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Mic-240: LABORATORY SKILLS IN MICROBIOLOGY (Dr. Sarfaraz Hadi )

Mic-240: LABORATORY SKILLS IN MICROBIOLOGY (Dr. Sarfaraz Hadi ). Mic-240: Laboratory skills in microbiology Marks allocation. Total marks: 100 marks Sessional Exams : 50 marks class room tests: 2 @ 10 = 20 Lab Exercises: 2 @ 15 = 30 Final exam : 50 marks.

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Mic-240: LABORATORY SKILLS IN MICROBIOLOGY (Dr. Sarfaraz Hadi )

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  1. Mic-240:LABORATORY SKILLS IN MICROBIOLOGY(Dr. SarfarazHadi)

  2. Mic-240: Laboratory skills in microbiologyMarks allocation Total marks: 100 marks Sessional Exams: 50marks class room tests: 2 @ 10 = 20 Lab Exercises: 2 @ 15 = 30 Final exam: 50marks

  3. Scope of the present course module - Hazards in microbiology lab - Precautions to be taken in a microbiology lab - Preparation of stock solutions - Preparation of media - Sterilization of media and solutions - Culturing bacteria and fungi - Use of laminar air flow hood - common media used for fungal and bacterial culture - Isolation of pathogenic fungi and bacteria from plant material - Isolation of comensal bacteria from human body - Microscopy - Staining and observation of fungi - Gram staining of bacteria - Extraction/isolation of DNA - Basic concept of polymerase chain reaction (PCR)

  4. Equipments used in a microbiology lab - Air sampler - Autoclave - Laminar air flow hood - Bead sterilizer/ Bunsen’s flame - Hand instruments - Incubator - Colony counter - Microscope - Thermal cycler (PCR) - Distillation unit - Precision balance - Heater/microwave oven - Stirrer - Lab ware: glass ware/plastic ware/petriplates

  5. Hazards in a microbiology lab. Microbes: - Bacteria - Fungi Chemicals: - Acids and alkalis – HCl, NaOH etc. - Fungicides and antibiotics - Other toxic substances - HgCl2 , Fumes: - Chlorine, formalin etc.

  6. Hazards in a microbiology lab.Cont… - UV light - Hot materials – media, instruments etc. - High pressure steam (autoclave) - Glass splinters, scalpel blades etc.

  7. Precautions(Good laboratory practices) • Wear a lab coat to protect clothing from contamination or accidental spillage of harmful chemicals and staining solutions. • Wash your hands properly before and after the work. • Use face mask and cap during the work. • Clean the work area (laboratory bench) with a recommended disinfectant such as 5% Lysol or 5% phenol before and after each work session. • Never eat, drink, or smoke in the laboratory.

  8. PrecautionsCont… • Keep the laboratory work area free from articles not in use and return all reagents, cultures, and glassware back to their appropriate places. • Keep your hands away from your mouth and eyes while in the laboratory. Do not place anything such as pencils, food, and fingers in your mouth. • Avoid contamination of benches, floor, and wastebaskets. • Place all discarded cultures, infectious materials, used glass slides and contaminated glassware into receptacles meant for special disposal.

  9. PrecautionsCont… • Place all used or contaminated pipettes in appropriate glass jar filled with a disinfectant such as 5% phenol. • If infectious material is spilled accidentally, cover it immediately with a disinfectant such as 5% Lysol or 5% phenol and notify your seniors at once. • Do not move with a loop or pipette containing infectious material through the laboratory. Flame wire loops and needles before and after use. • Label all your experimental material properly.

  10. Stock solutions • Working solution • Stock solution • Dilution of stock solution

  11. Percent solutions - Strength of the solution: percentage or parts per hundred. - Weight in grams – volume 100 ml. - Same percentage: variation in volume → variation in weight. Amount of solute in grams = Required %age × required volume (in liter) × 10 Example: Prepare10% solution of NaCl in 25 ml volume Amount of NaCl in gram = 10 × 0.025 × 10 = 2.5 g -Weigh 2.5 g NaCl , - Dissolve in ≈ 20 ml water, - Make volume 25 ml.

  12. Percent solutions -

  13. ppm (parts per million) solutions(mg per liter) Formula: Amount of solute in mg = Required concentration× required volume (in liter) Example: Prepare100 ppm solution of NaCl in 250 ml volume Amount of NaCl in mg = 100 × 0.25 = 25 mg -Weigh 25 mg NaCl , - Dissolve in ≈ 200 ml water, - Make volume 250 ml.

  14. Recording experimentscont… Exercise : Prepare……………. ppm solution of sodium chloride (NaCl) to make final volume …………….. ml. Objective:   To prepare……………. ppm solution of sodium chloride (NaCl) to make final volume …………….. ml. Materials used: 1. Sodium chloride (NaCl) – lab grade, manufactured by ………………. 2. Precision weighing balance, weighing boat, spatula 3. volumetric flask of known volume, glass funnel 4. distilled water, wash bottle Procedures: - Calculated the weight of NaCl required according to the formula: Weight of NaCl required = concentration required in ppm x volume required in liter. - Precisely weighed NaCl in a weighing boat on a precision balance using a spatula. - Transferred entire quantity of NaCl into the volumetric flask by washing the boat with dist. Water. - Poured about half the final volume distilled water and dissolved NaCl completely - Made the final volume to the mark of volumetric flask (bottom of the meniscus touching the mark). Results: Prepared ………….. ppm solution of NaCl to make final volume ………………. ml.

  15. ppm (parts per million) solutions(mg per liter) -

  16. Molar solutions Formula weight (FW)/Molecular weight (MW) in grams = 1mole → 6.0021415x1023 molecules (Avogadro’s number) → Dissolved per liter = 1 Molar (1M) solution = 1 mole per liter (K=39; Na=23; Cl=35; KCl= 74.55; NaCl= 58.44) Amount of solute in grams = Required Molarity × FW × required volume (in liter) Example: Prepare 200 mM (0.2M) solution of NaCl (FW: 58.44) in 25 ml Amount of NaCl in grams = 0.2 × 58.44 × 0.025 = 0.292 g (292.2mg) -Weigh 0.292 g NaCl , - Dissolve in ≈ 20 ml water, - Make volume 25 ml.

  17. Usage (dilution) of concentrated solutions - Stock solutions: high concentration. - Working solution: lower concentration. - Transfer portion of stock solution to working solution. S1 × V1 = S2 × V2 where, S1: Strength of stock solution (in ppm or %age, or Molarity) S2: Strength of working solution (in same unit as S1) V1: Volume of stock solution to be used (in ml or liter) V2: Volume of working solution (in same unit as V1)

  18. Usage (dilution) of concentrated solutionscont… Example: find the volume of 2M stock solution to be used for making 200mM (0.2M) working solution of 50 ml volume. S1 × V1 = S2 × V2 Strength of stock (2M) x volume required = Strength of working solution (0.2M) x volume of working solution (50ml) Volume required = (0.2 x 50) ÷ 2 = 10 ÷ 2 = 5 ml (Same procedure to be followed for %age, ppm, or ppb solutions)

  19. Lab exercise – 4Dilution of ppm solution -

  20. Lab exercise – 5Dilution of molar solution -

  21. Stock solutions with strength of solute in multiples Concentration → Multiple of known amount per liter Example: Required amount in working solution: Solute x 2 g/l Solute y 3 g/l Solute z 4 g/l 5X Strength of stock solution (in 1 liter): Solute x 2 g/l x 5 = 10 g Solute y 3 g/l x 5 = 15 g Solute z 4 g/l x 5 = 20 g Water ……………………. to make 1 liter Amount of solute in stock solutions = conc. of solute in working solution × Strength multiple of stock × volume of stock solution (same unit as for working solution).

  22. Stock solutions with strength of solute in multipleCont… Example: Medium / working solution - NaCl: 2 g/l Make 5x solution – 50 ml Amount of NaCl required (in gram) = 2g/l × 5 × 0.05 liter = 0.5 g Usage: 5x soln. – 50 ml Vol. of stock soln. to use (in ml): Vol. of working soln. ÷ Strength of stock solution (in x) Example: volume of above stock required for 100 ml working soln.? 100 ml ÷ 5x = 20 ml

  23. Lab exercise – 6Dilution of multiple strength solution -

  24. In-vitro culture of microorganisms(Fungi/Bacteria) 1. In-vitro: Inside the glass (Latin) – (tube – petri plate – flask) 2. Culture: Growth under controlled conditions (sterility – nutrition – temperature – humidity – light etc.) 3. All work to be performed under sterile conditions – on a laminar air flow hood – to avoid contamination 4. Hand instruments (loop – forceps – needle – scalpel etc.) to be sterilized in an autoclave or with a flame/bead sterilizer.

  25. 5. Culture media 1. Nutrition - Carbon - sucrose, glucose etc. - Nitrogen (N) – amino acids - Minerals – (Natural sources/synthetic form) - Phosphorus (P), Sulfur (S), Iron (Fe), Potassium (K), Copper (Cu), etc. - Essential amino acids - Vitamins 2. Support system/matrix – solid (Agar) or liquid 3. Aeration

  26. 5. Culture mediacont… Types of media: 1. Defined media: components are known/controlled 2. Undefined/complex media: components supplied through natural sources - yeast extract, peptone etc. Solidification/gelling of the medium: agar, phytagel, agarose

  27. 5. Culture mediacont… Preparation of media: 1. Weight and dissolve media components in water one by one (add agar in the last) 2. Adjust pH of the medium (if required) 3. Sterilize the medium – Autoclaving: 20 psi/1.4 kg/cm2 20 min. 4. Allow to cool down to ~60 °C 5. Pour in sterilized petri plates or tubes

  28. 5. Culture mediacont… 7. When making medium in unsterilized tubes: - Pour the medium in tubes before autoclaving - autoclave the tubes 8. Tilt the tubes with molten agar medium by 30-35° to make slants - To increase surface area - To make handling more convenient - To make a gradient of thickness and moisture

  29. 5. Culture mediacont… Frequently used media in microbiology lab PDA (Potato Dextrose Agar) medium: - Take 300 g potato and wash well with tap water - Cut the tubers to small pieces (~ 1 cm) without peeling - Boil in 1l distilled water till it remains 700 – 800 ml - Sieve the content - Add and dissolve 20 g dextrose (glucose) - Add 20 g agar and make volume 1000 ml - Autoclave at 20 psi for 20 minutes

  30. Lab Exercise Exercise : Prepare 250 ml PDA (Potato Dextrose Agar) medium , pour and solidify it in sterilized petri plates. Introduction/Objective:   To Prepare 250 ml PDA (Potato Dextrose Agar) medium , pour and solidify it in sterilized petri plates. Materials used: 1. Potato - ~100 g; knife/scalpel. 2. Dextrose (glucose), Agar powder, distilled water. 3. Weighing balance, conical flask, autoclave. 4. Laminar air flow hood, sterilized (disposable) petri plates

  31. Lab Exercise-Cont… Methods/Procedures: 1.Washed potatoes with tap water; cut into small pieces (~ 1 cm). 2. Weighed 100 g potato pieces, transferred into 500 ml flask, added 250 ml dist. water. 3. Boiled in autoclave at 20 psi for 15 min. 4. Sieved the broth in another 500 ml flask. 5. Added, dissolved 5 g glucose + 5 g agar powder. 6. Autoclaved: 20 psi – 15 min; allowed to cool to ~ 60 °C 7. Poured in petri plates in laminar flow, sealed with parafilm. Results: Prepared 250 ml PDA medium as above.

  32. 5. Culture mediacont…

  33. 5. Culture mediacont… • Media for culturing fungi • Media for culturing bacteria • - Defined media • - Undefined/complex media • V8 medium • Nutrient agar • Nutrient broth • LB broth • LB agar • Sabouraud Medium • Zapek-Dox

  34. Media for bacterial culture Nutrient agar: Peptone………………….5g Meat extract……………..3g Agar……………………..15g Distilled water to make….1 liter • Weigh the components/Premade medium • Mix in distilled water (~ 800 ml) • Make the final volume 1 liter • Autoclave at 20 psi for 20 minutes • Pour in petri plates on laminar flow

  35. 4. In-vitro culture of microorganismscont…Laminar air flow hood/bench 1. Laminar air flow hood/bench (laminar: in layers) Pre filter Blower HEPA filter (high efficiency particulate air filter) Pressure gauge (20 mm water/ 200 pa - Pascal) light source UV radiation source (sterilization) Provision for gas pipe/electrical power 2. Air velocity: 0.4 – 0.5 m/sec – 90 FPM (feet per minute)

  36. 4. In-vitro culture of microorganismscont…Laminar air flow hood/bench

  37. 4. In-vitro culture of microorganismscont…Laminar air flow hood/bench

  38. Cleaning the work bench 1. Laminar flow hood or work bench 2. Immediately before and after the work 3. 70% alcohol 4. Lacto phenol 5. Commercial preparation 6. Hands should also be regularly wiped with 70% alcohol or a disinfectant before and during the work.

  39. 3. Sterilization - Complete destruction or elimination of all forms of microorganisms from a material by physical or chemical methods. - Media and solutions - Hand instruments - glassware, and petri plates etc.

  40. 3. Sterilization Cont… 1. Application of dry heat Direct flaming – (Hand instruments: Loops, Forceps, Scalpel etc.) Glass bead sterilizer – (Ditto) Hot air oven – (Glassware, hand instruments etc.) 2. Application of moist heat Autoclaving (steam under pressure) - 20 pounds per square inch (psi)/ 1.4 kg per sq. cm - 121 °C - (Media, glassware, lab coats etc.)

  41. 3. SterilizationContApplication of moist heat Autoclaving (steam under pressure)

  42. 3. SterilizationCont… 3. Irradiation (200-280 nm) Ultra violet (UV) radiation: 200-280 nm (nano meter) - (Hospitals, OTs, Labs etc.) Gamma radiation (γ): 2-10 pm (peco meter) - (Food material, Lab ware, Liquids etc.) 4. Chemical treatment Mercuric chloride - (Host material etc.) Sodium hypochlorite - (Host material etc) Formalin, phenols - (Work bench, floor etc.) Ethanol - (Instruments, etc.) Isopropanol - (Instruments, etc)

  43. 3. SterilizationCont… 5. Filter sterilization Asbestos filters, membrane filters and sintered glass filters: pore size: 0.1µm , 0.22µm, 0.4µm – 10 µm - (Media, Heat labile solutions,)

  44. 3. SterilizationCont… Filtration

  45. Cleaning glassware 1. Use sterilized disposable plastic ware Or 2. Submerge the glassware in a cleaning solution: • Potassium dichromate ................... ............ 2 g • Distilled water ................................ ......... 200 ml • Dissolve dichromate in water • when cool, add very slowly: • Concentrated sulfuric acid ........................ ..9 parts • Potassium dichromate solution (2%)…... ..1 part

  46. Isolation of fungi Materials required: - Tape water - Ethyl alcohol - Scalpel/blade - PDA plates Procedures/Methods: - Clean/wash the source thoroughly - Wipe the surface with alcohol to kill external organisms - Cut a thin slice of tissue/material from the infected source - Plate it on PDA in a petri plate

  47. Isolation of bacteria 1. From infected plant material: Same as for isolation of fungi 2. From human body: (Mouth, Nose, ears etc.) Materials required: - Cotton buds - Nutrient broth/Nutrient agar Procedure/Methods: - Rub the bud on the probable site of bacterial colonization - Shake the bud in nutrient broth/streak on nutrient agar - Incubate at appropriate temperature (28-37 °C) for appropriate length of time (8-24 h)

  48. Isolation of bacteriaCont…2 3. From human body: (blood, sputum, urine, other fluids) Materials required: - Pipette - Nutrient broth/Nutrient agar Procedure/Methods: - Pipette a small portion in nutrient broth or plate on nutrient agar - Incubate at appropriate temperature (28-37 °C) for appropriate length of time (8-24 h) - Make pure cultures, if needed (contamination/mixture) - Observe and identify the cultures under a microscope

  49. Microscopy Microscopy: Technique of using microscopes for viewing magnified image of small objects. Microscope: Aninstrument to observe small objects not visible to naked eye. → Greek: ‘micros’ = small + ‘skopein’ = to look

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