Microbiology Laboratory Techniques: A Comprehensive Guide to Experiments and Procedures, Cheat Sheet of Pharmacy

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PRACTICAL GUIDE TO

PHARMACEUTICAL

MICROBIOLOGY

(BP307P)

For B. Pharmacy 3rd Semester As Per Latest PCI Syllabus for B. Pharmacy Course

… Alfred North Whitehead I would like to express my sincere gratitude and appreciation to all those who have contributed to the success of this endeavor. Their support, guidance, and encouragement have been invaluable throughout this journey. Writing a book is a journey that requires the support, encouragement, and inspiration of many individuals and institutions. I would like to extend my heartfelt gratitude to all those who have contributed to the creation of this book. First and foremost, I want to express my deep appreciation to my family for their unwavering support throughout this endeavor. I extend my heartfelt thanks to my academic advisors, Guide and mentor Dr. Rahul Pratap Singh for his guidance and expertise. Your wisdom and dedication have played a pivotal role in shaping my knowledge and skills. I am truly fortunate to have had the opportunity to learn from you. I am also thankful to Ms. Mahima Chauhan and Ms. Bhavna Yadav for offering their encouragement and understanding. Your camaraderie has made this endeavor a successful feat. I would like to acknowledge the support of my B. Pharmacy students Ms. Vidhi Mittal, Mr. Kartik Arora, Mr. Jaskaran Singh Pahawa and Mr. Manafa Paul Chuka for the support and efforts they have made to make this book a possible reality. I am indebted to the countless individuals who participated in this study, generously sharing their time and insights. Your contributions have enriched this work and advanced our understanding of the subject matter. I want to express my gratitude to Mr. Umakant for his support and contributions in finalizing the procedures of the contents. Lastly, I express my gratitude to all the faculty staff of Pharmacy Department of GD Goenka University for constant encouragement and support in continuing and achieving such endeavors. Thank you all for being a part of this journey. Your belief in me has been my motivation, and I am excited to see where the future leads with your continued support. With heartfelt thanks, Saurabh Shekhar Contents

S. no. Practical Title Page No.

1. To study different equipment and processing, e.g., B.O.D. incubator, laminar flow, aseptic hood, autoclave, hot air sterilizer,

microscopes used in experimental microbiology.

2. To carry out Sterilization of glassware and culture media. 29-
3. To prepare various buffers to be used in microbiology laboratory. 32-
4. To carry out preparation and sterilization of culture media in microbiology laboratory.

5. To carry out preparation Nutrient slab and agar slant culture of given sample of microorganisms.

6. To carry out sub-culturing of bacteria and fungus colonies. 40-
7. To carryout simple staining of given sample of microorganisms. 42-
8. To perform gram staining process of given sample of microorganisms.

9. To carryout simple staining of given sample of microorganisms. 49-
10. To carryout IMViC staining of given sample of microorganisms. 52-
11. To carryout motility determination of given sample of microorganisms by hanging drop method.

12. To carryout microbial analysis of given sample of water. 61-
13. To perform microbiological assay by the cylinder plate method. 63-
14. To perform isolation of bacteria by Streak Plate method of given sample of microorganisms.

15. To isolate and purify microorganism from given soil sample using Serial Dilution technique.

Aim 1: To study different equipments and processing, e.g., B.O.D. incubator, laminar flow, aseptic hood, autoclave, hot air sterilizer, microscopes used in experimental microbiology. Requirements: B.O.D. incubator, Laminar flow, autoclave, hot air sterilizer/hot air oven, Microwave oven, Centrifuge, VDRL Rotator, Mechanical Stirrer, Antibiotic Zone Reader. Theory/Procedure A) B.O.D. incubator:

According to specific incubation requirements, some BOD incubators are equipped with humidity control. These systems can add humidity to the room to maintain necessary humidity, which is important for certain applications such as plant growth or microbial culture.

5. Ventilation and Air Circulation: BOD incubators are designed with ventilation and ventilation systems to ensure even temperature distribution throughout the incubator. This prevents temperature variations and ensures that all samples in the chamber have the same conditions. 6. Insulation: Incubators are generally well insulated to reduce heat loss to the outside environment, ensuring energy efficiency and constant temperature control. 7. Control Panel: There is a user panel on the outside of the BOD incubator. This panel allows the user to set and adjust temperature, humidity, and other parameters as needed. B) Laminar flow Laminar air flow is a controlled, unidirectional, particle-free air flow used in many industries and laboratories, especially in applications that control contamination and sterile environments. Circulation is important. Laminar air flow instrumentation consists of a variety of equipment and monitoring tools to ensure proper operation and maintenance of laminar air flow systems.

Laminar air flow devices play an important role in control and sterilization in industries such as medicine, biotechnology, electronics manufacturing, healthcare and research laboratory.

1. HEPA (High-Efficiency Particulate Air) Filters: HEPA filters are the heart of laminar air flow systems. They remove airborne particles, including dust, bacteria, and other pollutants from incoming air. This filter has a minimum efficiency of 99.97% for particles 0.3 microns or larger in size. 2. Blower or Fan Unit: The blower or fan is responsible for drawing air from the environment and passing it through the HEPA filter. It creates a continuous, unidirectional flow of filtered air in a laminar (parallel) pattern in the work area. 3. Supply Plenum: A plenum device is a chamber that distributes filtered air evenly throughout the work area. Provides even laminar flow across the work surface. 4. Work Surface: A workspace is an area where processes, experiments or operations are carried out. It is designed to be smooth, non-porous and easy to clean to prevent contamination. 5. Exhaust System:

1. Horizontal Laminar Flow Cabinets (HLAF): The horizontal laminar flow cabinet has a work area where filtered air flows back to the user. They are frequently used in laboratories for activities such as microbiology, cell culture and assembly studies. 2. Vertical Laminar Flow Cabinets (VLAF): The vertical laminar flow cabinet is designed with low air flow. Air is filtered from the top of the cabinet and flows vertically into the work area. These cabinets are suitable for applications involving sensitive materials or samples that must be protected against contamination, such as semiconductor manufacturing or sterile compounding in the pharmacy. 3. Biological Safety Cabinets (BSC):

Biological Safety Cabinet is designed to protect the user and the environment. They have HEPA filters for visible air and have different levels of protection depending on the BSC category and type (Class I, II or III). BSC is used in microbiological research, laboratories and biological containment facilities.

4. Fan Filter Units (FFU): Fan Filter Unit is a device used to create air filters in clean rooms and control rooms. They are usually mounted on a grille on the ceiling or wall to ensure constant air flow. FFUs are mostly used in semiconductor manufacturing, aerospace and chemical industries. 5. Isolators: Isolators are closed systems that provide highly sterile control. They are used in the production and mixing of hazardous drugs, aseptic processes, and the examination of toxic substances. Insulators can incorporate laminar airflow systems into their designs. 6. Portable Laminar Flow Units: These are compact, portable laminar airflow systems that can be moved as needed. They are generally used in field applications or environments requiring a clean environment. C) Autoclave Autoclave is an important equipment used in many industries. and food processing, antibiotics, equipment and other products. This device uses high energy to kill bacteria and viruses. Working Principle of Autoclave The principle of the autoclave is that the pressure is proportional to the temperature. At atmospheric pressure, the temperature cannot rise above 100 degrees Celsius, so the equipment is used to increase the pressure so that the temperature of the water can rise above 100 degrees Celsius for sterilization, which is recommended in the autoclave. Standard parameters to achieve sterilization in autoclave is given below.

Schematic Diagram of Autoclave

1. Pressure vessel: The pressure vessel is the main compartment of the autoclave machine where the product needs to be sterilized. Designed to withstand pressure and temperature. Pressure vessels are usually made of stainless steel or other corrosion-resistant materials. 2. Air Tools: Autoclaves are equipped with electric or gas heating elements that create steam. These elements heat water to create steam used for disinfection. 3. Temperature Sensor: Autoclaves are equipped with a temperature sensor, usually in the form of a thermocouple or resistance thermometer (RTD). Sensors monitor the temperature in the pressure vessel and provide feedback to the control system. 4. Pressure Sensor: The pressure sensor or pressure sensor monitors the pressure in the autoclave chamber. They ensure that the chamber reaches and maintains the pressure required for sterilization. 5. Control System:

Control system is the brain of the autoclave. Allows the user to adjust temperature, pressure and sterilization time. The system controls heat and energy to meet the need.

6. Safety Valve: Safety valve is important to reduce the maximum pressure to prevent overpressurization of the autoclave. These are important safety features to prevent accidents. 7. Door Locking Mechanism: The autoclave door is equipped with a locking mechanism that will keep it closed during sterilization. Most autoclaves have interlocks that prevent the door from being opened while the sterilization chamber is under pressure or still hot. 8. Pressure Relief Valve: Pressure relief valve is another safety feature that relieves pressure in case of malfunction or overload. They help prevent damage to the autoclave and protect the user. 9. Water Reservoir: Autoclaves need a reservoir to create steam. Some autoclaves have an internal drain, while others are connected to an external drain. 10. Chamber Drain: The autoclave may have a drain to remove excess water from the chamber after the sterilization cycle. This prevents the product from getting wet and promotes drying. 11. Recorders and Loggers: Some autoclaves are equipped with loggers and loggers to record the sterilization process. This information is important for quality control and compliance purposes. 12. Display and Interface: Autoclave has a user-friendly and intuitive interface that allows the operator to monitor and control the sterilization process. They typically display temperature, pressure, and cycle progress. 13. A printer or data transfer: Many autoclaves have the ability to print sterile data or data transfer for storage and tracking purposes. 14. Cooling System (Optional):

7. Steam Sterilizers: Steam sterilizers are large autoclaves used in medical facilities, pharmaceuticals and biotechnology laboratories. Designed for high volume sterilization of surgical instruments, medical equipment and manufacturing equipment. 8. Pass-through Autoclaves: Pass-through autoclaves are designed for use in clean rooms and controlled environments. To maintain sterility, it has two separate rooms, one for loading and the other for unloading. 9. Front Loading Autoclaves: Front-loading autoclaves have a door at the front of the compartment that allows easy loading and unloading of products. They are frequently used in medicine and dentistry. 10. Top Loading Autoclaves: Stop autoclaves have a lid at the top of the chamber and are used to sterilize liquids, media, and small testing equipment. They are often found in research and commercial laboratories. 11. Benchtop Autoclaves: Benchtop autoclaves are compact and designed to be mounted on a bench. They are suitable for small sterile needs such as microbiology and research laboratories. 12. Portable Autoclaves: Portable Autoclaves are designed for office use and mobile clinics. They are compact, easy to carry and suitable for remote areas.

D) Hot air oven

Hot air oven, also known as forced convection oven or mechanical convection oven, is widely used for drying, curing and sterilization purposes in laboratories and various industrial applications. But they rely on hot air to cool in the oven. Heat exchangers are multi-purpose equipment used in drying, curing and other thermal treatments in various industries.

Fig.1. Schematic Diagram of Hot air oven Fig.1. Working of Hot air oven The main features often found in hot air ovens are:

1. Temperature controller: Temperature controller is an important part for the user to set and adjust the temperature of the oven. It monitors the temperature in the room and activates or deactivates the heating as needed to maintain the temperature. 2. Heating element:

12. Ventilation (optional): Sometimes, the heating stove will have a ventilation system that will release air or gases during heating to ensure operating safety. B) Microwave Oven A microwave oven is a household appliance that uses microwave energy to heat and cook food quickly and efficiently. Although most microwave ovens are designed for home use, there is also a built-in device that controls their operation. Fig.1. Schematic diagram of a microwave oven

Fig. 1. Instrumentation of microwave oven The main devices often found in microwave ovens are:

1. Magnetron: Magnetron is the heart of the microwave oven. It produces microwave energy at a specific frequency (usually around 2.45 GHz). This electricity is then supplied to the kitchen to heat the food. 2. Waveguide: The waveguide is a metal tube that carries microwave energy from the magnetron to the cooker. It ensures the distribution of microwaves within the oven. 3. Control Panel and User Interface: Control panel and user interface allows the user to adjust cooking time, power level and more. It usually includes buttons or a touch screen to operate the microwave. 4. Timer: Timer is used to set the cooking time. The microwave automatically turns off when the timer reaches zero. 5. Power control: Microwave ovens often have different power levels, allowing the user to adjust cooking usage. This control changes the microwave radiation emitted by the magnetron. 6. Turntable (optional): Most microwave ovens come with a turntable or rotating bowl, which helps distribute heat evenly throughout the food. It ensures that food remains balanced. 7. Temperature sensor (optional): Some microwave ovens are equipped with a temperature sensor that can measure the temperature of the food being cooked. This information can be used to adjust the cooking time and power level. 8. Safety Locking System: The microwave oven has a safety locking mechanism that prevents the microwave oven from operating when the door is open. This prevents exposure to microwave radiation. 9. Door and Latch: