Isolation of bacteria

Gram staining
 Gram staining or the Gram method is a method to distinguish bacterial
species into two large groups, namely gram-positive and gram-negative,
based on the chemical and physical properties of their cell walls
• In gram coloring, four reagents are needed, namely:
• Main color substance (violet crystal)
• Mordan (Iodine solution) is a compound used to intensify the main color.
• Dyestuff / alcohol cleanser (alcohol / acetone) which is an organic
solvent used to dissolve the main coloring agent.
• The second dye / cover paint (safranin) is used to re-dye cells that have
lost the main paint after alcohol treatment.
• Spore Coloring
Bacterial spores (endospores) cannot be stained with ordinary
coloring, special coloring techniques are needed. Klein's staining is
the most widely used spore coloring.
• Endospores are difficult to color with the Gram method. For
endspores staining, it is necessary to warm up so that green
malachite paint can enter the spores, as well as in Basil Tahan Acid
staining where carbol fuschsin paint must be heated to penetrate
the waxy layer of Mycolic acid from Mycobacterium.
• Flagellar staining
Coloring of the flagellum by giving a colloidal suspense of unstable
tanic acid salts, resulting in thick precipitates on the cell wall and
flagellum.
• Capsule coloring
This staining uses a hot crystal violet solution, then a copper sulfate
solution as a rinse produces a pale blue color on the capsule,
because if rinsing with water can dissolve the capsule. Copper salt
also gives color to the background. The dark blue color.
 Negative coloring

• Learning to use negative staining

procedures to observe the
morphology of organisms that are
difficult to color by simple dyes.
Bacteria are not colored, but color
the background. Intended for hard-
to-color bacteria, such as spirochaeta
• Negative staining requires acid dyes
such as eosin or negrosine. Acid dyes
have a negative charge of
chromogen, will not penetrate or
penetrate into cells due to negative
charge on the surface of bacteria.
therefore, colorless cells are easily
seen with a colored background