2. Gram Stain
Differential stain (Hans Christian Gram, a Danish doctor ). He developed a new method to stain bacteria so they can be visible in specimen samples.
Differentiate bacteria into two large groups (the Gram Positive and the Gram negative).
Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics.

3. Almost all bacteria are described by their Gram stain characteristics.
Based on differences of Cell wall structures
Classifies bacteria into 2 large groups: Gram-positive and Gram-negative. Gram-positive bacteria have a thick mesh-like cell wall made of 50% to 90% peptidoglycan, while Gram-negative bacteria only have around 10% of their cell wall made up of peptidoglycan.

4. Gram Positive & Negative Bacteria

7. Reagents for Gram Stain
Crystal Violet (purple).
Primary stain; positive stain
Stains cell wall purple
Iodine
Mordant
Combines with primary stain to form an insoluble complex that gets trapped in bacterial cell wall
Staining the slide:
Start with a heat-fixed slide of your material. Recall, Gram positive bacteria have a single cell membrane and hardy outer cell wall made of peptidoglycan. In contrast. Gram negative cells have two membranes. In between the two membranes is the *periplasmic space* which contains a layer of peptidoglycan. <-- Follow the images to the right to see how Gram positive and Gram negative cells would appear during each stage of the stain.
Crystal Violet: Flood the slide with Crystal violet. Let sit for 30 seconds, then rinse with tap water. If available, use forceps to hold the slide while rinsing. The crystal violet binds to the membrane of Gram positives, and the outer-membrane of Gram negative bacteria.
2. Lugol's Iodine: Now flood the slide with the Lugol's iodine, which forms a strong complex with bound crystal violet. Let sit for 30s, then rinse the slide with tap water. A this stage both Gram positive and Gram negative bacteria will stain purple.
3. Decolorization -- this is the most important step. Ethanol tends to be more forgiving than acetone, and may be worthwhile to try for beginners. Acetone or 95% ethanol dissolves the outer membrane of Gram negative bacteria, but not Gram positive bacteria. If done properly, Gram positive bacteria remain purple at this stage, while Gram negative bacteria become colorless. However, too much decolorization and all bacteria will appear Gram negative (all crystal violet+iodine will be washed away); too little, and all bacteria will appear Gram positive (not enough to remove the outer membrane of Gram negatives). In general, hold the slide at a slant and count for 3 seconds while squirting top edge of the slide with the decolorizing agent, so it runs down the length of the slide. Then immediately rinse in tap water to remove remaining decolorzing agent.
4. Counterstain with Saffranin: Flood the slide with the dye and let sit for 30s, then wash. Saffranin is a red dye and actually stains both Gram positive and negative bacteria. However, for Gram positive bacteria that are already purple, they remain an intense purple color while previously colorless Gram negative bacteria take up the red stain and will appear red by microscopy.
5. Interpretation (more below): Slides need to be read at 100X magnification on a microscope. Lenses at this magnification require immersion oil to obtain clear imaging of material on the slide. Gram positive organisms will be dark purple, while Gram negative will appear bright red.

8. Stains all cells, but only the negative ones actually appear pink.
Ethanol
Decolorizer
CV-I complex washed out of Gram negative organisms because it cannot be trapped by lippopolysaccharide layer; flows right through outer membrane
Gram +
Gram –
Primary stain:
Crystal violet
Purple
Mordant:
Iodine
Decolorizing agent:
Alcohol-acetone
Colorless
Counterstain:
Safranin
Pink
Safranin (pink)
Counterstain
Simple positive stain that provides contrasting dye for decolorized cells (Gram negative)
Stains all cells, but only the negative ones actually appear pink.

9. Gram Staining Procedure
After air drying and heat fixation.
If you don't perform it the bacteria may be washed off during staining
Heat-fix this slide. This step makes certain the bacteria stick to the glass during staining. If you don't perform it the bacteria may be washed off during staining. Bunsen Burner Method: Light the bunsen burner. Hold the slide with a pair of forceps (strong tweezers) so you don't burn your fingers. Pass it through the interface bewteen the blue flame and the yellow flame - this is the hottest region of the flame - 5 times. The slide should sit in this region for no more than a second, as if it gets too hot the bacterial will rupture, and you will not get a good stain.
Heating Plate: If using a heating plate, set it to 42°C (~108°F). Place your glass slide on the hotplate until all the liquid evaporates.

10. . It's important to not identify organisms as Gram positive or Gram negative at this magnification, as interpretation of the stain can be misleading. Always make your final interpretation at 100X magnification.
A drop over the area to be viewed is more than enough. Once immersion oil is added *never* go back to a 40X or 20X "high and dry" objective as oil will get on the lenses, rendering them useless. To refocus, use a 4X or 10X objective, being very careful not to touch the oil.

11. Explanation - Crystal violet stain dissociates in water to form CV+ and Cl- ions which penetrate through the cell wall and cell membrane of all cells. - Iodine is added and forms crystal complexes with CV+ to form CV-I, preventing removal of this CV+ ions and hence the colour of the cells. - Decolourisation with alcohol removes crystal violet from only Gram-negative cells, along with its outer membrane layer, due to the thin peptidoglycan layer. - Gram-positive cells only get dehydrated by the alcohol, hence trapping the large CV-I complexes in its multilayered peptidoglycan. - Gram-negative cells take up a counterstain - safranin (pink) or basic fuchsin (red). *Decolourisation process must be timed, if Gram-positive cells are exposed to alcohol for too long (a matter of seconds), they might lose colourisation as well,

13. Errors During Staining
Never ever used old culture.
Never ever used sample for patient take antibiotic.
Time of Decolorizer:
Over: G + see as G -.
Low: G- see as G +.
Time of fixation:
Low: no sample on slide.

14. Acid-fast stain (Ziehl-Neelsen stain)
The acid-fast stain is another differential staining method.
In this case, the target cells are usually members of the genus Mycobacterium.
The cell walls of these bacteria contain an unusually high concentration of waxy lipids, thus making conventional simple stains and Gram stains useless.
The genus Mycobacterium contains two important human pathogens, M. tuberculosis and M. leprae, which cause tuberculosis and leprosy, respectively.

15. The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol, and methylene blue.
Acid-fast bacilli will be bright red after staining.

16. Acid-fast stains are useful in identification of acid-fast bacilli (AFB)and rapid, preliminary diagnosis of tuberculosis (with greater than 90% predictive value from sputum samples).
It also can be performed on patient samples to track the progress of antibiotic therapy and determine their degree of contagiousness.
Acid-fast stain Binds strongly to bacteria that have waxy material(mycolic acid) in their cell walls. Heat can be used to increase permeability of stain. Identify bacteria in the genus Mycobacterium, including M. tuberculosis and M. laprae. Acid-fast organisms retain red colour of carbolfuchsin even after decolourization Non-acid-fast organisms become colourless. After staining with methylene blue, non-acid-fast cells appear blue.

17. Acid Fast Reagents
Carbolfuchsin (red), a phenolic stain: is the primary stain in the acid-fast test. It is soluble in the lipids of the mycobacterial cell wall.
Heating the specimen, or adding a wetting agent such as Tergitol, increases the penetration of the carbolfuchsin.
Following application of the carbolfuchsin, the specimen is cooled and decolorized with a solution of 3% hydrochloric acid and 95% ethanol (acid-alcohol).

18. Since carbolfuchsin is more soluble in waxy cell
lipids than in acid-alcohol, the acid-alcohol removes
the carbolfuchsin from non-acid-fast organisms, but
not from acid-fast organisms.
Following decolorization, the sample is counterstained with methylene blue which Cannot penetrate mycolic acid; provides contrast to non acid fast cells.

19. Procedures Prepare a smear organism and a on glass slides.
Allow the slides to air dry, and then heat fix the organisms.
Apply enough of carbolfuchsin with Tergitol to cover the bacteria. Allow it to set for five minutes.
(Alternate) If Tergitol is not available, apply enough carbolfuchsin to cover the bacteria. Place the slide on a pre-warmed hot plate set on low for 8 minutes. Do not allow the stain to evaporate or Boil. Add additional stain, if necessary. Remove the slide and allow it to cool.

20. Rinse the slide with acid-alcohol (15-20 sec), drop by drop, just until the alcohol runs clear.
Gently rinse the slide with water.
Apply enough methylene blue to cover the bacteria.
Allow it to set for 30 sec.
Gently rinse the slide with water.
Blot (don't wipe) the slide dry with bibulous paper.
Allow the slide to air dry.
Examine the slide under oil immersion.
Positive organisms will appear pink or red;
Negative organisms will appear blue.

24. Note
The reddish-pink color and “cording” (sticking together in long ropy masses) of the Mycobacterium cells due to the excess lipids of the cell wall