Artificial Intelligence use in Pharmaceutical Industry Microbiology Lab

Prions

Prions 

Elephantiasis explained: A guide to Lymphiatic Filariasis

 

 

Validation of Microbial Limit Test Method

 Microbial Limit Test 

Purpose

• The procedure applies to testing the microbiological quality of raw materials and batches of non-sterile pharmaceutical products such as tablets, capsules, ointments, and semi-solid dosage forms.

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Materials and Equipment

• Personal protective equipment: Head cap, full-sleeve apron, slippers, disposable sterilized gloves, safety goggles, liquid hand sanitizer (70% IPA).
• Media and solutions for microbial testing include:
  • Soyabean Casein Digest Agar, Sabouraud Dextrose Agar, Mac Conkey Broth/Agar, XLD Agar, Rappaport Vassiliadis Salmonella Enrichment Broth, Cetrimide Agar, Mannitol Salt Agar, GN Broth.
• Instruments: Incubator, autoclave, colony counter, microscope.
• Testing environment: Laminar Air Flow or Biosafety Cabinet.

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Procedure Overview

Sample Preparation

• Water soluble products: Dissolve 1 g or dilute 1 ml in Soyabean Casein Digest Medium (SCDM), volume adjusted to 10 ml (Solution A).
• Water insoluble products: Disperse 1 g or dilute 1 ml in SCDM with 1 g/L Polysorbate 80, volume adjusted to 10 ml (Solution A).
• Fatty products: Homogenize 10 g or 10 ml sample with 5 g sterilized Polysorbate 80, heat if necessary (≤40°C), add buffered sodium chloride peptone solution to total 100 ml (Solution A).
• Alternative diluents: Phosphate buffer pH 7.2 or buffered sodium chloride peptone solution pH 7.0 may be used for all product types.

Inoculation and Controls

• Add prepared sample to test media.
• Prepare positive controls with ≤100 cfu of inoculum.
• Prepare negative controls without sample or inoculum.

Inactivation of Antimicrobials

• If antimicrobial substances are present, inactivate using Polysorbate 80 (30 g/l), Lecithin (3 g/l), Sodium Lauryl Sulphate (4 g/l), or dilution techniques.

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Microbiological Tests

1. Microbial Enumeration Tests

• Total Aerobic Microbial Count (TAMC):

  • Sample aliquots plated in Soyabean Casein Digest Agar, incubated at 30-35°C for 3-5 days.
  • Controls: Bacillus subtilis ATCC 6633 (positive), no inoculum (negative).
  • Colony forming units (cfu) counted and calculated per g or ml of product.

• Total Yeast and Mould Count (TYMC):

  • Sample aliquots plated in Sabouraud Dextrose Agar, incubated at 20-25°C for 5-7 days.
  • Controls: Candida albicans ATCC 10231 (positive), no inoculum (negative).
  • Colony counts are reported similarly to TAMC.

Acceptance Criteria for Enumeration Tests ( as per IP 2018):

Product Type	TAMC (cfu/g or ml)	TYMC (cfu/g or ml)
Non-aqueous oral preparations	≤ 10³	≤ 10²
Aqueous oral preparations	≤ 10²	≤ 10¹
Raw materials & products	As per individual specifications

2. Tests for Specified Organisms

For each specified microorganism, the procedure involves sample enrichment, selective subculture, incubation, and colony identification based on morphology and color. Positive and negative controls are run simultaneously.

Organism	Sample Prep & Enrichment	Selective Medium & Incubation	Colony Characteristics	Compliance Criteria
Escherichia coli	1 g/1 ml in SCDM, incubate 18-24h at 30-35°C	Mac Conkey broth (42-44°C, 24-48h), then Mac Conkey agar (30-35°C, 18-72h)	Pink, non-mucoid colonies	Absent per g or ml (or as per specification)
Salmonella spp.	10 g/10 ml in SCDM, incubate 18-24h at 30-35°C	Rappaport Vassiliadis Salmonella Enrichment Broth (30-35°C, 18-24h), XLD agar (30-35°C, 18-48h)	Red colonies with/without black center	Absent per 10 g or 10 ml (or as per specification)
Pseudomonas aeruginosa	Same as E. coli enrichment	Cetrimide agar (30-35°C, 18-72h)	Greenish colonies	Absent per g or ml (or as per specification)
Staphylococcus aureus	Same as E. coli enrichment	Mannitol Salt Agar (30-35°C, 18-72h)	Yellow/white colonies with yellow zone	Absent per g or ml (or as per specification)
Shigella spp.	Same as Salmonella, then 1 ml to GN Broth (30-35°C, 24-48h)	XLD Agar (30-35°C, 24-48h)	Red translucent colonies without black center	Absent per 10 g or 10 ml (or as per specification)

Annexure - Media Details (per sample)

Day	Media	Volume Prepared	Purpose
Day I	Soyabean Casein Digest Agar (SCD Agar)	100 ml	TAMC
	Sabouraud Dextrose Agar	100 ml	TYMC
	Soyabean Casein Digest Medium / Buffered Peptone Water pH 7.0	10 ml	Diluent for TAMC and TYMC
	Soyabean Casein Digest Medium	10 ml	Sample prep and pre-incubation for E. coli, S. aureus, P. aeruginosa
	Soyabean Casein Digest Medium	200 ml	Sample prep and pre-incubation for Salmonella and Shigella
Day II	Mac Conkey Broth	300 ml	Enrichment of E. coli
	Rappaport Vassiliadis Salmonella Enrichment Broth	30 ml	Enrichment of Salmonella
	Gram Negative Broth	300 ml	Enrichment of Shigella
	Cetrimide Agar	75 ml	Enumeration of P. aeruginosa
	Mannitol Salt Agar	75 ml	Enumeration of S. aureus
Day III	Mac Conkey Agar	75 ml	Enumeration of E. coli
	XLD Agar	75 ml	Enumeration of Salmonella
	XLD Agar	75 ml	Enumeration of Shigella

Key Insights

• The Microbial Limit Test (MLT) ensures safety and quality of non-sterile pharmaceutical products through quantitative and qualitative microbial assessments.
• It integrates aseptic techniques, sample-specific preparation methods, and well-defined acceptance criteria aligned with global pharmacopeial standards.
• The procedure includes controls and inactivation methods to manage potential antimicrobial interference in samples.
• Rigorous testing for specified pathogens (E. coli, Salmonella, P. aeruginosa, S. aureus, Shigella) ensures compliance with safety standards.
• Documentation and traceability are emphasized via specific forms and revisions, supporting quality assurance and regulatory compliance.

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This summary encapsulates the full scope of the microbial limit test procedure as detailed in the source document without any external assumptions.

References

• USP 41
• Indian Pharmacopoeia (IP) 2018
• European Pharmacopeia 9.0
• British Pharmacopoeia (BP) 2020

History of Microbiology Application in Nepal: “10 Unknown facts”

History of  Microbiology style="color: #2b00fe;">in Nepal: “10 Unknown facts” 

 Microbiology may seem like a science reserved for laboratories and scientists, but in Nepal, it’s a fundamental part of daily life—embedded in traditions, food, health remedies, and even farming practices. At its core, microbiology explores the invisible realm of microorganisms—bacteria, viruses, fungi, and protozoa—that play a vital role in shaping our environments and health. While Louis Pasteur is known as the father of microbiology for his groundbreaking discoveries, countless Nepalese have been harnessing the power of microbes for centuries, often without realizing the science behind their customs. 

One classic example is the tradition of turning milk into curd, a staple in Nepalese households. By simply adding a spoonful of previous curd to fresh milk, families rely on beneficial bacteria to thicken the milk over several hours, creating a nutritious food that aids digestion and boosts gut health. This simple act of fermentation not only preserves milk but also enhances its nutritional value—a perfect synergy of tradition and science. 

                                        Fig: Curd preparation using curd itself as starter culture 

Another age-old practice is the making of Gundruk, a beloved fermented dish made from leafy greens such as mustard or radish leaves. Villagers pack greens into containers and allow natural bacteria to ferment them, resulting in a tangy, flavorful food rich in probiotics. Gundruk is especially important in rural diets, providing essential nutrients during lean seasons and contributing to a healthy digestive system.

                                                        Fig: Gundruk 

Bamboo shoots, known as Tama, undergo a similar transformation. Through fermentation, potentially harmful compounds are broken down, and the shoots become both safe and delicious. This process is a testament to how microbial action can turn raw, sometimes inedible materials into cherished delicacies, all while enhancing their shelf life. 

                                           Fig: Tama prepared from Bamboo shoot

 Even in agriculture, microbiology plays a quiet but crucial role. When processing jute plants, farmers submerge them in water, allowing natural microbes to break down the tough fibers. This process, called retting, makes the fibers pliable for weaving into ropes and mats. The understanding of timing and water conditions, passed down through generations, shows an intuitive grasp of microbial processes.

                                          Fig: Jute plant for biodegradation under water 

In beverage-making, the traditional drink Chayang is brewed from grains mixed with marcha, a starter culture rich in the yeast Saccharomyces cerevisiae. The yeast fuels fermentation, transforming starches into alcohol and giving the drink its distinctive taste. This homemade brewing process not only provides a source of social enjoyment but also exemplifies a deep rooted knowledge of fermentation.

                                         Fig: Prepared Chayang using grains and marcha 

Folk remedies in Nepal often draw upon microbial principles as well. For instance, egg whites are sometimes used to cover wounds, creating a natural, protective barrier that helps prevent bacterial infection and promotes healing. While modern medicine offers advanced solutions, these traditional approaches are still valued in rural areas.

                                                 Fig : Wound Treatment using egg white

 Jute plants continue to be useful even after harvest. The leftover plant matter is plowed back into the fields, enriching the soil as it decomposes. This practice harnesses the power of soil microbes to recycle nutrients, enhancing soil fertility and supporting sustainable agriculture—a vital strategy for smallholder farmers.

                                                     Fig: Jute plant in field 

Food preservation is another area where microbiology quietly works its magic. Salt is widely used to make pickles, such as tangy mango achar. The high salt concentration inhibits the growth of spoilage-causing microbes while allowing beneficial bacteria to flourish, ensuring the pickles last for months without refrigeration. 

                                                   Fig: Mango Pickle Preserved using salt. 

High in the Himalayan mountains, communities collect Yarshagumba, a rare fungus known scientifically as Ophiocordyceps sinensis. Revered as a natural remedy, it’s believed to strengthen immunity and vitality. While modern science is still unraveling its health benefits, generations of Nepalese have trusted in its restorative powers, showcasing the intersection of traditional knowledge and microbiology.

                                                      Fig: Yarshagumba

 Lastly, the herb Chirata (Swertia chirayita) is used to treat skin diseases, thanks to its antimicrobial properties. Local healers have long applied Chirata extracts to skin ailments, relying on the plant’s ability to inhibit harmful microbes and promote healing—a testament to the wisdom embedded in folk medicine.

                                                   Fig: Use of chirata for skin disorder 

These examples reveal that microbiology in Nepal is not confined to academic textbooks or research labs. Instead, it is woven into the fabric of everyday living—found in kitchens, fields, and medicine chests. By intuitively using microorganisms, Nepalese people have developed ingenious ways to improve health, preserve food, and sustain their environment, proving that the unseen world of microbes has a very visible impact on life.

Seroprevalance of Dengue virus infection

 This video is generated from my research article  Seroprevalence study of dengue virus infection

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July 2025