STAINING OF SPECIMENS

 Living microorganisms can be directly examined with the light microscope, but they need fixing and staining to increase visibility, highlight specific morphological features, and preserve them for future study.

The first step in most bacterial staining procedures is the preparation of a smear.

Smear Preparation

Smear preparation is a process in which bacterial culture is spread uniformly or as a thin layer on glass slide.

Bacterial Smear Preparation

The crucial step in staining is the preparation of a good smear. A good smear is important to get a good staining result.

Requirements:

• Culture of Bacteria

• Glass slides

• Bunsen burner

• Inoculating loop

• Glassware marking pencil/ Permanent Marker

Step I: Preparation of the glass slide:

• Clean, grease free slides are needed for smear preparation.

• Grease or oil from the fingers on slides must be removed by washing the slides with soap and water

• Finally rinse the slide with 95% alcohol and dry it.

 • Hold the slide by their edge.

Step II: Labeling of slides:

• Proper labelling of the slide is essential- every slide should be labelled clearly.

• A lead pencil /permanent CD marker is used to write on the one end of the glass slide.

 Step III: Preparation of smear:

• An evenly spread smear should be prepared covering area of 15-20mm diameter.

• Avoid thick and dense smear because thick smear prevent light penetration to visualize the morphology of cell.

• A good smear is one that, when dried, appears as a thin whitish layer or film. The print of textbook should be legible through the smear.

Different techniques are used for smear preparation depending upon culture media

i. Broth cultures (liquid medium)

• Re-Suspend the culture by tapping the tube with your finger.

• Depending on the size of the loop, one or two loopfuls should be applied to the center of the slide with a sterile inoculating loop and spread evenly over an area.

• Allow the smear to air-dry

ii. Culture plates (Solid medium)

• Suspension is accomplished by spreading the cells in a circular motion in the drop of water with the loop. This helps to avoid cell clumping.

• The finished smear should occupy an area about the size of a nickel and should appear as a translucent, or semitransparent, whitish film

• Place a drop of water into the circle that has been created on the slide.

• Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony from the culture palte.

• Gently mix the bacteria into the water drop.

Step IV: Air dry

• Smear should be allowed to dry completely at room temperature at safe place

  Step V: Fixation of smear:

• The purpose of fixation of smear is to preserve and prevent smear being washed away during staining.

• Heat fixation - After smear is air dried completely, rapidly pass the 3-4 times through flame of Bunsen burner or sprit lamp.  Avoid too much heating.

• After heat fix, allow the smear to cool before staining.

 

Fixation

The stained cells seen in a microscope should resemble living cells as closely as possible.

Fixation is the process by which the internal and external structures of cells and microorganisms are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microorganism usually is killed and attached firmly to the microscope slide during fixation.

There are two fundamentally different types of fixation.

Heat fixation is routinely used to observe procaryotes. Typically, a film of cells (a smear) is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology but not structures within cells.

Chemical fixation is used to protect fine cellular substructure and the morphology of  delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, making them inactive, insoluble, and immobile. Common fixative mixtures contain components such as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.