P Noakhali Science and Technology University Microbiology Practical Manual Course Code: PHAR 2206 Experiment No: 01 Name of the Experiment: Gram staining of bacterial cells. Theory: Danish microbiologist Christan Gram in 1884 discovered bacterial cell wall composition by one type of differential staining in which some bacterial cells are red (gram negative) and some are blue (gram positive) ‘The process of staining involves ion-exchange reactions between the stain and active sites at the surface of or within the cell. For example, [Bacterial Cell [Na*] + [MB*][Cl] = (Bacteria! Cell}[MB*] + [Na*][Cl] Where. Mb = Methylenr blue. Requirements: 1. Bacterial culture Reagents - Gram crystal violet solution - Gram iodine solution = Gram 95% Ethyl alcohol - Gram Safranine N Glass slide . Dropper . Distilled water .. Microscope . Loopful NOnEw~ Procedure: Preparation of fixed smear: One loopful of distilled water is placed on a clean slide in ord to smear for each loop. armen A portion of the colony is picked with a transfer needle and the cells are mixed in the water droplets until the mixtures become cloudy. = The slides are allowed to dry in air - The smear is fixed gently over the Bunsen burner for precise fixation. Gram staining usually consists of four steps a) Primary staining with crystal violet solution b) Mordatation with Gram iodine solution ©) Decolorization with 95% ethyl alcohol d) Counter staining with Safranine Primary staining with crystal violet solution: - After preparing the fixed smear on a glass slide, primary staining of the slide is done with crystal violet solution and stands for 30 sec. Mordatation with Gram iodine solution: ‘After discarding the excess crystal violet solution with water, the gram iodine solution of one or two drops is given on that slide and stands for 60 sec. - The slide is rinsed with distilled water - Blotting of the slide is done by blotting paper Decolorization with 95% ethyl alcohol: - After mordatation, 1 or 2 drops of 95% ethyl alcohol is added on the slide and stands for 15 sec - The slide is then rinsed with distilled water - Blotting of the slide is done by blotting paper Counter staining with Safranine: After the completion of decolorization process, one or two drops of Safranine is added to the smear slide and stands for 30 sec - Then the slide is rinsed with distilled water - Then the slide is allowed to dry in air - Finally observation is done under microscope at 10X or 40XResult: ; trod violet colour indicates Gram positive bacteria FA eae rod red colour indicates Gram negative bacteria 2. Figure: Gram staining of Gram negative bacteria,Experiment No: 02 Name of the Experiment: Preparation of pure culture of bacteria and its identification n and characterization Requirements: aurwne . Sample . Distilled water . Bacterial loop . Petriplate/Petriplate . Culture media . Laminar air flow Procedure: - Firstly, the sampl dosage form - The stock soluti condition - 0.1 mlofsto Spreading of sample is done with a glass spreader | Then the sample is incubated in an incubator at an inverted position, temperature 35-379C for 24-72 hours Observation of pure culture is then performed. le is collected in a sterile container from the pharmaceutical ion is prepared from sample and distilled water in an aseptic ck solution is put on bacterial culture media in a petriplate. at Identification of bacteria A. Identification of Escherichia coli Firstly, the pure culture is transferred in a MaCconkey broth (liquid media) Then the pure culture is incubated in an incubator at an inverted position, at temperature 35-37°C for 24-72 hours ‘Then streaking is done on MaCconkey Agar. Incubation is done in an incubator at an inverted position, at temperature 35- 37°C for 24-72 hours Observation: The colony in agar media is brick red colour. - Then the colony is transferred on Eosin Methy! yl Blue (EMB’ i spreading or streaking. : JTC: - Incubation is performed in an incubator at an ii in inverte iti 35-370C for 24-72 hours a’ position, at temperatureObservation: The appearance of bluish colony with metallic sheen indicate the presence of Escherichia coli Characteristics of Escherichia coli - Shortrod = Gram negative - Indole positive és - Brickred colour in Macconkey broth Bluish colour with metallic sheen in EMB Agar Production of H2S gas in Durhem’s Tube. B. Identification of Salmonella spp. - Firstly, the pure culture is enriched in a selective media such i i ae seek as Salanite Cysteine - Incubation is done in an incubator at an invert iti ed ett ene Position, at temperature 35- - Then streaking is done on Bismuth sulphate Agar (BSA)- Incubation is done in an incubator at an inverted position, at temperature 35- 37°C for 24-72 hours Observation: Green or black colony is observed. - Then the colony is transferred on Triple Sugar Iron (TSI) Agar Slant. - Then the preparation is incubated in an incubator at an inverted temperature 35-37°C for 24-72 hours Observation: i) Acidic but (yellow) ii) Alkaline slant (red) iii) Blackening the medium and cracking the medium due to pro' gas. This observation indicates the presence of Salmonella spp- position, at duction of H2S Characteristics of Salmonella spp - Short rod - Circular colony - Gram negative - Flagella present - In BSA Agar, Green or Black colony is observed - In TSI Agar Slant- i) Acidic but (yellow) ii) Alkaline slant (red) WG > eee ana t kB Neen oe -X Fig 3 Wa oMy nS Nh es ple Figure: Salmonella spp.Experiment No: 03 Name of the Experiment: Preparation, Dispensing and Sterilization of Nutrient Agar Media. Purpose: 1. To prepare and sterile a batch of nutrient agar media. 2. To check that the media is sterile. 3. To check that the media as to their ability to support the grow’ bacterial species. th of different Principle: Microorganisms vary widely in their requirement for growth. However all organism require - Suitable energy source = Suitable source of carbon and nitrogen - Water and Adequate amounts of certain mineral salts and trace elements. s require particular organic growth factors such as amino acids In addition, many organism: and vitamins. Nutrient agar although made from a simple recipe is made of complex and unidentified chemical composition and therefore useful for supporting the growth of a wide range of heterotrophic bacteria, which may have various growth requirements. ‘The source materials for making nutrient agar are: water extract of mined lean beef meat extract which contains all the low molecular weight substances that involved in the metabolism of animal tissues, all the intermediate and end products in the biosynthesis of protein, nucleic acids and carbohydrates and in energy generation, together with co-enzymes, vitami Is and minerals. Because of the biochemical reactions in all living thin , vitamins extract contains most of the growth factors, which most of the organi: igs, the meat ‘ganisms are linked a. Beef Extract: A to need. b. Peptone: this is an ill-defined mixture of polypeptides produc: ab 1 protein with the enzymes pepsin and trypsin. This i . carbon and nitrogen to that provided by beef ne sneredient a growth of microorganisms. and y the digestion of dds extra organic helps promote luxuriantc. Sodium Chloride: NaCl is used to make the medium isotonic with mammalian tissues. This is probably not absolutely necessary since the meat extract will contain some NaCl. However it is traditional to make culture media is osmotic with mammalian tissues and extra NaCl is added. d. Agar: Agar is used in nutrient agar medium. It is a glucose containing polysaccharide obtained from marine red alit is a valuable solidifying agent for bacteriological culture media because of its high melting temperature and its insusceptibility to attract by most microorganisms. Formula for Nutrient Agar: Ingredient Nutrient Agar (gm/100 ml) Beef Extract 0.3 gm 4 Peptone 0.5 gm. Sodium Chloride 0.5 gm ‘Agar 1.8 gm Water 100 ml pH 7.4 (approximately) Dissolve all nutrients agar media or suspend the commercially available ingredient in distilled water and then sterilized them by autoclave at 121°C for 15 minutes. Having made a batch of sterile culture medium, it is desirable to check it for sterility by incubating the sterilized medium at 37°C for overnight. Equipments: - Autoclave - Incubator - Balance - Hotplate - Magnetic stirrer - Laminar air flow cabinet - Bunsen burner - Inoculating loop and needle - Measuring cylinder - Spatula - Test tube rack - Conical flaskTest tubes Petridishes Autoclave tape Aluminum foil Pipette Procedures: 1. Appropriate quantities of the ingredients of nutrient agar for 100 ml medium are weighed in beaker. 2. The ingredients are dissolved in 100 ml distilled water by heating on a hot plate magnetic stirrer with continuous agitation. 3. About 6 ml molten agar mediums are dispensed in each test tube. 4, The screw caps are replaced and it should be made sure that the caps are loosed. 5. The tubes and flasks are sterilized by autoclaving at 121°C for 15 minutes. 6. After autoclaving the screw of each tube is closed tightly. 7. While in liquid state the tubes are placed in a slanted position to prepare agar slants. The agar slants are allowed to cool and harden.Experiment No: 04 Name of the Experiment: Microbial count in a supplied pharmaceutical sample. Requirement: 1 . Distilled water . Bacterial loop . Petriplate/Petridish . Culture media . Laminar air flow NQupnwn Sample Burner Procedure: 1) Firstly, the sample is collected from pharmaceutical dosage form. ; 2) Then, the stock solution is prepared from sample and distilled water in an aseptic condition. 3) Consecutive serial dilution of stock solution. 4) After dilution, from each dilution 1 ml of stock solution is taken ina petriplate by pour-plate method (first sample, then media). 5) Incubation is done in an incubator at inverted position at temperature 35-37°C for 24-72 hours. 6) Then, bacterial colony is counted by an appropriate method. Calculation: Total viable count (TVC) = cfu/plate X dilution factor Where, cfu = colony for unit If, bacterial colony 100 cfu/plate is found in third sta TVC= 100 x 103 [this calculation is done for 1 ml] = 105 cfu/ml ge of dilution, then, So, 1 ml contains 105 cfu So, 500 ml contains 105 x 500 =5x 107 cfu/tabletExp No-Y Name of the experiment: Cultivation, Isolation and {dentification of Bacteria from Human Skin (Palm of Hand) Principle: . The skin is the human body's largest organ, colonized by a diverse milieu of microorganisms, most of which are harmless or even beneficial to their host. Colonization is driven by the ecology of the skin surface, which is highly variable depending on topographical location, endogenous host factors and exogenous environmental factors. The cutaneous innate and adaptive immune Fesponses can modulate the skin microbiota, but the microbiota also functions in educating the immune system, In 1938, Price established that bacteria recovered from the hands could be divided into two categories, namely resident or transient. The resident flora (resident microbiota) consists of microorganisms residing under the superficial cells of the stratum corneum and can also be found on the surface of the skin. Staphylococcus epidermidis is the dominant species. Other resident bacteria include S. hominis and other coagulase-negative staphylococci, followed by coryneform bacteria (propionibacteria, corynebacteria, dermobacteria, and micrococci). Resident flora has two main protective functions: microbial antagonism and the competition for nutrients in the ecosystem. In general, resident flora is less likely to be associated with infections, but may cause infections in sterile body cavities, the eyes, or on non-intact skin. ‘Transient flora (transient microbiota), which colonizes the superficial layers of the skin, is more amenable to removal by routine hand hygiene (eg.S. aureus, Gram-negative bacilli). Some of them are pathogenic. Bacteria on the palm of hand can be cultivated in nutrient agar media. This culture can be purified and isolated of particular bacteria by a suitable method. Finally, the size, shape and nature of this isolated bacteria observed by microscope for characterization and identification of this bacteria. Apparatus and Materials Petri dishes Conical flask Flask stopper Weighing Balance Water bath Loop Bunsen Burner . Laminar hood Autoclave Culture media (Nutrient Agar) . Distilled water AMO EOmMMOOmDProcedure . Preparation of Nutrient agar media and pouring into petri dishes . Placing the tip of the finger on to solid media + Incubation of bacteria for 24 hours at a temperature 35-37 C Transfer of contamination free bacteria into fresh sterile media |. Further incubation of this bacterial culture for 24 hours at a temperature 35-37 C. Gram staining of this bacterial culture Final observation of bacteria. AYPweNE Observation: Gram Positive Present/ Absent Gram Negative Present/ Absent Size Tiny/Small/ Medium/ Large/Very Large Shape Circular/ Oval/ Rod/Filamentous Size distribution Number of different bacteria coccus | diplecoce! OGD Vibrio Comma form streptococe! m1 Bacilli Club Rod Halical for Confnebacierincese Heheobacter syleri \ Corkscrew form ) ‘errella burgdonert Streptobecilll Budding and appendaged bacteria ee. eo Pos hypha Comment:Have one/several types of Bacteria (Cocci, bacilli etc.) pup No- 87 Name of the Experiment: The Detection of Bacteriophage presence in Environmental sample (Tap water) by Phage Plaque Assay Principle A bacteriophage is a virus that infects and replicates within bacteria When a suspension of an infective phage (e.g. T4 phage) is spread over a susceptible bacterial cells (e.g. Escherichia coli), the phage attaches the bacterial cell, replicate inside it, and kills it during its lytic release. When this bacterium is cultured in a suitable media, lysis of the bacteriophage is indicated by the formation of a zone of clearing or plaque within the lawn of bacteria. Each plaque corresponds to the site where a single bacteriophage acted as an infectious unit and initiated its lytic cycle. The spread of infectious phage from the initially infected bacterial cell to the surrounding cells results in the lysis of the bacteria in the vicinity, eventually forming the plaque that is large enough to be visible to the naked eye. Plaques do not continue to spread indefinitely. The size of the plaque formed depends on the virus, the host, and conditions of culture The number of plaques that develop and the appropriate dilution factors can be used to calculate the number of bacteriophages i.e. plaque forming units (PFU) in a sample. Apparatus and Materials A. Petri dishes B. Conical flask C. Flask stopper D. Weighing Balance E, Water bath F. Loop G. Laminar hood H. Culture media (Nutrient Agar) 1. Distilled water J. Tap water Procedure 1. eee 2.8 gm of Nutrient agar and mixing with 100ml distilled water in a conical. Boiled and melt agar media applying heat of water bath. . Inoculum of £. coli bacteria is taken in five drops of tap water and mixed to make bacterial suspension. » 4, Melted media is cooled near 50-60 C and mixed the mixture mention under 3, 5. Pouring the mixture in a petri dishes in a thin layer. 6. Incubation of the petri dish for 24 hours at a temperature 35-37 C 7. Visual observation of plaque. 8. Counting plaque in unit area and calculating the number of Bacteriophage per liter of ‘water. Observation: Several plaque is visible or invisible in naked eye. Comment: ‘Tap water is contaminated by Bacteriophage or not contaminated by this virus. Mf plaque is observed, there will a chance to have other viruses and even pathogenic viruses.