STUDY OF THE MICROSCOPE AND ITS PARTS

 

STUDY OF THE MICROSCOPE AND ITS PARTS

Objective: to familiarize oneself with the parts and functions of a microscope and develop skills in using a microscope for microscopic observations.

         Theory: Microscopes are essential tools used in scientific research and various fields of study. "Micro" refers to tiny, "scope" refers to view or look at. Microscopes are tools used to enlarge small objects to be observed and studied. Microscopes range from a simple magnifying glass to the expensive electron microscope. They enable the visualization of objects that are too small to be seen by the naked eye. A typical compound microscope consists of several parts, including an eyepiece, objective lenses, a stage, a condenser, and a light source. The eyepiece magnifies the viewer, while the objective lenses offer different magnification levels for the specimen. The stage holds the specimen in place, and the condenser focuses light onto the specimen. By adjusting the focus and observing the specimen under different magnifications, one can gather valuable information and make detailed observations.

            The microscope operates on the principle of magnification and resolution:

·       Magnification: The process of enlarging the apparent size of an object.

·       Resolution (Resolving Power): The ability to distinguish two closely spaced points as separate entities.

The total magnification of a microscope is given by:

Total Magnification=Objective Lens Magnification × Eyepiece Magnification

For example, if the objective lens has a magnification of 40x and the eyepiece has 10x, the total magnification is 400x.

Based on the working principle, microscopes are of the following types.

Requirements:

1.     Compound microscope

2.     Specimens (e.g. prepared slides)

3.     Lens paper or a clean cloth

4.     Pen or marker

Procedure:

1.     Set up the microscope on a clean, sturdy surface near a power source, ensuring proper illumination.

2.     Clean the microscope lenses (eyepiece and objectives) using lens paper or a clean cloth.

3.     Place the prepared slide on the microscope stage and secure it using the slide holder.

4.     Start with the lowest magnification objective (e.g.10x) and position the slide on the stage.

5.     Look through the eyepiece and adjust the focus using the coarse and fine adjustment knobs until the image becomes clear.

6.     Observe the specimen, noting any structures or features of interest.

7.     Switch to higher magnification objectives (e.g., 40x or 100x) to observe the specimen in more detail.

8.     After completing the observations, turn off the microscope and clean the lenses before storing it properly.

Result:

Draw a well-labelled diagram of a microscope

Conclusion: 

Through this practical activity, we successfully studied the microscope and its functionalities. We gained hands-on experience in preparing slides, adjusting magnification, and observing specimens under a microscope. The microscopic observations allowed us to visualize and appreciate the intricate details of various specimens. The skills acquired during this exercise will be beneficial for future scientific endeavors that involve the use of a microscope.

Precautions

·        Always hold the microscope with both hands—one on the arm and the other supporting the base.

·         Ensure the microscope is on a flat, vibration-free surface to avoid slipping or shaking.

·        Use only lens paper (not tissue or cloth) to clean objective and eyepiece lenses.

·        Avoid touching the lenses with your fingers.

·         Always begin with the lowest objective lens (10x) to locate the specimen before switching to higher magnifications.

·        After use, lower the stage and switch to the lowest objective lens to avoid damage.

·         Prevent overheating by switching off the light source when not in use.

·        Always cover the microscope when not in use to prevent dust accumulation.

·        Store in a dry place, as humidity can cause fungal growth on lenses.

References

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual (11th ed.). Pearson.

 

General Rules and Regulations in the Microbiology Laboratory

 

General Rules and Regulations in the Microbiology Laboratory

1.     Always wear a clean laboratory coat to protect clothes and skin from spills and contamination.

2.    Disinfect the working bench with an appropriate disinfectant before starting any work and after finishing the work to maintain a clean and safe environment.

3.     Maintain an aseptic zone:

a.      Use a Bunsen burner to flame-sterilize inoculating loops, needles, and other tools before and after use.

b.     Work near a flame or laminar airflow to prevent contamination.

c.      Avoid unnecessary movement or talking during inoculation.

4.     Ensure all instruments and materials are sterile before use to avoid contamination of samples.

5.     Clearly label all samples, cultures, and chemicals to avoid mix-ups and ensure proper handling.

6.     Dispose of all biological waste in designated biohazard containers. Autoclave or otherwise properly sterilize waste before disposal.

7.     Tie long hair back and avoid touching your face, hair, or personal items during experiments

8.     Never pipette by mouth. Use mechanical pipetting devices.

9.     Do not eat, drink, or chew gum inside the laboratory.

10.  Dispose of all biological waste properly:

a.      Use designated containers for contaminated materials.

b.     Autoclave infectious waste before disposal.

11.  Report any spills or accidents to the instructor/lab supervisor immediately.

12.  Do not remove any materials (cultures, reagents, glassware) from the lab without permission.

13.  Wear gloves, face masks, or safety goggles if required for specific procedures involving hazardous organisms or chemicals.

14.  Turn off burners and electrical equipment after use.

15.  Follow proper handwashing and decontamination protocol even after wearing gloves.

16.  Be aware of the location of emergency exits, fire extinguishers, eye wash stations, and first aid kits.

 

 

Pharmaceutical Microbiology and its importance

 

Pharmaceutical Microbiology and its importance

                                   Anup Bajracharya

Microbiology is the scientific study of microorganisms such as bacteria, archaea, algae, fungi, protozoa, viruses, and certain helminths. This field focuses on understanding their structure, functions, classification, and the methods used to either utilize or regulate their behavior. When the principles, methods, and knowledge of microbiology are specifically applied to pharmaceutical processes, it becomes known as Pharmaceutical microbiology.

Pharmaceutical Microbiology is the applied science concerned with the study of microorganisms that are involved in:

• the production of pharmaceutical products,
• ensuring their safety,
• maintaining their quality and sterility,
• and preventing microbial contamination during drug development and production.

Pharmaceutical Microbiology can be defined as the study of microorganisms that are pertinent to the production of antibiotics, enzymes, vitamins, vaccines, and other pharmaceutical products; it also incorporates the study of microorganisms that cause pharmaceutical contaminations, and degradation, deterioration and spoil of pharmaceutical raw materials and finished products.

Importance of Pharmaceutical Microbiology

Pharmaceutical Microbiology is a vital discipline that plays a key role in ensuring the safety, quality, and effectiveness of pharmaceutical products. It is essential across every stage of drug development and production.

1. Production of Antibiotics, Vaccines, enzymes etc

Microorganisms are used in the development and production of antibiotics vaccines, enzymes, monoclonal antibodies, etc. It ensures biosafety and purity of these biological products.

·    Production of antibiotics- The most important use is the production of antibiotics, two third of antibiotics are produced from microorganisms. The pharmaceutical microbiology concerns with the isolation of antibiotic producing microorganisms from natural environments such as soil or water and use them for production of antibiotics through the process of fermentation. Thus, Microbiology helps in strain selection, fermentation monitoring, and antibiotic potency testing.

Production of vaccines- A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

Vaccines are often made from killed or weakened (attenuated) microorganisms, or specific components like proteins or toxins. Pharmaceutical microbiologists help to

• Select the right strain of bacteria or virus.
• Grow them in optimal culture media under controlled conditions (e.g., temperature, pH, aeration). Example: Using Salmonella typhi Ty21a strain for oral typhoid vaccine production.

 

·    Production of Enzymes-Microbial cells also produce intracellular and extracellular enzymes like amylase, proteases, lipases, invertase etc. Enzymes are collected either from the culture medium (extracellular enzymes) or from microbial cells (intracellular enzymes).

Examples- Amylase, protease, and lipase: Included in digestive formulations for patients with poor digestion. Streptokinase and urokinase helps to dssolve blood clots (used in heart attack treatment).Glucose oxidase is used in glucose biosensors for diabetics. DNA polymerases from Thermus aquaticus (Taq polymerase) is essential in PCR (Polymerase Chain Reaction) for genetic testing and disease diagnosis.

 

·     Production of Alcoholic products- Many microbial cells convert raw materials or substrates into valuable organic compounds such as butanol, ethanol, acetone etc. The production of alcoholic beverages and products is achieved through fermentation, a metabolic process in which microorganisms—mainly yeasts and bacteria—convert sugars into alcohol (ethanol) and other byproducts like carbon dioxide.

·      Production of Probiotics – Probiotics are live bacteria that may confer a health benefit on the host. Fuller in 1989 described probiotics as "live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance
Lactic acid bacteria (LAB) and Bifidobacteria are the most common types of microbes used as probiotics, but certain yeasts and bacilli may also be used. Probiotics are commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements.

 

2. Sterility Testing

Sterility testing is a critical quality control process used to ensure that sterile pharmaceutical products such as injectables, ophthalmic solutions, and surgical implants are completely free from any viable microorganisms. This test is especially important for products that are introduced directly into sterile areas of the body, like blood, eyes, or tissues, where even a single contaminating microbe can cause severe infection or sepsis. The process involves incubating samples of the product in specially prepared culture media, such as Fluid Thioglycollate Medium (FTM) and Soybean-Casein Digest Medium (SCDM), under controlled conditions for at least 14 days to observe microbial growth. For instance, an intravenous infusion must undergo sterility testing to confirm that it does not contain any bacteria or fungi before it can be released to the market.

3. Microbial Contamination Control

Microbial contamination control is essential for non-sterile pharmaceutical products like tablets, capsules, syrups, creams, and ointments, which may be exposed to the environment during manufacturing and packaging. This process involves regular environmental monitoring, personnel hygiene checks, and testing of raw materials, in-process materials, and finished products. The goal is to prevent the presence of objectionable microorganisms, such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella species, which can cause infections or degrade the product. For example, Pseudomonas aeruginosa in a topical ointment could infect wounds and delay healing, hence its presence is unacceptable in such formulations. Microbial contamination control ensures that the microbial content stays within acceptable limits and does not pose health risks to patients.

4. Microbial Limit Testing

Microbial Limit Testing (MLT) is used to determine the total number of viable aerobic microorganisms—both bacteria and fungi—present in a non-sterile pharmaceutical product. This test helps assess whether the microbial count falls within acceptable limits defined by pharmacopeias (like USP, BP, or IP). MLT consists of two components: Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC). Additionally, it includes specific tests to detect pathogenic organisms, such as E. coli, Salmonella, and Staphylococcus aureus. For example, a cough syrup might be tested for its total microbial load to ensure it contains fewer than 100 colony-forming units (CFU) per mL of bacteria and fewer than 10 CFU/mL of fungi, ensuring it is safe for oral consumption. MLT is crucial for maintaining the microbial quality of non-sterile products.

5. Preservative Effectiveness Testing (PET)

Preservative Effectiveness Testing, also known as antimicrobial preservative effectiveness testing, is performed to verify that the preservatives added to multi-use pharmaceutical products are effective enough to prevent microbial growth during storage and usage. This is especially important in products like eye drops, nasal sprays, creams, and syrups, which may be repeatedly exposed to air or come into contact with users. In this test, the product is intentionally inoculated with known microorganisms (e.g., Staphylococcus aureus, Candida albicans, Aspergillus brasiliensis) and observed over time to see whether the preservatives can eliminate or significantly reduce microbial growth. For instance, in a multi-dose eye drop, PET ensures that if bacteria are accidentally introduced during usage, they will not multiply and compromise the product's safety.

6. Plays a Role in Innovation

Pharmaceutical microbiology helps in the discovery of new antimicrobial agents and in developing rapid diagnostic techniques.

It is important for addressing antibiotic resistance and ensuring the continued effectiveness of medicines.

 Note-

The exploitation of microorganisms and their products has played an increasingly prominent role in the diagnosis, treatment and prevention of human diseases. The nonmedical uses are also of significance, Example, the use of bacterial spores (Bacillus thuringiensis) and viruses (baculoviruses) to control insect pests, the fungus Sclerotinia sclerotiorum to kill some common weeds, and improved varieties of Trichoderma harzianum to protect crops against fungal infections.

·    Diagnosis of diseases and treatment- Different tests are used to detect infectious microorganisms like ELISA, Widal test. Antimicrobial Susceptibility testing is mainly used for selection of antibiotics for the treatment of microbial infections.

·       Apart from drugs and bio products development, microbiology contributes towards quality control of a pharmaceutical laboratory. Regular environmental monitoring in manufacturing areas ensures early detection of contamination sources. Personnel hygiene monitoring ensures that staff do not introduce harmful microbes into clean areas.

References

1. Denyer, S. P., Hodges, N. A., & Gorman, S. P. (2004). Hugo and Russell's Pharmaceutical Microbiology (7th ed.). Blackwell Publishing.
2. WHO. (2002). Guidelines on Good Manufacturing Practices for Pharmaceutical Products. World Health Organization.

Acid-fast Staining

Perform acid-fast Staining for the given sputum sample

 
Objective: To detect the presence of acid-fast bacilli (AFB) such as Mycobacterium tuberculosis in a given sputum sample.

Theory:

Acid-fast staining is a differential staining technique commonly used to detect acid-fast organisms, such as Mycobacterium tuberculosis, which have a unique cell wall composed of waxy, lipid-rich material called mycolic acids. These bacteria retain the primary dye (carbol fuchsin) even after being washed with acid-alcohol, due to the presence of mycolic acid in their cell walls. Non-acid-fast bacteria get decolorized and take up the counterstain (methylene blue or malachite green)

The acid-fast staining or Ziehl-Neelsen staining method is based on:

• Staining the smear with carbol fuchsin while applying heat, which facilitates dye penetration.
• Decolorization with acid-alcohol, which removes the stain from non-acid-fast organisms.
• Counterstaining with methylene blue or malachite green to visualize non-acid-fast cells.

Acid-fast bacteria (AFB) appear red/pink rods, while non-acid-fast organisms and the background appear blue.

Requirements:

• Sputum sample
• Glass slide
• Bunsen burner
• Carbol fuchsin
• Acid-alcohol (3% HCl in ethanol or 25% sulfuric acid)
• Methylene blue
• Microscope
• Immersion oil

Procedure:

1. Take a small amount of sputum with an applicator stick and spread it evenly on a clean glass slide to make a thin smear. Allow it to air dry.
2. Pass the dried smear over a flame 2–3 times to heat fix the bacteria onto the slide.
3. Flood the slide with carbol fuchsin stain and keep it over steam for about 5 minutes
4. Continue heating for 5 minutes, ensuring that the stain does not dry out. Add more stain if necessary.
5. Remove the slide from the heat and allow it to cool for 2 minutes.
6. Rinse the slide gently with tap water to remove excess stain.
7. Decolorize the slide with acid-alcohol for about 1 minute
8. Rinse the slide with tap water to remove the acid-alcohol.
9. Counterstain the slide with methylene blue for 1 minute.
10. Rinse the slide with tap water and blot it dry using blotting paper.
11. Observe the slide under oil immersion microscopy using a 100x objective lens.
12. Record the presence or absence of acid-fast bacteria in the sample.

Observation:

S.N	Sample	Reagents used	Shape of bacteria	Color of bacteria	Inference

 

 Results: Acid-fast bacteria appear as red, rod-shaped cells against a blue background due to the counterstaining with methylene blue. Non-acid-fast bacteria appear blue.

Discussion:

Carbol fuchsin, the primary red dye, is lipid-soluble and, when heated, penetrates the waxy cell wall of acid-fast bacteria. Once inside, the dye binds tightly to the mycolic acid. When we apply acid-alcohol (decolorizer), acid-fast bacteria retain the red dye because their waxy wall prevents it from washing out. Non-acid-fast bacteria do not have this waxy layer, so the carbol fuchsin washes out easily. After decolorization, we apply a blue or green counterstain, which the non-acid-fast bacteria take up, making them appear blue or green under the microscope.

The presence of acid-fast bacilli (AFB) in a sputum sample indicates possible infection by Mycobacterium tuberculosis or other mycobacteria. The Ziehl-Neelsen technique is a crucial diagnostic tool, especially in developing countries with high TB prevalence. The specificity of the test is high, but its sensitivity can be limited if a bacterial load is low, so repeated samples or culture may be required.

 Conclusion:

The acid-fast staining procedure was successfully performed, and the presence or absence of acid-fast bacteria in the sample was determined. This staining technique is valuable for the identification of acid-fast organisms, particularly Mycobacterium tuberculosis, aiding in the diagnosis of tuberculosis and other related diseases.

Precautions:

• Always wear gloves, a mask, and a lab coat while handling sputum samples.
• Do not overheat the slide during heat fixation or staining.
• Use a biosafety cabinet while handling infectious samples.
• Dispose of all materials properly as per biosafety guidelines.

References:

1. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2. Cambridge University Press.
2. World Health Organization (WHO). (2013). Laboratory services in tuberculosis control.