EXPERIMENT I: ISOLATION AND IDENTIFICATION OF PROBIOTIC BACTERIA AND YEAST Introduction Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” Criteria to Qualify Microorganisms as “Probiotic” in Foods and Dietary Supplements Food and Agricultural organisation (FAO)/ World health organisation (WHO) definition of probiotics can be translated into four simple and pragmatic criteria allowing one to conclude if specific strains of microorganisms qualify as a probiotic for use in foods and dietary supplements. Probiotic strains must be (i) sufficiently characterized; (ii) safe for the intended use; (iii) supported by at least one positive human clinical trial conducted according to generally accepted scientific standards or as per recommendations and provisions of local/national authorities when applicable; and (iv) alive in the product at an efficacious dose throughout shelf life. Lactic acid bacteria (LAB) as protective cultures are common probiotic organisms that are considered safe due to having specific characteristics. Main genera of LAB are Leuconostoc, Enterococcus, Lactobacillus, Lactococcus, Bifidobacterium, Pediococcus, and Streptococcus. These bacteria cause reduction of gastrointestinal diseases by increasing benefit microorganisms’ growth and reducing pathogens’ population mechanisms. LAB are widely distributed in the environment that can prevent the growth of pathogenic microorganisms by producing particular substances. According to scientific reports, antiallergic and anticancer effects, increasing fat loss and immune response of the host, improvement symptoms of irritable bowel syndrome, intestinal inflammation, and antibiotic-induced diarrhoea are other useful effects of probiotics. Nowadays, probiotics are used not only as a driver of growth but also as a stimulator of the immune system and prevention of many diseases. Food probiotic products because of their nutritional value and health sector over the past parallel to the therapeutic effects are taken into consideration. Lactobacilli are Gram-positive and catalase-negative bacteria that are known as most important probiotics and desirable gut microflora. They are normal flora of mouth, intestine, and female genital tract with important role in the control of unde: microorganisms that can be considered as natural bio-preservatives. rable Aim- To isolate and identify probiotic bacteria and yeast Requirements- MRS agar, Petri plates, chloramphenicol, incubator, probiotic food sample ( yogur/kefir/probiotic chocolate etc),Isolation and Identification of probiotic bacteria Procedure + Two grams of probiotic food sample was transferred in a flask containing sterile MRS Broth as enrichment media and incubated in 37°C for 24 h + After 24 h of incubation, 100 pl of enriched samples were spread on MRS agar and incubated at 37°C and incubated for 48 h. Bacterial colonies were purified by subsequent subcultures. + The bacterial cultures were identified by Gram-staining and microscopic observation. Isolation and Identification of probit ic yeast Yeast strains were isolated by surface-streaking of the food sample onto yeast-extract glucose chloramphenicol (YGC) agar/ MRS agar with medium with chloramphenicol and incubated at 30°C for 2-5 days in aerobic conditions. “The pure cultures of isolates were preserved by freezing for longer preservation. + Isolated Yeasts were observed by using microscope. Observation and ResultEXPERIMENT 2: DETERMINATION OF MICROBIOLOGICAL QUALITY OF MILK By MBRT Aim: Determination of microbiological quality of milk samples by Methylene blue reduction test (MBRT) Principle: Milk is an excellent bacteriological medium. Non-boiled or unpasteurized milk contains a large number of bacteria. The milk in udder of a healthy cow is sterile, and the microorganisms that entered the teat canal through teat openings mix with milk during the process of milking. The udder surface contaminants, dairy utensils, water and other unhygienic conditions during milking, transport and handling result in contamination of milk with various microorganisms. Milk, being a very good nutrient, supports various groups of microorganisms. Hence to study the microbiological quality of milk, a number of tests have been devised. Methylene blue reduction test (MBRT) is one of the widely used tests. It is based on the oxidation-reduction activities of the bacteria present in milk. Methylene blue is ‘an oxygen sensitive dye and will be blue coloured in oxidized state and white in reduced state. When the dye is added to the milk it turns blue, since oxygen is presently in sufficient amount. On incubation, the milk may turn white depending upon the activity of the microorganisms present in the milk. The time for colour disappearance depends upon the number of microorganism’s present in the milk. More the bacterial population of the milk samples faster the rate of dye reduction. If the milk samples take six hours or more to decolorize the dye, they are considered as of good samples microbiologically. If decolorization occurs within 30 minutes, the samples are considered as of poor quality. This test was first employed by Barthel and Orla-Jensen in 1900 and later modified by Wilson and his colleagues. It is the official test for both untreated and pasteurized milks. Requirements: “Milk samples «> Methylene blue solution (1:25000) 4 Sterile screw capped bottles and pipettes (1 ml and 10 ml) Thermostatically controlled water bath maintained at 37°C 4 Sterile distilled water Procedure: Prepare fresh methylene 4 Collect unpasteurized milk samples from different soure distilled blue solution before the test, by dissolving 1 mg of the dye in 25 ml of sterile water. 4 Then distribute milk samples to sterile screw capped bottles or test tubes, at the rat 10 ml using sterile 10 ml pipettes. % Then add | ml of methylene blue solution into each 10 ml milk sample an shake so that the dye is uniformly mixed. id gently4 Keep the tubes in a suitable beaker and place them in water bath maintained at 37°C. # A number of samples can be prepared and incubated at a time. For each sample maintain at least three replicates, Observation: Observe the incubated milk samples regularly at 1 Sminute intervals. Record the time taken for decolorization of the dye in each sample, Result:EXPERIMENT-3 PREPARATION OF FERMENTED FOOD- YOGURT AND SAUERKRAUT AIM: To prepare fermented food- yoghurt Requirements: Pasteurized or sterilized milk, standard beaker, cultures of Streptococcus thermophilus and Lactobacillus bulgaricus. Procedure: The standard milk obtained from market is pasteurized in lab for 60-80C for 30 min. The milk is allowed cool to 42°C temperature. lactobacillus bulgaricus were inoculated 37°C-45°C for 6 to 8 hours, Streptococcus thermophilus and into pasteurized milk and is incubated between Result:10 ‘vim — To Prepare fermented food- Sauerkraut Sauerkraut is “acidic cabbage” produced by natural fermentation with Introduction- indigenous bacteria to cabbage in the presence of 2 to 3% salt. During fermentation lactic cid is formed as the major product which along with other minor products of fermentation, gives sauerkraut its characteristic flavour and texture, Salting of the cabbage serves two major purposes, Firs, it causes an osmotic imbalance resulting in the rel nutrients from the cabbage leaves. Second, the salt concentration used during fermentation inhibits the growth of many spoilage organisms and pathogens. It is critical 10 exclude oxygen throughout the fermentation because presence of oxygen would permit the growth of some spoilage organisms, particularly the acid-loving molds and yeasts. ase of water and ‘This is process is referred as a wild fermentation. Because there is no addition of staner cultures, The normal flora of the cabbage leaves is relied upon organisms responsible for desirable fermentation, as they enhance preservation and organoleptic acceptability. The succession of microbial flora is governed mainly by the pH of the growth medium. Initially, coliform starts the fermentation. Coliforms which contributes to sauerkraut fermentation are identified as Klebsiella pneumoniae, K. oxytoca and Enterobacter cloacae. Acid production makes the environment more favourable for Leuconostoc growth. As the coliform population declines, the Leuconostoc population builds. As Leuconostoc is a heterofermentative lactic acid bacterium, much gas (carbon dioxide) accompanies the acid production during this stage. The pH continues to drop, and a strain of Lactobacillus succeeds the Leuconostoc. (oceasionally a strain of Pediococcus arises instead of Lactobacillus.) The complete fermentation involves succession of three major groups of bacteria, wh decreasing pH. Sometimes accumulated acid has been found to involved in the fermentation. ich is governed by the off the organisms Procedure % Trim the cabbage heads, remove the outer leaves and all bruised or soiled tissue. “Wash the trimmed heads thoroughly with tap water. * Cul the heads in half and remove the hard, central core. 4 Shred the cabbage with the shredder. * & Weigh fee cabbage. Mix the salt to get a final concentration of 2.5%. Fe orough and even mixing of the salt is important for this process. rack the shredded cabbage into the crocks/glass bottles, 80% of total volume. ; i : ing to approximately 75- ‘ompress the mixture moderately while avoidi in ls 0 ately ing crushing bruising the cabbage tissue, Place the lid and wei le aa cheesecloth % Incubate the crocks at 21 to 24°C for 5-6 week ghts and finally cover with Observation and results12 Experiment. 4 Microbiological analysis of food- isolation, enumeration & detection of pathogens Introduction-The incidence of food borne diseases has increased over the years and resulted in major public health problem globally. It is important to detect food borne pathogens to provide safe food supply and prevent diseases. Culture-based methods are generally regarded as the ‘gold standard’ for microbiological analysis of food. Traditional culture relies on the ability of bacteria to grow and multiply on laboratory media and form visible colonies. These methods still represent the first choice for many food testing laboratories as they are sensitive, inexpensive, easy to use, and give either qualitative or quantitative information on the number and type of viable microorganisms present in the food samples Procedure: Isolation and enumeration of aerobic mesophilic organisms L distilled water) Il. Take Iml of the above mixture and serially dilute using sterile distilled water i. sterile Luria agar/ Nutrient agar. IV. Incubate the plates for 24-48 hrs at 37°C. V. Observe the cultural characteristics, microscopic morphology and calculate the Colony Forming Units. - incubation and calculated using the formula- CFU/ml = (no. of colonies x dilution factor) / volume of inoculum plated Observation and Result Prepare 1:10 dilution of given food sample using appropriate diluents (ex: sterile Inoculate 0.1 ml of sample from each of these samples by pour plate method using For enumeration, number of colonies growing on the agar medium were counted after no of microbial cells present in each 1 ml of stock mix were Se a i3 Detection of pathogens (any one) Pathogens present in food samples are detected using selective culture m staining and biochemical characteristics \edia followed by Bacillus cereus in Foods Inoculate each of three tubes of trypticase-soy-polymyxin broth with I ml food homogenate and its dilutions. Incubate at 30°C for 48 hours. Examine for dense growth typical of B.cereus Vortex-mix and using a 3mm loop transfer one loo} tube to dried MYP medium plates. Streak to obtain isolated colonies. v. Incubate at 30°C for 48 hours. vi. Pick one or more eosin pink (mannitol fermentati by precipitate zone (due to lecithinase activity) nutrient agar slants for confirmation tests i. pful from each growth positive iv. ion positive) colonies surrounded from each plate and transfer to MYP Agar (Mannitol Yolk Polymyxin) Preparation A- Meat extract .... Peptone D-mannitol . Sodium chloride .. Phenol red ... Agar Distilled water .. Preparation B- Egg Yolk Emulsion: 50%: Wash fresh eggs with stiff brush and drain, Soak 1 hour in 70% alcohol. Aseptically remove yolk and mix (I-+1) with sterile 0.859% sodium chloride solution. (Difco Egg Yolk Enrichment 50% is satisfactory). Preparation C— Polymyxin B sulfate, This selective agent is obtainable in sterile powdered form (500,000 units, ie, 50 mg per vial). Add aseptically, by syringe, 5.0 ml sterile distilled water. Mix to dissolve powder. Add 1.0 ml by syringe to a liter of final medium.14 Detection of S, aureus Inoculation for enrichment-Inoculate tryptose soy broth (with 10% sodium chloride and 1% sodium pyruvate) with Iml of food homogenate. Incubate at 35°C for 48h. Isolation of pure colonies-Vortex mix the tubes from above and then using 3mm loop transfer one loopful from each growth positive tube to dried Baird- Parker medium plates. Streak so as to obtain isolated colonies. Incubate at 35-37°C for 48 hours Colonies of S. aureus are typically grey black to jet black, circular, smooth, convex, and moist ; 2-3 mm diameter on uncrowned plates. Frequently there is a light coloured (off-white) margin, surrounded by opaque zone (precipitate) and frequently with outer opaque zone (Precipitate) and frequently with outer clear zone; colonies have buttery to gummy consistency when touched with the inoculating needle. Test for coagulase production on suspected colonies. Detection of Pathogenic Vibrios in Foods. Enrichment — Weigh 25g sample and transfer to 225ml of GPS broth. Incubate at 35°C for 6 to 8 hrs. Plating: Prepare dried plates of TCBS and GPS agar medium (Thiosulphate Citrate Bile Salts Sucrose Agar, Gelatin Phosphate Salt Broth (GPS) and Agar). Transfer a loopful of the surface Srowth of the broth culture to the surface of the two plating medium and streak in a manner that will yield isolated colonies. Incubate plating medium for 18 to 24h at 35°C. Interpretation: Typical colonies of V.cholerae on TCBS agar are lar; ge (2 to 3 mm in diameter) smooth, yellow (occasional slow sucrose fermentors are green), and slightly flattened with opaque centres and translucent peripheries. On GPS agar the colonies have a cloudy zone around them that becomes more definite after a few minutes of refrigeration. In oblique light, the colonies appear iridescent green to bronze coloured and finely granular. Typical colonies of V. parahaemolyticus on TCBS agar appear round, opaque, green or bluish colonies, 2 to 3 mm in diameter. Observation and Results16 Experiment-5 & 6 EXTRACTION AND DETECTION OF MYCOTOXIN Aim- To extract Aflatoxin (Mycotoxin) from food samples and detect mycotoxin using TLC. Introduction- A flatoxins occur in foods and feeds as a result of mold growth and in products such as milk meat and eggs as a result small of ingesting moldy feed by animals. Aflatoxins are potent carcinogens. Aflatoxins, produced mainly by Aspergillus flavus Aspergillus parasiticus, have been documented as one of the major food contaminants throughout the world. Because of their toxic nature, these food contaminants have acknowledged considerable attention in recent years. Among the different types of Aflatoxins, the most prevalent and predominant Aflatoxins are AFBI, AFB2, AFGI, AFG2, AFMI, AFM2 which are considered the more lethal as compared to others. Their control requires quantitative method of analysis that are sensitive to concentrations of micrograms to picograms per kilogram of food or feed. The aflatoxins are well suited for analysis by TLC since most of the compounds fluoresce strongly under long-wave UV light. The TLC technique serve as both purification and quantitation step. Before TLC analysis, the aflatoxins are extracted from the sample, usually with an aqueous organic solvent, and the extract is initially purified by one or more techniques such as solvent partition, heavy metal precipitation, column filtration, or chromatography. Requirements-Food sample, SMKY broth (Sucrose-200g, MgSOu-0.5g, KNOs- 0.3g and yeast extract- 7.0g in one litre of distilled water) Procedure for Extraction 1, Food samples (Chillies/Groundnuts/Wheat) to be brought to laboratory and stored in a sterile Petri dish with moist filter papers for 3 to 4 days to encourage visible fungal growth. 2. Food sample with fungal contamination should be inoculated in sterile SMKY or YES broth media for fungal multiplication and toxin production. Inoculated flasks must be incubated for 7 days at 25-27°C. 3. Aflatoxin is extracted by solvent extraction procedure 4. Mycelial mat was filtered using Whatman no.1 filter Paper. The filtrate was collected in a bottle and defatted using n hexane 5. Sodium chloride (80mg/ml) was added to the filtrate, 6. Further, to the filtrate, chloroform (half the volume of filtrate) was added and shook well at 100 rpm for Ihr. 7. The chloroform extract is separated using a separating funnel and dried at room temperature and stored for further analysisv7 Procedure for Detection 1.TLC plates coated with silica (0.75mm thickness) were purchased or prepared and pre heated at 110°C for 30 min. 2.The dried chloroform extracts(toxin) is made upto ml using benzene: Acetonitrile (98:2). This extract (20 -30 microliters) is spotted on TLC plate 3.Toluene: Ethylacetate: Formic acid (6:3:1) solvent mixture was utilized for development of TLC Observation The developed TLC plates are visualized using UV trans illuminator (365nm). Aflotoxin AFBI appeared as bright blue illumination. Result18 EXPERIMENT 7 ISOLATION OF MICROORGANSIMS IN AIR BY IMPINGEMENT METHOD AIM: To isolate the microorganisms from the air by impingement method. Principle: Airborne bacteria and fungi may cause several adverse effects, especially infectious, allergenic, and immunotoxic disorders. Microbiological air quality is an important criterion that must be taken into account when indoor workplaces are designed to provide a safe environment. Air microflora can be isolated and enumerated by various passive or active methods Passive monitoring uses “settle plates”, which are standard Petri dishes containing culture media, which are exposed to the air for a given time in order to collect biological particles which “sediment” out and are then incubated. Results are expressed in CFU/plate/time or in CFU/m2/hour In active monitoring a microbiological air sampler physically draws a known volume of air through or over a particle collection device which can be a liquid or a solid culture media or a nitrocellulose membrane and the quantity of microorganisms present is measured in CFU (colony forming units)/m3 of air. This system is applicable when the concentration of microorganisms is not very high, such as in an operating theatre and other hospital controlled environments. Requirements: agar strips, air sampler, incubator. Procedure ot Insert the agar strip in the air sampler machine Switch the on button to force the air entry into the sampling device Switch off the sampler after required time period Incubate the agar strips e oe Observe the agar strips regularly for the development of colonies. The bacterial colonies develop within 48 hours. The fungal colonies are usually indistinct upto 48 hours and it may take 4 to 5 days for sufficient sporulation useful for identification, Classify the colonies basing on their morphology and count the number of each type of colonies and tabulate the results20 EXPERIMENT 8 MICROBIOLOGICAL EXAMINATION OF WATER BY COLIFORM TEST (MULTIPLE TUBE FERMENTATION METHOD) ‘AIM: To examine the microbiological quality of a water sample by multiple tube fermentation test for the presence of coliform bacteria. Principle: Apparently clear looking water without any particulate matter may or may not be pure microbiologically. Some organisms naturally occur in water and some acquired from the soil. Usually the natural microflora of water is pathogenic to humans and animals. But some times the water is contaminated with sewage, especially domestic sewage and it contaminates the water with potentially pathogenic intestinal pathogens. Hence, drinking water supplies have to be treated before supply. The potability of water not only depends upon the chemical constituents of water but also by its microbiologists quality. The contamination of sewage is assessed by testing the water samples for the presence of coliform bacteria. The bacteria present in the colon or large intestine of humans are generally called coliforms, and mainly represented by Escherichria coli and other facultative bacteria. ‘The presence of coliforms in water samples is mainly tested by two methods viz. membrane filter method and multiple tube fermentation test. The second one is a statistical test to determine the coliform bacterial populations in water sample, and is commonly used for estimating the coliform bacteria in water samples. Coliform bacteria in water samples. Coliform bacteria are facultative anaerobic rod shaped organisms that ferment lactose with different concentrations of water sample, and observed for gas production. From the observations, the most probable number of coliform bacteria in water sample is estimated using Statistical Tables. Requirements: Water samples, test tubes, Durham’s tubes Procedure: > Prepare 100ml of single strength and S01 of double strength lactose broth medium by following the medium composition. Preparation of tubes: Disperse 10ml of single strength lactose broth (SSLB) into each of the 10 test tubes. Ina similar way, disperse 10m! of double strength lactose broth (DSLB) into each of the 5 test tubes. Introduce one Durham’s tube in an inverted position into each of the 15 test tubes with broth medium without any air bubbles in it. % Close the mouths of all the tubes with medium with cotton plugs and sterilize in an autoclave. After sterilization, let the tubes to cool to room temperature, % Label the 5 test tubes with DSLB as 10ml, 5 test tubes wi ing 5 SSLB as 0.1ml. ‘ith SSLB as Iml and the21 Inoculation and incubation of the tubes: Inoculate all the tubes with water sample as per labelling, i.e. inoculate 5 test tubes of DSLB medium each with 10ml of water sample, 5 tubes each with Iml of water sample, and remaining 5 tubes each with 0.1ml of water sample. Incubate all the inoculated tubes for 24hours at 37C in an electronically controlled water bath. Observations: + Observe the Durham tubes in all the incubated tubes for accumulation of gas. + Record the test tubes with more than 50% gas production as positive (+ve) tubes for gas production. “ Tabulate the results in the following table. Sample no. Number of Durham tubes +ve for gas with production 10m! inoculum Im| inoculum 0.1ml inoculum