Isolation of Pure Cultures by Dilution Techniques and Gram Staining Method

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Isolation of Pure Cultures by Dilution Techniques and Gram Staining Method


Table 1. Gram stain reaction and cellular features of the culture. Gram staining methods were applied on the given mixture of Bacillus cereus, Escherichia coli and Staphylococcus aureus and then examined microscopically. Results were recorded in Table 1.

Gram ReactionsCell shapesCell Ends and ArrangementSizeDistinctive CharactersPredicted Bacteria Bacteria 1Gram positive (purple)CocciRounded, clusters, singlySmall-Staphylococcus aureus Bacteria 2Gram negative (pink)Rods (shorter than B. cereus)Rounded, regular, pair, clustersMedium-Escherichia coli Bacteria 3Gram positive (purple)Long rodsRounded, irregular, groups, chainsLargeEndospores present; spherical, central, not distending the cellBacillus cereus

Table 2. The observation of the colonies on the nutrient agar plates after incubated Three isolation techniques were used; streak plate, spread plate and pour plate and the agar plates were then inverted and incubated at 370C for one day. The distributions of colonies were then observed. Observations were recorded.

Isolation TechniquesObservation on the nutrient agar plates Streak plateAt the first inoculum, all the bacterial colonies were overlapping with each other and it was difficult to distinguish the colonies. At the 4th and 5th inoculum, it was easier to distinguish the colonies as they grew apart from each other. The appearance of each colony type could be determined at the final inoculum. Spread plateThe colonies were distributed evenly over the surface of the nutrient agar but there were more overlapping of colonies. Bacterial colonies could still be distinguished from each other. Pour plateSome bacterial colonies are embedded in the agar medium and they are much smaller than those found on the surface. Bacterial colonies were difficult to distinguish from each other.

Table 3. The descriptions of the appearance of surface colonies on the nutrient agar after incubated In Session 2, the separated single colonies were observed and identified. The appearances of each colony type were described using suitable terminology and then recorded in a table.

ShapeCircular Circular Irregular
SurfaceSmooth; GlisteningSmooth; DullFinely to coarsely roughened; Dull OpacityOpaqueTranslucentTranslucent
ElevationConvex (dome-shaped)Convex (low)Flat
Predicted bacteriaStaphylococcus aureusEscherichia coliBacillus cereus

Table 4. Cellular and cultural features of Escherichia coli, Staphylcoccus aureus and Bacillus cereus Each colonies were isolated and cultured on the nutrient agar plates using the streak plate techniques and then, they were incubated at 370C for one day. Then, Gram stains from the three colonies were prepared and the morphology was observed microscopically. The appearances of the colonies were also observed and recorded in Table 4.

Cellular morphologyGram reactionsNegativePositive
Cell shapesRods (shorter than B. cereus)Long and thin rods
Cell arrangementRegular, pairs, groups, chainsIrregular, groups, chains
Other characteristics-Endospores present; spherical, central, not distending the cell Cultural morphologyShapeCircularIrregular
Size1.3 mm1.8mm
SurfaceSmooth; DullFinely to coarsely roughened; Dull
Predicted bacteriaEscherichia coliBacillus cereus


Gram staining is a technique used to differentiate bacterial species into two groups. The Gram staining technique was developed by a Danish doctor, Hans Christian Gram in...
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