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 Reactions Cell shapes Cell Ends and Arrangement Size Distinctive Characters Predicted Bacteria
Bacteria 1 Gram positive (purple) Cocci Rounded, clusters, singly Small - Staphylococcus aureus
Bacteria 2 Gram negative (pink) Rods (shorter than B. cereus) Rounded, regular, pair, clusters Medium - Escherichia coli
Bacteria 3 Gram positive (purple) Long rods Rounded, irregular, groups, chains Large
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A complex between crystal violet and iodine (CV-I) is formed within the cell. The structure that determines the Gram reaction is the cell wall structure and not that shape. Bacillus cereus and Staphylococcus aureus are stained purple in the Gram staining as they have a high amount of peptidoglycan which forms the outer layer of the cell. This thick peptidoglycan layer is able to trap the purple CV-I complex even after alcohol treatment. Escherichia coli is stained pink in the Gram staining and it is a Gram-negative bacteria. Gram-negative bacterias usually have a thin peptidoglycan layer compared to Gram-positive bacterias. The peptidoglycan layer is located between the plasma membrane and an outer membrane containing lipopolysaccharide and this outer layer is dissolved during the alcohol treatment which results in a loss of the CV-I complex, hence the pink safranin counterstain is trapped by the peptidoglycan layer (ASM, 2004). Gram staining allows us to observe the characteristics and cell size, shape and arrangement. For Bacillus cereus, endospores were also viewed during microscopic observation. During Gram staining, the most crucial step in determining the outcome is the decolourisation by alcohol step. If alcohol is applied too long, the alcohol may wash all the CV-I complex that is trapped in peptidoglycan layer. Therefore, alcohol is only applied for 30 seconds

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