Analysis of Bacteria Strains: Phonotypical Tests
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- Kunthavai Jeevananthan
Aims: To analyse and identify 10 different strains of bacteria by conducting 19 different tests phonotypical tests.
Bacteria also known as eubacteria are microorganisms that are invisible to the naked eye but exist in virtually all environments in the world. Bacteria are classified as part of the Monera kingdom which includes archaebacteria and cyanobacteria. Most bacteria are pathogenic or disease causing however not all bacteria are harmful as there are a number of bacteria that can be found in the human body that have positive benefit to their hosts such as help digest food, secrete hormones, chemicals and vitamins required in cell metabolism and even fight off other harmful bacteria. Bacteria exist in various rod, spiral and spherical shapes and are more numerous than any other living organisms. It is important to be able to identify microorganisms in medical clinic in order to help selection of antibodies. Some pharmaceutical products are also made using bacteria therefore many unknown and unidentified bacteria may be useful in the clinical industries. The taxonomy or a particular bacterial characteristic can be used to identify similarities that show relationships with disease related descriptions (Janda and Abbott, 2002). Various laboratory tests have been developed that are based on the type of nutrients a bacterium can grow on, the kind of toxins or waste products they produce or how much variation in growth temperature they can tolerate and their morphology can be used to distinguish closely related strains of bacteria. Rapid test kits have also been developed to identify bacteria in the Enterobacteriaceae genera and other gram negative bacteria.
Phonotypical approaches of identifying bacteria does not always provide sufficient information to set taxonomic boundaries between different species the repetition of some phenotypic characteristics make it difficult to separate them. Genotyping is however is more precise when it comes to differentiating bacteria within species that lead to the development of DNA hybridisation. This is a technique used measure the similarities in sequences between the DNA of an isolate and a known bacteria. Before conducting a phonotypical bacterial identification it is important to have a pure culture of the bacteria that needs to be identified so that all components of the cells have grown from a single cell and they are clones of one another also known as Holy Grail (Barrow and Feltham, 1993). The bacterial colonies formed for different types of bacteria have different cultural characteristics on agar plates known as colony morphology these include pigments, size edge, pattern, opacity and shine therefore macroscopically examining the colonies of bacterial cultures is one of the first important tests in bacterial identification. There may be drawbacks in this technique as the visual interpretation can differ from person to person therefore it might not produce reliable results and also mutations in the bacteria strains occur all the time that may provide slightly different characteristics than normal making difficult or incorrect identification.
A number of staining methods can be used to examine the cultures under a microscope such as negative staining which stains the background and leaves the cells clear so that the shape of the bacterial cells, presence of glistening capsule and presence of a diffusive extracellular substance (EPS) around the cell and the arrangement of cells can be determined. Differential staining is a test that divides bacteria into two large groups either gram negative or gram positive. Pink- red staining indicates gram negative bacteria and a blue purple staining shows gram positive bacteria cells. Unevenly stained clear surfaces can be formed on the surface of the bacterial cells during gram staining due to the presence of endospores that can be confirmed using spore stain. Acid fast staining is also used if cells appear long, slender and intertwined in order to confirm the presence of acid fast cells which are bacteria in the genera Mycobacteria and Nocardia that are resistant to gram staining.
Oxidase, catalase and the ability of the culture to grow in anaerobic conditions are three tests that are conducted during the first steps of identification. Catalase test is to dip an inoculating needle coated with culture into a droplet of hydrogen peroxide and if the bacteria possesses a catalase enzyme it will breakdown the hydrogen peroxide into water and oxygen that effervesce to form foam. Catalase positive bacteria are usually aerobic while catalase negative bacteria are anaerobic. Cytochrome oxidase is another enzyme found normally in the electron transport chains of an aerobic bacteria and this is tested by adding an artificial substrate such as para amino dimethylaniline that will produce a dark red to black product when oxidised (Cullimore, 2000). Alongside these test the cultures can be tested on their abilities to grow under anaerobic conditions which can further divide them into 4 major groups; strictly aerobic, reduced concentrations of oxygen, both aerobic and anaerobic and strictly anaerobic bacteria.
Urease synthesis, gelatin hydrolysis and citrate utilisation are tests that can be conducted to narrowly distinguish bacteria further to help identify their genera. Urease is produced the bacterial genera proteus, providentia and morgenella to break down urea into carbon dioxide and ammonia therefore it is a useful test to help distinguish these genera from other gram negative rods during identification. Urease test is carried out by incubating urea broth with samples of gram negative cultures. The presence of ammonia increases the pH hence turning the phenol red indicator to a pink-purple colour for a positive test (Harvey and Champe et al, 2001). The gelatin hydrolysis test identifies the ability of bacteria to produce gelatinases which can help identification of serratia and proteus. The citrate utilisation tests the ability of the bacteria to utilise citrate as its carbon and energy source used mostly to identify gram negative bacteria.
Indole, methyl red- Voges- protease test and fermentation of glucose, sucrose and lactose are also test that are conducted to help assist with identification of bacteria. Indole test is preformed to test the ability of bacteria to breakdown amino acid tryptophan and produce indole that can be detected using Kovac’s reagent. This method is important in the identification of gram negative enterobacteria. Methyl red-Voges- Proskauer on the other hand are two tests that are conducted together as they both require the use of the same medium. The methyl red test identifies the ability of the bacteria to carry out mixed acid fermentations whereas the VP test determines whether the bacteria fermenting sugars via the butanediol pathway by testing for the by-product acetoin. These tests are also useful in differentiating between members of the enterobacteria such as E. coli (Wong, 2005). The ability of bacteria to ferment carbohydrates is also a way to discriminate them during identification as fewer bacteria are able to use disaccharides like lactose and sucrose as a source of energy. This can be detected by checking for release of gaseous by products and metabolic chemicals that are released during the process of oxidation and fermentation of sugars. These tests described in the context above were carried out under standard conditions and results were recorded.
Table 1: shows test results for Colony Morphology for 10 unknown bacterial cultures A to J.
Table 2: shows test results for 18 different bacterial identification tests for unknown cultures A to J.
Urease, indole, citrate, oxidase, methyl red and Voges-Proskauer test were only carried out for gram negative strains of bacteria and the endospores were only tested for gram positive bacteria. Microphotographs showing cell morphologies and gram (+/-) strains for cultures C, D and F are shown in the appendix. For culture C it can be seen that the cells are arranged in “grape like” structures whereas C is arranged in packets of four. It can also be seen that culture F it can be seen that the cells were single and in chains.
Organism A and B are both gram positive rods that gave positive results for anaerobic, catalase and endospores test however they can both be differentiated as organism B is brown in colour and a glucose fermenter whereas organism A is orange in colour and a non-glucose fermenter. Organism B was in a cooked meat liquid broth which also indicates that the bacterium maybe part of the Clostridium species that have a few pathogenic bacteria that are responsible for food poisoning and tetanus. Organism A is therefore Bacillus Cereus some bacterium in this species are harmless whereas others are pathogenic that may cause foodborne illnesses such as nausea, vomiting and diarrhoea. Organism C has the morphology of pale yellow cocci clusters arranged in packets of four. It is a gram positive bacterium that also shows positive results for glucose fermentation and the catalase test show characteristics of Staphylococcus species which include pathogenic bacteria that causes skin infections, pneumonia and food poisoning. Organism C appeared in clusters that were “grape like” and is also a gram positive cocci and has a positive result for catalase however it does not ferment carbohydrates but shows positive test results for their metabolic chemical produced and hereby conveying characteristics of Micrococcus species these bacteria are very rarely disease causing and if so some may cause chronic cutaneous infections (Breed and Murray, 1957). Organism E has a white flat mycelial morphology and it is a gram positive glucose fermenter therefore it is a part of the Streptomyces genera that are known to inhibit the soil and causes the common scab in root vegetables.
Organism F is a clear gram negative rod bacterium that is glucose and lactose fermenter and produces metabolic chemicals for glucose, lactose. It also produced negative results for tests urease, gelatin, oxidase, Voges-Proskauer (VP) and indole however it showed positive results for catalase, citrate and methyl red and anaerobic tests as shown in figure 2. Analysing these results organism F can be identified as Escherichia coli that consist of many different strains some of which can cause urinary tract infections, diarrhoea, anaemia and even kidney failure. Organism G is a gram negative rod that produces metabolic chemicals for all three carbohydrates glucose, lactose and sucrose however bubbles were only present for lactose and sucrose. It also shows positive results for tests urease, indole anaerobic and VP but negative results for catalase, oxidase, gelatin and methyl red. Organism G showed similar results to organism F however unlike organism F it is urease positive therefore it can be a bacterium from the Proteus genera that also contain a few pathogenic bacteria that can cause urinary tract infections, kidney stones and cystitis. Organism H is only a glucose fermenter but does not produce any metabolic chemicals. It demonstrates positive test results for urease, gelatin, citrate, anaerobic catalase and oxidase but negative for both methyl red and VP as well as indole tests. It is possible that this bacterium maybe be a part of the Pseudomonas genera with different bacterial strains that may cause respiratory tract infections, dermatitis and bone and joint infections.
Organism I is also a gram negative rod and had a clear pigmentation on an agar medium that is a glucose fermenter but however it produces metabolic chemicals for glucose, lactose and sucrose. It also produces positive results when being tested for citrate, anaerobic, catalase and methyl red but not for urease, gelatin, indole, oxidase and VP this indicates that organism I could be Salmonella typhimurium a pathogenic bacteria that causes gastroenteritis that leads to diarrhoea. Organism J is another gram negative bacterium that produces no bubbles for glucose, lactose or sucrose fermentation but produces metabolic chemicals for glucose and sucrose. It is also positive for gelatin, citrate, anaerobic, catalase and VP however it is negative for urease, indole, oxidase and methyl red tests. The red pigmentation of the bacterial culture and the other tests results indicates that organism J maybe Serratia marcescens that are associated with many different types of diseases some of which include bacteraemia, sepsis and meningitis. In order to conduct more specific identification of these bacteria further tests can be carried out that will help to distinguish each bacterium more accurately. Other tests that can be carried out include starch hydrolysis, lipid hydrolysis, motility (SIM) deeps, beta galactosidase, nitrate, coagulase, mannitol salt, osmotic pressure and haemolysis.
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Culture C Culture D
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