Pharmaceutical Microbiology: A Laboratory Manual

By Dr. K. Srisailam and G. Shyam Prasad

Pharmaceutical Microbiology is an applied branch of Microbiology which deals with the study of microorganisms associated with manufacturing and quality control of pharmaceutical products. The present book “Pharmaceutical Microbiology: A Laboratory Manual” meets the syllabus designed by the Pharmacy Council of India and is written with a holistic approach encompassing various topics of Experimental Pharmaceutical Microbiology. This manual covers nearly 50 experiments based on General and Pharmaceutical Microbiology which would serve as a guide to B-Pharmacy students of all the Indian Universities. The main aim of writing this book is to cover theoretical and experimental parts of all the topics included in the curriculum for Experimental Pharmaceutical Microbiology given in the revised syllabus of B.Pharm students. The various concepts and phenomenon have been explained in simple terms with the help of appropriate diagrams. The experimental methods are tailor made to meet the modest facilities available in many of the colleges and universities within the country.

• Language: English
• Publisher: BSP BOOKS
• Release date: Nov 3, 2019
• ISBN: 9789388305020

Book preview

MICROBIOLOGY LABORATORY:

BASIC RULES AND REQUIREMENTS

Chapter 1

Pharmaceutical Microbiology and Basic Rules of Laboratory

Pharmaceutical Microbiology

Microbiology is a branch of biology which deals with the study of very minute living organisms such as bacteria, protozoa, fungi and similar organisms that can't be seen with the naked eye. Phannaceutical microbiology is an applied branch of microbiology which is responsible for the production of antibiotics, enzymes, vaccines, vitamins and other pharmaceutical substances. It also deals with microorganisms which contaminate pharmaceutical products, minimizing the number of microorganisms in a process environment, excluding microorganisms and microbial by-products like exotoxin and endotoxin from water and other starting materials. It ensures that medications do not contain harmful levels of microbes- such as bacteria, yeast and molds. It mainly focuses on the manufacturing techniques, process controls, and finished product attributes that limit the harmful effects of microorganisms on the drug product. Pharmaceutical products can save lives and bring back the health of patients, but what if these products are contaminated? The presence of a microbial contaminant in pharmaceutical products can reduce or even inactivate the therapeutic activity of the products and has the potential to adversely affect patients taking the medicines. The contaminating microorganisms may cause spoilage of the product with loss of its therapeutic properties and, if they are pathogenic, serious infections can arise. Furthermore, the presence of bad bugs in pharmaceutical products can lead to costly product recalls resulting in financial and image losses, loss of product sales, decreased customer confidence, and in many cases, legal proceedings.

Thus, it is important to know the microbial content of all dmgs and medicines, whether they are sterile or non-sterile and to implement strict microbial controls to ensure that the final products are consistent, safe, effective and predictable.

A. Microbiology Lab Practices and Safety Rules

1.   Leave your footwear in the rack provided before entering microbiology lab and wear lab footwear available at the entrance of the lab.

2.   Familiarize yourself with the location of instruments and safety equipment in the lab (e.g., eye-wash station, first aid kit etc)

3.   Wash your hands with disinfectant soap when you arrive at the lab and wear lab coat available inside the rack provided at the entrance of the lab.

4.   Absolutely do not eat drinks or smoke in the laboratory. Even do not put anything in mouth such as pencils, pens, labels, or fingers. Do not store food in areas where microorganisms are stored.

5.   Disinfect work areas before and after use with 70% ethanol or propanol.

6.   Ensure that all the instruments viz. refrigerator, incubator, oven, laminar air flow-, autoclave are in proper working conditions. And also check for availability of chemicals before starting the w ork.

7.   Long hair should be tied back to minimize contamination of cultures and fire hazards

8.   Protect yourself from exposure to eyes and skin to UV light by wearing goggles and clothing respectively.

9.   All cultures and prepared solutions should be labeled or marked clearly w ith the date of preparation.

10.   Before using any microbial culture ensure that you are using the right culture.

11.   Wear gloves and mask when working with pathogenic microbial cultures.

12.   Aseptic conditions should be strictly followed at all times to avoid contamination.

13.   If any culture has to be used, please check for purity and see that further stock of culture is available for future work.

14.   Never pipette out broth cultures or bacterial suspensions in saline, with the mouth.

15.   Always keep culture tubes in upright position in a rack or basket

16.   If live culture is spilled, cover the area with a disinfectant for 15min and then clean with 70% isopropyl alcohol or ethanol.

17.   Label all the culture plates, tubes properly before starting an experiment.

18.   Inoculating loops and needles should be flame sterilized in a Bunsen burner before you laying them down after inoculation.

19.   Wear disposable gloves when working with potentially infectious microbes or samples. If you are working with a sample that may contain a pathogen, then be extremely careful to use a good bacteriological technique.

20.   Turn off Bunsen burners when not in use. Long hair must be restrained if Bunsen burners are in use.

21.   Dispose off all used agar and broth cultures after incubation period in a biohazard bag and autoclave it before discarding in the regular trash. Do not pour any tiling down the sink.

22.   In the event of personal injuries such as cuts or bums inform your instructor immediately as bacteria enter open wounds

23.   Dispose of broken glass in the broken glass container.

24.   Replace caps on reagents, solution bottles, and bacterial cultures. Do not open Petri dishes with cultures in the lab unless absolutely necessary.

25.   Always clean microscope before and after use. Clean lenses with lens paper.

26.   Decontaminate laboratory equipment and work surfaces with an appropriate disinfectant on a routine basis, and especially after spills, splashes, or other contamination.

27.   Wash your hands and place the apron and footwear at the place provided before leaving the lab.

Chapter 2

Basic Requirements of Microbiology Laboratory

INSTRUMENTATION

Chapter 3

Microscope

A microscope (microscope: micro - small scope - look) is an optical instrument consisting of a lens or combination of lenses for making enlarged images of minute objects that are too small to be seen by naked eye. A compound microscope has optical and mechanical portions.

I. Optical Portion:

The optical portion has two types of lenses for greater magnification and consists of the ocular and objective lens. The magnification is due to eyepiece and the objective lens.

a. The ocular lens (eyepiece) that one looks into

The ocular lens or the eyepiece is the lens present at the top that you look through. The image magnified by the objective lens is further magnified bv the ocular lens. The ocular lenses are usually 10X, 15X etc.

b. The objective lens or the lens closest to the object.

The objective lens consists of several lenses to magnify an object and project a larger image. Most of the compound microscopes have three objective lens.

This lens when coupled with a 10X eyepiece the total magnification becomes 1000X (100X times 10X).

Total magnification = Magnifying power of objective x Magnifying power of eye piece

II. Mechanical Portion: The non- optical portion (mechanical portion) consists of the following parts

a. Body Tube: A hollow tube which connects eyepiece to the objective lens through which light travels from the objective to the ocular.

b. Arm: C-shaped arm is present between the foot and body tube. It is meant for the support of the body tube.

c. Coarse and fine adjustment knobs: The coarse adjustment knob is located on the arm of the microscope which moves the stage up and down to bring the specimen into focus.

d. Fine adjustment knob: This is also located on the arm and is used to bring the specimen into sharp focus.

e. Revolving Nose piece: It is provided with several objective lenses of varying magnification and numerical aperture.

f. Stage: This is the horizontal surface upon which the slide is placed. The slide is held in place by spring loaded clips and moved around the stage by turning the geared knobs on the stage.

i.   Devices for controlling light: Optimum amount of light should be provided and the microscopes are provided with light just below the stage. The sources of light are usually sun or an electric bulb.

ii.   Condenser: It is located immediately under the stage and condenses the irregular beams of light into the specimen.

iii.   Mirror: It can be adjusted in different planes to reflect light from the sources. The mirrors may be with a plane or concave surface. A plane surface is used when the rays are parallel i.e. when the source is very distant. The concave surface is used when the rays are from a nearby source as the electric bulb.

iv.   Diaphragm: It is a strong rotating disk under the stage. The diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upwards