MIC151 Manual for students.docx

**Lab Manual** **MSc. Microbiology Semester I** **Year I** **MSC151: Microbiology Laboratory 1** **Total Credit: 2** **\ ** **Certificate** This is to Certify that Mr./Ms.\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ having ID no. \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ of \_\_\_\_...

**Lab Manual** **MSc. Microbiology Semester I** **Year I** **MSC151: Microbiology Laboratory 1** **Total Credit: 2** **\ ** **Certificate** This is to Certify that Mr./Ms.\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ having ID no. \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ of \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ program has successfully completed all the Practicals in Course \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ pertaining to Semester \_\_\_\_\_\_. Therefore, it is hereby declared that student is allowed to appear for the End Semester Practical Examination of this course. **Signature of Course In-charge Signature of Program Chair** **(Name & Date) (Name & Date)** Remarks (if any): **\ ** **Index** **No.** **Title of Experiment** **Page No.** **Date of experiment** **Date of assessment** **Signature of Course in-charge** --------- ------------------------- -------------- ------------------------ ------------------------ ----------------------------------- **Experiment 1 Date:** **Aim: Good Laboratory Practices (GLP) for a Microbiology Lab** ***Write on your own*** ***You can also stick images for this exp.*** **Experiment 2 Date:** **Aim:** To study the various sterilization techniques **Introduction:** **Sterilization** **Disinfection** **Decontamination** **Sanitization** **[PHYSICAL METHODS OF STERILIZATION:]** 1. **Sunlight**: 2. **Heat:** **Factors affecting sterilization by heat are:** **Action of heat:** ### ### ### ### ### ### ### ### **DRY HEAT:** **Red heat:** **Flaming:** **Incineration:** **Hot air oven:** ### **MOIST HEAT:** ### At temperature below 100^o^C: - [Pasteurization:] - [Vaccine bath]: - [Serum bath]: At temperature 100oC: - [Boiling]: - [Steam at 100^o^C]: ### At temperature above 100^o^C: 3. **SONIC AND ULTRASONIC VIBRATIONS**: 4. ### **RADIATION:** - **Non-ionizing rays**: - **Ionizing rays**: 5. **FILTRATION:** ### **Different types of filters are:** **[CHEMICAL METHODS OF DISINFECTION:]** --------------------------------------------------- **Classification of disinfectants:** ------------------------------------ 1. Based on consistency 2. Based on spectrum of activity 3. Based on mechanism of action **Experiment 3 Date:** **Aim:** To study the minimizing liquid delivery Risk: Pipetting technique as a source of error For laboratory quality assurance, all critical tasks must be standardized, including liquid handling. This mainly focuses on pipette calibration, repair & maintenance, but rarely includes protocols on verifying operator techniques. Poor pipetting skills can result in inaccurate delivery volumes & alter results. Laboratory technicians as well as students must be trained on proper pipetting skills in order to minimize error & facilitate data comparability. Otherwise data integrity may be at risk. [Common pipetting errors include: ] Failure to Pre-Wet Pipette tip, Choosing the wrong pipette mode, working too quickly, Pipetting at an angle etc. For any class, the ability to measure learning & proficiency is essential, for these proper hands on coaching is required. [Easy tips to improve pipetting technique include:] Pre-wet the pipette tip- Aspirate & expel any sample liquid at least three times before aspirating a sample for delivery. Pre wetting increases humidity within the tip thus reducing any variation in sample delivery. Immerse the tip to the proper depth during aspiration- Too little immersion, particularly can lead to air aspiration, while too much immersion can cause sample to cling to outside of the tip. Touching the container bottom with the tip may resist aspiration. Pause consequently after aspiration & Use consistent plunger pressure & speed- Leaving the tip still in liquid for about one second is required as it takes a moment for liquid in the tip to finish moving after the plunger stops, so failure to do so will cause the volume to be too low. Pull the pipette straight out- During sample aspiration always hold the pipette vertically & avoid touching the sides of the container. Touching the sides of the container can cause wicking & a loss of volume due to the effect of surface tension. Use the appropriate Pipette- It is important to use a pipette with a volume range closest to the volume you plan to aspirate and dispense. Minimize pipette handling- Hold the pipette loosely. Draw the liquid sample to proper mark and release slowing. Return it to the pipette stand or set it down when you are not pipetting. **[Materials required:]** Pipette- 1ml, 2ml, 5ml, 10m etc., Glass measuring cylinder, glass beakers, Distilled water. **[Method:]** 1. Rinse properly all the glassware to be used. In a glass beaker take distilled water as a sample to be pipetted. 2. Using each pipette (1ml, 2ml, 5ml, 10ml, etc) draw distilled water to fill the glass cylinder with the appropriate volume mentioned. 3. Now count the times you have pipetted to get the final volume required. (For example, Required volume is 20ml and you are using 1ml pipette, so you have to pipette out 20 times) 4. Also note the volume showing on the glass measuring cylinder as cylinder is kept as a constant parameter to check pipette error. 5. Repeat the procedure thrice to get triplicate readings. 6. Calculate the average error observed for each pipette. 7. Compare the pipettes for error rate and pipette handling. **Observations:** Result and Conclusion: **Experiment 4 Date:** **Aim:** To study various types of culture medium used in microbiology **Bacterial culture media** **History**: **Classification of bacterial culture media on the basis of consistency** 1. Solid medium 2. Semisolid medium 3. Liquid (Broth) medium **Classification of culture media on the basis of composition** 1. Natural or Emperical media: 2. Synthetic or chemically defined medium 3. Non synthetic or chemically undefined medium **Classification of Bacterial Culture media on the basis of purpose/ functional use application** **Types of media** \(1) General purpose media. \(2) Anaerobic media. \(3) Transport media. \(4) Enriched media. \(5) Selective media. \(6) Differential media. \(7) Selective/Differential media. \(8) Assay media. **Experiment 5 Date:** **Aim:** To prepare solid media using Nutrient Agar **Introduction:** **Nutrient Agar** is a widely used general-purpose medium that supports the growth of a broad range of microorganisms, including bacteria and fungi. It\'s one of the most common culture media used in microbiology laboratories for routine cultivation, isolation, and maintenance of microbial cultures. Composition of Nutrient Agar: ### ### Properties and Uses: **Materials Required:** **Chemicals/Reagents: Nutrient medium, Agar powder, Distilled water.** **Glassware/Plastic ware:** Glass Flask (200 ml), test tubes. **Equipment:** Weighing machine, pH meter, Autoclave. **Miscellaneous:** Spatula, **Cotton plugs, Newspaper, B**unsen burner. **Procedure for preparation:** This protocol describes how to make 1 L of Nutrient agar for the culturing of bacteria on plates.  a. b. c. d. e. f.  **Results of Preparation of Media** Description of non-sterilized media: **Results of Preparation of Media** Description of non-sterilized media: **Experiment 6 Date:** **Aim:** To prepare agar slants using Nutrient medium **Introduction:**  ======================================================================= **Materials Required:** **Chemicals/Reagents:** Nutrient-Agar, Distilled water. **Glassware/Plastic ware:** Glass Flask (200 ml), test tubes. **Equipment:** Weighing machine, pH meter, Autoclave. **Miscellaneous:** Spatula, **Cotton plugs, Newspaper, B**unsen burner. **Procedure for preparation:** We prepare slants by preparing agar in a beaker, distributing it into tubes, sterilizing the capped tubes, and laying them at an angle to make a slanted surface as they cool. 1. Determine the volume of agar needed. We have found that 7 ml per 16 x 125 mm tube gives us a slanted surface with good surface area and a "butt" about 1 inch deep. Multiply the number of tubes needed by 7 ml and add 10% for waste. NOTE: You may be able to tilt a rack so that tubes need not be laid out individually. 2. Place the desired number of tubes in a rack and obtain an equal number of screw caps. Weigh out and mix the desired volume of agar in a beaker. The beaker should be close to double the volume of agar to prevent spilling. 3. Add a stir bar, place the beaker in a microwave, and heat until the agar begins to foam. Watch carefully so that the agar does not boil over. 4. Wearing gauntlet gloves for protection, carefully remove the beaker to a stir plate and stir to make a uniform mix. If the agar is not completely dissolved, heat some more. **\*\*\*CAUTION\*\*\* Hot agar that appears inactive may be superheated and may boil over violently if disturbed. Handle with care, keeping the face away from the agar and bare skin protected.** 5. While stirring to keep the agar mixed, use a large syringe (w/o needle) to distribute the required volume into each tube. 6. Place a cap on each tube but do not screw them down. That way the tubes are able to vent. 7. Sterilize on a liquid cycle (cycle 2 for our autoclave), remove the rack safely, then tilt the tubes to allow the agar to cure. **Results of Preparation of Media** Description of non-sterilized media: **Experiment 7 Date:** **Aim:** To study basic culture technique: Streak Plate **Introduction:** **Principle of Streak Plate Method** **Materials Required:** **Chemicals/Reagents: Luria-Agar and Nutrient Agar, Distilled water.** **Glassware/Plastic ware:** Glass Flask (250 ml), test tubes. **Equipment:** Weighing machine, pH meter, Autoclave, Nicrome loop. Sample Culture: *E. coli* pure culture and soil sample (1gm) **Miscellaneous:** Spatula, **Cotton plugs, Newspaper, B**unsen burner, marker/labelling tape etc. **Procedure or Protocol of Streak Plate Method** The general procedure of the streak plate method can be summarized as: 1. Arrange all the requirements, sterilize the work surface, and allow all the samples and media to come to room temperature if were refrigerated. 2. If the sample is very concentrated then dilution can be helpful to get the isolated colonies. (But it is not compulsory as the sample will be diluted during the streaking process.) 3. Sterilize the inoculating loop by flaming and allow it to cool. Pick a small portion of the isolated colony. (if the sample is in the suspension then take a loopful of the sample) The inoculating procedure is different according to the method of streaking, let us deal with each type: **Quadrant Streaking Procedure** 1. Lift the Petri plate in your left hand and hold it at an angle of 60°. (if you are left-handed, hold the plate in your right hand) 2. The sample is spread over about 1/4th of the media in the Petri plate from the rim to the center of the plate using a rapid, gentle, back and forth motion. (For ease, a beginner can draw two diameters intersecting each other diagonally at the back of the petri dish to divide the media into 4 equal sections) 3. Re-flame the loop and allow it to cool. Turn the Petri plate by 90° anticlockwise, and place the loop to the last streaks of the first quadrant. Move the loop back and forth to spread the inoculum over the last half of the streaks in the first quadrant into the empty second quadrant. (Be sure not to move the loop to the streaks in the first half of the first quadrant.) 4. Repeat the process (iii) for streaking the third quadrant and the fourth quadrant. 5. For the fourth quadrant similar step can be followed. However, many people prefer to draw a few (6 to 7 streaks) well-separated streaks by touching the second half of streaks in the third quadrant. Also, some prefer to make the final streak in a zigzag fashion making a tail. 6. (In a discontinuous fashion, the loop is sterilized after streaking each quadrant. In a continuous fashion, there is no need to flame the loop after streaking each quadrant. But, this is preferred only if the sample is very dilute.) 7. Incubate and observe the plates after 2 days. **T-Streaking Procedure** 1. Lift the Petri plate in your left hand and hold it at an angle of 600. (if you are left-handed, hold the plate in your right hand) 2. The sample is spread over about 1/3rd of the media in the Petri plate from the rim to the center of the plate using a rapid, gentle, back and forth motion. (For ease, a beginner can draw a letter "T" at the back of the petri dish to divide the media into 3 sections) 3. Re-flame the loop and allow it to cool. Turn the Petri plate by 900 anticlockwise, and place the loop to the last streaks of the first quadrant. Move the loop back and forth to spread the inoculum over the last half of the streaks in the first quadrant into the empty second quadrant. (Be sure not to move the loop to the streaks in the first half of the first quadrant.) 4. Repeat the process (iii) for streaking the third quadrant. As in quadrant streaking, you can follow any one of the streaking patterns at the 3rd quadrant. 5. Incubate and observe the plates after 2 days. **Observations:**