KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY

TITLE: PREPARATION AND EXAMINATION OF STAINED SMEARS

NAME: MONICA KAFUI ADOSSI INDEX NUMBER: 8850223

STUDENT ID: 21034654 COURSE CODE: Bio 251

Introduction
Gram staining is one of the most significant staining techniques in microbiology. It was named

after Danish bacteriologist Hans Christian Gram, who initially developed it in 1882, mainly to

identify organisms of pneumonia. Often the first test performed, gram staining uses crystal violet

or methylene blue as the primary color. The name of the organisms that retain the primary color

and appear purple-brown under a microscope is Gram-positive organisms. The organisms that do

not take up the primary stain appear red under a microscope and are Gram-negative. The first

step for gram staining is the use of crystal violet dye for the primary staining of the slide. The

second process, or the fixing of the dye, is the addition of iodine to form a crystal violet-iodine

complex to render dye removal troublesome. This is followed by the addition of a decolorizer,

often a solvent comprising ethanol and acetone, that extracts the dye. The overall principle of

gram staining relies on the retention ability of the bacterial cell wall for the crystal violet dye in

the face of solvent treatment. Gram-positive organisms have more peptidoglycan and gram-

negative organisms have more lipid. All bacteria stain initially with crystal violet dye, but when

treated with solvent, the lipid layer of gram-negative organisms is dissolved. When the lipid

layer is removed, gram negatives lose the primary stain. On the other hand, solvent dehydrates

gram-positive cell walls with the closure of pores inhibiting the diffusion of violet-iodine

complex, and hence, bacteria are not decolorized. The timing of decolorization is a critical step

in gram staining since longer exposure to a decolorizing agent will deprive both types of bacteria

of all stains. The final step in gram staining is staining with a basic fuchsin stain to leave a pink

stain on decolorized gram-negative bacteria for their easy identification. It is also known as

counterstain. We have used safranin as a counterstain here, but basic fuchsin stains gram-

negative organisms more strongly than safranin. Similarly, Hemophilus spp., Legionella app, and

some anaerobic bacteria also weakly stain with safranin. (Tripathi, Sapra., 2020). Most
bacteriological stains are done on grease-free slides. Slides are washed in chromic acid, rinsed

with water, and stored in a jar of alcohol. They need to be removed using forceps (replacing the

lid as soon as it is removed), drained, and flamed to incinerate the alcohol.

Materials

Grease-free slides, sterile/distilled water, sterile loop, Bunsen burner, gram stain(0.5% crystal

violet, Iodine, absolute alcohol, safranin), filter paper.

Methodology

Two agar plates, A and B, were provided, and the slides were also labeled as A and B. A very

small amount of sterile water was placed on each slide, and a colony of bacteria from the

corresponding agar plate was transferred into the drop using a sterile loop. The bacteria were

spread evenly in the water to make a smear.

Slides were permitted to air dry thoroughly before they were fixed by running them over the

flame of a Bunsen burner two or three times. Fixing was followed by smearing the smears with

0.5% crystal violet stain for a period of two minutes. Rinsing with water was then performed,

followed by the application of dilute iodine for two minutes. The iodine reacted with the crystal

violet to produce a purple-black complex within the bacterial cells.

To decolorize the smears, absolute alcohol was added in small increments and allowed to flow

over the surface until no further primary stain was released. The process was repeated three

times, then a final time with a water rinse. The slides were then counterstained for about one

minute with safranin, then gently washed with water again. The water was blotted off with filter

paper. Finally, the stained smears were examined under the microscope.
Results

BACTERIUM GRAM SHAPE POSSIBLE NAME

Discussion

The Gram stain result arises due to differences in cell wall structure. Gram-positive bacteria

(such as bacterium B) have thick layers of peptidoglycan in their walls while gram-negative

bacteria (such as bacterium A) have trace amounts. Gram-positive bacteria have teichoic cells

while Gram-negative bacteria lack them. If the bacteria are treated with the primary stain (crystal

violet) and fixed using the mordant (iodine), then the Gram-positive bacteria can hold onto the

primary stain, and the Gram-negative bacteria are decolorized using alcohol. Decolorization of

the cell dehydrates and condenses the peptidoglycan layer, closing the pores in the cell wall and

therefore preventing the leakage of the primary stain from the cell. This stains Gram-positive

bacteria purple. The alcohol, when applied to Gram-negative bacteria, breaks down the thick

outer layer of the cells and since it is composed of lipids, the crystal violet-iodine complex is

allowed to leach out of the cells. On retaining with safranin, they take up the stain which stains

them pink or red.

Conclusion

Gram stain is the most common staining technique used diagnostically both in the clinical setting
and in research laboratories to differentiate between Gram-positive and Gram-negative
microorganisms in all tissues. Gram-positive bacteria are colored purple and gram-negative
bacteria are colored red or pink. An important advantage of the modified Gram stain is that it can
demonstrate collagen, thereby being applicable to any tissues with collagen.

Errors and precautions

1. Stopwatches must be made available and time must be taken accurately while staining so as
not to over-stain.

2. Avoid allowing the filter paper to contact the surface of the slide during blotting in an attempt
not to contaminate the slide prior to viewing.

References

 Gram, H.C., 1884. Ueber die isolirte Färbung der Schizomyceten in Schnitt- und
Trockenpräparaten. Fortschritte der Medizin, 2, pp.185-189.
 Beveridge, T.J., 2001. Use of Gram Stain in Microbiology. Microbiology and Molecular
Biology Reviews, 65(4), pp. 593-620.
 Madigan, M.T., Bender, K.S., Buckley, D.H., Sattley, W.M. and Stahl, D.A., 2021. Brock
Biology of Microorganisms. 16th ed. New York: Pearson.
 Bartholomew, J.W., 1962. Variables Influencing Results, and the Precise Definition of
Steps in Gram Staining as a Basis for a Standardized Staining Procedure. Bacteriological
Reviews, 26(1), pp. 251-261.
 Prescott, L.M., Harley, J.P. and Klein, D.A., 2019. Microbiology. 11th ed. New York:
McGraw-Hill Education.
 Cappuccino, J.G. and Welsh, C.T., 2017. Microbiology: A Laboratory Manual. 11th ed.
Boston: Pearson.
 Murray, P.R., Rosenthal, K.S. and Pfaller, M.A., 2021. Medical Microbiology. 9th ed.
Philadelphia: Elsevier.