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Isolation of normal body flora was isolated from various parts of human body. They were isolated from armpit, body, fingers, head, mouth, and foot. Sterile technique was applied during the isolation of bacteria. A sterile cotton swab was dipped into sterile distilled water and swabbed at parts of human body. Then, the specimen was swabbed on the nutrient agar and incubated in the incubator for 24 hours at 37oC. After that, the colonies were observed and a single colony from each medium was recognized. Then, each single colony was transferred using sterile technique into slant nutrient agar stock and stored in a freezer at below 4oC. The slant nutrient agar is known as a stock culture and can be use when needed.
3.2.2 Identification of Bacteria
220.127.116.11 Macroscopic Cultural Characteristics
When grown on a variety of media, microorganism will exhibit differences in the macroscopic appearance of their growth. These differences, called cultural characteristics, are used as a basis for separating microorganism into taxonomic groups.
Using sterile technique, each of the appropriately labelled media listed below in the following manner:
Nutrient agar plates
By using sterile loop a streak-plate inoculation of each of the cultures was carried out for the isolation of discrete colonies. Then, the plates were incubated for 24 to 48 hours at 37oC in an incubator. After 24 to 48 hours, the cultural characteristics of the bacteria on the plates were observed. There are many shaped of bacteria. To be characterized by their forms of colonies, bacteria can be classified as circular, irregular or rhizoid. If to be classified according to their elevation, there are four of them which are flat, raised, convex or umbonate. According to the margins of the colonies form, bacteria can be classified as entire, lobate, undulate, serrate or filamentous.
18.104.22.168 Gram Stain of the Isolates
By using sterile technique, the smears of each of 24-hours bacteria cultures were prepared. A drop of water is placed on the slide and then each microorganism that grew on the nutrient agar was transferred separately to the drop of water with sterile, cooled loop. The cultures have been mixed and spread by means of a circular motion of the inoculating loop. The smears were flooded with crystal violet and let stand for one minute. Then, they have been gently washed with tap water. The smears were flooded with Gram's iodine and let stand for one minute. After that, they have been gently washed with tap water. Then, they have been decolorized with 95% ethyl alcohol, drop by drop until alcohol runs almost clear to avoid over-decolorized. After the process, they have been gently washed with tap water. Then, they have been counterstained with safranin for 45 seconds. After that step, they have been gently washed with tap water. Finally, air-dry the slides at room temperature and the light microscope is used for observation and examination for cell morphology, staining properties and aggregation patterns.
Colony morphology and pigmentation were also observed under light microscope. Cells from each culture were streaked on nutrient agar plates and incubated for 48 hours. Colonial characteristic pigment was observed daily throughout the incubation period.
22.214.171.124 Spore Stain of the Isolates
Obtain 20 clean glass slides and by using sterile technique, individual smears were made in the usual manner. The smear is allowed to air-dry and heat fixed in the usual manner. The smears is flooded with malachite green and was placed on top of a beaker of water sitting on a warm hot plate, the preparation is allowed to steam for two to three minutes. The slides is removed from hot plate, cooled and washed under running tap water. Then, safranin is used to counterstain for 30 seconds, and then washed with tap water. Finally, blotted dry with bibulous paper and is examined under oil immersion.
126.96.36.199 Biochemical Tests
The biochemical tests which were carried out are carbohydrate fermentation, IMViC test, hydrogen sulphide test, urease test, nitrate reduction test, catalase test, and oxidase test. All the procedures were referred to standard procedures (Cappuccino & Sherman, 2008). Procedures for determining the various keys morphological, physiological and biochemical were described as follows paragraphs.
Catalase test: This was tested by mixing one drop of 3% (v/v) hydrogen peroxide solution with one drop of culture suspension (Devriese and Oeding, 1975). The gas bubbles produced indicate positive result.
Carbohydrate fermentation: Acid production from sucrose, lactose and dextrose was observed under aerobic conditions by using phenol red carbohydrate broths. Complete colour changes occurring within 48 hours of incubation were recorded as positive and incomplete change as weakly positive.
Nitrate reduction test: Tubes containing 10 ml of trypticase nitrate broth were inoculated with a loopful of a culture, incubating for 48 hours and observing colour development after adding five drops of a sulfanilic acid reagent (0.8g/100 ml of 5N acetic acid) and 5 drops of an Î±-naphthylamine reagent (0.5g/100 ml of 5N acetic acid) to the broth culture.
Oxidase test: oxidase activity was estimated by placing an oxidase strip on an 24-hour culture colony that inoculated from stock culture on a clean glass slide. A blue violet colour appearing within a few seconds in the colonies was released as a positive result.
Urease test: Culture were inoculated into urea broth using loop and incubated at 35oC for up to 48 hours. Then, the bright pink coloration on broth was observed as positive result.
Starch hydrolysis: Cultures were streak by inoculating on the surface of the starch agar plate and incubated at 35oC overnight. After that, a few drops of Gram's iodine were flooded on plates. A clear zone around the area in which the organisms have grown indicated hydrolysis of the starch.
Gelatin hydrolysis: Cultures were inoculated into tubes of nutrient gelation and incubated at 37oC. Cultures were observed within two to seven days for hydrolysis. Hydrolysed gelatin will remain fluid even tubes were chilled in the chiller.
Lipid hydrolysis: Cultures were streak by inoculating on the surface of the tributyrin agar plate and incubated at 35oC for up to two days. A clear zone around the area in which the organisms have grown indicated hydrolysis of the lipid.
Hydrogen sulphide test: Cultures were aseptically inoculated by means of stab inoculation and incubated at 35oC for up to 48 hours. The hydrogen sulphide gas is colourless and therefore not visible. Ferrous ammonium sulphide in the medium serves as an indicator by combining with the gas, forming an insoluble black ferrous sulphide precipitate that is seen along the line of the stab inoculation and is indicative of H2S production. Absence of the precipitate is evidence of a negative reaction.
Bile esculin Test: An inoculum from a pure culture was transferred aseptically to a sterile tube of bile esculin agar and streaked along the slant. The inoculated tube was incubated at 37oC for 24 hours and the results are determined. Abundant growth on the slant indicates a positive test for growth in the presence of bile. If growth is present, esculin hydrolysis can be observed if the medium has taken on an intense, chocolate brown coloration.
Mannitol test: An inoculum from a pure culture was transferred aseptically to a sterile tube of phenol red mannitol broth. The inoculated tube is incubated at 37oC for 24 hours and the results are determined. A positive test consists of a colour change from red to yellow, indicating a pH change to acidic.
6.5% Salt tolerance test: An inoculum from a pure culture was transferred aseptically to a sterile tube of 6.5% NaCl broth. The inoculated tube is incubated at 37oC for 24 hours. A positive test is indicated by the presence of turbidity.
3.2.3 The Bacterial Growth Curve
The bacterial growth studies require inoculation of viable cells into a sterile broth medium and incubation of the culture under optimum temperature, pH and gases condition (Cappuccino & Sherman, 2008).
In this project, nutrient broth was used to cultivate the bacteria. When microorganisms are cultivated in liquid medium, they are incubated in a close culture vessel with a single batch of medium (Presscott et al., 2005).
To construct the growth curve, 200 Î¼l of each overnight culture were transferred into a 25 ml of fresh nutrient broth. Then, the broths were incubated in an incubator for 24 hours at 37oC. After each two hours 1.0 ml of the homogenized broth cultures were transferred into a sterile cuvette for optical density (OD) readings. The readings were based on the absorbance of 600 nm. The readings were taken every one hour for 24 hours of incubation.
3.2.4 Serial Dilution of Minimal Inhibitory Concentration Test
Into two test tube racks, 50 sterile 13 X 100-mm test tubes were placed. Then, from a set of 10 test tubes, labelled 1 through 10 for each candidate of bacteria. Labelled one set of test tube as Set 1 until Set 5. By performing aseptically technique, a sterile 1-ml pipette and mechanical pipetting device is used, 1-ml of Nutrient broth is added to the tubes labelled 2 through 10 in Set 1 and follow for the other sets. With a 1-ml sterile pipette, 1-ml of the hairspray's chemical solution is added to Tubes 1 and 2 in Sets I and other sets. From set 1 serial dilution, a sterile 1-ml pipette is used; transfer 1 ml from Tube 2 to Tube 3. One ml from Tube 3 to Tube 4 is mixed well and transferred. Continue this procedure through Tube 9 into beaker. One ml from Tube 9 is discarded. Tube 10 received no chemical and served as a positive control. These steps were repeated for the other sets. Both sets of tubes were incubated for 12 to 18 hours (common log phase of bacteria) at 37oC.
After incubation, the instructions for the use of the spectrophotometer to determine the absorbance readings for Tubes 2 through 10 in Sets 1 and other sets were followed. The Number 1 tubes, the negative controls, used as blanks to adjust the spectrophotometer. Nutrient agar plates were appropriately labelled. The spread-plate technique is used. By using micropipette, aseptically 0.1 of each bacteria candidate from each dilution is added, to its appropriately labelled agar plate. All plates were incubated in an inverted position for 24 hours at 37oC.
These steps were repeated by replacing hairspray's chemical to 70% ethanol to determine active ingredient activity for minimal inhibitory concentration against the test organism in presence of alcohol.
3.2.5 Antibiotic Sensitivity Test
The antibiotic discs were used which included Penicilin G, 10 Î¼g; Bacitracin, 10 Î¼g, Trimethoprim, 5 Î¼g; Sulfonamides, 250 Î¼g; Vancomycin, 30 Î¼g; Tetracycline, 30 Î¼g; Rifampin, 5 Î¼g and Ampicilin, 10 Î¼g. A sterile cotton swab was dipped into a culture and excess inoculum was removed by pressing the saturated swab against inner wall of the culture tube. The nutrient agar surface was allowed to dry and then, the entire surface was streaked horizontally, vertically and around the outer edge of the plate to ensure a heavy growth using dipped swab. All the culture plates were allowed to dry for several minutes. The discs were distributed at equal distances with forceps dipped in 95% ethanol and it was flamed for sterilization. Each disc was pressed down with the sterile forceps to ensure that the discs adhere to the surface of the agar. All culture plates were incubated in an inverted position for 24 to 48 hours.
3.2.5 Synergistic Effect of Drug Combinations Test
The inoculation of the nutrient agar plates, step 1 through 4 is followed as described under procedure in part Kirby-Bauer Antibiotic Sensitivity Test Procedure from Microbiology Laboratory Manual 8th Edition (Cappuccino & Sherman, 2008). The millimetre ruler is used, to determine the centre of the underside of each plate and mark with a glassware marking pencil. The glassware marking pencil is used to mark the underside of each agar plate culture at both sides from the centre mark at the distances specified. Sterile forceps is used, the antimicrobial discs is placed, in the combinations specified before, onto the surface of each agar plate culture at the previously marked positions. Each disc is gently pressed down with the sterile forceps to ensure that it adheres to the agar surface. All plate cultures is incubated in an inverted position for 24 to 48 hours at 37oC.