In Gram staining, a purple crystal violet stain is used to stain the cells for 30 seconds then it is washed with water and Gram’s iodine is applied for 30 seconds and wash of with water. 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 and then it was washed off with water. The dried smear was passed through the Bunsen flame so that the cells will adhere to the slide so that they can be stained.
The Essay on Gram Positive And Gram Negative Bacteria
Gram Positive and Gram Negative Bacteria It is a well-known fact that bacterial cells, like plant cells, are surrounded by a cell wall. However, few people know that their cell walls are quite different. Bacterial cell walls are made up of polysaccharide chains linked to amino acids. At the same time, plant cell walls are made up of cellulose, which contains no amino acids. In the same way, ...
Fixation also helps preserve the structure of stained cells by inactivating the enzymes that may disrupt the cell morphology and also to kill the cells (Hogg S. , 2005).
It was ensured not to use the whole colony to make the smear as it will affect the Gram stain results. If a whole colony is used to make the smear, the smear would be too thick and may not decolourize adequately with alcohol treatment. Gram-negative bacterias would not be stained by the safranin counterstain (ASM, 2004).
Each step in the Gram stain is crucial to produce accurate results.
If any of the steps is missed, it could seriously affect the Gram stain reactions/results. For a heat fixed smear slides containing B. cereus, E. coli and S. aureus, say if the staining with crystal violet step is missed, the B. cereus and S. aureus will be colourless and the E. coli will be stained red. If the staining with iodine step is omited, the B. cereus and S. aureus will be stained light purple while the E. coli will be stained red. If the decolorisation step is missed, the B. cereus, E. coli and S. aureus will be stained purple.
If the last step of Gram staining which is the fuchsin stain is omited, both the B. cereus and S. aureus will be stained purple while the E. coli will be colourless. Therefore, it is very important to follow the procedure of Gram staining in order to obtain accurate results. In Session 1, Part A, B and C, three different techniques of producing pure cultures were used; the streak plate, the spread plate and the pour plate. In the streak plate method, a relatively large concentration of microorganisms is diluted into a small, scattered population of cells.
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An inoculating loop is used for streaking the agar plates. The inoculating loop must be ensured that it is not badly oxidized or it will fail to dilute the inoculum. Observations on the colonies of the streak plate after incubation shows that at the 4th and 5th inoculum, the individual colonies are at least 2mm apart and could be distinguished easily. In Table 2, it is shown that for the spread plate technique, the colonies were evenly distributed on the plate but there was more overlapping but somehow individual colonies could still be distinguished.
In this spread plate technique, the mixture of the bacteria was diluted and a small aliquot was transferred to an agar plate and then it was incubated at 370C for one day. The suspension was distributed evenly over the surface by a special streaking technique using the bent glass rod. For the pour plate, it was observed that some bacterial colonies were embedded in the agar and it was difficult to distinguish the individual colonies. In all these three techniques, agar was used in preference to gelatin for making the nutrient media is because the agar once solidified will stay solid over a wide range of incubation conditions.
Agar medium also have resistance to degradation by bacteria. Another advantage of using agar as a solid medium is because of its relative clarity which permits easy viewing of growth on or in the medium. Gelatine is not preferred for making solid media as it liquidify at 250C and this prevents the plates from being incubated at higher temperature and gelatine is also hydrolysed by gelitinase enzyme that is produced by most proteolytic organisms (Perry et al. , 2002).
Amongst the three techniques used in producing pure cultures, it was observed that the streak plate technique was more suitable to produce well separated single colonies from a broth culture containing a mixture of organisms. In this technique, the inoculating loop is used to dilute the mixed culture around the agar plate. The loop is sterilized by flaming it in the Bunsen flame between each set of streaks so that the amount of mixed culture in set of streaks is progressively lessened, hence well isolated colonies were formed in the 4th and 5th inoculum.
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Resistance Is Futile: The Dilemma of Antibiotic-Resistance Has man pinned the arm behind the back of Mother Nature Have humans finally won this terrible pathogenic onslaught Apparently not the Era of Epidemics has risen once again, and it looks as if humans are being bumped off the top of the food chain. Bacteria are mutating into antibiotic-resistant monsters. One of the main reasons why this is ...
Specific colony can be insolated from the mixed culture. It was also observed that well separated colonies appear larger than those in areas of heavy growth. This is due to less competition for nutrient on the agar, thus increasing the growth rate of the bacteria in the colony. Individual colony does not always consist of one type of bacterium could be due to overlapping of colonies. Streaking method used could be wrong. Experimental errors could occur such as the inoculating loop is not properly sterilized during flaming before the 2nd, 3rd, 4th and 5th streaking.
Individual colony does not always originate from a single parent cell could be due to contamination of the environment. In this practical, experimental errors may have occurred such as the streak plate, pour plate and spread plate technique is not done in an aseptic environment. During the process of the streak plate method, the inoculating loop is rusty and pitted, hence the surface of the agar is scratched and bacteria colonies are found grown within the agar.
In Session 2 of the practical, only two types of colony are successfully isolated and the third colony is no where to be found on the streak plate agar. This could be due to incorrect streaking method and the flamed inoculating loop is not properly cool down before the next streaking thus killing the bacteria. Conclusion Gram staining is differential staining method which helps us to observe and compare the morphological cellular features of bacteria, the colonial features of isolated bacterial colonies were also observed and the streak plate technique is the most effective way to produce pure cultures.
