Stains
**Over-Decolorization: **
If alcohol is left too long, all cells may appear gram negative due to the loss of crystal violet.
Under-Decolorization:
Not allowing enough time for decolorization results in all cells appearing gram positive, as the alcohol cannot penetrate the thick walls sufficiently.
Gram Variability:
A phenomenon where both colors (purple from crystal violet and red from safranin) appear within a single sample.
Caused by:
Old cultures (> 24 hours) degrade, preventing retention of the primary dye.
Too thick smears impede uniform decolorization, yielding mixed results.
Practical Application: Recording Results
After observing samples under oil immersion, record:
Name of sample
Morphology
Arrangement (using majority rules)
Gram reaction (positive or negative)
A colored sketch or photo of representative cells observed.
Summary of Procedure for the Day
Create two slides ensuring each contains both a gram positive and a gram negative organism:
Slide 1: Neisseria and Staphylococcus aureus
Slide 2: Bacillus subtilis and E. coli
Gram stain both slides and compare smears to demo scopes provided.
Make sure to only gram stain one of each organism type.
After staining, place cleaned slides in a dedicated slide box for safe keeping.
Mini-Unknown #1 Activity
An unknown organism will be provided, labeled with a letter or number.
Stain this organism after the four known organisms, using previously stained slides for comparison.
Record identification on a submission form which can be sent physically or digitally via Canvas.
Final Notes on Gram Staining Procedure
Two slides with two heat-fixed smears from designated organisms should be prepared for gram staining, following the outlined procedural steps carefully. Compare stained unknown against known organisms for identification.