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Isolation of Gram Positive and Gram Negative Bacteria


Gram staining is a highly crucial step in the process of identifying unknown strains of bacteria. It helps to differentiate between gram-positive and gram-negative bacteria, as well as gives pertinent information on the size and shape of the bacterial cells. The next step in the identification criteria is based on the information obtained from gram-staining. Some gram-positive bacterial species usually appear gram-negative after they have aged. This is evident in the genus Bacillus. There are gram-negative bacterial strains that have also shown significant resistance to discoloration by ethanol thus appearing as gram-positive upon gram staining. This has been found in the Acinetobacter and Moraxella genera. This variant bacteria which can not be distinctively classified through gram staining forms the Gram variable and Gram indeterminate groups.

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Gram-positive bacteria have a thick peptidoglycan layer that absorbs the primary stain crystal violet and resists decolorization with acetone or alcohol. This makes the gram-positive bacteria stain purple. On the other hand, gram-negative bacteria have an outer lipopolysaccharide membrane which is lost upon decolorization exposing the inner thin peptidoglycan layer. Therefore, the gram-negative bacteria take up the counterstain, safranin staining pink.

Identification process

Gram staining the liquid culture was the first step in the identification process. This was done to prove that there was the presence of gram-negative and gram-positive bacteria in the culture. A thin smear of the culture was prepared on a clean glass slide and then fixed with heat after air drying. The next step was to stain the slides, whereby the fixed smear was flooded with the primary stain, crystal violet, and allowed to stain for 30 seconds. The slide was rinsed with running tap water, avoiding excessive rinsing, which could wash the crystal violet from gram-positive cells. The slide was then flooded with iodine solution, a mordant, and allowed to stand for 1minute. Iodine solution was rinsed followed by decolorization with acetone. Excess decolorizer is removed by rinsing with tap water and then counterstaining was done by flooding the slide with safranin and allowed to satin for 30seconds. Excess counterstain was removed by rinsing in tap water. Examination under the microscope was done after air-drying the slide.

Gram-positive cocci and gram-negative bacilli were observed under the microscope. The gram-positive cocci were arranged in pairs and clusters.

The catalase test was the next appropriate test in the identification of the gram-positive bacteria. This was to differentiate between Staphylococci and Streptococci which are both gram-positive cocci arranged in pairs. The catalase test was positive with the production of bubbles. Coagulase test was the next differential test in the identification process which is done to differentiate Staphylococcus aureus from other Staphylococcus species. Enzyme coagulase is found in S.aureus which makes a plasma clot. The coagulase test was positive.

To confirm that the unknown gram-positive bacteria is S.aureus, Mannitol salt plate test, a selective media, for the isolation of Staphylococci species was essential. Staph. species being osmotolerant can grow at a high salt concentration. The addition of mannitol sugar allows differentiating between S.aureus from coagulase-negative Staph species. S.aureus ferments mannitol causing a change in pH of the agar and subsequently a change of agar color to yellow. The mannitol salt plate produced a yellow color. This confirmed that the unknown gram-positive bacteria are S.aureus.

Culturing the gram-negative bacteria on McConkey agar was done to distinguish lactose fermenters from non-lactose fermenters. The cells grew with a yellow color indicating non-lactose fermenters. SIM agar was the next test performed. The SIM agar is a combination of hydrogen sulfide production, indole, and motility tests. The indole test is used to detect bacteria that have the capability of breaking down tryptophan. Kovac’s reagent is used in this test for the detection of the presence of indole. Hydrogen gas production occurs as a result of the anaerobic breakdown of sulfur to hydrogen sulfide gas. A black precipitate indicates a positive test. A motility test is a test of the organism’s mobility. If the organism is mobile, it will move from the stab region and spread all over the semi-solid agar. SIM agar test showed that the organism was motile, produced hydrogen sulfide gas, and was indole negative. This was positive isolation of Salmonella enteritidis.

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Biochemical tests are extremely valuable in making a positive diagnosis of disease-causing organisms in clinical samples. Gram-staining allows rapid differentiation of gram-positive and gram-negative bacteria. This helps the technician to decide on the most appropriate test to carry out to accurately the unknown bacteria.


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  2. Ralph, Feigin. Paediatric Infectious Diseases. New York: Garland science, 2004.
  3. Ochei, John. Medical Laboratory Science: Theory and Practice. London:Longman, 2000.

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