Bacteria are Prokaryotes, which are defined in terms of the structure of their cells and their biochemical properties. Prokaryotes are unique because of the way they organize DNA in the nucleus, they lack a nuclear membrane and other membrane-bound organelles, and because of their chemical components of their cell wall and plasma membrane. Specifically, they are classified by Genus and species and a large part comes from morphology, oxygen requirements, and staining (Gram positive, Gram Negative, Acid-fast, Endospore).
However, in order to thoroughly identify and classify bacteria, biochemical tests are typically used in order to find their phenotypic characteristics and to group or differentiate one bacterium from another. Biochemical tests also help classify groups of bacteria according to characteristics relating to their metabolism and any enzymes that may be presence. For many of these tests, different types of media are needed in order to test if the bacteria can survive in certain conditions. Most media can be grouped into two categories: selective and differential. Selective media are those that only allow certain bacteria to grow while prohibiting the growth of other bacteria. In order to make a media selective, various chemicals, such as types of dyes, are added to prevent other bacteria from growing. Differential media, on the other hand, differentiates between different bacteria based on their appearance (color, shape, or patterns of growth) and it usually exhibits this by color change (5).
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Some of these media used in lab include both differential and selective. For instance, Starch agar is a differential media that shows if bacteria have the ability to breakdown starch due to amylase production and therefore will show if bacteria have exoenzymes that hydrolyze starch. In order to perform this test, iodine must be added because color change will not occur with the medium itself. Iodine will turn the medium into a dark color and if a zone of clearing appears then it is a positive test, and therefore means that bacteria have the ability to hydrolyze starch (6). Agar in general is a growth media that supplies bacteria with nutrients of which other compounds can be added to test a bacteria's ability to have certain characteristics. Another differential media is the OF (oxidative fermentation) which is used to see if bacteria have the ability to break down sugar via fermentation or oxidation. Carbohydrates can be broken down by two types of processes: oxidation (aerobic) and fermentation (anaerobic). The upper half of the tube usually satisfies aerobic conditions, while the lower part of the test tube satisfies anaerobic conditions. If either of the two (the top half or bottom half of the test tube) change color from blue to yellow, then metabolism has occurred. In case of a fermentative organism, both the top and bottom will turn yellow. If only the top turns yellow then the organism is oxidative (Bacillus subtilis) and if neither turn yellow then the bacteria (for instance, Pseudomonas) cannot break down carbohydrates (7).
Other media used in lab include, for instance, the Simmons Citrate Slant test which is used to distinguish species of the Enterobacteriaceae. The test is used to observe whether certain bacteria have the ability to use sodium or ammonium citrate as only a carbon source. This test is done by inoculating a green slant with a loop that contains a bacterial colony and then incubating it for two days. After two days, if the slant appears to change colors from blue to green then it is a positive test. The Urea Hydrolysis media is a differential media which is used to distinguish bacteria that produce the enzyme urease from those that do not produce the enzyme. Urease hydrolyses urea to carbon dioxide and ammonia. The broth contains pH indicator phenol red and if there is a color change to pink (a positive result) then ammonia has been produced and an alkaline environment has been created. It phenol red turns yellow then an acidic environment has been created. This broth can be used to distinguish bacteria from the genus Proteus (7). Another selective and differential media is Bile Esculin Agar which shows if bacteria have the ability the hydrolyze esculin. It prohibits Gram positive bacteria from growing except for Enterococci and some Streptococci. A positive result for the test will show a dark phenolic complex and a negative result will not have a dark complex due to the organism's inability to hydrolyze esculin (1). The Phenol Red Broth is a differential media that is usually used to distinguish Enteric bacteria. There are three types of phenol red broths that are typically used: glucose, sucrose, and lactose. If bacteria are able to utilize one of these carbohydrates then an acid product is produced and the media will turn red to yellow (positive test). Also some bacteria are able to produce gas when they utilize carbohydrates; this is indicated by bubble production in the media. The Methyl Red (MR) and Voges-Proskauer (VP) Tests are two separate tests that are both used to distinguish species of Enteriobacteriaceae. Methy Red tests for acid-end products from glucose fermentation and Voges-Proskauer tests for acetoin production from glucose fermentation. The MR test is conducted by adding pH indicator methyl red to an inoculated tube of MR-VP broth. If bacteria use the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and create an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red, which indicates a positive test. On the other hand, the VP test detects organisms that use the butylene glycol pathway and creates acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with bacteria that uses the butylene glycol pathway, the acetoin end product is oxidized in the company of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then responds to produce a red color, which produces a red color and is a positive result. However, if after the reagents have been added and a copper color is present, the result is negative (7).
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The decarboxylase test is also useful to differentiate Enteriobacteriaceae. The test is used to see if bacteria have the ability to produce decarboxylase, which is an enzyme that removes a carboxyl group from an amino acid. This production of decarboxylase is specific to each bacterium and therefore this test shows if bacteria have the ability to produce arginine, lysine, and ornithine. The three broths are very similar and procedures are performed the same way, by inoculating the broth and incubating to observe for color change. If a color change is present from yellow to a dark color then the results are positive and if there is no apparent color change (stay yellow) then the results are negative (7).
A reduction test is classified as a differential media that is used to see if bacteria have the ability to reduce compounds such a nitrate. By inoculating the broth and incubating for two days, the results can either show a positive test (a color change) or a negative test (no color change). A Fluid Thioglycollate Media is used to test bacteria's aerotolerance by adding indicator Methylene blue. If the media appears colorless the bacteria prefers an anaerobic environment and if the media appears green/blue then the bacteria prefers aerobic conditions. Also, the position of where bacteria grow on the broth can further show aerotolerance behavior of the bacteria. For instance, obligate aerobes grow on the top layer while obligate anaerobes grow on the bottom layer. Facultative bacteria will grow all throughout the tube and microaerophiles will grow right below the top layer of the broth. Milk agar is used to test for a bacteria's ability to secrete extracellular proteases that catalyze the hydrolysis of the milk protein casein. This test is performed by inoculating the broth and after incubation a positive test will show a clear zone around the bacteria, which shows casein hydrolysis. Gelatin Agar, on the other hand, is used to distinguish bacteria that can produce an exoenzyme, gelatinase, from those bacteria that cannot. This test is primarily used for Gram negative bacteria, specifically bacilli. This test is performed by inoculating the broth and incubating for one to two days. A positive result will remain liquefied after incubation and a negative result will appear solidified after incubation. A DNase Agar tests for a bacteria's ability to produce an exoenzyme called DNase (which hydrolyses DNA-breaks down DNA into smaller pieces). This test to performed by swabbing the bacteria on the media and incubating for two days. If a clear zone appears then it is a positive result. Lipase Agar tests for a bacteria's ability to have lipase activity present. Just as the DNase test, the Lipase test is also performed by swabbing the bacteria on the media and incubating for two days. If a clear zone appears then it is a positive result. A SIM (Sulfide Indole Motility) medium is typically used to identify bacteria from the family Enterobacteriaceae by detecting sulfide production, motility, and indole formation. This test is performed by inoculating and then incubating for one to two days. When observing for motility, if growth only occurs where the bacteria were inoculated then this shows a negative result, whereas if growth is shown elsewhere then this would be a positive result. Sulfide production shows a color change to black and indole formation shows a color change to from yellow to pink. A Kligler Iron Agar is used to distinguish a bacteria's ability to produce hydrogen sulfide and their ability to ferment dextrose and lactose. This test is typically used on Gram negative bacilli. The test is done by inoculating the broth (streaking the slant) and then incubating. After incubation, if the slant appears to have both red and yellow then it ferments dextrose only. If the whole slant appears yellow then it only ferments dextrose and lactose and if the whole slant appears red then the bacteria ferment neither lactose nor dextrose (7).
CNA agar is a type of selective media that is used to inhibit the growth of Gram negative bacteria because it contains colistin and naladixic acid. This media also contains whole blood which allows for hemolysis differentiation. This media primarily allows for the growth of Gram positive cocci bacteria. Procedures for this media are performed by first inoculating and then incubating. After incubation, if there is a green discoloration of the medium then hemoglobin has reduced to methemoglobin. If a clear zone is produced then blood cells have been lysed out and if there is no change in medium then no hemolysis has occurred (3). Desoxycholate Agar is both a selective and differential media that is primarily used for Gram negative enteric bacilli, specifically for Salmonella and Shigella. They are tested to see if these types of bacteria can produce lactose by producing acid. By inoculating the media and then incubating for two days those that are able to form red colonies show a positive result and those that are not able to produce lactose are colorless (negative result). Methylene Blue Agar is also a selective and differential media used to inhibit the growth of Gram positive bacteria and to isolate fecal coliforms. The media will show those bacteria that are able to ferment sucrose and lactose by producing enough acid to change the color of the media to a dark color (positive result). Hektoen Enteric Agar is also another differential and selective media that also prevents Gram positive bacteria from growing. It is typically used to isolate Salmonella and Shigella from other members from Enterobacteriaceae. It indicates bacteria that can ferment lactose and produce hydrogen sulfide. By streaking the bacteria onto the media and then incubating for one to two days, bacteria that ferment lactose will show a salmon colored growth and bacteria that do not ferment lactose with not grow and appear the same before incubation. MacConkey Agars are selective for Gram negative bacteria and are tested to see bacteria have the ability to ferment lactose. By streaking the bacteria onto the media and then incubating for one to two days, bacteria that ferment lactose will show a pink color change and bacteria that do not ferment lactose with appear yellow. Mannitol Salt Agar (MSA) is typically used to differentiate Staphylococcus species from each other and from Micrococcus species by seeing if the bacteria can sustain in high concentrations of salt. It is done by inoculating a mannitol media with a loop that contains a bacterial colony and then incubating it for two days. After two days, if the broth appears yellow then it is a positive test and if is pink (as its original color) is it a negative test. Phenylethyl Alcohol Agar is used to inhibit the growth of Gram negative bacteria bacilli and is typically used to isolate Staphylococcus. By streaking the bacteria onto the media and then incubating for one to two days, a positive test with bacterial growth and a negative test with no bacterial growth. A Catalase test shows if there is a presence of catalase enzymes in bacteria. Most bacteria that do have catalase live in oxygenated areas and can break down hydrogen peroxide into water and oxygen. The test is done by applying a drop of hydrogen peroxide to the bacteria. If bubbles form right after hydrogen peroxide is added then it is a positive test and if there is no bubble formation then it is a negative test. The last test used in lab is Oxidase test which is used to see if bacteria produce the enzyme cytochrome oxidase (which takes part in the electron transport chain by transferring electrons from a donor molecule to an oxygen molecule). This test is performed by soaking a piece of filter paper into the reagent solution and then adding a colony on to the filter paper. After about fifteen minutes if the reagent turns blue/purple then it is a positive test and if there is no color change then it is a negative test (7).
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Many of the tests listed above were performed in lab. The citrate test was conducted on E. coli and showed a negative result due to no color change (remained green) and therefore E. coli do not have the ability to use sodium or ammonium citrate as only a carbon source. The lysine decarboxylate test was performed on Proteus vulgaris and appeared yellow after incubation which therefore is a negative test. Thus, Proteus vulgaris do not have the ability to produce decarboxylase. The Phenylalanine Deaminase Test was tested to see if Proteus vulgaris have the ability to produce the enzyme deaminase. After incubation, white colonies appeared as they were before incubation, which therefore means it is a negative test and they do not produce deaminase. The Bile Esculin test was used to test if Lactococcus lactis have the ability to hydrolyze eusculin. They do seem to hydrolyze eusculin because the broth was black colored after incubation, which indicates a positive test (1). The Starch test was used to see Bacillus cereus can breakdown starch due to amylase production and therefore have exoenzymes that hydrolyze starch. They did not show a clear zone and therefore is a negative result and do not have exoenzymes that hydrolyze starch (6). The next two tests, Urea and SMA, were both used to test E. coli. The first test (Urea) is used to differentiate bacteria that produce the enzyme urease. It showed a negative result due to no color change as well as the second test (SMA) also showed a negative test due to no clear zone. Gelatin agar can show if a bacteria (in this case Staphylococcus aureus) have the enzyme gelatinase. Staphylococcus aureus do have the enzyme gelatinase due to the media still being in liquid form. The DNAse test shows if organisms have the ability to produce an exoenzyme, DNAse. After incubation, there was no clear zone and therefore is a negative test and does not produce DNAse (4). The SIM medium was used to see is Salmonella typhimurium had the ability to produce sulfide. This bacterium does produce sulfide due to the medium appearing black after incubation. The Voges-Proskaur test was used to see if Enterobacter aerogenes for acetoin production from glucose fermentation. It tested negative due to no color change (remained yellow) and therefore does not produce acetoin. Enterobacter aerogenes was used again with Methyl Red to see if it can produce acid-end products from glucose fermentation. The test came out negative after incubation due to no color change (remained yellow instead of red). The Sucrose, Lactose, Glucose media was used in lab to see if Pseudomonas aeruginosa can ferment sucrose. There was no color change to yellow for all three media and therefore they all three are negative tests. Pseudomonas aeruginosa was also used for the Oxidative-Fermentative test, which shows if bacteria have the ability to break down sugar via fermentation or oxidation. After incubation, only the top half of the tube changed color from green to yellow, and therefore indicates that Pseudomonas aeruginosa is oxidative. A MSA test was used to see if Staphylococcus epidermis, which showed a negative result because there was no color change to yellow, which consequently show that they cannot sustain high salt concentrations. An EMB medium was used to see if Enterococcus faecalis can ferment lactose and produce acid to change the color of the medium. It showed a negative test because the colonies remained colorless instead of a green metallic color. A Mac media was also used to see if Entereococcus faecalis can ferment lactose. After incubation, the medium appeared pink (positive test) and which means that Entereococcus faecalis can ferment lactose. The last medium used is the Starch Agar medium, which shows that Salmonella typhimurium does not have the ability to breakdown starch due to amylase production and therefore will show if bacteria have exoenzymes that hydrolyze starch (6).