Title: Isolating and identifying different bacteria by using different media.
Introduction:
In this world, there are different type of microorganism which cannot be seen through naked eyes. Bacteria is a type of microorganism that is classified under prokaryotes. It has simple internal structures with no membrane bounded organelles, unlike the eukaryotes. Based on the textbook named ‘A Textbook of Microbiology’, it stated that there are approximately 4760 known species of bacteria and each bacteria have its own particular role in this world. Other than that, each species of bacterial have their own characteristic such as shape, presence of flagella, cell-wall and others, therefore different types of bacteria will have different preference of habitat. In order to identify and isolate each species, researchers have to use different media and techniques.
To isolate pure culture, researchers can use few methods which include streak plate, pour plate and spread plate. Among of all plating, the most common and simplest method used is the streaking-plate technique, which is getting a loopful of sample from a colony and grow onto a suitable agar plate. (1) Next is the aseptic techniques, which is required before carrying out any other techniques. It is important because it helps to prevent contamination of the agar plate and environment so that pure culture can be obtained (5). Aseptic technique can be carried out by flaming the wire loop befor̊e using and make sure the agar plate is kept near to the flame while streaking.
To identify each bacteria species, streak-plating technique helps to show the shape and colour of the bacteria colonies on the agar plate. Apart from that, microscope can be used to examine the bacteria morphology, such as shape, arrangement and the colour of gram stained. Before examine the bacteria through microscope, gram staining techniques is required to differentiate gram positive and gram negative bacteria. During the gram staining process, few solutions were added in order from crystal violet, iodine solution, alcohol to carbol fuchsin. Crystal violet solution will first be dissociated into CV+ and Cl-, then these ions will enter the bacteria cell wall and cell membrane. CV+ will then react with the negative side of the cell wall and cell membrane. Then when Iodine was added, it forms a complex with the CV+ ions and helps to strengthen the attachment to the cell wall and cell membrane. Next is the addition of alcohol which serve as a decolorizer, which the alcohol will react with the lipids of gram negative bacteria cell wall, therefore during this process the CV-I complex will be washed away. Since gram positive bacteria cells were less permeable than gram negative bacteria cell due to the thick peptidoglycan layer, the stain of gram positive bacteria will remain as purple colour (6). Lastly, is the carbol fuchsin which helps to stain the gram negative bacteria. Gram positive bacteria will have purple stain in colour while the gram negative bacteria will have pink stain (2). The difference between gram positive and gram negative bacteria is on the bacteria cell wall. Gram positive bacteria will have thicker peptidoglycan layer whereas the gram negative bacteria have thinner peptidoglycan layer and with a lipopolysaccharide layer.
Apart from all techniques, types of media used will affect the culture growth. Different type of agar plates can be used to grow different type of bacteria, based on the bacteria properties. Each type of agar plate contains different constituents, such as different nutrient base and different preferred incubation time and temperature. In this experiment, two types of agar plate will be used, which are nutrient agar plate and MacConkey agar plate. Nutrient agar contain peptone, beef or yeast extract that helps in bacteria growth, solidifying agent, sodium chloride which helps to maintain salt concentration of the medium and some distilled water. In the other hand, MacConkey contain peptone as source of nutrient for the bacteria, bile salt and crystal violet to inhibit growing of some bacteria (7), lactose which provide carbohydrate to the bacteria and some pH indicator.
Aim:
To determine types of bacteria from a given mixed bacterial culture by isolating and identifying each bacterial through using different types of medium.
Materials & Methods:
As per MIC2011-INTRODUCTION TO MICROBIOLOGY AND MICROBIAL BIOTECHNOLOGY Practical Class Notes 2018, page 59 to 63.
Gram staining method, please refer to MICROBIOLOGY TECHNIQUES MANUAL 2018/2019, page 28, ii) gram stained-modified.
Streak-plate technique, please refer to MIC2011-INTRODUCTION TO MICROBIOLOGY AND MICROBIAL BIOTECHNOLOGY Practical Class Notes 2018, page 30 to 31.
Results:
At the first lab session, both nutrient agar and MacConkey agar plates were stored at 4 ̊C until the day before lab, then incubated at 37̊C for less than 24 hours after conducting streak-plate techniques of the mix broth culture. After the first lab session, colony morphology of each bacterial which include Bacillus megaterium, Escherichia coli and Providencia rettgeri were observed and recorded. Next, to observe the cellular morphology of each bacterial, first the wire loop must be sterilized through aseptic technique then spread some colonies on the slides. After that, gram staining was performed on each bacterial on each slides and observed under microscope at x1000 magnification. The cellular morphology was then recorded
Table 1. The table shows the colony morphology and cellular morphology of Bacillus megaterium, Escherichia coli and Providencia rettgeri that was incubated in nutrient agar and MacConkey agar plates at 4 ̊C until the day before lab, then incubated at 37̊C for less than 24 hours.
Organism Colony Morphology Cellular Morphology
Nutrient Agar MacConkey Agar Gram Stain Shape Arrangement
Bacillus megaterium White, concave and smooth colonies No colonies Gram positive rod Chain
Escherichia coli Smooth, small and translucent colonies Red colonies Gram negative rod Random
Providencia rettgeri Glossy and white colonies Beige colonies Gram negative rod Random
The table above shows the Bacillus megaterium only appear on nutrient agar plate with white, concave and smooth colonies but not on MacConkey agar plate. As for the cellular morphology, it has rod shape and are arranged in chain. Next for Escherichia coli, translucent, smooth and small colonies appear on nutrient agar and red colonies appear on MacCokey agar plate. The cellular morphology for Escherichia coli is rod shape and randomly arranged. Lastly, for the Providencia rettgeri, glossy and white colonies appear on the nutrient plate and beige colonies appear on MacCokey agar plate. After observing it under microscope, it shows the cellular morphology of Providencia rettgeri is rod shape too with random arrangement. As for gram staining, both Escherichia coli and Providencia rettgeri are gram negative bacteria and Bacillus megaterium is gram positive.
Discussion:
In this experiment, two types of agar plates were used, which are nutrient agar and MacConkey agar plate. Both media is made from similar ingredients, such as peptone, meat and yeast extract, sodium chloride, distilled water and solidifying agent. Peptones are a source of amino acid for the bacterial, as it is the final products from source of protein, such as gelatin, meat and soy food substance which undergo proteolytic digestion through hydrolysis. In order to grow and maintain protein synthesis, bacteria require amino acid from peptones. Other than that, beef extract and yeast extract help in bacteria growth too as they provide nutrient such as vitamin B which can act as coenzyme or being a part of a functional group of some enzymes, nucleotide, amino acid, nitrogen and carbon compound to the bacteria. Next, the sodium chloride helps to maintain salt concentration or osmotic balance of the medium. Apart from that, water which take up more proportion of the agar plays an important role in bacteria growth, reproduction and transportation of nutrient between colonies. Lastly is the solidifying agent, which is the agar that is use to grow bacteria. Agar is use instead of gelatin because agar has higher melting point than gelatin to avoid melting of media, this is suitable for the incubation of bacteria.
In the other hand, there are some differences between both agar plates that affects the growth of bacteria. MacConkey agar is made from more ingredients compare with nutrient agar and the additional ingredients are bile salt, crystal violet, lactose and neutral red, which is a pH indicator. The function of bile salt is for the growth of selective bacteria, such as the enteric bacteria. Enteric bacteria are a group of gram-negative rods bacteria that live in human or animal intestinal tract (3). Crystal violet is the next ingredient that helps to inhibit growth of non-enteric bacteria. Next is lactose, which is a source of carbon for the bacteria to undergo fermentation but only bacteria with enzyme named β-galactosidase can undergo this fermentation process. Lastly, the function for the pH indicator is to differentiate the colour of colonies of bacteria that can ferment lactose and bacteria that cannot ferment lactose.
Based on the table 1 above, it shows that Bacillus megaterium only appear on MacConkey agar plate. There are few reasons that contribute to this result, first is because Bacillus megaterium is a non-enteric bacterium as the gram strain result shows that it is a gram positive rod bacteria. Bacillus megaterium is a large bacterium that contain thick cell wall which made up of peptidoglycan layer and without any lipopolysaccharide layer, therefore the stain will remain in the cell wall without being flush away by the alcohol during gram staining process. The second reason is that the crystal violet used as the ingredient for MacConkey agar will penetrate into the cell of Bacillus megaterium and the stain is toxic for the bacteria, therefore this inhibits the growth of the bacteria.
Next is Escherichia coli and Providencia rettger, both of the bacteria have the similar cell morphology which is gram negative, rod and random arrangement. As for the colony morphology, both bacteria can grow on both nutrient agar and MacConkey agar plate because both of the bacteria are enteric bacteria that can tolerate the bile salt. The only difference between both bacteria is the colour of the colonies for both bacteria is different in MacConkey agar plate. Based on the table 1 result, it shows that colonies of Escherichia coli are red in colour, this is due to the bacteria uses lactose for fermentation to produce carbon dioxide as Escherichia coli respires anaerobically (7 pg65). Escherichia coli is able to ferment lactose because it has β-galactosidase, an enzyme that helps to hydrolyze lactose.The production of carbon dioxide will reduce the pH of the MacConkey agar, therefore pH indicator changed to red which causes the colonies of Escherichia coli to be red in colour. As for Providencia rettger, the colour of the colonies are beige colour because it does not have any β-galactosidase to ferment the lactose, so no carbon dioxide will be produced that affect the pH of the MacConkey agar (4).
During the streak-plating process, nutrient agar was first inoculate before MacConkey agar to prevent the carrying of external source from MacConkey agar to nutrient agar, such as bile salt, lactose, crystal violet and pH indicator. For the incubation process, both nutrient agar and MacConkey agar plate was first incubated at 4 ̊C until the day before lab to inhibit the growth of bacteria. Then the day before lab, all the agar plates were incubated at 37 ̊C no longer than 24 hours because if the incubation occur more than 24 hours, Escherichia coli will use up all the lactose and will target peptone as source of nutrient. When no fermentation occurs, no carbon dioxide will be produced and then pH indicator will not change colour, therefore the colonies of Escherichia coli will be beige in colour. If both the Escherichia coli and Providencia rettger have colonies with the same colour, both bacteria are unable to identify.
Conclusion:
To conclude, different types of bacteria can be isolated and identified through different types of agar plates. All three bacteria, which include Bacillus megaterium, Escherichia coli and Providencia rettgeri were able to grow on nutrient agar due to the nutrient provided for growth and no inhibitor such as bile salt and crystal violet present that affect non-enteric bacteria growth. As for MacConkey agar plate, only Escherichia coli and Providencia rettgeri were able to grow because both of the bacteria are enteric bacteria that can tolerate bile salt. To identify Bacillus megaterium, cell morphology was compared, as Bacillus megaterium is the only gram positive bacteria due to the presence of thick peptidoglycan layer, while both Escherichia coli and Providencia rettgeri are gram negative bacteria. To identify Escherichia coli and Providencia rettgeri, the colonies colour for both bacteria were compared. Escherichia coli colonies are red in colour because fermentation of lactose occured which then carbon dioxide was produced that cause the pH indicator to change into red colour, while the colonies of Providencia rettgeri are beige in colour because no lactose fermentation occurred.