MICROBIOLOGY LAB 1 and 2 \u2013 Staining
USTMED \u201907 Sec C \u2013 AsM; Photos provided by JV.N. & A.M.
1. Air drying
2. Passing over flame of Bunsen burner
3. cover slide with methyl alcohol for 1 minute
2. distortion of cell size and shape are minimized
a. heat fixing is not necessary
b. cells do not pick up the stain
Acid fast stain Carbol fuchsin
Ziehl Neelsen stain
Schaefer Fulton method
Wirtz Conklin method
Microscopically, micrococci are larger than staphylococci and appear in tetrads
2. Inside the cells, the crystal violet and iodine combine to fome CV-1 which is larger than the crystal violet molecule that entered the cells.
- Provides valuable information for the treatment of disease
when stained with a modified Kinyoun stain using 2% H2SO4 as the decolorizing agent. This
this mircroorganism from other actinomycetes.
1. Acid fast organisms retain the red color because the
carbol fuchsin is more soluble in the cell wall lipids than
in the acid alcohol.
2. Non-acid fast organisms \u2013 cell walls lack the lipid
components; carbol fuchsin is rapidly removed during
the finding of acid fast bacilli in a sputum is a
presumptive evidence that the organisms is
rod in the center of the clear area. The purple color is from the basic stain, crystal violet.
The clear area is the capsule, and the background is colored by the negative, acidic stain (India ink).
B. Flagella stain \u2013 a tedious and delicate staining procedure using a mordant and the stain carbol fuchsin to build up the diameters of the flagella until they become visible.
Spore stain of Bacillus cereus. The arrows are pointed at green spores in a pink vegetative cell.
Gram stain of Bacillus cereus.
The arrow is pointed at a
spore, which is clear inside
the gram-positive vegetative
metachromatic granules when stained with Loeffler methylene blue stain or Neisser stain. The stain is best performed on colonies grown on a
Malachite green staining
Bacillus subtillis (gram positive bacilli in chains)
Streptococcus lactis (cocci in chains)
The Gram Stain Introduction
Gram\u2019s Stain is a widely used method of staining bacteria as
an aid to their identification. It was originally devised by
Hans Christian Joachim Gram, a Danish doctor.
Gram\u2019s stain differentiates between two major cell wall
types. Bacterial species with walls containing small amounts
of peptidoglycan and, characteristically, lipopolysaccharide,
are Gram-negative whereas bacteria with walls containing
relatively large amounts of peptidoglycan and no
lipopolysaccharide are Gram-positive.
Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram\u2019s stain appears to be unconnected with the wall
structure itself. The exact mechanism of the staining reaction is
not fully understood, however, this does not detract from its
1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium or from material containing bacteria eg faeces or soil.
2. The bacterial suspension is smeared unto a clean glass slide. If the bacteria have been removed from a culture or a solid media or it is from a soil or feces sample It will have to be mixed with a drop of bacteria-free saline solution
3. The bacterial smear is then dried slowly at first and the, when dry, heated for a few seconds to the point when the glass slide is too hot to handle. This fixes ie kills the bacteria making the slide safe to handle.
4. Once cool, the slide is transferred to a support over a sink and flooded with a stain called Gentian Violet (a dye consisting of a methyl derivative of pararosaniline). The stain is left on the slide for about 1 minute. This stains all the bacteria on the slide a dark purple colour. Note, this stain will not penetrate the waxy cell walls of some bacteria eg mycobacteria.
6.The bacterial smear is then treated with Gram\u2019s solution which consists of 1 part iodine, 2 parts potassium iodide, and 300 parts water. This iodine solution reacts with the Gentian Violet turning it a very dark shade of blue. It also causes it to be retained by certain types of bacteria in a way which is not really understood.
7.After about 30 seconds the slide is gently rinsed with ethyl alcohol which causes the dye-iodine complex to be washed out of some bacteria but not others. This is called decolourisation.
If we now look at the smear down a microscope, the bacteria which had retained the Gentian Violet-iodine complex will appear blue-black.
These are called Gram-positive. However wi would not be able to see those which had lost the dye-iodine complex which are called Gram-negative. The final step in the gram stain method is, therefore, to stain the Gram-negative cells so they can be seen.
8. This is achieved by treating the smear with a compound which stains the Gram- negative cells a colour which contrasts markedly with the blue-black colour of the gram-posiitve cells. The stain common used for this is either eosin or fuchsin, both of which are red. These are called counterstains.
Bacteria in the smear which are Gram-positive are unaffected by the counterstain.
9. The counterstain is left on the smear for about 30-60 seconds and then gently rinsed away with running water.
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