Applied Microbiology - Second Level Practical Exam Notes PDF

Applied microbiology Second level (practical part) Section six EXAMINATION OF MILK AND OTHER DAIRY PRODUCTS FOR COLIFORM BACTERIA 1  EXAMINATION OF MILK AND OTHER DAIRY PROD...

Applied microbiology Second level (practical part) Section six EXAMINATION OF MILK AND OTHER DAIRY PRODUCTS FOR COLIFORM BACTERIA 1  EXAMINATION OF MILK AND OTHER DAIRY PRODUCTS FOR COLIFORM BACTERIA The coliform group of bacteria are reliable indicator of fecal pollution and general unsanitary conditions of water, food, milk, and other dairy products. A finding of coliforms in foods is indicative of possible presence of enteropathogenic or toxigenic microorganisms which could constitute a public health hazard. The standard laboratory definition of the coliform group of bacteria is as follows:- G–ve, facultatively aerobic, short – non sporeforming rods-capable of fermenting lactose to acid and gas at 35 – 37 °C with in 24 hr. most culture media designed for coliform detection contain lactose as the role or principal carbohydrate source for enrichment. The two major coliform types constituting the group 1- E. Coli is fecal type. 2- Enterobacter aerogenes non-facal type (more widespread contamination of food products by soil, water, dust and air-born contamination). 2 Coliform detection:- The accurate identification of coliform type requires the isolation of the suspected coliform from presumptive test media in pure culture form. The culture should be confirmed on appropriate test media before an attempt is made to accurately identify it. A- Presumptive test (PT): 1. Transfer 1 ml of milk or decimal dilution into lactose broth medium (Macconky’s medium) 2. Incubation the tube at 37 °C / 24 – 48 hr. ; A (+) tube reaction is one where 10% or more gas formation has occurred in the Durham tube as a result of lactose fermentation following incubation. 3 4 B. Confirmed test caution must be exercised in the interpretation of (+) results obtained in liquid media designed for coliform detection. For example, in lactose broth it is known that lactose- fermenting microorganisms other than coliforms may flourish to produce gas, thereby yielding a “false” (+) presumptive test. Some of the reasons offered for such tests are:- 1. Bacterial synergisms 2. Lactose – fermenting (non – coliform) bacteria 3. Lactose – fermenting yeasts In order to exclude these possibilities, (+) presumptive tests must be confirmed in solid (EMB) confirmatory media held at 32°C for 24 – 48 hr. EMB agar streak- plates offer an excellent opportunity to confirm the presence of coliforms in (+) presumpative tests by accomplishing the following: 5 1. Isolation of “typical” coliform colonies. 2. Differentiation of coliform “ colony-type”. 1. fecal strains: dark, opaque, circular, 0.5 to 1.5 mm diameter, green-metallic sheen. 2. Nonfecal strains: pink to clear edge; red-brown center, convex, smooth, circular, 1.0 to 3.0 mm in diameter. 3. Production of “pure” colonies for further determination in the completed test. 6 7 C. COMPLETED TEST A “typical” coliform will produce the following results in the completed test: 1- Ferment lactose to gas (10% or more in Durham tube) 2- Slant culture appears as a “pure culture” of Gram-negative, short, thick, nonspore forming rods, in pairs and irregular masses. Procedural Diagram 1. (+) Presumptive test: lactose broth culture with 10% or more gas formation or “typical” VRBA colony. 2. Transfer to Levine’s EMB agar. Prepare streak plates. 8 9  Applied microbiology Third level (practical part) Section seven FERMENTED FOODS 10  FERMENTED FOODS Fermented foods play an important socio-economic role in developing countries as well as making a major contribution of the protein requirements of natural populations. In traditional fermented food preparation, microbes are used to prepare and preserve food products, adding to their nutritive value, the flavor and other qualities associated with edibility. 11  Soybean and different soybean products are known to contain phenolic compounds. Activities of these compounds in soybean were reported to increase after fermentation, for example, during the production of tempeh, miso, natto, furu and rice koji. Tempeh Miso Natto Furu Rice Koji 12  1- Tempeh Tempeh is now rapidly becoming more popular all over the world. In addition, vegetarians and vegans find tempeh as interesting food because of it's good functional properties. Procedure of Tempeh production 1. standard inoculums preparation (Fungus & Bacteria): A- Standard inoculum of fungus Rhizopus oligosporus suspension B- Standard inoculum of bacterial and yeast strains 13 2- Procedure of Tempeh production: Tempeh was made from some fermented seeds as soybean, chickpea, lupine, cowpea and faba bean according to the following method : 14 Flowchart of Tempeh Production 15 1- Tempah quality estimation: Parameters Deep-fried soybean tempeh Raw soybean tempeh Appearance Color Texture Taste Odor Mouth feeling Overall acceptability 2- Check Tempah fungal strain Rhizopus oligosporus: 16  tempeh Soybean Neurospors intermedia miso natto Fig. Some fermented foods and manufacture by fermentation process. 17  2- Natto Natto is a traditional fermented soybean food product with venerable history in Japan. By steaming soybeans and inoculating with Bacillus subtilis, natto spores to ferment it, and then it would become a characteristic odor, flavor and slimy food. The processing of natto includes soaking, steaming, cooling, inoculating, fermenting and aging. The higher proteins are hydrolyzed the more peptide bonds are broken and the free amino acids and lower molecular weight of oligopeptides are produced. 18 19  3- Miso Miso is produced in ordinary households all over Japan, and many people have their favorite local taste. Various kinds of soybean miso paste are possible, depending on the kind and amount of the cereal ingredients (soybean, rice or wheat) and the amount of salt in the fermentation of miso paste inoculated with Aspergillus oryzae. 20  4- Soy Sauce There are three stages in the fermentation : Lactic acid fermentation by lactic acid bacteria (Lactobacillus delbrueckii) from the koji, followed by more acid production by Pediococcus soyae. Alcoholic fermentation by yeasts such as Saccharomyces rouxii and completion of fermentation and aging. 21  Aspergilus oryzae Aspergilus oryzae Miso Soy sauce 22  Applied microbiology Third level (practical part) Section eight FERMENTED DRINKS (KOMBUCHA) 23  KOMBUCHA Kombucha is called fungus-tea composed of fermented different types of acetic acid bacteria and yeasts in symbiotic association with two portions, a floating cellulose pellicle layer and the sour liquid broth. Procedure of Kombucha fermented solutions (KFSs): 1. Boiling 1L of tap water for 15min. 2. 4g of black tea was added and infused for 15 min then removed by filtration. 3. 70 g/L of commercial sucrose was dissolved into the filtered infusion before it has cooled. 4. After cooling to room temperature, pour the solution into a glass jar has been 24 previously sterilized at 121°C for 20 min. 5. This solution was inoculated with 10% of the fermentation broth from the previous fermentation of black tea obtained under the same conditions. 6. The glass jars were covered with a clean piece of cloth and fixed with rubber bands. 7. The fermentation was carried out under room temperature (25°C) for 10 days. 8. The harvesting was done after 2, 4, 6, 8 and 10 days of fermentation. 9. Kombucha tea was centrifuged at 10.000 rpm for 15 min and the supernatant was used as Kombucha fermented solution (KFSs). Properties of kombucha: Kombucha tea is known to show a remarkable antimicrobial activity against a broad range of microorganisms of both Gram positive and Gram negative. 25 5. The growth inhibition was observed by the naked eye and the inhibition zone diameter (IZD) was measured using a ruler after 24 h of incubation. 6. The antimicrobial activity was evaluated by measuring the growth inhibition zone surrounding the wells. 7. Acetic acid sample was used as control at the same concentration as that of fermented solutions after 8 and 10 days were prepared and sterilized by filtration and then used for antimicrobial testing, as described previously. 8. Antibiotic was used as standard. 9. Each experiment was carried out in triplicate and the average diameter ± standard deviation of the IZD was recorded. 26 Antimicrobial Activity: The antimicrobial activity of KFSs was tested by agar well diffusion assay against pathogenic strains such as Listeria monocytogenes, Shigella dysenteriae, Salmonella enteritidis, Staphylococcus aureus and Esherichia coli. 27 28 Estimation of Kombucha: 1- Prepare a smear of Kombucha solution, and stain it with gram stain, then examine the different kinds of microorganisms using the microscope. 2- quality estimation: Parameters Deep-fried soybean tempeh Raw soybean tempeh Appearance Color Texture Taste Odor Mouth feeling Overall acceptability 29  30 Fig. Kombucha fermented solution and disk  Applied microbiology Third level (practical curriculum) Section nine MUSHROOM 31  MUSHROOM Mushroom is a fungi producing a fleshy fruiting body, especially one consisting of a stalk with an umbrella cap. It has two part: cap like structure is known as PILEUS, attached with thread like structure MYCELIA. Mycelia absorb nutrients from soil, it do not require sun light for their growth. Procedure of Mushroom (Spawn) Production : 1. Select good quality jowar or wheat grains free from pest and molds. 2. Boil the grains submerged in clean water for 20-30 minutes.when the grains become soft, 32 remove and spread evenly on a cotton cloth to drain out the water and cool the grains. 3. Mix 3% chalk powder (30g/kg of grain) for adjusting pH and to keep the grains loose. 4. Fill 250 gm of grain in cleaned and dried glucose bottle of 500ml capacity or propylene bags and plug the mouth of the bottle tightly with nonabsorbent cotton. 5. Sterilize the bottles in autoclave by exposing to 121°C and 15Ibs pressure/sq inch for 20 minute. 6. After cooling transfer the bottles to inoculation chamber. 7. Transfer few grains with mycelial growth into sterilized substrate bottle under aseptic condition and plug it with cotton. 8. Shift the inoculated bottles to spawn running room having temperature range of 25-30°C. 33 34 35 Precautions of Mushroom production: The key generic steps in mushroom production cycle that takes between one to three months from start to finish depending on species are: 1. Identifying and cleaning a dedicated room or building in which Temperature, moisture and sanitary conditions can be controlled to grow mushrooms. 2. Pasteurizing or sterilizing the medium (Rice straw) and bags in which, or tables on which, mushrooms will be grown ( to exclude other fungi that would compete for the same space – once the selected fungi has colonized the substrate it can fight off the competition). 3. Seeding the beds (Rice straw) with spawn (spores from mature mushrooms grown on sterile media). 4. Maintaining optimal temperature, moisture, hygiene and other conditions for mycelium growth and fruiting, which is the most challenging step ; adding water to the substrate to raise the moisture content. 5. Harvesting and eating or processing, packaging and selling the mushrooms. 36 A. Edible Mushroom - are consumed by humans for their nutritional value and they are occasionally consumed for their supposed medicinal value. - Edibility may be defined by criteria that include absence of poisonous effects on human and desirable taste and aroma. - Edible mushrooms include may fungal species that are either harvested wild or cultivated. - E.g. Agaricus bisporus & Volvariella volvacea B. Poisonous mushrooms - Poisonous mushroom look like edible mushroom in their morphology and lifecycle. However they can be distinguished by following features:- 1. Brightly colored fruit bodies. 2. Pink colored spores in gills. 3. Presence of Vulva and Annulus on the stalk 4. Unpleasant odor. 5. E.g. Amanita phalloides & Tricholoma muscarium 37  The Nutritional value of mushrooms: 1- Protein: Most mushrooms have a high protein content , usually around (20-30%) by dry weight. 2- Fiber: Helps lower cholesterol and is important for the digestive system. 3- Vitamin D: Essential for the absorption of calcium. 4- Copper : Aids in helping the body absorb oxygen and create red blood cells. 5- Selenium: An antioxidant that helps neutralize free radicals. 6- Potassium : regulates blood pressure. 7- Low levels of fat ,calories , sodium. 8- No cholesterol. 38 Fig. Structure of Mushroom 39