This document provides an overview of fundamental microbiology principles including definitions of microbiology and microorganisms. It describes bacteria and viruses, comparing their structures. Methods for isolating pure cultures, staining bacteria, and different staining techniques like Gram staining and acid fast staining are outlined. Key points covered include how Gram staining is used to classify bacteria as gram positive or negative based on cell wall structure and how acid fast staining identifies Mycobacterium species by their waxy cell walls.

1. Lecture No. 01
FUNDAMENTAL
PRINCIPLES
OF
MICROBIOLOGY
▪ By
▪ Mr. Sagar K. Gangurde
▪ M. Pharm (PHARMACOLOGY)
▪ Lecturer
▪ SGSS College of Pharmacy, Manur, Kalwan.
FUNDAMENTAL PRINCIPLES
OF
MICROBIOLOGY

2. 1 DEFINITION OF MICROIOLOGY
2
3
4
DEFINITION OF MICROORGANISM
CLASSIFICATION OF MICROORGANISM
STRUCTURE OF BACTERIA & VIRUS
5
6
7
ISOLATION OF PURE CULTURE
STAINING OF BACTERIA
TYPES OF STAINING

3. DEFINITION OF MICROBIOLOGY
 The word Microbiology is derived from-
Micros – Small
Bios – Life
Logos – To study or science
 Microbiology is the branch of science deals with the study of living micro-organisms.
Eg., Bacterial, Virus, Fungi etc.
 The study includes structure, function, reproduction and multiplication of micro-organisms.

4. DEFINITION OF MICRO-ORGANISMS
 These are the small groups of living organisms which can not be seen by
naked eyes and studied under microscope.
Eg., Bacteria like streptococcal, pneumococcal, salmonella typhi.
Virus like DNA or RNA virus, HIV.

6. BACTERIA
 These are the member of a large group of unicellular
(Prokaryotic) microorganisms which have cell wall
but lack of cell organelle like Golgi apparatus,
Mitochondria etc.
 Size: - 1 – 5 micron.
 Shape: - Cocci, Spherical, Rod-shaped, Spiral, Thread
shaped…..

7. VIRUS
 These are non cellular, ultramicroscopic highly infectious agent and posses only one type
of nucleic acid either DNA or RNA surrounded by protein (protective) coat called, Capsid.
 Size- 0.02 to 0.2 micron.
 Shape – As in image

8. Difference between Bacteria & Virus

9. Isolation of Pure Culture
 Growth of microbes on laboratory medium is known as Culture. A culture which may contains only one
species of microbe is called a pure culture and one which consist of several species is called mixed
culture.
 It is very difficult to obtain pure culture of bacterias in nature because they exist as mixed culture. To obtain
organisms in pure culture various techniques are used-
1. Streak plate method
2. Pour plate method
3. Spread plate method
4. Micromanipulator
5. Roll tube method

10. Staining Methods
 Stains are the organic dyes used for staining the micro-organisms.
 For ex, Crystal violet, methylene blue, safranin etc.
 Purpose of Staining: -
1. For greater visualization of cells.
2. For study of their structures.
3. To differentiate the cells.
4. To inhibit the growth of some organism so the others can be visualized.

11. Types of Staining
Simple Staining Differential Staining
Gram’s Staining
Acid Fast Staining

12. Simple Staining
 It is also called as Monochrome technique. In this method only one stain is used. It used to study morphology i.e.
size, shape and arrangement of microbes.
Prepare a smear and fixed on slide
Add stain for 30 sec to 3 min using
methylene blue
Wash with cool water
Air dry and examine under oil immersion
lens

13. Differential Staining
 Gram’s Staining: -
A differential staining technique used to classify bacteria i.e. gram positive or gram negative and their specific
structure.
Gram staining was discovered by a Danish Physician “Hans Christian Gram” while working in Berlin in
1883 and later procedure published in 1884. Hence, it is called Gram’s staining.
 Requirements: - Staining reagents like
1. Crystal violet- Primary Stain
2. Gram’s Iodine- Mordant - fixative agent
3. Acetone 95% or Alcohol- Decolorizer
4. Saffranine / dilute carbol fuchsin counter stain

14. Gram’s Staining
 Crystal Violet: - All bacteria takes crystal violet so all are appears violet colour.
 Iodine: - Crystal violet-iodine (CV-I) complex is formed.
 Acetone: - Bacteria with high lipid content loose CV-I complex and appears
colourless but bacteria with less lipid content retains CV-I complex and appears
violet.
 Saffranine: - Only colourless bacteria takes saffranine and appears pink

15. Procedure

16. Observation
If the bacteria shows violet or
purple then indicates Gram
Positive.
Eg., E. Coli, Pneumococci
If the bacteria shows pink or red
then indicates Gram Negative.
Eg., S. typhi, H. Influenzae

17. Acid fast staining technique
 This technique was discovered by the scientist Zeihl & Neelson,
 This technique is used to identify all the separation of Mycobacterium Group members from the others.
 It means it is used to identify the acid-fast micro-organism like Mycobacterium tuberculosis,
Mycobacterium Leprae etc.
 Acid –fast microorganism are characterized by wax like nearly impermeable cell walls means they
contain mycolic acid and large amount of fatty acids, waxes and complex lipids.
 Acid-fast organisms are highly resistant to disinfectants because of the cell wall is so resistant to most
compounds. Acid-fast microorganism required a special staining technique i.e. called Acid-fast staining
method.

18. Acid fast
staining
technique
For 10 Sec
For 30 Sec
Or Malachite green
20% Sulphuric
Acid for 1 min.

19. Observation
Those bacterias appears pinkish red
are Acid-fast bacteria
Those bacterias appears blue or
green are Non-Acid fast bacteria

20. Bacterial Structure

21. Virus Structure
1. Enveloped Icosahedral 2. Enveloped pliable helix

22. 22
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