Download General Microbiology

Microbial staining
Lab 6
Abeer Saati
Microbial staining
Since bacterial organisms are so minute, it is impossible to
view the organisms without compound microscope. In
order to imagine the cellular components and to
differentiate bacteria from other microbial agents, staining
techniques are used by scientists to categorize different
bacteria.
There are two types of stain:1- Simple staining
2- Differential staining
Microbial staining
1-Simple staining: using one stain only such as
methylene blue, carbol fuchsin or crystal violet to
determine the size, shape and arrangement of bacterial
cell.
2-Differential staining: (use more than one chemical
stain). Using multiple stains can better differentiate
between different microorganisms or structures/cellular
components of a single organism (Gram stain- flagella
stain, capsular stain, spore stain, nuclear stain).
Negative staining
Negative staining: Negative stain does not stain bacteria
but colors the background instead.
background = dark
bacteria = clear
What is the purpose of negative staining?
It can be used for the identification of certain bacteria or
structures (bacterial capsules), spores and difficult to
stain bacteria.
Simple staining
1- Preparation of smears:
o Put a drop of water on the middle of the slide using the
inoculating needle.
o With the sterile needle, collect bacteria from agar surface
by touching the bacterial growth.
o Rub the tip of the needle on the glass slide in the drop of
water in a circular motion till you get homogenous
smear.
o Allow the smear to dry.
Simple staining
2- Fixation of smears:
o Pass the slide with the smear side uppermost over the flame for 23 times.
o Do not overheat the smear. The slide should only be warm when
you touch it with hands.
Passage of the smear through heat has two
benefits:
Fixation of the smear to the slide.
Killing of the bacteria in the smear so it becomes non infectious.
Simple staining
3- Staining of smears:
o Cover the smear with methylene blue or carbol fuchsin or crystal
violet for 1 minute.
o Wash with water.
o Place the film at angle to air dry or with filter paper.
4- Examination of smears
o Place a drop of immersion oil on the smear.
o Use the oil lens.
o Lower the oil lens till the lens contacts the oil and almost touches
the smear.
Negative staining
 Put one or two drops of nigrosin on another slide. Use your sterilized
loop to pick up a loop-full of nigrosin. Carefully mix it in with the
drop of cells, without spreading the drop too much.
 Hold the right end of the slide in your right-hand; with your left–
hand take another slide at a 45 or less angle to the first slide
 Scoot the angled slide back along the surface of the first slide
 Set the stained slide aside to air dry before observing it under oil
immersion. Be sure to start examining your slide in the area with the
faintest gray background.
Negative staining
Negatively Stained Bacillus: (A) Vegetative
Cell (B) Endospore
Negatively Stained Cocci
Thank You