2 LOOP Smear Gram Stain

8/13/2019 2 LOOP Smear Gram Stain

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1.Sterilizing a bacteriological loop

2.Preparation of Smear for Staining3.Gram staining

Sayeed Ismail Khatib

LecturerMicrobiology & Immunology

Ibn Sina National College for Medical Studies

Jeddah.


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Learning this technique is essential in a microbiology lab.

Flame sterilization is a very quick simple method ofkilling microorganisms on an inoculating loop or needle.

The loop or needle is held inside a flame for a fewseconds to bring it to redness and then cooled.

Once cool, the loop or needle can be used for various

culture manipulations.

Also, be patient and let the loop cool down, this usuallytakes about 15-30 seconds.

Sterilizing a bacteriological loop


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Sterilizing a bacteriological loop

Nichrome Loop

Flame

Burner

Heat the Loop in the center of the flame till it turns Red Hot


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Smear preparation:

The preparation of a smear is required for many

laboratory procedures, including the Gram-stain.

The purpose of making a smear:

to fix the bacteria onto the slide

to prevent the sample from being lost during a staining

procedure.

A smear can be prepared from a solid or broth

medium.


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Procedure:

Place one needle of solid bacterialgrowth or two loops of liquid bacterial

growth in the center of a clean slide.


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2. If working from a solid medium, add one

drop of water to your specimen with awater bottle. If using a broth medium, do

not add the water.


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4. Place the slide on a slide warmer or pass

it over the flame a couple of times andwait for it to air dry. The smear is now

ready for the staining procedure.


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Gram staining (differential staining)

Developed by Dr. Hans Christian Gramin 1884.

It is the most important and widely used differential

staining technique.

The bacterial smear is subjected to four different

reagents - Crystal violet (primary stain), iodinesolution (mordant), alcohol (decolorizing agent) and

safranin (counter stain).

The bacteria which retainthe primary stain

(appear dark blue or violet) are called gram positive

Those which are counter stained by safranin (red)

are referred to as gram negative.


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Principle: The differences in staining are related to cell wall

composition. Gram negative bacterial cell wall is complex,

multilayered with relative high lipid content.

Lipid is readily dissolvable by alcohol, forming

large pores in the cell wall resulting in the easydecolorization of crystal violet - iodine (CV-1)complex.

Later they take up the counter stain and appear

red. In contrast, the Gram positive cell walls are thick

with less of lipids, so will not loose CV-1 andcells remain blue.


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Gram-staining Procedure:

Gram-staining is a four part procedure to make abacterial cell stand out against its background.

The specimen should be mounted and fixed on a

slide before you proceed to stain it.

The reagents you will need to successfully perform

this operation are:

1. Crystal Violet (the Primary Stain)2. Grams Iodine Solution (the Mordant)

3. Decolorizer (ethanol is a good choice)

4. Safranin (the Counterstain)


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STEP 1:Place your slide on a slide holder or a

rack. Flood (cover completely) the entire slide

with crystal violet. Let the crystal violet stand forabout 60 seconds. When the time has elapsed,

wash your slide for 5 seconds with water. The

specimen should appear blue-violet when

observed with the naked eye.


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STEP 2:Now, flood your slide with the

iodine solution. Let it stand about a minute

as well. Rinse the slide with water for 5

seconds. At this point, the specimen

should still be blue-violet.


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STEP 3: This step involves addition of the

decolorizer, ethanol. add the ethanol drop-

wise until the blue-violet color is no longer

emitted from your specimen. As in the

previous steps, rinse with the water for 5

seconds.


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After you have completed steps 1 through

4, you should blot the slide gently with filter

paper or allow it to air dry before viewing itunder the microscope.

[

DO NOT RUB THE SMEAR. Observe under Oil-immersion Microscope.

Gram-ve

Bacilli (Pink)Gram+ve

Cocci (Violet)

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2 LOOP Smear Gram Stain