Microorganism Cultivation Method in Liquid Media
Some bacteria do not stain easily by staining procedures. Staining of these organisms is facilitated by the application of heat during staining. Once stained, these organisms retain the dye, even when treated with a decolourization agent such as acid alcohol; They are said to be acid-fast. Another special stain method is spore staining.
Exercise 1 : Methods of cultivating microorganisms 1.1 Inoculation of liquid media Microorganism E.coli Pseduomonas aeruginosa Colour of the colonies Pale yellowish Bluish green Amount of growth Moderate Moderate Surface growth No pellicle formation Pellicle formation Turbidity Slightly turbid, uniform turbid Sediment granular flocculent Discussion : Broth media are usually used as enrichments, general cultivation and sterility testing. In this experiment, Escherichia coli is a facultative anaerobe. It can grow with or without the oxygen supply. Thus, there is no distinct layer of pellicle formation. However, Pseduomonas aeruginosa is a strict aerobe which can grow only with oxygen supply. Thus, the colonies formed pellicle as there was more oxygen supply at the top layer of the broth. The mild colorization of broth in greenish-blue was due to the presence of a greenish-blue pigment called pyocyanin which was produced by the bacteria Pseudomonas aeruginosa. 1.2 Inoculation on Solid Medium : Streak-plate Method Mixed culture Characteristics E.coli P.aeruginosa E.coli epidermis Shape Circular Circular Circular Circular Size 1mm 1-2mm 1-2mm 1-2mm Chromogenesis Pale yellowish, Bluish green Pale yellowish, Pale yellowish, insoluble pigment, insoluble insoluble soluble Opacity Opaque Translucent Opaque Opaque Elevation Convex Raised Convex Raised Surface Smooth Smooth Smooth Smooth Edge Entire Entire Entire Entire Discussion : Streaking is a technique used in microbiology to isolate a pure strain from a single species of microorganism. The inoculation loop is resterilized and dragged across the inoculated quadrant of the streak plate. Each time the loop gathers fewer and fewer bacteria until it gathers just one single bacterial cell that can grow into a colony. A bacteria can be grown by using this method so that the it can be identified, studied, or tested. There are different type of agar medium that can be used. For example, nutrient agar, minimum agar, blood agar, MacConkey and Thiosulfate Citrate Bile Sucrose ( TCBS ). Different kinds of bacteria cultured in different media will show different morphology and different results. TCBS and MacConkey are selective and differential media which selects and limits the growth of certain types of bacteria. For example, there are red colonies of E.coli grow on the MacConkey plate. This indicates that E.coli can utilize and ferment lactose, forming acidic products which turn the indicator in the growth medium red, thus causing the colonies to appear reddish as well. However, if other bacteria that cannot ferment lactose were grown in this medium, no colours will be observed because they cannot form acidic product that change the indicator into red colour. Besides, TCBS is suitable to be used in the selective isolation of vibrios. Different type of vibrios will show different colour depending on their ph because thymol blue is used as an indicator in this agar. Exercise 2 : Microbial Flora Skin Organ Hand Ear Shape Circular Circular Size Very small, few punctiform Relatively larger colonies ( 1–2mm ) Colour Off white Off white Opacity Opaque Opaque Elevation Convex Convex Surface Smooth Glistering Edge Entire Entire Haemolysis Absent Present Discussion : For this exercise, bacteria were swab from ear and hand and streak on the blood agar. There are very few bacteria colonies that swab from hand grow on the blood agar. It is believed that the experiment conducter had sterilize or wash her hand before doing this exercise. However, for the bacteria colonies that swab from ear, there is a big yellow circular colonies observed surrounding one of the off-white colony. It is Haemolytic colonies which can break down red blood cells. Exercise 3 : Characterization of Bacteria 3.1 Catalase Test 3.2 Oxidase Test Bacteria Type Catalase Test Oxidase Test E.coli + - Pseuduomonas aeruginosa + + Staphylococcus epidermis + - Staphylococcus aures + - Streptococcus - - Discussion : For catalase test, most aerobic or facultatively anaerobic that possesses catalase activity will give positive result by production of bubble or effervescence. Since Streptococcus is an anaerobic bacteria, it showed a negative result. For oxidase test, aerobic or facultatively anaerobic orgamisms will show positive results. In this experiment, Pseuduomonas aeruginosa is the only bacteria that shows positive result. Thus, it possesses oxidase activity and cytochrome oxidase system. This test can be used to differentiate Pseudomonas family and enterobacteria family. E. coli is one of the most important model organisms in enterobacteria family. Exercise 4 : Microscopy Bacteria Type Monochrome Staining Gram Staining Cocci / Rod shape, singular in Cocci / Rod shape, singular arrangement and blue arrangement / arranged in Mixed cluster, pink and purple Cocci, arranged in cluster and Cocci, arranged in cluster, blue purple Staphylococcus epidermis Rod shape, singular in Rod shaped, singular arrangement and blue arrangement, pink E.coli Discussion : Monochrome staining is not sufficient to observe detailed morphological characteristics. Under microscope observation, all the cells Escherichia coli and Staphylococcus epidermidis were stained blue and only certain aspects ( colour, shape and arrangement ) were able to be observed. However, gram staining is a better method to observe microorganism. In gram staining, Staphylococcus epidermidis are stained purple while Escherichia coli are stained pink. Hence, Staphylococcus epidermidis is gram positive whereas Escherichia coli is gram negative. The bacteria are first stained with crystal violet and next treated with iodine to promote dye retention. When gram positive bacteria are decolourized with ethanol, the alcohol is thougth to shrink the pores of the thick peptidoglycan. Thus the dye-iodine complex is retained during the short decolourization step and the bacteria remain purple. In contrast, gram negative peptidoglycan is very thin, not as highly cross-linked, and has larger pores. Alcohol treatment also may extract enough lipid from the gram negative wall to increase its porosity further. For these reasons, alcohol more readily removes the purple crystal violet-iodine complex from gram negative bacteria. The cell is stained pink with the counterstain carbol fushin. Some bacteria do not stain easily by staining procedures. Staining of these organisms is facilitated by the application of heat during staining. Once stained, these organisms retain the dye, even when treated with a decolourization agent such as acid alcohol; They are said to be acid-fast. Another special stain method is spore staining. With the help of heat treatment, a primary stain penetrates the coat and stains the spores whereas the cytoplasm will take up the secondary stain. The spores are stained green while the cytoplasm is stained red.