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

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). Microbial staining

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.

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 2- 3 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.

Negatively Stained Bacillus: (A) Vegetative Cell (B) Endospore Negatively Stained Cocci Negative staining

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