1. Place a slide on the centre supports of the staining rack, and flood the smear with a few drops of the methylene blue stain, and allow to act for 1-3 min.

2. Wash the smear with water (either from a wash bottle or a slow running tap) to remove dye.

3. Dry the slide using absorbent paper pressed lightly over the surface.

4. Examine the stained preparation under the x40 objective.

5. Sketch a picture of the microorganism.

6. Sign the picture and specify Total Magnification (tm).

Notice: No coverslip is required with stained preparations, but take due care when using high magnification that the objective lens does not touch the smear.

Gram Stain

• The Gram stain divides bacteria into two broad classes: those that test positive on the Gram stain, and those that don't. This distinction reflects a structural difference. Gram-positive bacteria have a thick cell wall that is rich in peptidoglycans, while Gram-negative bacteria have a thin layer of peptidoglycans sandwiched between an inner and outer membrane. The first step in Gram staining is to apply crystal violet, followed by Gram iodine; the bacteria soak up the crystal violet dye, which aggregates with the iodide ions. Next, a brief treatment with acetone removes the crystal violet from Gram-negative cells; it does not affect the stain in Gram-positive cells, because their peptidoglycan cell wall is much thicker. Finally, an application of safranin counterstain turns Gram-negative bacteria pink. The Gram-positive bacteria, by contrast, retain the crystal violet, and hence appear purple under the microscope.

4)What is the reason of using Gram staining? Describe this method of staining.

Microorganisms found in pharmaceutical and healthcare environments require identification in order to determine the species. This is important so that the origin of contamination can be assessed and the origin of contamination determined. This is commonly performed by using a standing technique called the Gram stain, which is based is a type of "phenotypic identification method" and it undertaken so that the microbiologist can understand the general profile for microorganisms.

The first step of most identification schemes is to describe the colony and cellular morphology of the microorganism. Colony morphology is normally described by directly observing growth on agar, where the colony will appear as a particular shape (such as raised, crenated, spherical and so on) and the colony will have a particular pigment. Some microbiologists will attempt to identify the microorganism based on such visual identification. This is not normally encouraged as considerable experience is required to do this and the variety of microflora cannot be characterised with any degree of accuracy. Furthermore, the characteristics of a microorganism are often dependent upon the type of culture medium used. Nevertheless, a description of the morphology can assist with further stages of identification.

Cellular staining provides important information relating to the composition of the microbial cell wall, as well as the shape of the organism. Of these, the most frequently used method is the Gram stain.

The Gram stain method employed includes the four-step technique: Crystal violet (primary stain); iodine (we used Lugol) (mordant); alcohol (decolorizer); and safranin (we used Fuksin) (counter stain). Done correctly, Gram-positive organisms retain the crystal violet stain and appear blue; Gram negative organisms lose the crystal violet stain and contain only the counter-stain safranin and thus appear red. Common pitfalls in this method are that heat fixation may cause Gram-positive cells to stain Gram-negative and older cultures may give Gram-variable reaction; using too much decolorizer could result in a false Gram-negative result and not using enough decolorizer may yield a false Gram-positive result.

The Gram reaction is based on the differences in the cell wall composition for the two cellular 'groups'. The bacteria that retained the stain (the Gram-positive bacteria) have a higher peptidoglycan and lower lipid content than those that do not retain the stain (the Gram-negative bacteria). The effect of the solvent is to dissolve the lipid layer in the cell wall of the Gram-negative bacteria, thereby causing the crystal violet to leach out; whereas for Gram-positive bacteria the solvent dehydrates the thicker cell walls, blocking any diffusion of the violet-iodine complex, which closes the pores of the cell and retains the stain. There are now several automated Gram stain devices available on the market that can reduce the labour requirement required when performing several multiple Gram stains and, possibly, improve accuracy.

In addition to the difference based on cell wall, microscopic examination of the stains allows the cellular shape to be determined. Bacteria commonly fall into the categories of coccus (spherical), rod, vibrio (curved), spirilla (spiral) and plemomorphic (variable).

Gram Staining.

In 1884 Hans Christian Gram, a Danish physician, developed the Gram stain. Gram-stain is a method for the differential staining of bacteria. Gram-positive microorganisms stain purple. Gram-negative microorganisms stain pink. Staphy-lococcus aureus, a common bacterium that causes food poisoning, is gram-positive. Escherichia coli is gram-negative.

Methodical instructions

In one glass of skim make strokes of different microorganisms in the center - a smear of cells studied culture, left and right - the control of microorganisms. The cells of one of the test organisms (eg, Staphylococcus aureus) Gram stain, and the cells of another (for example, Escherichia coli) - not colored. Smears must be thin so that the cells are uniformly distributed over the surface of the glass and did not form clusters, since the thickness of the stroke depends on the results of staining. Smears air-dried and fixed over the burner flame.

Gram Stain Procedure (variant A)

1. Prepare the specimen using the heat fixation process.

2. Place a drop of crystal violet stain on the specimen for 1-2 min. Poured into the dye, not washing the smear with water.

3. Apply Lugol on the specimen using an eyedropper for 1-2 minutes. The Lugol helps the crystal violet stain adhere to the specimen. It is a mordant, which is a chemical that fixes the stain to the specimen.

4. Wash the specimen with an ethanol during 0.5-1 min.

5. Wash the specimen with water to remove the dye.

6. Apply the fuksin stain to the specimen using an eyedropper.

7. Wash the specimen.

8. Use a paper towel and blot the specimen until the specimen is dry.

The specimen is ready to be viewed under the microscope. Gram-positive bacteria appear blue-violet and gram-negative bacteria appear pink.

When stained by Gram, the following errors:

a) All cells are blue due to lack of bleaching;

b) All cells are pale pink, gentian violet staining due to insufficient or excessive treatment with alcohol.

For comparison, you can use an accelerated test for determining membership gram-positive bacteria or gram-negative species.